Group Title: BMC Cancer
Title: Further evidence for increased macrophage migration inhibitory factor expression in prostate cancer
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Title: Further evidence for increased macrophage migration inhibitory factor expression in prostate cancer
Physical Description: Book
Language: English
Creator: Meyer-Siegler, Katherine
Iczkowski, Kenneth
Vera, Pedro
Publisher: BMC Cancer
Publication Date: 2005
 Notes
Abstract: BACKGROUND:Macrophage migration inhibitory factor (MIF) is a cytokine associated with prostate cancer, based on histologic evidence and circulating (serum) levels.Recent studies from another laboratory failed to document these results. This study's aims were to extend and confirm our previous data, as well as to define possible mechanisms for the discrepant results. Additional aims were to examine MIF expression, as well as the location of MIF's receptor, CD74, in human prostatic adenocarcinoma compared to matched benign prostate.METHODS:MIF amounts were determined in random serum samples remaining following routine PSA screening by ELISA. Native, denaturing and reducing polyacrylamide gels and Western blot analyses determined the MIF form in serum. Prostate tissue arrays were processed for MIF in situ hybridization and immunohistochemistry for MIF and CD74. MIF released into culture medium from normal epithelial, LNCaP and PC-3 cells was detected by Western blot analysis.RESULTS:Median serum MIF amounts were significantly elevated in prostate cancer patients (5.87 ± 3.91 ng/ml; ± interquartile range; n = 115) compared with patients with no documented diagnosis of prostate cancer (2.19 ± 2.65 ng/ml; n = 158). ELISA diluent reagents that included bovine serum albumin (BSA) significantly reduced MIF serum detection (p < 0.01). MIF mRNA was localized to prostatic epithelium in all samples, but cancer showed statistically greater MIF expression. MIF and its receptor (CD74) were localized to prostatic epithelium. Increased secreted MIF was detected in culture medium from prostate cancer cell lines (LNCaP and PC-3).CONCLUSION:Increased serum MIF was associated with prostate cancer. Diluent reagents that included BSA resulted in MIF serum immunoassay interference. In addition, significant amounts of complexed MIF (180 kDa under denaturing conditions by Western blot) found in the serum do not bind to the MIF capture antibody. Increased MIF mRNA expression was observed in prostatic adenocarcinoma compared to benign tissue from matched samples, supporting our earlier finding of increased MIF gene expression in prostate cancer.
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BMC CancerBioMed Central



Research article

Further evidence for increased macrophage migration inhibitory
factor expression in prostate cancer
Katherine L Meyer-Siegler*1,2, Kenneth A Iczkowski3,4 and Pedro L Veral,2


Address: 'Research and Development Service (151), Bay Pines Veterans' Administration Medical Center, Bay Pines, FL 33744, USA, 2Department
of Surgery, University of South Florida, Tampa, FL 33612, USA, 3Department of Pathology, Malcom Randall Veterans' Administration Medical
Center, Gainesville, FL 32608, USA and 4Department of Pathology, Immunology, and Laboratory Medicine, University of Florida College of
Medicine, Gainesville, FL 32610, USA
Email: Katherine L Meyer-Siegler* Katherine.Siegler@med.va.gov; Kenneth A Iczkowski iczkoka@pathology.ufl.edu;
Pedro L Vera pvera@hsc.usf.edu
* Corresponding author



Published: 06 July 2005 Received: 19 April 2005
BMC Cancer 2005, 5:73 doi:10.1 186/1471-2407-5-73 Accepted: 06 July 2005
This article is available from: http://www.biomedcentral.com/1471-2407/5/73
2005 Meyer-Siegler et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



Abstract
Background: Macrophage migration inhibitory factor (MIF) is a cytokine associated with prostate
cancer, based on histologic evidence and circulating (serum) levels.
Recent studies from another laboratory failed to document these results. This study's aims were
to extend and confirm our previous data, as well as to define possible mechanisms for the
discrepant results. Additional aims were to examine MIF expression, as well as the location of MIF's
receptor, CD74, in human prostatic adenocarcinoma compared to matched benign prostate.
Methods: MIF amounts were determined in random serum samples remaining following routine
PSA screening by ELISA. Native, denaturing and reducing polyacrylamide gels and Western blot
analyses determined the MIF form in serum. Prostate tissue arrays were processed for MIF in situ
hybridization and immunohistochemistry for MIF and CD74. MIF released into culture medium
from normal epithelial, LNCaP and PC-3 cells was detected by Western blot analysis.
Results: Median serum MIF amounts were significantly elevated in prostate cancer patients (5.87
3.91 ng/ml; interquartile range; n = I 115) compared with patients with no documented diagnosis
of prostate cancer (2.19 2.65 ng/ml; n = 158). ELISA diluent reagents that included bovine serum
albumin (BSA) significantly reduced MIF serum detection (p < 0.01). MIF mRNA was localized to
prostatic epithelium in all samples, but cancer showed statistically greater MIF expression. MIF and
its receptor (CD74) were localized to prostatic epithelium. Increased secreted MIF was detected
in culture medium from prostate cancer cell lines (LNCaP and PC-3).
Conclusion: Increased serum MIF was associated with prostate cancer. Diluent reagents that
included BSA resulted in MIF serum immunoassay interference. In addition, significant amounts of
completed MIF (180 kDa under denaturing conditions by Western blot) found in the serum do not
bind to the MIF capture antibody. Increased MIF mRNA expression was observed in prostatic
adenocarcinoma compared to benign tissue from matched samples, supporting our earlier finding
of increased MIF gene expression in prostate cancer.





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Background
Macrophage migration inhibitory factor (MIF) is a
cytokine initially isolated based upon its ability to halt the
in vitro random migration of macrophages [1,2]. Subse-
quently, it was defined as a proinflammatory cytokine
with a key regulatory role in inflammation and both spe-
cific and nonspecific immunity [3]. Currently MIF is rec-
ognized as being constitutively expressed in various cell
types, able to manifest itself as a cytokine, hormone or
enzyme, and as a mediator of many pathologic conditions
[4].

As a proinflammatory cytokine, MIF counter-regulates the
effects of glucocorticoids and stimulates the secretion of
other cytokines such as tumor necrosis factor (TNF)-a and
interleukin (IL)-lP [3], thus assuming a role in the patho-
genesis of inflammatory, immune diseases and cancer [5-
7]. MIF is directly associated with the growth of lym-
phoma, melanoma, and colon cancer [8]. Indications of
MIF's key role in tumor progression derive from studies
where treatments with either anti-MIF immunoglobulin
therapy and/or MIF antisense oligonucleotide confer anti-
tumor activity [9,10]. Although MIF is associated with
cancer angiogenesis, progression, and metastasis, the
exact mechanism of this cytokine's action is uncertain
since a receptor for MIF has only recently been identified
as the cell surface form of the invariant chain (CD74)
[11].

This laboratory was first in localizing MIF within prostatic
epithelium and in establishing that MIF is consistently
increased within both prostate tissue and serum in pros-
tate cancer patients [7,12,13]. The expression of CD74 in
the human prostate or its association with prostate cancer
has not been investigated, but if MIF affects prostate can-
cer cell growth, then it is predicted that MIF's receptor
should be localized within prostate tissue. Our recent
paper established that patients with prostate cancer had
elevated serum MIF levels [13]. However, a study from
Michael et al. failed to confirm these findings [14]. This
discrepancy is worth investigating since establishing the
exact role of MIF in prostate cancer may prove useful as a
diagnostic or prognostic indicator (in addition to the
well-established standard, prostate specific antigen PSA)
for this important disease entity.

The objectives of this study are: (1) to confirm and extend
our previous serum findings by analyzing serum samples
remaining following routine serum PSA analysis from
both prostate cancer and non-prostate cancer patients; (2)
to identify potential confounds within the ELISA protocol
that may account for the contradictory findings reported
[14]; (3) to localize, quantify, and compare MIF mRNA
and protein amounts in matched cancer and benign
biopsy samples using commercially available tissue


arrays; (4) to localize the MIF receptor, CD74, within
prostate tissue and (5) to compare MIF secretion by nor-
mal prostate epithelial cells and prostate cancer cells in
vitro.

Methods
Approval for all facets of this study was obtained from the
Bay Pines Veterans' Administration Medical Center
(VAMC) Institutional Review Board and the study proto-
cols comply with the Helsinki Declaration. The serum
samples were obtained with waiver of informed consent
under section 46.116(d) of the Department of Health and
Human Services human subject regulations at 45 CFR 46.

Comparison of MIF serum amounts in prostate cancer and
non-cancer patients
Random serum samples remaining following routine PSA
evaluation prior to being discarded were collected (n =
212) and stored at -80C prior to analysis. Clinical PSA
values for the serum samples (determined by the radioim-
munoassay laboratory at the Bay Pines VAMC) and docu-
mentation of prostate cancer were obtained through
analysis of computerized records. Normal patients were
defined as those without documented prostate cancer and
with serum PSA values at or below 2 ng/ml (n = 158).

Samples were assayed for MIF using the protocol
described by the manufacturer (DuoSet ELISA, R&D Sys-
tems, Minneapolis, MN) with the following modifica-
tions: Milk diluent, (500-92-01, Kirkegaard and Perry
Laboratories, Gaithersburg, MD) was used at a 1:20 dilu-
tion as the blocking agent and serum diluent. Our previ-
ously published data set of MIF serum levels in prostate
CaP patients was also included for comparison (n = 61)
[13]. Determined values for serum PSA and MIF were not
normally distributed, therefore differences in serum PSA
and MIF amounts were assessed by Kruskall-Wallis
ANOVA followed by post-hoc tests (Dunn's multiple
comparison test). Significance was defined as p < 0.05.
Data are reported as median interquartile range. Statisti-
cal analysis was performed using Prism statistical analysis
software version 4.02 (GraphPad Software, Inc., San
Diego, CA).

Comparison of different ELISA protocols on MIF serum
determination
Michael et al. used the MIF DuoSet ELISA system to deter-
mine serum MIF [14]. However, there were several devia-
tions from the manufacturer's protocol and from our own
protocol, principally the use of bovine serum albumin
(BSA) as a blocking agent and serum diluent. Therefore, to
test whether differences in ELISA protocols result in dis-
crepant findings, in a separate experiment 10 random
serum samples (n = 5 CaP; n = 5 non-CaP) were assayed
in parallel using either milk diluent (our established


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protocol in agreement with the manufacturer's sugges-
tions) or PBS with 1% BSA (as reported by Michael, et al.
[14] as the blocking agent and serum diluent). All other
steps in the protocol were as described by the manufac-
turer (R&D Systems). Comparisons between different
ELISA protocols were evaluated using two-tailed unpaired
nonparametric t-tests (Mann-Whitney). Data are reported
as median + interquartile range.

MIF forms in human serum
The discrepancy between these ELISA data could be attrib-
uted to blockage of the monoclonal capture antibody
(mAb) epitope due to MIF interaction with other serum
proteins. Changes in the dilution buffer composition may
alter these interactions resulting in unmasking of capture
antibody binding sites. To address this possibility, serum
proteins (diluted 1:20 with PBS) were separated by native,
denaturing and reducing polyacrylamide gel electro-
phoresis following the manufacturer's protocols (4-20%
Tris-glycine or 4-12 % NuPAGE Bis-Tris gels, Invitrogen,
Carlsbad, CA) and transferred to Invitrolon (Invitrogen).
Reducing conditions included addition of anti-oxidant
(Invitrogen) to running buffer and transfer buffer to pre-
vent oxidation of reduced cysteine, methionine and tryp-
tophan residues. Blots were incubated overnight with a
biotinylated MIF specific, affinity purified, antibody
(BAF289, R&D Systems) at 1:1000 dilution overnight at
4C as described previously [15].

Serum MIF was immunoprecipitated using agarose-
immobilized ELISA capture mAb (R&D Systems antibody;
Profound co-immunoprecipitation system, Pierce, Rock-
ford, IL) to characterize further mAb MIF binding forms.
For immunoprecipitation studies, 2 ml of serum was first
cleared of contaminating IgG by passage through a Pro-
tein G column (Pierce) and 1 ml PBS fractions collected.
The eluted fraction with the highest protein content was
batch incubated with 200 ptl immobilized capture mAb
(200 |tg MIF mAb per 100 ptl of packed resin) overnight
with continuous rocking at 4C. Following overnight
incubation the agarose resin was transferred to a spin col-
umn, proteins that did not stick were collected, and the
column washed with 5, 1 ml PBS washes. The bound pro-
teins were eluted and proteins in all fractions separated
under denaturing and reducing conditions on 4-12%
NuPAGE Bis-Tris gels, and then transferred to Invitrolon
membranes following the manufacturer's protocols (Inv-
itrogen). In some instances, fractions with low total pro-
tein content were first concentrated 800 fold (Eppendorf
vacuum concentrator, Westbury, NY).

In all instances, MIF Western blots were performed using
a polyclonal biotinylated antibody at 1:1000 dilution fol-
lowed by strepavidin-horseradish peroxidase conjugate
used at a 1:500 dilution (R&D Systems). Antibody specific


bands were detected using a chemiluminescent substrate
(Pierce) and bands quantified as described previously
[151. Data are expressed as net intensity values normal-
ized to the staining intensity of a recombinant MIF stand-
ard (7.5 ng).

Determination of MIF and CD74 in matched prostate
cancer and benign samples
Tissue array slides were obtained from Ambion (Low den-
sity; Austin, TX) and used for both in situ hybridization
(MIF only) and immunohistochemistry (IHC; MIF and
CD74). The slides contained paired samples of prostatic
adenocarcinoma and matched benign specimens from the
same patient. The present investigation was restricted to
comparison of expression in matched benign versus can-
cer samples. Samples that could not be matched (e.g.
missing section on the slide) were not included in the
analysis.

In situ hybridization
RNase inhibitor (Protect-RNA, Sigma, St. Louis, MO) was
included in all aqueous solutions. Tissue sections were
dewaxed and rehydrated through ethanol, then treated
with Proteinase K for 30 min at 37C and endogenous
peroxidase quenched by 10 min incubation in 3% hydro-
gen peroxide. MIF biotinylated probes were prepared by
reverse transcription, followed by amplification of a 254
base pair (bp) MIF fragment [13]. T7 RNA polymerase
promoters were ligated to the PCR product (Ambion) and
single stranded RNA oligonucleotide probes produced by
in vitro T7 transcription (Promega, Madison, WI) with
incorporation of biotinylated-dUTP. Hybridized probes
were detected with avidin-peroxidase (ISH-B1, Sigma).
Negative controls consisted of hybridization of tissue
arrays with sense biotinylated probes and hybridization of
test prostatic tissue sections without probe addition. A
poly-d(T) biotinylated probe was used as a positive con-
trol in test prostatic tissue sections. Tissue arrays were not
counterstained.

Immunohistochemistry
Paraffin sections (4 ptm) of formalin-fixed tissue arrays
were dewaxed and processed for antigen retrieval (citrate
buffer, pH 6.0; microwave 600 W to boiling, 2 min rest,
and repeat cycle 10 times for a total time of 20 min). Sec-
tions were treated with 3% H202 for 2 min to quench
endogenous peroxidase, preincubated in normal donkey
serum (3%; 30 min) and then exposed to MIF bioti-
nylated polyclonal antibody (BAF289, R&D Systems;
1:1000 in PBS, 0.3% Triton X-100) or CD74 antibody (sc-
5438, Santa Cruz Biotechnology, Santa Cruz, CA; 1:400 in
PBS, 0.3% Triton X-100) overnight at 4 C.

Previous experiments demonstrated the specificity of MIF
labeling of prostatic tissues [12]. Specificity of CD74


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30-


120-


10-


0.1


vv
vvv
V
a v '



v V
v vv


0.2009


Normal


0 0

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CaP


Figure I
Scatter plots of PSA and MIF in random serum sam-
ples obtained following routine PSA testing. Median
values for each group are represented by horizontal lines.
Diagnosis of prostate cancer was determined by chart
review. *** = p < 0.001 A) Serum MIF -All data points were
mean values of duplicate ELISA analyses. B) Serum PSA All
data were obtained from computerized records of clinical
laboratory analysis.





immunostaining was confirmed on a separate set of pros-
tatic tissue slides by omitting the primary antiserum (data
not shown). Bound primary antibody was detected using
biotinylated secondary antibody (Vector Laboratories,
Burlingame, CA) and diamidinobenzidine (DAB) accord-
ing to the manufacturer's protocol. The slides were coun-
terstained with hematoxylin, dehydrated, cleared in
xylene and coverslipped.

All tissue sections were scored for the location and inten-
sity of staining (0 = no staining detected through 3 = very
strong staining). MIF mRNA and MIF/CD74 immunos-
taining was examined in matched tissue samples. Differ-


ences were assessed in epithelial MIF staining intensity in
matched versus adenocarcinoma samples using paired,
two-tailed t-tests.

Determination of MIF expression in normal and prostate
cancer epithelial cells in vitro
Normal prostate epithelial cells (PrEC, Cambrex, Walkers-
ville, MD) and prostate cancer cell lines (LNCaP and PC-
3; American Type Culture Collection, Manassas, VA) were
cultured until 80% confluent according to previously
described protocols [16]. Media were exchanged for
serum free PrEGM (Cambrex) and following 48 h incuba-
tion, samples of culture medium containing equal total
protein concentrations were analyzed by denaturing
Western blot as described above. The resultant protein
bands from three separate Western blots from each cell
line were quantified by digital imaging (Kodak, Rochester,
NY). Data are expressed as fold change compared to nor-
mal cells and differences assessed by unpaired, two-tailed
t-tests.

Results
Comparison of MIF serum amounts in prostate cancer and
non-cancer patients
We determined MIF serum levels in normal (non-CaP)
and prostate cancer (CaP) patients using milk based
buffer as the ELISA blocking agent and sample diluent.
Using this protocol, the median MIF serum level in CaP
patients (Fig. 1, CaP 1; 4.70 4.32 ng/ml; interquartile
range) was significantly greater than that observed in non-
CaP patients (2.19 2.65 ng/ml; Fig 1; <0.001). Thus, we
confirm our previous observation of increased MIF serum
levels in CaP patients [13].

Furthermore, we compared our previously analyzed can-
cer patients with our current group to detect any possible
differences. Our previous CaP group had a median MIF
serum level of 6.50 + 3.10 ng/ml (Fig. 1, CaP 2) that was
not statistically different from our current CaP group (Fig.
1, CaP 1; p = 0.495) although it was still significantly ele-
vated when compared to the control group (Fig 1; p <
0.001).

Since no significant differences were observed between
the two cancer groups, we have pooled the serum MIF lev-
els to result in a combined prostate CaP group. The con-
trols from our first study were from serum samples used
for thyroid hormone analysis (n = 14) and were not com-
bined with this normal group since the corresponding
PSA values are unknown [13]. Thus, our data show that
CaP patients have a significantly higher MIF serum level
(5.87 3.91 ng/ml, n = 115) when compared to non-can-
cer patients (2.19 2.65 ng/ml, n = 158) and this differ-
ence is statistically significant (p < 0.0001). All serum
samples were obtained from patients at least 15 months


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15-







10-


I= Normal
M CaP


JI I


Figure 2
Comparison of Serum MIF amounts determined by
different ELISA conditions. A subset of random serum
samples were from pulled from our serum bank from
patients with no documented diagnosis of prostate cancer (n
= 5) and from patients with CaP (n = 5). The serum MIF
amounts were determined in parallel using either a milk
based serum blocking/diluent solution or phosphate buffered
saline pH 7.4 with I % bovine serum albumin. Data are
expressed as median interquartile range, ** = p < 0.01,
when compared to Milk normal serum samples.




post-CaP diagnosis and median PSA serum amounts were
significantly increased in the CaP (1.20 4.60 ng/ml,
interquartile range, n = 115) patients (Fig. IB, p < 0.001)
suggesting that this group of patients had recurrent or
metastatic prostate cancer.

Comparison of different protocols on MIF serum
determination
We noted several differences between the MIF ELISA pro-
tocol of Michael et al. [14] and our serum MIF ELISA pro-
tocol. Since MIF's interactions with albumin subfractions
have been described [17], we tested the possibility that use
of BSA in the MIF ELISA protocol would interfere in the
MIF ELISA assay. Thus, we compared a subset of serum
samples from prostate cancer patients (n = 5) and from
patients with no documented prostate cancer (n = 5),
which were assayed in parallel using either milk based
buffer (our protocol) or PBS with 1% BSA (Michael et al.
[14] protocol) as the serum diluent and blocking agent.
Serum MIF levels were significantly increased, even in this
small sample, when we used the milk-diluent based MIF
ELISA (our protocol), with non-CaP having 4.18 0.99
ng/ml compared to prostate cancer patients that had
13.06 6.15 ng/ml of MIF in the serum (Fig. 2). However,
the same samples, when run using the BSA diluent (as
reported by Michael et al. [14]) showed no significant dif-
ference between the two groups (Fig. 2), in agreement


with their findings. Moreover, when the same set of nor-
mal sera was compared using milk-diluent versus BSA
diluent, it was observed that the BSA protocol resulted in
a statistically significant lower MIF serum level (1.84 +
0.73 for BSA compared to 4.18 + 0.99 for milk). These
data demonstrate that addition of BSA to the MIF serum
ELISA protocol results in not only interference with the
ability to detect an increase in MIF serum levels, but it also
decreases the ability of the assay to detect MIF (as evi-
denced by the significantly lower MIF serum levels
observed in patients without prostate cancer with the BSA
protocol).

MIF forms in human serum
The success of antigen capture ELISA reactions is depend-
ent upon accessibility of the epitope. Detecting antigen
bound to endogenous proteins in serum is often difficult
because of epitope masking or sensitivity problems. MIF's
association with other proteins (either as carrier proteins
or activators) has not received much attention although it
may shed light on the mechanism of action of this com-
plex molecule. While many studies have detected serum
MIF, to date there is no information on the MIF form(s)
in human serum. MIF Western blots under native condi-
tions showed only a large high molecular weight band in
the range of 150 to 500 kDa. (Fig. 3A). No monomeric,
dimeric or trimeric MIF was observed. Under denaturing
conditions, a single MIF immunoreactive band was
detected (180 kDa). Again no monomeric, dimeric or
trimeric MIF was observed. Under reducing conditions
only a single 12 kDa MIF band was detected, suggesting
that this MIF-protein complex is either held by covalent
bonds or that the reducing conditions alters the complex
conformation allowing the MIF to be released. The same
banding pattern was observed using a well characterized
MIF monoclonal antibody (III.D.9), suggesting that this
banding pattern was not the result of antibody cross reac-
tivity or non-specific binding (data not shown).

The ability of the ELISA mAb to detect all the MIF in serum
samples was analyzed by affinity chromatography with
the ELISA capture antibody followed by Western blotting
(Fig. 3C). Following removal of immunoglobulin by Pro-
tein G chromatography, serum MIF was found in a 180
kDa complex (Fig. 3C, Denatured, NS Protein G). The MIF
capture monoclonal antibody (Fig. 3C, Denatured, NS
MIF mAb) did not recognize this 180 kDa MIF form.
Affinity captured MIF was not detectable by Western blot
without concentration of the eluted fraction (Fig. 3C,
Denatured, Elution, Elution conc). The concentrated (800
fold) mAb captured MIF was separated into four bands of
less than 40 kDa (12, 24, 30 and 36) with the 24 kDa
band being the predominant form detected. The serum
concentration of thel80 kDa MIF form was 220 ng/ml,
while the concentration of the 24 kDa band was 1.5 ng/ml


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Figure 3
MIF Western blot of serum proteins. A. Native serum MIF Serum was diluted 1:20 in PBS and proteins were separated
under native conditions then analyzed for MIF by Western blot analysis. Under native conditions, a broad band between 150 -
500 kDa was detected. B. Denatured and reduced serum MIF Serum was diluted as in A and proteins separated under dena-
turing or reducing conditions. Denaturing conditions resulted in a single prominent band at 180 kDa and some minor bands
between 80 and 120 kDa. Under reducing conditions, a single prominent 12-kDa band and a minor band at 24 kDa were
observed. C. mAb Affinity purified serum MIF MIF containing serum proteins were purified by sequential affinity chromatog-
raphy through Protein G agarose, followed by MIF mAb affinity chromatography. Nonsticking proteins as well as elution frac-
tions were separated by NuPAGE gel electrophoresis under denaturing or reducing conditions, transferred to PVDF and MIF
containing bands identified using a polyclonal antibody (R&D Systems, 1:1000 dilution). Lanes left to right: NS Protein G non-
sticking fraction from Protein G column; NS MIF mAb non-sticking fraction from MIF monoclonal antibody column; Elution -
third elution fraction; Elution conc third elution fraction concentrated 800 fold; MIF standard 7.5 ng of recombinant MIF
protein. Numbers and lines to the left of each Western blot indicate location of molecular weight markers.


as determined by normalization of band intensities to the
MIF recombinant protein. Therefore, the MIF ELISA
appears to detect less than 1% of the total serum MIF
detected by Western blot.

The 180 kDa MIF complex was disrupted by reducing
agents as only the 12 kDa monomeric MIF form was
detected with addition of dithiolthreitol to the sample
(Fig. 3C, Reduced). Under reducing conditions the MIF
captured by the monoclonal antibody could not be
detected without concentration (Fig. 3C, Reduced, Elu-
tion, Elution conc).


Determination of MIF and CD74 in matched prostate
cancer and benign samples
Only matched samples of benign and adenocarcinoma
tissue were included in the final analyses (MIF in situ, n =
20; MIF IHC analysis, n = 20; CD74 IHC, n = 18). The
patient ages ranged from 59 to 94 years, with a median of
72.5 years. All tumors were TNM classification TI a or Tib.

MIF mRNA in the prostate
In situ hybridization of 20 matched samples of benign and
cancerous prostate tissue revealed minimal MIF mRNA in
the epithelium of benign samples, which was restricted to
the cytoplasm (Fig. 4A). Increased MIF mRNA was
detected in the matched cancer samples (Fig. 4B), which
was localized to the cytoplasm with some evidence of


Page 6 of 12
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Figure 4
Matched prostatic tissue staining. All panels are 400 x total magnification. A) In situ hybridization MIF in benign tissue B)
In situ hybridization MIF in matched cancer tissue C) Immunohistochemistry MIF in benign tissue D) Immunohistochemistry
- MIF in matched cancer tissue E) Immunohistochemistry CD74 in benign tissue F) Immunohistochemistry CD74 in
matched cancer tissue




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Figure 5
MIF in situ hybridization and IHC staining intensity in
prostatic tissue arrays. Mean values for each group are
represented by horizontal lines. Values are expressed as
mean standard error. A) MIF in situ intensity of epithelial
cell staining of matched benign and tumor regions of prostate
biopsies from prostate cancer patients (n = 20). B) MIF IHC -
intensity of epithelial cell staining of matched benign and
tumor regions of prostate biopsies from prostate cancer
patients (n = 20). C) CD74 IHC intensity of epithelial cell
staining of matched benign and tumor regions of prostate
biopsies from prostate cancer patients (n = 18). (*** = p <
0.001).


nuclear or perinuclear localization of the signal. The mean
in situ hybridization intensity of prostate cancer was 1.5,
significantly higher than in matched benign tissues whose
mean score was 0.5 (Fig. 5A; p < 0.001).

MIF protein location and intensity in the prostate
MIF immunostaining was readily observed in both can-
cerous and benign prostate in the 21 matched samples
(Fig. 4C and 4D). MIF protein was localized predomi-
nantly within the epithelium in both benign prostate and
cancer (Figs. 4C and 4D). The mean staining intensity
within the epithelium in benign samples was 1.4, while
that of the epithelium in adenocarcinoma was 1.5 (Fig.
5B). Stromal staining was evident in both benign and ade-
nocarcinoma matched samples with no significant differ-
ence in the mean staining score (data not shown).

CD74 location and intensity in the prostate
Weak CD74 immunostaining was detected in both benign
and prostate cancer tissues (Fig. 4E and 4F). The staining
was patchy and localized to epithelial cells. There was no
significant difference noted in staining intensity when
comparing matched benign (mean intensity score = 0.6,
Fig. 5C) and prostate cancer specimens (mean intensity
score = 0.4).

Determination of MIF expression in normal and prostate
cancer epithelial cells in vitro
Western blotting of cell culture medium from prostate
epithelial cells showed that all cell lines secreted only 12
kDa MIF into the PrEGM serum-free culture media (Fig.
6A). However, prostate cancer cells (LNCaP and PC-3)
showed significantly greater MIF secretion when com-
pared to the normal cell line (Fig. 6A). Analysis of the
resulting band intensities documents an 8 to 12 fold
increase in MIF amounts secreted by prostate cancer cell
lines compared with normal prostate cells (Fig. 6B).

Discussion
Recently attention has been focused on the possibility that
epithelial cell injury as a result of chronic inflammation
plays an important role in prostate carcinogenesis [18].
Thus, we proposed that MIF, a proinflammatory cytokine
constitutively expressed by the prostatic epithelium and
upregulated in prostate cancer, plays an important role in
this disease entity [7]. Our present findings confirm our
hypothesis since: 1) increased MIF serum levels were
observed in an additional group of prostate cancer
patients when compared to patients with no documented
prostate cancer; 2) MIF mRNA was increased in prostate
cancer tissue suggesting increased MIF synthesis in pros-
tate cancer; 3) CD74, recently described as a receptor for
MIF[11], is localized to prostatic epithelia; 4) prostatic
epithelial cells in vitro secrete MIF, with much greater
secretion observed in prostate cancer cells. Moreover,


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0Z 0
Z (i 3
Zi O
en2


Figure 6
MIF secreted by in vitro prostate cells. A) Western blot
of 48 h culture medium from 80% confluent normal, LNCaP
and PC-3 prostate cells. B) MIF bands were quantified by dig-
ital imaging and are expressed as fold increase compared to
normal. Data are presented as the mean standard error of
three separate Western blots. (* = p < 0.05).




increased MIF expression in prostate cancer has been
independently documented by other laboratories, further
supporting our hypothesis [19,20]. The possible utility of
MIF as a prognostic marker for CaP is suggested by the
finding that serum MIF amounts are statistically increased
in patients with CaP as long as 15 months post diagnosis.

Our current findings also illustrate that different proto-
cols, or even seemingly small variations of the same
protocol (such as choice of diluent), can have large effects
on the ability to detect serum MIF. When we compared
the same group of patients using our milk-diluent proto-
col (as recommended by the manufacturer) or a BSA-dilu-
ent protocol (as reported by Michael et al. [14]), we
noticed that we were not able to detect differences in MIF
serum levels of prostate cancer patients using the BSA-
diluent protocol. This observation agrees with the find-
ings of Michael et al. [14]. Furthermore, addition of BSA
appears to diminish the ability of the ELISA protocol to
detect MIF, since even the values in the same normal
group were markedly smaller in the BSA-diluent protocol
(reduced by 66%) when compared to our milk-diluent


protocol. Thus, it is likely that the difference in the choice
of diluent for the ELISA protocol accounts in part for the
discrepant findings of Michael et al. [141, and their inabil-
ity to detect significantly elevated MIF serum levels in
their prostate cancer group. Additionally, the post-diagno-
sis duration was much later for our cancer group, so it is
also possible that there may also be intrinsic group differ-
ences. However, the rather large discrepancy observed in a
parallel test of both protocols (Fig. 2) suggests that differ-
ences in the ELISA procedure alone account for a large part
of their negative findings. Particularly since MIF is docu-
mented to bind to subfractions of human albumin [17]
and that albumin is a known interfering protein in serum
ELISA [21], which appears to interfere with the determina-
tion of serum MIF amounts. In the aforementioned study,
BSA at concentrations as high as 2.5 mg/ml was not
shown to bind to immobilized recombinant MIF [17].
However, the human serum MIF ELISA as performed by
both Michael et al. [14] and as reported here (Fig. 2) uti-
lizes 4-fold higher concentrations of BSA and the MIF-BSA
binding in the ELISA would occur in solution. BSA inter-
ference is a likely explanation for the discrepant findings
as there were no other major differences in the serum dilu-
ent/blocking agent used including pH and ionic strength.

Interference in immunoassay is a well-documented phe-
nomenon, especially prevalent when the analyte is
detected in serum [21]. Matrix effects (defined as the sum
of the effects of all of the components in a system with the
exception of the analyte to be measured and including the
reagents used in the assay) can increase or decrease the
measured result [21]. In fact, cross-platform fluctuations
in serum PSA have also been documented [22]. These
interassay variations were attributed to operator variabil-
ity, technical errors and/or inadequate estimation of this
heterogeneous molecule and its complexes with other
proteins [22,23]. All of these factors probably also affect
determination of MIF serum levels and thus, strict adher-
ence to established protocols aimed at standardization
should allow for comparison of results between different
laboratories.

The physiologically relevant form of MIF serum remains
to be determined. However, in the serum it does not
appear to be predominantly free uncomplexed MIF (mon-
omeric, dimeric or trimeric) since these MIF forms were
not detected by WB under native or denatured conditions
(Fig 3A and 3B). The monomeric MIF form (12 kDa) and
a small amount of the dimeric form (24 kDa) were
detected in serum under reducing conditions (Fig. 3B).
Crystallized recombinant bioactive MIF assumes a
trimeric structure [24]. However, mixtures of all three
forms of recombinant MIF have been found in solution
[25]. The active in vivo produced MIF form(s) have not
been determined. However, it was recently reported that


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the physiologically active form of MIF, purified from a
human B-cell line was primarily dimeric (24 kDa) [26].
Based upon the results reported here the monomer,
dimeric and trimeric MIF forms would account for less
than 1% of the total MIF present in the serum as detected
by Western blotting. In addition, our data suggest that
ELISA assay conditions can alter the amount of MIF cap-
tured by the monoclonal antibody.

Interestingly, the present study also documents that
majority of MIF in the serum is associated in a high molec-
ular weight complex (180 kDa under denaturing
conditions) that cannot be captured by the monoclonal
antibody. We recently documented that rat MIF released
by the urothelium into the bladder lumen is also part of a
high molecular weight complex [27]. The rat MIF binding
protein was identified as the acute phase protein ax-1-
inhibitor 3 [27]. a(-1-inhibitor 3 is a rodent member of the
ax-2-macroglobulin protease inhibitor family that is found
in high concentration in serum [28]. In general, the ax-2-
macroglobulin protease inhibitor family is recognized as
carriers of a numerous cytokines and growth factors [29]
and it is believed that interaction of cytokines and growth
factors with ax-2-macroglobulin family proteins modu-
lates cytokine and growth factor biological activity [30].
Thus, identification of the serum complex protein may
provide information about physiologically relevant MIF
forms. The identities of the protein(s) in the human
serum complex are presently under investigation.

The present study is the first report to document MIF
mRNA expression in prostate epithelial cells and to reveal
altered MIF expression in cancer using the complementary
techniques of in situ hybridization and immunostaining
within matched samples from the same patient. Low lev-
els of MIF mRNA were restricted to the cytoplasm of pro-
static epithelium in benign samples, but the MIF mRNA
signal was significantly more intense in the cytoplasm of
prostatic epithelial cells in matched cancer samples, sug-
gesting that increased cytoplasmic MIF mRNA is associ-
ated with tumorigenesis. Data from our previous study
(using a much smaller data set) gave some indication that
increased MIF mRNA was associated with more invasive,
aggressive tumor [13]. In that study, using laser capture
microscopy, we reported that invasive epithelial cells
exhibited higher MIF mRNA amounts than benign epithe-
lium within the same prostatic biopsy specimen. In the
present study, we demonstrate a significant increase in
MIF mRNA associated with prostatic tumors compared
with matched benign tissue from the same patients, doc-
umenting that upregulation of MIF gene expression is a
hallmark of prostatic tumorigenesis (Fig. 1A).

This report demonstrates that MIF protein is localized to
both the malignant and matched histologically benign


epithelia in prostate cancer patients. This observation con-
firms our earlier findings [12], as well as those of other
investigators [31,32]. del Vecchio et. al determined a
decrease in MIF immunostaining in high grade tumors
[31]. These authors suggested that this finding may be the
consequence of a reduced MIF synthesis or of an
enhanced and altered secretion by tumor cells into the
surrounding stroma [31]. However, these studies local-
ized and quantified MIF protein only. The alternative
explanation for reduced MIF immunostaining is sup-
ported by the data presented here, namely, there is
increased MIF secretion by aggressive prostate cancer cells.
This hypothesis is based upon two separate sets of find-
ings: (1) increased mRNA expression in prostate biopsy
samples without a concomitant increase in cytoplasmic
protein in prostate cancer epithelium suggests that tumor
cells may secrete more MIF and (2) documented increased
MIF secretion by prostate cancer cell lines compared with
normal prostate epithelial cells in culture. We recognize
that there is no correlation between MIF immunohisto-
chemistry and serum levels. In fact, this anomaly is
described for serum PSA versus PSA immunohistochemis-
try [33]. Based upon these findings it is tempting to spec-
ulate that the increased MIF secretion by prostate cancer
cells is responsible for the increased serum MIF levels
observed in prostate cancer patients. Prostate cancer dis-
rupts acinar structure and function resulting in "leakage"
of proteins normally released into the acinar ducts (e.g.
PSA), into the stroma and then absorption into the circu-
lation [34]. It is possible that a similar mechanism
accounts for the increased MIF secretion by prostate can-
cer epithelial cells and increased MIF serum levels in can-
cer patients.

CD74 was identified as the receptor for MIF using in vitro
models [111. The primary function of this protein is to reg-
ulate peptide loading of exogenously derived peptides
onto major histocompatibility class II heterodimers, but a
small portion of the total cell CD74 content is expressed
on the cell surface [35]. However, neither the exact func-
tion of the cell surface CD74, nor the physiological rele-
vance of the interaction of CD74 with MIF is known
[11,36]. We recently documented that MIF associates with
CD74 and CD44 in an urothelial cancer cell line [10] and
in an in vivo model of bladder inflammation [15] suggest-
ing that the effects of MIF on the bladder are mediated by
interaction with both CD74 and CD44. If MIF-CD74
interaction is needed for MIF to affect prostate tumor
growth then CD74 should be localized within the pros-
tate. To our knowledge, ours is the first report to show that
CD74 is present in human prostate tissue. However, these
data do not show any significant change in CD74 amount
in tumor compared to matched benign prostate. Thus, the
physiological relevance of CD74 in prostate cancer
remains to be determined.


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Conclusion
In summary, we confirmed and extended our observa-
tions of increased serum MIF and MIF mRNA are associ-
ated with CaP. Serum MIF is predominantly part of a
complex with other proteins that undetected by ELISA
using a commercially available MIF mAb. MIF mRNA was
observed in the cytoplasm of all prostatic epithelia, but
was increased in matched cancer samples. In addition,
this study documents the location of CD74, the MIF
receptor, within prostate epithelial cells. Whether
increased serum MIF has predictive value that is inde-
pendent of known predictive markers such as rising PSA
values or Gleason score in prostate cancer awaits a larger
trial that compares these variables prospectively with PSA
levels and patient outcomes.

Competing interests
The authors) declare that they have no competing
interests.

Authors' contributions
KLMS participated in the design of the study, ELISA, West-
ern blotting, immunoprecipitations, statistical analyses,
performed in situ hybridizations and carried out all cell
culture studies. KAL photographed all tissue slides, scored
all in situ hybridizations and immunohistochemical
slides. PLV participated in the design of the study, ELISA,
Western blotting, immunohistochemical analysis and sta-
tistical analyses. All authors read and approved the final
manuscript.

Acknowledgements
Supported by the VA Merit Review Program and the Bay Pines Foundation
(KLM-S, PLV) and VA MREP (KAI). Michael Bellino, Rachel Lyle and Gary A.
Smith, Jr. provided technical assistance.

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