loss of uteroglobin expression in prostate cancer: relationship … · adenocarcinoma of the...

Vol. 3, 2295-2300, Dece,nber 1997 Clinical Cancer Research 2295

Loss of Uteroglobin Expression in Prostate Cancer: Relationship to

Advancing Grade1

Ashani T. Weeraratna, Jaime A. Cajigas,

Arnold Schwartz, Erik G. Enquist,

Michael J. Manyak, and Steven R. Patierno2

Departments of Pharmacology [A T. W., S. R. P.], Pathology [A. S.],

and Urology [J. A. C., E. G. E., M. J. M,], George Washington

University Medical Center. Washington, DC 20037

ABSTRACT

We have shown previously that the secretory protein

uteroglobin (UG) is highly expressed in normal human pros-

tate tissue but this expression is either lost or altered in

human prostate cancer cell lines. Treatment of these cell

lines with recombinant human UG inhibits their ability to

invade human reconstituted basement membrane by up to

90%, implying that the loss of normal UG expression may be

related to the invasive potential of prostate cancer. Because

invasion represents a critical step in metastasis, the expres-

sion patterns of UG could provide a unique and relevant

indicator of cancer progression. In this study, we present the

immunohistochemical analyses of fresh frozen prostate tis-

sues from surgical specimens taken from 50 patients.

Eight slides per patient were analyzed for UG staining.

Slides from 26 patients showed evidence of prostate cancer,

whereas slides from the remaining 24 patients showed only

benign glands. The results demonstrate UG immunoreactiv-

ity in normal prostate, benign prostatic hyperplasia, and

prostatic atrophy; low but clearly positive expression in

prostatic intraepithelial neoplasia; positive expression in

cancerous glands of Gleason’s pattern �2; and complete loss

of UG immunoreactivity in cancerous glands of Gleason’s

pattern 3 or greater. In addition, in the one case of meta-

static prostate cancer that we examined, the prostate cancer

cells within the lymph node lacked UG expression. These

findings suggest that the loss of UG expression may be an

indicator of prostate cancer progression and possibly a com-

ponent of the molecular natural history of prostate cancer,

which may have prognostic value.

Received 6/4/97: revised 8/25/97; accepted 9/17/97.

The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby marked

advertisement in accordance with 18 USC. Section 1734 solely to

indicate this fact.

� This work was supported by an award from the Elaine A. Snyder

Cancer Research Trust,2To whom requests for reprints should be addressed. at Department of

Pharmacology. George Washington University Medical Center. 2300 IStreet NW.. Washington. DC 20037. Phone: (202) 994-3286: Fax:

(202) 994-2870: E-mail: [email protected].

INTRODUCTIONAdenocarcinoma of the prostate is the most commonly diag-

nosed form of cancer among men in the United States today. The

number of cases diagnosed in 1996 exceeded 260,000, and that

figure is expected to climb to around 334,500 in 1997 (1). It is

projected that about 41 ,800 of these diagnosed cancers will result in

death due to metastatic progression. Genetic alterations such as the

loss of heterozygosity at several loci, including 6p, 7q, 8q, lOq,

1 lp, l3q, 16q, 17q, and l8q (2-8), the loss of putative metastasis

suppressor genes such as KAI-l (9) and CD44 (10), as well as

changes in cell adhesion mechanisms such as E-cadherinla-catenin

( 1 1), are thought to contribute to this progression. However, despite

the recent strides made in this area of study. it is generally accepted

that the molecular alterations related to the malignant development

of PC� require better characterization.

One such alteration may involve a protein known as UG.

UG is a low molecular weight, homodimeric secretory protein,

also known as Clara cell 10 kDa (12) protein, which is found in

abundance in human uterus, lung. and prostate (13-15). The UG

locus has been localized to a single-copy gene on chromosome

1 lql2.3-l3.l (16). This chromosomal region is of interest in

several hormonally regulated cancers ( 1 7) and has recently been

identified as an additional site of loss of heterozygosity in PC

(18). We have reported previously that UG expression is mark-

edly decreased or absent in several PC cell lines and that

recombinant human UG modulates prostate tumor cell invasive-

ness, possibly by a mechanism related to the inhibition of the

actions of PLA2 in the production of eicosanoids (19, 20).

This study sought to determine the immunohistochemical

expression of UG in normal human prostate. BPH, PIN, and PC

tissue and to assess the possibility that alterations in UG ex-

pression may be associated with prostate cancer.

MATERIALS AND METHODSTissue Source. Informed consent was obtained to study

tissue from 50 patients who underwent radical prostatectomy for

PC, cystoprostatectomy for transitional cell carcinoma, or open

prostatectomy for BPH. Because a random sampling of cancer

patients was desired, there was no inclusion or exclusion criteria

before acquisition of these tissues. It is noteworthy. however,

that none of these patients had prior endocrine treatment. Ap-

proximately two longitudinal specimens, of 1-cm each, extend-

ing from base to apex were excised parallel to the urethra and

flash frozen in liquid N, and stored at -70#{176}C. Lymphatic tissue

containing PC was obtained from one laparoscopic pelvic lymph

node dissection prior to a planned radical prostatectomy. Of 46

specimens resected for prostatic carcinoma, 26 contained carci-

3The abbreviations used are: PC, prostate cancer: UG. uteroglobin:

PLA,, phospholipase A,; BPH, benign prostatic hyperplasia; PIN, pros-

tatic intraepithelial neoplasia.

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2296 Uteroglobin Expression in Prostatic Cancer

noma within the tissue samples obtained intraoperatively for UG

determination. Tissue from the remaining 24 specimens con-

tamed benign prostatic tissue, prostatic atrophy, or fibromuscu-

lar tissue. The 26 cases with sampled carcinoma in the intraop-

erative sample had a similar histopathological tumor pattern to

that present in the resected prostate. All 46 prostatectomy cases

with preoperative diagnosis of PC contained carcinoma in the

final surgical pathological review of the resected organ.

Immunohistochemical Procedures. Two hundred serial

sections of 5 p.m each were taken from each tissue and placed on

silanated slides (two sections per slide). Every tenth slide was

stained with hematoxylin and diagnosed by a pathologist to deter-

mine which serial sections exhibited BPH, PIN, or cancer. This was

to determine which slides contained both normal prostatic glands

(as a positive control) as well as adjacent cancerous glands. The

remaining slides were stored at -20#{176}Cuntil diagnosis was com-

plete. Selected slides were immunostained as follows. Sections

were fixed with 4% formaldehyde for 10 s and then blocked with

a 1:100 dilution of mouse serum (SIGMA Immunochemicals, St.

Louis, MO) for 30 mm at room temperature. Slides were then

incubated overnight at 4#{176}Cwith a 1:100 dilution of rabbit anti-

human UG antibody, and corresponding serial sections used as a

negative control were incubated with a 1 : 100 dilution of nonim-

munized rabbit serum (BABCO Laboratories, Inc., Berkeley, CA).

The sections were washed three times for 10 mm each in PBS and

then incubated with a dilution of 1:1000 biotin-conjugated mouse

anti-rabbit IgG (SIGMA Immunochemicals) for 30 mm at room

temperature. The sections were washed three times in PBS for 10

ruin, then incubated with streptavidin complex (DAKO, Carpinte-

rim, CA) for 30 mm, and washed again twice with PBS for 10 mm.

A 5-s immersion in chromogen (liquid DAB, DAKO) was fol-

lowed by counter staining with hematoxylin.

Antibody Preparation. A peptide comprised of the 20

amino acid residues 37-56 of human UG was synthesized by

Genemed (San Francisco, CA). This peptide was conjugated to

keyhole limpet hemocyanin and used to raise antibody in rabbits

by the Berkeley Antibody Company (Berkeley, CA). The spec-

ificity of this polyclonal serum to human UG was confirmed and

titred by ELISA and used in the immunostamning of prostate

tissue specimens and performance of Western blot analysis.

Western Blot Analysis. Slices of tissue (-50 mg) adja-

cent to the areas of tissue used for immunohistochemistry were

homogenized in protein lysis buffer [50 mrvi Tris Base, 350 mrvi

NaCl, 0.1% volume NP4O, 5 mr�i EDTA (pH 7.4), containing 2mM PMSF, 20 �xg/ml aprotinin, and 1 m�i NaOV] using an

Ultratorrax homogenizer (Tekmar, Cincinnati, OH), three times

for 30 5 each. Cell debris was pelleted out by centrifugation at

8000 rpm for 20 mm. The supernatant was then filtered through

a lOO-kDa molecular weight cut-off Centricon-100 spin filter

(Amicon, Beverly, MA) by centrifugation for 1 h at 2300 rpm at

4#{176}C.The lysates were then quantitated using a Bio-Rad protein

assay (Bio-Rad, Hercules, CA), and 75 �i.g of the proteins below

Mr l00�000 were heated for 3 mm at 95#{176}Cin an equal volume

of sample buffer (4% SDS, 100 mM Tris-HCI, 20% glycerol,

200 mst DTT, 0.2% w/v bromphenol blue, with 0.5% �-mer-

captoethanol) and loaded on to a 15% denaturing SDS-poly-

acrylamide gel and subjected to electrophoresis for 40 mm at 65

V. The proteins were transferred onto 0.2-p.m polyvinylidene

difluoride membrane (Bio-Rad, Hercules, CA) by electrotrans-

fer in 10 mM 3-(cyclohexylamino)ethanesulfonic acid buffer

(pH 1 1 .0) with 20% methanol for 10 mm at 350 mA. The blot

was then blocked overnight at 4#{176}Cin 1 % BSA. Rabbit anti-

human UG at a 1 : 100 dilution in 1% BSA in 1 X TBS-T (0.02

M Tris base, 0. 137 M NaCI, and 0.1 % Tween 20, pH 7.6) was

used to primary stain the blot for 1 h at room temperature. The

blot was then washed four times in 1 X TBS-T for 15 mm each.

It was then stained with secondary antibody. peroxidase-conju-

gated donkey anti-rabbit IgG (Amersham Corp., Arlington

Heights, IL) for 1 h at room temperature, and washed again as

above. The presence of UG was detected using the ECL system

(Dupont NEN, Boston MA). This method of detection was

modified by using a 1:2 dilution (in 1 X TBS-T) of the reagents

to minimize background staining. The blot was then exposed to

autoradiography film for 20 s.

RESULTSPatient age in the 28 Caucasian and 22 African-American

males ranged from 35 to 76 years with a median of 60.8 years.

Forty-six patients had a preoperative histopathological diagnosis

of prostate adenocarcinoma (26 Caucasians and 20 African-

Americans), whereas 4 patients (2 Caucasians and 2 African-

Americans) had no malignancy of the prostate and underwent

surgery either for bladder carcinoma ( I Caucasian and 1 Afri-

can-American) or for BPH (Table 1 ). These diagnoses were

confirmed in histopathological examination of the surgical spec-

imens. Of the 26 patients (15 Caucasians and 1 1 African-

Americans) with carcinoma within the sections we examined,

two had associated PIN. Two samples contained only fibromus-

cular tissue. The remaining 22 specimens tested contained only

BPH or prostatic atrophy in the sample. However, the final

surgical pathological diagnosis of these radical prostatectomies

was adenocarcinoma with Gleason scores ranging from 4 to 10,

with 16 patients determined to have moderately differentiated

tumors (Gleason score from 4 to 6) and 30 patients with poorly

differentiated tumors (Gleason score from 7 to 10). The latter

included the patient with lymph node metastasis (Gleason 10).

A total of 400 slides from the 50 specimens was evaluated,

and UG expression in each slide was scored by three investiga-

tors (A. T. W., J. A. C., and A. S.). The degree of UG expres-

sion was graded by assigning a value of 0 to an absence of stain

and assigning a value of 3+ to the most positive stain. Slides

that stain with intensity between these two extremes were ac-

corded intermediate values. Glands showing an absence of stain

were uniformly marked as 0 by all three investigators. Interme-

diate scores were usually identical and never differed by more

than a single value. Two of three scorers were blinded as to the

source of the tissue. Immunohistochemical stain was also eval-

uated and scored according to distribution as focal or diffuse. In

cases of suspected minimal disease, tissue sections were ob-

tamed from sites adjacent to the localized disease and did not

always contain tumor. Of the 50 patient samples examined, we

found we were able to analyze slides with cancer from 26. Two

of these latter slides contained only cancer, with no evidence of

any normal glands, and one contained cancer and PIN with no

evidence of any benign glands. Two samples we examined

contained only fibromuscular tissue, which lacks staining for

UG. Therefore, we found 45 samples that contained benign



Clinical Cancer Research 2297

Table I Patient data

Summary of patient characteristics according to age. race, and final

pathological diagnosis as determined by surgical pathological examina-

tion of the resected organ. Also included in this table is a summary ofthe differing pathologies seen among the various fields of view indifferent patients at the time of slide analysis.

Detail

a = 50 No. of patients

Age Range 35-76 yr

Median 60.8 yr

Race African-AmericanCaucasian

22

28

Final pathological BPH 4

diagnosis” Bladder carcinoma 2

PIN

PC Gleason’s grade 5






5

4

12

20

2

7

1

Slide diagnoses” BPH

Prostatic atrophy

PIN

PC Gleason’s pattern’ �3

PC Gleason’s pattern �2

Fibromuscular tissue only

42

3

2

20

6

2

(1 The two patients with bladder carcinoma had a final prostaticdiagnosis of BPH. PIN was associated with PC in all five cases; thus n =

PC + BPH = 50.

h Patterns of glands and cells in the field of view at the time of slide

analysis. The numbers here represent ii (patients) of a total of 50 in

which these pathologies were observed.‘ See footnote 4 in text.

tissue, of which 42 contained benign glandular hyperplasia and

three had prostatic glandular atrophy. Because of the difficulty

of both finding and diagnosing PIN on frozen sections, the

number of specimens in which PIN was detected remained low.

UG expression in benign tissue was predominantly diffuse

and moderate in most cases. In 42 cases of histologically con-

firmed BPH, there was one that stained 3+, 25 that stained 2+,

and 19 that stained 1 + for UG. In three cases of prostatic

atrophy, two stained 2+ and one stained 1 +. All were clearly

positive (1 + or greater). The staining pattern was cytoplasmic

and tended to be more pronounced at the luminal surface of the

acinar cells (Fig. 1 . A and B). Immunoreactivity was absent in

sections of the prostate that lacked epithelial elements, such as

fibromuscular tissue (Fig. 1A). The two cases of PIN examined

for the expression of UG were both clearly positive and were

scored as 1+ (Table 2, Fig. IC’).

Evaluation of slides containing PC contrasted sharply with

normal, BPH, or PIN samples. Twenty of 26 specimens con-

taming cancer did not stain for UG. Each of these cancerous

glands was diagnosed as Gleason’s pattern4 of 3 or above (Fig.

1, C and D). This absence of stain was consistent among all

4 The Gleason’s number referred to here is that of the particular glandswithin the field of view at the time of slide diagnosis and refers to the

pattern of the individual glands as opposed to the overall Gleason’s

grade of the patient.

cancerous glands of this Gleason’s pattern. Five samples con-

tamed cancerous glands that were graded with a Gleason’s

pattern 1 or 2, and these were positive for UG staining. One

sample contained several glands ofGleason’s pattern 3/4 as well

as glands that were Gleason’s pattern 2. The Gleason’s pattern

3/4 lacked staining for UG (Fig. 1E, left), whereas the Gleason’s

pattern 2 and the normal glands within the sample stained

positive for UG (Fig. 1E, right). The prostate tumor cells in the

metastatic lymph node likewise registered no signal (Fig. lF).

Western analysis was performed on prostate tissue from six

ofthe patients in this study presenting with cancers of Gleason’s

grade 8 or 9. The UG protein runs at around Mr I 0,000 on an

SDS polyacrylamide gel (21). This protein was present in nor-

mal tissue lysates but absent in the lysates taken from cancerous

tissue (Fig. 2).

DISCUSSION

We have previously reported our discovery of abundant

expression of UG in the epithelial cells of the human prostate

gland (15). In that preliminary description, there was no analysis

of the comparative expression of uteroglobin in tissue sections

exhibiting different pathological conditions. This study exam-

ines UG expression in fresh frozen cellular fields of BPH, PIN,

and prostate cancer. We report that UG was absent from pros-

tatic stromal tissue but was clearly expressed in normal glandu-

lar epithelial cells, in glandular areas exhibiting BPH and pros-

tatic atrophy. in acinar glands identified as PIN, and in

cancerous glands of Gleason’s pattern 2 or lower. In contrast,

UG expression in glands of Gleason’s pattern 3 or higher was

absent. In addition, in Western analysis of six patient samples of

Gleason’s grade 8 or higher, the expression of the UG protein is

undetectable as compared to normal tissue. This observation

links the loss of UG expression with the pathological pattern of

prostatic neoplasia.

The loss of normal UG expression in fresh frozen tissue

samples of PC shown here corroborates our observations of

human prostatic tumor cell lines derived from metastases ( I 9,

20). The cell lines DU14S. TSUprl, and PC-3 were analyzed for

UG mRNA expression by Northern blotting and compared with

normal tissue. Normal prostatic cells expressed an abundant

600-bp mRNA transcript, whereas UG expression in the cell

lines DU145 and PC-3 was absent. In TSUpr1 cells, an over-

expressed but aberrantly processed (> lO-kilobase pair) iran-

script was detected (20).

The absence of UG expression in prostatic tumor cells is

also consistent with reports on the expression of the Clara Cell

10 kDa protein (CC/U) gene. which is believed to be UG (21),

in lung tissue. Broers et al. (14) and Linnoila et al. (22) showed

that CC/U mRNA was abundantly expressed in normal bron-

chial tissue but was detectable in only I of 19 non-small cell

lung carcinoma specimens. A large percentage of non-small cell

lung carcinoma (primarily adenocarcinomas) exhibit ultrastruc-

tural and morphological characteristics indicative of peripheral

airway cell origin (22). Because CC/U was a strong positive

marker for normal peripheral airway cells, it is likely that the

low percentage of CC/U-positive tumors was due to the loss of

CCIO expression during tumorigenesis. Sandmoller et a!. (23)

also supported this observation by fusing the 5’ flanking region



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Fig. 1 Expression of UG in human prostate tissue. In A, UG expression in benign glandular hyperplasia is abundant and is found along the luminal surface

of the acinar cells. Fibromuscular elements lack staining for UG (X40). In B. UG expression is also abundant in normal prostatic glands undergoing atrophy(X200). In C, UG expression is present in PIN (gland on the left), although it is somewhat diminished from BPH (center). In glands that are PC of Gleason’spattern �3 (right). UG expression is absent (X 100). D, contrast between positive UG staining in benign tissue (left) and the lack of UG staining in PC glands

of Gleason’s pattern �3 (right, arrow; X 100). In E, in a single patient sample, regions of Gleason’s pattern �2 stained positive for UG (rig/it), whereas

glands on the same tissue section that were Gleason’s pattern �3 (left) were negative for UG expression ( X 200). In the patients studied, UG staining wasconsistently positive in sections of tissue that were of Gleason’s pattern �2 and consistently negative in those that were Gleason’s pattern �3. In F, UG

expression is absent in prostate tumor cells that have metastasized to the regional lymph nodes (X 100).

of the UG gene to SV4O T antigen to target SV-40-induced the UG gene may be transcriptionally silenced as a tumor

tumorigenesis to tissues that normally express abundant endog- progresses past its earliest stages.

enous UG such as lung, salivary gland, stomach, and prostate. The significance of the loss of UG expression in tumor

Endogenous production of UG in SV-40 T antigen-transformed cells is presently unknown. During development and in the adult

cells was clearly detectable during the early stages of tumori- genitourinary tract, its expression is a function of hormone

genesis but was undetectable in later stages. This suggests that receptor expression and hormonal status. Therefore, it is possi-



Clinical Cancer Research 2299

A. T, Weeraratna. J. Leyton. M. J. Manyak, and S. R. Patierno,

script in preparation.

Table 2 Staining for UG expression

Staining of the 50 patient samples ftr UG can be summarized as

follows. Of 50 samples. 2 contained only fibromuscular tissue, which

does not express UG. and therefore were excluded from this table. Two

samples contained cancer (Gleason’s pattern �3) but no benign tissue,

and one more sample contained cancer (Gleason’s pattern �3) and PIN

but no benign tissue. Thus, the 23 remaining cancers (17 Gleason’s

pattern �3 and 6 Gleasons pattern �2) form a subset of45 samples that

contain benign tissue. Cancers were divided into those of Gleasons

pattern �2. which show positivity for UG, and those of Gleasons

pattern �3, which lack staining for UG. Immunoreactivity is ranked as

in the previous table. where absence of staining is marked as negative,

or 0, low positivity is + . moderate positivity is + + . and strong posi-

tivity is +++

Negative

(0)

Low

(+)

Moderate

(++)

Strong

(+++)

Benign 19 25 1

PIN 2Cancer (�2) 5 1

Cancer (�3) 20

ble that the loss of UG expression may be an indicator of the

stage of differentiation of an evolving tumor. On the other hand,

there is a growing body of evidence that indicates that loss of

UG expression may be a functional component in the molecular

natural history of prostate cancer. We have reported that treat-

ment of human prostatic tumor cell lines (which lack normal

endogenous UG) with submicromolar nontoxic doses of recom-

binant human UG results in inhibition (by up to 90%) of the

ability of those cell lines to invade through reconstituted base-

ment membrane in response to fibroblast-conditioned medium

( 19). The ability of UG to inhibit invasiveness suggests that the

loss of normal UG expression may correlate with the acquisition

of metastatic potential because invasion of the local tissue

matrix is a necessary component of metastasis. Furthermore. the

genetic or pharmacological reconstitution of UG to UG-defi-

cient cells may have therapeutic value in suppressing metastasis.

Experiments to test this hypothesis are in progress.

Although the existence of UG has been known for almost

30 years (24). its physiological function and mechanism of

action remains unknown. We have found that recombinant UG

inhibited fibroblast conditioned medium-stimulated tumor cell

invasion but did not affect simple cell motility.5 Therefore, it is

possible that the ability of UG to interfere with cell motility may

be cell type specific or may itself be altered as a function of

tumorigenicity. It has also been shown that UG noncompeti-

tively inhibits the activity of PLA2 in vitro (25. 26), which may

account for the potential anti-inflammatory activity of UG, but

it is not known whether this inhibition occurs in viva. We have

shown that treatment of prostate tumor cell lines with recombi-

nant UG inhibits the biphasic release of arachidonic acid that

was also induced by fibroblast conditioned medium (19). This

may be due to inhibition of cell-membrane associated PLA2

activity. but presently the relationship between the inhibition of

arachidonic acid release and the inhibition of invasion is merely

p 14

1#{188}t 1#{188}#{149}d� 1#{188}1#{188}1#{188}NG9G9G9G9 G9 G8

Fig. 2 UG expression in human prostate tissue as determined by Western

analysis. The UG protein runs as a Mr 10,000 band on a 15% SDS-

polyacrylamide gel (N). In five patient samples of Gleason’s grade 9 (G9)

and one of Gleason�s grade 8 (G8). there is no detectable UG expression.

correlative. It remains to be seen whether either intracellular or

extracellular metabolites of arachidonic acid are the proximate

activators of invasive motility in these cells, or whether, alter-

natively, UG acts through a specific receptor and signaling

mechanism to regulate genes governing invasive motility. An

alternate mechanism is suggested by the observation by Zhang

et a!. (27) that UG binds to fibronectin. Thus, it is possible that

UG binding to fibronectin may also modulate the role of (1-

bronectin in cell-matrix attachment and invasive motility.

With an aging population. PC will continue to have an

impact on health care well into the next century. Over 50% of

patients undergoing radical prostatectomy have locally ad-

vanced disease, and 25% of these will eventually exhibit disease

progression. Clearly, there is a need to develop prognostic

markers that will help predict individual outcome and thereby

direct appropriate therapy. If UG proves to be an autocrine/

paracrine inhibitor of localized cellular invasiveness, then the

degree of loss of UG expression in biopsy specimens may be

predictive of increased potential for metastasis. In contrast,

positive expression of UG, such as we observed in PIN, may

indicate an earlier stage in the continuum from neoplastic

change to wide dissemination. Differential UG expression by

PIN may distinguish those cases with a predilection for invasion

from those with a less aggressive course. This hypothesis is

presently being tested by expanding this study to include ar-

chived specimens, where PIN can be readily selected from

among paraffin sections used for pathological diagnosis.

ACKNOWLEDGMENTSWe thank Steven A. Seltzer for generous support; Drs. Gary B.

Loden, Erin McCormick, David Koota, Frederick Hendricks, andThomas Jarrett for assistance with tissue collection: Dr. Sana Tabbara

for assistance with pathological diagnosis; Eileen Feeney for assistance

with patient data collection: Caliope Sarago. Susan Gani. Kristina

Kligys. and Levi Norton for technical assistance; and Dr. Mary-Ann

Stepp for valuable advice.

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1997;3:2295-2300. Clin Cancer Res A T Weeraratna, J A Cajigas, A Schwartz, et al. to advancing grade.Loss of uteroglobin expression in prostate cancer: relationship

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