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Identification of differentiation markers in normal and virally transformed avian hematopoietic cells

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Title:
Identification of differentiation markers in normal and virally transformed avian hematopoietic cells
Creator:
Liu, Juinn-Lin G., 1960-
Publication Date:
Language:
English
Physical Description:
xiv, 119 leaves : ill. ; 29 cm.

Subjects

Subjects / Keywords:
Antigens ( jstor )
Bone marrow ( jstor )
Bone marrow cells ( jstor )
Cell lines ( jstor )
Cells ( jstor )
Chickens ( jstor )
Erythrocytes ( jstor )
Stem cells ( jstor )
Tumors ( jstor )
Yolk sac ( jstor )
Antibodies, Monoclonal ( mesh )
Antigens, Differentiation ( mesh )
Birds ( mesh )
Cell Transformation, Viral ( mesh )
Department of Pathology and Laboratory Medicine thesis Ph.D ( mesh )
Disease Models, Animal ( mesh )
Dissertations, Academic -- College of Medicine -- Department of Pathology and Laboratory Medicine -- UF ( mesh )
Hematopoiesis ( mesh )
Research ( mesh )
Retroviridae ( mesh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida, 1990.
Bibliography:
Bibliography: leaves 111-117.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Juinn-Lin G. Liu.

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University of Florida
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University of Florida
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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
001476235 ( ALEPH )
22519072 ( OCLC )
AGY8080 ( NOTIS )

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Full Text












IDENTIFICATION OF DIFFERENTIATION MARKERS
IN NORMAL AND VIRALLY TRANSFORMED
AVIAN HEMATOPOIETIC CELLS












By

JUINN-LIN G. LIU


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


1990




IDENTIFICATION OF DIFFERENTIATION MARKERS
IN NORMAL AND VIRALLY TRANSFORMED
AVIAN HEMATOPOIETIC CELLS
By
JUINN-LIN G. LIU
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1990


DEDICATION
This dissertation is dedicated to my parents. Without
their understanding, support and encouragement, it would
have never been possible for me to accomplish any goals in
my life.


ACKNOWLEDGEMENTS
Words cannot express my appreciation to my mentor, Dr.
Carlo Moscovici. Four years ago, I went to his office to
discuss my rotation project. Suddenly I was touched by a
poster on the wall, which depicted a newly hatched baby
chick, excited and curious about its new life, asking What
do I do ? It was nearly the mirror image of myself. The
very same question had struck me from time to time since I
arrived in the United States. Fortunately, he has guided
me, spiritually and intellectually, during the past four
years not only to become an independent researcher but a
more mature person as well. Nevertheless, we still have
some disagreement about my own differentiation pathway,
e.g., he never gives up the idea that jazz should be a
"growth factor" for me.
My work would not be complete without the great impact
of Dr. Giovannella Moscovici. She has enthusiastically
shared her knowledge and experience with me. I have learned
from her many techniques regarding cell biology and tumor
virology and have a better vision about life.
I also sincerely appreciate Dr. Paul Klein's help. His
generosity allowed me to use the space and materials in his
laboratory to complete the work on RIA and SDS-PAGE. He
iii


also helped me interpret the results and select the MAbs.
In addition, I am indebted to Dr. Paul Linser for providing
me the opportunity to establish the SDS-PAGE and
immunostaining techniques in his laboratory at the Whitney
Lab. The atmosphere there was so wonderful that I used to
walk along the beach and listen to the songs of the ocean at
night. It really eased a lot of pressure built up during my
project. By the way, the enjoyment of reading a science
fiction novel "The Rapture Effect," a gift from Dr. Linser,
may also enhance my "healing" process.
I would like to extend my appreciation to the other
members of my committee, Dr. Edward Wakeland and Dr. Ammon
Peck, for their helpful discussions and constructive
comments on my work.
Finally, I am also grateful to our technician, Mr.
Gordon Thompson, for his technical assistance and
preparation of the materials; to Mrs. Linda Green, Sue
Hammack, Janice Odebralski and Sandra Wotoweic in the
Hybridoma Laboratory for helping me develop the hybridomas;
and to Mrs. Melissa Chen for her assistance with FACS
sorting and analysis.
iv


TABLE OF CONTENTS
page
ACKNOWLEDGEMENTS iii
LISTS OF TABLES vii
LISTS OF FIGURES viii
ABBREVIATIONS X
ABSTRACT xii
CHAPTERS
1 INTRODUCTION AND BACKGROUND 1
Introduction 1
Normal Avian Hematopoiesis 3
Interaction of the Avian Leukemia Viruses with
Hematopoietic Cells 19
Identification of Cell Surface Markers in Normal
Hematopoietic Cells and Tumor Cells by MAbs.... 30
2 PRODUCTION OF MONOCLONAL ANTIBODIES AND THEIR
CELL-TYPE SPECIFICITIES 38
Introduction 38
Materials and Methods 39
Results 49
Discussion 61
3 IDENTIFICATION OF TARGET CELLS RECOGNIZED BY MABS
IN THE YOLK SAC AND THE BONE MARROW 72
Introduction 72
Materials and Methods 73
Results 75
Discussion 81
4 BIOCHEMICAL CHARACTERIZATION OF THE
DIFFERENTIATION MARKERS RECOGNIZED BY MABS 85
Introduction 85
Materials and Methods 86
v


Results 89
Discussion 98
5 CONCLUDING REMARKS 105
REFERENCES Ill
BIOGRAPHICAL SKETCH 118
vi


LIST OF TABLES
page
Table 1-1. Chicken hematopoietic precursor cells 12
Table 1-2. The three different lineages of the avian
erythroid compartment 15
Table 1-3. Avian defective leukemia viruses 21
Table 2-1. The cell-type specificities of MAbs from
BM2 and BM2/L fusions 51
Table 2-2. The isotypes of selected MAbs 51
Table 2-3. RIA binding indexes of MAbs to different
cell types 52
Table 2-4. Summary of the cell-type specificities
of MAbs 60
Table 2-5. Comparison of the proliferating potential
of BM2/C3A cells and 2.5 /g/ml
PMA-differentiated BM2/C3A cells 62
Table 2-6. Comparison of the proliferating potential
of 6C2 cells and ImM butyric acid-treated
6C2 cells 65
Table 2-7. RIA binding indexes of MAbs to normal CEF
and RAV-infected CEF 68
Table 3-1. Characterization of the target cells
sorted with 3D7-1C9 from the bone marrow... 78
Table 3-2. Characterization of the target cells
sorted with 2E10-1E10 from the bone marrow. 80
Table 3-3. Characterization of the negative
populations isolated with MAbs from the
yolk sac 80
Table 4-1. The biochemical nature and antigenic
determinants of the differentiation
markers recognized by MAbs 97
vii


LIST OF FIGURES
page
Figure 1-1. Schematic illustration of the chick
embryo at the 3rd day of embryogenesis.... 7
Figure 1-2. Diagram of hematopoiesis 11
Figure 1-3. Inerference of the AEV with cells of
the erythroid lineage 28
Figure 1-4. Interference of the AMV with cells of
the monocytic lineage 32
Figure 2-1. Flow cytometric analysis of the 20%/50%
interface of a percoll gradient of 2-week-
old bone marrow cells 53
Figure 2-2. APAAP immunoenzymatic staining of 20%/50%
interface of a percoll gradient of 2-week-
old bone marrow cells 54
Figure 2-3. Flow cytometric analysis of the 50%/70%
interface of a percoll gradient of 2-week-
old bone marrow cells 56
Figure 2-4. Flow cytometric analysis of the 30%/50%
interface of a percoll gradient of 4-day-
embryo yolk sac cells 57
Figure 2-5. Flow cytometric analysis of the 20%/50%
interface of a percoll gradient of 2-week-
old bone marrow cells tagged with
2E10-1E10 58
Figure 2-6. Flow cytometric analysis of the 50%/70%
interface of a percoll gradient of 2-week-
old bone marrow cells tagged with
2E10-1E10 59
Figure 2-7. Reactivitities of MAbs to BM2 cells and
PMA-differentiated BM2 cells 64
viii


Figure 2-8. Reactivities of MAbs to 6C2 cells and
butyric acid-treated 6C2 cells 67
Figure 3-1. Flow cytometric analysis of 3D7-1C9
tagged bone marrow cells before sorting... 76
Figure 3-2. Flow cytometric analysis of 3D7-1C9
tagged bone marrow cells after sorting.... 77
Figure 4-1. Reactivities of MAbs to BM2/C3A cells with
enzymatic digestion and chemical
deglycosylation 92
Figure 4-2. Reactivities of MAbs to 6C2 cells with
enzymatic digestion and chemical
deglycosylation 94
Figure 4-3. Reactivities of MAbs to MSB1 cells with
enzymatic digestion and chemical
deglycosylation 96
Figure 4-4. Silver stain of 10% SDS-PAGE analysis
of immunoprecipitates obtained from
unlabeled BM2/C3A cells 99
Figure 4-5. Fluorography of 10% SDS-PAGE analysis
of immunoprecipitates obtained from
35 S-Methionine-labeled BM2/C3A cells 100
Figure 4-6. Fluorography of 10% SDS-PAGE analysis
of immunoprecipitates obtained from
35 S-Methionine-labeled 6C2 cells 101
Figure 4-7. Fluorography of 10% SDS-PAGE analysis
of immunoprecipitates obtained from
35 S-Methionine-labeled MSB1 cells 102
Figure 5-1. Diagram of the specificities of MAbs
for the hematopoietic cells 109
ix


ABBREVIATIONS
ACS
AEV
AHS
ALV
AMV
APAAP
BFU-E
BPA
CEF
CFU-E
CFU-M
cpm
CSF
DLV
DMSO
DMEM
EGF
ELISA
FACS
FBS
FITC
Anemic chicken serum
Avian erythroleukemia virus
gamma globulin-free horse serum
Avian leukemia virus
Avian myeloblastosis virus
Alkaline phosphatase and monoclonal anti-alkaline
phosphatase
Burst-forming unit-erythroid
Burst-promoting activity
Chicken embryo fibroblast
Colony-forming unit-erythroid
Colony-forming unit-marrow
Counts per minute
Colony-stimulating factor
Defective leukemia virus
Dimethyl sulfoxide
Dulbecco's modified Eagle's medium
Epidermal growth factor
Enzyme-linked immunosorbent assay
Fluorescence-activated cell sorter
Fetal bovine serum
Fluorescein-isothiocyanate
x


GAM
Goat anti-mouse polyvalent IgA, G & M
HBSS
Hank's balanced salt solution
LLV
Lymphoid leukosis virus
LPS
Lipopolysaccharide
MAb
Monoclonal antibody
MAV
Myeloblastosis-associated virus
MG-CFC
Macrophage-granulocyte colony-forming cell
m.o.i.
Multiplicity of infection
PBS
Phosphate-buffered solution
PMA
Phorbol 12-myristate-13-acetate
PMSF
Phenylmethylsulfonylfluoride
PNPP
P-nitrophenyl phosphate
PPD
P-phenylene diamine
RaMIG
Rabbit anti-mouse IgG
RAV
Rous-associated virus
RIA
Radioimmunoassay
SDS-PAGE
Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis
TBS
Tris-buffered saline
TSA
Tris/saline/azide
xi


Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
IDENTIFICATION OF DIFFERENTIATION MARKERS
IN NORMAL AND VIRALLY TRANSFORMED
AVIAN HEMATOPOIETIC CELLS
By
JUINN-LIN G. LIU
May 1990
Chairman: Dr. Carlo Moscovici
Major Department: Pathology and Laborotary Medicine
Avian hematopoiesis has been an excellent model
for resolving numerous enigmas about growth and
development. The interaction of avian retroviruses with the
avian system has created a relatively new discipline of
onco-development which allows us to analyze abnormal tissue
growth and hematological disorders in a more sophisticated
fashion.
The specific aim of this project is to identify
lineage-specific differentiation markers in normal avian
hematopoietic cells and transformation-associated
antigens in retrovirus-transformed cells by utilizing
monoclonal antibody techniques. Four groups of MAbs were
selected among nearly 5,000 supernatants from 10 fusions.
Characterization of the cell-type specificities was achieved
xii


by radioimmunoassay, immunofluorescence staining, flow
cytometry and immunoenzymatic staining as well as FACStar
sorting or immunomagnetic bead separation followed by
colony-forming assays and transforming assays. Analysis of
MAbs revealed that 1) MAbs 1H10-1F9, 2H1-2A10 and 3D7-1C9
are specific for transformation-associated antigens present
preferentially on BM2 cell lines rather than on normal
monocytic cells. The expression of these antigens was
diminished after BM2 cells were induced to differentiate.
2) MAb 1F7-1A3 recognizes BFU-E and CFU-E, AEV-transformed
yolk sac cells and MSB1 cells. 3) MAb 3F6-1E7 reacts with
the embryonic stem cell and precursor cell populations. The
expression of the marker recognized by MAb 3F6-1E7 was also
observed on some tumor cells, e.g., AEV-transformed yolk sac
cells, BM2 cells and MSB1 cells. 4) MAb 2E10-1E10 defines a
marker present on proliferating hematopoietic cells instead
of terminally differentiated cells, however, it starts
appearing only after the 4th day of embryogenesis.
Trypsinization, neuraminidase digestion and
deglycosylation treatment reduced the binding
specificities of MAbs 1H10-1F9, 2H1-2A10, 3D7-1C9 and
2E10-1E10. This suggests that the markers recognized by
these MAbs are glycoproteins and that sialic acids with or
without carbohydrates are contributing to the conformation
of the antigenic determinants. Conversely the antigenic
xiii


determinants for MAbs 1F7-1A3 and 3F6-1E7 must be strictly
proteins, since only trypsinization was able to inhibit
their binding specificities.
This study will permit investigations focusing on the
expression of these markers to bring us a step closer
toward the understanding of the mechanisms involved in
regulating proliferation and differentiation of
normal cells versus tumor cells.
xiv


CHAPTER 1
INTRODUCTION AND BACKGROUND
Introduction
Proliferation and differentiation of normal cells is
controlled by the interactions with other cell types, with
extracellular matrix and with regulatory molecules such as
growth factors and differentiation factors. Although the
regulatory mechanisms are extremely complicated, they have
been programmed in such a way as to maintain proliferation
and differentiation of the cells in a harmonic state. In
other words, the loss of cells from the stem cell
compartment by differentiation into committed progenitor
cells must be balanced by replenishment via self-renewal of
the stem cells. If too many stem cells undergo
differentiation, the stem cell reserve will rapidly become
exhausted; if too many stem cells undergo self-renewal
rather than differentiation, the production of mature cells
will drastically fall.
Cancer is believed to be a molecular disease resulting
from the deregulation of proliferation and differentiation,
i.e., the harmony has been short-circuited by the
1


2
constitutively triggered self-renewal machinery with or
without the blockage of the differentiation pathway.
It has been noticed that AEV-transformed avian
embryonic yolk sac cells can eventually undergo spontaneous
differentiation into mature erythrocytes (Jurdic et al.,
1985) and that spontaneous regression and differentiation of
human neuroblastomas are observed occasionally (Evans et
al., 1976). In addition, a variety of tumor cells have also
been shown to revert to a normal state under different
conditions despite the continued expression of activated
oncogenes. For example, the tumorigenicity of hybrids
formed between normal and tumor cells has been completely
suppressed (Stanbridge et al., 1982); embryonal carcinoma
(Pierce et al., 1979), neuroblastoma (Podesta et al., 1984),
B16 melanoma (Pierce et al., 1984) and murine leukemia
(Gootwine et al., 1982) have been converted into benign cell
lineages by their appropriate embryonic environments;
naturally occurring substances such as colony-forming
factors (Sachs, 1986), glia maturation factor (Lim et al.,
1986) and transforming growth factors (Sporn et al., 1986)
have been shown to be able to induce differentiation of
tumor cells; while a number of chemical agents such as
hexamethylene bisacetamide, retinoid acid, 5-azacytidine and
DMSO etc. can also induce terminal differentiation and/or
reverse the neoplastic phenotype of malignant cells (Bloch,
1984; Fresney, 1985). Moreover, in some cases, terminally


3
differentiated cells such as macrophages can still serve as
the target cells for transformation by a group of
retroviruses, namely AMV, MC29 and MH2 (Pessano et al.,
1979). All the information mentioned above suggests that
self-renewal and differentiation of cells are regulated by
separate mechanisms. The roles of various protooncogenes
and antioncogenes in these processes are yet to be
elucidated.
Normal Avian Hematopoiesis
Introduction
The avian hematopoietic system has provided a unique
and interesting model to study mechanisms of the regulation
of cell proliferation and differentiation in normal versus
tumor cells. It has several distinct features compared to
that of mammals, including the presence of the bursa of
Fabricius which is involved in the differentiation of B
lymphocytes, the expression of class IV MHC antigens, coded
by the B-G region, on the surface of the mature erythrocytes
(Miller et al., 1982). In addition, the erythrocytes are
nucleated and oval-shaped, and the nucleated thrombocytes,
instead of the platelets, are responsible for the hemostasis
in the avian system. There are several advantages in using
the avian models. For instance, tolerance to foreign
antigens can be developed during early ontogeny (Hasek and
Hraba, 1955), and hematopoiesis can be studied both in vivo


and in vitro by using retroviruses as indicators for
specific precursors present within each lineage.
The most studied avian hematopoietic system is in the
chicken. The outbred SPAFAS line has been used in our
laboratory to carry out all the experiments for my
dissertation project. My work has focused mainly on the
erythroid and monocytic lineage and their interaction with
the avian retroviruses.
The Blood-Forming Organs
The first blood cells appear after 18 hours of
incubation in the blood islands disseminated in the
blastoderm. During embryogenesis, the yolk sac represents
the major hematopoietic organ until day 15. Erythropoiesis
in the spleen starts around day 9 and continues through day
16 to 18 with a peak at day 15. The bone marrow begins its
function on day 12 and becomes the main site of
hematopoiesis throughout adult life (Dieterlen-Livre,
1988) .
The volk sac
The yolk sac becomes established during the third day
of embryogenesis and it originates in the extraembryonic
region consisting of a peripheral "area vitellina" which is
made up of ectoderm and endoderm only, and a central "area


5
vasculosa" consisting of all three germ layers (Figure 1-
1). The area vasculosa contains the blood islands and its
boundary is delineated by a circular blood vessel, the sinus
marginalis. As the area vitellina grows over the yolk, the
area vasculosa also increases in size and invades the area
vitellina. Eventually the latter disappears completely, and
the entire yolk sac is vascularized.
The bone marrow
In the adult chicken, the bone marrow remains the major
source for erythropoiesis, granulopoiesis and lymphopoiesis.
The spleen does not appear to play a role in hematopoiesis
in the adult. In the avian bone marrow, erythropoiesis
occurs in the lumen of the medullary sinuses, while
granulopoiesis and lymphopoiesis are compartmentalized
within the extravascular spaces (Campbell, 1967). In
addition, masses of lymphatic tissue with germinal centers
are also present (Campbell, 1967; Payne and Powell, 1984).
However, in the mammalian bone marrow, erythropoiesis is
confined to the extravascular spaces and there is no
lymphatic tissue at all.
The development of the bone marrow during ontogeny has
been studied in the chick embryo by Sorrell and Weiss (1980)
using light, scanning and transmission electron microscopy.
The bone marrow cells can be obtained from the chick embryo
as early as 12 days of incubation. Marrow at this stage is


Figure 1-1. Schematic illustration of the chick
embryo at the 3rd day of incubation. The yolk sac
is composed of the area vitellina and the area
vasculosa. As the area vitellina grows over the
yolk, the area vasculosa also increases in size
and invades the area vitellina. Eventually the
latter disppears completely and the entire yolk
sac is vascularized. 1, embryo; 2, area
pellucida; 3, area vasculosa; 4, sinus terminus;
5, area vitellina interna; 6, area vitellina
externa.


7
2
I
5


8
richer in stem cells and non-committed progenitor cells than
the older bone marrow, but it already harbors cells which
are committed to specific hematopoietic lineages. The
latter cells are less numerous than in the adult bone marrow
and consist essentially of hemocytoblasts.
Other blood-forming organs
The spleen, the bursa, the liver and the thymus
function as additional hematopoietic organs. In the adult,
lymphopoiesis occurs mainly in the thymus and in the spleen,
whereas the bursa, from which the precursors of B
lymphocytes originate, is a transient granulopoietic organ.
Recent investigations (Cormier and Dieterlen-Livre,
1988) report that some intraembryonic sites may be another
source of hematopoietic stem cells in the developing embryo.
At 3-4 days of incubation, the wall of the dorsal aorta
surrounding the intraembryonic mesenchyme is found to be the
site from which hematopoietic progenitor cells emerge, i.e.,
M-CFC, G-CFC, GM-CFC and BFU-E.
The Differentiation Pathway of Hematopoietic Cells
The cells of the hematopoietic system arise by
proliferation and differentiation of the progenitor cells.
This process begins with multipotential stem cells which can
self-renew as well as undergo progressive differentiation to
progenitor cells committed to the particular lineages,


9
ultimately yielding mature blood cells (Metcalf and Moore,
1971).
Analysis of the stem cells and progenitor cells in the
different hematopoietic tissues has been useful in
clarifying the differentiation pathway as well as in
exploring the regulatory mechanisms of hematopoiesis.
Hematopoietic stem cells
The stem cell population is the fundamental base from
which all the major hematopoietic cell lines are derived.
This population is thus considered to be pluripotent in its
differentiation potential, giving rise to erythroid,
granulocytic, monocytic, megakaryocytic and lymphoid
lineages (Figure 1-2). However, the stem cells account only
for 0.01% of the total bone marrow cells in a normal mouse,
and for 0.003-0.004% in a normal chicken (Table 1-1). In
addition, since they are morphologically indistinguishable,
their existence can only be inferred by the progeny they
produce.
Efforts to identify chicken hematopoietic stem cells
have followed the protocol of the transplantation
experiments by Till and McCulloch (1961) whereby they have
identified the mouse hematopoietic stem cells. Samarut, et
al. (1976) transplanted normal chicken bone marrow into
irradiated chickens. Six days later, erythrocytic colonies
were observed only on the surface of the tibial marrow.


Figure 1-2. Diagram of hematopoiesis. The entire pool of mature hematopoietic
cells is derived from a single pluripotent stem cell. The more differentiated
they become, the less self-renewal potential they possess. In the avian system,
thromboblast and thrombocytes stand for megakaryocyte and platelets
respectively. Abbreviations used: BFU-E, burst-forming unit-erythroid; CFU-E,
colony-forming unit-erythroid; Eo-CFC, eosinophil-colony forming cell; GM-CFC,
granulocyte & monocyte-colony forming cell; MEG-CFC, megakaryocyte-colony
forming cell.


Self-renewal
Pluripotent
Stem Cell
t t T
Erythroid Myeloid Lymphoid
Precursor Precursor Precursor
t
BFU-E GM-CFC Eo-CFC MEG-CFC

CFU-E

Erythroblast Myeloblast Monoblast Eosinophilic Megakaryoblast
Myeloblast (Thromboblast) Lymphoblast Lymphoblast
I I I I V
Peripheral
Blood
Erythrocyte Myelocyte Monocyte Eosinophil Platelets T Cell B Cell
y 1 (Thrombocytes) 1
Granulocyte f
Tissue
Macrophage Plasma
H y Cell


12
Table 1-1. Chicken hematopoietic precursor cells
Hematopoietic
precursor cells
Frequency*
Bone marrow Yolk sac
Progeny
BFU-E
110-160
300-600
1,
000-2,000
CFU-E
500-2,000
1,500-1,800
8-150
GM-CFC
(early progenitor)
250-400
100-200
50-1,000
GM-CFC
(late progenitor)
1,000-1,300
100-200
3-50
Source: Modified
Expressed as per
from Moscovici
105 cells.
and Gazzolo
(1982)



13
Each colony was originated from one single cell, i.e.,
colony-forming unit in the marrow (CFU-M). However, neither
macrophage-granulocytic colonies nor mixed types of colonies
were observed in the marrow of the irradiated chickens. It
is still unclear whether the medullar environment does not
favor the development of colonies other than the ones of the
erythroid lineage or if the CFU-M represents only the stem
cells at the earliest step of commitment in the erythroid
lineages.
Committed progenitor cells
Committed progenitor cells are directly derived from
the stem cells and are each committed to a specific
differentiation pathway or lineage. Commitment is an
irreversible step whereby these cells have lost the
potential to generate hematopoietic cells of other lineages.
Most of the proliferative activity in the bone marrow seems
to occur in the progenitor cells committed to the production
of single or restricted ranges of hematopoietic cell types.
Only a small proportion of the pluripotential stem cells is
cycling at a given time. The progenitor cells of both
erythroid and myeloid lineages will be discussed in detail.
The erythroid lineage
The avian erythroid compartment consists of three
distinct lineages, namely the primitive, the intermediate


14
and the definitive lineage, respectively (Table 1-2). The
cells from the primitive lineage are produced by the early
blood islands and mature "in cohort" (Ingram, 1972). These
cells are released at a very immature state, but they
continue to divide and mature synchronously within the blood
vessels between 2 and 5 days of incubation. These cells are
called megalocytes because of their large size. They are
spherical with round nuclei and synthesize hemoglobins E
(embryonic) and P (primitive) which are specific for the
primitive lineage. At 5 days, cells of the intermediate
erythroid lineage begin entering the blood and eventually
supersede the primitive cells. At 7 days, the primitive
cells account for only 5% of the red blood cells, and after
12 days of incubation they are rarely encountered. The
cells of the intermediate lineage are observed from 5 to 6
days until 18 to 20 days. They synthesize specific
hemoglobin H (hatching). It is not until 18 to 21 days of
incubation that mature erythrocytes of the definitive
lineage start to appear in the blood circulation. They are
oval-shaped with oval nuclei and have hemoglobins A (adult)
and D (definitive) (Bruns and Ingram, 1973).
The progenitor cells of the definitive lineage are
morphologically unrecognizable. However, by the use of in
vitro colony forming assays in methylcellulose, two classes
of erythroid progenitor cells have been identified,
the colony-forming unit-erythroid (CFU-E) and the burst-


15
Table 1-2. The three different lineages of the avian
erythroid compartment
Primitive
Intermediate
Definitive
Appearance
Day 2-7
Day 5-20
Day 18-Hatched
Progenitor
Cell
Megaloblast?
BFU-E, CFU-E
BFU-E, CFU-E
Mature Cell
Megalocyte
Erythrocyte
Erythrocyte
Hemoglobin*
E: aA + e
P: 7t + p
H: aA + pH
A: aA + /3a
D: aD + /3a
Source: Modified from Bruns and Ingram (1973).
a-like globin: aA, n, aD; /3-like globin: e, p, /3H, /3A.
Abbreviation: E, embryonic; P, primitive; H, hatching;
A, adult; D, definitive.


16
forming uint-erythroid (BFU-E). The BFU-E give rise, after
six days in culture, to large aggregates made of several
benzidine-positive clusters containing about 1,000
erythrocytes (Samarut and Bouabdelli, 1980). These BFU-E
are highly sensitive to burst-promoting activity (BPA), and
are also dependent on high concentrations of erythropoietin.
The CFU-E proliferate to form one compact colony of 8
to 150 benzidine-positive erythrocytes after three days of
incubation (Samarut et al., 1979). The requirement for
erythropoietin in the development of CFU-E is lower than
that for BFU-E. The BFU-E and CFU-E can be detected in the
yolk sac as well as in the embryonic and adult bone marrow.
The BFU-E are also found in the blastoderm at the primitive
streak stage (18 hours of incubation), whereas the CFU-E are
not yet detectable (Samarut and Bouabdelli, 1980). The
BFU-E and CFU-E can be also distinguished by the expression
of two different cell surface antigens which are recognized
by polyclonal antisera (Gazzolo et al., 1980; Samarut et
al., 1979). An antigen specific to immature red blood cells
is present on the CFU-E but not detectable on the BFU-E.
Conversely, a chicken brain-related antigen is expressed on
the BFU-E and less expressed on the CFU-E.
In the murine system, subpopulations of the BFU-E at
different degrees of maturation have been observed (Gregory
and Eaves, 1978). This is not the case, however, for the
BFU-E in the chickens.


17
The myeloid lineage
Hematopoietic cells of granulocytic and monocytic
lineages are referred to as myeloid cells. In response to
infection, the progenitor cells in the bone marrow, i.e.,
granulocytic-macrophage colony-forming cells (GM-CFC), would
rapidly produce a large amount of mature granulocytes and
monocytes under the control of a variety of colony-
stimulating factors (CSFs). In the murine and human system,
four kinds of CSFs involved in myelopoiesis have been
characterized as IL3, GM-CSF, G-CSF and M-CSF. Conversely,
the specific CSFs in the chicken have yet to be identified.
Nevertheless, sources of CSFs can be furnished by using an
underlayer of macrophages (Graf et al., 1981), or by adding
to the semi-solid medium either egg albumin (Szenberg,
1977), or serum from endotoxin-injected chickens (Dodge and
Hansell, 1978) or a conditioned medium from chicken
fibroblast cultures (Dodge and Moscovici, 1973; Dodge et
al., 1975; Gazzolo et al., 1979). AMV-transformed cells
were shown also to be capable of producing CSFs (Silva et
al., 1974). Recently a myelomonocytic growth factor was
isolated from medium conditioned by a transformed macrophage
cell line (Leutz et al., 1984). This factor (MGF) promotes
the growth of macrophage colonies together with a minor
proportion of granulocytes.
The existence of chicken GM-CFC can be detected when
bone marrow cells are cultured in soft agar or


18
methylcellulose media. The wide range of the colony size
obtained suggests that these progenitor cells display
different degrees of maturity. The less mature CFC give
rise to clusters containing from 50 to more than 2,000 cells
(Dodge and Moscovici, 1973; Dodge et al., 1975), while the
more mature CFC produce colonies from 3 to 50 cells (Gazzolo
et al., 1980). Colonies which are composed mostly of
macrophages readily develop in a semi-solid medium
containing chicken serum and fibroblast-conditioned-medium
(Dodge and Hansell, 1978). Granulocytic colonies will
develop if the chicken serum is depleted from the medium and
the fibroblast-conditioned-medium is replaced by spleen-
conditioned-medium (Dodge and Sharma, 1985). This finding
suggests the existence of a factor similar to M-CSF in the
mouse and human. The monocytic colonies are composed of
scattered cells, whereas the granulocytic colonies are
dense. The cells from both colonies contain granules.
The GM-CFC have been observed in various chicken
hematopoietic tissues of embryonic and adult stages (Dodge
and Moscovici, 1973; Dodge et al., 1975; Szenberg, 1977).
Moreover, the CFC can be enumerated in the blastoderms
incubated for 24 hours (Moscovici et al., unpublished
results). A higher percentage of CFC was also found in the
embryonic spleen and bone marrow. The frequency dropped
rapidly once the chickens hatched. Interestingly, a peak of
CFC occurred in the bursa at 14 and 15 days of incubation,


19
indicating that the stem cells colonizing the bursa
differentiate first into myeloid elements and subsequently
into lymphoid ones (Szenberg, 1977).
Interaction of the Avian Leukemia Viruses with
Hematopoietic Cells
Introduction
There have been many comprehensive reviews to date on
the molecular biology and the pathogenesis of the avian
leukemia viruses (ALVs) (Moscovici and Gazzolo, 1982; Graf
and Sthelin, 1982; Bishop, 1983; Enrietto and Wyke, 1983;
Bister and Jansen, 1986). The ALVs belong to a taxonomic
subfamily termed oncovirinae (specifically, avian leukosis-
sarcoma group of type C RNA tumor viruses) within the family
of retroviridae (retroviruses) (Fenner, 1976). They
contribute to a variety of avian hematopoietic as well as
non-hematopoietic disorders. The ALVs can be divided into
two groups according to the pathological features: the
defective leukemia viruses (also known as acute leukemia
viruses) and the avian leukosis viruses (Hanafusa, 1977).
The defective leukemia viruses (DLVs)
These viruses induce various types of acute leukemia
within a few weeks after inoculation. They also cause
sarcomas and carcinomas in some cases. All the strains
known can transform the cells of specific hematopoietic
lineages in vitro. In addition, most of them transform


20
fibroblasts in culture as well, with the exception of AMV
and E26. Another distinct aspect of the DLVs is that they
are all replication-defective due to total or partial
deletions of the essential virion genes: gag, pol and env.
Consequently, they can produce infectious progeny only in
the presence of the avian leukosis helper viruses. The
deleted sequences are replaced by the viral oncogenes
(v-onc), i.e., v-myc, v-mil, v-erbA, v-erbB, v-myb, and
v-ets, which originated from transduction of mutated or
truncated forms of protooncogenes. Their gene products are
responsible for the transformation of the hematopoietic
cells. Based on the predominant response of the
hematopoietic system of the infected host and the major
types of oncogenes which they carry, three subgroups of
DLVs and their respective representatives can be
distinguished: i) the MC29 subgroup: myelocytomatosis
(v-myc), ii) the AEV subgroup: erythroblastosis (v-erb),
iii) and the AMV subgroup: myeloblastosis (v-myb) (Table
1-3). However, a more detailed description of the
interaction of the AEV and the AMV with the hematopoietic
system will be given since more data have been obtained in
the last decade.
The Lymphoid leukosis viruses (LLVs)
In contrast to the DLVs, the LLVs do not contain any
v-onc and they are fully competent for replication. Because


21
Table 1-3. Avian defective leukemia viruses
Subgroups
and
virus strains
Viral
oncogenes
Neoplasms
induced
in vivo
Cell types
transformed
in vitro
MC29 subgroup
Strain MC29
Strain CMII
Strain OKIO
Strain MH2
v-myc
v-myc
v-myc
v-myc, v-mil
Myelocytomatosis,
endothelioma,
carcinoma
Myeloid,
macrophage,
epithelioid,
fibroblastic
AEV subgroup
Strain AEV-R
Strain AEV-H
v-erbA, v-erbB
v-erbB
Erythroblastosis,
fibrosarcoma
Erythroid,
fibroblastic
AMV subgroup
Strain AMV
v-myb
Monoblastosis
Monocytic
Strain E26
v-myb, v-ets
Erythroblastosis,
monoblastosis
Erythroid,
monocytic
Source: Modified from Bister and Jansen (1985).


22
of the absence of v-onc, the LLVs don't transform cells in
vitro and most strains induce predominantly lymphoid
leukosis in vivo only after a long latent period of several
months or longer by the activation of protooncogenes. In
the cases of chicken B-cell lymphomas induced by the LLVs,
the viral LTR regions containing an enhancer and a promoter
were found to be integrated very close to the c-myc gene
(Hayward et al., 1981). Most integrations of LLVs result in
the separation of exons II and III of the c-myc gene from
the normal promoter and exon I (Payne et al., 1982; Shih et
al., 1984), causing a fifty-fold higher transcription of c-
myc RNA under the control of the LTR promoter than the 5
copies found in normal cells. In a minority of tumors, the
LTR integrated in the opposite orientation to that of the c-
myc and in one case the provirus was actually located at the
3' end of the c-myc (Payne et al., 1982). It is thought in
these rare events that the enhancer element in the viral LTR
probably increases the transcription from the normal c-myc
promoter. More recently, the LLVs have also been found
integrated at the c-erbB locus in chicken erythroblastosis
(Fung et al., 1983). An elevated level of c-erbB
transcripts was observed.
LLVs under different conditions, for example, chicken
genotype, site of injection, age of host, etc., may induce a
larger spectrum of diseases including osteopetrosis, anemia,


nephroblastoma and occasional fibrosarcomas and
endotheliomas.
23
The Avian Ervthroleukemia Virus (AEV)
The oncogenes of the AEV
The AEV can induce erythroblastic leukemia and sarcomas
in infected birds within a short period of time (Graf and
Beug, 1978; Moscovici and Gazzolo, 1982). The virus also
transforms chicken fibroblasts and hematopoietic precursor
cells of the erythroid lineage in vitro (Graf et al., 1981).
It carries two oncogenes, namely v-erbA and v-erbB. The
v-erjbB encodes a protein of 61 Kd which is glycosylated in
infected cells to higher molecular weight forms (Privalsky
et al., 1983). The gp65erbB and gp68erbs are localized in the
intracellular membrane, while the mature gp74erbB is found in
the plasma membrane (Privalsky and Bishop, 1984). The
latter protein represents a truncated form of the receptor
for epidermal growth factor (EGF) (Downward et al., 1984),
where the extracellular EGF-binding domain has been deleted,
but the region for tyrosine-specific protein kinase is
preserved (Gilmore et al., 1985). It has been postulated
that the transforming potential of v-erbB is due to the lack
of regulation by EGF resulting in the constitutive
activation of the tyrosine kinase (Hayman and Beug, 1984;
Privalsky and Bishop, 1984). However, the exact mechanism


24
of transformation by the tyrosine kinase activity is still
unknown.
The v-erbA is a mutated truncation transduced from a
cellular multigene family encoding the thyroid hormone
receptor (Sap et al., 1986). The v-erbA protein,
p759a9"erbA, is linked with the gag product, is unglycosylated
and has no kinase activity. It appears to be a DNA-binding
protein exhibiting distinct nuclear and cytoplasmic
subcellular locations (Boucher et al. 1988). Nevertheless,
it doesn't seem to bind thyroid hormone (Sap et al., 1986).
The transforming potential of v-erbA and v-erbB
It has been shown that v-erbB alone is necessary and
sufficient to induce cell transformation (Frykberg et al.,
1983; Sealy et al., 1983) by constructing a series of
deletion mutant viruses. The data corroborate the ability
of the product of v-erbB gp65-68erbB to independently induce
erythroleukemia in chickens and transform fibroblasts in
vitro. In contrast, constructs which produce the v-erbA
p759a9'erbA only were incompetent for transforming activity in
vitro. However, other observations from studies of
temperature sensitive mutants (Beug and Hayman, 1984),
transductants of the c-erbB gene alone (Fung et al., 1983;
Yamamoto et al., 1983), suggest that v-erbA may play a
distinct role in maintaining the proliferation and
transformed phenotype of AEV-infected cells. It has been


25
shown that v-erbA possesses the capability to potentiate the
erythroid transformation not only by v-erbB but also by
other oncogenes (Frykberg et al., 1983; Kahn et al., 1986).
These phenomena could be partially due to the suppression of
the transcription of the anion transporter (band 3) gene by
the v-erbA proteins (Zenke et al., 1988). It is not until
recently, however, that the transforming potential of
v-erbA has been reevaluated. The XJ12 vector which carries
v-erbA oncogene in association with Neo R (neomycin
resistance) gene is shown to be able to transform bone
marrow cells in vitro (Gandrillon et al., 1989; Moscovici
and Moscovici, unpublished results).
The target cells for AEV
The target cells for AEV infection in the bone marrow
of the hatched chick are recruited within the BFU-E (Burst
forming unit-erythroid) compartment (Gazzolo et al., 1980).
After AEV infection, the transformed cells continue to
proceed within the differentiation pathway until they are
blocked at the stage of CFU-E (colony forming unit-
erythroid) (Samarut and Gazzolo, 1982). These cells express
the erythroid markers of the CFU-E, but have gained the
self-renewal potential to forego the fate of terminal
differentiation. Conversely, the target cells for AEV in
the embryo are within either the CFU-M or the pre-BFU-E or
in both compartments (Jurdic et al., 1985). The embryonic


26
transformed colonies are partially hemoglobinized even after
subcloning, which suggest that the AEV-transformed embryonic
cells can escape the blockage and undergo spontaneous
differentiation in contrast to the AEV-transformed adult
cells (Figure 1-3).
The Avian Myeloblastosis Virus (AMY)
The oncogene of the AMV
The AMV induces rapid myeloblastic leukemia in the
chickens and transforms hematopoietic cells of monocytic
lineages in vitro. It can be divided into subgroups A and
B, depending on the expression of envelope glycoproteins of
the helper viruses (Moscovici et al., 1975). Besides the
AMV transforming agent, this strain contains two
nondefective helper leukosis viruses, the myeloblastosis
associated virus 1 and 2 (MAV-1 & MAV-2) of subgroups A and
B, respectively (Moscovici and Vogt, 1968).
The oncogene of AMV, v-myb, encodes a transforming
protein p45myb which is located in the nucleus (Klempnauer et
al., 1984). It has been shown that p45myb has DNA- binding
activity (Boyle et al., 1985). The v-myb proteins are
associated with their nuclear sublocation. The exact
function of v-myb proteins remains to be clearly
established. However, recent results indicate that v-myb
along with c-myb may act as transcriptional activators
(Weston and Bishop, 1989).


Figure 1-3. Interference of the AEV with cells of the
erythroid lineage. The AEV only transforms the cells
at the BFU-E stage from the adult bone marrow.
Transformed cells are frozen at the CFU-E stage and
capable of self-renewal. Only cells at the pre-BFU-E
stage are the target cells for AEV transformation in
the embryonic yolk sac. Although the transformed cells
display the phenotypes of CFU-E, they will eventually
escape the blockage and spontaneously differentiate
into erythrocytes. The brain antigens (Br) are
expressed on the BFU-E more than on the CFU-E, the
immature antigens (Im) are found on the CFU-E and
erythroblasts, meanwhile only erythroblasts and
erythrocytes are capable of synthesizing the
hemoglobins (Hb).


28
Normal
AEV Infection
Erythropoiesis
Adult
Bone Marrow
Embryonic
Yolk Sac


29
The target cells for the AMY
The characterization of AMV target cells by density,
velocity sedimentation, adherence and phagocytic activity
indicate that they are recruited among a wide range of cells
within the monocytic lineage from the stage of the
myelomonocytic progenitors, i.e., CFC (colony-forming cells)
(Gazzolo et al., 1979) committed toward the macrophage
lineage (Boettiger and Durban, 1984) to the terminally
differentiated macrophages (Moscovici and Gazzolo, 1982).
Regardless of the origin of the target cells, the AMV-
transformed cells are morphologically identical, and possess
the same functional and surface properties (Gazzolo et al.,
1979; Beug et al., 1979; Durban and Boettiger, 1981). They
are mostly nonadherent and round which diametered about 10
micrometers. Their large and eccentric nucleus is
surrounded by a rim of cytoplasm containing small granules.
The receptors for the Fc region of immunoglobulins are
expressed on the cell surface, whereas C3 receptors are not
present. Normal avian macrophages express both receptors.
Although the AMV-transformed cells can engulf latex
particles mediated by nonspecific receptors, phagocytosis
mediated by Fc receptors does not occur, i.e., these Fc
receptors are not functional. Acid phosphatase and
adenosine triphosphatase are also found in the cytoplasm and
on the membrane of the transformed cells. Moreover, after
treatment with phorbol 12-myristate-13-acetate (PMA), a


30
tumor promoter, the AMV-transformed cells became adherent to
the surface of the culture flask and eventually
differentiated into macrophages (Pessano et al., 1979).
There were no obvious alterations in terms of the expression
of the w-myb proteins in these PMA-differentiated cells.
However, the w-myb proteins were found to be located in the
cytoplasm instead of in the nucleus (Symonds et al., 1984).
The differentiation into macrophages was also obtained with
a temperature-sensitive mutant of AMV when the transformed
cells were shifted to a non-permissive temperature
(Moscovici and Moscovici, 1983).
In conclusion, cells from all stages of monocytic
lineages, from the committed progenitors to the mature
macrophages, may serve as the target cells for AMV. Once
the cells are transformed, they become frozen at a stage
between monoblasts and monocytes (Figure 1-4).
Identification of Cell Surface Markers in Normal
Hematopoietic Cells and Tumor Cells by MAbs
Introduction
The cellular microenvironment plays a crucial role in
regulating the proliferation and differentiation of normal
cells. A cell will interact with adjacent cells and with
structural components of the extracellular matrix via the
external surface of its plasma membrane. Although little is
known about the mechanisms of cellular interactions, it is
well established that cell development requires that the


Figure 1-4. Interference of the AMV with cells of the monocytic lineage. Cells
from different stages of the monocytic lineage can serve as the target cells for
AMV transformation. The transformed cells are arrested at the stage between
monoblast and monocyte via either partial differentiation or
de-differentiation. These cells are non-adherent and round-shaped. The ATPases
are expressed on their cell surface, whereas C3 receptors are not. Although
they have Fc receptors, no immune-phagocytosis can be mediated by these non
functional receptors.


GM-CFC Monoblast
Non-adherent Non-adherent
Fc Receptor -
C3 Receptor -
Phagocytosis -
AMV
Partial
Dedifferentiation
V
AMV
transformed
cells
Macrophages
Non-adherent
Fc Receptor +
C3 Receptor -
Immune
Phagocytosis -
ATPase +
Adherent
Fc Receptor +
C3 Receptor+
Immune
Phagocytosis +
ATPase +
w
to


33
surface membrane should be capable of receiving and
transmitting regulatory signals, i.e., growth factors and
differentiation factors from the microenvironment. Cancer
is a disease resulting from abnormalities in both cell
proliferation and differentiation characterized by increased
growth rate, prolonged survival, decreased adhesion, loss of
contact inhibition, increased invasiveness and motility,
expression of repressed antigens and escape from immune
surveillance (Wallach, 1968). All these phenomena have been
shown to be associated with alterations in structure and
function, and in particular with an aberrant glycosylation
of the cell surface membrane. As a result, cancer can be
regarded as a molecular disease of cell surface
glycoconjugates (Abe et al., 1983).
Cell surface glycoconjugates comprise a heterogeneous
group of compounds, all of which contain carbohydrate
N-glycosidically or O-glycosidically linked to protein
(glycoproteins) or O-glycosidically to lipid (glycolipids).
The predominant glycoconjugates are glycoproteins containing
at least 80% of all cell surface-located carbohydrate. It
is assumed that all membrane-bound proteins but only 10% of
membrane lipids are glycosylated (Shinitzky, 1984).
However, it is extremely difficult to identify specific
glycoconjugates expressed exclusively on tumor cell surface.
All identified tumor-associated markers to date are found to


34
be more or less expressed on normal cells at particular
stages of the differentiation pathway (Old, 1981).
The identification and characterization of cell surface
markers can provide us with invaluable information on
various aspects of differentiation and oncogenesis. First,
the identification of cell surface markers that are specific
for particular stages of differentiation and maturation will
enable us to follow the differentiation pathway in molecular
terms. Mechanisms regulating the expression of cell surface
markers will allow us to understand how functionally mature
cells are formed and what kind of structural elements are
necessary for functional differentiation. Secondly, they
could be used to study whether tumor cells are arrested at a
certain stage of differentiation. In addition, the studies
could also enable us to determine how the control of
proliferation and differentiation in tumor cells differs
from that of normal cells. Thirdly, differentiation markers
can be used for diagnostic and therapeutic purposes (Fukuda,
1985).
The introduction of hybridoma technology by Kohler and
Milstein (1975) has made a significant contribution in the
study of both normal and malignant cell surface markers.
MAbs have been used as powerful tools for the detection,
isolation and characterization of cell surface markers.
Compared to polyclonal antibodies, MAbs exhibit three major
advantages. First, they can be produced against relatively


35
impure antigens. Secondly, MAbs can be produced in much
larger (theoretically unlimited) quantities. Thirdly, they
are monospecific (i.e., they bind to a single epitope) and
thus MAbs recognized markers can be identified and
characterized individually.
Brief synopsis on the MAbs recognizing chicken cells of
ervthroid and monocytic lineages
Cell surface markers of normal or transformed
hematopoietic cells in the human and murine systems have
been extensively studied by using monoclonal antibody
techniques. There are only a handful of MAbs which have
been developed against chicken hematopoietic cells, and none
of them are specific for embryonic precursor cells.
Hayman et al. (1982) developed a panel of MAbs against
the temperature-sensitive mutant of ts34 AEV-transformed
erythroblasts which had been grown at 41.5C for five days.
Three MAbs were chosen for further characterization. MAb
4.2A5 recognized erythrocytes and AEV-transformed
erythroblasts as well as granulocytes; MAb 4.5A5 reacted
with erythroblasts and retrovirus-transformed producer
cells, however, the authers did not address the specificity
tests on other types of normal cells; MAb 4.6C1 was specific
for erythroid cells at all stages.
Jurdic et al. (1982) produced a MAb, Sl-37, from the
fusion of spleen cells of a mouse immunized with AMV-
transformed cells. Sl-37 was shown to be specific for the


36
cells of the monocytic lineage. Its binding specificities
revealed by radioimmunoassay and flow cytometric analysis
were too low for further identification and
characterization.
Miller et al. (1982) immunized mice with 1-day-old
erythrocytes from inbred line 003 and hybrid strain
Shaver-Starcross 288 and raised a MAb, MaEEl, against a 48Kd
antigen which was expressed on the erythrocytes of 1-day-
old peripheral blood and adult bone marrow. It was also
present in the retina, muscle tissues, liver and on
epithelia and lymphoid cells of young and adult chickens.
Sanders et al. (1982) were able to generate a MAb
(190-4) against 1-day-old erythrocytes from the SC strain
which recognized a 50Kd molecule expressed on the cell
surface of erythrocytes, reticulocytes, chicken embryo cells
and reticuloendotheliosis virus (REV)-transformed lymphoid
cells, but not on the AEV-transformed erythroleukemia cells.
Kornfeld et al. (1983) obtained five different groups
of MAbs by immunizing mice with normal macrophages and
myeloid cells transformed by MC29, AMV and E26. Only one
group was specific for myeloid lineage, predominantly
reacting with immature myeloid cells. However, the authers
failed to show the reactivities to normal cells except the
macrophages.
Trembicki and Dietert (1985) produced 4 MAbs against 1-
day Cornell K-strain white leghorn chickens. MAb 10C6


37
detected a chicken fetal antigen (CFA) on 1-day-old chick
erythrocytes. MAbs 3F12 and 4C2 recognized chicken adult
antigen (CAA) on adult erythrocytes, whereas 9F9 reacted
with all peripheral erythrocytes from both Japanese quail
and chicken regardless of age.
Schmidt et al. (1986) immunized mice with plasma
membranes from the ts34 AEV cell line HD3 induced to
differentiate at 42C for five days. Only one out of eight
groups of MAbs was specific for erythroid lineage, reacting
only with reticulocytes.
The following chapters in this dissertation describe
the first systematic attempt using MAbs in combination with
other techniques to identify embryonic differentiation
markers on normal avian hematopoietic cells and
transformation-associated antigens on retrovirus-transformed
ones.


CHAPTER 2
PRODUCTION OF MONOCLONAL ANTIBODIES
AND THEIR CELL-TYPE SPECIFICITIES
Introduction
The lack of MAbs recognizing embryonic differentiation
markers on avian hematopoietic precursor cells may be due to
the use of inappropriate antigens for immunization. Because
in most cases 1-day-old erythrocytes were chosen as antigens
instead of embryonic cells, the MAbs developed were not
specific for the embryonic precursor cells. Therefore it
was decided to use different strategies for the immunizing
protocols in order to produce MAbs which identify embryonic
differentiation markers present on normal avian
hematopoietic cells of the erythroid and monocytic lineages.
Moreover the approaches used were designed to identify
transformation-associated antigens in retrovirus transformed
avian hematopoietic cells and to characterize the
biochemical properties as well as to study the biological
functions of these markers.
Several types of cells were used to immunize 6-week-
old BALB/c BYJ female mice: 1) normal 3-day-embryo yolk sac
cells, 2) normal 3-day-embryo megalocytestogether with AEV-
38


39
transformed nonproducer cells from 3-day-embryo yolk sac, 3)
BM2 cells (AMV-transformed nonproducer cells from embryonic
bone marrow), and 4) BM2/L cells (leukemogenic variant of
BM2 cell line). Theoretically, 3-day-embryo yolk sac cells
are rich in embryonic hematopoietic precursor
cells, and the AMV/AEV nonproducer cells possess not only
the transformation-associated antigens but also
oncodevelopmental markers which are present normally only on
the precursor cells. 10-14 days after each cell fusion,
supernatants of hybridomas were screened against a panel of
different types of cells as well as against yolk by indirect
radioimmunoassay (RIA) and enzyme-linked immunosorbent assay
(ELISA). Only hybridomas showing continuous production of
MAbs of potential interest were subcloned by the limiting
dilution method. The isotypes of the MAbs were determined
by the Ouchterlony (double diffusion) test using isotype
specific antisera. MAbs were purified from culture media of
the cloned hybridomas or from mouse ascites by using high-
salt protein A-sepharose chromatography.
Material and Methods
Normal Cells
17-somite blastoderm. The blastoderm cells were
obtained from the 17-somite stage at 2 days of incubation by
mechanical dissociation with gentle pipetting and dispersed
in a-MEM (GIBCO) containing 10% fetal bovine serum (FBS).


40
Cells were then filtered through a 1.5/i nylon mesh (Tetko
Inc., New York) to remove any clumps (Moscovici et al.,
1983) .
Yolk sac cells. The yolk sacs from 3rd or 4th day of
embryogenesis were dissected free of other embryonic
membranes, pooled and rinsed extensively with Tyrode's
solution (8.0 g/L NaCl, 0.2 g/L KC1, 0.05 g/L NaH2P04-H20,
1.0 g/L Glucose and 1.0 g/L NaHC03) to remove as much yolk
as possible. The tissue were minced with scalpels and
dispensed in a-MEM/10% FBS by gentle pipetting. The
resulting cell suspension was then washed by centrifugation
to remove residual yolk, after which the cells were
resuspended in media and passed through nylon mesh to obtain
a single cell suspension. The yolk sacs from 6th or 12th
day of embryogenesis were first minced thoroughly with
scalpels and then digested with 0.125% trypsin for 10-15
minutes at 37C (Moscovici et al., 1975). The cell
suspension was then washed by centrifugation and passed
through nylon mesh as above.
Bone marrow cells. Bone marrow cells were flushed out
of the tibias with BT-88 medium (GIBCO) containing 10%
tryptose phosphate broth, 5% calf serum and 5% chicken serum
by passing the cells through a syringe with a 22-gauge
needle 3 times (Jurdic et al., 1982). The cells were washed
once, resuspended in medium and then filtered through nylon
mesh.


41
Buffv coat. The buffy coat (WBC) from peripheral blood
was harvested by Ficoll-Hypaque (Lymphocyte Separation
Medium; Litton Bionetics) gradient centrifugation at 2,000
rpm for 20 minutes.
Macrophages. The buffy coat obtained from heparinized
blood was seeded in BT-88 complete medium and 48 hours later
the attached cells differentiated into macrophages. These
cells were then detached from the petri dishes by adding the
C-PEG solution (8.0 g/L NaCl, 0.29 g/L KCl, 0.2 g/L KH2P04,
0.763 g/L Na2HP04, 0.2 g/L EDTA, 3.7 g/L NaHC03 and 1.0 g/L
Glucose, pH 8.0) for 5 minutes.
Chicken embryo fibroblasts (CEF). The fibroblasts from
10-day embryos were prepared according to the procedure
described by Vogt (1969).
Transformed Cells
BM2/C3A cells. An AMV-transformed monoblastic
nonproducer cell line, GM727, was generated by in vitro
infection of 17-day-embryo bone marrow cells with AMV-B at a
low multiplicity of infection (m.o.i. of 10"2 to 103)
(Moscovici & Moscovici, 1980). GM727 cells were then
injected into 13-day embryos via the chorioallantoic vein.
Four weeks after the injection, no overt case of leukemia
was observed unless the chickens were challenged with helper
viruses such as MAV-2 or RAV-7 (Moscovici et al., 1982).
However, the injected transformed cells could be retrived


42
from the bone marrow, and cloned by an in vitro colony
assay. A cell line namely BM2/C3A was established. The
cells in this line all express the v-myb proteins but are
nonproducers and are nonleukemogenic.
BM2/L cells. The BM2/L cell line is a variant of
BM2/C3A cell line. It was obtained from a BM2/C3A-injected
bird which came down with leukemia involving liver, spleen
and heart (Moscovici and Moscovici, unpublished results).
The leukemic cells were reisolated and a new line was
established, namely BM2/L, which when reinjected into
chicken embryos induced a 90-100% incidence of leukemia.
6C2 cells. 6C2 is an AEV(RAV-2)-transformed
erythroleukemia producer cell line obtained from infection
of adult bone marrow cells in vitro (Beug et al., 1982).
MSB 1 cells. MSB 1, obtained from U.S.D.A. (East
Lansing, MI), is a lymphoblastoid producer cell line derived
from a splenic lymphoma of chicken with Marek's Disease
(Akiyama and Kato, 1974).
Viruses
AMV-B. The AMV subgroup B (AMV-B) was derived from
standard laboratory stocks as described (Moscovici et al.,
1975).
AEV-A. The AEV subgroup A (AEV-A) is the ES-4 strain
of AEV (RAV-1) originally obtained from Dr. J.M. Bishop (San
Francisco, CA).


43
RAV-1 and RAV-2. The RAVs were prepared from our
laboratory stocks.
Discontinuous Percoll Gradient
2 ml of cell suspension was layered on top of a
discontinuous percoll gradient (20%/50%/70% for bone marrow
cells and 30%/50%/70% for yolk sac cells) and centrifuged 15
minutes at 2,500 rpm. It has been demonstrated under these
conditions that the 20%/50% interface from the bone marrow
cells and the 30%/50% interface from the yolk sac cells are
rich in the precursor cells and mononucleated cells, whereas
the 50%/70% interface consists mostly of erythroblasts and
other types of differentiated cells, and the cells from the
pellet are terminally differentiated erythrocytes.
Hvbridoma Production
Hybridomas were produced according to the protocol
established in the Hybridoma Laboratory, Interdisplinary
Center for Biotechnology Research, University of Florida.
Spleen cells harvested from immunized mice were fused with
SP2/0 myeloma cells at a ratio of 7.5:1 (spleen cells :
myeloma cells) using polyethylene glycol 1540. The
supernatants of growth-positive hybridomas from 96-well flat
bottom tissue culture plates were screened 12-14 days later
by the indirect RIA and ELISA methods. Hybridomas which


44
produced monoclonal antibodies with potential interest were
subcloned by the limiting dilution method.
Radioimmunoassay (RIA)
The cells used as targets in immunoassays were washed
with PBS buffer containing 1% AHS (gamma globulin-free horse
serum) and 0.02% sodium azide and resuspended to a final
concentration of 20 x 106 cells/ml in PBS/azide/5% AHS. 50
/Ltl of cell suspension and 100 ¡il of hybridoma supernatant
was added to each well of 96-well flexible polyvinyl round-
bottom microtiter plates (Dynatech) for 45 minutes at 4C.
At the end of this incubation, plates were washed with
PBS/azide/1% AHS and centrifuged at 1,100 rpm 3 times.
50 jul of 125I-rabbit anti-mouse IgG (RaMIG) containing 1 x
105 cpm was then added and incubated with the cells for 45
minutes at 4C. Plates were washed with PBS/azide/AHS and
centrifuged 3 times again. Individual wells were cut free
from each plate with a hot-wire cutter, transferred into
plastic tubes and counted in a gamma counter (LKB-Wallace
Ria Gamma 1274, Pharmacia).
Binding index = mean com bound with specific MAbs
mean cpm bound with negative control MAb
Enzvme-Linked Immunosorbent Assay (ELISA)
96-well flat bottom immunoplates (Nunc, Denmark) were
coated with 50 il of yolk at a 1:40 dilution overnight and
then blocked with IX PBS containing 0.02% sodium azide and


45
1% BSA for 1 hour at room temperature. 100 il of hybridoma
supernatants were then incubated in wells for 45 minutes at
room temperature followed by 100 1 alkaline phosphatase-
conjugated rabbit anti-mouse IgG (Sigma) at a 1:1000
dilution in PBS/azide/BSA for 45 minutes at room
temperature. The plates were finally incubated with 200 pi
p-nitrophenyl phosphate (P-NPP) (lmg/ml) (Sigma) in pH 9.0
bicarbonate substrate buffer for 30 minutes to 2 hours in
the dark and read on an ELISA reader (Molecular Devices; V
Max). The plates were washed three times with PBS/azide/1%
Tween-20 in between the steps.
Immunofluorescence staining
Live cells. The cells were incubated with 1 ml MAb
supernatant for 30 minutes at 4C and washed before fixation
with 30 pi 37% formaldehyde in 1 ml PBS for 20 minutes at
4C. The cells were washed again after fixation. 200 pi
fluorescein-isothiocyanate (FITC) conjugated goat anti
mouse polyvalent IgG, A and M (FITC-GAM) at 1:50 dilution in
PBS containing 1% normal goat serum was then added for 30
minutes at room temperature. After washing in PBS twice,
the cell pellet was resuspended with 200 /I PPD-Glycerol/
PBS (1 ml 10X PBS, 3 ml deionized water, 6 ml glycerol and 1
to 2 flakes of p-phenylene diamine) and dropped onto slides
to observe under the fluorescence microscope coverslipped.


46
Frozen sections. Chicken embryos from 3 and 6 days of
incubation were dissected. Tissues of no more than a few mm
thick were fixed in 4% paraformaldehyde fix (in 0.1 M sodium
cacodylate buffer, pH 7.2-7.4) for 5 hours and shifted into
30% sucrose in IX PBS overnight. Afterwards, the tissues
were embedded in OCT compound (Lab-Tek; Miles) and frozen
with liquid nitrogen. 10 |im Cryostat sections were then
mounted onto slides which were precoated with Histostick
(Accuraqe Biochemicals) and stored at -20C overnight prior
to use. The slides were incubated with primary antibodies
containing 0.3% triton-X in PBS for 30 minutes at room
temperature. They were then washed in a PBS bath for 5
minutes and incubated with FITC-GAM/0.3% triton-X/1% normal
goat serum for another 30 minutes at room temperature. The
slides were again washed in PBS for 5 minutes followed by
addition of a few drops of PPD-Glycerol/PBS and then
examined under fluorescence microscope coverslipped.
Flow Cytometry
Cells to be examined were washed with IX PBS/azide/AHS
and incubated with 1 ml MAb supernatant for 1 hour on a
rocker at 4C followed by incubation with 1:10 dilution of
FITC-conjugated sheep anti-mouse IgG [F(ab')2 fragment]
(Sigma) for 30 minutes on a rocker at 4C. Cells were
washed twice and resuspended in Hank's balanced salt
solution (HBSS) containing 1% FBS at a concentration of 2 to


47
3 x 106 cells/xnl. 1 x 104 cells were then analyzed on a
FACStar-plus fluorescence-activated cell sorter (Becton-
Dickinson, Mountain View, CA) by the parameters of forward
light scatter and fluorescein fluorescence. Cellular
excitation was obtained with an emission wavelength of 488
nm at an output power of 0.25W for fluorescein fluorescence.
The FITC fluorescence emitted was filtered with a 530 nm
long pass interface filter and a 530 band pass filter. The
data were collected and analyzed by a Becton/Dickinson
Consort 30 Computer program (Braylan et al., 1982).
Immunoenzvmatic Staining by APAAP (Alkaline Phosphatase and
Monoclonal Anti-Alkaline Phosphatase) Complex
2 x 105 cells in 0.2 ml were cytofuged onto slides by
Cytospin (Shandon Southern) at 300 rpm for 7 minutes. The
slides were air-dried at room temperature for 2 hours
followed by fixation with equal parts of acetone and
methanol for 5 minutes at 4C. 1 ml MAb supernatants were
added to the slides and incubated in a moist chamber for 30
minutes at room temperature. Anti-mouse immunoglobulins
(DAKOPATTS; at 1:25 dilution) were then incubated for 30
minutes followed by APAAP complex (DAKOPATTS; at 1:50
dilution) for another 30 minutes. The slides were washed in
a tris-buffered saline (TBS) bath for 1 minute in between
the steps. Finally, alkaline phosphatase substrate was
added onto the slides for 15-20 minutes and then washed off
first with TBS and then with tap water (Cordell et al.,


48
1984). Slides were counter-stained with Giemsa stain at
1:20 dilution for 5 minutes.
Benzidine Staining
10 /Lil H202 (30%) was added to one ml of the benzidine
solution (0.5 g benzidine in 100 ml 70% ethanol) immediately
prior to use. Just a few drops of the staining mixture were
deposited on the cell sample and left at room temperature in
the dark for 5-10 minutes.
Induction of Cell Differentiation
BM2 cells. 1 x 107 BM2 cells were treated with 10
/zg/ml lipopolysaccharide (LPS) and 0.25 /xg/ml phorbol
12-myristate-13-acetate (PMA) or 2.5 nq/ml PMA alone in a
100-mm petri dish. 3 days later, most of BM2 cells had
attached to the petri dish and had differentiated into
macrophages.
6C2 cells. 6C2 cells were treated with 1.0 mM butyric
acid for 3 days. Although 6C2 cells did not
differentiate into mature erythrocytes with butyric acid,
their proliferating potential had been arrested.
High-Salt Protein A-Sepharose Chromatography
Ascites (1:10 dilution) or hybridoma supernatants were
adjusted to contain 1.5M glycine, 3M NaCl (pH 8.9), filtered
through a 0.22 n millipore filter and run through a protein


49
A-sepharose column (Sigma) twice to allow binding of IgG to
the column. 5-10 column volumes of binding buffer (1.5M
glycine, 3M NaCl, pH 8.9) was then percolated through the
column to get rid of unbound proteins. Elution buffer (100
mM citric acid, pH 6.0) was then added to the column to
elute the IgG, followed by regeneration buffer (100 mM
citric acid, pH 3.0) to wash the column. Samples were
collected by a fraction collector, neutralized to pH 7.0-
7.2 with 1M tris buffer (pH 9.0) and read with a U.V.
spectrophotometer at wavelength 280 nm. The concentration
of IgG was calculated as: mg/ml = O.D. 280 nm / 1.4.
Collected fractions were then dialyzed against lx PBS/azide
buffer overnight.
Results
Production. Subcloninq and Isotvoinq of the MAbs
The rationale for MAb production is simple and
straightforward, however, the goals turned out to be much
tougher to achieve than we originally expected, especially
from the fusions with spleens from mice immunized against 3-
day-embryo yolk sac cells and megalocytes. Of nearly 2,500
hybridomas derived from 5 fusions, the predominant antibody
specificities detected were for yolk components due to the
fact that the unavoidably large amount of yolk was
associated with the yolk sac cells. Only three MAbs
exhibited potential interest, namely, 2E10, 3F6 and 1F7.


50
Fusions of spleens from mice immunized against BM2
cells and BM2/L cells yielded a panel of MAbs with various
specificities against different types of hematopoietic cells
rather than just MAbs specific for BM2 or BM2/L cells (Table
2-1). Three MAbs, 1H10, 2H1 and 3D7, from group VI, which
displayed specificity for normal monocytic cells and AMV-
transformed cells, were chosen for further studies.
These selected hybridomas were then subcloned by
limiting dilution methods and isotyped by Ouchterlony double
diffusion tests (Table 2-2). MAbs 1H10-1F9, 2H1-2A10, 3D7-
1C9 and 2E10-1E10 as well as 3F6-1E7 are all IgGl (k); only
1F7-1A3 is an IgM (/c) .
Cell-Type Specificities of MAbs
As demonstrated by RIA, flow cytometry and
immunofluorescence staining, MAbs 1H10-1F9, 2H1-2A10 and
3D7-1C9 exhibited specificity for monocytic cells with a
preferential reaction against BM2 lines. The RIA results
also revealed a slight reaction of these MAbs with cells
from the 20%/50% interface of a discontinuous percoll
gradient from 2-week-old bone marrow cells (Table 2-3).
Further analysis by flow cytometry (Figure 2-1) and APAAP
immunoenzymatic staining (Figure 2-2) confirm that about
10%-20% of the cells are expressing different degrees of the
differentiation markers recognized by this group of MAbs.
Conversely, no expression of these markers was observed in


51
Table 2-1. The cell-type specificities of MAbs from BM2 and
BM2/L fusions
Monoclonal Antibody Group
Cell Type
II
II
III
IV
V
VIa
Normal Cells
Granulocytic
++b
+
-
-
+
-
Monocytic
++
++
+
+
++
+
Erythroid
++
+
+
-
-
-
Lymphoid
++
-
-
-
+
-
Transformed Cells
AMV
++
++
++
++
++
++
AEV
++
++
+
+
-
-
aOnly three MAbs
from group VI
were selected
for further
characterization because of their specificities for
normal monocytic cells and AMV-transformed cells.
b++: RIA binding indexes >10.0; +; RIA binding indexes >5.0;
-: RIA binding indexes <2.0.
Table 2-2. The
isotypes of selected
MAbs
MAb
Isotype
1H10-1F9
IgGl
(/c)
2H1-2A10
IgGl
()
3D7-1C9
IgGl
(k)
2E10-1E10
IgGl
(k)
3F6-1E7
IgGl
(k)
1F7-1A3
IgM
(k)


52
Table 2-3. RIA binding indexes of MAbs to different cell types
Monoclonal
Antibodv
Cells*
1H10-1F9
2H1-2A10
3D7-1C9
2E10-1E10
3F6-1E7
1F7-1A3
TRANSFORMED
6C2 1.19b
1.21
1.66
26.35
3.03
3.96
3DYS-AEV
N.T.
N.T.
1.00
6.48
5.38
3.04
6DYS-AEV
N.T.
N.T.
N.T.
25.94
12.52
5.27
MSB1
1.26
0.59
0.34
14.06
7.78
5.17
BM2/C3A
43.55
38.80
38.01
9.91
9.61
1.25
BM2/REC1
34.09
50.83
57.77
8.03
N.T.
N.T.
BM2L/A1
29.98
35.05
41.72
8.26
9.72
1.29
NORMAL
M0
4.89
6.85
5.87
N.T.
N.T.
N.T.
RBC(PB)
0.77
1.52
1.14
2.58
1.09
1.31
WBC(PB)
1.15
2.25
1.63
2.24
1.25
1.11
CEF
0.83
0.97
0.88
0.87
0.75
N.T.
2DYS
0.52
0.94
1.13
2.34
2.01
N.D.
3DYS
1.52
1.40
1.16
2.18
2.11
5.72
6DYS
0.68
1.60
0.84
3.27
1.12
N.T.
12DYS
0.69
1.28
0.62
7.59
0.52
N.T.
BM 20/50
2.88
3.48
2.89
4.76
1.98
N.T.
BM 50/70
1.62
2.39
1.91
7.49
1.63
N.T.
a6C2, AEV-transformed producer cell line from adult bone marrow;
3DYS-AEV, AEV-transformed 3-day-embryo yolk sac cells; MSB1,
Marek's Disease Virus-transformed lymphoblastoid producer cell
line; BM2, AMV-transformed non-producer cell line from embryonic
bone marrow; BM2/C3A, a subclone of BM2; BM2/REC1, a subclone
recovered from the bone marrow of BM2/C3A injected chick;
BM2L/A1, a leukemogenic variant of BM2/C3A; M peripheral blood; CEF, chicken embryo fibroblast; BM 20/50,
20%/50% interface of a percoll gradient of 2-week-old bone
marrow cells; N.T., not tested.
bRIA binding indexes; see Materials and Methods for details.


Cell Number
53
Log Fluorescence Intensity
Figure 2-1. Flow cytometric analysis of the 20%/50%
interface of a percoll gradient of 2-week-old bone marrow
cells. The cells were labeled with negative control MAb,
1H10-1F9, 2H1-2A10 and 3D7-1C9 respectively. The background
fluorescence resulting either from nonspecific binding or
autofluorescence was present in 10% of the cells. 10-20% of
the cells were positive for MAbs 1H10-1F9, 2H1-2A10 and 3D7-
1C9.


54
Figure 2-2. APAAP immunostaining of 20%/50% interface of a
percoll gradient of 2-week-old bone marrow cells (600x).
The cytofuges were treated with (A) negative control MAb,
(B) 1H10-1F9, (C) 2H1-2A10 and (D) 3D7-1C9 respectively.
Positive cells were labeled with purple-reddish products
around the cell surface and/or inside the cytoplasm.


55
the cells from the 50%/70% interface (Table 2-3 and Figure
2-3) .
1F7-1A3 recognizes the AEV-transformed yolk sac cells,
MSB1 cells and 20% of the cells in the 30%/50% interface of
a discontinuous percoll gradient of yolk sac cells (Table 2-
3 and Figure 2-4). Interestingly enough, its reaction to 6C2
cells is amplified after the treatment with neuraminidase
(discussed in Chapter 4 in more detail).
3F6-1E7 detects a differentiation marker present mainly
on AEV-transformed yolk sac cells, BM2 cell lines and MSB1
cells as well as 10% of cells from the 30%/50% interface of
a percoll gradient of normal yolk sac cells (Table 2-3 and
Figure 2-4).
2E10-1E10 possesses a variety of cell-type
specificities such as 6C2 cells, AEV-transformed yolk sac
cells, MSB1 cells, BM2 cells, 6 & 12-day-embryo yolk sac
cells and 2-week-old bone marrow cells (Table 2-3, Figure
2-5 & Figure 2-6). However, there is no reaction to the
terminally differentiated hematopoietic cells.
The cell-type specificities of MAbs are summarized in
Table 2-4.
Induction of Cell Differentiation
Treatment of BM2/C3A cells with 0.25 xg/ml PMA and 10
jug/ml LPS or 2.5 /g/ml PMA alone for 3 days caused the
BM2/C3A cells to attach to the petri dishes and become


Cell Number
56
Figure 2-3. Flow cytometric analysis of the 50%/70%
interface of a percoll gradient of 2-week-old bone marrow
cells. The cells were labeled with negative control MAb,
1H10-1F9, 2H1-2A10 and 3D7-1C9 respectively. The results
showed no reaction of MAbs with these cells at all.


57
Figure 2-4. Flow cytometric analysis of the 30%/50%
interface of a percoll gradient of 4-day-embryo yolk sac
cells. Cells were tagged with negative control MAb, 1F7-1A3
and 3F6-1E7 respectively. The analysis showed that 3F6-1E7
label 10% of the cells, whereas 20% of the cells are
positive for 1F7-1A3.


58
Figure 2-5. Flow cytometric analysis of the 20%/50%
interface of a percoll gradient of 2-week-old bone marrow
cells tagged with MAb 2E10-1E10. Almost 80% of the cells
were positive for MAb 2E10-1E10.


59
Figure 2-6. Flow cytometric analysis of the 50%/70%
interface of a percoll gradient of 2-week-old bone marrow
cells tagged with MAb 2E10-1E10. 2E10-1E10 recognized
nearly 70% of the cells.


60
Table 2-4. Summary of the cell-type specificities of MAbs
Groups
MAbs
Specificities
I
3D7-1C9
2H1-2A10
1H10-1F9
BM2 cell lines
Normal monocytic cells
II
3F6-1E7
AEV-transformed yolk sac cells
BM2 cell lines
MSB1 (Marek's Disease)
10% of cells from the 30%/50%
interface of a percoll fraction
of 3-day-embryo yolk sac cells
III
1F7-1A3
AEV-transformed yolk sac cells
MSB1 (Marek's Disease)
20% of cells from the 30%/50%
interface of a percoll fraction
of 3-day-embryo yolk sac cells
IV
2E10-1E10
6C2 cells
AEV-transformed yolk sac cells
MSB1 (Marek's Disease)
BM2 cell lines
6-day-embryo yolk sac cells
12-day-embryo yolk sac cells
2-week-old bone marrow cells


61
differentiated into macrophage-like cells. In addition,
their proliferating potential (Table 2-5) and ability to
bind MAbs 1H10-1F9, 2H1-2A10 and 3D7-1C9 were dramatically
decreased (Figure 2-7). Therefore, the differentiation
markers recognized by these MAbs may be candidates for
transformation-associated antigens in this system.
Although 6C2 cells were not induced to terminally
differentiate into erythrocytes with the treatment of ImM
butyric acid for 3 days, their proliferating potential had
been impaired (Table 2-6) and the expression of the 2E10-
lE10-recognized marker was also reduced (Figure 2-8).
Reactivity to the Envelope Proteins of Retroviruses
The RAV-1 or RAV-2 infected CEF cells were used as
control to exclude the possibility that MAbs might react
with the envelope proteins of retroviruses which were
expressed on the cell surface. 3F6-1E7 and 2E10-1E10 were
shown to have no reaction to either CEF cells or RAV-
infected CEF cells (Table 2-7).
Discussion
Four groups of MAbs were selected among nearly 5,000
hybridoma supernatants from ten fusions. Characterization
of their cell-type specificities was achived by RIA,
immunofluorescence staining, flow cytometry and
immunoenzymatic staining. 1) MAbs 1H10-1F9, 2H10-2A10 and


Table 2-5. Comparison of the proliferating potential of
BM2/C3A cells and 2.5nq/ml PMA-diffentiated BM2/C3A cells
BM2/C3Ab
BM2/C3A + PMAb
Number of cells
after 3 days of
incubation
58 x 106
6 X 106
a10 x 106 cells were seeded per 100-mm petri dish.
bThe number of cells was expressed as the average from 6
dishes.


Figure 2-7. Reactivities of MAbs with BM2 cells and differentiated BM2 cells.
BM2 cells were differentiated into macrophage-like cells by the treatment of
0.25 /ig/m 1 PMA and 10 ng/ml LPS for three days. The reactivities are expressed
as the MAb binding indexes as determined by RIA.


1H10-1F9 2H1-2A10 3D7-1C9 2E10-1E10 3F6-1E7
Control PMA&LPS
(Ti


65
Table 2-6. Comparison of the proliferating potential of 6C2
cells and ImM butyric acid-treated 6C2 cells
6C2b
6C2 + Butyric acidb
Number of cells
after 3 days of 40 x 106
incubation3
ao ^ -i
11.5 x 106
a10 x 106 cells were seeded in 60-mm petri dish.
bThe number of cells was expressed as the average from 6
dishes.


Figure 2-8. Reactivities of MAbs with 6C2 cells and butyric acid-treated 6C2
cells. 6C2 cells were treated with l.OmM butyric acid for three days. Though
6C2 cells were not differentiated into erythrocytes, the proliferating potential
had been impaired. The reactivities are expressed as MAb the binding indexes as
determined by RIA.


30
25
20
15
10
5
0
ON
'J


68
Table 2-7. RIA binding indexes of MAbs to normal CEF and
RAV-infected CEF
Cells
Monoclonal
2E10-1E10
Antibody
3F6-1E7
CEF
0.87
0.75
CEF
(RAV-1)
1.08
0.75
CEF
(RAV-2)
1.10
0.80


69
3D7-1C9 are specific for transformation-associated antigens
present preferentially on BM2 cell lines rather than on
normal monocytic cells. 2) MAb 1F7-1A3 recognizes AEV-
transformed yolk sac cells, MSB1 cells and 20% of cells from
the 30%/50% interface of a discontinous percoll gradient of
4-day-embryo yolk sac cells. 3) MAb 3F6-1E7 reacts with
AEV-transformed yolk sac cells, BM2 cells and MSB1 cells as
well as 10% of cells from the interface of a discontinous
percoll gradient of 4-day-embryo yolk sac cells. 4) MAb
2E10-1E10 defines a marker present on different tumor cells,
yolk sac cells and bone marrow cells instead of terminally
differentiated cells.
The expression of transformation-associated antigens
recognized by MAbs 1H10-1F9, 2H1-2A10 and 3D7-1C9 is
possibly up-regulated by v-myb proteins, but is diminished
after the BM2 cells are differentiated by PMA. Future
investigators may want to determine 1) whether the
expression of these antigens is regulated at the
transcriptional and/or the translational level or is due to
posttranslational modification, such as aberrant
glycosylation and/or sialylation, and 2) whether the
expression of these markers is essential for the
transforming process or if they are merely the by-products
of transformation.
Although 2E10-1E10 displays a wide range of cell-type
specificities, it labels primarily 6C2 cells and


70
AEV-transformed 6-day-embryo yolk sac cells and it does not
recognize terminally differentiated cells such as
erythrocytes, macrophages, lymphocytes and PMA-
differentiated BM2/C3A cells. These results lead us to
speculate that 2E10-1E10 may react only with proliferating
hematopoietic cells. This possibility was supported by the
observation that the binding specificities of 2E10-1E10 to
6C2 cells was diminished after treatment with butyric acid
which inhibited the proliferating potential of 6C2 cells
without the induction of differentiation. Moreover, this
marker starts appearing at the 4th day of embryogenesis
(discussed in Chapter 3 in more detail) and its expression
is enhanced after the cells are transformed by AEV.
LPS only induces the BM2 cells to attach to the petri
dishes but does not induce differentiation. As a matter of
fact, PMA alone can induce both the attachment and
differentiation of the BM2 cells even at a low concentration
of 0.25 ig/ml. The reason why we added 10 ^g/ml LPS to the
PMA treatment was simply because we didn't want to see "any
fish sneak out of the net", as the Chinese proverb says.
Butyric acid has been shown to induce the
differentiation of the AEV strain R (RAV-2)-transformed
erythroleukemia cells from SC strain chickens (Nelson et
al., 1982), but was incapable of differentiating 6C2 cells
even though their proliferating potential had been
apparently impaired. This is possibly due to the


71
difference in the susceptibilities to butyric acid of
various avian erythroleukemia cell lines and/or sublines.
Immunoperoxidase staining procedures were not used
because significant amounts of endogenous peroxidase present
in granulocytes, erythroid cells and macrophages would give
rise to unwanted background staining, and it is rather
difficult to abolish this activity by exposing samples to
peroxidase inhibitors (such as H202 and methanol) without
causing antigenic denaturation (Cordell et al., 1984). In
contrast endogenous alkaline phosphatase activity survives
poorly in cytofuge slide preparation and any residual
activity may be selectively inhibited by including
levamisole in the enzyme substrate solution (Ponder and
Wilkinson, 1981). Therefore the APAAP immunostaining
technique was chosen for our studies. Wet slides without
any mounting fluid were then photographed becaused it was
observed that the nonaqueous mounting solution dissolved the
reaction product, whereas an aqueous mounting medium will
disrupt the Giemsa counter-stain.
The antigens recognized by MAbs 1F7-1A3, 2E10-1E10 and
3F6-1E7 could only be detected by RIA and immunofluorescence
staining and not by APAAP immunostaining technique. It is
very likely that these markers may be so fragile that they
were destroyed during the fixation (acetone and ethanol)
and/or any subsequent steps.


CHAPTER 3
IDENTIFICATION OF TARGET CELLS FOR MABS
IN THE BONE MARROW AND YOLK SAC
Introduction
The avian hematopoietic precursor cells represent only
a small population in the blood-forming organs, however, it
is this small population that builds up the entire
hematopoietic repertoire of various types of specialized
blood cells with different functions. The nature of self
renewal and commitment remains an enigma, but if the
precursor cells can be isolated from the heterogenous
population of hematopoietic cells for direct studies, it
would provide us with invaluable pieces of information to
solve the enigma. However, these cells are morphologically
unrecognizable, and until now no specific markers had been
identified to facilitate the purification of these cells.
One of the specific aims of this project was to develop MAbs
against differentiation markers specifically present on the
surface membranes of the avian hematopoietic precursor
cells. These cells have been purified and enriched by using
FACS or immunomagnetic beads as follows. Bone marrow cells
or yolk sac cells are treated with specific MAbs followed by
FITC-conjugated or magnetic bead coated with secondary
72


73
antibodies. Fluorescence-positive and -negative populations
are then separated by the FACS, whereas the magnetic bead-
bound cells are separated from the negative population by a
magnetic field (negative selection). The separated
fractions are then identified by indirect methods, such as
AMV/AEV transforming assays and BFU-E/CFU-E colony forming
assays. For example, if MAb-positive fractions produce
colonies derived from the CFU-E instead of the BFU-E, the
antigen must be expressed later than the BFU-E stage; if it
produces AMV-transformed colonies, but neither AEV-
transformed colonies nor BFU-E/CFU-E colonies are developed,
the MAb ought to be specific for the target cells for AMV,
i.e., cells of the monocytic lineage.
These approaches helped us to confirm the cell-type
specificities of the MAbs that we developed.
Materials And Methods
FACS Sorting
After being analyzed on a FACStar-plus sorter,
according to the procedure described in the previous
chapter, the cells were sorted into positive and negative
fractions at a rate of 1,000 cells/sec.
Immumomagnetic Beads Separation
10 x 106 Cells were tagged with 1 ml of MAb
supernatants for 1 hour at 4C on a rocker and then washed


74
with PBS/azide/1% AHS followed by incubation with magnetic
beads coated with sheep anti-mouse IgG, (Fc) (Dynabeads M-
450; Dynal Inc.) or magnetic particles coated with goat
anti-mouse IgM (Advanced Magnetics) at a bead to cell ratio
of 4 to 1 for 30 minutes at 4C on a rocker. Cells bound to
the beads were separated from the negative population that
remained in suspension by applying a cobalt steel magnetic
force (Dynal MPC-1; Dynal Inc.) for 1 minute (Cruikshank et
al., 1987). This process was repeated twice. It was not
possible to separate viable cells (positive selection) from
the magnetic beads because of technical limitations.
Retrovirus Transforming Assays
The transforming assays were performed according to the
protocol described by Moscovici et al. (1983). Briefly,
cells were infected with retroviruses at high m.o.i. The
virus adsorption was carried out 30 minutes at 4C and then
30 minutes at room temperature. 1 xlO5 AEV-infected cells
was seeded in 2 ml semi-solid a-medium containing 20% FBS,
10% ACS, 1% BPA, 0.1% 10_1M 0-mercaptoethanol, 0.1%
gentamycin and 25% methocellulose per 35-mm dish. Whereas,
1 xlO5 AMV-infected cells was mixed with 1 ml F12 overlay
medium containing 20% 2X F12, 6% calf serum, 2% chicken
serum, 10% tryptose phosphate, 1% 100X vitamins, 1% 100X
folic acid and 40% fibroblast-conditioned medium as well as
20% of 1.8% Bacto agar, and then overlayed on top of 2 ml


75
3.6% hard agar base in 35-nun petri dishes. The transformed
colonies were scored after 6-12 days of incubation.
BFU-E/CFU-E Colony Assays
0.5 ml of cell suspension were mixed with a-medium
containing 20% FBS, 10% ACS, 1% BPA, 0.1% lO^M /3-
mercaptoethanol, 0.1% gentamycin and 25% methocellulose and
seeded at 1x10s cells per 35-mm dish. The CFU-E were scored
after 3-4 days of incubation, whereas BFU-E were scored
after 6-7 days of incubation (Samarut and Bouabdelli, 1980).
Results
FACS Sorting
In order to identify the target cells for the MAbs in
the bone marrow, FACS was utilized to separate the MAb-
positive population and MAb-negative population. The bone
marrow cells were analyzed before and after the FACS sorting
(Figure 3-1 and 3-2). Transforming assays and colony
forming assays were performed on both negative and positive
populations.
The results obtained in the transforming assays and
colony-forming assays (Table 3-1) confirm that the 3D7-1C9
positive population (10%-20% of the cells from the 20%/50%
interface of a discontinuous percoll gradient of 2-week-old
bone marrow cells) represents the target cells for AMV,
i.e., cells of the monocytic lineage.


76
Figure 3-1. Flow cytometric analysis of 3D7-lC9-tagged bone
marrow cells before sorting. 20%/50% interface of a percoll
gradient of 2-week-old bone marrow cells were tagged with
3D7-1C9 MAb. The analysis showed that 10-20% of the cells
are positive for MAb 3D7-1C9.


77
Figure 3-2. Flow cytometric analysis of 3D7-lC9-tagged bone
marrow cells after sorting. 20%/50% interface of a percoll
gradient of 2-week-old bone marrow cells were sorted into
3D7-1C9 positive fraction and 3D7-1C9 negative fraction
followed by the FACS analysis. The 3D7-1C9 positive
fraction still contained about 20% negative cells due to
contamination during the sorting.


78
Table 3-1. Characterization of the target cells isolated
with MAb 3D7-1C9 from the bone marrow
Colonies 3D7-1C9
(lxlO5 cells) positive
population
3D7-1C9
negative
population
CFU-E
34.0
+
5.0
1620.5
+
52.5
BFU-E
12.0

3.0
512.0
+
132.0
AEV-A
26.0
+
2.0
144.0
+
7.0
AMV-B
592.5
+
20.5
86.5
+
13.5
20%/50% interface of a percoll gradient of 2-week-old bone
marrow cells were tagged with 3D7-1C9 and then sorted by
FACStar followed by transforming assays and colony
forming assays. The results show that 3D7-1C9 positive
population contain the target cells for AMV, i.e.,
cells of monocytic lineage, rather than BFU-E/CFU-E or the
target cells for AEV.


79
On the other hand, the results of colony-forming assays
and retrovirus transforming assays suggest that MAb 2E10-
1E10 not only recognize the target cells for AEV and AMV
but also the BFU-E and CFU-E (Table 3-2). This finding
prompted us to suggest in Chapter 2 that MAb 2E10-1E10 react
with proliferating hematopoietic cells.
Immunomaqnetic Bead Separation
Cells from 30%/50% interface of a discontinuous percoll
gradient of 4-day-embryo yolk sacs were incubated with MAbs
3F6-1E7 or 1F7-1A3 followed by magnetic beads conjugated
with secondary antibodies. Only the negatively selected
population of MAbs were used for the assays. There was
about 60% reduction in the BFU-E and the CFU-E colonies from
1F7-1A3 negative population. However, no significant
difference in transformed colonies was found (Table 3-3).
In other words, MAb 1F7-1A3 recognizes erythroid cells at
the BFU-E and the CFU-E stages but not the pre-BFU-E stage,
since it does not recognize cells at the pre-BFU-E stage
which are the target cell for AEV in the yolk sac.
Conversely MAb 3F6-1E7 seems not to react with either the
BFU-E/CFU-E or the target cells for the retroviruses. It
probably recognizes a differentiation marker expressed on
the stem cell and precursor cell populations at an earlier
stage than pre-BFU-E one.


Table 3-2. Characterization of the target cells isolated
with MAb 2E10-1E10 from the bone marrow
80
Colonies 2E10-1E10
(lxlO5 cells) positive
population
2E10-1E10
negative
population
CFU-E
474.0
+
27.0
o

o

o

o
BFU-E
282.0
+
2.0
0.0
+
o

o
AEV-A
59.0
+
1.0
0.5
+
0.5
AMV-B
403.0
+
3.0
o

in
+
2.0
20%/50% interface of a percoll gradient of 2-week-old bone
marrow cells were tagged with 2E10-1E10 and then sorted by
FACStar followed by transforming assays and colony
forming assays. The results reveal that MAb 2E10-1E10
recognize not only BFU-E/CFU-E, but also the target cells
for AEV and AMV.
Table 3-3. Characterization of the target cells isolated
with MAbs from the yolk sac
Colonies
(1 x 105
Monoclonal
Antibodv
Cells) Control
3F6-
1E7
2E10-1E10
1F7-
1A3
CFU-E 3313.1142.5
2576.21232.4
1597.0178.4
1487.51103.4
BFU-E 1211.9
11.9
899.21
67.3
684.8173.3
501.11
23.8
AEV-A 135.6
6.4
115.01
7.0
108.11 5.2
102.01
11.0
AMV-B 50.0
5.0
46.01
2.0
36.81 2.4
44.01
3.0
30%/50% interface of a percoll gradient of 4-day-embryo yolk
sac cells were treated respectively with MAbs 1F7-1A3, 2E10-
1E10 or 3F6-1E7 followed by immunomagnetic bead separation.
The MAb-negative populations were collected to perform
colony-forming assays and transforming assays. See text for
more details.


81
The MAb 2E10-1E10 negative population in the 4-day-
embryo yolk sac cells exhibited nearly 50% reduction in the
BFU-E/CFU-E colonies and 30% in the AMV-transformed colonies
repectively (Table 3-3). Moreover, I have shown in Chapter
2 that MAb 2E10-1E10 has no binding specificities for 2- or
3-day-embryo yolk sac cell. These results indicate that the
marker identified by MAb 2E10-1E10 starts appearing after
the 4th day of embryogenesis.
Discussion
The FACS sorting and immunomagnetic bead techniques
allow us to perform direct studies on MAb-positive and/or
MAb-negative populations. The results from colony-forming
assays and transforming assays showed that MAb 3D7-1C9 can
purify the target cells for AMV, i.e., cells of the
monocytic lineage, MAb 2E10-1E10 define a marker present on
proliferating hematopoietic cells and it starts appearing
only after the 4th day of embryogenesis and that MAb 1F7-1A3
recognize erythroid cells at BFU-E and CFU-E stages.
MAb 3F6-1E7 recognizes only some tumor cell lines and
10% of cells from the 30%/50% interface of a discontinuous
percoll gradient of 3- and 4-day-embryo yolk sac cells, and
it does not possess the specificities for the terminally
differentiated cells and the lineage-committed progenitor
cells. However there is no direct evidence yet to support
the idea that the 10% of the cells recognized by MAb 3F6-1E7


82
represent the stem cell and precursor cell populations in
the yolk sac. In order to prove this, there are two
obstacles that need to be overcome. One is to improve the
FACS sorting condition to prevent yolk sac cells from
lysing. The other is to establish a long-term culture of
normal avian yolk sac cells. Once these problems are
solved, it will become possible to obtain the long-term
culture of the FACS-sorted 3F6-1E7 positive cells and then
we can compare the results from the transforming assays and
colony-forming assays between the 3F6-1E7 positive
population with and without the long-term culture.
Theoretically, in the long-term culture, the stem cell and
precursor cell populations will proliferate and undergo the
normal differentiation program to become the committed
progenitor cells and mature cells. If the 3F6-1E7 positive
cells indeed represent these populations, the increased
number of retrovirus-transformed colonies and BFU-E/CFU-E
colonies will then be observed in the 3F6-1E7 positive cells
with long-term culture. Ultimately, 3F6-1E7 positive cells
should be capable of repopulating the bone marrow of
irradiated chicks.
It is still a puzzle why MAb 1F7-1A3 reacts with
lymphoblastoid cell line-MSBl. Maybe the marker recognized
by 1F7-1A3, which is normally present on the embryonic
BFU-E/CFU-E only, can be regarded as an onco-fetal antigen,
i.e., its expression being turned on in the AEV-transformed


83
yolk sac cells as well as in the MSB1 cells instead of in
other types of transformed cells. This interesting finding
also implies that the relationship between the erythroid and
lymphoid lineage may be closer than we originally thought.
The bone marrow cells were sorted into fluorescence
positive and -negative populations using the FACStar-plus at
a rate of 1,000 cells/sec. At this rate it took
approximately 2-3 hours to collect the minimum workable
number of cells, i.e., 5xl05 MAb-positive cells which
account for 10-20% of cells from the 20%/50% interface of a
discontinuous percoll gradient of 2-week-old bone marrow
cells. The results of flow cytometry, retrovirus
transforming assays and colony-forming assays showed that
there is a slight contamination of MAb-negative cells in the
positive population. To increase the purity of the
collection by reducing the analysis rate or by processing a
"two-run" procedure would be too time-consuming and the
viability or the behavior of the sorted cells might be
influenced.
The yolk sac cells are extremely delicate and fragile
compared to other types of cells. Suffering from the
"abuse" of scalpel mincing, extensive washing, percoll
fractionation, as well as FACS sorting, the cell membranes
could have been damaged in such a way that only less than
10% of the sorted cells stayed intact, while the rest were
all lysed. We have tried different approaches such as


84
changing the sheath fluid, minimizing the laser power and
electric charge, reducing the centrifugation speed and
changing the collecting tubes etc. None so far gave us
satisfactory results.
Nevertheless, this is the pioneer study of the avian
hematopoietic system by FACS analysis. Once all the
conditions are standardized, it will become a tremendously
powerful tool to identify the stem cell and precursor cell
populations of the avian hematopoietic system, especially in
the yolk sac.
There are two disadvantages in using the immunomagnetic
beads for cell separation. First, its sensitivity is much
lower than that of the FACS sorting, i.e., it yields less
pure separation than FACS does. Second, because of the
strength of positive cells binding to magnetic beads,
trypsinization is needed to free the magnetic beads from the
cell surface. Cell surface receptors for retroviruses and
growth factors will be destroyed by the trypsinization, not
to mention the possibility that the yolk sac cells may
become more susceptible to lysis. As a result, few viable
cells remain for further study. Nevertheless, compared to
the FACS sorting, immunomagnetic bead separation is not as
time-consuming and has the advantage of not damaging the
yolk sac cells. Therefore, immunomagnetic beads separation
represents the most useful technique so far to study the
influence of MAbs in the yolk sac system.


CHAPTER 4
BIOCHEMICAL CHARACTERIZATION OF
THE DIFFERENTIATION MARKERS RECOGNIZED BY MABS
Introduction
There are two kinds of changes that contribute to the
expression of differentiation markers specific for a
particular cell type or lineage on normal hematopoietic
cells or to the expression of transformation-associated
antigens on retrovirus-transformed hematopoietic cells. One
is the appearance of new surface markers due to enhanced
synthesis at the transcriptional and/or translational level.
The other is the alteration in the structure of carbohydrate
groups attached to proteins or lipids at the
posttranslational level. These changes probably are
involved in the alteration of the interaction of particular
hematopoietic cells with other types of hematopoietic cells,
with the extracellular matrix or with stromal cells and in
the different response to growth and differentiation
factors.
Our initial attempt was to determine the nature and
molecular weights of these differentiation markers in order
to elucidate what kind of changes have occurred to these
markers recognized by MAbs. The experimental approach
85


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IDENTIFICATION OF DIFFERENTIATION MARKERS
IN NORMAL AND VIRALLY TRANSFORMED
AVIAN HEMATOPOIETIC CELLS
By
JUINN-LIN G. LIU
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1990

DEDICATION
This dissertation is dedicated to my parents. Without
their understanding, support and encouragement, it would
have never been possible for me to accomplish any goals in
my life.

ACKNOWLEDGEMENTS
Words cannot express my appreciation to my mentor, Dr.
Carlo Moscovici. Four years ago, I went to his office to
discuss my rotation project. Suddenly I was touched by a
poster on the wall, which depicted a newly hatched baby
chick, excited and curious about its new life, asking " What
do I do ? " It was nearly the mirror image of myself. The
very same question had struck me from time to time since I
arrived in the United States. Fortunately, he has guided
me, spiritually and intellectually, during the past four
years not only to become an independent researcher but a
more mature person as well. Nevertheless, we still have
some disagreement about my own differentiation pathway,
e.g., he never gives up the idea that jazz should be a
"growth factor" for me.
My work would not be complete without the great impact
of Dr. Giovannella Moscovici. She has enthusiastically
shared her knowledge and experience with me. I have learned
from her many techniques regarding cell biology and tumor
virology and have a better vision about life.
I also sincerely appreciate Dr. Paul Klein's help. His
generosity allowed me to use the space and materials in his
laboratory to complete the work on RIA and SDS-PAGE. He
iii

also helped me interpret the results and select the MAbs.
In addition, I am indebted to Dr. Paul Linser for providing
me the opportunity to establish the SDS-PAGE and
immunostaining techniques in his laboratory at the Whitney
Lab. The atmosphere there was so wonderful that I used to
walk along the beach and listen to the songs of the ocean at
night. It really eased a lot of pressure built up during my
project. By the way, the enjoyment of reading a science
fiction novel "The Rapture Effect," a gift from Dr. Linser,
may also enhance my "healing" process.
I would like to extend my appreciation to the other
members of my committee, Dr. Edward Wakeland and Dr. Ammon
Peck, for their helpful discussions and constructive
comments on my work.
Finally, I am also grateful to our technician, Mr.
Gordon Thompson, for his technical assistance and
preparation of the materials; to Mrs. Linda Green, Sue
Hammack, Janice Odebralski and Sandra Wotoweic in the
Hybridoma Laboratory for helping me develop the hybridomas;
and to Mrs. Melissa Chen for her assistance with FACS
sorting and analysis.
iv

TABLE OF CONTENTS
page
ACKNOWLEDGEMENTS iii
LISTS OF TABLES vii
LISTS OF FIGURES viii
ABBREVIATIONS X
ABSTRACT xii
CHAPTERS
1 INTRODUCTION AND BACKGROUND 1
Introduction 1
Normal Avian Hematopoiesis 3
Interaction of the Avian Leukemia Viruses with
Hematopoietic Cells 19
Identification of Cell Surface Markers in Normal
Hematopoietic Cells and Tumor Cells by MAbs.... 30
2 PRODUCTION OF MONOCLONAL ANTIBODIES AND THEIR
CELL-TYPE SPECIFICITIES 38
Introduction 38
Materials and Methods 39
Results 49
Discussion 61
3 IDENTIFICATION OF TARGET CELLS RECOGNIZED BY MABS
IN THE YOLK SAC AND THE BONE MARROW 72
Introduction 72
Materials and Methods 73
Results 75
Discussion 81
4 BIOCHEMICAL CHARACTERIZATION OF THE
DIFFERENTIATION MARKERS RECOGNIZED BY MABS 85
Introduction 85
Materials and Methods 86
v

Results 89
Discussion 98
5 CONCLUDING REMARKS 105
REFERENCES Ill
BIOGRAPHICAL SKETCH 118
vi

LIST OF TABLES
page
Table 1-1. Chicken hematopoietic precursor cells 12
Table 1-2. The three different lineages of the avian
erythroid compartment 15
Table 1-3. Avian defective leukemia viruses 21
Table 2-1. The cell-type specificities of MAbs from
BM2 and BM2/L fusions 51
Table 2-2. The isotypes of selected MAbs 51
Table 2-3. RIA binding indexes of MAbs to different
cell types 52
Table 2-4. Summary of the cell-type specificities
of MAbs 60
Table 2-5. Comparison of the proliferating potential
of BM2/C3A cells and 2.5 /¿g/ml
PMA-differentiated BM2/C3A cells 62
Table 2-6. Comparison of the proliferating potential
of 6C2 cells and ImM butyric acid-treated
6C2 cells 65
Table 2-7. RIA binding indexes of MAbs to normal CEF
and RAV-infected CEF 68
Table 3-1. Characterization of the target cells
sorted with 3D7-1C9 from the bone marrow... 78
Table 3-2. Characterization of the target cells
sorted with 2E10-1E10 from the bone marrow. 80
Table 3-3. Characterization of the negative
populations isolated with MAbs from the
yolk sac 80
Table 4-1. The biochemical nature and antigenic
determinants of the differentiation
markers recognized by MAbs 97
vii

LIST OF FIGURES
page
Figure 1-1. Schematic illustration of the chick
embryo at the 3rd day of embryogenesis.... 7
Figure 1-2. Diagram of hematopoiesis 11
Figure 1-3. Inerference of the AEV with cells of
the erythroid lineage 28
Figure 1-4. Interference of the AMV with cells of
the monocytic lineage 32
Figure 2-1. Flow cytometric analysis of the 20%/50%
interface of a percoll gradient of 2-week-
old bone marrow cells 53
Figure 2-2. APAAP immunoenzymatic staining of 20%/50%
interface of a percoll gradient of 2-week-
old bone marrow cells 54
Figure 2-3. Flow cytometric analysis of the 50%/70%
interface of a percoll gradient of 2-week-
old bone marrow cells 56
Figure 2-4. Flow cytometric analysis of the 30%/50%
interface of a percoll gradient of 4-day-
embryo yolk sac cells 57
Figure 2-5. Flow cytometric analysis of the 20%/50%
interface of a percoll gradient of 2-week-
old bone marrow cells tagged with
2E10-1E10 58
Figure 2-6. Flow cytometric analysis of the 50%/70%
interface of a percoll gradient of 2-week-
old bone marrow cells tagged with
2E10-1E10 59
Figure 2-7. Reactivitities of MAbs to BM2 cells and
PMA-differentiated BM2 cells 64
viii

Figure 2-8. Reactivities of MAbs to 6C2 cells and
butyric acid-treated 6C2 cells 67
Figure 3-1. Flow cytometric analysis of 3D7-1C9
tagged bone marrow cells before sorting... 76
Figure 3-2. Flow cytometric analysis of 3D7-1C9
tagged bone marrow cells after sorting.... 77
Figure 4-1. Reactivities of MAbs to BM2/C3A cells with
enzymatic digestion and chemical
deglycosylation 92
Figure 4-2. Reactivities of MAbs to 6C2 cells with
enzymatic digestion and chemical
deglycosylation 94
Figure 4-3. Reactivities of MAbs to MSB1 cells with
enzymatic digestion and chemical
deglycosylation 96
Figure 4-4. Silver stain of 10% SDS-PAGE analysis
of immunoprecipitates obtained from
unlabeled BM2/C3A cells 99
Figure 4-5. Fluorography of 10% SDS-PAGE analysis
of immunoprecipitates obtained from
35 S-Methionine-labeled BM2/C3A cells 100
Figure 4-6. Fluorography of 10% SDS-PAGE analysis
of immunoprecipitates obtained from
35 S-Methionine-labeled 6C2 cells 101
Figure 4-7. Fluorography of 10% SDS-PAGE analysis
of immunoprecipitates obtained from
35 S-Methionine-labeled MSB1 cells 102
Figure 5-1. Diagram of the specificities of MAbs
for the hematopoietic cells 109
ix

ABBREVIATIONS
ACS
AEV
AHS
ALV
AMV
APAAP
BFU-E
BPA
CEF
CFU-E
CFU-M
cpm
CSF
DLV
DMSO
DMEM
EGF
ELISA
FACS
FBS
FITC
Anemic chicken serum
Avian erythroleukemia virus
gamma globulin-free horse serum
Avian leukemia virus
Avian myeloblastosis virus
Alkaline phosphatase and monoclonal anti-alkaline
phosphatase
Burst-forming unit-erythroid
Burst-promoting activity
Chicken embryo fibroblast
Colony-forming unit-erythroid
Colony-forming unit-marrow
Counts per minute
Colony-stimulating factor
Defective leukemia virus
Dimethyl sulfoxide
Dulbecco's modified Eagle's medium
Epidermal growth factor
Enzyme-linked immunosorbent assay
Fluorescence-activated cell sorter
Fetal bovine serum
Fluorescein-isothiocyanate
x

GAM
Goat anti-mouse polyvalent IgA, G & M
HBSS
Hank's balanced salt solution
LLV
Lymphoid leukosis virus
LPS
Lipopolysaccharide
MAb
Monoclonal antibody
MAV
Myeloblastosis-associated virus
MG-CFC
Macrophage-granulocyte colony-forming cell
m.o.i.
Multiplicity of infection
PBS
Phosphate-buffered solution
PMA
Phorbol 12-myristate-13-acetate
PMSF
Phenylmethylsulfonylfluoride
PNPP
P-nitrophenyl phosphate
PPD
P-phenylene diamine
RaMIG
Rabbit anti-mouse IgG
RAV
Rous-associated virus
RIA
Radioimmunoassay
SDS-PAGE
Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis
TBS
Tris-buffered saline
TSA
Tris/saline/azide
xi

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
IDENTIFICATION OF DIFFERENTIATION MARKERS
IN NORMAL AND VIRALLY TRANSFORMED
AVIAN HEMATOPOIETIC CELLS
By
JUINN-LIN G. LIU
May 1990
Chairman: Dr. Carlo Moscovici
Major Department: Pathology and Laborotary Medicine
Avian hematopoiesis has been an excellent model
for resolving numerous enigmas about growth and
development. The interaction of avian retroviruses with the
avian system has created a relatively new discipline of
onco-development which allows us to analyze abnormal tissue
growth and hematological disorders in a more sophisticated
fashion.
The specific aim of this project is to identify
lineage-specific differentiation markers in normal avian
hematopoietic cells and transformation-associated
antigens in retrovirus-transformed cells by utilizing
monoclonal antibody techniques. Four groups of MAbs were
selected among nearly 5,000 supernatants from 10 fusions.
Characterization of the cell-type specificities was achieved
xii

by radioimmunoassay, immunofluorescence staining, flow
cytometry and immunoenzymatic staining as well as FACStar
sorting or immunomagnetic bead separation followed by
colony-forming assays and transforming assays. Analysis of
MAbs revealed that 1) MAbs 1H10-1F9, 2H1-2A10 and 3D7-1C9
are specific for transformation-associated antigens present
preferentially on BM2 cell lines rather than on normal
monocytic cells. The expression of these antigens was
diminished after BM2 cells were induced to differentiate.
2) MAb 1F7-1A3 recognizes BFU-E and CFU-E, AEV-transformed
yolk sac cells and MSB1 cells. 3) MAb 3F6-1E7 reacts with
the embryonic stem cell and precursor cell populations. The
expression of the marker recognized by MAb 3F6-1E7 was also
observed on some tumor cells, e.g., AEV-transformed yolk sac
cells, BM2 cells and MSB1 cells. 4) MAb 2E10-1E10 defines a
marker present on proliferating hematopoietic cells instead
of terminally differentiated cells, however, it starts
appearing only after the 4th day of embryogenesis.
Trypsinization, neuraminidase digestion and
deglycosylation treatment reduced the binding
specificities of MAbs 1H10-1F9, 2H1-2A10, 3D7-1C9 and
2E10-1E10. This suggests that the markers recognized by
these MAbs are glycoproteins and that sialic acids with or
without carbohydrates are contributing to the conformation
of the antigenic determinants. Conversely the antigenic
xiii

determinants for MAbs 1F7-1A3 and 3F6-1E7 must be strictly
proteins, since only trypsinization was able to inhibit
their binding specificities.
This study will permit investigations focusing on the
expression of these markers to bring us a step closer
toward the understanding of the mechanisms involved in
regulating proliferation and differentiation of
normal cells versus tumor cells.
xiv

CHAPTER 1
INTRODUCTION AND BACKGROUND
Introduction
Proliferation and differentiation of normal cells is
controlled by the interactions with other cell types, with
extracellular matrix and with regulatory molecules such as
growth factors and differentiation factors. Although the
regulatory mechanisms are extremely complicated, they have
been programmed in such a way as to maintain proliferation
and differentiation of the cells in a harmonic state. In
other words, the loss of cells from the stem cell
compartment by differentiation into committed progenitor
cells must be balanced by replenishment via self-renewal of
the stem cells. If too many stem cells undergo
differentiation, the stem cell reserve will rapidly become
exhausted; if too many stem cells undergo self-renewal
rather than differentiation, the production of mature cells
will drastically fall.
Cancer is believed to be a molecular disease resulting
from the deregulation of proliferation and differentiation,
i.e., the harmony has been short-circuited by the
1

2
constitutively triggered self-renewal machinery with or
without the blockage of the differentiation pathway.
It has been noticed that AEV-transformed avian
embryonic yolk sac cells can eventually undergo spontaneous
differentiation into mature erythrocytes (Jurdic et al.,
1985) and that spontaneous regression and differentiation of
human neuroblastomas are observed occasionally (Evans et
al., 1976). In addition, a variety of tumor cells have also
been shown to revert to a normal state under different
conditions despite the continued expression of activated
oncogenes. For example, the tumorigenicity of hybrids
formed between normal and tumor cells has been completely
suppressed (Stanbridge et al., 1982); embryonal carcinoma
(Pierce et al., 1979), neuroblastoma (Podesta et al., 1984),
B16 melanoma (Pierce et al., 1984) and murine leukemia
(Gootwine et al., 1982) have been converted into benign cell
lineages by their appropriate embryonic environments;
naturally occurring substances such as colony-forming
factors (Sachs, 1986), glia maturation factor (Lim et al.,
1986) and transforming growth factors (Sporn et al., 1986)
have been shown to be able to induce differentiation of
tumor cells; while a number of chemical agents such as
hexamethylene bisacetamide, retinoid acid, 5-azacytidine and
DMSO etc. can also induce terminal differentiation and/or
reverse the neoplastic phenotype of malignant cells (Bloch,
1984; Fresney, 1985). Moreover, in some cases, terminally

3
differentiated cells such as macrophages can still serve as
the target cells for transformation by a group of
retroviruses, namely AMV, MC29 and MH2 (Pessano et al.,
1979). All the information mentioned above suggests that
self-renewal and differentiation of cells are regulated by
separate mechanisms. The roles of various protooncogenes
and antioncogenes in these processes are yet to be
elucidated.
Normal Avian Hematopoiesis
Introduction
The avian hematopoietic system has provided a unique
and interesting model to study mechanisms of the regulation
of cell proliferation and differentiation in normal versus
tumor cells. It has several distinct features compared to
that of mammals, including the presence of the bursa of
Fabricius which is involved in the differentiation of B
lymphocytes, the expression of class IV MHC antigens, coded
by the B-G region, on the surface of the mature erythrocytes
(Miller et al., 1982). In addition, the erythrocytes are
nucleated and oval-shaped, and the nucleated thrombocytes,
instead of the platelets, are responsible for the hemostasis
in the avian system. There are several advantages in using
the avian models. For instance, tolerance to foreign
antigens can be developed during early ontogeny (Hasek and
Hraba, 1955), and hematopoiesis can be studied both in vivo

and in vitro by using retroviruses as indicators for
specific precursors present within each lineage.
The most studied avian hematopoietic system is in the
chicken. The outbred SPAFAS line has been used in our
laboratory to carry out all the experiments for my
dissertation project. My work has focused mainly on the
erythroid and monocytic lineage and their interaction with
the avian retroviruses.
The Blood-Forming Organs
The first blood cells appear after 18 hours of
incubation in the blood islands disseminated in the
blastoderm. During embryogenesis, the yolk sac represents
the major hematopoietic organ until day 15. Erythropoiesis
in the spleen starts around day 9 and continues through day
16 to 18 with a peak at day 15. The bone marrow begins its
function on day 12 and becomes the main site of
hematopoiesis throughout adult life (Dieterlen-Liévre,
1988) .
The volk sac
The yolk sac becomes established during the third day
of embryogenesis and it originates in the extraembryonic
region consisting of a peripheral "area vitellina" which is
made up of ectoderm and endoderm only, and a central "area

5
vasculosa" consisting of all three germ layers (Figure 1-
1). The area vasculosa contains the blood islands and its
boundary is delineated by a circular blood vessel, the sinus
marginalis. As the area vitellina grows over the yolk, the
area vasculosa also increases in size and invades the area
vitellina. Eventually the latter disappears completely, and
the entire yolk sac is vascularized.
The bone marrow
In the adult chicken, the bone marrow remains the major
source for erythropoiesis, granulopoiesis and lymphopoiesis.
The spleen does not appear to play a role in hematopoiesis
in the adult. In the avian bone marrow, erythropoiesis
occurs in the lumen of the medullary sinuses, while
granulopoiesis and lymphopoiesis are compartmentalized
within the extravascular spaces (Campbell, 1967). In
addition, masses of lymphatic tissue with germinal centers
are also present (Campbell, 1967; Payne and Powell, 1984).
However, in the mammalian bone marrow, erythropoiesis is
confined to the extravascular spaces and there is no
lymphatic tissue at all.
The development of the bone marrow during ontogeny has
been studied in the chick embryo by Sorrell and Weiss (1980)
using light, scanning and transmission electron microscopy.
The bone marrow cells can be obtained from the chick embryo
as early as 12 days of incubation. Marrow at this stage is

Figure 1-1. Schematic illustration of the chick
embryo at the 3rd day of incubation. The yolk sac
is composed of the area vitellina and the area
vasculosa. As the area vitellina grows over the
yolk, the area vasculosa also increases in size
and invades the area vitellina. Eventually the
latter disppears completely and the entire yolk
sac is vascularized. 1, embryo; 2, area
pellucida; 3, area vasculosa; 4, sinus terminus;
5, area vitellina interna; 6, area vitellina
externa.

7
2
I
5

8
richer in stem cells and non-committed progenitor cells than
the older bone marrow, but it already harbors cells which
are committed to specific hematopoietic lineages. The
latter cells are less numerous than in the adult bone marrow
and consist essentially of hemocytoblasts.
Other blood-forming organs
The spleen, the bursa, the liver and the thymus
function as additional hematopoietic organs. In the adult,
lymphopoiesis occurs mainly in the thymus and in the spleen,
whereas the bursa, from which the precursors of B
lymphocytes originate, is a transient granulopoietic organ.
Recent investigations (Cormier and Dieterlen-Liévre,
1988) report that some intraembryonic sites may be another
source of hematopoietic stem cells in the developing embryo.
At 3-4 days of incubation, the wall of the dorsal aorta
surrounding the intraembryonic mesenchyme is found to be the
site from which hematopoietic progenitor cells emerge, i.e.,
M-CFC, G-CFC, GM-CFC and BFU-E.
The Differentiation Pathway of Hematopoietic Cells
The cells of the hematopoietic system arise by
proliferation and differentiation of the progenitor cells.
This process begins with multipotential stem cells which can
self-renew as well as undergo progressive differentiation to
progenitor cells committed to the particular lineages,

9
ultimately yielding mature blood cells (Metcalf and Moore,
1971).
Analysis of the stem cells and progenitor cells in the
different hematopoietic tissues has been useful in
clarifying the differentiation pathway as well as in
exploring the regulatory mechanisms of hematopoiesis.
Hematopoietic stem cells
The stem cell population is the fundamental base from
which all the major hematopoietic cell lines are derived.
This population is thus considered to be pluripotent in its
differentiation potential, giving rise to erythroid,
granulocytic, monocytic, megakaryocytic and lymphoid
lineages (Figure 1-2). However, the stem cells account only
for 0.01% of the total bone marrow cells in a normal mouse,
and for 0.003-0.004% in a normal chicken (Table 1-1). In
addition, since they are morphologically indistinguishable,
their existence can only be inferred by the progeny they
produce.
Efforts to identify chicken hematopoietic stem cells
have followed the protocol of the transplantation
experiments by Till and McCulloch (1961) whereby they have
identified the mouse hematopoietic stem cells. Samarut, et
al. (1976) transplanted normal chicken bone marrow into
irradiated chickens. Six days later, erythrocytic colonies
were observed only on the surface of the tibial marrow.

Figure 1-2. Diagram of hematopoiesis. The entire pool of mature hematopoietic
cells is derived from a single pluripotent stem cell. The more differentiated
they become, the less self-renewal potential they possess. In the avian system,
thromboblast and thrombocytes stand for megakaryocyte and platelets
respectively. Abbreviations used: BFU-E, burst-forming unit-erythroid; CFU-E,
colony-forming unit-erythroid; Eo-CFC, eosinophil-colony forming cell; GM-CFC,
granulocyte & monocyte-colony forming cell; MEG-CFC, megakaryocyte-colony
forming cell.

Self-renewal
Pluripotent
Stem Cell
t t t
Erythroid Myeloid Lymphoid
Precursor Precursor Precursor
t
BFU-E GM-CFC Eo-CFC MEG-CFC
Í
CFU-E
Í
Erythroblast Myeloblast Monoblast Eosinophilic Megakaryoblast
Myeloblast (Thromboblast) Lymphoblast Lymphoblast
I I ! I ! f V
Peripheral
Blood
Erythrocyte Myelocyte Monocyte Eosinophil Platelets T Cell B Cell
y 1 (Thrombocytes) 1
Granulocyte f â–¼
Tissue
Macrophage Plasma
H y Cell

12
Table 1-1. Chicken hematopoietic precursor cells
Hematopoietic
precursor cells
Frequency*
Bone marrow Yolk sac
Progeny
BFU-E
110-160
300-600
1,
000-2,000
CFU-E
500-2,000
1,500-1,800
8-150
GM-CFC
(early progenitor)
250-400
100-200
50-1,000
GM-CFC
(late progenitor)
1,000-1,300
100-200
3-50
Source: Modified
‘Expressed as per
from Moscovici
105 cells.
and Gazzolo
(1982)
•

13
Each colony was originated from one single cell, i.e.,
colony-forming unit in the marrow (CFU-M). However, neither
macrophage-granulocytic colonies nor mixed types of colonies
were observed in the marrow of the irradiated chickens. It
is still unclear whether the medullar environment does not
favor the development of colonies other than the ones of the
erythroid lineage or if the CFU-M represents only the stem
cells at the earliest step of commitment in the erythroid
lineages.
Committed progenitor cells
Committed progenitor cells are directly derived from
the stem cells and are each committed to a specific
differentiation pathway or lineage. Commitment is an
irreversible step whereby these cells have lost the
potential to generate hematopoietic cells of other lineages.
Most of the proliferative activity in the bone marrow seems
to occur in the progenitor cells committed to the production
of single or restricted ranges of hematopoietic cell types.
Only a small proportion of the pluripotential stem cells is
cycling at a given time. The progenitor cells of both
erythroid and myeloid lineages will be discussed in detail.
The erythroid lineage
The avian erythroid compartment consists of three
distinct lineages, namely the primitive, the intermediate

14
and the definitive lineage, respectively (Table 1-2). The
cells from the primitive lineage are produced by the early
blood islands and mature "in cohort" (Ingram, 1972). These
cells are released at a very immature state, but they
continue to divide and mature synchronously within the blood
vessels between 2 and 5 days of incubation. These cells are
called megalocytes because of their large size. They are
spherical with round nuclei and synthesize hemoglobins E
(embryonic) and P (primitive) which are specific for the
primitive lineage. At 5 days, cells of the intermediate
erythroid lineage begin entering the blood and eventually
supersede the primitive cells. At 7 days, the primitive
cells account for only 5% of the red blood cells, and after
12 days of incubation they are rarely encountered. The
cells of the intermediate lineage are observed from 5 to 6
days until 18 to 20 days. They synthesize specific
hemoglobin H (hatching). It is not until 18 to 21 days of
incubation that mature erythrocytes of the definitive
lineage start to appear in the blood circulation. They are
oval-shaped with oval nuclei and have hemoglobins A (adult)
and D (definitive) (Bruns and Ingram, 1973).
The progenitor cells of the definitive lineage are
morphologically unrecognizable. However, by the use of in
vitro colony forming assays in methylcellulose, two classes
of erythroid progenitor cells have been identified,
the colony-forming unit-erythroid (CFU-E) and the burst-

15
Table 1-2. The three different lineages of the avian
erythroid compartment
Primitive
Intermediate
Definitive
Appearance
Day 2-7
Day 5-20
Day 18-Hatched
Progenitor
Cell
Megaloblast?
BFU-E, CFU-E
BFU-E, CFU-E
Mature Cell
Megalocyte
Erythrocyte
Erythrocyte
Hemoglobin*
E: aA + e
P: 7t + p
H: aA + pH
A: aA + /3a
D: aD + /3a
‘Source: Modified from Bruns and Ingram (1973).
a-like globin: aA, n, aD; /3-like globin: e, p, /3H, /3A.
Abbreviation: E, embryonic; P, primitive; H, hatching;
A, adult; D, definitive.

16
forming uint-erythroid (BFU-E). The BFU-E give rise, after
six days in culture, to large aggregates made of several
benzidine-positive clusters containing about 1,000
erythrocytes (Samarut and Bouabdelli, 1980). These BFU-E
are highly sensitive to burst-promoting activity (BPA), and
are also dependent on high concentrations of erythropoietin.
The CFU-E proliferate to form one compact colony of 8
to 150 benzidine-positive erythrocytes after three days of
incubation (Samarut et al., 1979). The requirement for
erythropoietin in the development of CFU-E is lower than
that for BFU-E. The BFU-E and CFU-E can be detected in the
yolk sac as well as in the embryonic and adult bone marrow.
The BFU-E are also found in the blastoderm at the primitive
streak stage (18 hours of incubation), whereas the CFU-E are
not yet detectable (Samarut and Bouabdelli, 1980). The
BFU-E and CFU-E can be also distinguished by the expression
of two different cell surface antigens which are recognized
by polyclonal antisera (Gazzolo et al., 1980; Samarut et
al., 1979). An antigen specific to immature red blood cells
is present on the CFU-E but not detectable on the BFU-E.
Conversely, a chicken brain-related antigen is expressed on
the BFU-E and less expressed on the CFU-E.
In the murine system, subpopulations of the BFU-E at
different degrees of maturation have been observed (Gregory
and Eaves, 1978). This is not the case, however, for the
BFU-E in the chickens.

17
The myeloid lineage
Hematopoietic cells of granulocytic and monocytic
lineages are referred to as myeloid cells. In response to
infection, the progenitor cells in the bone marrow, i.e.,
granulocytic-macrophage colony-forming cells (GM-CFC), would
rapidly produce a large amount of mature granulocytes and
monocytes under the control of a variety of colony-
stimulating factors (CSFs). In the murine and human system,
four kinds of CSFs involved in myelopoiesis have been
characterized as IL3, GM-CSF, G-CSF and M-CSF. Conversely,
the specific CSFs in the chicken have yet to be identified.
Nevertheless, sources of CSFs can be furnished by using an
underlayer of macrophages (Graf et al., 1981), or by adding
to the semi-solid medium either egg albumin (Szenberg,
1977), or serum from endotoxin-injected chickens (Dodge and
Hansell, 1978) or a conditioned medium from chicken
fibroblast cultures (Dodge and Moscovici, 1973; Dodge et
al., 1975; Gazzolo et al., 1979). AMV-transformed cells
were shown also to be capable of producing CSFs (Silva et
al., 1974). Recently a myelomonocytic growth factor was
isolated from medium conditioned by a transformed macrophage
cell line (Leutz et al., 1984). This factor (MGF) promotes
the growth of macrophage colonies together with a minor
proportion of granulocytes.
The existence of chicken GM-CFC can be detected when
bone marrow cells are cultured in soft agar or

18
methylcellulose media. The wide range of the colony size
obtained suggests that these progenitor cells display
different degrees of maturity. The less mature CFC give
rise to clusters containing from 50 to more than 2,000 cells
(Dodge and Moscovici, 1973; Dodge et al., 1975), while the
more mature CFC produce colonies from 3 to 50 cells (Gazzolo
et al., 1980). Colonies which are composed mostly of
macrophages readily develop in a semi-solid medium
containing chicken serum and fibroblast-conditioned-medium
(Dodge and Hansell, 1978). Granulocytic colonies will
develop if the chicken serum is depleted from the medium and
the fibroblast-conditioned-medium is replaced by spleen-
conditioned-medium (Dodge and Sharma, 1985). This finding
suggests the existence of a factor similar to M-CSF in the
mouse and human. The monocytic colonies are composed of
scattered cells, whereas the granulocytic colonies are
dense. The cells from both colonies contain granules.
The GM-CFC have been observed in various chicken
hematopoietic tissues of embryonic and adult stages (Dodge
and Moscovici, 1973; Dodge et al., 1975; Szenberg, 1977).
Moreover, the CFC can be enumerated in the blastoderms
incubated for 24 hours (Moscovici et al., unpublished
results). A higher percentage of CFC was also found in the
embryonic spleen and bone marrow. The frequency dropped
rapidly once the chickens hatched. Interestingly, a peak of
CFC occurred in the bursa at 14 and 15 days of incubation,

19
indicating that the stem cells colonizing the bursa
differentiate first into myeloid elements and subsequently
into lymphoid ones (Szenberg, 1977).
Interaction of the Avian Leukemia Viruses with
Hematopoietic Cells
Introduction
There have been many comprehensive reviews to date on
the molecular biology and the pathogenesis of the avian
leukemia viruses (ALVs) (Moscovici and Gazzolo, 1982; Graf
and Stéhelin, 1982; Bishop, 1983; Enrietto and Wyke, 1983;
Bister and Jansen, 1986). The ALVs belong to a taxonomic
subfamily termed oncovirinae (specifically, avian leukosis-
sarcoma group of type C RNA tumor viruses) within the family
of retroviridae (retroviruses) (Fenner, 1976). They
contribute to a variety of avian hematopoietic as well as
non-hematopoietic disorders. The ALVs can be divided into
two groups according to the pathological features: the
defective leukemia viruses (also known as acute leukemia
viruses) and the avian leukosis viruses (Hanafusa, 1977).
The defective leukemia viruses (DLVs)
These viruses induce various types of acute leukemia
within a few weeks after inoculation. They also cause
sarcomas and carcinomas in some cases. All the strains
known can transform the cells of specific hematopoietic
lineages in vitro. In addition, most of them transform

20
fibroblasts in culture as well, with the exception of AMV
and E26. Another distinct aspect of the DLVs is that they
are all replication-defective due to total or partial
deletions of the essential virion genes: gag, pol and env.
Consequently, they can produce infectious progeny only in
the presence of the avian leukosis helper viruses. The
deleted sequences are replaced by the viral oncogenes
(v-onc), i.e., v-myc, v-mil, v-erbA, v-erbB, v-myb, and
v-ets, which originated from transduction of mutated or
truncated forms of protooncogenes. Their gene products are
responsible for the transformation of the hematopoietic
cells. Based on the predominant response of the
hematopoietic system of the infected host and the major
types of oncogenes which they carry, three subgroups of
DLVs and their respective representatives can be
distinguished: i) the MC29 subgroup: myelocytomatosis
(v-myc), ii) the AEV subgroup: erythroblastosis (v-erb),
iii) and the AMV subgroup: myeloblastosis (v-myb) (Table
1-3). However, a more detailed description of the
interaction of the AEV and the AMV with the hematopoietic
system will be given since more data have been obtained in
the last decade.
The Lymphoid leukosis viruses (LLVs)
In contrast to the DLVs, the LLVs do not contain any
v-onc and they are fully competent for replication. Because

21
Table 1-3. Avian defective leukemia viruses
Subgroups
and
virus strains
Viral
oncogenes
Neoplasms
induced
in vivo
Cell types
transformed
in vitro
MC29 subgroup
Strain MC29
Strain CMII
Strain OKIO
Strain MH2
v-myc
v-myc
v-myc
v-myc, v-mil
Myelocytomatosis,
endothelioma,
carcinoma
Myeloid,
macrophage,
epithelioid,
fibroblastic
AEV subgroup
Strain AEV-R
Strain AEV-H
v-erbA, v-erbB
v-erbB
Erythroblastosis,
fibrosarcoma
Erythroid,
fibroblastic
AMV subgroup
Strain AMV
v-myb
Monoblastosis
Monocytic
Strain E26
v-myb, v-ets
Erythroblastosis,
monoblastosis
Erythroid,
monocytic
Source: Modified from Bister and Jansen (1985).

22
of the absence of v-onc, the LLVs don't transform cells in
vitro and most strains induce predominantly lymphoid
leukosis in vivo only after a long latent period of several
months or longer by the activation of protooncogenes. In
the cases of chicken B-cell lymphomas induced by the LLVs,
the viral LTR regions containing an enhancer and a promoter
were found to be integrated very close to the c-myc gene
(Hayward et al., 1981). Most integrations of LLVs result in
the separation of exons II and III of the c-myc gene from
the normal promoter and exon I (Payne et al., 1982; Shih et
al., 1984), causing a fifty-fold higher transcription of c-
myc RNA under the control of the LTR promoter than the 5
copies found in normal cells. In a minority of tumors, the
LTR integrated in the opposite orientation to that of the c-
myc and in one case the provirus was actually located at the
3' end of the c-myc (Payne et al., 1982). It is thought in
these rare events that the enhancer element in the viral LTR
probably increases the transcription from the normal c-myc
promoter. More recently, the LLVs have also been found
integrated at the c-erbB locus in chicken erythroblastosis
(Fung et al., 1983). An elevated level of c-erbB
transcripts was observed.
LLVs under different conditions, for example, chicken
genotype, site of injection, age of host, etc., may induce a
larger spectrum of diseases including osteopetrosis, anemia,

nephroblastoma and occasional fibrosarcomas and
endotheliomas.
23
The Avian Ervthroleukemia Virus (AEV)
The oncogenes of the AEV
The AEV can induce erythroblastic leukemia and sarcomas
in infected birds within a short period of time (Graf and
Beug, 1978; Moscovici and Gazzolo, 1982). The virus also
transforms chicken fibroblasts and hematopoietic precursor
cells of the erythroid lineage in vitro (Graf et al., 1981).
It carries two oncogenes, namely v-erbA and v-erbB. The
v-erjbB encodes a protein of 61 Kd which is glycosylated in
infected cells to higher molecular weight forms (Privalsky
et al., 1983). The gp65erbB and gp68erbs are localized in the
intracellular membrane, while the mature gp74erbB is found in
the plasma membrane (Privalsky and Bishop, 1984). The
latter protein represents a truncated form of the receptor
for epidermal growth factor (EGF) (Downward et al., 1984),
where the extracellular EGF-binding domain has been deleted,
but the region for tyrosine-specific protein kinase is
preserved (Gilmore et al., 1985). It has been postulated
that the transforming potential of v-erbB is due to the lack
of regulation by EGF resulting in the constitutive
activation of the tyrosine kinase (Hayman and Beug, 1984;
Privalsky and Bishop, 1984). However, the exact mechanism

24
of transformation by the tyrosine kinase activity is still
unknown.
The v-erbA is a mutated truncation transduced from a
cellular multigene family encoding the thyroid hormone
receptor (Sap et al., 1986). The v-erbA protein,
p759a9"erbA, is linked with the gag product, is unglycosylated
and has no kinase activity. It appears to be a DNA-binding
protein exhibiting distinct nuclear and cytoplasmic
subcellular locations (Boucher et al. , 1988). Nevertheless,
it doesn't seem to bind thyroid hormone (Sap et al., 1986).
The transforming potential of v-erbA and v-erbB
It has been shown that v-erbB alone is necessary and
sufficient to induce cell transformation (Frykberg et al.,
1983; Sealy et al., 1983) by constructing a series of
deletion mutant viruses. The data corroborate the ability
of the product of v-erbB gp65-68erbB to independently induce
erythroleukemia in chickens and transform fibroblasts in
vitro. In contrast, constructs which produce the v-erbA
p759a9'erbA only were incompetent for transforming activity in
vitro. However, other observations from studies of
temperature sensitive mutants (Beug and Hayman, 1984),
transductants of the c-erbB gene alone (Fung et al., 1983;
Yamamoto et al., 1983), suggest that v-erbA may play a
distinct role in maintaining the proliferation and
transformed phenotype of AEV-infected cells. It has been

25
shown that v-erbA possesses the capability to potentiate the
erythroid transformation not only by v-erbB but also by
other oncogenes (Frykberg et al., 1983; Kahn et al., 1986).
These phenomena could be partially due to the suppression of
the transcription of the anion transporter (band 3) gene by
the v-erbA proteins (Zenke et al., 1988). It is not until
recently, however, that the transforming potential of
v-erbA has been reevaluated. The XJ12 vector which carries
v-erbA oncogene in association with Neo R (neomycin
resistance) gene is shown to be able to transform bone
marrow cells in vitro (Gandrillon et al., 1989; Moscovici
and Moscovici, unpublished results).
The target cells for AEV
The target cells for AEV infection in the bone marrow
of the hatched chick are recruited within the BFU-E (Burst
forming unit-erythroid) compartment (Gazzolo et al., 1980).
After AEV infection, the transformed cells continue to
proceed within the differentiation pathway until they are
blocked at the stage of CFU-E (colony forming unit-
erythroid) (Samarut and Gazzolo, 1982). These cells express
the erythroid markers of the CFU-E, but have gained the
self-renewal potential to forego the fate of terminal
differentiation. Conversely, the target cells for AEV in
the embryo are within either the CFU-M or the pre-BFU-E or
in both compartments (Jurdic et al., 1985). The embryonic

26
transformed colonies are partially hemoglobinized even after
subcloning, which suggest that the AEV-transformed embryonic
cells can escape the blockage and undergo spontaneous
differentiation in contrast to the AEV-transformed adult
cells (Figure 1-3).
The Avian Myeloblastosis Virus (AMY)
The oncogene of the AMV
The AMV induces rapid myeloblastic leukemia in the
chickens and transforms hematopoietic cells of monocytic
lineages in vitro. It can be divided into subgroups A and
B, depending on the expression of envelope glycoproteins of
the helper viruses (Moscovici et al., 1975). Besides the
AMV transforming agent, this strain contains two
nondefective helper leukosis viruses, the myeloblastosis
associated virus 1 and 2 (MAV-1 & MAV-2) of subgroups A and
B, respectively (Moscovici and Vogt, 1968).
The oncogene of AMV, v-myb, encodes a transforming
protein p45myb which is located in the nucleus (Klempnauer et
al., 1984). It has been shown that p45myb has DNA- binding
activity (Boyle et al., 1985). The v-myb proteins are
associated with their nuclear sublocation. The exact
function of v-myb proteins remains to be clearly
established. However, recent results indicate that v-myb
along with c-myb may act as transcriptional activators
(Weston and Bishop, 1989).

Figure 1-3. Interference of the AEV with cells of the
erythroid lineage. The AEV only transforms the cells
at the BFU-E stage from the adult bone marrow.
Transformed cells are frozen at the CFU-E stage and
capable of self-renewal. Only cells at the pre-BFU-E
stage are the target cells for AEV transformation in
the embryonic yolk sac. Although the transformed cells
display the phenotypes of CFU-E, they will eventually
escape the blockage and spontaneously differentiate
into erythrocytes. The brain antigens (Br) are
expressed on the BFU-E more than on the CFU-E, the
immature antigens (Im) are found on the CFU-E and
erythroblasts, meanwhile only erythroblasts and
erythrocytes are capable of synthesizing the
hemoglobins (Hb).

28
Normal
AEV Infection
Erythropoiesis
Adult
Bone Marrow
Embryonic
Yolk Sac

29
The target cells for the AMY
The characterization of AMV target cells by density,
velocity sedimentation, adherence and phagocytic activity
indicate that they are recruited among a wide range of cells
within the monocytic lineage from the stage of the
myelomonocytic progenitors, i.e., CFC (colony-forming cells)
(Gazzolo et al., 1979) committed toward the macrophage
lineage (Boettiger and Durban, 1984) to the terminally
differentiated macrophages (Moscovici and Gazzolo, 1982).
Regardless of the origin of the target cells, the AMV-
transformed cells are morphologically identical, and possess
the same functional and surface properties (Gazzolo et al.,
1979; Beug et al., 1979; Durban and Boettiger, 1981). They
are mostly nonadherent and round which diametered about 10
micrometers. Their large and eccentric nucleus is
surrounded by a rim of cytoplasm containing small granules.
The receptors for the Fc region of immunoglobulins are
expressed on the cell surface, whereas C3 receptors are not
present. Normal avian macrophages express both receptors.
Although the AMV-transformed cells can engulf latex
particles mediated by nonspecific receptors, phagocytosis
mediated by Fc receptors does not occur, i.e., these Fc
receptors are not functional. Acid phosphatase and
adenosine triphosphatase are also found in the cytoplasm and
on the membrane of the transformed cells. Moreover, after
treatment with phorbol 12-myristate-13-acetate (PMA), a

30
tumor promoter, the AMV-transformed cells became adherent to
the surface of the culture flask and eventually
differentiated into macrophages (Pessano et al., 1979).
There were no obvious alterations in terms of the expression
of the v-myb proteins in these PMA-differentiated cells.
However, the w-myb proteins were found to be located in the
cytoplasm instead of in the nucleus (Symonds et al., 1984).
The differentiation into macrophages was also obtained with
a temperature-sensitive mutant of AMV when the transformed
cells were shifted to a non-permissive temperature
(Moscovici and Moscovici, 1983).
In conclusion, cells from all stages of monocytic
lineages, from the committed progenitors to the mature
macrophages, may serve as the target cells for AMV. Once
the cells are transformed, they become frozen at a stage
between monoblasts and monocytes (Figure 1-4).
Identification of Cell Surface Markers in Normal
Hematopoietic Cells and Tumor Cells by MAbs
Introduction
The cellular microenvironment plays a crucial role in
regulating the proliferation and differentiation of normal
cells. A cell will interact with adjacent cells and with
structural components of the extracellular matrix via the
external surface of its plasma membrane. Although little is
known about the mechanisms of cellular interactions, it is
well established that cell development requires that the

Figure 1-4. Interference of the AMV with cells of the monocytic lineage. Cells
from different stages of the monocytic lineage can serve as the target cells for
AMV transformation. The transformed cells are arrested at the stage between
monoblast and monocyte via either partial differentiation or
de-differentiation. These cells are non-adherent and round-shaped. The ATPases
are expressed on their cell surface, whereas C3 receptors are not. Although
they have Fc receptors, no immune-phagocytosis can be mediated by these non¬
functional receptors.

GM-CFC Monoblast
Non-adherent Non-adherent
Fc Receptor -
C3 Receptor -
Phagocytosis -
AMV
Partial
Dedifferentiation
V
AMV
transformed
cells
Macrophages
Non-adherent
Fc Receptor +
C3 Receptor -
Immune
Phagocytosis -
ATPase +
Adherent
Fc Receptor +
C3 Receptor+
Immune
Phagocytosis +
ATPase +
CJ
to

33
surface membrane should be capable of receiving and
transmitting regulatory signals, i.e., growth factors and
differentiation factors from the microenvironment. Cancer
is a disease resulting from abnormalities in both cell
proliferation and differentiation characterized by increased
growth rate, prolonged survival, decreased adhesion, loss of
contact inhibition, increased invasiveness and motility,
expression of repressed antigens and escape from immune
surveillance (Wallach, 1968). All these phenomena have been
shown to be associated with alterations in structure and
function, and in particular with an aberrant glycosylation
of the cell surface membrane. As a result, cancer can be
regarded as a molecular disease of cell surface
glycoconjugates (Abe et al., 1983).
Cell surface glycoconjugates comprise a heterogeneous
group of compounds, all of which contain carbohydrate
N-glycosidically or O-glycosidically linked to protein
(glycoproteins) or O-glycosidically to lipid (glycolipids).
The predominant glycoconjugates are glycoproteins containing
at least 80% of all cell surface-located carbohydrate. It
is assumed that all membrane-bound proteins but only 10% of
membrane lipids are glycosylated (Shinitzky, 1984).
However, it is extremely difficult to identify specific
glycoconjugates expressed exclusively on tumor cell surface.
All identified tumor-associated markers to date are found to

34
be more or less expressed on normal cells at particular
stages of the differentiation pathway (Old, 1981).
The identification and characterization of cell surface
markers can provide us with invaluable information on
various aspects of differentiation and oncogenesis. First,
the identification of cell surface markers that are specific
for particular stages of differentiation and maturation will
enable us to follow the differentiation pathway in molecular
terms. Mechanisms regulating the expression of cell surface
markers will allow us to understand how functionally mature
cells are formed and what kind of structural elements are
necessary for functional differentiation. Secondly, they
could be used to study whether tumor cells are arrested at a
certain stage of differentiation. In addition, the studies
could also enable us to determine how the control of
proliferation and differentiation in tumor cells differs
from that of normal cells. Thirdly, differentiation markers
can be used for diagnostic and therapeutic purposes (Fukuda,
1985).
The introduction of hybridoma technology by Kohler and
Milstein (1975) has made a significant contribution in the
study of both normal and malignant cell surface markers.
MAbs have been used as powerful tools for the detection,
isolation and characterization of cell surface markers.
Compared to polyclonal antibodies, MAbs exhibit three major
advantages. First, they can be produced against relatively

35
impure antigens. Secondly, MAbs can be produced in much
larger (theoretically unlimited) quantities. Thirdly, they
are monospecific (i.e., they bind to a single epitope) and
thus MAbs recognized markers can be identified and
characterized individually.
Brief synopsis on the MAbs recognizing chicken cells of
ervthroid and monocytic lineages
Cell surface markers of normal or transformed
hematopoietic cells in the human and murine systems have
been extensively studied by using monoclonal antibody
techniques. There are only a handful of MAbs which have
been developed against chicken hematopoietic cells, and none
of them are specific for embryonic precursor cells.
Hayman et al. (1982) developed a panel of MAbs against
the temperature-sensitive mutant of ts34 AEV-transformed
erythroblasts which had been grown at 41.5°C for five days.
Three MAbs were chosen for further characterization. MAb
4.2A5 recognized erythrocytes and AEV-transformed
erythroblasts as well as granulocytes; MAb 4.5A5 reacted
with erythroblasts and retrovirus-transformed producer
cells, however, the authers did not address the specificity
tests on other types of normal cells; MAb 4.6C1 was specific
for erythroid cells at all stages.
Jurdic et al. (1982) produced a MAb, Sl-37, from the
fusion of spleen cells of a mouse immunized with AMV-
transformed cells. Sl-37 was shown to be specific for the

36
cells of the monocytic lineage. Its binding specificities
revealed by radioimmunoassay and flow cytometric analysis
were too low for further identification and
characterization.
Miller et al. (1982) immunized mice with 1-day-old
erythrocytes from inbred line 003 and hybrid strain
Shaver-Starcross 288 and raised a MAb, MaEEl, against a 48Kd
antigen which was expressed on the erythrocytes of 1-day-
old peripheral blood and adult bone marrow. It was also
present in the retina, muscle tissues, liver and on
epithelia and lymphoid cells of young and adult chickens.
Sanders et al. (1982) were able to generate a MAb
(190-4) against 1-day-old erythrocytes from the SC strain
which recognized a 50Kd molecule expressed on the cell
surface of erythrocytes, reticulocytes, chicken embryo cells
and reticuloendotheliosis virus (REV)-transformed lymphoid
cells, but not on the AEV-transformed erythroleukemia cells.
Kornfeld et al. (1983) obtained five different groups
of MAbs by immunizing mice with normal macrophages and
myeloid cells transformed by MC29, AMV and E26. Only one
group was specific for myeloid lineage, predominantly
reacting with immature myeloid cells. However, the authers
failed to show the reactivities to normal cells except the
macrophages.
Trembicki and Dietert (1985) produced 4 MAbs against 1-
day Cornell K-strain white leghorn chickens. MAb 10C6

37
detected a chicken fetal antigen (CFA) on 1-day-old chick
erythrocytes. MAbs 3F12 and 4C2 recognized chicken adult
antigen (CAA) on adult erythrocytes, whereas 9F9 reacted
with all peripheral erythrocytes from both Japanese quail
and chicken regardless of age.
Schmidt et al. (1986) immunized mice with plasma
membranes from the ts34 AEV cell line HD3 induced to
differentiate at 42°C for five days. Only one out of eight
groups of MAbs was specific for erythroid lineage, reacting
only with reticulocytes.
The following chapters in this dissertation describe
the first systematic attempt using MAbs in combination with
other techniques to identify embryonic differentiation
markers on normal avian hematopoietic cells and
transformation-associated antigens on retrovirus-transformed
ones.

CHAPTER 2
PRODUCTION OF MONOCLONAL ANTIBODIES
AND THEIR CELL-TYPE SPECIFICITIES
Introduction
The lack of MAbs recognizing embryonic differentiation
markers on avian hematopoietic precursor cells may be due to
the use of inappropriate antigens for immunization. Because
in most cases 1-day-old erythrocytes were chosen as antigens
instead of embryonic cells, the MAbs developed were not
specific for the embryonic precursor cells. Therefore it
was decided to use different strategies for the immunizing
protocols in order to produce MAbs which identify embryonic
differentiation markers present on normal avian
hematopoietic cells of the erythroid and monocytic lineages.
Moreover the approaches used were designed to identify
transformation-associated antigens in retrovirus transformed
avian hematopoietic cells and to characterize the
biochemical properties as well as to study the biological
functions of these markers.
Several types of cells were used to immunize 6-week-
old BALB/c BYJ female mice: 1) normal 3-day-embryo yolk sac
cells, 2) normal 3-day-embryo megalocytestogether with AEV-
38

39
transformed nonproducer cells from 3-day-embryo yolk sac, 3)
BM2 cells (AMV-transformed nonproducer cells from embryonic
bone marrow), and 4) BM2/L cells (leukemogenic variant of
BM2 cell line). Theoretically, 3-day-embryo yolk sac cells
are rich in embryonic hematopoietic precursor
cells, and the AMV/AEV nonproducer cells possess not only
the transformation-associated antigens but also
oncodevelopmental markers which are present normally only on
the precursor cells. 10-14 days after each cell fusion,
supernatants of hybridomas were screened against a panel of
different types of cells as well as against yolk by indirect
radioimmunoassay (RIA) and enzyme-linked immunosorbent assay
(ELISA). Only hybridomas showing continuous production of
MAbs of potential interest were subcloned by the limiting
dilution method. The isotypes of the MAbs were determined
by the Ouchterlony (double diffusion) test using isotype
specific antisera. MAbs were purified from culture media of
the cloned hybridomas or from mouse ascites by using high-
salt protein A-sepharose chromatography.
Material and Methods
Normal Cells
17-somite blastoderm. The blastoderm cells were
obtained from the 17-somite stage at 2 days of incubation by
mechanical dissociation with gentle pipetting and dispersed
in a-MEM (GIBCO) containing 10% fetal bovine serum (FBS).

40
Cells were then filtered through a 1.5/i nylon mesh (Tetko
Inc., New York) to remove any clumps (Moscovici et al.,
1983) .
Yolk sac cells. The yolk sacs from 3rd or 4th day of
embryogenesis were dissected free of other embryonic
membranes, pooled and rinsed extensively with Tyrode's
solution (8.0 g/L NaCl, 0.2 g/L KC1, 0.05 g/L NaH2P04-H20,
1.0 g/L Glucose and 1.0 g/L NaHC03) to remove as much yolk
as possible. The tissue were minced with scalpels and
dispensed in a-MEM/10% FBS by gentle pipetting. The
resulting cell suspension was then washed by centrifugation
to remove residual yolk, after which the cells were
resuspended in media and passed through nylon mesh to obtain
a single cell suspension. The yolk sacs from 6th or 12th
day of embryogenesis were first minced thoroughly with
scalpels and then digested with 0.125% trypsin for 10-15
minutes at 37°C (Moscovici et al., 1975). The cell
suspension was then washed by centrifugation and passed
through nylon mesh as above.
Bone marrow cells. Bone marrow cells were flushed out
of the tibias with BT-88 medium (GIBCO) containing 10%
tryptose phosphate broth, 5% calf serum and 5% chicken serum
by passing the cells through a syringe with a 22-gauge
needle 3 times (Jurdic et al., 1982). The cells were washed
once, resuspended in medium and then filtered through nylon
mesh.

41
Buffv coat. The buffy coat (WBC) from peripheral blood
was harvested by Ficoll-Hypaque (Lymphocyte Separation
Medium; Litton Bionetics) gradient centrifugation at 2,000
rpm for 20 minutes.
Macrophages. The buffy coat obtained from heparinized
blood was seeded in BT-88 complete medium and 48 hours later
the attached cells differentiated into macrophages. These
cells were then detached from the petri dishes by adding the
C-PEG solution (8.0 g/L NaCl, 0.29 g/L KCl, 0.2 g/L KH2P04,
0.763 g/L Na2HP04, 0.2 g/L EDTA, 3.7 g/L NaHC03 and 1.0 g/L
Glucose, pH 8.0) for 5 minutes.
Chicken embryo fibroblasts (CEF). The fibroblasts from
10-day embryos were prepared according to the procedure
described by Vogt (1969).
Transformed Cells
BM2/C3A cells. An AMV-transformed monoblastic
nonproducer cell line, GM727, was generated by in vitro
infection of 17-day-embryo bone marrow cells with AMV-B at a
low multiplicity of infection (m.o.i. of 10"2 to 10‘3)
(Moscovici & Moscovici, 1980). GM727 cells were then
injected into 13-day embryos via the chorioallantoic vein.
Four weeks after the injection, no overt case of leukemia
was observed unless the chickens were challenged with helper
viruses such as MAV-2 or RAV-7 (Moscovici et al., 1982).
However, the injected transformed cells could be retrived

42
from the bone marrow, and cloned by an in vitro colony
assay. A cell line namely BM2/C3A was established. The
cells in this line all express the v-myb proteins but are
nonproducers and are nonleukemogenic.
BM2/L cells. The BM2/L cell line is a variant of
BM2/C3A cell line. It was obtained from a BM2/C3A-injected
bird which came down with leukemia involving liver, spleen
and heart (Moscovici and Moscovici, unpublished results).
The leukemic cells were reisolated and a new line was
established, namely BM2/L, which when reinjected into
chicken embryos induced a 90-100% incidence of leukemia.
6C2 cells. 6C2 is an AEV(RAV-2)-transformed
erythroleukemia producer cell line obtained from infection
of adult bone marrow cells in vitro (Beug et al., 1982).
MSB 1 cells. MSB 1, obtained from U.S.D.A. (East
Lansing, MI), is a lymphoblastoid producer cell line derived
from a splenic lymphoma of chicken with Marek's Disease
(Akiyama and Kato, 1974).
Viruses
AMV-B. The AMV subgroup B (AMV-B) was derived from
standard laboratory stocks as described (Moscovici et al.,
1975).
AEV-A. The AEV subgroup A (AEV-A) is the ES-4 strain
of AEV (RAV-1) originally obtained from Dr. J.M. Bishop (San
Francisco, CA).

43
RAV-1 and RAV-2. The RAVs were prepared from our
laboratory stocks.
Discontinuous Percoll Gradient
2 ml of cell suspension was layered on top of a
discontinuous percoll gradient (20%/50%/70% for bone marrow
cells and 30%/50%/70% for yolk sac cells) and centrifuged 15
minutes at 2,500 rpm. It has been demonstrated under these
conditions that the 20%/50% interface from the bone marrow
cells and the 30%/50% interface from the yolk sac cells are
rich in the precursor cells and mononucleated cells, whereas
the 50%/70% interface consists mostly of erythroblasts and
other types of differentiated cells, and the cells from the
pellet are terminally differentiated erythrocytes.
Hvbridoma Production
Hybridomas were produced according to the protocol
established in the Hybridoma Laboratory, Interdisplinary
Center for Biotechnology Research, University of Florida.
Spleen cells harvested from immunized mice were fused with
SP2/0 myeloma cells at a ratio of 7.5:1 (spleen cells :
myeloma cells) using polyethylene glycol 1540. The
supernatants of growth-positive hybridomas from 96-well flat
bottom tissue culture plates were screened 12-14 days later
by the indirect RIA and ELISA methods. Hybridomas which

44
produced monoclonal antibodies with potential interest were
subcloned by the limiting dilution method.
Radioimmunoassay (RIA)
The cells used as targets in immunoassays were washed
with PBS buffer containing 1% AHS (gamma globulin-free horse
serum) and 0.02% sodium azide and resuspended to a final
concentration of 20 x 106 cells/ml in PBS/azide/5% AHS. 50
/Ltl of cell suspension and 100 ¡il of hybridoma supernatant
was added to each well of 96-well flexible polyvinyl round-
bottom microtiter plates (Dynatech) for 45 minutes at 4°C.
At the end of this incubation, plates were washed with
PBS/azide/1% AHS and centrifuged at 1,100 rpm 3 times.
50 jul of 125I-rabbit anti-mouse IgG (RaMIG) , containing 1 x
105 cpm was then added and incubated with the cells for 45
minutes at 4°C. Plates were washed with PBS/azide/AHS and
centrifuged 3 times again. Individual wells were cut free
from each plate with a hot-wire cutter, transferred into
plastic tubes and counted in a gamma counter (LKB-Wallace
Ria Gamma 1274, Pharmacia).
Binding index = mean com bound with specific MAbs
mean cpm bound with negative control MAb
Enzvme-Linked Immunosorbent Assay (ELISA)
96-well flat bottom immunoplates (Nunc, Denmark) were
coated with 50 ¿il of yolk at a 1:40 dilution overnight and
then blocked with IX PBS containing 0.02% sodium azide and

45
1% BSA for 1 hour at room temperature. 100 ¿il of hybridoma
supernatants were then incubated in wells for 45 minutes at
room temperature followed by 100 ¿¿1 alkaline phosphatase-
conjugated rabbit anti-mouse IgG (Sigma) at a 1:1000
dilution in PBS/azide/BSA for 45 minutes at room
temperature. The plates were finally incubated with 200 pi
p-nitrophenyl phosphate (P-NPP) (lmg/ml) (Sigma) in pH 9.0
bicarbonate substrate buffer for 30 minutes to 2 hours in
the dark and read on an ELISA reader (Molecular Devices; V
Max). The plates were washed three times with PBS/azide/1%
Tween-20 in between the steps.
Immunofluorescence staining
Live cells. The cells were incubated with 1 ml MAb
supernatant for 30 minutes at 4°C and washed before fixation
with 30 pi 37% formaldehyde in 1 ml PBS for 20 minutes at
4°C. The cells were washed again after fixation. 200 pi
fluorescein-isothiocyanate (FITC) conjugated goat anti¬
mouse polyvalent IgG, A and M (FITC-GAM) at 1:50 dilution in
PBS containing 1% normal goat serum was then added for 30
minutes at room temperature. After washing in PBS twice,
the cell pellet was resuspended with 200 /¿I PPD-Glycerol/
PBS (1 ml 10X PBS, 3 ml deionized water, 6 ml glycerol and 1
to 2 flakes of p-phenylene diamine) and dropped onto slides
to observe under the fluorescence microscope coverslipped.

46
Frozen sections. Chicken embryos from 3 and 6 days of
incubation were dissected. Tissues of no more than a few mm
thick were fixed in 4% paraformaldehyde fix (in 0.1 M sodium
cacodylate buffer, pH 7.2-7.4) for 5 hours and shifted into
30% sucrose in IX PBS overnight. Afterwards, the tissues
were embedded in OCT compound (Lab-Tek; Miles) and frozen
with liquid nitrogen. 10 |im Cryostat sections were then
mounted onto slides which were precoated with Histostick
(Accuraqe Biochemicals) and stored at -20°C overnight prior
to use. The slides were incubated with primary antibodies
containing 0.3% triton-X in PBS for 30 minutes at room
temperature. They were then washed in a PBS bath for 5
minutes and incubated with FITC-GAM/0.3% triton-X/1% normal
goat serum for another 30 minutes at room temperature. The
slides were again washed in PBS for 5 minutes followed by
addition of a few drops of PPD-Glycerol/PBS and then
examined under fluorescence microscope coverslipped.
Flow Cytometry
Cells to be examined were washed with IX PBS/azide/AHS
and incubated with 1 ml MAb supernatant for 1 hour on a
rocker at 4°C followed by incubation with 1:10 dilution of
FITC-conjugated sheep anti-mouse IgG [F(ab')2 fragment]
(Sigma) for 30 minutes on a rocker at 4°C. Cells were
washed twice and resuspended in Hank's balanced salt
solution (HBSS) containing 1% FBS at a concentration of 2 to

47
3 x 106 cells/xnl. 1 x 104 cells were then analyzed on a
FACStar-plus fluorescence-activated cell sorter (Becton-
Dickinson, Mountain View, CA) by the parameters of forward
light scatter and fluorescein fluorescence. Cellular
excitation was obtained with an emission wavelength of 488
nm at an output power of 0.25W for fluorescein fluorescence.
The FITC fluorescence emitted was filtered with a 530 nm
long pass interface filter and a 530 band pass filter. The
data were collected and analyzed by a Becton/Dickinson
Consort 30 Computer program (Braylan et al., 1982).
Immunoenzvmatic Staining by APAAP (Alkaline Phosphatase and
Monoclonal Anti-Alkaline Phosphatase) Complex
2 x 105 cells in 0.2 ml were cytofuged onto slides by
Cytospin (Shandon Southern) at 300 rpm for 7 minutes. The
slides were air-dried at room temperature for 2 hours
followed by fixation with equal parts of acetone and
methanol for 5 minutes at 4°C. 1 ml MAb supernatants were
added to the slides and incubated in a moist chamber for 30
minutes at room temperature. Anti-mouse immunoglobulins
(DAKOPATTS; at 1:25 dilution) were then incubated for 30
minutes followed by APAAP complex (DAKOPATTS; at 1:50
dilution) for another 30 minutes. The slides were washed in
a tris-buffered saline (TBS) bath for 1 minute in between
the steps. Finally, alkaline phosphatase substrate was
added onto the slides for 15-20 minutes and then washed off
first with TBS and then with tap water (Cordell et al.,

48
1984). Slides were counter-stained with Giemsa stain at
1:20 dilution for 5 minutes.
Benzidine Staining
10 /Lil H202 (30%) was added to one ml of the benzidine
solution (0.5 g benzidine in 100 ml 70% ethanol) immediately
prior to use. Just a few drops of the staining mixture were
deposited on the cell sample and left at room temperature in
the dark for 5-10 minutes.
Induction of Cell Differentiation
BM2 cells. 1 x 107 BM2 cells were treated with 10
/zg/ml lipopolysaccharide (LPS) and 0.25 /xg/ml phorbol
12-myristate-13-acetate (PMA) or 2.5 nq/ml PMA alone in a
100-mm petri dish. 3 days later, most of BM2 cells had
attached to the petri dish and had differentiated into
macrophages.
6C2 cells. 6C2 cells were treated with 1.0 mM butyric
acid for 3 days. Although 6C2 cells did not
differentiate into mature erythrocytes with butyric acid,
their proliferating potential had been arrested.
High-Salt Protein A-Sepharose Chromatography
Ascites (1:10 dilution) or hybridoma supernatants were
adjusted to contain 1.5M glycine, 3M NaCl (pH 8.9), filtered
through a 0.22 /i millipore filter and run through a protein

49
A-sepharose column (Sigma) twice to allow binding of IgG to
the column. 5-10 column volumes of binding buffer (1.5M
glycine, 3M NaCl, pH 8.9) was then percolated through the
column to get rid of unbound proteins. Elution buffer (100
mM citric acid, pH 6.0) was then added to the column to
elute the IgG, followed by regeneration buffer (100 mM
citric acid, pH 3.0) to wash the column. Samples were
collected by a fraction collector, neutralized to pH 7.0-
7.2 with 1M tris buffer (pH 9.0) and read with a U.V.
spectrophotometer at wavelength 280 nm. The concentration
of IgG was calculated as: mg/ml = O.D. 280 nm / 1.4.
Collected fractions were then dialyzed against lx PBS/azide
buffer overnight.
Results
Production. Subcloninq and Isotvpinq of the MAbs
The rationale for MAb production is simple and
straightforward, however, the goals turned out to be much
tougher to achieve than we originally expected, especially
from the fusions with spleens from mice immunized against 3-
day-embryo yolk sac cells and megalocytes. Of nearly 2,500
hybridomas derived from 5 fusions, the predominant antibody
specificities detected were for yolk components due to the
fact that the unavoidably large amount of yolk was
associated with the yolk sac cells. Only three MAbs
exhibited potential interest, namely, 2E10, 3F6 and 1F7.

50
Fusions of spleens from mice immunized against BM2
cells and BM2/L cells yielded a panel of MAbs with various
specificities against different types of hematopoietic cells
rather than just MAbs specific for BM2 or BM2/L cells (Table
2-1). Three MAbs, 1H10, 2H1 and 3D7, from group VI, which
displayed specificity for normal monocytic cells and AMV-
transformed cells, were chosen for further studies.
These selected hybridomas were then subcloned by
limiting dilution methods and isotyped by Ouchterlony double
diffusion tests (Table 2-2). MAbs 1H10-1F9, 2H1-2A10, 3D7-
1C9 and 2E10-1E10 as well as 3F6-1E7 are all IgGl (k); only
1F7-1A3 is an IgM (/c) .
Cell-Type Specificities of MAbs
As demonstrated by RIA, flow cytometry and
immunofluorescence staining, MAbs 1H10-1F9, 2H1-2A10 and
3D7-1C9 exhibited specificity for monocytic cells with a
preferential reaction against BM2 lines. The RIA results
also revealed a slight reaction of these MAbs with cells
from the 20%/50% interface of a discontinuous percoll
gradient from 2-week-old bone marrow cells (Table 2-3).
Further analysis by flow cytometry (Figure 2-1) and APAAP
immunoenzymatic staining (Figure 2-2) confirm that about
10%-20% of the cells are expressing different degrees of the
differentiation markers recognized by this group of MAbs.
Conversely, no expression of these markers was observed in

51
Table 2-1. The cell-type specificities of MAbs from BM2 and
BM2/L fusions
Monoclonal Antibody Group
Cell Type
II
II
III
IV
V
VIa
Normal Cells
Granulocytic
++b
+
-
-
+
-
Monocytic
++
++
+
+
++
+
Erythroid
++
+
+
-
-
-
Lymphoid
++
-
-
-
+
-
Transformed Cells
AMV
++
++
++
++
++
++
AEV
++
++
+
+
-
-
aOnly three MAbs
from group VI
were selected
for further
characterization because of their specificities for
normal monocytic cells and AMV-transformed cells.
b++: RIA binding indexes >10.0; +; RIA binding indexes >5.0;
-: RIA binding indexes <2.0.
Table 2-2. The
isotypes of selected
MAbs
MAb
Isotype
1H10-1F9
IgGl
(/c)
2H1-2A10
IgGl
(«)
3D7-1C9
IgGl
(k)
2E10-1E10
IgGl
(k)
3F6-1E7
IgGl
(k)
1F7-1A3
IgM
(k)

52
Table 2-3. RIA binding indexes of MAbs to different cell types
Monoclonal
Antibodv
Cells*
1H10-1F9
2H1-2A10
3D7-1C9
2E10-1E10
3F6-1E7
1F7-1A3
TRANSFORMED
6C2 1.19b
1.21
1.66
26.35
3.03
3.96
3DYS-AEV
N.T.
N.T.
1.00
6.48
5.38
3.04
6DYS-AEV
N.T.
N.T.
N.T.
25.94
12.52
5.27
MSB1
1.26
0.59
0.34
14.06
7.78
5.17
BM2/C3A
43.55
38.80
38.01
9.91
9.61
1.25
BM2/REC1
34.09
50.83
57.77
8.03
N.T.
N.T.
BM2L/A1
29.98
35.05
41.72
8.26
9.72
1.29
NORMAL
M0
4.89
6.85
5.87
N.T.
N.T.
N.T.
RBC(PB)
0.77
1.52
1.14
2.58
1.09
1.31
WBC(PB)
1.15
2.25
1.63
2.24
1.25
1.11
CEF
0.83
0.97
0.88
0.87
0.75
N.T.
2DYS
0.52
0.94
1.13
2.34
2.01
N.D.
3DYS
1.52
1.40
1.16
2.18
2.11
5.72
6DYS
0.68
1.60
0.84
3.27
1.12
N.T.
12DYS
0.69
1.28
0.62
7.59
0.52
N.T.
BM 20/50
2.88
3.48
2.89
4.76
1.98
N.T.
BM 50/70
1.62
2.39
1.91
7.49
1.63
N.T.
a6C2, AEV-transformed producer cell line from adult bone marrow;
3DYS-AEV, AEV-transformed 3-day-embryo yolk sac cells; MSB1,
Marek's Disease Virus-transformed lymphoblastoid producer cell
line; BM2, AMV-transformed non-producer cell line from embryonic
bone marrow; BM2/C3A, a subclone of BM2; BM2/REC1, a subclone
recovered from the bone marrow of BM2/C3A injected chick;
BM2L/A1, a leukemogenic variant of BM2/C3A; M peripheral blood; CEF, chicken embryo fibroblast; BM 20/50,
20%/50% interface of a percoll gradient of 2-week-old bone
marrow cells; N.T., not tested.
bRIA binding indexes: see Materials and Methods for details.

Cell Number
53
Log Fluorescence Intensity
Figure 2-1. Flow cytometric analysis of the 20%/50%
interface of a percoll gradient of 2-week-old bone marrow
cells. The cells were labeled with negative control MAb,
1H10-1F9, 2H1-2A10 and 3D7-1C9 respectively. The background
fluorescence resulting either from nonspecific binding or
autofluorescence was present in 10% of the cells. 10-20% of
the cells were positive for MAbs 1H10-1F9, 2H1-2A10 and 3D7-
1C9.

54
Figure 2-2. APAAP immunostaining of 20%/50% interface of a
percoll gradient of 2-week-old bone marrow cells (600x).
The cytofuges were treated with (A) negative control MAb,
(B) 1H10-1F9, (C) 2H1-2A10 and (D) 3D7-1C9 respectively.
Positive cells were labeled with purple-reddish products
around the cell surface and/or inside the cytoplasm.

55
the cells from the 50%/70% interface (Table 2-3 and Figure
2-3) .
1F7-1A3 recognizes the AEV-transformed yolk sac cells,
MSB1 cells and 20% of the cells in the 30%/50% interface of
a discontinuous percoll gradient of yolk sac cells (Table 2-
3 and Figure 2-4). Interestingly enough, its reaction to 6C2
cells is amplified after the treatment with neuraminidase
(discussed in Chapter 4 in more detail).
3F6-1E7 detects a differentiation marker present mainly
on AEV-transformed yolk sac cells, BM2 cell lines and MSB1
cells as well as 10% of cells from the 30%/50% interface of
a percoll gradient of normal yolk sac cells (Table 2-3 and
Figure 2-4).
2E10-1E10 possesses a variety of cell-type
specificities such as 6C2 cells, AEV-transformed yolk sac
cells, MSB1 cells, BM2 cells, 6 & 12-day-embryo yolk sac
cells and 2-week-old bone marrow cells (Table 2-3, Figure
2-5 & Figure 2-6). However, there is no reaction to the
terminally differentiated hematopoietic cells.
The cell-type specificities of MAbs are summarized in
Table 2-4.
Induction of Cell Differentiation
Treatment of BM2/C3A cells with 0.25 /¿g/ml PMA and 10
jug/ml LPS or 2.5 /¿g/ml PMA alone for 3 days caused the
BM2/C3A cells to attach to the petri dishes and become

Cell Number
56
Figure 2-3. Flow cytometric analysis of the 50%/70%
interface of a percoll gradient of 2-week-old bone marrow
cells. The cells were labeled with negative control MAb,
1H10-1F9, 2H1-2A10 and 3D7-1C9 respectively. The results
showed no reaction of MAbs with these cells at all.

57
Figure 2-4. Flow cytometric analysis of the 30%/50%
interface of a percoll gradient of 4-day-embryo yolk sac
cells. Cells were tagged with negative control MAb, 1F7-1A3
and 3F6-1E7 respectively. The analysis showed that 3F6-1E7
label 10% of the cells, whereas 20% of the cells are
positive for 1F7-1A3.

58
Figure 2-5. Flow cytometric analysis of the 20%/50%
interface of a percoll gradient of 2-week-old bone marrow
cells tagged with MAb 2E10-1E10. Almost 80% of the cells
were positive for MAb 2E10-1E10.

59
Figure 2-6. Flow cytometric analysis of the 50%/70%
interface of a percoll gradient of 2-week-old bone marrow
cells tagged with MAb 2E10-1E10. 2E10-1E10 recognized
nearly 70% of the cells.

60
Table 2-4. Summary of the cell-type specificities of MAbs
Groups
MAbs
Specificities
I
3D7-1C9
2H1-2A10
1H10-1F9
BM2 cell lines
Normal monocytic cells
II
3F6-1E7
AEV-transformed yolk sac cells
BM2 cell lines
MSB1 (Marek's Disease)
10% of cells from the 30%/50%
interface of a percoll fraction
of 3-day-embryo yolk sac cells
III
1F7-1A3
AEV-transformed yolk sac cells
MSB1 (Marek's Disease)
20% of cells from the 30%/50%
interface of a percoll fraction
of 3-day-embryo yolk sac cells
IV
2E10-1E10
6C2 cells
AEV-transformed yolk sac cells
MSB1 (Marek's Disease)
BM2 cell lines
6-day-embryo yolk sac cells
12-day-embryo yolk sac cells
2-week-old bone marrow cells

61
differentiated into macrophage-like cells. In addition,
their proliferating potential (Table 2-5) and ability to
bind MAbs 1H10-1F9, 2H1-2A10 and 3D7-1C9 were dramatically
decreased (Figure 2-7). Therefore, the differentiation
markers recognized by these MAbs may be candidates for
transformation-associated antigens in this system.
Although 6C2 cells were not induced to terminally
differentiate into erythrocytes with the treatment of ImM
butyric acid for 3 days, their proliferating potential had
been impaired (Table 2-6) and the expression of the 2E10-
lE10-recognized marker was also reduced (Figure 2-8).
Reactivity to the Envelope Proteins of Retroviruses
The RAV-1 or RAV-2 infected CEF cells were used as
control to exclude the possibility that MAbs might react
with the envelope proteins of retroviruses which were
expressed on the cell surface. 3F6-1E7 and 2E10-1E10 were
shown to have no reaction to either CEF cells or RAV-
infected CEF cells (Table 2-7).
Discussion
Four groups of MAbs were selected among nearly 5,000
hybridoma supernatants from ten fusions. Characterization
of their cell-type specificities was achived by RIA,
immunofluorescence staining, flow cytometry and
immunoenzymatic staining. 1) MAbs 1H10-1F9, 2H10-2A10 and

Table 2-5. Comparison of the proliferating potential of
BM2/C3A cells and 2.5nq/ml PMA-diffentiated BM2/C3A cells
BM2/C3Ab
BM2/C3A + PMAb
Number of cells
after 3 days of
incubation®
58 x 106
6 X 106
a10 x 106 cells were seeded per 100-mm petri dish.
bThe number of cells was expressed as the average from 6
dishes.

Figure 2-7. Reactivities of MAbs with BM2 cells and differentiated BM2 cells.
BM2 cells were differentiated into macrophage-like cells by the treatment of
0.25 /xg/ml PMA and 10 nq/ml LPS for three days. The reactivities are expressed
as the MAb binding indexes as determined by RIA.

40
1H10-1F9 2H1-2A10 3D7-1C9 2E10-1E10 3F6-1E7
â–¡ Control â–¡ PMA&LPS
(Ti
.P*

65
Table 2-6. Comparison of the proliferating potential of 6C2
cells and ImM butyric acid-treated 6C2 cells
6C2b
6C2 + Butyric acidb
Number of cells
after 3 days of 40 x 106
incubation3
ao ^ _ -i •
11.5 x 106
a10 x 106 cells were seeded in 60-mm petri dish.
bThe number of cells was expressed as the average from 6
dishes.

Figure 2-8. Reactivities of MAbs with 6C2 cells and butyric acid-treated 6C2
cells. 6C2 cells were treated with l.OmM butyric acid for three days. Though
6C2 cells were not differentiated into erythrocytes, the proliferating potential
had been impaired. The reactivities are expressed as MAb the binding indexes as
determined by RIA.

2E10-1E10
SI Control
Butyric Acid
1F7-1A3

68
Table 2-7. RIA binding indexes of MAbs to normal CEF and
RAV-infected CEF
Cells
Monoclonal
2E10-1E10
Antibody
3F6-1E7
CEF
0.87
0.75
CEF
(RAV-1)
1.08
0.75
CEF
(RAV-2)
1.10
0.80

69
3D7-1C9 are specific for transformation-associated antigens
present preferentially on BM2 cell lines rather than on
normal monocytic cells. 2) MAb 1F7-1A3 recognizes AEV-
transformed yolk sac cells, MSB1 cells and 20% of cells from
the 30%/50% interface of a discontinous percoll gradient of
4-day-embryo yolk sac cells. 3) MAb 3F6-1E7 reacts with
AEV-transformed yolk sac cells, BM2 cells and MSB1 cells as
well as 10% of cells from the interface of a discontinous
percoll gradient of 4-day-embryo yolk sac cells. 4) MAb
2E10-1E10 defines a marker present on different tumor cells,
yolk sac cells and bone marrow cells instead of terminally
differentiated cells.
The expression of transformation-associated antigens
recognized by MAbs 1H10-1F9, 2H1-2A10 and 3D7-1C9 is
possibly up-regulated by v-myb proteins, but is diminished
after the BM2 cells are differentiated by PMA. Future
investigators may want to determine 1) whether the
expression of these antigens is regulated at the
transcriptional and/or the translational level or is due to
posttranslational modification, such as aberrant
glycosylation and/or sialylation, and 2) whether the
expression of these markers is essential for the
transforming process or if they are merely the by-products
of transformation.
Although 2E10-1E10 displays a wide range of cell-type
specificities, it labels primarily 6C2 cells and

70
AEV-transformed 6-day-embryo yolk sac cells and it does not
recognize terminally differentiated cells such as
erythrocytes, macrophages, lymphocytes and PMA-
differentiated BM2/C3A cells. These results lead us to
speculate that 2E10-1E10 may react only with proliferating
hematopoietic cells. This possibility was supported by the
observation that the binding specificities of 2E10-1E10 to
6C2 cells was diminished after treatment with butyric acid
which inhibited the proliferating potential of 6C2 cells
without the induction of differentiation. Moreover, this
marker starts appearing at the 4th day of embryogenesis
(discussed in Chapter 3 in more detail) and its expression
is enhanced after the cells are transformed by AEV.
LPS only induces the BM2 cells to attach to the petri
dishes but does not induce differentiation. As a matter of
fact, PMA alone can induce both the attachment and
differentiation of the BM2 cells even at a low concentration
of 0.25 ¿ig/ml. The reason why we added 10 ^g/ml LPS to the
PMA treatment was simply because we didn't want to see "any
fish sneak out of the net", as the Chinese proverb says.
Butyric acid has been shown to induce the
differentiation of the AEV strain R (RAV-2)-transformed
erythroleukemia cells from SC strain chickens (Nelson et
al., 1982), but was incapable of differentiating 6C2 cells
even though their proliferating potential had been
apparently impaired. This is possibly due to the

71
difference in the susceptibilities to butyric acid of
various avian erythroleukemia cell lines and/or sublines.
Immunoperoxidase staining procedures were not used
because significant amounts of endogenous peroxidase present
in granulocytes, erythroid cells and macrophages would give
rise to unwanted background staining, and it is rather
difficult to abolish this activity by exposing samples to
peroxidase inhibitors (such as H202 and methanol) without
causing antigenic denaturation (Cordell et al., 1984). In
contrast endogenous alkaline phosphatase activity survives
poorly in cytofuge slide preparation and any residual
activity may be selectively inhibited by including
levamisole in the enzyme substrate solution (Ponder and
Wilkinson, 1981). Therefore the APAAP immunostaining
technique was chosen for our studies. Wet slides without
any mounting fluid were then photographed becaused it was
observed that the nonaqueous mounting solution dissolved the
reaction product, whereas an aqueous mounting medium will
disrupt the Giemsa counter-stain.
The antigens recognized by MAbs 1F7-1A3, 2E10-1E10 and
3F6-1E7 could only be detected by RIA and immunofluorescence
staining and not by APAAP immunostaining technique. It is
very likely that these markers may be so fragile that they
were destroyed during the fixation (acetone and ethanol)
and/or any subsequent steps.

CHAPTER 3
IDENTIFICATION OF TARGET CELLS FOR MABS
IN THE BONE MARROW AND YOLK SAC
Introduction
The avian hematopoietic precursor cells represent only
a small population in the blood-forming organs, however, it
is this small population that builds up the entire
hematopoietic repertoire of various types of specialized
blood cells with different functions. The nature of self¬
renewal and commitment remains an enigma, but if the
precursor cells can be isolated from the heterogenous
population of hematopoietic cells for direct studies, it
would provide us with invaluable pieces of information to
solve the enigma. However, these cells are morphologically
unrecognizable, and until now no specific markers had been
identified to facilitate the purification of these cells.
One of the specific aims of this project was to develop MAbs
against differentiation markers specifically present on the
surface membranes of the avian hematopoietic precursor
cells. These cells have been purified and enriched by using
FACS or immunomagnetic beads as follows. Bone marrow cells
or yolk sac cells are treated with specific MAbs followed by
FITC-conjugated or magnetic bead coated with secondary
72

73
antibodies. Fluorescence-positive and -negative populations
are then separated by the FACS, whereas the magnetic bead-
bound cells are separated from the negative population by a
magnetic field (negative selection). The separated
fractions are then identified by indirect methods, such as
AMV/AEV transforming assays and BFU-E/CFU-E colony forming
assays. For example, if MAb-positive fractions produce
colonies derived from the CFU-E instead of the BFU-E, the
antigen must be expressed later than the BFU-E stage; if it
produces AMV-transformed colonies, but neither AEV-
transformed colonies nor BFU-E/CFU-E colonies are developed,
the MAb ought to be specific for the target cells for AMV,
i.e., cells of the monocytic lineage.
These approaches helped us to confirm the cell-type
specificities of the MAbs that we developed.
Materials And Methods
FACS Sorting
After being analyzed on a FACStar-plus sorter,
according to the procedure described in the previous
chapter, the cells were sorted into positive and negative
fractions at a rate of 1,000 cells/sec.
Immumomagnetic Beads Separation
10 x 106 Cells were tagged with 1 ml of MAb
supernatants for 1 hour at 4°C on a rocker and then washed

74
with PBS/azide/1% AHS followed by incubation with magnetic
beads coated with sheep anti-mouse IgG, (Fc) (Dynabeads M-
450; Dynal Inc.) or magnetic particles coated with goat
anti-mouse IgM (Advanced Magnetics) at a bead to cell ratio
of 4 to 1 for 30 minutes at 4°C on a rocker. Cells bound to
the beads were separated from the negative population that
remained in suspension by applying a cobalt steel magnetic
force (Dynal MPC-1; Dynal Inc.) for 1 minute (Cruikshank et
al., 1987). This process was repeated twice. It was not
possible to separate viable cells (positive selection) from
the magnetic beads because of technical limitations.
Retrovirus Transforming Assays
The transforming assays were performed according to the
protocol described by Moscovici et al. (1983). Briefly,
cells were infected with retroviruses at high m.o.i. The
virus adsorption was carried out 30 minutes at 4°C and then
30 minutes at room temperature. 1 xlO5 AEV-infected cells
was seeded in 2 ml semi-solid a-medium containing 20% FBS,
10% ACS, 1% BPA, 0.1% 10_1M 0-mercaptoethanol, 0.1%
gentamycin and 25% methocellulose per 35-mm dish. Whereas,
1 xlO5 AMV-infected cells was mixed with 1 ml F12 overlay
medium containing 20% 2X F12, 6% calf serum, 2% chicken
serum, 10% tryptose phosphate, 1% 100X vitamins, 1% 100X
folic acid and 40% fibroblast-conditioned medium as well as
20% of 1.8% Bacto agar, and then overlayed on top of 2 ml

75
3.6% hard agar base in 35-nun petri dishes. The transformed
colonies were scored after 6-12 days of incubation.
BFU-E/CFU-E Colony Assays
0.5 ml of cell suspension were mixed with a-medium
containing 20% FBS, 10% ACS, 1% BPA, 0.1% lO^M /3-
mercaptoethanol, 0.1% gentamycin and 25% methocellulose and
seeded at 1x10s cells per 35-mm dish. The CFU-E were scored
after 3-4 days of incubation, whereas BFU-E were scored
after 6-7 days of incubation (Samarut and Bouabdelli, 1980).
Results
FACS Sorting
In order to identify the target cells for the MAbs in
the bone marrow, FACS was utilized to separate the MAb-
positive population and MAb-negative population. The bone
marrow cells were analyzed before and after the FACS sorting
(Figure 3-1 and 3-2). Transforming assays and colony¬
forming assays were performed on both negative and positive
populations.
The results obtained in the transforming assays and
colony-forming assays (Table 3-1) confirm that the 3D7-1C9
positive population (10%-20% of the cells from the 20%/50%
interface of a discontinuous percoll gradient of 2-week-old
bone marrow cells) represents the target cells for AMV,
i.e., cells of the monocytic lineage.

76
Figure 3-1. Flow cytometric analysis of 3D7-lC9-tagged bone
marrow cells before sorting. 20%/50% interface of a percoll
gradient of 2-week-old bone marrow cells were tagged with
3D7-1C9 MAb. The analysis showed that 10-20% of the cells
are positive for MAb 3D7-1C9.

77
Figure 3-2. Flow cytometric analysis of 3D7-lC9-tagged bone
marrow cells after sorting. 20%/50% interface of a percoll
gradient of 2-week-old bone marrow cells were sorted into
3D7-1C9 positive fraction and 3D7-1C9 negative fraction
followed by the FACS analysis. The 3D7-1C9 positive
fraction still contained about 20% negative cells due to
contamination during the sorting.

78
Table 3-1. Characterization of the target cells isolated
with MAb 3D7-1C9 from the bone marrow
Colonies 3D7-1C9
(lxlO5 cells) positive
population
3D7-1C9
negative
population
CFU-E
34.0
+
5.0
1620.5
+
52.5
BFU-E
12.0
±
3.0
512.0
+
132.0
AEV-A
26.0
+
2.0
144.0
+
7.0
AMV-B
592.5
+
20.5
86.5
+
13.5
20%/50% interface of a percoll gradient of 2-week-old bone
marrow cells were tagged with 3D7-1C9 and then sorted by
FACStar followed by transforming assays and colony¬
forming assays. The results show that 3D7-1C9 positive
population contain the target cells for AMV, i.e.,
cells of monocytic lineage, rather than BFU-E/CFU-E or the
target cells for AEV.

79
On the other hand, the results of colony-forming assays
and retrovirus transforming assays suggest that MAb 2E10-
1E10 not only recognize the target cells for AEV and AMV
but also the BFU-E and CFU-E (Table 3-2). This finding
prompted us to suggest in Chapter 2 that MAb 2E10-1E10 react
with proliferating hematopoietic cells.
Immunomagnetic Bead Separation
Cells from 30%/50% interface of a discontinuous percoll
gradient of 4-day-embryo yolk sacs were incubated with MAbs
3F6-1E7 or 1F7-1A3 followed by magnetic beads conjugated
with secondary antibodies. Only the negatively selected
population of MAbs were used for the assays. There was
about 60% reduction in the BFU-E and the CFU-E colonies from
1F7-1A3 negative population. However, no significant
difference in transformed colonies was found (Table 3-3).
In other words, MAb 1F7-1A3 recognizes erythroid cells at
the BFU-E and the CFU-E stages but not the pre-BFU-E stage,
since it does not recognize cells at the pre-BFU-E stage
which are the target cell for AEV in the yolk sac.
Conversely MAb 3F6-1E7 seems not to react with either the
BFU-E/CFU-E or the target cells for the retroviruses. It
probably recognizes a differentiation marker expressed on
the stem cell and precursor cell populations at an earlier
stage than pre-BFU-E one.

Table 3-2. Characterization of the target cells isolated
with MAb 2E10-1E10 from the bone marrow
80
Colonies
(lxlO5 cells)
2E10-1E10
positive
population
2E10-1E10
negative
population
CFU-E
474.0
+
27.0
o
•
o
±
o
•
o
BFU-E
282.0
+
2.0
0.0
+
o
•
o
AEV-A
59.0
+
1.0
0.5
+
0.5
AMV-B
403.0
+
3.0
o
•
in
+
2.0
20%/50% interface of a percoll gradient of 2-week-old bone
marrow cells were tagged with 2E10-1E10 and then sorted by
FACStar followed by transforming assays and colony¬
forming assays. The results reveal that MAb 2E10-1E10
recognize not only BFU-E/CFU-E, but also the target cells
for AEV and AMV.
Table 3-3. Characterization of the target cells isolated
with MAbs from the yolk sac
Colonies
(1 x 105
Monoclonal
Antibodv
Cells) Control
3F6-
1E7
2E10-1E10
1F7-
1A3
CFU-E 3313.1±142.5
2576.21232.4
1597.0178.4
1487.51103.4
BFU-E 1211.9±
11.9
899.21
67.3
684.8173.3
501.11
23.8
AEV-A 135.6±
6.4
115.01
7.0
108.11 5.2
102.01
11.0
AMV-B 50.0±
5.0
46.01
2.0
36.81 2.4
44.01
3.0
30%/50% interface of a percoll gradient of 4-day-embryo yolk
sac cells were treated respectively with MAbs 1F7-1A3, 2E10-
1E10 or 3F6-1E7 followed by immunomagnetic bead separation.
The MAb-negative populations were collected to perform
colony-forming assays and transforming assays. See text for
more details.

81
The MAb 2E10-1E10 negative population in the 4-day-
embryo yolk sac cells exhibited nearly 50% reduction in the
BFU-E/CFU-E colonies and 30% in the AMV-transformed colonies
repectively (Table 3-3). Moreover, I have shown in Chapter
2 that MAb 2E10-1E10 has no binding specificities for 2- or
3-day-embryo yolk sac cell. These results indicate that the
marker identified by MAb 2E10-1E10 starts appearing after
the 4th day of embryogenesis.
Discussion
The FACS sorting and immunomagnetic bead techniques
allow us to perform direct studies on MAb-positive and/or
MAb-negative populations. The results from colony-forming
assays and transforming assays showed that MAb 3D7-1C9 can
purify the target cells for AMV, i.e., cells of the
monocytic lineage, MAb 2E10-1E10 define a marker present on
proliferating hematopoietic cells and it starts appearing
only after the 4th day of embryogenesis and that MAb 1F7-1A3
recognize erythroid cells at BFU-E and CFU-E stages.
MAb 3F6-1E7 recognizes only some tumor cell lines and
10% of cells from the 30%/50% interface of a discontinuous
percoll gradient of 3- and 4-day-embryo yolk sac cells, and
it does not possess the specificities for the terminally
differentiated cells and the lineage-committed progenitor
cells. However there is no direct evidence yet to support
the idea that the 10% of the cells recognized by MAb 3F6-1E7

82
represent the stem cell and precursor cell populations in
the yolk sac. In order to prove this, there are two
obstacles that need to be overcome. One is to improve the
FACS sorting condition to prevent yolk sac cells from
lysing. The other is to establish a long-term culture of
normal avian yolk sac cells. Once these problems are
solved, it will become possible to obtain the long-term
culture of the FACS-sorted 3F6-1E7 positive cells and then
we can compare the results from the transforming assays and
colony-forming assays between the 3F6-1E7 positive
population with and without the long-term culture.
Theoretically, in the long-term culture, the stem cell and
precursor cell populations will proliferate and undergo the
normal differentiation program to become the committed
progenitor cells and mature cells. If the 3F6-1E7 positive
cells indeed represent these populations, the increased
number of retrovirus-transformed colonies and BFU-E/CFU-E
colonies will then be observed in the 3F6-1E7 positive cells
with long-term culture. Ultimately, 3F6-1E7 positive cells
should be capable of repopulating the bone marrow of
irradiated chicks.
It is still a puzzle why MAb 1F7-1A3 reacts with
lymphoblastoid cell line-MSBl. Maybe the marker recognized
by 1F7-1A3, which is normally present on the embryonic
BFU-E/CFU-E only, can be regarded as an onco-fetal antigen,
i.e., its expression being turned on in the AEV-transformed

83
yolk sac cells as well as in the MSB1 cells instead of in
other types of transformed cells. This interesting finding
also implies that the relationship between the erythroid and
lymphoid lineage may be closer than we originally thought.
The bone marrow cells were sorted into fluorescence¬
positive and -negative populations using the FACStar-plus at
a rate of 1,000 cells/sec. At this rate it took
approximately 2-3 hours to collect the minimum workable
number of cells, i.e., 5xl05 MAb-positive cells which
account for 10-20% of cells from the 20%/50% interface of a
discontinuous percoll gradient of 2-week-old bone marrow
cells. The results of flow cytometry, retrovirus
transforming assays and colony-forming assays showed that
there is a slight contamination of MAb-negative cells in the
positive population. To increase the purity of the
collection by reducing the analysis rate or by processing a
"two-run" procedure would be too time-consuming and the
viability or the behavior of the sorted cells might be
influenced.
The yolk sac cells are extremely delicate and fragile
compared to other types of cells. Suffering from the
"abuse" of scalpel mincing, extensive washing, percoll
fractionation, as well as FACS sorting, the cell membranes
could have been damaged in such a way that only less than
10% of the sorted cells stayed intact, while the rest were
all lysed. We have tried different approaches such as

84
changing the sheath fluid, minimizing the laser power and
electric charge, reducing the centrifugation speed and
changing the collecting tubes etc. None so far gave us
satisfactory results.
Nevertheless, this is the pioneer study of the avian
hematopoietic system by FACS analysis. Once all the
conditions are standardized, it will become a tremendously
powerful tool to identify the stem cell and precursor cell
populations of the avian hematopoietic system, especially in
the yolk sac.
There are two disadvantages in using the immunomagnetic
beads for cell separation. First, its sensitivity is much
lower than that of the FACS sorting, i.e., it yields less
pure separation than FACS does. Second, because of the
strength of positive cells binding to magnetic beads,
trypsinization is needed to free the magnetic beads from the
cell surface. Cell surface receptors for retroviruses and
growth factors will be destroyed by the trypsinization, not
to mention the possibility that the yolk sac cells may
become more susceptible to lysis. As a result, few viable
cells remain for further study. Nevertheless, compared to
the FACS sorting, immunomagnetic bead separation is not as
time-consuming and has the advantage of not damaging the
yolk sac cells. Therefore, immunomagnetic beads separation
represents the most useful technique so far to study the
influence of MAbs in the yolk sac system.

CHAPTER 4
BIOCHEMICAL CHARACTERIZATION OF
THE DIFFERENTIATION MARKERS RECOGNIZED BY MABS
Introduction
There are two kinds of changes that contribute to the
expression of differentiation markers specific for a
particular cell type or lineage on normal hematopoietic
cells or to the expression of transformation-associated
antigens on retrovirus-transformed hematopoietic cells. One
is the appearance of new surface markers due to enhanced
synthesis at the transcriptional and/or translational level.
The other is the alteration in the structure of carbohydrate
groups attached to proteins or lipids at the
posttranslational level. These changes probably are
involved in the alteration of the interaction of particular
hematopoietic cells with other types of hematopoietic cells,
with the extracellular matrix or with stromal cells and in
the different response to growth and differentiation
factors.
Our initial attempt was to determine the nature and
molecular weights of these differentiation markers in order
to elucidate what kind of changes have occurred to these
markers recognized by MAbs. The experimental approach
85

86
toward the characterization of these differentiation markers
was via enzymatic digestion and chemical deglycosylation of
the cell surface. Trypsin was used to test if proteins were
portions of the antigens and endoglycosidase F and sodium-M-
periodate were used to determine whether they were
glycosylated. In addition, neuraminidase digestion was
performed to test for the presence of sialic acid. Western
blotting and biosynthetic labeling/immunoprecipitation
techniques were utilized to determine the molecular weights
of these markers.
Materials and Methods
Enzymatic Digestion
Trypsin. Cells were treated with 0.125% trypsin
(Sigma) in TBS buffer, pH 5.0, for 1 hour at 37°C. Cells
were then washed, incubated with MAb supernatants followed
by RIA.
Neuraminidase♦ Cells were treated with neuraminidase
(from Clostridium perfringens; Sigma) at a concentration of
20 x 106 cells/U in TBS buffer, pH 5.0, for 1 hour at 37°C.
Endoglycosidase F. Cells were treated with Endo F at a
concentration of 20 x 106 cells/U in TBS buffer, pH 5.0, for
1 hour at 37°C.
Chemical Deglycosylation
2.5 mM sodium-M-periodate in PBS was added to BM2

87
cells, 1.25 mM to 6C2 cells and 1.25 mM to MSB1 cells in TBS
buffer, pH 5.0, for 1 hour at 37°C.
Cell Membrane Solubilization
Unlabeled or 35S-Methionine-labeled 50 x 106 cells were
lysed in 1 ml lysis buffer containing 1.0% Triton X-100,
0.15 M NaCl, 0.01 M Tris-HCl, pH 8.0, 0.02% NaN3, 2 mM PMSF
and 100 KIU/ml aprotinin for 25 minutes on ice followed by
the centrifugation at 12,000 x g for 15 minutes to get rid
of the nuclear pellet.
Biosynthetic Labeling
50 x 106 cells were incubated with Methionine-free DMEM
and 500 n Ci L-35S-Methionine for 7 hours at 37°C. After two
washes with cold TSA buffer (0.15 M NaCl, 0.01 Tris-HCl, pH
8.0, 0.02% NaN3) the cells were then resuspended in 1 ml of
lysis buffer.
Immunoprecipitation
Cell lysates were precleared with 50 /¿I protein
G-sepharose 4B or protein A-sepharose 4B (Zymed) and 50 jul
unrelated MAb supernatant overnight at 4°C. The supernatant
fluid of the cell lysates were recovered after
centrifugation. 100 /xl of precleared lysate was incubated
with 100 nl MAb supernatant for 4 hours at 4°C followed by
the addition of 40 nl protein G-sepharose or protein

88
A-sepharose for 2 hours at 4°C. The lysates were washed
twice with washing buffer containing 0.1% Triton X-100/TSA,
once with TSA buffer and then 0.05 M Tris-HCl, pH 6.8.
After the last wash, the pellets were resuspended with 50 ijlI
2x SDS/sample buffer (2% SDS and 5% 2-mercaptoethanol) and
boiled at 100°C for 5 minutes.
SDS-PAGE
The supernatant fluids which were recovered from the
centrifugation of the boiled immunoprecipitates in sample
buffer were loaded at 20 K cpm per lane of a 10% acrylamide
denaturing (SDS) discontinuous mini-gel. The SDS-PAGE was
performed according to the method described by Laemmli
(1970). Briefly, the gel was run at 25 mA of constant
current per 0.75 mm slab gel on a Mini-PROTEIN II cell (BIO¬
RAD) for 45 minutes to 1 hour. Rainbowâ„¢ protein molecular
weight markers (Amersham) were used for unlabeled cell
lysates, whereas uC-methylated protein molecular weight
markers (Amersham) were used for radiolabeled cell lysates.
Fluorographv
After the electrophoresis was complete, the gel was
fixed in 50% methanol and 10% acetic acid for 1 hour and
then incubated with EN3HANCE (Dupont) for 1 hour followed by
two changes of tap water. The gel was dried in a slab-gel
dryer (Hoefer Scientific Instruments) at 60°C for 1 hour

89
onto a filter paper and exposed on a Kodak XAR-5 film for 1-
3 days.
Silver Staining
The unlabeled proteins were detected by silver
staining. Briefly, the polyacrylamide gel was fixed in
fixing solution (50% methanol and 10% acetic acid) for 30
minutes and in destaining solution (5% methanol and 7%
acetic acid) for 1 hour followed by 10% glutaraldehyde for
30 minutes. After 4 washes with water for 30 minutes each
wash, the gel was stained with silver nitrate solution for
15 minutes and washed 5 times with water. Developer (25 ml
of 0.5% w/v sodium citrate and 0.5% v/v 37% formaldehyde
with 500 ml water) was then added and the gel was shaken
vigorously until the bands appeared as desired. The gel was
transferred to Kodak Rapid Fix (solution A) for 5 minutes
and washed with water extensively. Eventually, the gel was
dried in a gel dryer at 80°C for 1 hour (Oakly et al.,
1980) .
Results
Enzymatic Digestion and Chemical Deqlvcosvlation
Three different cell lines, BM2/C3A, 6C2 and MSB1 were
treated with trypsin, neuraminidase, Endo F and periodate
oxidation respectively and then incubated with MAbs followed

90
by RIA to reveal whether or not the markers recognized by
MAbs remained intact (Figure 4-1, 4-2 and 4-3).
All of the reactions of MAbs were knocked out by
trypsinization, which indicates that proteins probably are
the structural backbones of these markers. Deglycosylation
with either periodate oxidation or Endo F digestion could
decrease the binding specificities of MAbs 1H10-1F9, 2H1-
2A10, 3D7-1C9 and 2E10-1E10 to different degrees suggesting
that these MAbs may be recognizing carbohydrate groups. In
addition, treatment with neuraminidase showed that sialic
acid also may contribute to the conformation of the
antigenic determinants. Altogether, these data imply that
the differentiation markers recognized by MAbs 1H10-1F9,
2H1-2A10, 3D7-1C9 and 2E10-1E10 may be glycoproteins.
Conversely, binding of 3F6-1E7 and 1F7-1A3 to the cells was
not affected by deglycosylation and neuraminidase digestion,
which suggests that the antigenic binding sites for 1F7-1A3
and 3F6-1E7 are localized on the polypeptide chain without
the involvement of carbohydrate or sialic acid.
Interestingly, the reaction of MAb 1F7-1A3 with 6C2
cells was enhanced by neuraminidase digestion (Figure 4-2).
This is possibly due to the masking of the antigenic
determinant for MAb 1F7-1A3 by sialic acid in 6C2 cells.
The nature and antigenic determinants of the
differentiation markers are summarized in Table 4-1.

Figure 4-1. Reactivities of MAbs with BM2 cells following enzymatic digestion
and chemical deglycosylation. BM2 cells were treated with neuraminidase (20x10
cells/U), 0.125% trypsin, Endo F (20xl06 cells/U) and 2.5mM sodium-M-periodate
respectively for 1 hr at 37°C. The reactivities of MAbs to the cells are
expressed as the MAb binding indexes as determined by RIA.

50
40
30
20
10
0
1H10-1F9 2H1-2A10
3D7-1C9 2E10-1E10
M Control
HI Neuraminidase
Trypsin
Endo F
Periodate
VO
CO

Figure 4-2. Reactivities of MAbs with 6C2 cells following enzymatic digestion
and chemical deglycosylation. 6C2 cells were treated with neuraminidase (20x10
cells/U), 0.125% trypsin, Endo F (20xl06 cells/U) and 1.25mM sodium-M-periodate
respectively for 1 hr at 37°C. The reactivities of MAbs to the cells are
expressed as the MAb binding indexes as determined by RIA.

30
25
20
15
10
5
0
Control [\! Neuraminidase [//] Trypsin
Endo F
Periodate
vo
4^

Figure 4-3. Reactivities of MAbs with MSB1 cells following enzymatic digestion
and chemical deglycosylation. MSB1 cells were treated with neuraminidase
(20x10° cells/U), 0.125% trypsin, Endo F (20xl06 cells/U) and 1.25mM sodium-M-
periodate respectively for 1 hr at 37°C. The reactivities of MAbs to the cells
are expressed by the MAb binding indexes as determined by RIA.

RIA Binding Indexes
16
14
12
2E10-1E10 3F6-1E7 1F7-1A3
HI Control
Neuraminidase
S3 Trypsin
Endo F
Periodate
VO
crv

97
Table 4-1. The biochemical nature and antigenic determinants
of the differentiation markers recognized by MAbs
MAb
Differentiation markers
Biochemical nature
Antigenic determinant
1H10-1F9
Glycoprotein
sialic acid +/-a CHOb
2H1-2A10
Glycoprotein
sialic acid +/- CHO
3D7-1C9
Glycoprotein
sialic acid +/- CHO
2E10-1E10
Glycoprotein
sialic acid +/- CHO
1F7-1A3
Glycoprotein ?
Protein
3F6-1E7
Glycoprotein ?
Protein
a+/-, with or without
bCHO, carbohydrate

98
Characterization of the Molecular Weights of These Markers
The attempts to characterize the molecular weights of
these markers have not been very successful. The results of
dot blots showed that the binding specificities of the MAbs
to the cell lysates was decreased quite a bit after SDS
denaturation (data not shown), which may explain why western
blotting did not work.
Although the MAbs could be precipitated by protein
G-sepharose (Figure 4-4), no specific bands of antigens were
detected from the immunoprecipitates of 35S-Methionine-
labeled cell lysates (Figure 4-5, 4-6 and 4-7).
Discussion
Trypsinization, neuraminidase digestion and
deglycosylation treatment could reduse the binding
specificities of MAbs 1H10-1F9, 2H1-2A10, 3D7-1C9 and 2E10-
1E10. This suggests that the markers recognized by these
MAbs are glycoproteins and that sialic acids with or without
carbohydrates are contributing to the conformation of the
antigenic determinants. Conversely, the antigenic
determinants for MAbs 1F7-1A3 and 3F6-1E7 must be strictly
localized on the polypeptide chain, since only
trypsinization was able to inhibit their binding
specificities. In addition, the antigenic determinant for
MAb 1F7-1A3 is masked by sialic acid in 6C2 cells and

99
M.W. (Kd) ABC
D E F
Figure 4-4. Silver staining of 10% SDS-PAGE analysis of
immunoprecipitates obtained from unlabeled BM2/C3A cells.
(A) protein G-sepharose alone, (B) 1H10-1F9 MAb sup +
protein G-sepharose, (C) 2H1-2A10 MAb sup + protein
G-sepharose, (D) 3D7-1C9 MAb sup + protein G-sepharose,
(E) anti-BM2/C3A serum + protein G-sepharose, (F) BM2/C3A
cell lysate only. The results showed that antibodies can be
precipitated by ptrotein G-sepharose.

100
M.W.(Kd) ABC
200 —
92.5
69 -
46
30
Figure 4-5. Fluorography of 10% SDS-PAGE analysis of
immunoprecipitates obtained from 35S-Methionine-labeled
BM2/C3A cells. (A) 1H10-1F9 MAb sup + protein G-sepharose,
(B) 2H1-2A10 MAb sup + protein G-sepharose, (C) 3D7-1C9 MAb
sup + protein G-sepharose. No specific bands of cell
lysates were precipitated by either MAb.

101
Figure 4-6. Fluorography of 10% SDS-PAGE analysis of
immunoprecipitates obtained from 35S-Methionine-labeled
6C2 cells. (A) 3F6-1E7 MAb sup + protein A-sepharose,
(B) 2E10-1E10 MAb sup + protein A-sepharose, (C) 1F7-1A3
MAb sup + protein A-sepharose, (D) 1A8-1A2 MAb sup + protein
A-sepharose, (E) 3F6-1E7 MAb sup + protein G-sepharose,
(F) 2E10-1E10 MAb sup + protein G-sepharose, (G) 1F7-1A3
MAb sup + protein G-sepharose, (H) 1A8-1A2 MAb sup + protein
G-sepharose. No specific bands of cell lysates were
precipitated by either MAb.

102
Figure 4-7. Fluorography of 10% SDS-PAGE analysis of
immunoprecipitates obtained from 35S-Methionine-labeled
MSB1 cells. (A) 1F7-1A3 MAb sup + protein A-sepharose,
(B) 1F7-1A3 MAb sup + protein G-sepharose, (C) 2E10-1E10
MAb sup + protein A-sepharose, (D) 2E10-1E10 MAb sup +
protein G-sepharose, (E) 3F6-1E7 MAb sup + protein A-
sepharose, (F) 3F6-1E7 MAb sup + protein G-sepharose.
No specific bands of cell lysates were precipitated by
either MAb.

103
possibly also in the normal BFU-E and CFU-E of the erythroid
definitive lineage.
Cell surface sialic acid, comprising a group of N- and
O-acyl as well as O-methyl and O-sulfate derivatives of
neuraminic acid, is linked differently to galactose or
N-acetylgalactosamine of glycoproteins and glycolipids
(Schauser, 1988). Sialic acid has been shown to participate
in several biological roles, such as the physicochemical
properties of glycoproteins, the transport of glycoproteins
inside the cell, the prevention of degradation of
glycoconjugates, the survival of circulating cells and has
been attributed a function in receptors for various
substances infectious agents such as viruses, mycoplasmas
and pseudomonas. In addition, several studies on
transformed cells also suggest that cell surface sialic acid
may play an important role in tumorigenicity and metastatic
potential via its different properties including strong
influences on platelet aggregation, tumor cell adhesion, the
ability of tumor cells to implant in various organs, tumor
cellular motility and deformability as well as invasiveness
and immunogenicity of tumor cells (Yogeeswaran, 1983).
There are several ways in which sialic acid may
contribute to the immunogenicity of normal cells and tumor
cells. For instance, sialic acid may act as an antigen by
itself; it may participate in the formation of antigenic

104
determinants along with other residues; or it can even mask
antigen-binding sites on proteins, lipids and carbohydrates.
The deglycosylation experiments performed by Endo F
digestion and periodate oxidation didn't result in the full
inhibition of the reactions to MAbs. This may be due to:
1) Endo F doesn't work as well with the carbohydrate
residues on cell surface glycoproteins as those on free
glycoproteins. 2) The sublethal dosages of sodium-M-
periodate, i.e., 2.5mM for BM2/C3A cells, 1.25mM for 6C2 and
MSB1 cells, are at a much lower concentration compared to
the amount (10-50mM) was given to other cell types (Stein
and Goldenberg, 1988; Mandeville et al., 1987).
It was rather frustrating that these markers could not
be precipitated by the MAbs at this point. This could be
due to many factors, such as the alteration of the secondary
and/or tertiary structure of the antigenic determinants by
detergent solubilization, the decrease of binding affinity
of MAbs by inappropriate pH in the lysis buffer and the
interference of the electrophoresis pattern of specific
immunoprecipitates by the nonspecifically bound cell lysates
and the heavy chains of MAbs etc.

CHAPTER 5
CONCLUDING REMARKS
The major achievement in my project has been the
generation of MAbs which are either specific for
transformation-associated antigens or normal differentiation
markers on avian hematopoietic cells. 1) MAbs 1H10-1F9,
2H1-2A10 and 3D7-1C9 define transformation-associated
antigens which are present preferentially on the
AMV-transformed BM2 cell lines rather than on normal
monocytic cells. The expression of these antigens is
diminished after BM2 cells are induced to differentiate by
PMA. 2) MAbs 1F7-1A3, 3F6-1E7 and 2E10-1E10 exhibit
different specificities for normal differentiation markers.
MAb 1F7-1A3 reacts with normal BFU-E and CFU-E, the AEV-
transformed yolk sac cells and MSB1 cells. MAb 3F6-1E7
probably is specific for the stem cell and precursor cell
populations during embryogenesis. In addition, the marker
recognized by MAb 3F6-1E7 is also expressed on some
transformed cells such as the AEV-transformed yolk sac
cells, BM2 cell lines and MSB1 cells. MAb 2E10-1E10 defines
a differentiation marker present on proliferating
hematopoietic cells rather than terminally differentiated
105

106
cells, however, it starts appearing after the 4th day of
embryogenesis and persists thereafter.
The specificities of these MAbs for avian hematopoietic
cells is illustrated in Figure 5-1.
The biochemical characterization of the nature of these
differentiation markers was conducted by enzymatic digestion
and chemical deglycosylation. The binding of MAbs was
knocked out by trypsinization, which indicates that
proteins are the structural backbones of these markers. The
removal of sialic acid by neuraminidase digestion as well as
the deglycosylation with either periodate oxidation or
Endo F could also reduce the binding specificities of MAbs
1H10-1F9, 2H1-2A10, 3D7-1C9 and 2E10-1E10 to different
degrees. This suggests that the differentiation markers
recognized by these MAbs are glycoproteins and that sialic
acids with or without carbohydrates are contributing to the
conformation of the antigenic determinants. In contrast,
the binding specificities of MAbs 1F7-1A3 and 3F6-1E7 were
not diminished either by deglycosylation or by neuraminidase
treatment suggesting that a protein moiety contains the
antigenic determinants for 1F7-1A3 and 3F6-1E7. However, it
is yet to be determined whether or not these proteins are
glycosylated.
These MAbs will be useful for the diagnosis of the
naturally occurring leukemia in the chicken. For instance,
monocytic leukemia cells can be detected by monocytic cell-

107
specific MAbs such as 1H10-1F9, 2H1-2A10 and 3D7-1C9, and
these MAbs provide us with the opportunity to test the
therapeutic possibility of utilizing MAb-immunoconjugates
for AMV-induced leukemia in vivo, whereas erythroleukemia
cells will be recognized by 2E10-1E10 and not by other MAbs.
Some important questions remain to be elucidated by
future investigators: 1) Is the expression of the normal
differentiation markers recognized by MAbs 1F7-1A3, 2E10-
1E10 and 3F6-1E7 essential for specific lineages and/or
stages within the hematopoitic differentiation pathway?
2) I have demonstrated that the expression of
transformation-associated antigens defined by MAbs 1H10-1F9,
2H1-2A10 and 3D7-1C9 is amplified in the AMV-transformed
cells. Are these antigens the integral part of, or are
merely associated with the myb-orchestrated transforming
process? 3) How is the expression of these markers
controlled? Is it due to the amplification at the
transcriptional and/or translational level, or is it induced
by posttranslational modifications of other differentiation
markers such as aberrant glycosylation and/or sialylation?
To answer the above questions, it will be necessary to
purify these differentiation markers followed by protein
sequencing and structural characterization of their
carbohydrate moieties by direct chemical methods such as NMR
spectroscopy, mass spectrometry and methylation analysis.
Once the protein sequences and the carbohydrate structures

Figure 5-1. Diagram of the specificities of MAbs for the hematopoietic cells.
3D7-1C9 is specific for the cells of monocytic lineage, 1F7-1A3 recognizes BFU-E
and CFU-E, 2E10-1E10 detects a differentiation marker expressed on the
proliferating hematopoietic cells after the 4th day of embryogenesis, whereas
3F6-1E7 probably reacts with the embryonic stem cell and precursor cell
populations.

Self-renewal
Pluripotent
Stem Cell
â–² 3F6-1E7
• 3D7-1C9
â–  1F7-1A3
* 2E10-1E10
1 A ' â–² t .
Erythroid Myeloid Lymphoid
Precursor Precursor Precursor
â–¼ â–  *
BFU-E GM-CFC Eo-CFC MEG-CFC
^ â– *
CFU-E
^ it F ^ #* ^
Erythroblast Myeloblast Monoblast Eosinophilic Megakaryoblast
Myeloblast (Thromboblast) Lymphoblast Lymphoblast
I Í I Í I I Í
» u:i T
Peripheral
Erythrocyte Myelocyte
Monocyte® Eosinophil
Platelets T Cell
B Cell
Blood
Granulocyte
Í
(Thrombocytes)
1
Tissue
Macrophage®
Plasma
Cell
109

110
have been determined, their expression at the
transcriptional, translational and posttranslational levels
in normal versus tumor cells can be evaluated. The quest to
understand tumor cells is definitely linked to our desire to
know more precisely how the cell is able to direct a panel
of cellular genes for normal proliferation and
differentiation. The data presented in this dissertation
represent for the first time a systematic attempt to compare
how differentiation markers are expressed during
hematopoietic development and how they may vary during the
"chaos" provoked by oncoviruses.
" We cannot always assault the present problems of
biology at will; we must remain alert to nature's clues and
seize on them whenever and whereever they may appear—even
if it be in a chicken."
—J.M. Bishop, Amer. Zool., 29 (1989), p665

REFERENCES
Abe, K., McKibbin, J.M. and Hakomori, S. (1983) J. Biol.
Chem. 258, 11793-11797.
Akiyama, Y. and Kato, S. (1974) Bikens J., 17, 105-116.
Beug, H., Doederlein, G., Freudenstein, C. and Graf, T.
(1982) J. Cell. Physiol. Suppl., 1, 195-207.
Beug, H. and Hayman, M.J. (1984) Cell, 36, 963-972.
Beug, H., von Kirchbach, A., Doederlein, G., Conscience,
J.D. and Graf, T. (1979) Cell, 18, 375-390.
Bishop, J.M. (1983) Ann. Rev. Biochem., 52, 301-354.
Bister, K. and Jansen H.W. (1986) Adv. Cancer Res., 47, 99-
187.
Bloch, A. (1984) Cancer Treat. Rep., 68,199-205.
Boettiger, D. and Durban, E.M. (1984) J. Virol., 49, 841-
847.
Boucher, P., Koning, A. and Privalsky, M.L. (1988) J.
Virol., 62, 534-544.
Boyle, W.J., Laxnpert, M.A. , Li, A.C. and Baluda, M.A. (1985)
Mol. Cell Biol., 5, 3017-3023.
Braylan, R.C., Benson, N.A., Nourse, V. and Kruth, H.S.
(1982) Cytometry, 2, 337-343.
Bruns, G.A.P. and Ingram, W.M. (1973) Phil. Trans. Royal
Soc. London, 266, 227-305.
Campbell, F. (1967) J. Morph., 103, 405-440.
Cordell, J.L., Falini, B., Ecber, W.N., Ghosh, A.K.,
Abdulaziz, Z., MacDonald, S., Pulford, K.A.F., Stein, H. and
Mason, D.Y. (1984) J. Immunochem. Cytochem., 32, 219-229.
Cormier, F. and Dieterlen-Liévre, F. (1988) Develop., 102,
279-285.
Ill

112
Cruikshank, W.W., Berman, J.S., Theodore, A.C., Bernardo, J.
and Center, D.M. (1987) J. Immunol., 138, 3817-3823.
Dieterlen-Liévre, F. (1988) In Vertebrate blood cells,
Rowley, A.F. and Ratcliffe, N.A. (ed.), Cambridge University
Press, London, pp. 257-336.
Dodge, W.H. and Hansell, C.C. (1978) Exp. Hemat., 6, 661-
672.
Dodge W.H. and Moscovici, C. (1973) J. Cell. Physiol., 81,
371-386.
Dodge, W.H. and Sharma, S. (1985) J. Cell. Physiol., 123,
264-268.
Dodge, W.H., Silva, R.F. and Moscovici, C. (1975) J. Cell.
Physiol., 85, 25-30.
Downward, J., Yarden, Y., Mayes, E., Scrace, G., Stockwell,
P., Ullrich, A., Schlessinger, J. and Waterfield, M.D.
(1984) Nature, 307, 521-527.
Durban, E.M. and Boettiger, D. (1981) Proc. Natl. Acad. Sci.
USA, 78, 3600-3604.
Enrietto, P.J. and Wyke J. A. (1983) Adv. Cancer Res., 39,
269-314.
Evans, A.E., Gerson, J. and Schnanfer, L. (1976) Natl.
Cancer Inst. Monogr., 44, 49-54.
Fenner, F. (1976) Intervirol., 7, 1-115.
Fresney, R.I. (1985) Anticancer Res., 5, 111-130.
Frykberg, L., Palmieri, S., Beug, H., Graf, T., Hayman, M.J.
and Vennstróm, B. (1983) Cell, 32, 227-238.
Fukuda, M. (1985) Biochim. Biophys. Acta, 780, 119-150.
Fung, Y.-K.T., Lewis, W.G., Crittenden, L.B. and Kung, H.-
J. (1985) Cell, 33, 357-368.
Gandrillon, 0., Jurdic, P., Pain, B., Desboies, C., Madjar,
J.J., Moscovici, M.G., Moscovici, C. and Samarut, J. (1989)
Cell, 58, 115-121.
Gazzolo, L., Moscovici, C., Moscovici, M.G. and Samarut, J.
(1979) Cell, 16, 627-638.

113
Gazzolo, L., Samarut, J., Bouabdelli, M. and Blanchet, J.P.
(1980) Cell, 22, 683-691.
Gilmore, T., DeClue, J.E. and Martin, G.S. (1985) Cell, 40,
609-618.
Gootwine, E., Webb, C.G. and Sachs, L. (1982) Nature, 299,
63-65.
Graf, T. and Beug, H. (1978) Biochem. Biophys. Acta, 516,
269-299.
Graf, T. and Stéhelin, D. (1982) Biochim. Biophys. Acta,
651, 245-271.
Graf, T., von Kirchbach, A. and Beug, H. (1981) Exp. Cell
Res., 131, 331-343.
Gregory, C.J. and Eaves, A.C. (1978) Blood, 51, 527-537.
Hanafusa, H. (1977) In Comprehensive virology, 10,
Franenkel-Conrat, H. and Wagner R. (ed.), Plenum Press, New
York, pp. 401-483.
Hasek, M. and Hraba, T. (1955) Nature, 175, 764-765.
Hayman, M.J. and Beug, H. (1984) Nature, 309, 460-462.
Hayman, M.J., Beug, H. and Savin, K.W. (1982) J. Cell.
Biochem., 18, 351-362.
Hayward, W.S., Neel, B.G. and Astrin, S.M. (1981) Nature,
290, 475-479.
Ingram, V.M. (1972) Nature, 235, 338-339.
Jurdic, P., Bouabdelli, M., Moscovici, M.G. and Moscovici,
C. (1985) Virology, 144, 73-79,
Jurdic, P., Moscovici, C., Pessano, S., Bottero, L. and
Rovera, G. (1982) J. Cell. Physiol. Suppl., 2, 85-95.
Kahn, P., Frykberg, L., Brady, C., Stanley, II, Beug, H.,
Vennstróm, B. and Graf, T. (1986) Cell, 45, 349-356.
Klempnauer, K.-H., Symonds, G., Evan, G.I. and Bishop, J.M.
(1984) Cell, 37, 537-547.
Kohler, G. and Milstein, C. (1975) Nature, 256, 495-497.
Kornfeld, S. Beug, H., Doederlein, G. and Graf, T. (1983)
Exp. Cell Res., 143, 383-394.

114
Laemmli, U.K. (1970) Nature, 227, 680-685.
Leutz, A., Beug, H. and Graf, T. (1984) EMBO J., 3, 3191-
3197.
Lim, R., Hicklin, D.J., Ryken, T.C., Han, X-M., Liu, K-N.,
Miller, J.F. and Baggenstoss, B.H. (1986) Cancer Res. 46,
5241-5247.
Mandeville, R., Dumas, F., Amarouch, A., Sidrac-Ghali, S.,
Walker, M.C., Zelechowska, M., Adjukovic, I. and Grouix, B.
(1987) Hybridoma, 6, 441-451.
Metcalf, D. and Moore, M.A.S. (1971) In Frontiers of
biology, 24, Neuberger, A. and Tatum, E.L. (ed.), North-
Holland Publishing Company, Amsterdam, pp. 1-9.
Miller, M.M., Goto, R. and Clark, D. (1982) Dev. Biol., 94,
400-414.
Moscovici, C. and Gazzolo, L. (1982) Adv. Viral Oncol., 1,
83-106.
Moscovici, C., Gazzolo, L. and Moscovici, M.G. (1975)
Virology, 68, 173-181.
Moscovici, C. and Vogt, P.K. (1968) Virology, 35, 487-497.
Moscovici, C., Zeller, N. and Moscovici, M.G. (1982) In
Expression of differentiated functions in cancer cells,
Revoltella, R.F., Basilico, C., Gallo, R.C., Potori, G.M.
Rovera, G. and Subak-Sharpe, J.H. (ed.), Raven Press, New
York, pp. 435-449.
Moscovici, M.G., Jurdic, P., Samarut, J., Gazzolo, L. Mura,
C.V. and Moscovici, C. (1983) Virology, 129, 65-78.
Moscovici, M.G. and Moscovici, C. (1980) In In vivo and in
vitro erythropoiesis: The friend system, Rossi, G.B. (ed.),
Elsevierl North-Holland Biochemical Press, Amsterdam, pp.
503-514.
Moscovici, M.G. and Moscovici, C. (1983) Proc. Natl. Acad.
Sci. USA, 80, 1421-1425.
Nelson, C.H., Allison, J.P., Kline, K. and Sanders, B.G.
(1982) Cancer Res., 42, 4625-4630.
Oakley, B.R., Kirsch, D.R. and Morris, N.R. (1980) Anal.
Biochem., 105, 361-363.
Old, L.J. (1981) Cancer Res., 41, 361-375.

115
Payne, G.S., Bishop, J.M. and Varmus, H.S. (1982) Nature,
295, 209-213.
Payne, L.N. and Powell, P.C. (1984) In Physiology and
biochemistry of the domestic fowl. Freeman, B.M. (ed.),
Academic Press, Inc., London, pp. 277-321.
Pessano, S., Gazzolo, L. and Moscovici, C. (1979)
Microbiologica, 2, 379-392.
Pierce, G.B., Aguilar, D., Hood, G. and Wells, R.S. (1984)
Cancer Res., 44, 3987-3996.
Pierce, G.B., Lewis, S.H., Miller, G., Moritz, E. and
Miller, P. (1979) Proc. Natl. Acad. Sci. USA, 76, 6649-665.
Podesta, A.H., Mullins, J., Pierce, G.B. and Wells, R.S.
(1984) Proc. Natl. Acad. Sci. USA, 81, 7608-7611.
Ponder, B.A. and Wilkinson, M.M. (1981) J. Histochem.
Cytochem., 29, 981-984.
Privalsky, M.L. and Bishop, J.M. (1984) Virology, 135, 356-
368.
Privalsky, M.L., Sealy, L, Bishop, J.M., McGrath, J.P. and
Levinson, A.D. (1983) Cell, 32, 1257-1267.
Sachs, L. (1986) Sci. Am. 254, 30-37.
Samarut, J., Blanchet, J.P., Godet, J. and Nigon, V. (1976)
Ann. Inst. Pasteur (Paris), 127C, 873-888.
Samarut, J., Blanche, J.P. and Nigon, V. (1979) Dev. Biol.,
72, 155-166.
Samarut, J. and Bouabdelli, M. (1980) J. Cell. Physiol.,
105, 553-565.
Samarut, J. and Gazzolo, L. (1982) Cell, 28, 921-929.
Sanders, B.G., Allison, J.P., and Kline, K. (1982) Cancer
Res., 42, 4532-4539.
Sap, J., Munoz, A., Damm, K., Goldberg, Y., Ghysdael, J.,
Leutz, A., Beug, H. and Vennstróm, B. (1986) Nature, 324,
635-640.
Schauer, R. (1988) In The molecular immunology of complex
carbohydrates. Wu, A.M. (ed.), Plenum Press, New York, pp.
47-72.

116
Schmidt, J.A., Marshall, J., Hayman, M.J., Doderlein, G. and
Beug, H. (1986) Leuk. Res., 10, 257-272.
Sealy, L., Privalsky, M.L., Moscovici, M.G., Moscovici, C.
and Bishop, J.M., (1983) Virology, 130, 155-178.
Shih, C.-K., Linial, M., Goodenow, M.M. and Hayward, W.S.
(1984) Proc. Natl. Acad. USA, 81, 4697-4701.
Shinitzky, M. (1984) Biochem. Biophys. Acta, 738, 251-261.
Silva, R.F., Dodge, W.H. and Moscovici, C. (1976) J. Cell.
Physiol., 83, 187-192.
Sorrell, J.M. and Weiss, L. (1980) Anat. Res., 197, 1-19.
Sporn, M.B., Roberts, A.B., Wakefield, L.M. and Assoian,
R.K. (1986) Science, 233, 532-534.
Stanbridge, E.J. Der, C.J., Doersen, C-J., Mishimi, R.Y.,
Puhl, D.M., Weissman, B.E. and Wilkinson, J.E. (1982)
Science, 215, 252-259.
Stein, R. and Goldenberg D.M. (1988) Hybridoma, 7, 555-567.
Symonds, G., Klempnauer, K.-H., Evan, G.I. and Bishop, J.M.
(1984) Mol. Cell. Biol., 4, 2587-2593.
Szenberg, A. (1977) Adv. Exp. Med. Biol., 88, 3-11.
Till, J.E. and McCulloch, E.A. (1961) Radiat. Res., 14, 213-
222.
Trembicki, K.A. and Dietert, R.R. (1985) J. Exp. Zool., 235,
127-134.
Vogt, P.K. (1969) In Fundamental techniques in vorology,
Habel, K. and Salzman, N.P. (ed.), Academic Press, New York,
pp. 198-211.
Wallach, D.F.H. (1968) Proc. Natl. Acad. Sci. USA, 61, 868-
874.
Weston, K. and Bishop, J.M. (1989) Cell, 58, 85-93.
Yamamoto, T., Hihara, H., Nishida, T., Kawai, S. and
Toyoshima, K. (1983) Cell, 34, 225-232.
Yogeeswaren, G. (1983) Adv. Cancer Res., 38, 289-350.

117
Zenke, M., Kahn, P. Disela, C., Vennstróm, B., Leutz, A.,
Keegan, K., Hayman, M.J., Choi, H.-R., Yew, N. Engel, J.D.
and Beug, H. (1988) Cell, 52, 107-119.

BIOGRAPHICAL SKETCH
Juinn-Lin G. Liu was born in Kao-Shong, Taiwan, on
October 23, 1960. He grew up with cartoons, baseball and
traffic. He went to the Department of Veterinary Medicine,
National Taiwan University, in September, 1978. During his
fifth year as an intern, he discovered two interesting cases
which were never been found in Taiwan before. One was a
female Akita dog which suffered from congenital intestinal
lymphangiectasia; the other case was a male Pit bulldog with
the TVT (transmissible venereal tumor) developed on the
skin instead of in the genital regions. It was possibly due
to been bitten by another fight dog which had the
involvement of the TVT in the mouth cavity. Shortly
after he received his D.V.M. degree in June, 1983, there was
another female Pit bulldog had exactly the same skin-type
TVT. Before he had any opportunity to do further
investigation, he was drafted by the Chinese Marine Corps to
fulfill his two-year military service and he was overwhelmed
with military and physical training as well as brain wash.
Somehow he managed to remain intact with the exception that
he became allergic to beer and then came to the University
of Florida to pursue his Ph.D. degree in the Department of
Pathology and Laboratory Medicine, in August, 1985 until
118

119
now. He has accepted a postdoctoral position in the
Department of Molecular Biology and Microbiology, School of
Medicine, Case Western Reserve University, Cleveland, Ohio.
He is scheduled to "launch" in January, 1990 despite the
forecast of one foot of snow waiting for him. Yes! Your
guess is as good as mine, he has never seen snow before.

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
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Carlo Moscovici, Chair
Professor of Pathology and
Laboratory Medicine
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Vl. tOV^QuUJU y
mT Gibvannella Moscovici, Cochair
Assistant Professor of Pathology
and Laboratory Medicine
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
—
Paul A. Klein
Professor of Pathology and
Laboratory Medicine
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Edward K. Wakeland
Associate Professor of Pathology
and Laboratory Medicine

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
__Amm©n-B.—Peck
Associate Professor of Pathology
and Laboratory Medicine
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
and Cell Biology
This dissertation was submitted to the Graduate Faculty
of the College of Medicine and to the Gradute School and was
accepted as partial fulfillment of the requirements for the
degree of Doctor of Philosophy.
May 1990
Dean, College of Medicine
Dean, Gradute School

UNIVERSITY OF FLORIDA
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UNIVERSITY OF FLORIDA
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