Lack of Association between Enhanced TRPM-2/Clusterin Expression and Increased Apoptotic Activity in Sex-Hormone-Induced Prostatic Dysplasia of the Noble Rat
1998; Elsevier BV; Volume: 153; Issue: 1 Linguagem: Inglês
10.1016/s0002-9440(10)65553-8
ISSN1525-2191
AutoresShuk‐Mei Ho, Irwin Leav, Shibnath Ghatak, Frederick B. Merk, Vehda S. Jagannathan, Kevin Mallery,
Tópico(s)Cancer, Lipids, and Metabolism
ResumoAlthough the functional role of TRPM-2/clusterin in the prostate remains controversial, it has been postulated that transcriptional activation of the gene is an important mechanism in castration-induced prostatic involution and perhaps is a means for prostatic cells to escape apoptotic induction. In the present study, we have measured expression levels of TRPM-2/clusterin and apoptotic activities in the prostates of castrated Noble (NBL) rats and those treated with testosterone (T) and estradiol-17β (E2) for 16 weeks. We have previously shown that the combined sex hormone treatment (T+E2) induces dysplasia, a purported preneoplastic lesion, exclusively in the dorsolateral prostates (DLPs) of all treated rats. In the present study, we demonstrate that, as expected, castration readily induced enhanced TRPM-2/clusterin expression, which was accompanied by increased apoptotic activity in the epithelia of DLP and ventral prostate (VP). The increase in TRPM-2/clusterin expression appeared earlier and was more dramatic in the VP than in the DLP. In sharp contrast, treatment of rats with T+E2 for 16 weeks induced augmentation of TRPM-2/clusterin expression selectively in the dysplastic lesions of the DLP but not in the lesion-free VP. The enhanced expression of TRPM-2/clusterin in the dysplastic epithelium was, however, not attended by an increase in apoptotic activity within the lesion. Thus, the observed up-regulation of TRPM-2/clusterin expression in the dysplastic foci of T+E2-treated rats occurred in animals whose androgen status remained normal and, despite the increased level of expression of this gene, apoptotic activity in these lesions was unchanged from basal values measured in the DLPs of untreated rats. These findings suggest that TRPM-2/clusterin expression in dysplastic lesions was no longer repressed by androgen nor was it associated with apoptosis. We propose that overexpression of the gene is likely a phenotype of neoplastic transformation. In addition, we speculate that TRPM-2/clusterin may serve as a survival factor, which could favor accumulation of transformed cells in dysplastic foci and thus promote the carcinogenic process. Although the functional role of TRPM-2/clusterin in the prostate remains controversial, it has been postulated that transcriptional activation of the gene is an important mechanism in castration-induced prostatic involution and perhaps is a means for prostatic cells to escape apoptotic induction. In the present study, we have measured expression levels of TRPM-2/clusterin and apoptotic activities in the prostates of castrated Noble (NBL) rats and those treated with testosterone (T) and estradiol-17β (E2) for 16 weeks. We have previously shown that the combined sex hormone treatment (T+E2) induces dysplasia, a purported preneoplastic lesion, exclusively in the dorsolateral prostates (DLPs) of all treated rats. In the present study, we demonstrate that, as expected, castration readily induced enhanced TRPM-2/clusterin expression, which was accompanied by increased apoptotic activity in the epithelia of DLP and ventral prostate (VP). The increase in TRPM-2/clusterin expression appeared earlier and was more dramatic in the VP than in the DLP. In sharp contrast, treatment of rats with T+E2 for 16 weeks induced augmentation of TRPM-2/clusterin expression selectively in the dysplastic lesions of the DLP but not in the lesion-free VP. The enhanced expression of TRPM-2/clusterin in the dysplastic epithelium was, however, not attended by an increase in apoptotic activity within the lesion. Thus, the observed up-regulation of TRPM-2/clusterin expression in the dysplastic foci of T+E2-treated rats occurred in animals whose androgen status remained normal and, despite the increased level of expression of this gene, apoptotic activity in these lesions was unchanged from basal values measured in the DLPs of untreated rats. These findings suggest that TRPM-2/clusterin expression in dysplastic lesions was no longer repressed by androgen nor was it associated with apoptosis. We propose that overexpression of the gene is likely a phenotype of neoplastic transformation. In addition, we speculate that TRPM-2/clusterin may serve as a survival factor, which could favor accumulation of transformed cells in dysplastic foci and thus promote the carcinogenic process. Testosterone-repressed prostatic message-2 gene (TRPM-2) was first identified and cloned from the regressing rat prostate after castration.1Montpetit ML Lawless KR Tenniswood MP Evidence for an androgen repressed mRNA in rat ventral prostate.Prostate. 1995; 8: 25-36Crossref Scopus (216) Google Scholar The gene encodes a sulfated glycoprotein that is closely related to sulfated glycoprotein-2 (SGP-2) secreted by rat Sertoli cells,2Collard MW Griswold MD Biosynthesis and molecular cloning of sulfated glycoprotein 2 secreted by rat Sertoli cells.Biochemistry. 1987; 26: 3297-3303Crossref PubMed Scopus (272) Google Scholar, 3Griswold MD Roberts K Bishop P Purification and characterization of a sulfated glycoprotein secreted by Sertoli cells.Biochemistry. 1986; 25: 7265-7270Crossref PubMed Scopus (94) Google Scholar clusterin in ram rete testicular fluid,4Blaschuk O Burdzy K Fritz IB Purification and characterization of a cell-aggregating factor (clusterin), the major glycoprotein in ram rete testis fluid.J Biol Chem. 1983; 258: 7714-7720Abstract Full Text PDF PubMed Google Scholar glycoprotein III in bovine adrenal chromaffin granules,5Palmer DJ Christie DL The primary structure of glycoprotein III from bovine adrenal medullary chromaffin granules: sequence similarity with human serum protein-40,40 and rat Sertoli cell glycoprotein.J Biol Chem. 1990; 265: 6617-6623Abstract Full Text PDF PubMed Google Scholar and apolipoprotein J, SP-40,40, and complement lysis inhibitor in human serum.6de Silva HV Harmony JA Stuart WD Gil CM Robbins J Apolipoprotein J: structure and tissue distribution.Biochemistry. 1990; 29: 5380-5389Crossref PubMed Scopus (290) Google Scholar, 7Jenne DE Tschopp J Molecular structure and functional characterization of a human complement cytolysis inhibitor found in blood and seminal plasma: identity to sulfated glycoprotein 2, a constituent of rat testis fluid.Proc Natl Acad Sci USA. 1989; 86: 7123-7127Crossref PubMed Scopus (349) Google Scholar The TRPM-2/clusterin gene product and its related proteins share similar primary amino acid sequences but undergo significantly different post-translational modifications.8Tenniswood MP Guenette RS Lakins J Mooibroek M Wong P Welsh JE Active cell death in hormone-dependent tissue.Cancer Metastasis Rev. 1992; 11: 197-220Crossref PubMed Scopus (244) Google Scholar, 9Buttyan R Olsson CA Pintar J Chang C Bandyk M Ng PY Sawczuk IS Induction of the TRPM-2 gene in cells undergoing programmed death.Mol Cell Biol. 1989; 9: 3473-3481Crossref PubMed Scopus (423) Google Scholar Apart from having a broad tissue distribution, these related proteins have been implicated in a wide variety of cellular processes and functions (for reviews see 8Tenniswood MP Guenette RS Lakins J Mooibroek M Wong P Welsh JE Active cell death in hormone-dependent tissue.Cancer Metastasis Rev. 1992; 11: 197-220Crossref PubMed Scopus (244) Google Scholar, 10May PC Finch CE Sulfated glycoprotein 2: new relationships of this multifunctional protein to neurodegeneration.Trends Neurosci. 1992; 15: 391-396Abstract Full Text PDF PubMed Scopus (166) Google Scholar). Perhaps the most common postulated role for TRPM-2/clusterin is its involvement in the regulation of apoptosis. In this regard, expression of TRPM-2/clusterin is induced or greatly enhanced in a number of tissues undergoing apoptosis.11Leger JG Montpetit ML Tenniswood MP Characterization and cloning of androgen repressed mRNAs from rat ventral prostate.Biochem Biophys Res Commun. 1987; 147: 196-203Crossref PubMed Scopus (212) Google Scholar, 12Kyprianou N Isaacs JT Expression of transforming growth factor-β in the rat ventral prostate during castration-induced programmed cell death.Mol Endocrinol. 1989; 3: 1515-1522Crossref PubMed Scopus (395) Google Scholar, 13Sensibar JA Griswold MD Sylvester SR Buttyan R Bardin CW Cheng CY Dudek S Lee C Prostatic ductal system in rats: regional variation in localization of an androgen-repressed gene product, sulfated glycoprotein-2.Endocrinology. 1991; 128: 2091-2102Crossref PubMed Scopus (91) Google Scholar, 14Russo P Warner JA Huryk R Perez G Heston WD TRPM-2 gene expression in normal rat ventral prostate following castration and exposure to diethylstilbestrol, flutamide, MK-906 (finasteride), and coumarin.Prostate. 1994; 24: 237-243Crossref PubMed Scopus (24) Google Scholar, 15Norman DJ Feng L Cheng SS Gubbay J Chan E Heintz N The lurcher gene induces apoptotic death in cerebellar Purkinje cells.Development. 1995; 121: 1183-1193PubMed Google Scholar, 16Conner J Buttyan R Olsson CA D'Agati V O'Toole K Sawczuk IS SGP-2 expression as a genetic marker of progressive cellular pathology in experimental hydronephrosis.Kidney Int. 1991; 39: 1098-1103Crossref PubMed Scopus (66) Google Scholar, 17May PC Lampert-Etchells M Johnson SA Poirier J Masters JN Finch CE Dynamics of gene expression for a hippocampal glycoprotein elevated in Alzheimer's disease and in response to experimental lesions in rat.Neuron. 1990; 5: 831-839Abstract Full Text PDF PubMed Scopus (290) Google Scholar, 18Gu Y Jow GM Moulton BC Lee C Sensibar JA Park-Sarge OK Chen TJ Gibori G Apoptosis in decidual tissue regression and reorganization.Endocrinology. 1994; 135: 1272-1279Crossref PubMed Scopus (93) Google Scholar, 19Hurwitz A Ruutiainen-Altman K Marzella L Botero L Dushnik M Adashi EY Follicular atresia as an apoptotic process: atresia-associated increase in the ovarian expression of the putative apoptotic marker sulfated glycoprotein-2.J Soc Gynecol Invest. 1996; 3: 199-208Crossref PubMed Scopus (8) Google Scholar In the prostate, TRPM-2/clusterin was first considered to be an androgen-repressed gene with its protein product playing a role in regression of the gland after castration.1Montpetit ML Lawless KR Tenniswood MP Evidence for an androgen repressed mRNA in rat ventral prostate.Prostate. 1995; 8: 25-36Crossref Scopus (216) Google Scholar, 11Leger JG Montpetit ML Tenniswood MP Characterization and cloning of androgen repressed mRNAs from rat ventral prostate.Biochem Biophys Res Commun. 1987; 147: 196-203Crossref PubMed Scopus (212) Google Scholar, 12Kyprianou N Isaacs JT Expression of transforming growth factor-β in the rat ventral prostate during castration-induced programmed cell death.Mol Endocrinol. 1989; 3: 1515-1522Crossref PubMed Scopus (395) Google Scholar, 13Sensibar JA Griswold MD Sylvester SR Buttyan R Bardin CW Cheng CY Dudek S Lee C Prostatic ductal system in rats: regional variation in localization of an androgen-repressed gene product, sulfated glycoprotein-2.Endocrinology. 1991; 128: 2091-2102Crossref PubMed Scopus (91) Google Scholar A number of past studies have demonstrated that a close association exists between castration-induced apoptosis and the induction of TRPM-2/clusterin expression in the epithelium of the rat ventral prostate (VP).1Montpetit ML Lawless KR Tenniswood MP Evidence for an androgen repressed mRNA in rat ventral prostate.Prostate. 1995; 8: 25-36Crossref Scopus (216) Google Scholar, 11Leger JG Montpetit ML Tenniswood MP Characterization and cloning of androgen repressed mRNAs from rat ventral prostate.Biochem Biophys Res Commun. 1987; 147: 196-203Crossref PubMed Scopus (212) Google Scholar, 12Kyprianou N Isaacs JT Expression of transforming growth factor-β in the rat ventral prostate during castration-induced programmed cell death.Mol Endocrinol. 1989; 3: 1515-1522Crossref PubMed Scopus (395) Google Scholar, 13Sensibar JA Griswold MD Sylvester SR Buttyan R Bardin CW Cheng CY Dudek S Lee C Prostatic ductal system in rats: regional variation in localization of an androgen-repressed gene product, sulfated glycoprotein-2.Endocrinology. 1991; 128: 2091-2102Crossref PubMed Scopus (91) Google Scholar However, in several recent investigations, enhanced TRPM-2/clusterin expression was found to be dissociated from apoptosis and/or from androgen regulation. For example, prostatic involution, induced by treating rats with anti-androgens, 5α-reductase inhibitors, or luteinizing hormone releasing hormone (LHRH) agonists, was not attended by the induction of TRPM-2/clusterin expression in the rat VP.14Russo P Warner JA Huryk R Perez G Heston WD TRPM-2 gene expression in normal rat ventral prostate following castration and exposure to diethylstilbestrol, flutamide, MK-906 (finasteride), and coumarin.Prostate. 1994; 24: 237-243Crossref PubMed Scopus (24) Google Scholar, 20Fleshner NE Trachtenberg J Sequential androgen blockade: a biological study in the inhibition of prostatic growth.J Urol. 1992; 148: 1928-1931PubMed Google Scholar Similarly, in several prostatic neoplasms and cancer cell lines21Brandstrom A Westin P Bergh A Cajander S Damber JE Castration induces apoptosis in the ventral prostate but not in an androgen-sensitive prostatic adenocarcinoma in the rat.Cancer Res. 1994; 54: 3594-5601PubMed Google Scholar, 22Akakura K Bruchovsky N Rennie PS Coldman AJ Goldenberg SL Tenniswood M Fox K Effects of intermittent androgen suppression on the stem cell composition and the expression of the TRPM-2 (clusterin) gene in the Shionogi carcinoma.J Steroid Biochem Mol Biol. 1996; 59: 501-511Crossref PubMed Scopus (43) Google Scholar, 23Rennie PS Bruchovsky N Akakura K Goldenberg SL Otal N Akakura S Wong P Tenniswood M Effect of tumor progression on the androgenic regulation of the androgen receptor, TRPM-2 and YPT1 genes in the Shionogi carcinoma.J Steroid Biochem Mol Biol. 1994; 50: 31-40Crossref PubMed Scopus (24) Google Scholar as well as in the VPs of aging rats,24Marinelli M Quaglino D Bettuzzi S Strocchi P Davalli P Corti A Increased levels of clusterin mRNA in the ventral prostate of the aging rat are associated to increases in cuboidal (atrophic) cell population and not to changes in apoptotic activity.Biochem Cell Biol. 1994; 72: 512-521Crossref Scopus (23) Google Scholar TRPM-2/clusterin up-regulation occurred in the absence of increased apoptotic activity or diminished androgen stimulation. It has therefore been suggested that TRPM-2/clusterin expression, under these physiological or pathological conditions, reflects the escape of prostatic cells from androgen regulation or apoptotic control. We have reported that treatment of Noble (NBL) rats with testosterone (T) and estradiol-17β (E2) for 16 weeks induces dysplasia, a purported preneoplastic lesion, exclusively in the dorsolateral prostates (DLPs) of all treated rats.25Leav I Ho SM Ofner P Merk FB Kwan PW Damassa D Biochemical alterations in sex hormone-induced hyperplasia and dysplasia of the dorsolateral prostates of Noble rats.J Natl Cancer Inst. 1988; 80: 1045-1053Crossref PubMed Scopus (178) Google Scholar, 26Leav I Merk FB Kwan PW Ho SM Androgen-supported estrogen-enhanced epithelial proliferation in the prostates of intact Noble rats.Prostate. 1989; 15: 23-40Crossref PubMed Scopus (133) Google Scholar The sex-hormone-induced lesion is morphologically similar to a premalignant lesion, prostatic intraepithelial neoplasia (PIN), in the human gland.27McNeal JE Bostwick DG Intraductal dysplasia: a premalignant lesion of the prostate.Hum Pathol. 1986; 17: 64-71Abstract Full Text PDF PubMed Scopus (482) Google Scholar, 28Bostwick DG Pacelli A Lopqez-Beltran A Molecular biology of prostatic intraepithelial neoplasia.Prostate. 1996; 29: 117-134Crossref PubMed Scopus (175) Google Scholar Longer-term treatment with T+E2 has been reported to induce adenocarcinomas in the DLPs of a majority of treated animals.29Drago JR The induction of NB rat prostatic carcinomas.Anticancer Res. 1984; 4: 255-256PubMed Google Scholar, 30Bosland MC Ford H Horton L Induction at high incidence of ductal prostate adenocarcinomas in NBL/Cr and Sprague-Dawley Hsd: SD rats treated with a combination of testosterone and estradiol 17β or diethylstilbestrol.Carcinogenesis. 1994; 16: 1311-1317Crossref Scopus (164) Google Scholar In the present study, we now report a marked elevation of TRPM-2/clusterin expression, localized in dysplastic foci but not observed in adjacent normal epithelia of the DLP, nor was it found in the lesion-free VP. Moreover, high levels of TRPM-2/clusterin expression were not attended by increased apoptotic activity in these DLP lesions. Male NBL rats were purchased from Charles River Laboratories (Wilmington, MA) at 5 to 6 weeks old. Animals were housed at the departmental animal facility until they reached a size of 280 to 300 g or 11 to 12 weeks of age before they were used in these studies. All surgery was done under light isoflurane (Ohmeda Caribe, Guayama, PR) anesthesia. Animals were separated into four groups (n = 6 animals per group). The first group was surgically implanted with two 2-cm Silastic capsules (catalog number 602–205; 1.0-mm inner diameter × 2.2-mm outer diameter, Dow Corning Corp., Midland, MI) filled with T (Sigma Chemical Co., St. Louis, MO) and one 1-cm capsule filled with E2 (Sigma) as previously described.26Leav I Merk FB Kwan PW Ho SM Androgen-supported estrogen-enhanced epithelial proliferation in the prostates of intact Noble rats.Prostate. 1989; 15: 23-40Crossref PubMed Scopus (133) Google Scholar, 27McNeal JE Bostwick DG Intraductal dysplasia: a premalignant lesion of the prostate.Hum Pathol. 1986; 17: 64-71Abstract Full Text PDF PubMed Scopus (482) Google Scholar We previously showed that this treatment maintained normal levels of circulating T but caused a moderate elevation in plasma E2.25Leav I Ho SM Ofner P Merk FB Kwan PW Damassa D Biochemical alterations in sex hormone-induced hyperplasia and dysplasia of the dorsolateral prostates of Noble rats.J Natl Cancer Inst. 1988; 80: 1045-1053Crossref PubMed Scopus (178) Google Scholar These rats were killed by an overdose of isoflurane (Ohmeda Caribe) followed by decapitation 16 weeks after implantation. The second group consisted of untreated age-matched rats that served as controls. The last two groups were orchiectomized via the scrotal route.25Leav I Ho SM Ofner P Merk FB Kwan PW Damassa D Biochemical alterations in sex hormone-induced hyperplasia and dysplasia of the dorsolateral prostates of Noble rats.J Natl Cancer Inst. 1988; 80: 1045-1053Crossref PubMed Scopus (178) Google Scholar One group of castrates was killed on day 3, and the others were killed on day 7 after castration. Three animals from each group were used for tissue total RNA preparation, and the remaining three were used for histology. The VPs and DLPs were excised from individual animals and used to generated total RNA and multiple serial histology sections. All animal treatment protocols were previously approved by Tufts Animal Care and Usage Committee and are in accordance with National Institutes of Health animal usage guidelines. All chemicals used were of reagent grade and were purchased from Sigma or from Fisher (Pittsburgh, PA) unless otherwise indicated. Restriction enzymes were from New England Biolabs (Beverly, MA). The 10X borate buffer (pH 8.0) contained 0.5 mol/L boric acid, 50 mmol/L sodium borate, 100 mmol/L sodium sulfate, 10 mmol/L EDTA. The 1 mol/L phosphate buffer (pH 7.0) was 1 mol/L dibasic sodium phosphate adjusted to pH 7.0 using phosphoric acid. Total RNA was prepared by a modified single-step method31Chomczynski P Sacchi N Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.Anal Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63357) Google Scholar using RNAzol B (Tel-Test, Friendswood, TX). Ten micrograms of total RNA from each sample was size fractionated through a 0.8% agarose (electrophoresis grade, Gibco-BRL, Gaithersburg, MD)/1 mol/L formaldehyde gel in 1X borate buffer, transferred to a Nytran membrane (Schleicher & Schuell, Keene, NH) by a downward alkaline capillary transfer method,32Chomczynski P One-hour downward alkaline capillary transfer for blotting of DNA and RNA.Anal Biochem. 1992; 201: 134-139Crossref PubMed Scopus (497) Google Scholar and ultraviolet cross-linked onto the membrane using a UV Stratalinker 1800 (Stratagene, La Jolla, CA). A cDNA probe, pG21–04, containing a 300-bp insert corresponding to the 3′ end of rat TRPM-2/clusterin sequence,1Montpetit ML Lawless KR Tenniswood MP Evidence for an androgen repressed mRNA in rat ventral prostate.Prostate. 1995; 8: 25-36Crossref Scopus (216) Google Scholar, 12Kyprianou N Isaacs JT Expression of transforming growth factor-β in the rat ventral prostate during castration-induced programmed cell death.Mol Endocrinol. 1989; 3: 1515-1522Crossref PubMed Scopus (395) Google Scholar was kindly provided by Dr. Martin Tenniswood at the University of Ottawa, Canada. The cDNA (20 to 40 ng) was routinely radiolabeled to a specific activity of approximately 1 × 109 cpm/μg of cDNA. Northern blots were prehybridized for 2 to 3 hours in 0.5 mol/L phosphate buffer, 7% sodium dodecyl sulfate SDS, 1 mmol/L EDTA, 100 μg/ml low molecular weight salmon testis DNA (pH 7.0) at 60°C to 62°C. The blots were then hybridized with 106 cpm/ml32P-labeled cDNA probes for approximately 16 hours at 62°C in a buffer similar to the prehybridization buffer with the exclusion of the low molecular weight salmon testis DNA. After hybridization, the blots were washed in 40 mmol/L phosphate buffer, 1% SDS, and 1 mmol/L EDTA, twice at room temperature for 15 minutes each and then once at the hybridization temperature for 15 minutes, and rinsed once in 40 mmol/L phosphate buffer without SDS. The washed blots were wrapped in plastic and exposed with Fuji x-ray film at −70°C with intensifying screens for autoradiography. To verify equal loading of RNA in each lane, blots were stripped in boiling diethylpyrocarbonate-treated H2O for 15 minutes and reused for hybridization with an end-labeled 30-mer oligonucleotide (5′-d(CGGCATGTATTAGC-TCTAGAATTACCACAG)-3′)33Clements JA Matheson BA Wines DR Brady JM MacDonald RJ Funder JW Androgen dependence of specific kallikrein gene family members expressed in rat prostate.J Biol Chem. 1988; 263: 16132-16137Abstract Full Text PDF PubMed Google Scholar complementary to part of the 18 S rRNA at 45°C. Hybridization signals were quantitated by densitometric scanning and normalized with respect to the corresponding 18 S rRNA signal to correct for loading variations. Normalized signal intensities of samples obtained from the DLPs of untreated rats were designated as controls and arbitrarily assigned a value of 1. Signal intensities of samples obtained from the VPs of control and treated animals and DLPs of T+E2-treated rats were compared with a contiguous DLP control sample within the same Northern blot and expressed as folds of the control value (set as 1) to obtain relative mRNA levels. The prominent 4.5-kb TRPM-2/clusterin transcript signal was used for quantitation of this message. Data points in Figure 1, Figure 2 (histograms) are group mean values derived from three separate experiments. In each experiment, total RNA was prepared from individual animals and used to obtain at least two Northern blots. After hybridization, signal intensities were quantitated for each blot, and values from the two blots were averaged to give the mean signal intensity for each sample.Figure 2Effects of 16-week T+E2 treatment on prostatic TRPM-2/clusterin mRNA expression. Northern blot analysis was used to estimate relative message levels in the DLPs and VPs of untreated and T+E2-treated NBL rats. Experimental protocol and message quantitation methods were similar to those described for Figure 1. A: A representative autoradiograph. B: Histograms are relative message levels expressed as group means ± SE derived from three separate experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) A modified procedure adapted from Höfler et al34Höfler H Childers H Montminy MR Lechan RM Goodman RH Wolfe HJ In situ hybridization methods for the detection of somatostatin mRNA in tissue sections using antisense RNA probes.Histochem J. 1986; 18: 597-604Crossref PubMed Scopus (161) Google Scholar and Komminoth et al35Komminoth P Merk FB Leav I Wolfe HJ Roth J Comparison of 35S- and digoxigenin-labeled RNA and oligonucleotide probes for in situ hybridization: expression of mRNA of the seminal vesicle protein II and androgen-receptor genes in the rat prostate.Histochemistry. 1992; 98: 217-218Crossref PubMed Scopus (118) Google Scholar was used on 6- to 10-μm-thick frozen sections. To detect the TRPM-2/clusterin, a35S-labeled riboprobe was prepared from linearized pG21–04 using a Riboprobe R system kit from Promega (Madison, WI) following the manufacturer's protocol. The riboprobe was diluted in hybridization buffer to deliver 6 × 105 cpm/20 μl/slide. As negative controls, an unrelated riboprobe, derived from a 550-bp cDNA encoding the human preproparathyroid hormone (prepro-PTH) sequence,36Hendy GN Kronenberg HM Potts Jr, JT Rich A Nucleotide sequence of cloned cDNAs encoding human preproparathyroid.Proc Natl Acad Sci USA. 1981; 78: 408-413Crossref Scopus (134) Google Scholar was similarly applied to consecutive sections. Photomicrographs were taken on a Zeiss universal microscope. The dark-field images were produced with the aid of a dark-field fiberoptics illuminator (Microvideo Instrument, Avon, MA). For immunohistological detection of the translation product of the TRPM-2/clusterin gene, a polyclonal antibody against rat sulfated glycoprotein-2 was used as the primary anitbody. The antibody was a generous gift from Dr. C. Y. Cheng of the Population Council, Center for Biomedical Research, New York, NY. It had been characterized37Grima J Zwain I Lockshin RA Bardin CW Cheng CY Diverse secretory of clusterin by epididymis and prostate/seminal vessels undergoing cell repression following orchiectomy.Endocrinology. 1990; 126: 2989-2997Crossref PubMed Scopus (64) Google Scholar and previously used to localize TRPM-2/clusterin translational product in rat prostatic tissues.38Ahuja HS Tenniswood M Lockshin R Zakeri ZF Expression of clusterin in cell differentiation and cell death.Biochem Cell Biol. 1994; 72: 523-529Crossref PubMed Scopus (44) Google Scholar Prostatic tissues were fixed in 10% buffered formalin, routinely processed, and embedded in paraffin. Sections were cut at 6 μm and deparaffinized through a graded series of xylenes and ethanol and then rehydrated in water and phosphate-buffered saline (PBS). After deparaffinization, sections were treated for 30 minutes with normal rabbit serum. The blocking serum was drained off, and the sections were rinsed twice in PBS. The primary antibody, at a dilution of 1:1000, was applied to the sections. Incubation was carried out overnight at 4°C in humid chambers. After rinsing off the primary antibody with PBS, the sections were incubated with a biotinylated goat anti-rabbit antiserum (ABC Kit, Vector Laboratories, Burlingame, CA) at a 1:200 dilution for 30 minutes at room temperature. After a rinse in PBS, the sections were covered with peroxidate-conjugated streptavidin for 30 minutes at room temperature, washed with PBS, and incubated with diaminobenzidine/H2O2 substrate until the desired density of the reaction product was achieved (2 to 10 minutes). Sections were then lightly counterstained with 5% hematoxylin. Negative controls were processed in parallel with normal rabbit IgG substituted for the primary antibody. Estimates of the intensity of immunostaining was evaluated by F. Merk and K. Mallery in a double-blinded manner. Intensity was scored as 1+ to 4+ with the higher number indicating the strongest positivity. Apoptotic cells were detected in either paraffin or frozen sections (5 to 6 μm thick) using the ApopTag in situnuclear DNA fragmentation detection kit (Oncor, Gaithersburg, MD), following instructions described in the company's literature. This assay uses terminal deoxynucleotide transferase to catalytically link digoxigenin-labeled nucleotides to 3′-OH ends of DNA, fragmented during apoptosis. Digoxigenin-labeled nuclei were recognized by immunoperoxidase staining. Four replicate sections from each prostatic specimen were stained with the ApopTag protocol. Using a 25× objective, 500 cells were counted for each specimen. The number of positively stained cells were then divided by 500 to estimate the percentage of apoptotic cells in each specimen. Apoptotic indices (AIs) were calculated as group means ± SD derived from values obtained from sections of individual animals of the group (data were derived from individual animals; n = 3 animals per group). TRPM-2/clusterin message levels in untreated intact VP were approximately 0.8 ± 0.3-fold (80%) of that found in the DLP (Figure 1). Three days after castration a 5.5 ± 1.0-fold increase in TRPM-2/clusterin expression was observed in the DLP whereas a 27 ± 5-fold elevation occurred in the VP. At 7 days after castration, the level of TRPM-2/clusterin mRNA in the DLP continued to rise to 10.5 ± 2.0-fold whereas values in the VP declined to approximately 15.5 ± 3.0-fold. Thus, castration induced a slower increase of TRPM-2/clusterin mRNA in the DLP than in the VP. The magnitude of increase in the DLP was also of a lower magnitude than was the level of expression in the VP. Values reported were group means ± SD with three animals in a group. After 16 weeks of T+E2 treatment, a 21 ± 4-fold increase in TRPM-2/clusterin mRNA levels was observed in the DLPs harboring dysplasia whereas no increase in expression was found in the lesion-free VPs of
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