Localization of Transforming Growth Factor-β1 and Receptor mRNA after Experimental Spinal Cord Injury

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 DOI: 10.1177/002215549804600312

1998 46: 379J Histochem CytochemMichael J. Gerdes, Melinda Larsen, Lauren McBride, Truong D. Dang, Bing Lu and David R. Rowley

Prostate and Carcinoma Tissues1 and Type II Receptor in Developing Normal HumanβLocalization of Transforming Growth Factor-

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Volume 46(3): 379–388, 1998The Journal of Histochemistry & Cytochemistry

http://www.jhc.org

© The Histochemical Society, Inc.

0022-1554/98/$3.30

379

ARTICLE

Localization of Transforming Growth Factor-

b

1

and Type II Receptor in Developing Normal Human Prostate andCarcinoma Tissues

Michael J. Gerdes, Melinda Larsen, Lauren McBride, Truong D. Dang, Bing Lu,and David R. Rowley

Department of Cell Biology (MJG,LM,TDD,BL,DRR) and Cell and Molecular Biology Program (ML,DRR), Baylor College of Medicine, Houston, Texas 77030

SUMMARY

Transforming growth factor-

b

1

(TGF-

b

1

) is implicated in prostate develop-ment, and elevated expression of TGF-

b

1

has been correlated with prostate carcinogenesis.In this study, cell type specificity of TGF-

b

1

and TGF-

b

receptor Type II (RcII) protein expres-sion was determined by immunocytochemistry in human normal prostate and compared toprostate carcinoma tissues. Heterogeneous localization patterns of LAP-TGF-

b

1

(TGF-

b

1

pre-cursor) and RcII were observed in both epithelial and mesenchymal cells in fetal prostate,with LAP-TGF-

b

1

localizing to more basal epithelial cells. Homogeneity of LAP-TGF-

b

1

stain-ing was increased in neonatal, prepubertal, and adult prostate, with elevated immunoreac-tivity noted in epithelial acini relative to stromal tissue for both LAP-TGF-

b

1

and RcII pro-teins. In stromal tissues, RcII cell localization exhibited staining patterns nearly identical tosmooth muscle

a

-actin. In prostate carcinoma, LAP-TGF-

b

1

localized to carcinoma cells withan increased staining heterogeneity relative to normal prostate. In contrast to normal epi-thelial cells, carcinoma epithelial cells exhibited low to nondetectable RcII staining. Stromalcell staining patterns for LAP-TGF-

b

1

and RcII in carcinoma, however, were identical tothose of normal prostate stromal cells. These studies implicate both epithelial and stromalcells as sites of TGF-

b

1

synthesis and RcII localization in the developing and adult normalhuman prostate. In addition, these data indicate a loss of epithelial expression of RcII con-current with altered LAP-TGF-

b

1

expression in human prostate carcinoma cells.

(J Histochem Cytochem 46:379–388, 1998)

T

GF-

b

1

is a member

of an extended gene family in-volved in regulation of growth and differentiation(Kingsley 1994). TGF-

b

1

is synthesized as a 390 amino-acid precursor which contains an amino-terminal prosegment (latency-associated peptide, LAP) and a car-boxy-terminal 112 amino-acid TGF-

b

1

monomer (Miya-zono et al. 1991; Kingsley 1994). TGF-

b

1

transducessignal through interaction with hetero-oligomeric com-plexes of Type I and Type II receptors that are mem-bers of the serine/threonine kinase family (Lin et al.1992; Ebner et al. 1993; Franzen et al. 1993). TGF-

b

1

regulates diverse biological activities in developingand adult tissues, including cell proliferation, epithe-lial and stromal cell differentiation, and modeling ofextracellular matrix (Pepper et al. 1990; Torre–Ami-one et al. 1990; Yang and Moses 1990; Sporn andRoberts 1992; Kingsley 1994). However, the biologi-cal consequence of TGF-

b

1

action in the developinghuman prostate is not yet known.

TGF-

b

1

has been implicated in several aspects ofcarcinoma progression, including an elevated metastaticpotential and masking of tumor cells from immunesurveillance (Welch et al. 1990; Steiner and Barrack1992; Chang 1995; Pierce et al. 1995). Expression ofTGF-

b

1

message and protein is elevated in manytransformed cell lines and carcinomas, including pros-tate cancer, and elevated expression has been corre-

Correspondence to: David R. Rowley, PhD, Dept. of Cell Biol-ogy, 466A, Baylor College of Medicine, One Baylor Plaza, Houston,TX 77030.

Received for publication March 27, 1997; accepted September30, 1997 (7A4284).

KEY WORDS

TGF-

b

1

TGF-

b

receptor type II

prostate

human

cancer

development

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380

Gerdes, Larsen, McBride, Dang, Lu, Rowley

lated with metastatic progression (Merz et al. 1991;Thompson et al. 1992; Eklov et al. 1993; Truong et al.1993; Steiner et al. 1994; Van Laethem et al. 1995).However, many carcinoma cell lines exhibit a decreasedresponse to TGF-

b

1

(Huggett et al. 1991; Chang et al.1993; Rodeck et al. 1994; Herman and Katzenellen-bogen 1994), shown in some cases to result from de-fects in the TGF-

b

receptor Type II (Rc II) (Park et al.1994). Hence, it has been postulated that paracrine ef-fects of carcinoma-derived TGF-

b

1

on adjacent stromacould lead to an increased metastatic capability throughregulation of stromal cell-mediated events (Pierce etal. 1995). Accordingly, the cell type localization ofTGF-

b

1

and TGF-

b

receptors is important to under-stand TGF-

b

1

mechanisms in normal developmentand cancer progression.

Localization of TGF-

b

1

mRNA and protein in therodent has shown epithelial cell expression in bothnormal prostate gland and the Dunning R3327 PAPtumor (Lindstrom et al. 1996). In human subjects,previous studies to assess the cell-type specificity ofTGF-

b

1

protein expression in prostate carcinoma havemet with equivocal results. The use of the LC anti-body to TGF-

b

1

(Carruba et al. 1994) has shown in-tracellular staining in prostate carcinoma epithelialand stromal cells (Truong et al. 1993; Steiner et al.1994). However, this antibody may recognize onlycells positive for cell-bound or intracellularly activatedTGF-

b

1

(Barcellos–Hoff et al. 1994). Use of the CCantibody, which recognizes extracellular TGF-

b

1

(Car-ruba et al. 1994), has shown extracellular localizationprimarily in stromal regions adjacent to carcinoma acini.Because TGF-

b

1

is secreted as a latent form, thesestudies have not yet established the cell type(s) synthe-sizing TGF-

b

1

in human prostate carcinoma. How-ever, the development of an antibody to the LAP re-gion of human TGF-

b

1

precursor (Barcellos–Hoff etal. 1994) makes possible the definitive localization ofcells synthesizing TGF-

b

1

protein.Similar studies to examine expression of TGF-

b

re-ceptors in prostate carcinoma has been recently re-ported by several groups (Kim et al. 1996; Williams etal. 1996; Guo et al. 1997). These studies correlate aloss of receptor expression coordinate with carcinomaprogression. An alteration in receptor expression pat-tern in carcinoma relative to receptor and TGF-

b

1

ex-pression patterns in developing human fetal and neo-natal prostate and in normal adult prostate, however,have not been examined in a comprehensive manner.

The present study was conducted to determine cell-type specificity and differential staining patterns ofcellular LAP-TGF-

b

1

precursor and RcII protein in de-veloping and normal adult human prostate tissue incomparison with prostate carcinoma. The results pre-sented here characterize fetal to adult expression pat-terns and show alterations in LAP-TGF-

b

1

and RcII

staining patterns in carcinoma cells relative to normalprostate epithelial cells, with no apparent alteration instromal cell staining patterns for either protein.

Materials and Methods

Tissue Preparation

Carcinoma tissue was removed from patients undergoingprostatectomy for treatment of prostate cancer. Prostate tis-sues were provided by the Scott Department of Urology andthe Department of Pathology, The Methodist Hospital, Bay-lor College of Medicine. Normal adult prostate glands wereremoved from cadaver donors. Consent and tissue process-ing procedures followed human tissue use protocols estab-lished in the SPORE (Specialized Program of Research Ex-cellence) in prostate cancer at Baylor College of Medicine.Fetal and neonatal prostate tissues were processed and pro-vided by Dr. Edith Hawkins (Texas Children’s Hospital,Houston, TX). After removal, whole adult glands were re-producibly sectioned in samples, regions P1–P15 as previ-ously described (Wheeler and Lebovitz 1994), and all tissueswere fixed in 10% neutral buffered formalin at room tem-perature (RT) overnight, processed through dehydration,and embedded in paraffin blocks according to routine histo-logical procedures (Bratthauer 1994). Serial sections (5

m

m)were made by routine microtomy from multiple regions ofan individual sample, applied to poly-

l

-lysine-coated glassslides, and baked at 37C following standard histologicaltechniques (Bratthauer 1994). Representative sections fromeach set of serial sections were stained with hematoxylin–eosin for orientation and histopathological examination be-fore immunohistochemistry. Continuous sets of serial sec-tions were used for all subsequent immunohistochemistryand compared with hematoxylin–eosin-stained slides.

Immunohistochemistry

Antibody to human LAP-TGF-

b

1

(no. AB-246-PB) was ob-tained from R & D Systems (Minneapolis, MN). This antise-rum was prepared to recombinant human LAP as immuno-gen and is monospecific for the human LAP pro-segmentTGF-

b

1

precursor with no crossreactivity to LAP-TGF-

b

2

precursor or other cytokines or proteins (Barcellos–Hoff etal. 1994). This antibody has been shown to specifically rec-ognize intracellular LAP-TGF-

b

1

precursor in human cellsby immunohistochemistry and Western analysis (Barcellos–Hoff et al. 1994). Antibody specific to human RcII (no. 06-227) was obtained from Upstate Biotechnology (Lake Placid,NY). This antibody was made to the first 28 amino-acid res-idues of human RcII, using a synthetic peptide as immuno-gen, and is monospecific for human p75 type II TGF-

b

re-ceptor isoform with no crossreactivity to other receptors(Lin et al. 1992). This antibody has been used to localizeRcII receptor protein in a cell type-specific manner in humanuterine tissue (Chegini et al. 1994). Anti-smooth muscle

a

-actinantibody (Boehringer Mannheim, Mannheim, Germany; cloneasm-1) was made to the first 10 residues with specificity forhuman, murine, and chicken smooth muscle

a

-actin with nocrossreactivity with skeletal or cardiac

a

-actin. Secondaryantibodies conjugated to biotin and specific to either goat,mouse, or rabbit IgG, were obtained from either Cappel

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Localization of TGF-

b

1

and RCII in Human Prostate

381

(anti-goat; Cochranville, PA) or Sigma (anti-mouse and anti-rabbit; St Louis, MO).

Tissue sections on slides were deparaffinized in Hemo-Declearing agent (xylene substitute; Fisher Scientific; FairLawn, NJ) for 15 min at RT and step-rehydrated through agraded series of ethanol. Tissue sections were equilibrated inPBS (pH 7.4) and permeabilized with 0.1% Triton X-100 inPBS for 5 min. For RcII immunolocalization, the microwavefixation antigen retrieval method (Shi et al. 1991) was foundto provide optimal and most consistent staining. For LAP-TGF-

b

1

, the antigen retrieval method produced identical re-sults to standard procedures. For the microwave antigen re-trieval procedure, slides were rehydrated as above, placed ina 2-L glass beaker containing 0.01 M citrate buffer (pH 5.9),and microwaved at full power (800 W) for 30 min beforecooling and equilibration in PBS (Shi et al. 1991). To neu-tralize endogenous peroxidase activity, slides were pretreatedwith 3% hydrogen peroxide for 5 min. Primary antibodieswere applied at the following concentrations: anti-humanLAP-TGF-

b

1

(0.5

m

g/ml); anti-human RcII (5

m

g/ml); andanti-smooth muscle

a

-actin (5

m

g/ml), per manufacturer’srecommended concentrations in PBS plus 1% BSA (PBS-BSAbuffer, 40

m

l per section under a plastic coverslip) for 1 hr ina humidified chamber at 37C. Tissue sections were washedwith agitation consecutively in PBS followed by PBS/0.1%Triton X-100, and PBS for 10 min each. Immunoreactivitywas detected using biotinylated secondary antibodies (1:500rabbit anti-goat and 1:1000 dilution goat anti-rabbit in PBS-BSA buffer) incubated for 45 min as above, followed by a30-min incubation with ExtrAvidin–peroxidase (Sigma, 1:500dilution in PBS-BSA buffer) and visualized by a 7-min incu-bation with 0.7 mg/ml 3,3

9

-diaminobenzidene tetrahydrochlo-ride as the chromagen (Sigma, in 1.6 mg/ml urea hydrogenperoxide, 60 mM Tris buffer, pH 7.6, RT). Control parallelslides were prepared either lacking primary antibody or lack-ing primary and secondary antibodies, or stained with nor-mal sera to control for background reactivity. The color re-action was terminated by immersion in PBS for 5 min, followedby rinsing in water. Coverslips were mounted on slides with40

m

l Biomedia gel mount (Fisher) and slides analyzed bylight microscopy using a Nikon Labophot-2 system fittedwith a Nikon N6000 camera and photographed with Ek-tachrome ASA 400 slide film.

Results

Analysis of LAP-TGF-

b

1

and RcII protein localizationwas performed in normal human prostate glands in-cluding fetal, neonatal, prepubertal, postpubertal, andadult prostate glands (

n

5

12). Donor ages were 37-week fetal, 6.5-month-old, 11.5-month-old, 10.5-year-old, 14-year-old, 19-year-old, 29-year-old, 38-year-old,and one adult donor of unknown age. Each was can-cer-free (histopathology analysis) and multiple regionsof each sample were analyzed for localization studies.

Human fetal prostate was typified by cords of rudi-mentary epithelial islands and glandular acini lined bytwo or three layers of cuboidal cells, as shown in Fig-ure 1A. Immunolocalization of LAP-TGF-

b

1

protein

exhibited a spotty, focal distribution, restricted to thecuboidal epithelial cells of acini (Figure 1B) and devel-oping ducts (data not shown). Low to nondetectableimmunoreactivity was observed in adjacent stromalmesenchymal cells. Epithelial cells exhibited a hetero-geneous staining pattern, with apparent LAP-TGF-

b

1

-negative cells adjacent to LAP-TGF-

b

1

-positive cells,which tended to be oriented more towards the basalsurface of the ducts or acini. No extracellular immu-noreactivity was observed. Control sections in allcases showed no specific immunoreactivity, as shownin Figure 1C.

In contrast to LAP-TGF-

b

1

, localization of RcIIprotein was observed in both epithelial and stromalcells in fetal prostate, as shown in Figures 1D and 1E.Islands of epithelial cell clusters and distinct acini werepositive for RcII immunoreactivity, with some cellsexhibiting differential staining intensity relative to ad-jacent cells. Stromal cells in close apposition to epithe-lial acini exhibited a comparable or higher staining in-tensity for RcII. The stromal cell staining pattern forRcII (Figure 1E) was almost identical to the stainingpattern for smooth muscle

a

-actin, as shown in serialsections (Figure 1F). To further illustrate this co-local-ization, Figure 1G shows the localization of RcII in astromal region void of epithelial acini. Figure 1H is adirect serial section stained for smooth muscle

a

-actin.Antibody positive staining for RcII was seen in smoothmuscle tunica media of a centrally located muscularartery (Figure 1G) and in other stromal cells positivefor smooth muscle

a

-actin. The similarity of stromalRcII localization with

a

-actin localization in serial sec-tions was a pattern observed in all sets of tissues ex-amined from fetal to adult carcinoma.

Localization of LAP-TGF-

b

1

and RcII in neonataltissue exhibited a more homogeneous staining patternrelative to fetal tissue. Both LAP-TGF-

b

1

and RcII ex-hibited an increase in staining intensity in epithelialcells of glandular acini, as shown in Figures 2A and 2Bfrom a 6.5-month-old neonate. Compared with fetaltissue, epithelial cells were more uniformly positive forLAP-TGF-

b

1

. Basal cells exhibited an elevated LAP-TGF-

b

1

staining intensity relative to columnar acinicells, as shown in Figures 2A and 2C. LAP-TGF-

b

1 waspresent in stromal cells but was weaker in immunore-activity compared with epithelial cells and showed amore focal and heterogeneous pattern. RcII proteinappeared to be uniformly expressed in all epithelialcells and in isolated stromal cells, as shown in Figure2B. Localization patterns in epithelial cells were simi-lar in an 11.5-month-old neonatal prostate (Figures2C and 2D). Stromal regions, however, showed an ap-parent increase in LAP-TGF-b1-positive stromal cells(Figure 2C). Epithelial acini were uniformly RcII-posi-tive, as were isolated stromal cells (Figure 2D). Con-nective tissue trabeculae, observed between regions of

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382 Gerdes, Larsen, McBride, Dang, Lu, Rowley

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Localization of TGF-b1 and RCII in Human Prostate 383

glandular acini, exhibited lower immunoreactivity forRcII, whereas stromal cells immediately adjacent toepithelial acini were RcII-positive, as shown in Figure2D, where stromal cells appear in cross-section. LAP-TGF-b1 and RcII localizations in the prostate of a10.5-year-old were similar to those in neonatal tissue(data not shown).

Figures 2E and 2F show representative regions ofexpression patterns in normal disease-free adult tis-sues (29-year-old). These prostate glands contained amore extensive development of glandular acini, exhib-iting a more well-developed, mature secretory pheno-type typical of the normal adult human prostategland. LAP-TGF-b1 (Figure 2E) was uniformly local-ized in epithelial acini and exhibited a more heteroge-neous staining pattern in stromal cells. RcII (Figure2F) staining patterns were uniformly homogeneous inepithelial acini in all tissues. RcII exhibited a spottypattern in stromal cells and, as in fetal tissue, showeda staining distribution identical to that of a-actin (datanot shown). Staining patterns for both LAP-TGF-b1

and RcII were essentially identical in prostate tissuesamples from a 14-, 19-, and a 38-year-old (data notshown) compared with the 29-year-old (Figures 2Eand 2F). A comparison of fetal with adult tissuesshowed that immunoreactivity for RcII in stromalcells increased with age from neonatal to adult. In allsamples analyzed, the staining patterns and relativestaining intensity per cell for both LAP-TGF-b1 andRcII showed no apparent differences in regions oftransition zone compared with peripheral zone of themature prostate gland. In addition, the localizationpattern for LAP-TGF-b1 and RcII in normal regions ofprostate carcinoma specimens (n 5 23) were identicalto patterns observed in disease-free normal adult pros-tate tissue as described above.

LAP-TGF-b1 and RcII protein expression in hu-man prostate carcinoma tissues exhibited fundamentalchanges in staining intensity and patterns of cell typelocalization. Multiple regions of excised whole pros-tate gland were examined from 23 patients with pe-ripheral zone prostatic carcinoma in regions rangingfrom well to more poorly differentiated (Gleason 3–4)in each of 23 patients. Figure 3A shows LAP-TGF-b1

localization in a well- to moderately differentiated

Gleason 3 region. LAP-TGF-b1 staining intensity washigh in carcinoma epithelial acini but exhibited a morevariable pattern of cell-to-cell staining relative to nor-mal glandular tissue. Some acini exhibited an obviousincrease in staining intensity relative to normal pros-tate. Figure 3B shows a representative staining profileof a region of a less moderately differentiated carci-noma (Gleason 3–4). The staining patterns of LAP-TGF-b1 in smaller acini consistently exhibited a morevariable staining intensity relative to normal prostategland. This pattern of variable staining intensity wasobserved in all Gleason 3–4 carcinoma samples exam-ined. In addition, individual cells within an acinus ex-hibited differential staining intensities. Figure 3C showsa representative LAP-TGF-b1 staining pattern from amore poorly differentiated region composed of small,angular acini invading the stroma. As shown here,small acini typically exhibited an apparent increasedstaining intensity relative to larger fused glands. Veryfew stromal cells were positive for LAP-TGF-b1 in car-cinoma tissue, regardless of differentiation status orGleason score and, in general, showed an apparentlower staining intensity relative to carcinoma epithe-lial cells (Figures 3A–3C).

In contrast to LAP-TGF-b1 localization patterns,carcinoma epithelial acini exhibited low to nondetect-able RcII staining. Figure 3D shows a representativeRcII staining pattern in a well-differentiated carci-noma (Gleason grade 3). An apparent lack of specificRcII staining in carcinoma epithelial cells was noted inacini, and a relatively strong staining intensity was ob-served in adjacent stromal cells. Figure 3E shows arepresentative staining pattern observed in regions ofsmaller, closely packed carcinoma acini (moderatelydifferentiated, Gleason 3–4). Epithelial cells exhibitedlow RcII staining intensity, whereas individual stromalcells between acini showed high RcII staining inten-sity. Notable RcII staining was observed in some smallcarcinoma acini, as shown in Figure 3F (arrow) and inFigure 3G, whereas in larger acini RcII was not detect-able (see Figure 3F, acini in left side). In stromal re-gions containing arteries, the tunica media smoothmuscle layer was consistently positive for RcII stain-ing, as shown in Figure 3G (arrow), whereas the outertunica adventitia (fibroblasts) was negative.

Figure 1 Localization of LAP-TGF-b1 and RcII in human fetal prostate. (A) Hematoxylin–eosin-stained section of a 37-week fetal prostategland with rudimentary acini–ductal network. Acini were composed of one or two layers of simple columnar-cuboidal epithelial cells andsurrounding stroma. (B) Immunohistochemical localization of LAP-TGF-b1 in a serial section. LAP-TGF-b1 exhibited a heterogeneous expres-sion pattern in acinar epithelial cells, with a preferential localization to the basal cells and low immunoreactivity in stromal cells. (C) Normalserum (heterologous serum) negative control in a serial section. (D) Immunohistochemical localization of TGF-b receptor type II (RcII) in a37-week human fetal prostate. RcII is localized to both epithelial acini and stromal cells. (E) Localization of RcII in a region of epithelial is-lands with high stromal immunoreactivity. (F) Companion serial section (to E) stained for smooth muscle a-actin. (G) Localization pattern ofRcII in a stromal region containing a central artery. (H) Companion serial section (to G) showing the similar saining pattern for smooth mus-cle a-actin. Note that the arterial tunica media smooth muscle cells in both G and H stain positive for RcII and smooth muscle a-actin. Bar 550 mm.

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384 Gerdes, Larsen, McBride, Dang, Lu, Rowley

DiscussionThis study used immunocytochemistry to localize theLAP-TGF-b1 precursor and the RcII receptor to deter-mine the spatial and cell-type specificity of synthesis

patterns in developing normal human prostate forcomparison with prostate cancer tissue. These studiesshow localization of LAP-TGF-b1 primarily in isolatedbasal epithelial cells during development, and less fre-

Figure 2 Localization of LAP-TGF-b1 and RcII in neonatal through normal adult prostate gland. (A) Immunoreactivity of LAP-TGF-b1, show-ing increased intensity in secretory epithelial cells and basal cells in a 6.5-month-old prostate. Positive expression is observed in stromal cellswith increased immunoreactivity in acinar basal cells. (B) Immunoreactivity of RcII is localized to both normal glandular acini and stromalcells in the 6.5-month-old prostate. (C) Localization of LAP-TGF-b1 in an 11.5-month-old prostate gland. The staining pattern was essentiallysimilar to the 6.5-month-old, with a slight increase in stromal cell immunoreactivity. (D) RcII localization in the 11.5-month-old prostate. RcIIis preferentially localized in epithelial acini cells and stroma adjacent to acini. Trabeculae of supportive connective tissue between groups ofacini were essentially negative for RcII. (E) Localization patterns of LAP-TGF-b1 in a 29-year-old normal prostate. The staining pattern was es-sentially identical to 14-, 19-, and 38-year-old normal prostates (data not shown). (F) RcII staining patterns in the 29-year-old normal humanprostate. Localization was typically observed in both epithelial cells and stromal cells as noted in all normal tissues. Bar 5 50 mm.

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Localization of TGF-b1 and RCII in Human Prostate 385

quently in stromal cells, whereas RcII is present inboth epithelial and stromal cells. In fetal tissue, local-ization of LAP-TGF-b1 was observed in basal cells orin the second layer of cells in rudimentary acini. Stro-

mal cells in fetal prostates were essentially negative forLAP-TGF-b1 protein, implicating a subset of epithelialcells as a site of TGF-b1 synthesis during human pros-tate development. In contrast, stromal cells in fetal

Figure 3 Localization of LAP-TGF-b1 and TGF-b receptor type II in prostatic carcinoma. (A) LAP-TGF-b1 expression in a well- to moderatelydifferentiated prostatic carcinoma (Gleason 3). Expression was observed in both epithelial cells and stroma. A more heterogeneous stainingintensity pattern in carcinoma acini relative to normal was observed. (B) LAP-TGF-b1 expression in a region of less moderately differentiatedcarcinoma (Gleason 3–4). An increased staining heterogeneity in some acini was observed, with a more intense staining pattern in smallacini relative to normal. (C) LAP-TGF-b1 expression in a more poorly differentiated region of small carcinoma acini. An elevated staining in-tensity is associated with smaller, more angular acini. (D) RcII in a well-differentiated region of human prostatic carcinoma (Gleason 3). De-creased carcinoma epithelial cell staining for RcII in acini is in contrast to the relatively intense stromal cell staining pattern. (E) RcII localiza-tion in a moderately differentiated human prostatic carcinoma (Gleason grade 3–4). Decreased expression is noted in small acini, with amore intense staining apparent in adjacent stromal cells. (F) RcII protein localization in a moderately differentiated carcinoma (Gleasongrade 3–4). Some small acini (arrow) exhibit increased staining intensity relative to larger acini in the same field (lower and upper left). (G)RcII protein localization in a moderately differentiated carcinoma (Gleason 3–4). The tunica media (arrow) of arteries is RcII-positive, as arestromal cells in adjacent carcinoma acini, which exhibit low to nondetectable staining. Bar 5 50 mm.

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386 Gerdes, Larsen, McBride, Dang, Lu, Rowley

prostate stained positive for RcII, compared withmore moderate staining For RcII in epithelial cells.Comparison of neonate with adult showed an increasein the apparent number of positive cells and overallstaining homogeneity for both proteins. However, nomajor apparent changes were noted in postpubertaltissue compared with prepubertal tissues.

Neonatal, prepubertal, and adult human prostateglands showed localization of LAP-TGF-b1 and RcIIin both epithelial acini and adjacent stromal cells. Epi-thelial cells exhibited an apparent increased stainingrelative to stromal cells. Notably, the stromal cell lo-calization of both LAP-TGF-b1 and RcII was almostidentical to the pattern of smooth muscle a-actin lo-calization. These data are in close agreement with thepreferential localization of androgen receptor in a-actinpositive cells (Prins et al. 1991; Prins and Birch 1993)and is consistent with hypotheses as to the central roleof a-actin positive cells (likely smooth muscle) in stro-mal-epithelial interactions in prostate development(Prins et al. 1991; Prins and Birch 1995). Such stro-mal–epithelial interactions are likely important in car-cinoma progression as well.

Of fundamental significance were the altered pat-terns of LAP-TGF-b1 and RcII staining in prostate car-cinoma. Carcinoma cells were heterogeneous in LAP-TGF-b1 staining intensity, with heavy staining notedin many cells relative to normal prostate epithelialcells. Our studies are consistent with data in other re-ports indicating that elevated TGF-b1 message and ex-tracellular protein is associated with prostate carcinomaprogression, albeit localized in stroma (Thompson etal. 1992; Truong et al. 1993; Steiner et al. 1994). Stro-mal cell staining intensity and the pattern of LAP-TGF-b1 and RcII were essentially identical in both car-cinoma and normal tissues in our study. These datasuggest that the elevated extracellular accumulation ofTGF-b1 is primarily the result of elevated expressionby carcinoma epithelial cells as opposed to stromalcells.

Significantly, RcII protein staining in prostate carci-noma epithelial cells was almost undetectable relativeto normal epithelial cells. However, stromal cell stain-ing for RcII was not altered in carcinoma relative tonormal tissue stomal cells. These data suggest that adecrease in RcII in epithelium is associated with hu-man prostate carcinoma progression. Three recentstudies have reported that prostate carcinoma epithe-lial cells exhibit a decreased expression of TGF-b RcIand RcII receptors (both mRNA and protein) (Kim etal. 1996; Williams et al. 1996; Guo et al. 1997). Thepresent study corroborates and extends these previousobservations in describing a maintained stromal cellexpression of RcII in carcinoma progression relativeto normal prostate. This finding was not reported inthe previous studies. A second observation unique to

this study was the expression of RcII in select smallcarcinoma acini in moderately differentiated regions.The significance of this observation relative to carci-noma progression is not yet known. The present studyutilized the antigen retrieval method for the RcII local-ization, which exhibited a significant increase in thesensitivity of antibody-specific reactivity. Moreover,differing sources of antibodies were used in the respec-tive studies. The precise role of TGF-b1 in prostatecarcinoma progression and the biological significanceof altered RcII staining in carcinoma cells remain to bedetermined. However, our data and those of othersmust be interpreted with caution owing to the non-quantitative nature of immunocytochemistry, and al-ternative explanations may exist. Differential stainingintensities may relate to altered protein turnover or se-cretion rates. Hence, it is difficult to conclude withcertainty that one cell type necessarily synthesizesmore protein than another, using immunocytochemistry.Although descriptive by nature, immunocytochemistryremains the only method to study native protein local-ization in specific cell types. Therefore, immunocy-tochemistry is of value to determine cell types synthesiz-ing the protein of interest and apparent pattern changesassociated with disease progression.

An overexpression of TGF-b1 concomitant with al-tered RcII expression and altered cell response appearsto be a general observation in many neoplasias andderived cell lines. Melanoma cell lines, spontaneoustransformants of high-passage liver cells, E1A-trans-formed 293 cells, breast carcinoma cells, and variantsof Meth A sarcoma cells all expressed TGF-b1 at ele-vated levels and demonstrated enhanced tumorigene-sis in vivo (Huggett et al. 1991; Arrick et al. 1992;Chang et al. 1993; Herman and Katzenellenbogen 1994;Rodeck et al. 1994). Consistently, these lines were re-sistant to TGF-b1 downstream responses, typicallymeasured through inhibition of cell growth. Thesestudies together suggest that resistance to TGF-b1 maybe an integral step in spontaneous transformationleading to tumorigenesis. Several human gastric cancercell lines resistant to TGF-b1 exhibit genetic alter-ations in RcII, including truncated message or no de-tectable mRNA expression (Park et al. 1994). Thesestudies theorized that escape from proliferation con-trol by TGF-b1 during carcinogenesis could be the re-sult of changes in RcII expression.

In this regard, an overproduction of TGF-b1 inprostate cancer, concurrent with a variable resistanceof carcinoma cell response through RcII alteration,could result in several stroma-associated alterationsleading to a more favorable local environment for car-cinoma progression. Angiogenesis, for example, is di-rected by the stroma, is induced by TGF-b1, and isnecessary for tumor progression (Pepper et al. 1990;Yang and Moses 1990). This is consistent with TGF-

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Localization of TGF-b1 and RCII in Human Prostate 387

b1-induced effects in experimental prostate carcino-mas (Steiner and Barrack 1992). Moreover, becauseTGF-b1 action alters extracellular matrix (ECM) re-modeling (Chang 1995), alterations in stroma-derivedECM remodeling may also enhance carcinoma pro-gression patterns, as is typically seen in breast andprostate metastases (Welch et al. 1990; Steiner andBarrack 1992). Moreover, it is well established thatdifferent carcinomas induce different patterns of “fi-broblastic response” in vivo (Lieubeau et al. 1994)and that stromal cell responses, including expressionof growth factors, are mediated by paracrine-actingfactors derived from carcinoma cells (Singer et al. 1995).

Normal prostate development as well as carcinomainitiation and progression in the human prostate glandlikely involves a complex interplay between andro-gens, growth factors, and stromal–epithelial interac-tions. In addition to other factors, TGF-b1 is knownto affect normal cell proliferation and differentiationand influences carcinoma phenotype and malignantprogression. Further studies to address the specificrole of TGF-b1 action in prostate stromal and epithe-lial cells will allow a more comprehensive understand-ing of the role of this growth factor in prostate devel-opment and carcinoma progression.

AcknowledgmentsSupported by NIH DK45909, CA58093, CA58204, and

by a grant from Sheffield Biomedical Technologies Inc.We wish to thank Dr Tom Wheeler, Department of Pa-

thology, Baylor College of Medicine and Dr Edith Hawkins,Texas Children’s Hospital, for providing fixed tissue speci-mens and pathological analysis of tissue sections, and theSPORE in Prostate Cancer Research at Baylor College ofMedicine for providing fixed tissue samples. We also wish tothank Ms Liz Hopkins for microtomy.

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