Title: Isolation, characterization, and mechanism of action of a complement inhibitor derived from Erhlich ascites tumor cells /
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Title: Isolation, characterization, and mechanism of action of a complement inhibitor derived from Erhlich ascites tumor cells /
Physical Description: x, 155 leaves : ill. ; 28cm.
Language: English
Creator: Renk, Clifford Michael, 1948-
Publication Date: 1975
Copyright Date: 1975
 Subjects
Subject: Immune complexes   ( lcsh )
Antigens   ( lcsh )
Complement (Immunology)   ( lcsh )
Microbiology thesis Ph. D
Dissertations, Academic -- Microbiology -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
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Thesis: Thesis--University of Florida.
Bibliography: Bibliography: leaves 148-154.
Statement of Responsibility: by Clifford Michael Renk.
General Note: Typescript.
General Note: Vita.
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Bibliographic ID: UF00099400
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: alephbibnum - 000167709
oclc - 02864509
notis - AAT4100

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ISOLATION, CHARACTERIZATION, AND MECHANISM OF ACTION
OF A COMPLEMENT INHIBITOR DERIVED FROM
EHRLICH ASCITES TUMOR CELLS




By





Clifford Michael Renk


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


UNIVERSITY OF FLORIDA
1975















ACKNOWLEDGEMENTS


The author wishes to express his appreciation to Dr.

Edward M. Hoffmann, chairman of his supervisory committee,

for his concern, encouragement, guidance, suggestions, and

criticisms in the conduct of the research and in the

preparation of the manuscript.

He would also like to thank the members of his

committee, Dr. Paul A. Klein, Dr. James F. Preston, Dr.

L. William Clem, and Dr. Arnold S. Bleiweis, for their

advice throughout the conduct of the research and in the

preparation of the manuscript. The author would also like

to thank Dr. Lonnie O. Ingram for his concern and advice

during this investigation. The author wishes to express

his appreciation to Joanne Hall for typing the manuscript.

Finally, the author wishes to express his deepest

gratitude to his wife, Patti, for her encouragement,

patience, and understanding.


















TABLE OF CONTENTS





ACKNOWLEDGEMENTS ................................ ii

LIST OF TABLES .................................. iv

LIST OF FIGURES ................................. v

GLOSSARY OF ABBREVIATIONS ....................... viii

ABSTRACT ........................................ ix

INTRODUCTION .................................... 1

MATERIALS AND METHODS .... ........................ 13

RESULTS ........................................ 33

DISCUSSION ...................................... 137

BIBLOGRAPHY ..................................... 148

BIOGRAPHICAL SKETCH ............................ 155















LIST OF TABLES


TABLE PAGE

1. Effect of EATC Extract on the Hemolytic
Susceptibility of Sheep E, EA, EAC1,
EAC14, EAC142, EAC1-3, EAC1-5, EAC1-6,
EAC1-7, and EAC1-9 ....................... 58

2. Comparison of the Relative Numbers of
Effective C1 Molecules Fixed by EA
Treated with EATC Extracts and by
Untreated EA .............................. 60

3. Effect of Phenol Extracts on the
Hemolytic_Susceptibility of EA, EAC1,
EAC4, EAC14, and EAC142 ....... .. ......... 99

4. Comparison of the Relative Numbers of
Effective C! Molecules Capable of
Transfer from EAC1 Treated with EATC
Phenol Extracts ........................... 101

5. Comparison of the Relative Numbers of
Effective Cl Molecules Inactivated in
the Fluid Phase and on EAC1 by EATC
Phenol Extracts ......................... 102

6. Inactivation of Guinea Pig Complement
Components in Whole Sera by EATC
Extracts ................................... 104

7. Release of Complement Inhibitory Substances
from EATC with time at 40C ............... 117

8. Extraction of Complement Inhibitory
Activity from Spleen, Liver, and EAT
Cells with Phenol ................ ........ 122

9. Inhibition of Complement Mediated Lysis
of EA by Phenol Extracts of Ascitic Fluid
from Ehrlich Ascites Tumor Bearing Mice .. 130

10. Extraction of Complement Inhibitory
Material from P815 and EL4 Tumor Cells ... 132

11. Inhibition of Cytoxicity of EATC with
Ribonuclease ............................. 133















LIST OF FIGURES


FIGURE PAGE

1. Inhibition of immune hemolysis by various
salt extracts of Ehrlich ascites tumor
cells ....................................... 35

2. Inhibition of immune hemolysis by EATC
extracts .................................... 37

3. Inhibition of guinea pig complement........ 40

4. Inhibition of rabbit complement ........... 42

5. Inhibition of mouse complement ............ 44

6. Inhibition of human complement ............ 46

7. Titration of adsorbed guinea pig complement
after treatment with EATC extracts.......... 49

8. Effect of EATC extracts on hemolytic
antibody titration........................ 51

9. Decay of EAC142 at 300 in the presence of
tumor cell extracts................ ......... 54

10. Effect of EATC extract on the stability
of the intermediate EAC14................. 57

11. Titration of residual C4 activity after
treatment with EACT and EACIC cells........ 62

12. Effect of EATC extracts on the generation
of SAC142 .................................. 65

13. Titration of Cl treated with EATC extracts
and with PBS ................................ 67

14. Titration of C2 treated with EATC extracts
and with PBS ................................ 69

15. Gel filtration of PBS extracts from EATC
on G-200 Sephadex ....... ............... ... 72

16. DEAE chromatography of the 20 percent
ammonium sulfate precipitated inhibitory
material from the EATC extracts .......... 75

v











17. Gel filtration of DEAE purified tumor cell
extract....................................... 77

18. Inactivation of EATC extract by trypsin
treatment................................... 80

19. Inactivation of EATC extract by protease..... 83

20. Inactivation of EATC extract by ribonuclease
A............................................. 85

21. Removal or inactivation of EATC extract
inhibitory material with streptomycin
sulfate........................................ 88

22. DEAE chromatography of PBS extract from
EATC ......................................... 90

23. Inhibition of immune hemolysis caused by
a phenol extract of the crude sodium
chloride extract from EATC..................... 95

24. Heat stability of the crude tumor cell
extract and the phenol extract at 56C........ 97

25. Precipitation of tumor cell extract by
human Clq...................................... 107

26. Inhibition of guinea pig complement lysis
of EA by an RNA preparation from E. coli..... 109

27. Inhibition of guinea pig complement lysis
of EA by yeast RNA............................ 111

28. Sucrose gradient centrifugation of phenol
extracted inhibitory material................ 114

29. Sucrose gradient centrifugation of E. coli
RNA. ........................................ . 116

30. Inhibition of immune hemolysis by dilutions
of PBS extracts obtained from EATC, liver,
and spleen cells............................. 120

31. Sucrose gradient centrifugation of complement
inhibitory material obtained by extracting
whole tumor cells with phenol................ 124

32. Sucrose gradient centrifugation of phenol
extract of normal mouse liver cells........... 126










33. Sucrose gradient centrifugation of phenol
extract of normal mouse spleen cells........ 128

34. Inhibition of EATC cytotoxicity by
EATC extracts................................ 136

















GLOSSARY OF ABBREVIATIONS


E: Erythrocyte

A: Antibody

C: Complement

Cl, C2---C9: Complement components. Horizontal bars
over complement components indicate that
the components in question are in the
biologically active state.

S: Single site of complement activation.

EAT: Ehrlich Ascites Tumor

EATC: Ehrlich Ascites Tumor Cells

RNA: Ribonucleic Acid









Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment
of the Requirements for the Degree of Doctor of Philosophy

ISOLATION, CHARACTERIZATION, AND MECHANISM OF ACTION
OF A COMPLEMENT INHIBITOR DERIVED FROM
EHRLICH ASCITES TUMOR CELLS

By

Clifford Michael Renk

June, 1975

Chairman: Edward M. Hoffmann
Major Department: Microbiology

Tumors possess specific surface antigens and tumor

specific antibodies can be shown to exist in many tumor

bearing hosts. However, antibodies arising in response to

the tumor frequently are noncytotoxic in the presence of

complement. Anticomplementary factors in the sera or on

the tumor cells may play a role in preventing complement

mediated lysis of some tumors. This investigation was

initiated to determine if a complement inhibitory substance

could be extracted from Ehrlich ascites tumor cells. The

anticomplementary effect of Ehrlich ascites tumor cell

extracts was investigated using a sheep erythrocyte target

cell assay procedure with known amounts of hemolytic anti-

bodies and various complement sources. Experiments were

conducted to define the site of action of the inhibitory

material. Partial purification of the inhibitory substance

was obtained using a combination of phenol extraction and

DEAE chromatography. Normal mouse cells were compared with

Ehrlich ascites tumor cells to determine if normal mouse

cells possess a complement inhibitory material. The










possibility is discussed that the possession of a cell

associated complement inhibitor could play a role in the

resistance of that tumor to immune rejection.
















INTRODUCTION


Complement is a term used to describe a system of

nine interacting serum proteins which are normally present

in serum as inactive precursors. The complement components

are designated Cl, C4, C2, C3, C5, C6, C7, C8, and C9, in

the order of their reactivity. It is now recognized that

the complement system may be activated by two different

pathways. The so-called classical pathway is initiated

by the interaction of antigen antibody complexes with Cl,

the first component of complement. As a result of the

interaction, Cl is converted to an active form, Cl, which

in turn activates and cleaves C4 and C2 (1,2,3). This

process generates a second complement complex with enzyme-

like activity; the C42 complex or classical C3 convertase.

The alternate pathway of complement action bypasses the

early complement components (Cl, C2, and C4) and the com-

plement sequence is initiated beginning at C3. The alter-

nate pathway is activated by a number of substances such

as inulin,bacterial lipopolysaccharide, zymosan, and a

factor derived from Cobra venom. Serum factors, other

than complement components, react with the foregoing sub-

stances which ultimately leads to the activation of C3

without Cl, C4, or C2 participation (4,5). The activation

of C3 by either pathway leads to cleavage and activation











of the terminal complement components C5, C6, C7, C8 and

*C9 (6). Products of the complement system formed in the

activation steps are able to mediate a number of biologi-

cal phenomena such as: increased vascular permeability,

leucocyte chemotaxis, enhanced phagocytosis and cell lysis.

The first component of complement is a complex of

three different proteins, Clq, Clr, and Cls which exists

as a macromolecular complex in vivo (7). Activation of

Cl follows the attachment of the component to various

substances via the Clq subunit. Antibody is probably the

most important activator of Cl. The binding site for Clq

on antibody is located on the Fc fragment of IgG and IgM.

The acceptor site for Cl is present on the monomer of the

immunoglobulin IgG (8). The binding of Cl to monomeric

IgG in solution is very unstable (8) and Augener suggests

that Cl binding is increased by aggregation (9). Borsos

and Rapp, however, showed that it takes a doublet of IgG to

initiate complement lysis on the cell surface (10). The

binding of antibody to antigen, however, may subsequently

reveal or alter additional Cl binding sites on the Fc

region so Cl then binds more efficiently to aggregated or

fixed immunoglobulin (11,12,13).

Binding of Cl usually leads to Cl activation although

both processes can be separated. Modification of trypto-

phan on IgG with 2-hydroxy-5-nitrobenzyl bromide leads to

Clq binding but does not activate Cl (14). The internal

activation of Cl occurs by a conformational change of Clq











which induces a change in Clr. Valet and Cooper have

shown that the proenzyme Clr is activated in the process

so that it acquires enzymatic activity which in turn

activates or converts Cls to Cis (15). Calcium ions are

required to hold the complex Clqrs together and these

components can be dissociated in EDTA solution (6,7).

Activation of Cl can also occur by its attachment to

various substances such as polyanions and certain lympho-

cyte and viral membranes. For example, Clq has been shown

to react with DNA and RNA (16,17), polyribonucleotides and

dextran sulfate (18, 19), and carrageenin (20). Yachnin

has reported that certain polynucleotides do not affect

complement hemolytic activity (21). C1 activation does

not always accompany binding of the component to some

substance. Carrageenin interacts with Cl but does not

lead to activation of the molecule since Borsos has shown

that there is no loss in serum C4 activity when carra-

geenin is added to serum (20). Ordinarily, attachment

of the Cl complex by Clq to immunoglobulin or other sub-

stances such as DNA initiates the activation of Clr and

ultimately Cls. Once Cl is activated, however, its

activity can be inhibited by a normal serum glycoprotein,

ClINH (22). C1INH has been shown to inhibit the hemo-

lytic and esterolytic activity of Cls (23,24). Cls, if it

is not inhibited by C1INH, in turn activates the next two

steps in the complement cascade.

The second phase of complement activation leads to











the formation of two enzymes, C3 convertase (C4b2a) and

C5 convertase (C4b2a3b). The formation of C42 enzyme is

mediated by the action of Cl. CT reacts with C4 and

cleaves it into two fragments, C4b, which can interact

with Cl, and C4a, which is released (3,25). Cls next

activates and splits C2 into C2a and C2b (26,27). Gigli

and Austen have reported that C4 is required for effective

activation of C2 by Cis (28,29). The activation process

then permits the completing of C4b and C2a to form the

enzyme C3 convertase.

The C42 complex brings about the enzymatic cleavage

of C3 into C3a and C3b (30). The C3a part of C3 has been

shown to possess anaphalatoxic and chemotactic activities

(31,32,33). The fragment C3b has the ability to bind to

membranes, and specific receptors for C3b have also been

shown to be present on erythrocytes, polymorphonuclear

leukocytes, platelets and on B lymphocytes. The binding

of C3b has been postulated to play a role in B cell

activation as well (34). There are two naturally occur-

ring inhibitors of the fragment C3b in serum. Tamura

and Nelson described a substance in guinea pig and rabbit

serum that could block C3b activity (35). This material

C3bINA, has also been found in human sera (36). Treat-

ment of cell bound C3b by C3bINA releases a fragment C3c

or B1A. A portion of C3 remaining on the cell surface

contains the D antigenic determinant of C3 or a2D (37).

The reaction of C3b with C3bINA prevents immune cytolysis










if C3b is on the cell membrane and also inhibits the

immune adherence reactivity and the enhanced phagocytic

properties associated with C3b. Human sera has also been

shown to contain an anaphylatoxin inactivator (AI) which

destroys the histamine releasing capacity of C3a as well

as C5a (38). The major fragment C3b reacts with the C42

complex to form a C5 convertase C423 which activates the

membrane attack system. C5 is cleaved in this reaction

into two fragments, C5b and C5a (39). C5a has been shown

to have chemotactic and anaphylatic activities (32,40,41).

The complement components C6 and C7 interact with C5b to

form a trimolecular complex that can bind to a membrane

site if present (42,43). This complex also has chemo-

tactic activity (44). C8 and C9 react sequentially with

the C567 complex to form a large multimolecular complex

that, if it is present on the cell membrane, can cause

membrane disruption (45). The sequential interaction of

the complement components on the cell surface causes

ultrastructural lesions to occur on the membrane that

can become evident after C5 addition (46,47). However,

studies of complement activity on artificial phospholipid

membranes indicate that the cell damage might occur through

alteration of the membrane lipid (48,49).

The second or alternate pathway of complement can be

activated by a munber of substances such as yeast cell walls,

inulin, agar, aggregates of immunoglobulins such as IgA, and

a factor from cobra venom (40,50,51). The alternate










pathway leads to the direct activation of C3 without the

participation of Cl, C2, or C4 and then merges with the

classical pathway to activate C5 through C9. The alternate

pathway is composed of a number of proteins, one of which

is C3b which interacts to split C3 into C3a and C3b. These

proteins are C3NeF or initiating factor, properdin, C3

proactivation on Factor B, C3 activator or Factor B and C3

PA Convertase or Factor D (52). These factors have been

shown to interact in a sequential fashion, upon activation,

to split C3 and generate the membrane attack sequence of

complement C5-C9) as well as the biological fragments C3a

and C5a.

The recognition of foreign substances in a host

usually leads to the elimination of these substances by

immune lymphocytes or phagocytic cells or by the concerted

effort of specific antibody and complement. If antibodies

are made against cell surface antigens, complement could

be activated to cause cell lysis or to augment cell medi-

ated immune functions through the activated components and

fragments. Tumors possess specific surface antigens, and

tumor specific antibodies can be shown to exist in animals

and humans with primary tumors (53,54,55,56). However,

antibodies arising in response to the tumor frequently are

noncytotoxic in the presence of complement (57,58). Tu-

mors may be categorized by their ability to react with

antibody and complement. Leukemias are characterized as

being susceptible to cytolysis by antibody and complement,










whereas sarcomas are resistant (59,60,61,62). The reason

for the low susceptibility of some tumor cells to the lytic

action of antibody and complement may be due to low anti-

gen density on the tumor cells, inaccessability of the

humoral factors to the tumor itself, stimulation of non-

complement fixing antibodies by the tumor or the presence

of complement inhibitors associated with the tumor.

The distribution of tumor antigens might be such that

the chances for complement mediated cytotoxicity are re-

mote. Linscott has shown that cells with a low number of

antigen sites were not readily susceptible to immune hemo-

lysis even when excess antibody was present (63). This

idea is reasonable in view of the report that it takes the

random association of about 1000 IgG molecules to generate

a doublet needed for complement activation (64,9). How-

ever, for mouse leukemia cells, Baker has reported that it

takes approximately 1200 IgM molecules to generate an

active lytic site which should be more than sufficient to

cause lysis if one accepts the sheep E model (65).

Although the density of tumor antigens on the cell

surface plays an important role with regard to the effec-

tiveness of cytotoxic antibody and complement, the exposure

of the tumor cells to these latter agents is also critical.

Serum glycoproteins may form a mucoid barrier that prevents

recognition or availability of tumor antigens to antibody

and complement (66). For example, plasma from patients

immunized with specific tumor antigens was found to be










cytotoxic only when it was injected directly into sub-

cutaneous cancer lesions (67).

The type of antibody made in response to a tumor can

vary. Mice have been shown to possess five immunoglobulin

classes: IgM, IgA, IgG, IgG2a, IgG2b (68). Only the IgM

and IgG2 fractions have been shown to fix complement (69,

70). Ehrlich ascites tumor cells have been shown to possess

bound immunoglobulin in both normal and irradiated C3H mice

(71). We have also demonstrated that Ehrlich ascites tumor

cells taken from AKR and ICR mice have bound immunoglobulin

on their surface (72). Immunoglobulins of IgG2 complement

fixing class have been eluted from chemically induced

tumors (73). Hartveit has reported that Ehrlich ascites

and Bergen A4 ascites tumor cells, supposedly sensitized

with antibody, lysed in the presence of fresh human sera

in vitro, although fixation of natural antibodies to tumor

cells in human sera with the resultant fixation of comple-

ment was not ruled out (74). Another report has shown

that the availability of complement may be critical since

tumor cells sensitized with specific antibody are sensitive

to lysis by complement in vitro, but may not be lysed when

placed in perfusion chambers in vivo (75). If specific

complement fixing antibodies are present on tumor cells,

why then does the host fail to lyse these cells in vivo?

Work by Ohanian et al. has shown that tumor cells

resist lysis even when many Cl molecules have fixed to

their surfaces(76). In this report, there was a difference










in the susceptibility of two cell lines of rat hepatoma

to complement mediated cytotoxcity when the different cell

types were reacted with equivalent amounts of complement

components. Cytotoxcity of Maloney virus induced tumors

by antibody and complement has been shown to be confined

to the G phase of cell growth even though antibody and

complement could fix to the cells throughout the cell

cycle (77,78). Lerner (77) suggests that there might be

some cell cycle changes in the membrane that render it

resistant to cytotoxicity or that the ability of the cell

to repair damage to the membrane may differ during the

cycle. The failure of some tumor cells to be lysed by

antibody and complement, therefore, might not be due simply

to the unavailability of complement fixing immunoglobulins

on the cells.

Anticomplementary factors in the sera of tumor

bearers or the tumor itself may play a significant role

in preventing complement mediated lysis of some tumors.

Evidence that anticomplementary substances are present in

sera has been presented in several laboratories. A factor

isolated from human and guinea pig sera has been shown to

inhibit the sheep EAC142 intermediate (79). In vitro lysis

of mouse tumor cells by peritoneal fluid can be inhibited

by normal mouse serum (80). Serum from tumor bearing mice

can inhibit lysis of Ehrlich ascites tumor cells by rabbit

antibody and guinea pig complement (81). Likewise, lysis

of Ehrlich ascites tumor cells by human sera can be inhibited










by ascites fluid (82). Recently, Cl and C3 inactivators

have been found in mouse sera as well (83). Dauphinee et

al. reported that lysosomal extracts from tumor cells were

capable of decreasing the ability of alloantibodies to

mediate complement-dependent cytoxicity. The alloanti-

bodies were unaffected by the lysosomal extract in blocking

specific killing by alloimmune lymphocytes (84). Cell free

Ehrlich ascites fluid has been reported to contain a factor

or factors that are capable of blocking immune adherence

produced by Ehrlich cells by anti Ehrlich sera. This

blocking factor was not tumor specific and was not correlated

with the antibody or antibody-antigen complex blocking

factors that have been reported (85).

The resistance of tumor cells to the lytic action of

antibody and complement might also be due to the presence

of a complement inhibitory substance on their surface as

well as in serum. There are differences in the sensitivity

of red cells from various species to the lytic action of

complement (63). Mollison has shown that human erythrocytes

are resistant to the action of complement when coated with

specific antibody (86). Hoffmann has shown that factors

derived from human, rabbit, and guinea pig erythrocytes

can inhibit the cytotoxic action of guinea pig and human

complement by interfering with C3 convertase (87,88,89).

Osther and Linnemann have shown that Cl inactivator is

present on human tumor cells in vivo (90). Klein,

Harris, and coworkers have reported that Ehrlich ascites











cells might elaborate a factor which makes the cells

resistant to cytotoxicity. They also showed in cell

fusion studies that the characteristic was genetically

controlled and dominant (91).

The relative susceptibility of a tumor to the action

of complement might be due, in part, to the association of

a complement inhibitor with that tumor. The association of

a complement inhibitory substance would also lower the cell

mediated responses that are complement dependent such as

increased phagocytosis and chemotoxis of leukocytes. A

tumor cell associated complement inhibitor might correlate

with the persistence of certain tumors.

This investigation was initiated in an attempt to

answer the question, "Do tumor cells possess a factor or

factors that can interfere with complement mediated cyto-

toxicity?" A sheep erythrocyte target cell assay procedure

was utilized using known amounts of hemolytic antibodies and

various complement sources. In this way, anticomplementary

effects would be easily detected. In subsequent studies

a tumor cell target system was utilized.

Ehrlich ascites tumor cells were chosen as a model

since they are easily obtainable and growth is not restricted

to a particular mouse strain (91). Extracts from Ehrlich

ascites tumor cells have been found to inhibit complement

mediated lysis of sensitized sheep red blood cells. Experi-

ments were conducted to define the site of action of the

inhibitory material, and partial purification of the






12


inhibitory material has been obtained. Normal mouse cells

were compared with Ehrlich ascites tumor cells to determine

if normal mouse cells possess a complement inhibitory

material. The possession of cell associated complement

inhibitory substance could have a relationship with

persistence of that tumor in a given host.















MATERIALS AND METHODS


Solutions

Stock Veronal buffered saline (Stock VBS). A stock

five times concentrated sodium chloride-Veronal buffer

solution was prepared according to the method described

by Mayer (92).

Veronal buffer with CaCl,, and MqCl2 (VB++). This

solution was prepared by mixing 100 ml of Stock VBS, 2.5

ml of 0.2M MgCl2, 2.5 ml of 0.03M CaCl2 with enough dis-

tilled water to bring the volume to 500 ml.

Gelatin Veronal buffer (GVB). This solution was

prepared by mixing 100 ml of Stock VBS and 25 ml of 2

percent (w/v) gelatin with enough distilled water to

bring the volume to 500 ml.

Gelatin Veronal buffer with CaC1l and MgCl2 (GVB++.

This solution was prepared by mixing 100 ml of Stock VBS,

25 ml of 2 percent gelatin, 2.5 ml of 0.2M MgCl2, 2.5 ml

of 0.03M CaC12 with enough distilled water to bring the

volume to 500 ml.

Gelatin Veronal buffer with double concentrations of

gelatin, CaCl, and MgC12(2XGVB++). 2XGVB++ was prepared

by mixing 100 ml of Stock VBS, 50 ml of 2 percent gelatin,

5 ml of 0.2M MgCl2, 5 ml of 0.03M CaCI2 with distilled











water to bring the volume to 500 ml.

Dextrose Gelatin Veronal buffer (DGVB ). DGVB+

was prepared by mixing equal volumes of 5 percent (w/v)

D-Glucose and 2XGVB++

0.04M EDTA-GVB This solution was prepared by

mixing equal volumes of 0.08M isotonic stock trisodium

ethylenediaminetetraacetate (EDTA) solution (pHl 7.5) and

GVB.

Phosphate buffered saline (PBS). Sodium chloride

solution (3M) was diluted with 0.005M, pH 7.5 potassium

phosphate buffer. The solution was adjusted to an ionic

strength of 0.15 with water. A conductivity bridge

(Model RC 16 B2) was used to measure the electrical con-

ductance of the solutions and ionic strength was estimated

by comparison with conductance values from a standard

sodium chloride calibration curve.

Complement, Human (HuC). Fresh human blood was

obtained from the Gainesville Plasma Corporation,

Gainesville, Florida. The blood was allowed to clot at

room temperature for about 60 min, and the serum was

separated by centrifugation at 500G at 0C. The sera

were collected and stored at -700C.

Complement, Rabbit (RC). Fresh rabbit blood was

obtained from New Zealand White strain animals by cardiac

puncture. The blood was allowed to clot at room

temperature for about 30 min and the serum was separated

at 500G at 00. The sera were collected and stored at -700C.










Complement, Mouse (MC). Mouse blood was obtained

by puncture of the retroorbital socket with a pasteur

pipette. The blood was transferred to test tubes and

allowed to clot at 0C. The sera were collected and

stored at -700C.

Complement Components. Purified guinea pig Cl, C2,

and C4 were prepared according to Nelson et al. (93) and

Ruddy and Austen (36,94). Purified guinea pig C3, C5, C6,

C7, C8, and C9 were purchased from Cordis Laboratories

(Miami, Florida). Purified Human Clq was prepared by

method of Yonemasu and Stroud (95).

Erythrocytes. Sheep blood was taken by venipuncture

from a single animal maintained at the Animal Research

Laboratory of the J. Hillis Miller Health Center (Gaines-

ville, Florida). One volume of blood was mixed with an

equal volume of sterile modified Alsever's solution (92),

and the blood was stored at 4C for up to 3 weeks.

Antibody sensitized sheep E (EA). Rabbit anti-sheep

erythrocyte (E) stromata was obtained from Cordis

Laboratories (Miami, Florida). Sensitization was performed

as recommended by the manufacturer.

Complement component intermediates (cell intermediates).

Sheep E in various states of complement component fixation

were used in this study. All complement components listed

are guinea pig components unless indicated otherwise.

EAC1, EAC4, EACH4, and EAC142 were prepared by the methods

described by Borsos and Rapp (96). EAC14235 and EAC142356










were prepared by the method described by Hoffmann (88).

The intermediate EAC1Hu423567 was purchased from Cordis

Laboratories (Miami, Florida). EAC1Hu42356789 was pre-

pared by mixing limiting amounts of C8 and C9 with

EAC1 Hu423567.
Hu
Animals. Male ICR mice were purchased from Flow

Laboratories. Male DBA/2J and C57B1/6J were purchased

from Jackson Laboratories (Bar Harbor, Maine). Male,

New Zealand White rabbits were obtained from Animal

Research Laboratories of the J. Hillis Miller Health

Center (Gainesville, Florida).

Tumor Cells. Ehrlich ascites tumor cells (EATC)

were obtained from Dr. Paul Klein (University of Florida,

Department of Pathology) and maintained by weekly passage

of 106 cells into ICR mice. P815 and El-4 tumor cells

were also obtained from Dr. Paul Klein.

Preparation of EATC Extracts. Ehrlich ascites tumor

cells were harvested from the peritoneal cavities of mice

seven days after inoculation with tumor cells. The cells

were washed with PBS at 50G for 10 min until no red cells

were detectable. A crude membrane fraction of EATC was

prepared by freeze-thawing the cells in 10 volumes of

distilled water. The insoluble portion was extensively

washed with ice cold distilled water until the supernate

was clear. The insoluble portion (1000G, 10 min pellet)

was then used as a crude membrane fraction.

Three different methods of extraction were used.











For the first method, the crude membrane fraction of EATC

was suspended in an equal volume of 0.005M potassium

phosphate, pH 7.5 and extracted with n-butanol using the

method described by Hoffmann for extraction of human red

cell stromata (87). The second method consisted of

extraction of crude EATC cell membranes three times with

0.5M sodium chloride buffered at pH 7.5 with 0.005M

potassium phosphate according to the methods that have

been previously described (87). The third method involved

extraction of intact EATC. Washed packed tumor cells were

shaken slowly overnight on Burrell Wrist Action Shaker

(Burrell Corporation, Pittsburgh, Pennsylvania) at 40C

with 3 volumes of various concentrations of sodium

chloride buffered with 0.005M potassium phosphate at pH

7.5. The cell suspensions were centrifuged first at 500G

for 10 min to sediment the cells and then at 10,000G for

30 min. The supernates were dialyzed against PBS overnight

at 40C.

Inhibition of immune hemolysis by cell extracts.

Extracts from Ehrlich ascites tumor cells and from other

sources were tested for the ability to inhibit whole

complement activity. Extracts were serially diluted in

PBS, and 0.2 ml of each dilution was added to a series of

test tubes to which 0.2 ml of EA, at a concentration of

1 X 108/ml, and 0.6 ml of complement at a concentration

to give approximately 75 percent hemolysis, were added.

In a control tube, 0.2 ml of PBS was added in place of











the extract. After 60 min incubation at 370C with con-

tinuous shaking, 2.0 ml of ice cold PBS were added to each

tube. The mixtures were centrifuged and the optical

densities of the supernates were determined at a wave-

length of 414nm. The inhibition of hemolysis was calcu-

lated for each dilution of extract used.

Complement fixation by tumor cell extracts. Ten

volumes of guinea pig complement were adsorbed 3 times at

0C with 1 volume of washed, packed EATC. One volume of

adsorbed complement and 1 volume of tumor cell extract

were added to a test tube and incubated at 30C for 15

min. The guinea pig complement was then titrated in the

standard complement titration assay described by Mayer

(92).

Inhibition of hemolytic antibody titration by tumor

cell extracts. In order to determine if extracts from

EATC contained any shared antigens with sheep red cell

stroma, a hemolytic antibody titration was performed.

Equal volumes of EATC extracts and rabbit anti-sheep E

stromata (Cordis Laboratories, Miami, Florida) were

incubated for 15 min at 30C and at 0C for 15 min. A

control consisted of incubating equal volumes of PBS and

anti-sheep E for the prescribed length of time. The

antibody was titered using limiting amounts of comple-

ment as described by Mayer (97).

Decay of SAC142 by tumor cell extracts. To deter-

mine if there was any influence on the rate of decay of










SAC142,extracts from EATC were incubated with EAC142 and

assayed according to the method described by Hoffmann (88).

Decay of SAC14 by tumor cell extracts. To determine

if there was any influence on the stability of SAC14,

extracts from EATC were incubated with EAC14 and assayed

according to the method described by Hoffmann and Etlinger

(89).

Hemolytic inhibition assays using sheep E, EA, and

various cell intermediates. Analysis of inhibition at the

cellular level was examined using the methods described by

Hoffmann(88). This method consisted of incubating one

volume of the intermediate to be tested with an equal

volume of EATC extract at 300C for 15 min. The cells were

washed two times at 0C and resuspended to a concentration

of 1 X 108/ml and the necessary reagents were then added,

as described, in concentrations sufficient to cause between

50 and 80 percent hemolysis.

Generation of EAC142 in the presence of EATC extracts.

In order to determine if tumor cell extracts had any

effect on SAC142 generation, Tmax experiments were per-

formed. Tmax is the time required for the generation of

the maximum number of SAC142 per cell (98). Equal volumes

of EAC14 and tumor cell extracts were incubated at 300C

for 15 min. A control tube consisted of PBS instead of

tumor extract. After incubation, the cells were washed

two times and resuspended to a concentration of 1 X 108

cells per ml. The cells were prewarmed to 300C and a










Tmax experiment was performed as described by Borsos et

al. (98). The absorbancies of the supernates were read at

a wavelength of 414nm, and the data plotted as number of

SAC142 per cell (Z number) versus time (99).

Cl fixation and transfer. The number of Cl mole-

cules bound to antibody-antigen complexes can be measured

by the Cl fixation and transfer test described by Borsos

and Rapp (64). Their procedure was used here in an

attempt to quantitate the number of Cl molecules fixed

to EA which had been previously treated with tumor cell

extract. This Cl test was carried out in different stages.

Equal volumes of EA (1 X 108/ml) and tumor cell extract

were incubated at 300C for 15 min, washed twice with

DGVB and resuspended to 1 X 108 cells/ml in DGVB

A control consisted of treating EA with PBS. Equal

volumes of EA treated with tumor cell extract (EA ) or

EA treated with PBS (EA ) and Cl were incubated at 30C

for 10 min in DGVB. The cell mixtures were washed

twice with DGVB resuspended in GVB at a concentra-

tion of 1 X 108/ml, 5 X 107/ml, 1 X 106/ml, 5 X 105/ml,

and 1 X 105/ml. One volume of each cell concentration

was added to one volume of EAC4 to permit transfer of Cl

from EAC1 to EAC4. The cells were incubated at 300C for

10 min. C2 and C-EDTA were then added in relative excess

as described.

In another experiment, EAC1 were treated with tumor

cell extract or with PBS at 300C for 15 min. The cells










were washed twice in DGVB++ and resuspended in GVB++ at

a cell concentration of 1 X 108/ml. The amount of Cl

capable of transfer was then measured in the Cl transfer

test described above.

Consumption of C4 by tumor cell extract treated

EAC1. In order to measure the activity of Cl on the

intermediate EAC1 that had been treated with tumor cell

extract, a C4 consumption experiment was performed. EACl

at a concentration of 1 X 108/ml were generated as described

(96) and treated with tumor cell extract at 300C for 15 min,

washed twice and resuspended to a cell concentration of

5 X 108/ml. EACT treated with tumor cell extract are desig-

nated EACIT. Control cells treated with PBS are designated

EAC1C.

EACI cells were separately mixed with an equal volume

of C4. The mixtures were incubated at 300C for 10 min.

The cells were centrifuged at 500G for 5 min, the super-

natent fluids were serially diluted in DGVB and 0.2 ml

of each dilution was added to 0.2 ml of EAC1 that had not

been treated with extract or PBS. The mixtures were incu-

bated at 300C for 20 min. An equal volume of C2 (0.2 ml)

was added to each tube and the mixtures incubated for an

additional 20 min at 30C. C-EDTA (0.4 ml) was added to

each tube and the mixtures were incubated at 370C for 60

min. After the incubation period, 2.0 ml of ice cold PBS

were added to each tube. The reaction mixtures were cen-

trifuged at 500G for 5 min at 0C. The absorbancy of the











supernates was read at a wavelength of 414nm, and the

data plotted as described by Rapp and Borsos (99).

Titration of individual complement components. In

order to determine if extracts from tumor cells had any

inhibitory effect on individual complement components,

whole guinea pig sera or each component to be examined

were incubated with extract or buffer at 30C for 15 min.

The sera were then titrated for the component in question or

the individual component was titrated as described (93). In

each case, the dilution of tumor cell extract used in the

initial incubation was inhibitory in the hemolytic assay,

but the inhibitory effect of the extract was diluted out in

the titration range of the component being tested.

Ammonium sulfate fractionation of crude tumor cell

extract. Tumor cell extracts were initially fractionated

by using ammonium sulfate. A saturated ammonium sulfate

solution was added to extracts of EATC to bring the final

concentration of ammonium sulfate to 20, 40, 60, and 80

percent saturation. The mixtures were incubated at 0C

for 1 hr and centrifuged at 10,000G for 30 min. The

supernates were transferred directly to dialysis tubing and

dialyzed overnight versus PBS with two changes of the

dialysis buffer. The pellets obtained after centrifugation

were redissolved in a minimal amount of PBS, transferred to

dialysis tubing and dialyzed overnight as above. The

dialyzed fractions were then tested for inhibitory activity

using the immune hemolysis inhibition test.










Gel filtration and ion exchange chromatography. For

gel filtration, crude tumor cell extracts were applied to

a 2.5 X 80/cm column of G-200 Sephadex (Pharmacia Fine

Chemicals, Piscataway, New Jersey) which had been equili-

brated with PBS. The column was eluted with PBS and

fractions were assayed for the ability to inhibit whole

complement mediated lysis of EA.

For Bio-Gel A-1.5m separations, partially purified

EATC extracts were applied to a 1.5 X 80/cm column of Bio-

Gel A-1.5m (Bio-Rad Laboratories, Richmond, California)

which had been equilibrated with PBS. The column was eluted

and assayed as described for G-200 Sephadex.

For DEAE chromatography, standard capacity Cellex D

(.67meq/gram) was prepared according to the manufacturer's

instructions (Bio-Rad Laboratories, Richmond, California).

Tumor cell extract was adjusted to an ionic strength of 0.3

pH 7.5 (0.005M potassium phosphate sodium chloride buffer)

and applied to a 4.0 X 40cm DEAE column that had been

equilibrated to an ionic strength of 0.3 pH 7.5. After

application of the extract, the column was washed with the

initial buffer and fractions were collected until the

absorbancy at 280nm was near zero. A linear salt gradient

buffered with 0.005M potassium phosphate pH 7.5, was then

initiated and fractions were collected as described in the

results section. The fractions were adjusted to 0.15 ionic

strength using distilled water and assayed for the ability

to inhibit complement mediated lysis of EA.










Enzyme sensitivity. Tumor cell extracts were sub-

jected to various enzyme treatments in order to determine

which class of compounds was responsible for the inhibitory

activity. Deoxyribonuclease, ribonuclease free, (Worthington

Biochemical Corporation, Freehold, New Jersey) was used at a

concentration of 100lg/ml in VB One volume of deoxyribo-

nuclease was added to four volumes of tumor cell extract.

Controls consisted of mixing one volume of enzyme with

four volumes of PBS and by mixing one volume of VB with

four volumes of tumor cell extract. The mixtures were

incubated for 30 min at 250C and each mixture was titrated

for the ability to inhibit whole complement mediated lysis

of EA. Ribonuclease A (Worthington Biochemical Corporation,

Freehold, New Jersey) was used at a concentration of 100lg/

ml in VB+ and Ribonuclease A, type III (Sigma Chemical

Company, St. Louis, Missouri) was used at a concentration

of 1.0mg/ml in VB One volume of ribonuclease and four

volumes of tumor cell extract were mixed together and in-

cubated for 30 min at 370C. The controls used and the

inhibition test are described above. One volume of trypsin,

type III (Sigma Chemical Company, St. Louis, Missouri) at a

concentration of 2.5mg/ml was mixed with four volumes of

tumor cell extract. The controls are the same as indicated

for deoxyribonuclease. The mixtures were incubated for 60

min at 370C. One volume of lima bean trypsin inhibitor

(Sigma Chemical Company, St. Louis, Missouri) at ten times

equimolar amounts of trypsin was added to each tube to










stop trypsin enzymatic activity. The mixtures were then

titrated for inhibitory activity in the whole complement

inhibition test. The protease, obtained from Streptomyces

griseus, was purchased from Miles Laboratories (Kanakee,

Illinois) and was used as an insolubilized enzyme in a 2.5

ml syringe type column. The void volume of the column was

approximately 2 ml. The column was equilibrated with VB+

and the buffer was allowed to drain to the top of the

column. One volume (1.0 ml) of tumor cell extract was

applied and allowed to drain to the top of the column.

The column was closed and incubated for 60 min at 25C.

After incubation, 2 ml of VB was applied to the column

to elute the tumor cell extract. The dilution of extract

on the protease column was approximately twofold. The

extract was then compared with a 1:2 dilution of untreated

extract for its ability to inhibit complement mediated

lysis of EA. Controls consisted of adding one volume of

PBS to the column as described for the tumor extract.

Streptomycin sulfate fractionation. Streptomycin

sulfate was purchased from Sigma Chemical Company, St.

Louis, Missouri. The fractionation of the crude tumor

cell extract with streptomycin sulfate was performed

according to the procedure of Stern and Mehler (100). Three

columes of a 6 percent streptomycin sulfate solution were

added, over a 10 min period with stirring, to 10 volumes

of crude extract. The precipitate was allowed to coagu-

late and settle overnight. The mixture was centrifuged at










500G for 10 min and the supernate was dialyzed overnight

versus PBS. The supernate was then assayed for its ability

to inhibit complement mediated hemolysis of EA.

Heat sensitivity of tumor cell extracts. Crude and

partially purified Ehrlich ascites tumor extracts were

placed in a 560C water bath. At timed intervals, samples

were taken and transferred to an ice bath. Each sample

was then assayed for its ability to inhibit lysis of EA

by whole guinea pig complement.

Sucrose gradient centrifugation. Two-tenth ml samples

were applied to a preformed buffered sucrose gradient(ionic

strength = 0.05, pH 7.6, 5 percent to 20 percent w/v sucrose)

of 5.0 ml. The tubes were centrifuged at 38,000 rpm for 5

hr in a Sw-39 rotor of a Model L-2 Beckman Ultracentrifuge

(Beckman Instruments, Inc., Palo Alto, California). After

centrifugation, the mixture was pumped on a Buchler Poly-

staltic Pump (Buchler Instruments, Fort Lee, New Jersey) at

a flow rate of 0.6 ml/min from the bottom of each tube

through a Gilford Model 2400 Spectrophotometer (Gilford

Instrument Laboratories, Inc., Oberlin, Ohio) continuously

monitored at 260nm and recorded. Fractions were collected

and assayed for inhibition of immune hemolysis.

Viability of EATC and release of complement inhibi-

tory material. One volume of washed intact EATC was

incubated with 3 volumes of PBS at 40C with mild shaking.

At timed intervals, 5 ml samples were taken and the via-

bility of the cells determined by trypan blue exclusion

(62) on 0.2 ml samples. The remainder of the sample was










centrifuged at 500G for 10 min to sediment the cells and

the supernate was centrifuged at 10,000G for 30 min. The

ability of the supernate to inhibit complement mediated

hemolysis of EA was titered.

Extraction of normal mouse tissue cells and whole

tumor cells for nucleic acid. One volume of liver and

spleen cells from ICR mice and Ehrlich ascites tumor cells

was suspended in one volume of PBS. The cells were frozen

and thawed rapidly 3 times. The cells were then placed at

0C and extracted using a modification of the phenol extrac-

tion procedure (101). One volume of 88 percent aqueous

phenol (Fisher Scientific, Pittsburgh, Pennsylvania) was

added to 1 volume of cells at 0C with constant stirring.

After 10 min, the solution was centrifuged at 10,000G for

10 min. The aqueous phase was carefully removed and trans-

ferred to another tube. An equal volume of PBS was added

to the phenol phase and the mixture was stirred at 0C for

10 additional min and centrifuged. The second aqueous layer

was removed and added to the first aqueous phase. Two and

one half volumes of ice cold 95 percent ethanol were added

to the pooled aqueous layer, stirred briefly, and the mix-

ture was stored overnight at -200C. The mixtures were cen-

trifuged at 1000G for 30 min at 0C. The supernate was dis-

carded and the pellet was washed 2 additional times with 95

percent ethanol. After the last washing, the supernate was

discarded and the tube was allowed to drain dry briefly. A

small volume of PBS was added to the pellet. The mixture was










stirred carefully with a glass rod to dissolve the pellet.

Two and one half volumes of 95 percent ethanol were added to

the dissolved pellet and the procedure repeated as before.

After washing the second precipitate, the precipitate was

dissolved in PBS, transferred to dialysis bag and dialyzed

overnight versus 20 volumes of PBS at 4C. The dialyzed

extract was then frozen at -700C.

Inhibition of immune hemolysis by RNA. RNA from

Escherchia coli and yeast were used to determine if they

were capable of inhibiting guinea pig complement mediated

lysis of EA. Total E. coli RNA was obtained from Dr. James

Preston (University of Florida, Department of Microbiology).

Yeast RNA was purchased from Sigma Chemical Company (St.

Louis, Missouri). The RNA was resuspended in PBS and titrated

for its ability to inhibit immune hemolysis.

Extraction of tumor cell extract with phenol. Crude

extracts obtained by incubating whole EATC with 0.15M NaCl

pH 7.5 were further extracted with aqueous phenol as out-

lined above.

Ascitic fluid extraction. Cell free ascitic fluid

obtained from harvesting EATC from ICR mice was also sub-

jected to phenol extraction as outlined above.

Extraction of P815 and EL4 tumors. The tumor lines

P815 and EL4 were obtained from Dr. Paul Klein (University

of Florida, Department of Pathology). P815 cells were

maintained by weekly passage of 106 cells into DBA/2J mice

and the EL4 tumor line was maintained in C57B1/6J. The











tumors were harvested after one week and washed extensively

with PBS as outlined for EATC. Washed, packed tumor cells

were shaken overnight at 4C with 3 volumes of PBS. The

cell suspensions were centrifuged first at 500G for 10 min

to sediment the cells and then at 10,000G for 30 min. The

.supernates were then assayed for their ability to inhibit

lysis of sensitized sheep erythrocytes with guinea pig

complement that had previously been adsorbed with each

tumor line investigated.

Extraction of liver and spleen cells. Spleen and

liver cells obtained from non-tumor bearing ICR mice were

extracted with PBS as outlined for P815 and EL4 tumors.

Cytotoxicity tests. Antisera to EATC were prepared as

outlined previously (73). EATC were harvested from ICR mice
++
and washed extensively, first in PBS and then in DGVB

The cells were standardized to 5 X 106 cells/ml. In the

first set of experiments, 1 volume of EATC (5 X 106 cells/

ml) was incubated for 15 min at 30C with an equal volume

of ribonuclease A (Worthington Biochemical Corporation,

Freehold, New Jersey). The ribonuclease was used at a

concentration of 200 ug/ml. After incubation, the cells

were centrifuged, washed 2 times with DGVB and restan-

dardized to 5 X 106 cells/ml. A control consisted of

treating EATC with DGVB One volume (0.025 ml) each of

the cell suspensions was added to separate wells of plastic

microtiter plates (Cooke Engineering Company, Alexandria,

Virginia). ne volume (0.025 ml) of DGVB was added to
Virginia). One volume (0.025 ml) of DGVB was added to











each well, as well as one volume (0.025 ml) of anti-EATC

sera and one volume (0.025 ml) of a 1:20 dilution of guinea

pig serum which had been previously adsorbed with EATC. The

plates were incubated for 60 min at 37C with shaking. The

test was read by placing the microtiter plates in ice,

adding 0.025 ml of trypan blue solution to each well and

counting the proportion of stained (dead) cells in a hemo-

cytometer. The trypan blue solution was prepared by dissolv-

ing 1.0gm of trypan blue in 100 ml of distilled water. At

the time of use, 4 parts of this solution were diluted with

one part of a 4.25 percent NaCl solution.

In another set of experiments, EATC were not pretreated

with ribonuclease but the enzyme was substituted for DGVB

in the reaction mixture outlined above.

Extracts from EATC were also assayed for their ability

to inhibit cytotoxicity of EATC by antibody and complement.

One volume (0.025 ml) of tumor cell extract was incubated

with an equal volume of PBS and serially diluted in plastic

microtiter plates. Ehrlich ascites tumor cells at a concen-

tration of 5 X 106 cells/ml were incubated with 1 volume of

anti-Ehrlich antiserum (1:5 dilution) at 37C for 10 min and

at 0C for 15 min. The cells were centrifuged and washed

2 times with DGVB+ and restandardized to 5 X 106 cells/ml.

One volume (0.025 ml) of the sensitized EATC was then added

to each dilution extract. One volume (0.025 ml) of EATC

was also added to control wells containing 0.025 ml PBS.

Two volumes (0.05 ml) of guinea pig serum diluted 1:40 in










DGVB were added to each well. The plates were incubated

at 37C for 60 min. After incubation, the dead cells were

scored as before. In each experiment, each test was per-

formed in triplicate.

Immunodiffusion analysis and reaction of Clq with

tumor cell extracts. Clq was tested for its ability to

agglutinate gamma globulin coated latex particles (Hyland

Laboratories, Los Angeles, California) according to the

method of Ewald and Schubart (102). Tumor cell extracts

were serially diluted in PBS. Clq was diluted in glycine-

saline buffer as described by Ewald and Schubart. One

volume of each dilution of tumor cell extract was mixed

with an equal volume of each dilution of Clq. The mixtures

were incubated for 15 min at 300C. After incubation, the

mixtures were titered for their ability to agglutinate

gamma globulin coated latex particles. Ouchterlony double

diffusion was carried out in 0.6 percent agarose in .005M

potassium phosphate containing 0.01M EDTA, relative salt

concentration (RSC) = 0.09, pH 7.2 as supporting medium

according to the method of Agnello et al. (16). Anti-human

sera and anti-human IgG, IgM, and IgA were purchased from

Hyland Laboratories (Los Angeles, California). Anti-human

Clq was purchased from Behring Diagnostics (Somerville,

New Jersey).

Clq purified according to the method of Yonemasu and

Stroud (95), did not contain any detectable IgG, IgM, or

IgA by Ouchterlony double diffusion. Clq reacted with







32


anti-human sera and anti-Clq with a band of identity.

There was a trace contaminant, however, when Clq was

reacted against anti-human sera. The purified Clq was

then tested for its ability to react with dilutions of

tumor cell extract in Ouchterlony double diffusion,

according to the method of Agnello (16).
















RESULTS


Extraction of complement inhibitory material from

EATC with various salt solutions. Washed packed tumor cells

were incubated overnight at 40C in various concentrations

of buffered sodium chloride solutions in order to determine

if a complement inhibitory substance would be released from

Ehrlich ascites tumor cells. The extracts were assayed for

their ability to cause inhibition of immune hemolysis using

a limiting amount of guinea pig complement which had been

adsorbed previously with EATC. The inhibitory effects of

the various salt extracts of EATC are shown in Figure 1.

Extraction of EATC with buffered 0.15M sodium chloride

(PBS) yielded the highest relative amount of inhibitory

material using the parameters measured.

Inhibition of immune hemolysis by extracts from

whole tumor cells and crude membranes of EATC. Since

extracts from human erythrocyte membranes have been shown

to inhibit complement activity (87), extracts from crude

membrane fractions of EATC were compared with the PBS

extract from whole tumor cells for their inhibitory effects

on the lysis of EA by guinea pig complement. In each case,

guinea pig serum which had been adsorbed with EATC was used

as the source of complement. Figure 2 shows the inhibition

of lysis of EA caused by the tumor cell extracts. Since the
































Figure 1. Inhibition of immune hemolysis by various salt
extracts of Ehrlich ascites tumor cells. The
curves represent the relative salt concentration
of the solutions used for the extraction of EATC.
All extracts were adjusted to an ionic strength
of 0.15M before assaying. The ionic strengths
(p) of the solutions used were (--0), 0.05p;
(D-O ), 0.15p; (A-A), 0.2p; (--*), 0.3p;
(A- M), 0.4p; ( A-A), 0.5j.

































100







- 60



i 40



20




1 16 1:8 1:4 1:2 1I

Dilution of Extract



































Figure 2. Inhibition of immune hemolysis by EATC extracts.
Extracts obtained with the 0.15M salt solution
were obtained from washed, packed tumor cells.
The extracts obtained with 0.5M NaC1 and butanol
were obtained by treating a crude membrane frac-
tion of EATC.










































* 0.5 M Salt Extract
SButanol Extract











extractions of intact tumor cells with PBS resulted in

relatively good yields under the mildest conditions, the

PBS extract was used in subsequent experiments unless other-

wise noted.

Effects of EATC extracts on rabbit, mouse, and human

complement. Since extracts from EATC were capable of in-

hibiting the lysis of EA by guinea pig serum, the extracts

were tested to determine if they could inhibit complement

from other sources. Rabbit, mouse, and human sera were

adsorbed with Ehrlich ascites tumor cells before use. A

0.15M sodium chloride extract from EATC was diluted and

tested for its ability to inhibit immune hemolysis with

various complement sources. The inhibition of guinea pig

complement by this extract is shown in Figure 3. The in-

hibitory capacity of the extract was then tested in the

other complement systems. Figure 4 shows the inhibition of

rabbit complement by this extract. It appears that the

tumor cell extract inhibits rabbit complement to a slightly

greater extent than guinea pig complement. A maximun of

60 percent inhibition of mouse complement was obtained by

this EATC extract as shown in Figure 5. It is not clear

why mouse serum could not be inhibited beyond 60 percent.

Finally, Figure 6 shows the relative inhibition of human

complement caused by the extract.

Complement fixation by tumor cell extracts. The

inhibition of whole complement activity caused by tumor

cell extract could have conceivably been due to complement

































Figure 3. Inhibition of guinea pig complement activity
by Ehrlich ascites tumor cell extracts.











































60
o


40
2 40



&" 20


1'32 1 16 1:8 1'4 1.2

Dilution of TCE



































Figure 4. Inhibition of rabbit complement activity by
Ehrlich ascites tumor cell extracts.





















































1:64 1:32


Dilution of TCE

































Figure 5. Inhibition of mouse complement activity by
Ehrlich ascites tumor cell extracts.










44
























i00



80



60-
I-.^--- --- ^-- ------ ---------------


S40
0.

20-




1.16 8 1:4 1:2 1:1
Dilution of TCE



































Figure 6. Inhibition of human complement activity by
Ehrlich ascites tumor cell extract.


























































C
0
C

- 60-
c4



s 40
C
03
Q-


1 64 1 32 1 16 1 8

Dilution of TCE











fixation resulting from natural antibodies in the com-

plement source reacting with tumor antigens in the tumor

cell extract even though the complement was routinely

adsorbed with intact EATC. A 1:2 dilution of adsorbed

guinea pig complement was incubated with a 1:5 dilution

of EATC extract for 15 min at 30C. The complement was

then diluted to 1:300 and titrated as by Mayer (92).

The inhibitory activity of the tumor extract used showed

100 percent inhibition at 1:5 dilution and no inhibition

at a 1:256 dilution. Figure 7 shows that there is no

apparent complement fixation by EATC extracts under the

described conditions. Figure 7 also shows that the PBS

extract does not seem to have any fluid phase effect on

whole complement activity in guinea pig serum.

Inhibition of hemolytic antibody titration by tumor

cell extracts. Extracts from EATC were incubated with anti-

serum against sheep erythrocytes to see if anti-sheep

erythrocyte antibodies reacted with EATC extracts. The

inhibition of whole complement could be a result of the

antibodies reacting with the tumor extract and interfering

in some way with lysis of sheep erythrocyte target cells.

EATC tumor extracts and PBS were each incubated with

separate samples of anti-sheep erythrocyte hemolysin and

the antiserum was titered as described by Mayer (97). The

titration of the antiserum as shown in Figure 8 shows

that extracts from EATC did not affect the titration

of antibodies to sheep red cell antigens. If the

































Titration of adsorbed guinea pig complement
after treatment with EATC extracts. The closed
circles (0-- ) show the titration of guinea
pig serum after incubation with PBS and the
open circles (O--0) denote the titration of
guinea pig serum after incubation with PBS
extract of EATC.


Figure 7.

































Absorbed GPS with Buffer
CH50= 137 units

o Absorbed GPS with Extract
CHo= 140 units

3.0-
2.5

2.0 -





.2 .3 .5 .7 .9 1.0 2.0 4.0 60 80
Y
I -Y





































Effect of EATC extracts on hemolytic antibody
titration. Incubation of tumor cell extracts
( *-4 ) with antiserum to sheep erythrocytes shows
no apparent effect on the titration of the anti-
serum. Titration of control treated antiserum is
given by the open circles (0-0) .


Figure 8.


~
















o Titration with Buffer
* Titration with Extract


Dilution of Antibody


100

80


-j
C,

CL20


0
0b 0.
00 ~


/










tumor cell extract did cross react with sheep erythrocyte

antigens the titer of the antiserum would be lower in the

presence of the extract.

Decay of SAC142 in the presence of EATC extracts.

Extracts from human erythrocytes have been shown to

accelerate the decay of the sheep erythrocyte, rabbit anti-

body, complement component intermediate, EAC142 (88). EATC

extracts were incubated with EAC142 to see what effect this

material had on the decay rate of the intermediate. One

volume each of tumor cell extract or PBS was mixed with an

equal volume of the intermediate EAC142 at 0C. The

mixtures were placed at 30C and the rates of decay were

followed. Figure 9 shows that EATC extracts did not accel-

erate the decay of the intermediate EAC142 since the two

EAC142 decay curves are approximately parallel. The extract

does impair the ability of the cells in this intermediate

state to undergo lysis in the presence of C-EDTA (source

of C3 through C9) since there was less lysis of EAC142 in

the presence of EATC extract.

Effect of tumor extract on the stability of SAC14.

Extracts from rabbit and guinea pig stromata have been

shown to cause a time dependent inactivation of EAC14 at

30C (89). The crude PBS extract of EATC was mixed with

an equal volume of EAC14 (1 X 108/ml) and placed at 30'C.

The control consisted of incubating PBS with EAC14.

Samples were taken at timed intervals, the cells were

washed twice in DGVB and assayed for SAC14 (98). As can
































Decay of EAC142 at 30 in the presence of tumor
cell extracts. The upper curve (0- ) shows
the decay of the intermediate treated with PBS.
The lower curve (C0- ) shows the decay of the
intermediate treated with a PBS extract of
Ehrlich ascites tumor cells. Z = average number
of SAC142 per cell.


Figure 9.






















2.0



I .0
.8
S.6
.5
.4

.3

.2


5 10 15 25 40


Time in Minutes










be seen in Figure 10, there is no effect of crude tumor cell

extract on the stability of the EAC14 intermediate. It is

evident, however, that EAC14 treated with the tumor extract

undergo less lysis as compared to controls. The rate of

inactivation must be rapid since the inactivation was com-

plete within two min at 300C.

Site of hemolytic inhibition by EATC extracts. In

order to find out what steps in the complement sequence

were affected by the crude salt extract of Ehrlich ascites

tumor cells, sheep E, EA, and all of the cellular inter-

mediates in the complement sequence were incubated with the

tumor extracts. Each intermediate to be tested was adjusted

to 1 X 108/ml in DGVB mixed with an equal volume of the

PBS extract from EATC and incubated for 15 min at 300C.

After incubation, the cells were washed twice and assayed

for reactivity as compared to PBS treated cells. The

results shown in Table 1 indicate that the early steps in

the complement sequence were inhibited by EATC extracts.

The inhibitory material showed no inactivation of sheep

erythrocytes, but did show marked inactivation of EA, EACI,

EAC4, and EAC14. The intermediate EAC142 was inhibited to

a lesser extent and once C2 was on the cells, the EATC

extracts had no effect on the later reacting components.

Effect of tumor cell extract on the uptake of Cl by

EA. Treatment of EA with tumor cell extract led to an

impairment of the ability of complement to lyse the cells.

Therefore, the ability of extract treated and untreated EA
































Effect of EATC extract on the stability of
the intermediate EAC14. The upper curve (-- )
shows the stability of EAC14 at 300C in the
presence of PBS. The lower curve (0--0)
shows the stability of EAC14 in the presence
of a PBS extract of EATC. Z = average number
of SAC14 per cell.


Figure 10.























2.0



1.0 -O- -
.8

.6 'o 0
.5
.4

.3

.2




5 10 15 25 40 60
5 10 15 25 40 60


Time in Minutes















TABLE 1


Effect of EATC Extract on the Hemolytic Susceptibility
of Sheep E, EA, EAC1, EAC14, EAC4, EAC142, EACI-3,
EAC1-5, EAC1-6, EAC1-7 and EAC1-9




Percent Percent
Intermediate Hemolysis Inhibition

Treated Control


E 53 55 3

EA 25 73 65

EACl 30 77 61

EAC4 27 61 55

EAC14 42 64 34

EACl42 59 73 19

EAC1-3 50 52 4

EAC1-5 62 62 0

EAC1-6 73 75 2

EACl-7 74 76 2

EAC1-9 54 55 1









to fix Cl was assayed using the method of Cl fixation and

transfer. EA (1 X 108/ml) were treated with tumor cell

extract or with PBS; the cells were washed and resuspended

to a cell concentration of 1 X 108/ml. The cells were then

reacted with guinea pig Cl and the amount of Cl fixed in

each case was measured in the Cl transfer test. Table 2

shows that EA treated with tumor cell extract (EAT) fixed

more Cl than did PBS treated EA (EA ). The Cl fixed to the

tumor cell extract treated EA was not inactivated since

active Cl was being measured with the Cl fixation and trans-

fer procedure. Although tumor cell extract treated EA were

capable of fixing more Cl than control cells, the cells were

inhibited from lysing with guinea pig complement as shown in

Table 1.

Consumption of C4 by tumor cell extract treated EAC1.

EAC1 were treated with the tumor cell extract to determine

if the extract had any effect on the reactivity of Cl on

EACI cells. This experiment was performed to investigate

the effects of tumor cell extract on the ability of EAC1 to

consume C4. EACl were generated and then treated with tumor

cell extract or PBS. EACl treated with extract are desig-

nated EACT EACT treated with PBS are designated EACIC

The treated EAC1 cells were then used to measure consumption

of C4. EAC1 were reacted with C4; the mixtures centrifuged,

and the residual C4 activity present in the supernatent

fluids was titrated. Figure 11 shows that EAC1 treated with

tumor cell extract (EAC T) consumed less C4 than did EACIC

since more C4 was titratable in the supernatent after

treatment.















TABLE 2



Comparison of the Relative Numbers of Effective Cl
Molecules Fixed by EA Treated with EATC Extracts and
by Untreated EA




Experiment Effective Number of Standard
Sample Number Cl Molecules Fixed/Cell Deviation


S1 245
EA 1 245 + 4


b
EA
C


a) EA treated
min before


with tumor cell
addition of Cl.


extract at 300C for 15


b) EA treated with PBS at 300C for 15
addition of Cl.


min before

































Figure 11. Titration of residual C4 activity after
treatment with EAC1 and EACI cells. The
T C
upper curve represented by the open squares
(----) represents the titration of untreated
C4. The middle curve (0---) shows the titra-
tion of residual C4 after treatment with EAC1 .
The lower curve represents the titration of
residual C4 after treatment with PBS treated
cells (EAC1 ). Z = average number of SAC14
per cell.


































20



1 6



1 2



8


1 256 1 128


1 64 1 32
Dilution of C4










Effect of EATC extracts on the generation of SAC142.

Tumor cell extracts were reacted with cells in the inter-

mediate state of EAC14 to determine if the extract was

capable of affecting the generation of SAC142 from SAC14.

Equal volumes of EAC14 (1 X 108/ml) were incubated with

equal volumes of either EATC extracts or PBS for 15 min at

300C. The cells were washed and resuspended to 1 X 108/ml

and the Tmax was determined. The results given in Figure 12

show that EAC14 which had been treated with tumor cell extract

had a longer Tmax than did a buffer treated control, thus

indicating that the treatment caused a decrease in the

available number of SAC14 on the cells (98).

Effect of tumor cell extracts on individual complement

components. The tumor cell extract was incubated with Cl

and C2 to determine if the extract was capable of inactivaing

fluid phase Cl and C2. Equal volumes of Cl at a 1:5 dilu-

tion were incubated with EATC extracts or PBS for 15 min at

300C and the Cl was titrated in the standard assay system (93)

Figure 13 shows that treatment of Cl with tumor extracts

had no effect on titer of the component using an incubation

time and temperature equivalent to those used when EATC

extract was incubated with EACT. C2 at a dilution of 1:4

was incubated with tumor cell extracts in the same manner

as was Cl and the C2 was titered as described. Figure 14

shows that the titration curves of C2 treated with PBS or

with the tumor cell extract were essentially the same. No

evidence was found for fluid phase inactivation of C2 using
































Effect of EATC extracts on the generation of
SAC142. The curve denoted by the closed
circles (0-0) represents SAC142 generation
on EACl4 cells treated with PBS. The curve
denoted by the open circles (0-- ) represents
the generation of SAC142 on EAC14 treated with
tumor cell extract. Z = average number of
SAC142 per cell.


Figure 12.





























1.6


z .2-


.8


.4-



10 20 30 40 50 60 70


Time in Minutes

































Figure 13. Titration of Cl treated with EATC extracts
(0---) and with PBS (0-- ) Z = average
number of SACT per cell.



























1.2


1.0



.8


.6



.4



.2


1:40,000 1:20,000 1:10,000 1:5,000

Dilution of CI

































Figure 14. Titration of C2 treated with EATC extracts
(0--) and with PBS (O--) Z = average
number of SAC142 per cell.












































1:1024 1:512 1:256 1:128


Dilution of C2











incubation conditions which were the same as when EAC142

were incubated with EATC extract.

Preliminary purification attempts. Since the tumor

cell extract could inhibit at several points in the early

sequence of complement activation, it was difficult to come

up with a model that could explain all of the phenomena

that were observed. Therefore, it was necessary to attempt

to purify the inhibitory substance or substances in the

tumor cell extract.

A combination of gel filtration, ammonium sulfate

fractionation and DEAE chromatography was utilized initially

in an attempt to purify the active inhibitory material in

the PBS extracts of EATC. Crude tumor cell extract was

first applied to a G-200 Sephadex column and the inhibitory

activity eluted immediately after the void volume (Figure 15).

Since Sephadex gave no additional purification, the next

approach to the partial purification of the tumor cell extract

was fractional ammonium sulfate precipitation. The inhibi-

tory activity precipitated at a concentration of 20 percent

saturation with ammonium sulfate and less than 10 percent of

the inhibitory activity was found in the other ammonium

sulfate fractions. The 20 percent ammonium sulfate preci-

pitate was redissolved and dialyzed overnight versus PBS.

The volume of the dialyzed fraction (50 ml) represented

one-half of the original starting volume. The crude tumor

cell extract was capable of causing 50 percent inhibition

at a 1:512 dilution. The redissolved ammonium sulfate

































Gel filtration of PBS extract from EATC on
G-200 Sephadex. The column was equilibrated
with PBS. Five milliliter volumes of the crude
tumor cell extract were applied. The fractions
were collected and the absorbancy at 280nm was
determined, as shown by the open circles (--0).
The fractions capable of inhibiting immune
hemolysis are shown by the cross-hatched area.
Void volume = 100ml.


Figure 15.



























































10 20 30 40 50 60 70 80 90
Fraction Number (5ml)










precipitate caused 50 percent inhibition at a 1:400 dilu-

tion. One half of the redissolved precipitate ( 25 ml) was

adjusted to an ionic strength of 0.3, pH 7.5, and applied

to a DEAE column that was equilibrated at an ionic strength

of 0.3, pH 7.5. After application, the column was washed

with the equilibrating buffer and a linear sodium chloride

gradient was initiated. The inhibitory material bound to

the DEAE column at an ionic strength of 0.3 and it began to

elute at an ionic strength of 0.4. The elution peak was

at an ionic strength of 0.55. The absorbancy of each frac-

tion was determined at a wavelength of 280nm. The fractions

were then adjusted to an ionic strength of 0.15 and assayed

for inhibitory activity as shown in Figure 16. The input

material caused 50 percent inhibition of complement mediated

lysis of EA at a 1:400 dilution.

The entire inhibitory peak obtained after DEAE

chromatography was pooled (fractions 37 through 70), preci-

pitated with saturated ammonium sulfate. The precipitate

was redissolved in 8 ml of PBS, dialyzed and 2 ml was

applied to a Bio-Gel A-1.5m column. The column was eluted

with PBS and the optical density at 280nm of each fraction

was determined. The inhibitory activity was retained by

the gel and all of the inhibitory activity eluted in a

single peak which coincided with the optical density at

280nm (Figure 17). The input material caused 50 percent

inhibition of EA lysis at a dilution of 1:16. The frac-

tions containing inhibitory activity were pooled(fractions


































DEAE chromatography of the 20 percent ammonium
sulfate precipitated inhibitory material from
the EATC extracts. The inhibitory activity is
given by the cross-hatched area. The open
circles (0--0) show the optical density at
280nm, and the dashed line shows the sodium
chloride gradient.


Figure 16.




































u A 280
.24

.20

.16

-- .14

.9 .12

.8 .10

.7 .08

.6 .06

.5 .04

.4 .02


60
-o



40


C30
30


10 20 30 40 50 60 70 80 90

Fraction Number (5 ml.)



































Gel filtration of DEAE purified tumor cell
extract on Bio-Gel A-1.5m. The optical density
at 280nm is given by the open circles (0--).
The inhibitory activity coincided with the peak
of A280 absorbing material. After concentration,
the purified extract was capable of causing 50
percent inhibition at a 1:2 dilution. Void
volume = 60 ml.


Figure 17.





















































20 40 60 80 100 120 140 160

Fraction Number (2 ml )










72 through 80) and concentrated 8-fold. After concentration,

a 1:2 dilution of this purified material was capable of

causing 50 percent inhibition of complement mediated lysis

of EA. Although these methods could achieve purification

and recover inhibitory material, the yield of inhibitory

material was less than one percent and therefore the

procedure was generally unsatisfactory.

Sensitivity of tumor cell extracts to enzyme treatment.

The tumor cell extracts were subjected to enzyme treatment

in order to determine what class of compound was responsible

for the inhibitory activity. Determining the class of com-

pounds responsible for the activity would also provide an

insight as to the type of purification scheme that should be

developed. The PBS extract from EATC was not sensitive to

the action of deoxyribonuclease since there was no loss of

activity after treatment with the enzyme. Trypsin treat-

ment of the tumor cell extract caused only a partial reduc-

tion in the inhibitory activity as shown in Figure 18.

The inactivation of the extract could not be increased

even when the concentration of trypsin was increased 10-

fold. The control containing trypsin plus trypsin inhibi-

tor showed no complement inhibitory activity.

The sensitivity of the EATC extract to protease was

tested using an enzyme bound to agarose by the azyl azide

procedure (103). Crude tumor cell extract was incubated

with the enzyme on the column and eluted with VB+. The

inhibitory activity of the extract after treatment was
































Figure 18. Inactivation of EATC extract by trypsin treat-
ment. Tumor cell extracts were incubated with
trypsin and with buffer. The mixtures were
then titrated for inhibitory activity. The
inhibitory curve for tumor cell extract treated
with buffer is given by the open circles (0--) .
The inhibition curve of the tumor cell extract
after treatment with trypsin is given by the
closed circles (0--).



















































1 320 1 160 1 80 1 40
Dilution of TCE











compared with an extract that was diluted 1:2 with VB++ to

correct for the dilution of the extract on the column. The

buffer control consisted of incubating VB with the enzyme

instead of the tumor extract. Figure 19 shows that there

is only a small amount of inactivation of the tumor cell

extract by the insolubilized protease. Increasing the

time of enzyme treatment did not increase the amount of

inhibitor inactivated. The differences in the susceptibi-

lity of the extract to trypsin and protease treatment could

reflect the differences in the specific activities of the

enzymes used.

The tumor cell extract was also tested for its suscep-

tibility to the action of ribonuclease A. Figure 20 shows

that the treatment of tumor cell extract with ribonuclease

inactivated all of the inhibitory activity associated with

the tumor cell extract. Interaction of ribonuclease with

EA and complement did not show any effects by the enzyme

on complement mediated lysis of EA. These findings indicated

that the active inhibitory material contained RNA as a

functional entity.

Streptomycin sulfate fractionation. Streptomycin

sulfate has been used to precipitate nucleic acids from

enzyme preparations (100). Therefore, a PBS extract from

EATC was incubated with streptomycin sulfate to deter-

mine if the inhibitory activity of the tumor extract could

be removed by this treatment. EATC extract was incubated

with streptomycin sulfate for 10 min at 250C and then for
































Inactivation of EATC extract by protease.
Inhibition of complement mediated lysis by
untreated tumor cell extract is given by the
closed circles (0--* ). The curve denoted by
the open circles (0--0) shows the inhibitory
activity after treatment with protease. The
closed squares (--a ) show the inhibition
caused by the buffer control.


Figure 19.






















































1 640 1 320 ) 160


1 80
Dilution of TCE


1 40
































Inactivation of EATC extract by ribonuclease
A. Tumor cell extracts treated with ribo-
nuclease and with buffer were titrated for
inhibitory activity in the immune hemolysis
inhibition test. The curve represented by the
open circles (O-0- ) shows the inhibitory
activity of the extract with buffer. The closed
circles (0--*) show the inhibitory activity of
the extract treated with ribonuclease.


Figure 20.
















































c
0



- 60
6
2 40




20


1:1280 1:640


1:320

Dilution of TCE


1:160










18 hours at 4C. An insoluble precipitate formed and was

removed by centrifugation at 10,000G for 30 min. The

supernate was dialyzed overnight versus PBS and titrated

for inhibitory activity. Figure 21 shows that the super-

nate remaining after streptomycin treatment did not possess

any inhibitory activity as compared to untreated EATC

extracts when tested in the immune hemolysis inhibition

test, thus providing additional evidence that the inhibitor

was nucleic acid.

Partial purification of a complement inhibitory

material from EATC using DEAE chromatography and phenol

extraction. Since the original attempt to purify the

inhibitory component from the EATC extract was not fruit-

ful, another attempt was made to partially purify the

inhibitory substance. A combination of DEAE chromatography

and phenol extraction was used since the original DEAE

chromatography gave good separation of inhibitory material

from inactive contaminants and phenol extraction procedures

are used to purify RNA (101).

A PBS extract from EATC capable of causing 50 per-

cent inhibition of complement mediated lysis of EA at a

1:2000 dilution was adjusted to an ionic strength of 0.3,

pH 7.5, and applied to DEAE which was equilibrated at the

same pH and ionic strength. The column was washed with

buffer (ionic strength = 0.3, pH 7.5) and a linear salt

gradient was initiated. Fractions were collected and

the absorbancy of each fraction was determined at wave -


































Removal or inactivation of EATC extract inhi-
bitory material with streptomycin sulfate.
The curve depicted by the open circles (0-- )
shows the inhibition caused by untreated tumor
extracts. The closed circles (&-- ) represent
the inhibition of complement mediated lysis after
the extract was treated with streptomycin
sulfate.


Figure 21.






















































c

60
-o
c

; 40


oj
0-


1.64 1:32 1:16 1:8 1.4

Dilution of TCE

































DEAE chromatography of PBS extract from EATC.
The open circles (0--0 ) represent the optical
density at 280nm and the closed circles (0--4)
represent the absorbancy at 260nm. The dashed
line shows the sodium chloride gradient. The
input was 10.0 ml of crude extract which had an
optical density at 280nm of 6.10 and at 260nm
of 10.44. The crude extract was capable of
causing 50 percent inhibition of EA lysis at a
dilution of 1:2000.


Figure 22.














































-6



-5



-.4


-/+ .9- 3


140 160 180 200
(5m1)




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