Does the lack of regression-associated mRNA expression render a rat ventral prostate epithelial cell line androgen independent?

A series of rapidly dividing epithelial (RDE) cell lines have been isolated from primary cultures of rat ventral prostate (RVP) epithelial cells. Unlike androgen-dependent secretory epithelial cells, the RDE cells in culture do not express the androgen-dependent secretory proteins, nor do they express the androgen-repressed cell death sequences (TRPM-2) found in the epithelial cells during prostatic regression. Screening of a cDNA clone library established from RDE cell mRNA has yielded a number of RDE cell-specific sequences. One of these, RDE-.25 is a 250-base mRNA. The sequence of RDE-.25 shows considerable homology with the rat growth hormone gene and two murine oncogene sequences. We believe that the absence of androgen-repressed cell death sequence expression confers androgen independence for survival and growth, while the expression of RDE-.25 may represent an autocrine growth stimulus which greatly increases the rate of cell division in these cells.     

Ductal heterogeneity of cytokeratins, gene expression, and cell death in the rat ventral prostate.

The rat ventral prostate is a complex gland composed of numerous ducts. The epithelial cells that line the lumen of the ducts are surrounded by stromal cells. The epithelial cells display a characteristic morphology that is dependent on their anatomical location within the ducts; the cells that line the lumen in the region of the ducts close to the urethra (the proximal region) are cuboidal, while those in the distal regions of the ducts are tall columnar cells. We have examined the regional expression of two genes that are expressed in the prostate: prostate steroid-binding protein (PSBP; a marker for androgen-dependent protein synthesis) and TRPM-2 (a marker for programmed cell death). We have demonstrated that the expression of PSBP, in the presence of androgens, and TRPM-2, in the absence of androgens, is restricted to the luminal epithelial cells in the distal regions of the prostatic ducts. Neither of the genes is expressed in the proximal regions of the ducts. In view of the probable effects of the epithelial-stromal interactions in the gland we have also characterized the cytokeratin composition of the epithelial cells lining the prostatic ducts. We have established that the basal epithelial cells of the prostate are primarily localized in the proximal region of the ducts. We propose that these cells may attenuate the influence of the stromal cells on the luminal epithelium and exert a negative influence on the cytodifferentiation of the secretory epithelial cells. The results also suggest that PSBP, which has been considered to be an androgen-dependent gene may, in fact, be a sequence that is constitutively expressed in the luminal cells that die in the absence of androgens. This has significant implications on the mechanism of androgen action in the rat ventral prostate.     

Intravesicular Fas localization in epithelial cells of castrated rat prostate glands

Androgenic steroids regulate the development and size of mammalian prostate epithelial cells. To evaluate the relationship between Fas-Fas ligand system and apoptosis in prostate epithelial cells of the castrated rats, we have examined immunocytochemical localization of Fas antigen in the castrated rat prostate glands at a series of different times. We used a rabbit polyclonal anti-Fas antibody with a streptavidin-biotin method and confocal laser scanning method or an immunogold method. Fas immunolocalization was examined in ventral lobes of prostate glands taken from intact or castrated adult male Wistar rats on day 1, 2, 3, 4 and 5 by light or electron microscopy. At a light microscopic level, the castrated prostate epithelial cells showed mostly Fas immunolocalization in their apical parts of cytoplasm on day 2 after the castration. In addition, their extent of the Fas expression was expanded throughout the cytoplasm in proportion to the androgen ablation periods, and later the Fas expression was detected at luminar or basolateral sides of the epithelial cells. Both immunogold labeling with ultrathin sections and immunoperoxidase technique with cryostat sections demonstrated that Fas was localized mainly in secretory granules of the castrated prostate epithelial cells and some parts of their cell membranes at later stages. Our immunocytochemical findings showed that Fas expression was time-dependently induced in most of the prostatic epithelial cells after castration of rats. The rate of Fas-expressing epithelial cells was too high and inconsistent with the previously reported rate of TUNEL-positive ones. The membrane-associated Fas may have little effect on the apoptosis in the present case, bacause a lot of soluble Fas was secreted from the prostatic epithelial cells. A further study is needed to clarify some significance of the secretory Fas in the prostatic epithelium after the rat castration.     

Differential regulation of protooncogene c-myc expression in rat ventral prostate after castration

To evaluate the possibility that the protooncogene c-myc plays a role in ventral prostate, the effects of castration have been investigated at a beginning of a period by Northern blot hybridization and the levels of c-myc mRNA were also compared with mRNA of androgen-regulated genes, C1 and TRPM-2. Levels of c-myc mRNA in ventral prostate increased with maximal stimulation reached at 6 hours (early induction) and 48 hours (late induction) after castration, respectively. The level of C1 mRNA did not change and TRPM-2 was not detected at early induction of c-myc mRNA after castration. The level of early induction of c-myc mRNA after castration was increased in ventral prostate treated with cycloheximide, but it was almost reduced by actinomycin-D pretreatment. Administration of androgen at the time of castration prevented early induction of c-myc mRNA. These results suggest that protooncogene c-myc is differentially regulated in ventral prostate after castration.     

Autologous down-regulation of androgen receptor messenger ribonucleic acid

Autoregulation of androgen receptor (AR) mRNA was investigated using Northern blot analysis with AR cDNA fragments as probes. The amount of AR mRNA increased 2- to 10-fold with androgen withdrawal and decreased below control levels after androgen stimulation in rat ventral prostate, coagulating gland, epididymis, seminal vesicle, kidney, and brain, and in a human prostate cancer cell line, LNCaP. In rat ventral prostate, AR mRNA increased 2- to 3-fold within 24 h after castration and remained elevated for 4 days. Treatment with testosterone propionate beginning 24 h after castration reduced ventral prostate AR mRNA 4-fold within 8 h of androgen replacement. Administration of estradiol 24 h after castration had no significant effect on prostatic AR mRNA. Androgens, including testosterone and the synthetic androgen methyltrienolone (R1881), or the antiandrogen cyproterone acetate down-regulated AR mRNA in vitro in LNCaP cells, whereas estradiol was without effect. Administration of testosterone propionate to rats with androgen insensitivity did not decrease AR mRNA. Down-regulation of AR mRNA by androgen is therefore a receptor-mediated process which occurs in vivo in rat tissues that differ in androgen responsiveness and in cultured human prostate cells.     

Oxidative stress signalling in the apoptosis of Jurkat T-lymphocytes

Within the first 24 h after castration of an adult male rat, the vascular system of the ventral prostate gland undergoes a degenerative process that drastically reduces blood flow to the tissue. Since the vascular degeneration precedes the loss of the prostatic epithelium (by apoptosis), we have proposed that the onset of epithelial cell apoptosis in this tissue is caused by an ischemic/hypoxic environment resulting from the loss of blood flow. In order to further evaluate the extent to which ischemia/hypoxia might be a factor in apoptosis of the prostate epithelium after castration, we analyzed for biomarkers of cellular hypoxia in rat ventral prostates during the first 3 days following castration. Ventral prostate tissues removed from hypoxyprobe-1-treated adult male rats (uncastrated controls; surgically castrated for 24, 48 or 72 h, or sham-castrated for equivalent times) were directly analyzed for evidence of hypoxia by in situ immunohistochemical evaluation of hypoxyprobe-1 adduct formation in the prostate cells. Protein extracts from these tissues were also tested for expression of the 120 kDa hypoxia-inducible factor-1-alpha (HIF-1-alpha) protein as well as for expression of mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) proteins using a Western blot assay. The tyrosine phosphorylation status of the latter signaling molecules was also evaluated by Western blotting using anti-tyrosine phosphate antibodies. Our results showed that epithelial cells of the rat ventral prostate stained positively for hypoxyprobe-1 adducts at all times after castration, whereas cells in control tissues were unstained by this procedure. In addition, the prostatic expression of HIF-1-alpha protein was increased approximately 20-fold at 48 h after castration compared to control tissues. Finally, although prostatic MAPK and JNK protein expression was unaltered during the early period after castration, phosphorylation of the JUN kinase protein was significantly elevated, indicating that this stress-activated cellular signaling pathway becomes more active subsequent to castration. These results support our proposal that early castration-induced degeneration and constriction of the vascular system of the rat ventral prostate gland leads to reduced oxygenation of prostatic epithelial cells and the activation of hypoxic cellular signaling in these cells through upregulation of HIF-1-alpha expression and stimulation of the JUN kinase signaling pathway.     

Androgen regulation of FLICE-like inhibitory protein gene expression in the rat prostate

In hope of eventually identifying defects in human prostatic neoplasias that render them insensitive to anti-androgen therapy, we have examined the regulation of components of ligand-induced cell death pathways during castration-induced regression of the prostate. Rat prostates were obtained after surgical castration with or without subsequent androgen replacement. The mRNA levels of genes encoding components of the apoptotic pathway were measured from individual prostates. Whole prostates 1-10 days after castration did not show a significant change in mRNA levels encoding either Fas or FasL, which some studies suggest are necessary for regression to occur. However, the mRNA encoding a catalytically inactive cysteinyl aspartate-specific protease (caspase) analog, FLICE-like inhibitor protein (FLIP), decreases during the first day following castration. In the most apoptotically responsive ventral lobe of the rat prostate, the reduction in FLIP mRNA levels is evident within 12 h of castration. The mRNA levels of the principal target of FLIP inhibition, caspase-8, do not change during the period preceding the onset of detectable DNA fragmentation. Androgen administration to castrated rats reverses prostate regression, and restores FLIP mRNA to normal levels. By acting as an inhibitor of caspase-8, FLIP may protect prostatic epithelium from apoptosis. Androgen withdrawal, by reducing FLIP mRNA levels, might leave the cells vulnerable to as yet unidentified cell death signals.     

A secretory protease inhibitor requires androgens for its expression in male sex accessory tissues but is expressed constitutively in pancreas.

A full length cDNA clone encoding a mouse prostatic secretory glycoprotein (p12) whose synthesis is dependent upon testicular androgens has been cloned and characterized. The predicted amino acid sequence of p12 shares extensive homology with several members of the Kazal family of secretory protease inhibitors, in particular the pancreatic secretory trypsin inhibitors. In agreement with sequence data, prostatic secretory p12, purified from mouse ventral prostate secretion, exhibits anti-trypsin activity. Steady-state levels of protease inhibitor mRNA in ventral prostate are reduced from approximately 0.06% in normal mice to undetectable after androgen withdrawal but are inducible within 4 h by re-administration of testosterone. Androgen-dependent expression of the secretory protease inhibitor mRNA was also observed in coagulating gland and seminal vesicle. In seminal vesicle, a tissue of different embryonic origin to the prostate, the kinetics of secretory protease inhibitor mRNA loss after castration are not as rapid as in the ventral prostate and coagulating gland. Low-level androgen independent expression was also observed in the pancreas. There appears to be a single gene for this secretory protease inhibitor and yet expression is markedly stimulated by testosterone in the sex accessory tissues and unaffected by this hormone in the pancreas.     

"Thymineless" death in androgen-independent prostatic cancer cells

The molecular mechanism of "thymineless" death induced by 5-fluorodeoxyuridine or trifluorothymidine, in androgen-independent rat prostatic adenocarcinoma AT-3 cells was investigated. Fragmentation of genomic DNA into discrete multiples of a nucleosomal unit (i.e. 180bp subunit) and induction of expression of TRPM-2, a programmed cell death-associated gene, temporally correlated with the activation of programmed cell death in this system. In contrast, killing of AT-3 cells by osmotic lysis, or membrane-targeted metabolic inhibitors results in neither the stereotypic DNA fragmentation into nucleosomal oligomers nor the elevation of TRPM-2 mRNA levels but to non-specific biochemical changes characteristic of necrosis. These results suggest that androgen-independent prostatic cancer cells retain a major portion of the programmed cell death cascade which can be activated by non-androgen ablative cytotoxic drugs that induce "thymineless" death.     

Glyceraldehyde-3-phosphate dehydrogenase expression during apoptosis and proliferation of rat ventral prostate.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional enzyme known to play a critical role in neuronal apoptosis. We undertook the current studies to determine whether GAPDH also plays a role in prostate epithelial cell apoptosis in response to androgen deprivation. To do so, we analyzed GAPDH staining by immunohistochemistry during castration-induced involution and androgen-induced regeneration of rat ventral prostate. We found that GAPDH was undetectable in secretory epithelial cells at baseline and that staining did not increase in the epithelium during the period of peak apoptosis from 1 to 3 days after castration. However, GAPDH levels did increase within nuclei of some basal epithelial cells 5 days after castration and within the cytoplasm of all secretory epithelial cells 7 days after castration. GAPDH was also abundant within the cytoplasm of secretory epithelial cells during the period of maximal cell proliferation from 2 to 3 days after androgen replacement and was clearly apparent within nuclei of some epithelial cells 4 days after androgen replacement. Our studies suggest that GAPDH plays multiple roles during prostate epithelial cell apoptosis and proliferation.     

Immunochemical characterization of the androgen-dependent spermine-binding protein of the rat ventral prostate.

A solid-phase radioimmunoassay was developed to measure the level of the androgen-dependent spermine-binding protein (SBP) in the cytosol fraction of the rat ventral prostate during endocrine manipulation. The concentration of SBP and immunologically cross-reacting material (CRM) in the ventral prostate was at least 5000 times higher than the level of CRM detected in rat serum or cytosol from other rat tissues. Cytosol from the ventral prostate of intact rats was separated by DEAE-cellulose chromatography into three major fractions of CRM. One of these fractions corresponded to the elution position of SBP. Cytosol prepared from rats 48 h after castration lacked SBP and one of the two other fractions of CRM. This loss coincided with an increase in CRM in the remaining fraction. No significant difference was detected in the total level of CRM when intact and 48 h-castrated rats were compared. Injection of rats with 5 alpha-dihydrotestosterone (DHT) immediately after castration prevented these changes in the profile of CRM. Several proteins cross-reacting with antibodies to purified SBP were detected in cytosol by using an immunoblot procedure. The highest-Mr band corresponded to SBP. The effect of short- and long-term castration and subsequent DHT treatment on CRM was studied by using the immunoblot technique. Short-term castration (2 days) led to the disappearance of CRM coinciding with SBP (Mr 35 000-38 000) and an increase in smaller forms of CRM (Mr 24 000 and 22 000). Injection of rats with DHT 2 days after castration led to the reappearance of CRM corresponding to SBP, which returned to normal levels within 4 to 5 days of treatment. Long-term castration (up to 14 days) led to a gradual disappearance of all CRM; subsequent DHT treatment led to the reappearance of all forms of CRM and normal levels were attained within 5 days. We have identified SBP and the various forms of CRM as a secretory product of the rat ventral prostate by immunohistochemical staining and by DEAE-cellulose fractionation of prostatic fluid. Prostatic fluid is rich in proteolytic activity and these proteinases may be responsible for processing SBP to small forms of CRM.     

Androgen repression of the production of a 29-kilodalton protein and its mRNA in the rat ventral prostate

The regression of the ventral prostate, after a rat is deprived of androgens by castration, is accompanied by a marked decrease in the prostate's ability to synthesize RNA and major proteins. Surprisingly, in vitro translation of prostate RNA, isolated from rats 2 days after castration, detects four proteins with Mr of approximately 29,000, 37,000, 46,000, and 49,000 whose message levels increased 4- to 12-fold compared to results from normal rats. According to cDNA dot hybridization analysis, the increase after castration in the level of the 29-kDa protein-mRNA (per unit amount of DNA) was reversed within 6 h by androgen treatment of castrated rats. In contrast, the level of a mRNA in male rat liver, which hybridized to a cloned probe for the prostate 29-kDa protein-mRNA was reduced by castration and increased by androgen treatment. During an in vitro incubation, the ventral prostates of normal rats were much less efficient than the prostates of rats castrated 2 days earlier in synthesizing a 29-kDa protein. Despite the fact that androgenic manipulation of rats induced very rapid and significant changes in the production of the 29-kDa protein and in the level of its mRNA, the cellular level of this protein in the prostate, as determined by radioimmunoassay, was maintained at near normal values throughout the 2-week experimental period. Thus, the prostate appears to have a mechanism, based on androgen repression of certain genes, to maintain the cellular levels of the 29-kDa protein and possibly other structurally or functionally important proteins during both the periods of androgen-dependent growth and the castration-induced regression. The loss of such a regulatory mechanism may result in androgen-independent abnormal prostate growth.     

Molecular cloning of rat prostate transglutaminase complementary DNA. The major androgen-regulated protein DP1 of rat dorsal prostate and coagulating gland.

Complementary DNA (cDNA) that codes for a major androgen-dependent secretory protein of rat coagulating gland and dorsal prostate, dorsal protein 1 (DP1), was isolated by molecular cloning. Recombinant DP1 cDNA clones were identified from a bacteriophage lambda gt11 rat coagulating gland expression library using an affinity purified polyclonal antibody. Amino acid sequence deduced from DNA contained sequences identical with several DP1 cyanogen bromide cleavage fragments. Northern blot hybridization of poly(A) RNA isolated from intact rat dorsal prostate and coagulating gland revealed a predominant messenger RNA (mRNA) species of approximately 3200 nucleotides. Tissue-specific expression of DP1 mRNA was indicated by the absence of DP1 mRNA in ventral prostate and other tissues of the rat. Expression of DP1 mRNA was androgen-dependent, decreasing approximately 80% 7 days after castration and increasing rapidly following androgen replacement. Southern blot analysis of restriction enzyme-digested rat DNA indicated that DP1 is encoded by a single gene and that no major genomic rearrangements accounted for its lack of expression in the dorsal prostate-derived rat Dunning tumor. Sequence comparisons revealed that rat prostate DP1 shares sequence identity with Factor XIIIa and tissue transglutaminase, including the active center, GQCWVF, indicating that DP1 is a member of the transglutaminase gene family.