| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Male Reproductive Tract |
Institut National de la Santé et de la Recherche Médicale (INSERM U 407),3 Faculté de Médecine Lyon-Sud, 69921, Oullins, France
Biochemistry Laboratory,4 University-Hospital Sahloul, Sousse, Tunisia
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
apoptosis, gene regulation, mechanisms of hormone action, prostate, steroid hormones
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
It is commonly believed that execution of apoptosis is brought about by the caspase family of proteins. Although very obvious and clearly expected, almost no published evidence links caspase activation with prostate apoptosis under androgen control in vivo [15]. Indeed, the chief effectors of the apoptotic cell death pathway are the caspase family of cysteine proteases [16]. Caspases are synthesized as inactive precursors that are cleaved at specific aspartate residues to generate the active subunit [17]. Procaspase cleavage can occur by several mechanisms, including proximity-induced autoprocessing or cleavage by other caspases, revealing a caspase cascade with upstream initiator caspases such as caspases-8, -9, and -10 and downstream effector caspases such as caspases-3, -6, and -7 [18].
In the present study, by using as a model castrated male rats treated with androgens, we have examined whether caspase expression and activation during the apoptotic cell death process in the ventral, dorsolateral, and anterior prostate lobes are targeted by androgens. Specifically, the expression and activation of two effector caspases (caspases-3 and -6) and of one initiator caspase (caspase-8) were evaluated in terms of mRNA and protein (including procaspase and active cleaved caspase) levels in ventral, dorsolateral, and anterior rat prostate lobes following androgen withdrawal (castration) and replacement.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
TRIzol and deoxynucleotide triphosphates (dNTPs) were obtained from Life Technologies, Inc. (Eragny, France). Protease-inhibitors cocktail, calf thymus deoxynucleotidyl terminal transferase, and biotin 16-deoxyuridine triphosphate (dUTP) were obtained from Roche Molecular Biochemicals (Mannheim, Germany). Sigma (Meylan, France) was the source for random hexanucleotides, Biomax MR films, BSA, actin polyclonal antibody (catalog no. A5060), diaminobenzidine (DAB), nickel chloride, cobalt chloride, sodium cacodylate, CAPS buffer, and ExtrAvidine peroxidase. Taq polymerase was purchased from Promega Life Science (Madison, WI). Horseradish peroxidase-labeled goat anti-rabbit immunoglobulin (Ig) G, rabbit polyclonal antibody raised against human caspase-3 (PA-cas 3) that detects only caspase-3 precursor (32 kDa), and Covalight kit were obtained from CovalAb (Lyon, France). Rabbit polyclonal antibody raised against human cleaved caspase-3 (catalog no. 9961), which detects only the large fragment of activated caspase-3 that results from cleavage after ASP 175 (17 kDa), and rabbit polyclonal antibody raised against caspase-6 (catalog no. 9762), which detects procaspase-6 (34 kDa) and activated caspase-6 (12 kDa), were obtained from Ozyme (Saint Quentin en Yvelines, France). Rabbit polyclonal antibody raised against human caspase-6 (catalog no. AAP-106), which detects only the caspase-6 precursor (34 kDa), was obtained from Stressgen Biotechnologies Corp (Victoria, BC, Canada). Goat polyclonal antibody raised against human caspase-8 (catalog no. sc6134), which reacts with both procaspase-8 (52 kDa) and cleaved caspase-8 (p20), was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). DAKO (Trappes, France) was the source for Faramount, Envision+ kit, antibody diluent, and hematoxylin. Oligonucleotides primers were purchased from Genset (Paris, France). Testosterone heptylate was purchased from Theramex (Monaco, Monaco) and pentobarbital from Centravet (Plancoët, France).
Animals
Male Sprague-Dawley rats (age, 60 days) were obtained from IFFA Credo (l'Arbresle, France). Rats were housed in controlled conditions of lighting (photoperiod, 12L:12D), temperature (22 ± 2°C), humidity (55% ± 15%), and ventilation (
15 air changes/h) and were given free access to water and feed (certified rodent pellet diet, AO4C; UAR, Villemoisson-sur-orge, France). Animals were killed by CO2 inhalation.
Eighteen adult (age, 60 days) male Sprague-Dawley rats were castrated by a scrotal incision under pentobarbital anesthesia (60 mg/kg body wt). Two groups of nine castrated animals were considered. In the first group, rats were injected s.c. with testosterone heptylate at a dose of 10 mg/kg body weight immediately and at 2, 4, and 6 days after castration. In the second group, rats were not treated. Three animals of each group and three intact animals were killed on Days 1, 3, and 7 after castration. The experiment was repeated three times. Ventral, dorsolateral, and anterior prostatic lobes were separated from one another, excised, and weighed. A portion of each lobe was frozen at -80°C for reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis; the second portion was fixed in Bouin liquid followed by routine paraffin embedding for TUNEL and immunohistochemical analyses. All studies on animals were conducted in accordance with current regulations and standards approved by the INSERM animal care committee.
TUNEL Analysis
Paraffin sections (thickness, 5 µm) of Bouin-fixed prostatic lobes were mounted on glass slides. The sections were deparaffinized and rehydrated (xylene for 5 min; 100%, 95%, and 70% ethanol for 1 min each), then washed in distilled water before beginning the TUNEL reaction. The slides were transferred to a plastic jar containing 0.01 M citrate buffer (pH 6) and microwave irradiated for 5 min (370 W). After a wash with single-strength PBS, endogenous peroxidases were blocked for 5 min with 2% H2O2. Sections were washed three times with single-strength PBS. The specimens were then incubated for 60 min at 37°C in a moist chamber with the TUNEL mix, which consisted of 0.3 U/µl of calf thymus terminal deoxynucleotidyl transferase, 0.007 nmol/µl of biotin dUTP, 1 mM coblat chloride, 30 mM Tris (pH 7.2), and 140 mM sodium cacodylate. After washing (four PBS baths of 5 min each at room temperature), the sections were saturated for 10 min in 2% BSA at room temperature. Sections were treated for 30 min at 37° in a moist chamber with a 1:20 dilution of ExtrAvidin peroxidase. After three washes in PBS, detection was performed with DAB (1.25 mg of DAB, 25 µl of 3% nickel chloride, and 152 µl of 1 M Tris-HCl [pH 7.5] in a final volume of 2 ml). Slides were mounted in Faramount.
Immunohistochemistry
Paraffin sections of Bouin-fixed prostatic lobes were sectioned (thickness, 5 µm). The sections were mounted on positively charged glass slides (Superfrost Plus; Menzel-Glaser, Braunschweig, Germany), deparaffinized, hydrated, treated for 20 min at 93%–98°C in 0.01 M citric buffer (pH 6), rinsed in osmosed water (twice for 5 min each), and washed (twice for 5 min each) in Tris-buffered saline (TBS). For anti-procaspase-3 and anti-cleaved caspase-3, the Envision+ kit was used. For anti-procaspase-6 and anti-caspase-8, the UltraVision Detection System (Lab Vision Corporation, Fremont, CA) was used. Briefly, endogenous peroxidases were blocked with 3% H2O2 for 15 min. The sections were then incubated overnight at 4°C with the primary antibody (1:300 anti-caspase-3, 1:50 anti-cleaved caspase-3, 1:200 anti-procaspase-6, and 1:200 anti-caspase-8) diluted in antibody diluent. After incubation with the primary antibody, the sections were washed (twice for 5 min each) in TBS. For the Envision+ kit, the sections were incubated for 30 min at 37°C in the presence of the secondary antibody. Next, the sections were rinsed (twice for 5 min each) in TBS, incubated for 10 min at room temperature with 3-amino-9-ethylcarbazole, which generated a red color at the site of peroxidase activity, and then rinsed (twice for 5 min each) in osmosed water. For the UltraVision Detection System, the sections were incubated with the biotinylated secondary antibody; after two, 5 min washes, a peroxidase-streptavidin complex was applied. The DAB was used as a peroxidase chromogen. For both kits, slides were counterstained with Meyer hematoxylin. Finally, sections were mounted in Faramount. As a negative control, the primary antibody was replaced by the antibody diluent. The same antibodies were used for immunohistochemistry and Western blot analyses.
RT-PCR Analysis
Total RNAs were extracted from rat prostatic tissues with TRIzol reagent. The amount of RNA was estimated by spectrophotometry at 260 nm.
The cDNAs were obtained from RT of 5 µg of total RNAs using random hexanucleotides as primers (5 µM) in the presence of dNTP (0.2 mM), dithiothreitol (10 mM), and Moloney murine leukemia virus (10 U/µl) for 1 h at 37°C. For PCR analysis, the target gene (caspase-3, -6, or -8) was coamplified with an endogenous standard gene (ß-actin). The stock reactions (20 µl) were prepared on ice and contained 0.02 U/µl of Taq polymerase, 1.5 mM MgCl2, 200 µM dNTPs, 1 µM caspase primers, 10 nM ß-actin primers, and 2 µl of RT mixture (cDNA). The PCR conditions were 94°C for 5 min; 25 cycles of 94°C for 30 sec, 58°C for 30 sec, 72°C for 1 min; and then 72°C for 7 min. After amplification, the PCR products were separated by electrophoresis on 2% agarose gels containing 0.005% ethidium bromide visualized by ultraviolet light. Band intensities were estimated by densitometric scanning using a GelDoc scanner (Bio-Rad Life Science, Marnes la Coquette, France). Data were expressed as caspase:ß-actin mRNA ratios. Primers used were as follows: for caspase-3: upstream primer, 5'-ACGGTACGCGAAGAAA-AGTGAC-3'; downstream primer, 5'-TCCTGACTTCGTATTTCAGGGC-3' (282 base pairs [bp]); for caspase-6: upstream primer, 5'-AACCACATTTACGCATACGATG-3', downstream primer, 5'-CGGTGAGAGTAATACCCTTCTG-3' (289 bp); for caspase-8: upstream primer, 5'-GGGACAGGAATGGAACACAC-3'; downstream primer, 5'-CAGCAAGGGAAGGGCA CTTC-3' (275 bp); and for ß-actin: upstream primer, 5'-TTGCTGATCCACATCTGCTG-3'; downstream primer, 5'-GACAGGATGCAGAAGGAGAT-3' (146 bp). The PCR analysis for all PCR products was carried out from the logarithmic phase of amplification; PCRs with different cycle numbers were realized for each primer couple to determine the minimum number of cycles necessary to detect the PCR product. The PCR-amplified products were checked by direct sequencing. The RT-PCR primers were designed inside separate exons to avoid any bias caused by residual genomic contamination. Moreover, for all primers, no amplification was observed when PCR was performed on RNA preparations.
Western Blot Analysis
Prostatic tissue was homogenized in 200 µl of ice-cold hypotonic buffer (25 mM Tris-HCl [pH 7.4] and protease-inhibitor cocktail). Tissues were further homogenized by sonication (10 sec). Protein concentration was determined by the Bradford assay.
Proteins (100 µg) were resolved on 10% SDS/polyacrylamide gels and electrophoretically transferred to nitrocellulose membranes using CAPS buffer (1x, pH 11) containing 20% methanol at a constant voltage of 100 V for 60 min. Following transfer, the membrane was incubated in TBS containing 10% fat-free dry milk and 0.1% Tween-20 for 2 h at room temperature. The membrane was rinsed three times with TBS/0.1% Tween-20 (three times for 10 min each) and then incubated with the first antibody (in TBS containing 2% fat-free dry milk) overnight at 4°C (1:100 procaspase-3, 1:100 cleaved caspase-3, 1:250 caspase-6, and 1:250 caspase-8). The membrane was then rinsed with TBS/0.1% Tween-20 (three times for 10 min each) and incubated with horseradish peroxidase-labeled anti-rabbit IgG (1:2500) or anti-goat IgG (1:2000) in TBS containing 2% fat-free dry milk and 0.1% Tween-20 for 1 h at room temperature. The membrane was thoroughly washed with TBS/0.1% Tween-20 (three times for 10 min each) and then with TBS. Bound antibodies were detected by chemiluminescence using a CovalAb detection kit and Biomax MR films. The protein loading was checked by reprobing the blot with an rabbit IgG antiactin (1:500).
Data Analysis
Data are expressed as the mean ± SD. At least three different adult male rats (n = 3–7 per condition) were utilized. For statistical analysis, one-way ANOVA was performed to determine whether differences existed between all groups (P < 0.05), and then the Bonferroni/Dunn posttest was performed to determine the significance of the differences between the pair of groups. A P value of less than 0.05 was considered to be significant. The statistical tests were performed on StatView software(version 5.0; SAS Institute, Inc., Cary, NC) on a Macintosh computer (Apple, Cupertino, CA).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
To clarify the effect of testosterone on caspase expression and activation in prostate, we used, as a model, castrated adult rats treated (or not) with testosterone. In a first set of experiments, we validated this model by showing that castration induces a significant (P < 0.0002) loss (30%) of prostate ventral weight as early as 1 day after castration (Fig. 1A). By 7 days, more than 70 % of ventral prostate weight was lost. Testosterone supplementation to the castrated rats prevented the ventral prostate weight loss by 1 day after castration, whereas by 3 and 7 days, it allowed the complete ventral prostate weight to be retained. In control experiments, it was shown that dorsolateral and anterior prostate lobe weight was not affected in adult rats by castration or by castration plus testosterone replacement (Fig. 1A).
|
These changes in ventral prostate weight appear to be related to a cell death process, as shown by the TUNEL approach. Apoptotic cells were neither observed in the ventral lobe from intact rats nor in castrated animals (7 days) treated by testosterone, whereas apoptotic cells were evidenced in the ventral prostate of rats by 7 days after castration (Fig. 1B). Moreover, TUNEL-positive (i.e., apoptotic) cells were exclusively located to epithelial cells of prostate (Fig. 1B). After rat castration, a significant (P < 0.0007) increase in TUNEL-positive cells in the ventral prostate was first observed by 1 day, reaching maximal levels (P < 0.0001) by 3 days postcastration (Fig. 1C). In contrast, in castrated rats receiving testosterone, no TUNEL-positive cells were observed in ventral prostate at the different days postcastration (Fig. 1C). No TUNEL-positive cells were observed in dorsolateral and anterior prostate lobes after castration or after castration plus testosterone replacement (Fig. 1C).
In the next experiments, caspase-3, -6, and -8 immunostaining as well as mRNA and protein levels were determined in the ventral and dorsolateral rat prostate after castration supplemented (or not) with testosterone. Immunostaining of caspase-3, -6, and -8 was performed in ventral prostate from intact rats, castrated rats, and castrated rats supplemented with testosterone. In intact animals, caspase-3 (Fig. 2A), -6 (Fig. 2E), and -8 (Fig. 2I) immunostaining was detected at low intensity. Caspase-6 (Fig. 2, E–G) and -8 (Fig. 2, I–K) immunostaining was detected in their particular punctiform aspect [19]. It is generally assumed that this punctiform immunolocalization could be related (although not exclusively) to a sublocalization to the Golgi apparatus. By 7 days postcastration, an apparent increase in the intensity of caspase-3 (Fig. 2B) and -6 (Fig. 2F) immunostaining was observed in ventral prostate. Administration of testosterone to the castrated rats prevented the increase in the intensity of caspase-3 (Fig. 2C) and -6 (Fig. 2G) immunostaining, which remained at levels comparable to those observed in intact rats. Caspase-8 immunostaining apparently remained unchanged in the different experimental conditions (Fig. 2, I–K).
|
Caspase-3 mRNA levels were evaluated through RT-PCR (Fig. 3A), whereas caspase-3 protein was identified and evaluated in Western blot analysis using two different antibodies. The first antibody recognizes exclusively procaspase-3 protein (32 kDa) (Fig. 3B); the second antibody recognizes exclusively the cleaved active form (17 kDa) (Fig. 3C). Caspase-3 mRNA levels were increased as early as 1 day postcastration (P < 0.0001) (Fig. 3A). The maximal increase in caspase-3 mRNA levels was observed by 3 days after castration (3-fold, P < 0.0001) (Fig. 3A). Procaspase-3 protein was not detected in the ventral prostate of intact animals, whereas it was observed by 1 day after castration and reached maximal levels by 7 days after castration (Fig. 3B). Administration of testosterone to castrated rats prevented a increase in caspase-3 mRNA and procaspase-3 protein in the ventral prostate. For example, caspase-3 mRNA (Fig. 3A) and procaspase-3 (Fig. 3B) levels were not different in intact rats and in castrated rats supplemented with testosterone over 7 days. Furthermore, castration induced caspase-3 activation, whereas testosterone inhibited it (Fig. 3C). Indeed, active cleaved caspase-3 was not detected in ventral prostate from control intact rats, but it was visible by 1 day after castration and further increased by 7 days. In contrast, active cleaved caspase-3 was no longer detected by 3 and 7 days after castration in rats treated with testosterone (Fig. 3C).
|
Caspase-6 mRNA levels were also evaluated using RT-PCR (Fig. 4A), and caspase-6 protein was identified and evaluated by Western blot analysis using two different antibodies. The first antibody recognizes exclusively procaspase-6 (34 kDa) (Fig. 4B); the second antibody recognizes both procaspase-6 and the cleaved active caspase-6 (12 kDa) (Fig. 4C). In the ventral prostate of adult rats, caspase-6 mRNA levels were significantly (P < 0.0001) (Fig. 4A) increased as early as 1 day postcastration. The maximal increase in caspase-6 mRNA levels (4-fold, P < 0.0001) (Fig. 4A) was observed by 7 days postcastration. Whereas procaspase 6 was not detected in the ventral prostate of intact rats, it was evidenced after castration and reached maximal levels 7 days later (Fig. 4B). In castrated animals supplemented with testosterone, caspase-6 mRNA and protein levels remained low and comparable to control levels. For example, caspase-6 mRNA (Fig. 4A) and procaspase-6 protein (Fig. 4B) levels were not different in intact rats and in castrated rats treated with testosterone over 7 days. Castration also induced procaspase-6 activation in the rat ventral prostate. Indeed, active caspase-6 (12 kDa) was detected following castration, with a maximal increase observed at day 7, but it was not detected in castrated rats treated with testosterone (Fig. 4C).
|
Caspase-8 mRNA levels were evaluated by RT-PCR, and caspase-8 protein was identified and evaluated by Western blot analysis using an antibody designed to recognize both procaspase-8 (52 kDa) and activated caspase-8 (20 kDa) (Fig. 5). The data obtained show that in the ventral prostate of adult rats, caspase-8 mRNA (Fig. 5A) and caspase-8 protein (Fig. 5B) levels were not affected by castration or testosterone replacement. Furthermore, as the anti-caspase-8 antibody used was designed to recognize both activated caspase-8 and procaspase-8, the present data also indicate that procaspase-8 was not cleaved (and thus activated) in ventral prostate of adult castrated rats supplemented (or not) with testosterone (Fig. 5B).
|
Effect of Testosterone on Caspase-3, -6, and -8 mRNA and Proteins Levels in Anterior and Dorsolateral Prostate of Rats
Caspase-3, -6, and -8 mRNA and protein levels were also evaluated in the dorsolateral and anterior prostate. No changes were observed in caspase-3 (Fig. 6) and -6 (Fig. 7) mRNA and proteins levels in dorsolateral prostate in castrated rats or in castrated rats treated with testosterone. As expected, no changes in caspase-8 mRNA and protein levels were observed in dorsolateral prostate of castrated rats supplemented (or not) with testosterone (data not shown). No cleaved active caspase-3 (Fig. 6), -6 (Fig. 7), and -8 (data not shown) was detected in dorsolateral or anterior prostate lobes.
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
During apoptotic cell death, cells undergo a regulated autodigestion, which involves the disruption of cytoskeletal integrity, cell shrinkage, nuclear condensation, and activation of endonucleases [21]. The chief effectors of the apoptotic cell death pathway are the caspase family of cysteine proteases. Caspases are synthesized as inactive precursors that are cleaved at specific aspartate residues to generate the active subunits. It has been suggested that caspases are at play during prostate regression after castration [15], because inhibition of the caspase activities by the overexpression of crmA, a viral inhibitor of caspase activity, prevented apoptosis induced in vitro following androgen withdrawal [22]. To our knowledge, however, it remained to identify the members of the caspase family involved in the prostate apoptosis and the level of control exerted by androgens on caspase expression and/or activation. Thus, in the present study, we have used a model of castrated rats treated (or not) with androgens. We validated our experiments by weighing the prostate lobes. Ventral prostate lobe weight decreased as early as 1 day after castration, and maximal effect was observed after 7 days. In contrast, the anterior and dorsolateral prostate lobe weight was unaffected by androgen deprivation.
The results concerning ventral prostate are in accordance with those reported in the literature [12, 13]. However, concerning the anterior and dorsolateral prostate weight, other authors have observed a decrease, although with a slow kinetic (after 7 days of castration) in the weight of these prostate lobes in castrated rats [13]. Although the reasons for these discrepancies are unknown, it could be attributable to the use of different experimental approaches. Indeed, in the study of Banerjee et al. [13], lateral and dorsal lobes were weighed separately, whereas in the present study, the lobes were weighed together. Additionally, in the present study, the lobes were weighed just after dissection; in the study of Banerjee et al. [13], the prostate lobes were weighed after immersion in ice-cold buffer. However, despite these discrepancies, one should note that both studies are consistent in that the apoptotic process was observed in ventral lobe prostate but not in the other lobes in this organ.
The process of apoptosis in prostate lobes of castrated animals was analyzed. Three days after castration, apoptotic cell numbers in ventral prostate reached a maximal level and remained high 7 days after androgen deprivation. Banerjee et al [13] showed that TUNEL-positive cells reached maximal levels by 2 and 3 days after castration and returned to control levels by 7 days. The reasons for these discrepancies are not known.
In the present study, we show that inhibition of the apoptotic cell death process by testosterone in ventral prostate is related to a decrease in both caspase-3 and -6 mRNA production and active caspase levels. Two observations support the existence of relationships between testosterone action and caspase-3 and -6 expression and activation during the apoptotic cell death process in the prostate. First, castration of adult rats induced an increase in caspase-3 and -6 mRNA and protein (procaspase and cleaved active caspase) levels in the ventral prostate but not in the dorsolateral prostate. Second, testosterone administration to castrated rats prevented this increase in caspase-3 and -6 mRNA and protein levels in the ventral lobe. Although the effector caspases were identified in the whole ventral prostate, the possibility exists that the expression and activation of these effector caspases may differ depending on the different segments of the ductal system.
Together, these observations would suggest that the steroid hormone may act on at least three levels. First, testosterone may inhibit caspase-3 and -6 transcription gene activity and/or increase caspase-3 and -6 mRNA degradation. Although different responsive elements in the rat caspase-3 promoter gene have been identified (including Sp1 and Ets-like elements) [23], androgen-responsive elements in this promoter are not present, suggesting a possible indirect control of androgens on caspase-3 gene promoter. This would means that androgen action on caspase-3 gene expression involves a cascade of intermediates, the natures of which remain to be identified. In addition, an increase in caspase-3 and -6 mRNA levels also has been recently shown in prostate carcinoma developed in transgenic rats with the SV40 T antigen under probasin promoter control 7 days after castration; however, procaspase and active caspase-3 and -6 levels have not been studied [24]. Second, testosterone may act at the protein level (translation/protein turnover) at 7 days after castration; the increase in the levels of the proteins was clearly more pronounced compared to that of the mRNAs. Third, testosterone prevented caspase-3 and -6 protein cleavage (i.e., activation). Specifically, both the cellular and molecular mechanisms underlying caspase activation under testosterone control in prostate remain to be investigated. The normal prostate has a low turnover of cells, indicating the existence of basal apoptotic cell death process in intact rat prostate [13]. These observations suggest that the nondetection of apoptotic (i.e., TUNEL-positive) cells as well as procaspase and active caspase-3 and -6 proteins in intact animals in the present study could be related to the sensitivity threshold of the techniques used.
Procaspase cleavage can occur by several mechanisms, including proximity-induced autoprocessing or cleavage by other caspases, revealing a caspase cascade with upstream initiator caspases such as caspases-8, -9, and -10 and downstream effector caspases such as caspases-3, -6, and -7 [18, 25]. The activity of these caspases is regulated by several families of both pro- and antiapoptotic cellular proteins [26, 27]. Classically, two major apoptotic pathways leading to effector caspase activation have been identified in mammalian cells, the Fas/tumor necrosis factor (TNF)-R1 death receptors and the mitochondrial pathways [25]. The first pathway involves the engagement of the death receptors by soluble ligand and results in a further recruitment of upstream signaling caspases-2, -8, and -10. It occurs through formation of specific death complexes formed by the binding of a death receptor, via a death domain-containing protein, with the death effector domains of caspase-8 or 10. Such an activation of initiator caspase-8 or -10 leads ultimately to activation of effector caspases. The second pathway, the mitochondrial pathway, is thought to be triggered by translocation into the mitochondria of proapoptotic members of Bcl-2 family such as Bax. The Bcl-2 family consists of pro- and antiapoptotic proteins, which compete through dimerization and regulate apoptosis mainly by controlling the release of cytochrome c and other mitochondrial apoptotic events [28]. The involvement of death receptor pathway in ventral prostate apoptosis is supported by several studies reporting that prostatic epithelial cells express soluble Fas and Fas receptor [29–31], by immunohistochemical studies that have revealed an upregulation of the Fas receptor following castration [32, 33], and by lpr -/- mutant mice, which lack functional Fas receptor and exhibit a lack of regression in prostate tumor after androgen withdrawal [34].
Whether the death receptor pathway is involved in caspase-3 and -6 activation in ventral prostate following rat castration reported here remains to be investigated. Because caspase-8 mRNA and protein levels were not affected in the ventral prostate of castrated rats treated (or not) with testosterone (present study), this pathway may not be at play. However, among at least the 14 mammalian caspases identified so far, caspase-10 shares homologous death effector domains with caspase-8, suggesting that caspase-10 may also function by interacting with death receptors and that caspase-10 can function independently of caspase-8 in initiating Fas and TNF-related apoptosis-inducing ligand (Trail) receptor-mediated apoptosis [35]. Because Trail and its receptors have been recently identified in adult rat prostate [36], it will be interesting to study caspase-10 expression in our present experimental model before excluding the involvement of the death receptor pathway in testosterone action on effector caspase expression and activation. Concerning the mitochondrial pathway, different studies have suggested that the Bcl-2 family of proteins could be involved in the prostate cell death apoptotic process. First, a raise in the Bax:Bcl-2 ratio has been observed in ventral prostate during apoptotic cell death process induced by androgen withdrawal [37, 38]. Second, an increase in Bax and Bcl-x mRNA following castration has been observed in prostate carcinoma induced in transgenic rats with the SV40 T antigen under probasin promoter control [24]. Third, Bcl-2 or Bcl-xL transfection in prostate cancer cell lines are associated with increased resistance to apoptosis, with a concomitant decrease in mitochondrial membrane disruption and cytochrome c release [39]. Whether the mitochondrial pathway is involved in caspase-3 and -6 activation during the apoptotic prostate cell death process under androgen control is currently being investigated.
Finally, one interesting point relates to the lack of responsiveness of dorsolateral and anterior lobes of rat prostate to castration. Indeed, although epithelial cells of dorsolateral and anterior lobes expressed, as in the ventral lobe, androgen receptors [11, 40; unpublished data], the present findings indicate that testosterone was unable to affect both caspase-3 and -6 expression and activation. These observations would suggest that the absence of response to androgens of the dorsolateral and anterior prostate, in terms of caspase-3 and -6 expression, could probably relate to one or more biochemical/molecular steps located beyond the androgen receptor.
In summary, we report here, using a model of castrated rats supplemented (or not) with testosterone, that the apoptotic cell death process in ventral prostate involves at least the effector caspases-3 and -6. After castration, both caspase-3 and -6 mRNA and procaspase-3 and -6 levels were increased. In contrast, testosterone administration to castrated rats prevented such an increase. Furthermore, castration also induced an activation of the two effector caspases in the ventral prostate of adult rats, whereas the concomitant administration of testosterone prevented such an activation of the two caspases.
|
FOOTNOTES |
|---|
2 Correspondence: Mohamed Benahmed, INSERM U.407, Communications cellulaires en biologie de la reproduction, Faculté de Médecine Lyon-Sud, 165 chemin du grand Revoyet, BP 12, 69921 Oullins Cedex, France. FAX: 33 4 78 86 31 16; benahmed{at}grisn.univ-lyon1.fr
Received: 17 October 2002.
First decision: 31 October 2002.
Accepted: 15 April 2003.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-reductase. Endocrinology 1999 140:4509-4515-reductase inhibition and castration on androgen-regulated gene expression in rat prostate. Mol Endocrinol 1991 5:1023-1029[Abstract]-reductase inhibitor, SK&F 105657: role for testosterone in the androgenic response. Endocrinology 1992 130:685-694[Abstract]-related apoptosis-inducing ligand (TRAIL) and its receptors in adult rat ventral prostate. Mol Cell Endocrinol 2002 198:115-121[CrossRef][Medline]This article has been cited by other articles:
|
|
 |
|
  L. K. Potter, M. G. Zager, and H. A. Barton Mathematical model for the androgenic regulation of the prostate in intact and castrated adult male rats Am J Physiol Endocrinol Metab, November 1, 2006; 291(5): E952 - E964. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S Morimoto, C A Mendoza-Rodriguez, M Hiriart, M E Larrieta, P Vital, and M A Cerbon Protective effect of testosterone on early apoptotic damage induced by streptozotocin in rat pancreas J. Endocrinol., November 1, 2005; 187(2): 217 - 224. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. G. Rudmann, I. R. Cohen, M. R. Robbins, D. E. Coutant, and J. W. Henck Androgen Dependent Mammary Gland Virilism in Rats Given the Selective Estrogen Receptor Modulator LY2066948 Hydrochloride Toxicol Pathol, October 1, 2005; 33(6): 711 - 719. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. V. Desai, A. M. Michalowska, P. Kondaiah, J. M. Ward, J. H. Shih, and J. E. Green Gene Expression Profiling Identifies a Unique Androgen-Mediated Inflammatory/Immune Signature and a PTEN (Phosphatase and Tensin Homolog Deleted on Chromosome 10)-Mediated Apoptotic Response Specific to the Rat Ventral Prostate Mol. Endocrinol., December 1, 2004; 18(12): 2895 - 2907. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. L. Carey, L. M. Sasur, H. Kawakubo, V. Gupta, B. Christian, P. M. Bailey, and S. Maheswaran Mutually Antagonistic Effects of Androgen and Activin in the Regulation of Prostate Cancer Cell Growth Mol. Endocrinol., March 1, 2004; 18(3): 696 - 707. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
