Experimental Gerontology 34 (1999) 275–287 Proliferative disorders of the aging human prostate: Involvement of protein hormones and their receptors G. Untergassera, H. Rumpolda, M. Hermanna, S. Dirnhofera, G. Jilgb, P. Bergera,* a Institute for Biomedical Aging Research of the Austrian Academy of Sciences Rennweg 10, A-6020, Innsbruck, Austria b Department of Urology, General Hospital, Hall, Austria Manuscript received July 30, 1998; manuscript accepted October 12, 1998 Abstract The majority of elderly men is affected by benign and malignant diseases of the prostate. Both proliferative disorders, i.e., benign hyperplasia of the prostate (BPH) and prostate cancer (PCa)—which has recently emerged as the most common male malignancy in industrialized countries—seem to be governed by endocrine factors such as sex steroid hormones, but auto/paracrine factors are involved as well. Age-related changes in levels and ratios of endocrine factors as androgens, estrogens, gonadotropins, and prolactin (PRL) and changes in the balance between auto/paracrine growth-stimulatory and growth-inhibitory factors such as insulin-like growth factors (IGFs), epidermal growth factor (EGF), nerve growth factor (NGF), IGF-binding proteins (IGFBPs), and transforming growth factor  (TGF) are meant to be responsible for abnormal prostatic growth. We investigated the existence of putative local regulatory circuits involving the protein hormones, human growth hormone (hGH), human placental lactogen (hPL), and hPRL, and their corresponding receptors in prostatic tissue specimens (transurethral resections of the prostate, TURP; n ϭ 11), in the prostatic cancer cell lines PC3, Du145, LnCap, a virus-transformed BPH cell line (BPH-1), and in a normal healthy prostate by RT-PCRs and highly specific and sensitive immunofluorometric assays (IFMA). Neither hPRL nor hGH was detected at the mRNA or protein levels in prostatic tissue and cell lines, with the exception of 2 of 11 prostatic TURP-samples, which showed weak expression of the PL-A/B genes. PRL- and GH-receptors were expressed in all normal and pathological prostatic specimens. Surprisingly, * Corresponding author. Peter Berger, PhD, Institute for Biomedical Aging Research, Austrian Academy of Sciences, Rennweg 10, A-6020 Innsbruck, Austria. Tel: ϩ43-512-583919-24; Fax: ϩ43-512-583919-8; E-mail: peter.berger@oeaw.ac.at 0531-5565/99/$–see front matter © 1999 Elsevier Science Inc. All rights reserved. PII: S 0 5 3 1 - 5 5 6 5 ( 9 8 ) 0 0 0 6 3 - 1 276 G. Untergasser et al./Experimental Gerontology 34 (1999) 275–287 PRL-receptor expression was not detectable in prostatic cancer cell lines. The trophic effects of exogenous hGH, hPL, and hPRL were investigated by cell proliferation assays (WST-1) in prostatic primary cell cultures and PCa cell lines. hGH significantly ( p Ͻ 0.005) increased cell proliferation up to 138 Ϯ 3.2% (1 nM hGH), while hPL and hPRL revealed only moderate effects. Our data suggest that local auto/paracrine networks of protein hormone actions are not involved in the pathology of BPH or prostatic cancer. On the other hand, systemic pituitary-derived hGH can increase the proliferative response of BPH and PCa, acting directly on the target organ prostate, via the hGH-R. In this case, envisaged GH substitution in elderly people must be viewed at with caution because age-related declines in GH/IGF-I could act as a protective mechanism against abnormal cell growth. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Growth hormone; Prolactin; Placental lactogen; Benign prostatic hyperplasia; Prostate cancer Enlargement of the aging prostate affects one of every two men over 50 years of age and more than 95% of the male population after 70 years of age (Berry et al., 1984; Glyn et al., 1985). The development of this benign prostatic hyperplasia (BPH) during senescence is correlated to alterations in the endocrine status. In contrast to age-related decreases of free testosterone (Vermeulen, 1991), pituitary-derived GH (Veldhuis and Leim, 1995) and, consequently, hepatic IGF-I (Juul et al., 1994), an increase of gonadotropins, i.e., luteinizing hormone (LH) and follicle stimulating hormone (FSH) (Neaves et al., 1984; Madersbacher et al., 1993) and PRL (Vekemans and Robyn, 1975) can be observed in elderly men. In addition to systemic endocrine changes, local changes of growth factors and hormones also seem to be involved in the development of BPH (Byrne et al., 1996; Culig et al., 1996; Griffiths, 1996; Dirnhofer et al., 1998). Stromal– epithelial interactions via steroid hormones (androgens, estrogens) and growthstimulatory factors such as insulin-like growth factors (IGFs), epidermal growth factor (EGF), nerve growth factor (NGF), keratinocyte growth factor (KGF), and fibroblast growth factors (FGFs), as well as growth-inhibitory factors as transforming growth factor  (TGF) and IGF-binding proteins (IGFBPs) (Rajah et al., 1997) play an essential role in regulating prostatic growth during embryonic and pubertal development and differentiation (Griffiths, 1996). After pubertal maturation, prostate homeostasis is mainly achieved by a balance between growth-inhibitory and -stimulatory factors (Fig. 1). With aging, a disturbance of this balance leads to abnormal prostatic growth and BPH. Which endocrine or local hormonal factors induce this imbalance is still a matter of debate. There are several possibilities, including increased availability of local prostatic estrogens (Krieg et al., 1993) or overexpression of ectopically produced growth factors (Dirnhofer et al., 1998). Recently, it has been shown that transgenic mice overexpressing the rat PRL gene develop dramatic enlargements of the prostate gland (Wennbo et al., 1997). Transgenic bovine GH mice presented increased prostatic sizes, due to elevated IGF-I levels (Wennbo et al., 1997). Interestingly, young patients with untreated acromegaly show a remarkable increase in prostatic diameter and volume and a higher prevalence of micro- and macrocalifications compared to healthy subjects (Colao et al., 1998). Thus, a possible involvement of protein hormones in growth regulation of the prostate is conceivable. G. Untergasser et al./Experimental Gerontology 34 (1999) 275–287 277 Fig. 1. Growth-inhibitory factors such as transforming growth factor  (TGF-), insulin-like growth factor binding proteins (IGFBPs), or glycoprotein hormone ␣ (unpublished data), and growth-stimulating factors, such as epidermal growth factor (EGF), insulin-like growth factors (IGFs), and keratinocytes/fibroblast growth factors (KGF/FGF) are involved in the local growth regulation of the human prostate. These two types of factors provide a balance apparently regulated by systemic factors, such as androgens, estrogens, and pituitary-derived hormones as prolactin (PRL), growth hormone (GH), and gonadotropins. As a consequence of aging, the altered systemic or local hormonal situation leads to an imbalance favoring growth-stimulatory factors, which ultimately leads to the benign growth of the human prostate (BPH). The aim of this study was to analyze endocrine and local prostatic networks, including human (h) protein hormones, because hGH, hPL, and hPRL are potentially involved in aberrant prostatic growth. Coexpression of GH- and PRL-receptors and the respective ligands in prostatic tissue was investigated, and possible systemic or auto/paracrine functions of these somatotrophic hormones elucidated by studying proliferative effects on human BPH/PCa primary cultures and human prostate carcinoma cell lines Du 145, PC3, LnCap, and BPH-1. 1. Materials and methods 1.1. Tissues and RNA isolation BPH (n ϭ 5) and PCa-tissue (n ϭ 2) collected from transurethral resections of the prostate (TURP; n ϭ 7; age range: 58 – 84 years), a prostate biopsy (45 years), Du145, PC3, LnCap prostatic carcinoma cells (obtained from the American Type Culture Collection, Rockville, MD), BPH-1 cells (obtained from the German Collection of Microorganisms and Tissue Cultures, Braunschweig, Germany), and a human term placenta were snap frozen in liquid nitrogen and kept at Ϫ70°C until use for RNA extraction and analysis by the reverse-transcription polymerase chain reaction (RT-PCR). Written informed consent was obtained from all patients before surgery. Peripheral blood mononuclear cells (PBMCs) were obtained from healthy subjects (n ϭ 4) by Ficoll/Hypaque gradient centrifugation, as described elsewhere (Boyum, 1968). 278 G. Untergasser et al./Experimental Gerontology 34 (1999) 275–287 Total RNA was extracted from 100 mg of homogenized tissue (Ultra-Turrax, Janke & Kunkel, Germany) or 5 ϫ 106 cells by the single-step acid guanidinium thiocyanate phenol/chloroform method of Chomczynski and Sacchi (Chomczynski and Sacchi, 1987). Integrity of RNA was assessed by analysis of 28S and 18S rRNA on ethidium bromidestained 1% agarose gels. 1.2. Cytosolic extracts One hundred milligrams of BPH (n ϭ 4) and PCa (n ϭ 3) tissue specimen (TURP; n ϭ 7; age range: 61–75 years) or 5 ϫ 106 prostatic carcinoma cells or PBMCs were homogenized on ice in 1 mL PBS (Ultra-Turrax, Janke & Kunkel, Stauffen, Germany). Phenylmethane–sulfonyl fluoride and aprotinin (Merck, Darmstadt, Germany) were added as protease inhibitor (1 mM). After centrifugation (10,000 ϫ g, 20 min, 4°C), the aqueous cytosolic extract was stored at Ϫ20°C until analyses for hGH/hPRL/hPL content by immunofluorometric assays (IFMA). 1.3. Prostatic cell cultures Two hundred milligrams of TURP tissue were cut into to small pieces with a sterile razor and digested with 50 mg collagenase (Sigma-Aldrich, Milwaukee, WI) in 20 mL RPMI 1640 (BioWhittaker, Boehringer-Ingelheim, Belgium) medium without fetal calf serum (FCS) for two hours at 37°C in a head-over-head shaker. The cell suspensions were centrifuged at 250 ϫ g for 5 min, and cell pellets resuspended in 20 mL fresh RPMI 1640 containing 10% FCS. After five days attached cells were passaged twice to obtain adequate amounts of cells, which in their vast majority are of stromal origin. 1.4. Reverse transcriptase polymerase chain reaction (RT-PCR) Extreme care was taken to avoid specimen contamination during RNA extraction and RT-PCR. Filtered pipette tips (Biorad, Richmond, CA) were used, and mock-transcribed RNA from each cDNA sample served as control for contamination in the RT-PCR reaction. One microgram of total RNA was diluted in 10 L Diethylpyrocarbonate (DEPC, Sigma-Aldrich) water in a 0.5-mL PCR tube (Perkin-Elmer Cetus, Norwalk, CT), denatured at 65°C for 10 min in a programmable thermal cycler (TC; Techne, Cambridge, UK), rapidly cooled to 4°C, and reverse transcription performed in a final volume of 50 L under the following reaction conditions: 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 1 mM each of dATP, dCTP, dGTP, and dTTP (Promega, Madison, WI), 10 U human placental ribonuclease inhibitor (Promega), 200 pmol random hexamer oligonucleotide (Boehringer-Mannheim, Germany), and 50 U MMLV-Reverse Transcriptase (Promega). The reaction mix was incubated for eight minutes at 20°C and eight minutes at 25°C to achieve optimal hybridization between the RNA and the hexamer primers. The reverse transcriptase was then allowed to proceed at 37°C for 30 min. Five microliters (100 ng) of cDNA were used for PCR amplification in a final volume of 25 L under the following conditions: 10 mM Tris-HCl (pH 9), 50 mM KCl, 1.5 mM MgCl2, G. Untergasser et al./Experimental Gerontology 34 (1999) 275–287 279 Table 1 Primer sequences, primer localization, cDNA fragment lengths and annealing temperatures used for RT-PCR Primer Sequence 5Ј-3Ј Localization cDNA Fragment Annealing Temp. PRL-R 5Ј PRL-R 3Ј GH-R 5Ј GH-R 3Ј GH/PL 5Ј GH/PL 3Ј PRL 5Ј PRL-3Ј LDL-R 5Ј LDL-R 3Ј ACTTACATAGTTCAGCCAGAC TGAATGAAGGTCGCTGGACTCC GGTATGGATCTCTGGCAGCTG GGAGGGCAATGGGTGGATCTG CACTCAGGGTCCTGTGGACAG ACAGAGCGGCACTGCACGATG GGGTTCATTACCAAGGCCATC TTCAGGATGAACCTGGCTGAC CAATGTCTCACCAAGCTCTG TCTGTCTCGAGGGGTAGCTG 363–384 652–673 41–62 489–509 (Ϫ)35-(Ϫ)14 620–631 228–249 483–504 n.d. n.d. 310 bp 55°C 468 bp/402 bp 59°C 666 bp 63°C 276 bp 55°C 258 bp 57°C Localization referred to the start codon of the cDNA sequence encoding for th protein: PRL-R, Boutin et al., 1989; PRL, Cooke et al., 1981; GH/PL, Chen et al., 1989; GH-R, Leung et al., 1987. Due to their location in highly conserved regions of exon 1 and exon 5 the GH/PL primer pair amplifies mRNAs from all five GH and PL genes (GH-N, GH-V, PL-A, PL-B, PL-L). In addition to the full length transcripts, potential alternatively spliced products can also be detected. GH-N/V and PL-A/B transcripts were distinguished by digestion with Rsa I (GH-N/V) and Xba I (PL-A/B). n.d.: not determined. 0.1% Triton X-100, 0.2 mM each of dATP, dCTP, dGTP, and dTTP, 4 pmol each of the two oligonucleotide primers specific for each gene-product (PRL, GH/PL, GH-R, PRL-R; Table 1), and 0.125 U Taq polymerase (Promega). After initial denaturation for two minutes at 95°C, 35 amplification cycles were performed. PCR products were run on a 2% agarose gel containing 4 g/100 mL ethidiumbromide at 60 V/100 mA for two hours and visualized and photographed under UV light. 1.5. Immunofluorometric assays (IFMAs) for hPL-A/B, hGH-N, and hPRL Specific IFMAs for pituitary-derived hGH-N and hPRL and hPL-A/B were established based on our own panel of well-characterized monoclonal antibodies (MCA) as described previously (Untergasser et al., 1997). IFMA technology provided the highest sensitivity and widest measuring range, and were performed in analogy to our assays for glycoprotein hormones (Madersbacher et al., 1993). hPRL 81/541, hGH 66/217 (NIBSB, South Mimms, UK), and hPL (NIDDK, Bethesda, MD) were used as hormone standard preparations. Briefly, 10 g of highly purified MCA, coded as INN-hPL-37, INN-hGH-2, or INNhPRL-9, respectively, were diluted in 100 L phosphate-buffered saline (PBS) pH 7.2 and incubated for two hours at 37°C in a microtiter plate (Nunc, Roskilde, Denmark). Remaining binding sites were blocked with 200 L of 1% bovine serum albumin (BSA) in PBS for 30 min at 37°C. The plates were then washed three times with PBS containing 0.5 mL Tween 20 and 5 g of thiomersal/liter. For the assay, we used an incubation volume of 100 L/well and an assay buffer consisting of 50 mM Tris-HC1 (pH 7.75), 9 g/liter NaCl, 5 g BSA/liter, 0.1 g/liter Tween 40, 0.5 g/liter bovine gamma-globulin, and 20 mM Diethylenetriaminepentaacid (Sigma-Aldrich). Graded amounts of the hormone standards or homogenized tissue/cells (1:2 in assay buffer) were allowed to react on a orbit shaker (500 rpm, 90 min, 280 G. Untergasser et al./Experimental Gerontology 34 (1999) 275–287 20°C) followed by three washes and subsequent incubation with 100 ng of europium-labeled detection MCA INN-hPL-5, INN-hGH-5, or INN-hPRL-1, respectively (30 min, 20°C, orbit shaker). After extensive washing, enhancement solution was added (Pharmacia, Uppsala, Sweden) and incubated for 5 min on a orbit shaker. Time-resolved fluorescence was measured for 1 s in a fluorometer (1232 Delfia-fluorometer; Wallac, Turku, Finland). 1.6. Cell proliferation (WST-1 assay) 4-[3-(4-Iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) is a slightly red tetrazolium salt reduced to the dark-red formazan by the mitochondrial succinate–tetrazolium–reductase system, which gains substrate reduction by dehyrogenizing NADH. Proliferation of cells results in an increased mitochondrial reductase system activity, which leads to increasing amounts of dark-red formazan. The change of color from slightly red, to dark red assessed by measuring optical density, directly correlates to the number of viable cells in the sample. DU 145, PC 3, BPH-1, and prostatic short-term cultured cells, were seeded into 96-well plates at a density of 5000 cells/well, LnCap at 8000 cells/well in 100 L RPMI 1640 containing 1% FCS, 10 mg/mL penicillin, 100 units/mL streptomycin, and 10 mg/mL L-glutamin (PSG). Cells were starved for 12 h and then the medium was changed to 100 L RPMI 1640 containing 1% FCS and hGH (Humatrope 4I.E, Eli Lilly GmbH, Vienna, Austria), hPL (NIDDK), and hPRL (hPRL-SI AFP-B-2, NIH) dilutions (0.01–10 nM). To assess cell proliferation, 10 L WST-1 (Boehringer-Mannheim, Germany) was added to each well after a 48-h growing period. Optical density was measured after a further three hours at a wavelength of 450 nm in an ELISA reader (Dynatech, Burlington, MA). 2. Results 2.1. GH/PRL-receptor expression in the prostate PRL-R transcripts were present in the normal prostate and on all seven BPH/PCa samples (Fig. 2D). Surprisingly, this expression was not observed in the prostatic carcinoma cell lines PC3, LnCap, or BPH-1 (data not shown); only Du145 cells showed a weak expression of PRL-R. In contrast, the GH-R transcripts were present in all analyzed BPH/PCa tissue samples (Fig. 2E) and prostatic cell lines. While PC3, LnCap, and BPH-1 expressed only the mRNA encoding for the full length GH-R, Du145 cells showed in addition to the classical transcripts the exon 3 missing mRNA form (Ϫ66 bp). 2.2. Protein hormone expression in the prostate Even when using the highly sensitive RT-PCR method, 35 cycles of amplification were not sufficient to detect PRL transcripts in any of the analyzed cell lines (PC3, Du145, LnCap, and BPH-1), the seven BPH/PCa samples (Fig. 2B), and the normal prostate. In contrast, the positive control specimens (placenta and PBMCs) showed PRL expression. In a sensitive and G. Untergasser et al./Experimental Gerontology 34 (1999) 275–287 281 Fig. 2. Expression of GH/PL, PRL, PRL-receptor, and GH-receptor genes in human prostates. cDNAs were amplified in 35 cycles and specificities of the amplified products were verified by restriction enzyme digestion of the cDNA fragments. Five BPH and two PCa samples were analyzed for LDL-R (A), PRL (B), GH/PL (C), GH-R (D), and PRL-R (E). Human placenta (plac.) known to express PRL, GH/PL, PRL-receptors, and GH-receptors served as a positive control. GH-R cDNA includes or skips exon 3 (66 bp) (Urbanek et al., 1993). Both Isoforms of the GH-R bind hGH with identical affinity (Sobrier et al., 1993) and their expression is regulated in an interindividual rather than a tissue-specific manner (Wickelgren et al., 1995). In addition to the PRL-receptor mRNA (E), both isoforms of GH-receptor were expressed in all analyzed BPH/PCa specimens (D), but no transcripts encoding for hPRL (C) and hGH were observed. Only BPH#1 and PCa#6 showed a very weak PL gene expression (B). Molecular weight marker ϭ 100 bp DNA ladder (Biorad, Hercules, CA). specific IFMA for hPRL all prostatic tissues and cell lines contained cytosolic hPRL levels lower than 0.02 ng/mL (Table 2), whereas cytosolic extracts of freshly prepared PBMCs (5 ϫ 106 cells) contained 1.46 ng PRL/mL (n ϭ 4). All prostate carcinoma cell lines, as well as the normal prostate and PBMCs, were also negative in the GH/PL RT-PCR. The GH/PL gene cluster consists of five highly homologous genes coded as GH-N, GH-V, PL-L, PL-A, and PL-B. In this particular PCR, full length and/or alternatively spliced transcripts originating from any of these five GH/PL genes can be amplified. Interestingly, two of seven BPH/PCa samples, one BPH and one PCa, showed 282 G. Untergasser et al./Experimental Gerontology 34 (1999) 275–287 Table 2 GH/PL/PRL contents in cytosolic extracts of BPH and PCa tissue specimens and cell lines determined by sensitive IFMAs Tissue (code)/cell line hGHa hPLa hPRLa Testis PBMCs BPH #4, #5, #8, #10 PCa #7, #9, #11 PC3, Du 145, LnCap, BPH-1 0.51 Ͻ0.005 Ͻ0.005 Ͻ0.005 Ͻ0.005 0.10 Ͻ0.030 Ͻ0.030 Ͻ0.030 Ͻ0.030 5.87 1.46 Ͻ0.020 Ͻ0.020 Ͻ0.020 Tissue: 100 mg/mL Cells: 5 ϫ 106/mL Italic: prostatic cancer cell lines. a ng/mL. a very weak expression of the full length mRNAs for GH/PL, comprising all five exons (Fig. 2C). Restriction enzyme analysis of the generated cDNA fragment with GH- (Rsa I) and PL(Xba I) specific nucleases revealed two specific fragments for PL A/B gene transcripts, as did the positive control placenta (data not shown). Cytosolic concentrations of hGH and hPL were lower than 0.005 or 0.030 ng/mL in all analyzed carcinoma cell lines BPH-1, Du 145, PC3, LnCap, in the 7 BPH/PCa samples and in PBMC (Table 2). 2.3. Effects of GH, PL and PRL on prostate cell proliferation Effects of exogenous systemic hGH on cell proliferation were studied in vitro in prostatic cell lines and BPH/PCa tissue specimens expressing the GH-receptor. In addition to the cell lines Du 145, LnCap, PC3, and BPH-1, prostatic cell cultures of BPH- (BPH#10) or PCa(PCa#12) tissue were also incubated with hGH or hPL ranging from 0.01 to 10 nM in the presence of 1% FCS. Prostatic cell cultures showed dose-dependent increases in proliferation (Fig. 3), with greatest responses (138 Ϯ 3.2, BPH#10; 124 Ϯ 2.8, PCa#12; mean Ϯ % SD referred to the 1% FCS controls) at a doses of 1 nM hGH (Table 3). hPL showed a proliferative effect (113 Ϯ 6.0) only on the BPH#10 at a concentration of 0.01 nM (Fig. 3), but not on the PCa#12. Interestingly, the LnCap cell line was sensitive to hGH (130 Ϯ 4.4) and hPL (133 Ϯ 8.2), whereas the other prostatic cell lines Du 145, PC3, and BPH-1 did not respond to hGH or hPL. hPRL increased the proliferation of the BPH#10 (120 Ϯ 4.7) at a concentration of 0.1 nM, but showed no significant effects in the PCa#12 or in the prostatic carcinoma cell lines Du145, PC3, LnCap, or BPH-1 (Table 3). 3. Discussion Involvement of GH and PRL in the physiology of the rodent prostate has recently been demonstrated (Costello and Franklin, 1994; Reiter et al., 1995), but their roles in the G. Untergasser et al./Experimental Gerontology 34 (1999) 275–287 283 Fig. 3. Effects of hGH, hPL, and hPRL on BPH primary culture cell proliferation as determinated by the WST-1 assay. Cells were seeded into 96-well plates at a density of 5,000 to 8,000 cells per well in RPMI 1640 containing 10% FCS. After seven hours, medium was changed to RPMI 1640 containing 1% FCS and the respective hormone concentration ranging from 0.01 to 10 nM. Proliferation was measured after 48 h. Bars indicate means of triplicates Ϯ SD. Levels of significance are referred to 1% FCS control group ( * p Ͻ 0.025). pathology of human BPH or PCa are still debatable. BPH occurs only in humans and dogs; thus the data obtained in rats and mice may not reflect the appropriate situation in humans, because only the dorsolateral lobe of rodent prostates is ontogenetically comparable to the human prostate. It has been shown that systemic administration of GH and PRL can increase the number of androgen receptors, IGF-I and IGF-I receptors, and 5␣ reductase type I and II in the prostate of immature hypophysectomized rats (Prins, 1987; Reiter et al., 1992, 1995). Whether these effects are mediated directly or indirectly via systemic IGF-I produced in the liver remains unclear. Transgenic mice overexpressing rat PRL or bovine GH develop an enlarged prostate gland, but both transgenes, in addition to elevated IGF-I, also had increased androgen levels (Wennbo et al., 1997). In these animal models direct and indirect effects of protein hormones on prostate development and physiology cannot be distinguished. There are only few reports regarding direct effects of protein hormones on human BPH tissue (Costello and Franklin, 1994; Nevalainen et al., 1997). Immunochemistry and RT-PCR showed hPRL and the hPRL-R expression in the epithelial cells of human BPH tissue (Nevalainen et al., 1997). Moreover, hPRL revealed a proliferative effect (conc. 45 nM; 100-fold physiological concentration) on epithelial cell growth in seven-day organ cultures of BPH tissue. 284 G. Untergasser et al./Experimental Gerontology 34 (1999) 275–287 Table 3 Effects of hGH, hPL and PRL on proliferation of prostatic primary cell cultures and cancer cell lines determined by WST-1 assay Tissue/cell line hGHa hPLa hPRLa BPH#10 PCa#12 LnCap Du145, PC3, BPH-1 138 Ϯ 3.2 124 Ϯ 2.8 130 Ϯ 2.4 n.s. 113 Ϯ 6.0 n.s. 133 Ϯ 8.2 n.s. 120 Ϯ 4.7 n.s. n.s. n.s. n.s. ϭ nonsignificant increase of proliferation refereed to 1% FCS control (100 Ϯ 3.4%). Italic: prostatic cancer cell lines. a % mean Ϯ % SD. Conversely, we did not detect mRNA coding for the 23 K hPRL in any examined BPH/PCa sample (n ϭ 8) or in human prostatic cell lines (n ϭ 4), although the primer pair used, located in exon 3 and exon 4/5, amplifies, as expected, placental-, PBMC-, and pituitary-derived PRL mRNAs. Even with highly sensitive and specific IFMAs (Untergasser et al., 1997; Madersbacher et al., 1998), we could not detect significant amounts of hPRL protein in any prostatic cell line or tissue, in contrast to fresh cytosolic extracts of PBMCs containing significant amounts of hPRL (1.46 ng/mL; Table 2). Moreover, cytosolic extracts of all prostatic specimens and cell lines were negative for hGH and hPL, indicating that no auto/paracrine axes, including ectopically produced hPRL or hGH, are involved in the pathogenesis of BPH or prostate cancer. Two of seven analyzed BPH/PCa samples showed weak hPL expression at the mRNA level (Fig. 2C), which is not very surprising, because hPL, normally a trophoblast hormone, is occasionally produced by extratrophoblastic tumors (Sheth et al., 1977; Campo et al., 1989; Dirnhofer et al., 1998). Employing different concentrations of hPL (0.01 to 10 nM) in the WST-1 assay, a significant increase of cell proliferation in BPH#10 and in the androgensensitive prostate cancer cell line LnCap (Table 3) has been observed. Because the receptor for hPL is still unknown, it is generally believed that this hormone binds to hGH- and/or hPRL-receptors. hPL binds 2300-fold weaker than hGH to the hGH-receptor (Lowman et al., 1991; Sobrier et al., 1993; Urbanek et al., 1993). Interestingly, we observed the best proliferative responses with low concentrations of this somatotrophic hormone (0.01– 0.1 nM), indicating that there is a distinct receptor or signalling pathway for hPL. hPRL-R receptor mRNA was present in all analyzed BPH/PCa samples (Fig. 2E) and in the normal prostate, but not in the human prostatic cancer cell lines Du145, PC3, LnCap, and BPH-1 in our tissue culture approach. Dedifferentiation of the cell lines or genetic instabilities are probably responsible for the loss of the transcripts encoding for full-length receptors. Only Du145 showed weak expression, but lower than BPH/PCa tissue specimens. Proliferation of the BPH primary culture was significantly increased (Fig. 3, Table 3) at hPRL concentrations ranging from 0.01 to 0.1 nM. Normal serum values for hPRL in nonpregnant, nonlactating human adults range between 0.12 nM and 0.6 nM, indicating, that the slightly increasing physiological PRL values observed in the elderly (Vekemans and Robyn, 1975), could influence prostatic growth and support BPH development. Because all BPH/PCa samples express the hGH-receptor gene (Fig. 2D), it is conceivable G. Untergasser et al./Experimental Gerontology 34 (1999) 275–287 285 that pituitary-derived endocrine hGH affects differentiation and function of the prostate gland, as shown in animal models (Reiter et al., 1992, 1995). In this study, we report that in vitro proliferation of human prostatic cell cultures and the androgen-sensitive human prostatic cell line LnCap can be increased by dose-dependent administration of hGH (Fig. 3). This endocrine effect is direct, and does not involve testicular sex steroid hormones or increased hepatic IGF-I. Future studies will include more patients, and should yield further information on the effects of hGH administration and the in vivo growth of prostatic carcinoma and BPH. Our findings are relevant in the context of discussed GH substitution therapies in elderly people, because the age-related decrease of the GH/IGF-I axis might be a natural mechanism of protection for preventing abnormal growth of prostate and mammary cells (Siu et al., 1997). Acknowledgment This work was supported by the Austrian National Bank (#6120). 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