Early Castration-Induced Upregulation ofTransforming Growth Factor b1 and Its

Receptors Is Associated With Tumor CellApoptosis and a Major Decline in Serum

Prostate-Specific Antigen in ProstateCancer Patients

Pernilla Wikstrom,1 Patrick Westin,2 Par Stattin,1 Jan-Erik Damber,1* andAnders Bergh2

1Department of Urology and Andrology, Umeå, Sweden2Department of Pathology, Umeå University, Umeå, Sweden

BACKGROUND. The mechanism behind castration-induced apoptosis in prostate cells isunknown, but data from other species suggest that transforming growth factor b1 (TGF-b1)may be involved.METHODS. By using quantitative RT-PCR and immunohistochemistry, expression of TGF-b1 and its receptors type I and II (RI and RII) was studied in normal and tumor areas of corebiopsies taken before and 2–11 days after castration therapy. The TGF-b responses wererelated to changes in apoptotic index and to changes in serum prostate-specific antigen (PSA).RESULTS. In normal prostate tissue, apoptosis was generally increased by castration, andapoptosis was accompanied by an increase in TGF-b1 and RII mRNA levels (P < 0.05). Intumors, apoptosis was seen only in 44% of the cases and in these, but not in the others,TGF-b1, RI, and RII mRNA levels were increased (P < 0.05). In the patients showing aprognostically favorable PSA response (nadir PSA <5 ng/ml), but not in the others, RI and RIImRNA levels were significantly upregulated (P < 0.05).CONCLUSIONS. Short-term upregulation of TGF-b1 and its receptors is associated withapoptosis in human prostate and prostate cancer, and possibly with a favorable clinicaloutcome after castration therapy. Prostate 38:268–277, 1999. © 1999 Wiley-Liss, Inc.

KEY WORDS: TGF-b1; apoptosis; prostate cancer; castration therapy

INTRODUCTION

Castration treatment has remained the first-linetherapy for metastatic prostate cancer over the past 50years [1], since it initially relieves clinical symptomsfor most patients. However, almost all tumors relapseto an androgen-independent stage within a fewmonths or up to several years later. This wide time-span in progression-free survival indicates tumor dif-ferences at the cellular level. At present, the best wayto predict clinical outcome after castration is to mea-sure the serum level of prostate-specific antigen (PSA)3–6 months after therapy initiation [2,3]. Patients with

normalized PSA values at that time (PSA-responders)have longer predicted progression-free survival timesthan non-PSA-responders. However, a recent studyshowed that it may be possible to predict clinical re-

Grant sponsor: Swedish Cancer Society; Grant number: Project 1760;Grant sponsor: University Hospital of Northern Sweden; Grantsponsor: Maud and Birger Gustavsson Foundation; Grant sponsor:Lions Cancer Research Foundation; Grant sponsor: Umeå Univer-sity; Grant sponsor: Swedish Society for Medical Research.*Correspondence to: J.-E. Damber, Department of Urology and An-drology, Umeå University, 901 85 Umeå, Sweden.Received 27 March 1998; Accepted 30 July 1998

The Prostate 38:268–277 (1999)

© 1999 Wiley-Liss, Inc.

sponse at an earlier time point by examining short-term cellular effects induced by castration therapy [4].

In the rat ventral prostate (VP), androgen depriva-tion causes massive cell death within days due to in-duction of apoptosis [5,6]. Castration-induced apopto-sis in the prostate is believed to be mediated by dif-ferent intermediate factors. It has been suggested thattransforming growth factor b1 (TGF-b1) is a crucialfactor for the apoptotic process in the VP [7]. TGF-b1is a multifunctional growth factor with a number ofeffects in the development, differentiation, andgrowth of epithelial tissues [8]. The expression of TGF-b1 and its receptors, TGF-b receptor type I and type II(RI and RII), is negatively regulated by androgens andincreases during castration-induced apoptosis in therat VP [9–11]. Moreover, TGF-b1 has been shown toinduce apoptosis in normal prostatic epithelial cellsboth in vivo and in vitro [12,13]. In the rat prostaticDunning R3327 PAP adenocarcinoma, on the otherhand, neither apoptosis nor TGF-b1 or RII expressionincrease shortly after castration treatment [11,14,15].Lack of TGF-b induction after castration has also beendemonstrated in other Dunning tumor sublines [16].Taken together, these results suggest that castration-induced apoptosis in androgen-dependent prostatictissues involves a TGF-b1 response that might be lostin androgen-independent prostatic tumors.

In earlier studies, we found that human prostatetumors respond to castration in a highly heteroge-neous way. Only approximately one third of tumors,mainly belonging to PSA-responding patients,showed increased apoptotic index 1 week after treat-ment [4,17]. This could be compared to a sevenfoldincrease in apoptotic cells in adjacent normal prostaticglands [17]. However, it is not known if castration-induced apoptosis in human prostate and prostate tu-mors involves a TGF-b response. Neither is it knownif the lack of short-term apoptosis induction in someprostate tumors after androgen withdrawal could bedue to some failure in mediating such a TGF-b re-sponse. Human prostate tumors often express highlevels of TGF-b1 [18,19], and overproduction of TGF-b1 has been shown to be associated with angiogenesis,metastasis, and short survival in prostate cancer [20].This may be due to possible tumor-stimulating prop-erties of TGF-b1 such as inhibition of immune re-sponses [21] and stimulation of angiogenesis and cellmotility [22,23]. Furthermore, low expression of RIand RII has been associated with short prostate can-cer-specific survival [20,24], which may indicate somefailure of prostate tumor epithelial cells in respondingto TGF-b1 growth-inhibiting and/or apoptotic signals.

The aim of this study was to examine if short-term

effects on TGF-b1, RI, and RII expression are associ-ated with apoptosis and clinical response after castra-tion therapy in patients with advanced prostate can-cer.

MATERIALS AND METHODS

Tissue Collection and Processing

Several ultrasound-guided core biopsies were takenshortly before and 2–11 days after castration therapyin a series of patients with advanced prostate cancer.Local tumor stage was evaluated by digital rectal ex-amination, according to the 1992 UICC classification[25], and radionuclide bone scan was performed formetastasis staging at the time of diagnosis (Table I).Biopsies were immediately frozen in liquid nitrogenor fixed in phosphate-buffered formalin, before beingembedded in TissueTek (Miles, Inc., Elkhart, IN) andparaffin, respectively. The biopsies were cut into4-mm-thick sections and stained with hematoxylin andeosin (HE). The tumors were classified into high (G1),moderate (G2), and low (G3) differentiation by evalu-ating the HE-stained slides from the fixed biopsies,according to the World Health Organization (WHO)classification system (Table I) [26]. The HE-stainedcryosections were used for localizations of normal ar-eas (biopsy areas where no malignant cells were de-tected) and tumor areas (Fig. 1). Biopsy parts corre-sponding to these areas were microdissected from thefrozen biopsies, by using a sterile scalpel blade, andfurther processed for total RNA extraction. By usingthis procedure, total RNA from normal and tumorprostate tissue were isolated from 9 and 18 pairs offrozen pre- and posttherapy biopsies, respectively.

Clinical Classification

Response to castration therapy was defined, as pre-viously described [4], as a serum level of PSA of ø5ng/ml 3–6 months posttreatment (PSA-responders),and nonresponse as a nadir PSA ù10 ng/ml (non-PSA-responders, Table I).

Determination of Apoptotic Indexes

Apoptotic indexes (number of apoptotic cells per1,000 cells) in the HE-stained sections from the frozentumors were determined by counting approximately250–1,000 and 300–2,500 normal and tumor cells, re-spectively, at 400× magnification. Apoptotic cells weredefined as single rounded cells or fragments with

TGF-b1 and Apoptosis in Prostate Cancer 269

densely aggregated chromatin and condensed cyto-plasm, often lying in ‘‘halos’’ of extra cellular space(Fig. 2) [27]. If more than one apoptotic body was seenper ‘‘halo,’’ these were considered to originate fromthe same cell and were counted as one.

Apoptotic response to castration therapy was de-fined as increased apoptotic index in the posttherapybiopsy compared to the pretherapy biopsy (Ap-responders), and apoptotic nonresponse as unchangedor decreased apoptotic index after treatment (non-Ap-responders, Table I).

Total RNA Preparation and Competitive ReverseTranscription-Polymerase Chain Reaction

(RT-PCR)

Total RNA was isolated from the frozen prostatictissues by the TRIzol extraction method (Life Tech-nologies AB, Taby, Sweden). The mRNA levels forTGF-b1, RI, RII, and the housekeeping gene cy-clophilin [28] were quantified, by using competitiveRT-PCR and PCR primers as previously described[11,15]. Briefly, 50 ng of total RNA were reverse tran-scribed together with appropriate amounts of internalRNA standards (IS) [11,15] for TGF-b1, RI, RII, andcyclophilin. Each RNA sample was titrated with threeamounts (double samples) of IS, in ranges of 8.5–170,0.52–10, 2.3–46, and 34–680 amol for TGF-b1, RI, RII,and cyclophilin, respectively. After RT completion,samples were divided into four PCR tubes, in order toamplify cDNA for TGF-b1 (248 bp), RI (177 bp), RII(215 bp), and cyclophilin (362 bp) separately. During32 cycles of PCR (95°C, 30 sec; 63°C, 30 sec; and 72°C,45 sec), the TGF-b templates were competitively am-plified with cDNA for their corresponding IS (266,163, 199, and 315 bp, respectively). Resulting PCRproducts were analyzed in an automatic laser fluores-cence system (ABI PRISM™ 377 DNA sequencer, Per-kin Elmer, Askim, Sweden). The data were processedby the ABI PRISM™ GeneScan software (PerkinElmer), and RNA levels were calculated from tem-

TABLE 1. Clinical Characteristics of Patients With Advanced Prostate Cancer Treated With Castration Therapy andIncluded in the mRNA Experiments

Ap-responders(n = 8)a

Non-Ap-responders(n = 10)

PSA-responders(n = 10)b

Non-PSA-responders(n = 7)

Tumor stagec

T1–T2 1 2 2 1T3–T4 7 8 8 6

Tumor graded

G1 1 1G2 4 5 5 3G3 3 5 4 4

Metastasis (bone scan) 7 8 7 7PSA before therapye 1,400 (1,000) 550 (210) 1,100 (850) 720 (230)PSA nadir 26 (21) 25 (11) 2.3 (0.44) 61 (23)AI before therapy 8.8 (1.5) 17 (2.0) 12 (1.5) 14 (3.4)AI after therapy 19 (3.6) 13 (1.8) 14 (1.9) 16 (4.4)Time for biopsyf 6.4 (1.1) 6.3 (0.47) 5.6 (0.92) 7.3 (0.29)

aApoptotic (Ap) response defined as increased apoptotic index (AI) in post- compared to pretherapy biopsy. Indexes calculated asnumber of apoptotic cells per 1,000 cells in HE-stained sections.bResponse defined as serum prostate-specific antigen (PSA) ø5 ng/ml, and nonresponse as PSA ù10 ng/ml 3–6 months after therapy.One patient had a nadir PSA value of 8 ng/ml.cAccording to UICC [25].dAccording to WHO [26].eValues expressed as means and SEM (in parentheses).fDays between therapy and posttherapy biopsy.

Fig. 1. Identification of normal (N) and tumor (T) areas in HE-stained cryosection from a pretherapy prostate cancer core bi-opsy (×200).

270 Wikstrom et al.

plate- to IS-cDNA ratios, as previously described [15].The TGF-b1, RI, and RII levels were corrected for thecorresponding cyclophilin levels in each RNA sampleand expressed as relative levels (amol/amol cy-clophilin mRNA) in the resulting figures.

Immunohistochemistry

In addition to quantify the mRNA levels for TGF-b1and its receptors before and after castration, wewanted to use immunohistochemistry (IHC) to exam-ine TGF-b1, RI, and RII protein expression in responseto castration therapy. Unfortunately, the biopsy mate-rial used in the mRNA experiment was not sufficientfor these studies, and we had to use core biopsies fromanother series of patients. The two series of patientswere similar according to tumor and metastasis clas-sification and PSA levels before and after therapy, and

the biopsies were collected and processed in the sameway (Tables I and II).

Four-micron sections from formalin-fixed tumor bi-opsies were deparaffinated and rehydrated accordingto standard procedures, washed with phosphate-buffered saline (PBS), and heated in a microwave ovenat 600 W for 2 × 7.5 min and 1 × 5 min in 0.01 M citratebuffer, pH 6.0, as earlier described [29]. To quenchendogenous peroxidase activity, slides were im-mersed in 3% H2O2 in methanol for 20 min. Slideswere incubated overnight, at 4°C, with the followingantibodies: anti-TGF-b1 (10 mg/ml, anti-TGF-b1 neu-tralizing antibody, R&D Systems, Oxon, UK), anti-RI(0.25 mg/ml, V-22, Santa Cruz Biotechnology, SantaCruz, CA), and anti-RII (1 mg/ml, anti-human TGF-bRII neutralizing antibody, R&D Systems). For detec-tion, the ABC technique was used, with aminoethyl-carbazole as chromogen, according to the manufactur-er’s instructions (Vector Laboratories, Burlingame,CA). Sections were counterstained with Mayer’s he-matoxylin solution. Specificity of TGF-b immunoreac-tions was examined by preincubation of the primaryantibodies with a 25-fold (w/w) excess of the corre-sponding control peptides (recombinant human TGF-b1 and TGF-b1 soluble receptor II, R&D Systems, andV-22P, Santa Cruz Biotechnology).

The immunoreactivity in tumor and normal areas

Fig. 2. Cryosections showing castration-induced apoptosis (ar-rows) in tumor epithelial cells of a prostate cancer core biopsy(×400). A: Tumor cells before therapy. B: Tumor cells aftertherapy.

TABLE II. Clinical Characteristics of Patients WithAdvanced Prostate Cancer Treated With Castration

Therapy and Included in theImmunohistochemistry Experiments

PSA-responders

(n = 12)a

Non-PSA-responders

(n = 9)

Tumor stageb

T1–T2 4 1T3–T4 8 8

Tumor gradec

G1 3 2G2 7 4G3 2 3

Metastasis (bone scan) 6 8PSA before therapyd 770 (720) 1,600 (460)PSA nadir 2.0 (0.38) 84 (22)Time for biopsye 7.0 (0.64) 6.8 (0.40)

aResponse defined as serum prostate-specific antigen (PSA) ø5ng/ml, and nonresponse as PSA ù10 ng/ml 3–6 months aftertherapy. One patient had a nadir PSA value of 8 ng/ml.bAccording to UICC [25].cAccording to WHO [26].dValues expressed as means and SEM (in parentheses).eDays between therapy and posttherapy biopsy.

TGF-b1 and Apoptosis in Prostate Cancer 271

was evaluated without knowledge of any patient data.Immunoreactivities for TGF-b1, RI, and RII were clas-sified as negative (−), moderate (+), or intense (++),and assessed as either increased or unchanged by cas-tration therapy.

Statistics

Groups were compared by the Mann-Whitney U-test and paired observations by the Wilcoxon pairedtest. To test correlations, the Spearman rank sum testwas applied. P ø 0.05 was considered statistically sig-nificant.

RESULTS

Effects of Castration Therapy on Apoptosis, andon TGF-b1, RI, and RII Expression

Apoptotic indexes were determined and TGF-b1,RI, and RII mRNA levels were quantified in normaland tumor parts of prostate cancer biopsies taken be-fore and 2–11 days after castration therapy (Table III).Furthermore, TGF-b1 and TGF-b receptor protein ex-pression was examined in pre- and posttherapy biop-sies by using IHC (Table IV).

In normal prostatic tissue, apoptotic indexes wereincreased after castration in 8 of 9 cases (89%), andTGF-b1, RI, and RII mRNA levels were increased in 7(78%), 5 (56%), and 7 (78%) cases, respectively. In theone normal case where no induction of apoptosis wasseen after treatment, there was also no increase inTGF-b1 or TGF-b receptor mRNA levels. The average

TGF-b1 mRNA levels and apoptotic indexes were sig-nificantly higher in the post- than in the pretherapybiopsies (3.1- and 2.8-fold; P = 0.028 and 0.011, respec-tively), while the increase in RI and RII mRNA levelswere nonsignificant (P = 0.21 and 0.14, Table III).

In the tumor tissue, apoptotic response to castrationwas more heterogeneous than in normal tissue, withonly 8 of 18 cases (44%) showing increased apoptoticindexes after therapy. For the whole group, castrationdid not induce a significant increase in apoptosis. TheTGF-b1 and RI mRNA levels were increased by cas-tration in 12 (67%, P = 0.031 and 0.102, respectively),and the RII mRNA levels in 13 (72%, P = 0.006), of thetumors (Table III).

Immunoreactivity for TGF-b1, RI, and RII wasfound mainly in normal and tumor epithelial cells(Fig. 3), which is in line with recent results demon-strating protein expression of TGF-b1 and its recep-tors, and mRNA expression of TGF-b1, preferentiallyin prostate epithelial cells [20]. The epithelial immu-nostaining in the pretherapy biopsies was classified asnegative (−), moderate (+), or intense (++), and wasfound to be unchanged or intensified (Fig. 3) in theposttherapy biopsies (Table III). In accordance withthe mRNA results (Table III), protein levels of TGF-b1,RI, and RII seemed to be heterogeneously upregulatedby androgen ablation in the prostate tissues (Table IV).TGF-b1 and TGF-b receptor protein induction weremore frequently seen in normal than in tumor tissue(Table IV). The specificity of the immunoreactions haspreviously been determined [20], and control slidesincubated with preblocked antibodies showed nostaining (results not shown).

TABLE III. Short-Term Effects Induced by Castration Therapy on Apoptosisand TGF-b1, RI, and RII mRNA Levels in Patients With Advanced

Prostate Cancer

Normal tissue (n = 9) Tumors (n = 18)

Untreatedmean (SEM)

Castratedmean (SEM)

Untreatedmean (SEM)

Castratedmean (SEM)

AIa 5.0 (0.64) 14.0 (2.2)* 13.0 (1.6) 16.0 (2.0)TGF-b1b 0.12 (0.017) 0.37 (0.15)* 0.11 (0.012) 0.21 (0.045)*RI 0.022 (0.0034) 0.060 (0.032) 0.014 (0.0023) 0.020 (0.0025)RII 0.23 (0.085) 0.71 (0.36) 0.094 (0.014) 0.17 (0.032)**Time for biopsyc 7.0 (0.65) 6.3 (0.55)

aApoptotic indexes (AI) calculated as number of apoptotic cells per 1,000 cells in HE-stained sections.bRelative mRNA levels expressed as amol/amol cyclophilin mRNA (see Materials andMethods).cDays between therapy and post-therapy biopsy.*P < 0.05, significant increase after castration therapy.**P < 0.01, significant increase after castration therapy.

272 Wikstrom et al.

TGF-b1, RI, and RII mRNA Expression in Relationto Castration-Induced Apoptosis

Normal tissue with increased apoptotic index aftercastration showed three- and fivefold inductions ofTGF-b1 and RII mRNA, respectively (P = 0.025, Fig.4A). The RI mRNA level was increased twofold, butthis was not statistically significant (P = 0.123, Fig. 4A).

In the tumors, the relative changes in TGF-b1, RI,and RII mRNA levels observed after castrationtherapy were correlated to the corresponding changesin apoptosis indexes (rs = 0.60, 0.63, and 0.61; P = 0.016,0.005, and 0.008, respectively). TGF-b1, RI, and RIImRNA levels were increased in the Ap-respondingtumors after castration (2.5-, 1.8-, and 2.3-fold; P =0.036, 0.036, and 0.017, respectively, Fig. 4B), whileneither TGF-b1 nor the TGF-b receptor mRNA levelswere significantly increased in the non-Ap-respond-ing tumors (Fig. 4C).

TGF-b1, RI, and RII mRNA Expression in Relationto PSA Response After Castration Therapy

There was no significant correlation found betweenthe relative changes in tumor TGF-b1, RI, or RIImRNA levels and the corresponding changes in pa-tient PSA levels after castration therapy. In accordancewith the Ap-responding patients, however, the PSA-responding patients showed increased RI and RIImRNA levels after treatment (1.7- and 1.5-fold, P =0.047 and 0.022, respectively, Fig. 5A). There was alsoa tendency for TGF-b1 induction in this group, al-though the increase was nonsignificant (P = 0.114, Fig.5A). Neither TGF-b1, nor the TGF-b receptor mRNAlevels, were significantly increased in the non-PSA-responding tumors after therapy (Fig. 5B).

DISCUSSION

Androgen ablative therapy today, due to its abilityto initially relieve clinical symptoms for most patients,

is the most frequently used method in the treatment ofadvanced prostate cancer. The beneficial therapy re-sponses are believed to be achieved by castration-induced apoptosis and decreased cell proliferationamong androgen-dependent epithelial cells [5,6,30].Only a minority of human prostate tumors, however,respond with increase in apoptosis. Recent studieshave shown increased, unchanged, or even decreasedapoptosis in human prostate cancer 1 week after cas-tration [17], and this heterogeneity in short-term apop-totic response was demonstrated to be differential forsubsequent clinical outcome [4]. The molecularmechanism for castration-induced apoptosis in pros-tate epithelial cells is not clear, but it has been thoughtto involve a TGF-b1-dependent pathway (see Intro-duction).

Several studies have demonstrated a relationshipbetween TGF-b1 and castration-induced apoptosis inanimal models [9,10,30,31]. However, the presentstudy is the first to report short-term effects on TGF-b1, RI, and RII expression in the human prostate andadvanced prostate cancer after castration. Induction ofTGF-b1, RI, and RII expression was found to be re-lated to the apoptotic response in tumor cells aftercastration therapy. The TGF-b1, RI, and RII mRNAlevels were significantly increased in the Ap-responding, but not in the non-Ap-responding tumorsafter treatment. This is in line with previous resultsshowing increased expression of TGF-b1 and its re-ceptors in apoptosis-responding model systems, suchas the rat VP and the human PC-82 tumor [9,10,30,31],but not in apoptosis-nonresponding Dunning tumorsafter castration [11,15,16]. Taken together, these resultssuggest that TGF-b1 is involved in mediating castra-tion-induced apoptosis in the prostate, and further-more, that androgen-independent tumors may evadecastration-induced apoptosis due to defects in theirTGF-b1 response to androgen ablation.

Defects in TGF-b receptor expression have been as-sociated with TGF-b1 insensitivity in many systems,

TABLE IV. Short-Term Effects Induced by Castration Therapy on TGF-b1, RI,and RII Immunoreactivity in Patients With Advanced Prostate Cancer

No. of cases(normal/tumor)

Time for biopsy(normal/tumor)a

Increased immunoreactivityafter therapy

Normal tissueno. (%)

Tumorno. (%)

TGF-b 13/21 6.5 (0.27)/6.8 (0.44) 11 (85) 9 (43)RI 13/21 7.3 (0.36)/6.7 (0.42) 9 (69) 11 (52)RII 16/22 7.3 (0.55)/7.0 (0.33) 10 (63) 12 (55)

aDays between therapy and posttherapy biopsy. Values expressed as means and SEM (inparentheses).

TGF-b1 and Apoptosis in Prostate Cancer 273

including the prostate [32–36], and loss of epithelialexpression of RI and RII has been shown with humanprostate cancer progression [37,38]. Lack of RI or RIIexpression in the epithelial cells would be one obviousexplanation for the absence of castration-inducedTGF-b1 response in advanced prostate cancer. In thisstudy, however, TGF-b receptor expression in the in-tact tumors was not predictable for the apoptotic re-

sponse to castration. All untreated tumors expressedsimilar amounts of RI and RII mRNA, and the Ap-responding group contained intact tumors with nega-tive as well as intense receptor immunoreactivity. Ourresults suggest that the ability of androgen-indepen-dent tumors to avoid castration-induced apoptosis isnot simply due to loss of RI or RII expression in thetumor cells, but may be influenced by the inability of

Fig. 3. Sections from Ap-responding tumors before (A, C, E) and after (B, D, F) castration therapy, showing increased immunoreactivityfor TGF-b1 (A, B), RI (C, D), and RII (E, F) after castration (×400).

274 Wikstrom et al.

some tumors to upregulate the expression of these re-ceptors in response to castration. The TGF-b1 signal-ing pathway in prostate cancer cells therefore needs tobe thoroughly investigated.

In addition to being associated with apoptosis, thetumor TGF-b response to castration therapy seemed tobe predictable for the subsequent PSA-response. TheRI and RII mRNA levels were significantly increasedby castration in the group of PSA-responding tumors,

but not in the group of non-PSA-responding tumors.These results are consistent with results by Stattin etal. [4], indicating the possibility of predicting clinicaloutcome at an early time-point by studying short-termeffects of castration therapy. There was, however, nosignificant induction of TGF-b1 in the PSA-responding tumors after castration, although an over-all induction of TGF-b1 was seen in 67% of the tumorcases. Speculatively, induction of TGF-b1 in tumorcells that do not go into apoptosis after castration maybe nonbeneficial to the patient, due to tumor-promoting effects of TGF-b1 such as inhibition of im-mune responses [21] and stimulation of angiogenesis[20,22], cell motility [23], and metastasis [20].

Apoptosis was more frequently seen in normalprostate tissue than in tumors after castration. Onlyone normal case showed lack of castration-inducedapoptosis, and in this tissue there were also no in-creases in TGF-b1 or TGF-b receptor expression. Theaverage increase of TGF-b1, RI, and RII mRNA in thenormal Ap-responding tissue was more pronounced

Fig. 4. Competitive RT-PCR results, showing relative TGF-b1,RI, and RII mRNA levels before (open columns) and after (shadedcolumns) castration therapy in Ap-responding normal prostateareas (A), and in Ap-responding (B) and non-Ap-responding (C)prostate tumors. TGF-b1 and RII mRNA levels were increased bycastration in the Ap-responding normal areas, and the TGF-b1, RI,and RII mRNA levels were increased in the Ap-responding tumors(*P < 0.05). No significant induction of TGF-b1, RI, or RII was seenin the non-Ap-responding tumors after castration.

Fig. 5. Competitive RT-PCR results, showing relative TGF-b1,RI, and RII mRNA levels before (open columns) and after (shadedcolumns) castration therapy in PSA-responding (A) and non-PSA-responding (B) prostate tumors. RI and RII mRNA levels wereincreased by castration in the PSA-responding tumors (*P < 0.05).No significant induction of TGF-b1, RI, or RII was seen in thenon-PSA-responding tumors after castration.

TGF-b1 and Apoptosis in Prostate Cancer 275

than the increase in the Ap-responding tumors, al-though the increase in RI mRNA was not statisticallysignificant. Lack of statistical confirmation of RI in-duction in this group, as well as the big standard de-viations seen for TGF-b1, RI, and RII in normal tissue,suggest that normal, nonmalignant prostate tissuecould be rather heterogeneous. Possible reasons forthis are premalignant cellular defects in normal-looking epithelial cells, but also regional differences innormal epithelial cell function and/or functionalchanges related to the proximity of cancer tissue.

CONCLUSIONS

The present study suggests that increased expres-sion of TGF-b1, RI, and RII is associated with castra-tion-induced apoptosis in the human prostate and inadvanced prostate cancer. Moreover, short-term ef-fects on RI and RII seem to be predictable for PSAresponse and thus probably also for the long-termclinical outcome after castration therapy.

ACKNOWLEDGMENTS

Mrs. Birgitta Ekblom, Mrs. Elisabeth Dahlberg, andMs. Pernilla Andersson have skillfully contributed tothis paper by their technical assistance.

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