Genetic change in transforming growth factor beta (TGF-beta) receptor type I gene correlates with insensitivity to TGF-beta 1 in human prostate cancer cells.

Transforming growth factor beta 1 (TGF-beta 1), a potential regulator of growth of prostate cancer cells, exerts its effects through interaction with membrane receptors. In the present study, an attempt was made to establish a correlation between TGF-beta 1 sensitivity and TGF-beta receptor expression in three prostate cancer cell lines (PC3, DU145, and LNCaP). In a dose-dependent manner, TGF-beta 1 inhibited the proliferation of PC3 and DU145 cells but not LNCaP cells. Since TGF-beta signals through a heteromeric complex composed of TGF-beta receptors type II and type I, the expression of these receptors was investigated by Western blot analysis and reverse transcriptase-PCR. These studies demonstrated that all three prostate cancer cell lines express type II receptor. In contrast, type I receptor was detected only in the TGF-beta 1-sensitive PC3 and DU145 cells but not in the TGF-beta 1-insensitive LNCaP cells. To investigate the possibility that the undetectable expression of type I receptor in LNCaP cells is due to a change in the respective gene, Southern blot analysis was performed. The result demonstrated that there was a genetic change in type I receptor gene in these cells. Subsequently, when LNCaP cells were transiently transfected with T beta R-I cDNA, sensitivity to TGF-beta 1 was restored. These observations indicate that LNCaP cells contain a defective T beta R-I gene which rendered these cells insensitive to the action of TGF-beta 1.

Prevention of cell death induced by tumor necrosis factor alpha in LNCaP cells by overexpression of sulfated glycoprotein-2 (clusterin).

Sulfated glycoprotein-2 (SGP-2) expression has been associated with programmed cell death in the prostate, but its exact role remains unclear. The present study was carried out in an attempt to establish the function of SGP-2 in programmed cell death using tumor necrosis factor (TNF) alpha-induced cytotoxicity in LNCaP cells as the model system. LNCaP is an androgen-sensitive, human prostatic cancer cell line that responds to TNF in culture by undergoing programmed cell death, as determined by the loss of cell number, failure to exclude trypan blue, detection of DNA fragmentation, and increased release of previously incorporated [3H]thymidine. Immunocytochemical staining for SGP-2 was weak but evident in LNCaP cells. Following treatment with TNF alpha, there was a time-dependent increase in SGP-2 staining, the intensity of which peaked at 2 h and declined thereafter. SGP-2 staining in LNCaP cells was undetectable prior to the onset of DNA fragmentation at 6 h of TNF treatment. This observation indicated that TNF-induced cell death in LNCaP cells was characterized by an initial transient elevation of SGP-2, followed by a period of SGP-2 depletion that preceded cell death. Transfection of LNCaP with a 21-base oligonucleotide antisense to SGP-2 resulted in a significant increase in cell death that was sequence specific and was accompanied by a reduction in SGP-2 biosynthesis. These findings supported the concept that SGP-2 depletion, rather than its expression, was associated with cell death. Finally, stable transfection and subsequent overexpression of SGP-2 in LNCaP cells resulted in resistance to the cytotoxic effect of TNF. These results have provided evidence to indicate that SGP-2 plays a role in the protection of TNF-induced cell death in LNCaP cells.

Intracellular levels of SGP-2 (Clusterin) correlate with tumor grade in prostate cancer.

Our previous observations in LNCaP cells in vitro demonstrated an association between apoptotic cell death resistance and SGP-2 (Clusterin) overexpression. Accordingly, we hypothesized that high levels of cellular SGP-2 would aid in identifying biologically aggressive prostate cancer cells with unique survival advantages. To test this hypothesis, 40 archival radical prostatectomy and/or biopsy specimens of varying grades of prostate cancer were subjected to immunohistochemical SGP-2 staining. The resulting epithelial stains were quantified subjectively on a scale of 1-3 by four independent observers. Benign prostatic epithelial cells from young donors served as controls and showed a consistently weak staining intensity. In contrast, prostate cancer specimens showed varying degrees of staining intensity that correlated with a Gleason pattern (P = 0.006). This correlation supports the hypothesis that protection from apoptotic death may account, in part, for biologically aggressive tumor behavior.

Expression and localization of transforming growth factor-beta receptors type I and type II in the rat ventral prostate during regression.

Of the three ubiquitously expressed transforming growth factor-beta (TGF beta) receptors, only type I and type II receptors contain serine/threonine kinase activity and have a direct role in TGF beta signal transduction. In the prostate, it has been reported that the level of type III receptor expression increases transiently after castration. However, the relationship between the TGF beta signaling receptors, type I and type II, and androgen is currently unclear. Thus, in the present study, we made an initial attempt to elucidate the effect of androgen on type I and type II receptor expression in the rat ventral prostate by measuring the levels of messenger RNA (mRNA) and protein at specific time points after castration up to 10 days. Within 3 days after castration, an increase in type II receptor mRNA was observed in the prostate, and the level continued to rise until 7 days postcastration (approximately 8-fold increase). Between days 7-10 postcastration, no significant change in the level of type II receptor mRNA was observed. Testosterone administration immediately after castration abolished the induction of type II receptor mRNA during the same 10-day period. Western blot analysis performed for type II receptor showed a similar result, in that the level of type II receptor protein increased approximately 5-fold by day 10 postcastration. In a similar manner to the expression of type II receptor mRNA, the level of type I receptor mRNA increased steadily until day 7 postcastration (approximately 6-fold increase). Between days 7-10 postcastration, the level of type I receptor mRNA did not change significantly. As with type II receptor mRNA, the induction of type I receptor mRNA was suppressed when testosterone was administered immediately after castration. To localize the expression of TGF beta receptor type II, immunohistochemical studies were performed. The results of these studies demonstrated a preferential localization of type II receptor in the prostatic epithelial cells and an increased staining intensity for the receptor after castration. Taken together, these data indicate that TGF beta signaling receptors, type I and type II, are under negative androgenic regulation at the transcriptional level and that TGF beta may be an important regulator of a stromal-epithelial interaction in the rat ventral prostate.

Transforming growth factor-beta1-induced proliferation of the prostate cancer cell line, TSU-Pr1: the role of platelet-derived growth factor.

The results of our previous study revealed that transforming growth factor-beta1 (TGFbeta1) stimulated proliferation of the prostate cancer cell line, TSU-Pr1. This observation is unexpected, for TGFbeta usually inhibits proliferation in prostate cancer cells. The present study examines possible mechanisms through which TGFbeta1 induces this proliferation. We postulate that TGFbeta1 action is mediated through an indirect mechanism by inducing the expression of platelet-derived growth factor (PDGF), which, in turn, stimulates proliferation. The TGFbeta1-induced proliferation can be abrogated by treatment with a PDGF-neutralizing antibody. Treatment with exogenous PDGF significantly increased TSU-Pr1 proliferation. Finally, treatment of TSU-Pr1 cells with TGFbeta1 resulted in an increase in PDGF secretion. These results indicate that TGFbeta1-induced proliferation in TSU-Pr1 cells is at least mediated through an increased secretion of PDGF.

Prostatic ductal system in rats: regional variation in localization of an androgen-repressed gene product, sulfated glycoprotein-2.

The rat prostate is a complex ductal system with branches and subbranches extending from one end to another. Owing to the relative distance of various regions from the urethra, the entire length of the ductal system can be divided into three segments, i.e. the proximal, intermediate, and distal segments. The present study was carried out to examine the pattern of localization of sulfated glycoprotein-2 (SGP-2), a marker protein associated with programmed cell death, in various regions of the prostatic ductal system under normal conditions and during castration-induced regression. SGP-2 has been considered an androgen-repressed gene product in the rat prostate and has previously been known as castration-induced protein or TRPM-2. In the normal rat prostate, immunoreactive SGP-2 was localized in epithelial cells lining the proximal segment in which signs of programmed cell death were apparent. Cells lining the distal and intermediate segments were, however, devoid of SGP-2. This observed regional variation in SGP-2 localization did not support an earlier hypothesis which stated that SGP-2 was constitutively expressed by all prostatic epithelial cells in the normal rat prostate. After castration in adult rats, there was a shift in the location of cells containing SGP-2 from the proximal segment toward the distal segment. Therefore, there is a regional variation in the distribution of SGP-2 in the rat prostate both before and after castration in the host. These findings are likely to be associated with a regional variation in cellular responsiveness to androgen stimulation and androgen depletion in the prostatic ductal system. Results also support the view that SGP-2 localization is associated with an early manifestation of programmed cell death in the rat prostate.

Apoptosis in decidual tissue regression and reorganization.

During blastocyst implantation, cellular degeneration of decidual tissue spreads from the antimesometrial region to the mesometrial region to accommodate the developing conceptus. To identify the mechanism of decidual regression and its tissue localization, decidual formation was induced in either intact pseudopregnant rats or ovariectomized rats treated with steroids. Antimesometrial and mesometrial cells were separated by elutriation and cultured for 48 h before DNA analysis. Nucleosomal DNA fragmentation was observed in antimesometrial cells, whereas DNA fragmentation was less marked in mesometrial cells. To determine whether a similar pattern of apoptosis occurred during decidual development in vivo, decidua tissues from antimesometrial and mesometrial regions were obtained on days 8-14. Nucleosomal DNA fragmentation was first detected on day 10 in the antimesometrial region, with fragmentation in the mesometrial region delayed by at least 24 h. DNA fragmentation increased in both tissues with time, but was always more pronounced in antimesometrial cells. Sulfated glycoprotein-2 (SGP-2) has been associated with apoptosis, and its expression was examined by Northern analysis. Levels of SGP-2 messenger RNA (mRNA) were detected in the antimesometrial region on day 9 and increased markedly by day 10; mesometrial expression was delayed 24 h. Levels of SGP-2 mRNA in both regions decreased before the onset of DNA fragmentation. In contrast, cathepsin-D expression, detected by immunohistochemistry, was localized to few mesometrial and antimesometrial cells between days 9-12, with all cells showing extensive staining by day 14. To determine whether changes in progesterone control mechanisms initiated decidual apoptosis, plasma progesterone levels were measured by RIA, and progesterone receptor mRNA levels in decidual tissues were examined by reverse transcription-polymerase chain reaction. Both parameters remained unchanged during decidual growth and regression. These results provide biochemical evidence that decidual regression occurs by apoptosis initiated in the antimesometrial region and with progression to the mesometrial region. Apoptotic cell death occurs despite high levels of plasma progesterone and high levels of progesterone receptor message in decidual tissue.

Regulation of proliferation and production of prostate-specific antigen in androgen-sensitive prostatic cancer cells, LNCaP, by dihydrotestosterone.

LNCaP is an androgen-sensitive human prostatic cancer cell line. The effect of androgen on these cells is characterized by a bell-shaped growth response and a dose-dependent induction of prostate-specific antigen (PSA) production. The present study was carried out to gain further insight into the effect of androgen on LNCaP. Cells were cultured in phenol red-free RPMI-1640 supplemented with 10% charcoal-stripped fetal bovine serum, with concentrations of dihydrotestosterone (DHT) ranging from 0-10(-7) M, in a 4-day culture system. A bell-shaped growth response was reproduced with a peak level of cell count at 10(-10) M DHT. PSA secretion from these cells did not increase significantly until the DHT level in the medium reached 10(-9) M. A progressive increase in PSA secretion was observed at higher DHT concentrations accompanied with a progressive decline in cellular proliferation. The results of immunocytochemical analysis of PSA localization indicated that the proportion of cells with positive staining for PSA also increased with increasing concentrations of DHT. Analysis of androgen receptors, as determined by both immunocytochemistry and Western blot analysis, showed a decline in nuclear androgen receptor at low concentrations of DHT and an increase in the amount of receptor protein at high concentrations. These results indicated that the androgen-induced bell-shaped growth response in LNCaP cells represented the manifestation of two different cellular events in dose-related manner: cellular proliferation at low DHT concentrations and increased production of PSA at high DHT concentrations.

Transforming growth factor-beta1 is a mediator of androgen-regulated growth arrest in an androgen-responsive prostatic cancer cell line, LNCaP.

LNCaP is an androgen-responsive prostatic cancer cell line that exhibits a bell-shaped growth response to increasing doses of dihydrotestosterone (DHT) in culture. Although the precise mechanism responsible for this growth response to androgen stimulation remains unclear, many studies have suggested that androgen modulates the level of various growth factors. In the present study, the role of transforming growth factor-beta (TGF-beta) in mediating the androgen-regulated growth arrest of LNCaP cells was investigated. The following concentrations of DHT were used: 0, 10(-12), 10(-10), and 10 (-7) M. These concentrations were selected because they represent the zero DHT control, the low-proliferative dose, the high-proliferative dose, and the growth arrest dose, respectively. Results of RT-PCR showed that LNCaP cells express TGF-beta1 but not -beta2 and -beta3 messenger RNA. Competitive quantitative RT-PCR demonstrated that the level of TGF-beta1 messenger RNA increased approximately 7-fold when cells were treated with 10(-7) M DHT. Results of Western blot analysis showed a dramatic increase in the level of latent TGF-beta1 protein in cell lysates with increasing concentrations of DHT. In addition, results of enzyme-linked immunoadsorbent assay for TGF-beta1 indicated that treatment of LNCaP cells with DHT led to a dose-dependent increase in both total and biologically active TGF-beta1 in the conditioned media. To determine the role of TGF-beta1 in regulating LNCaP proliferation, the action of TGF-beta1 was blocked by two different but complementary approaches. First, TGF-beta1 neutralizing antibody was added to the culture medium with varying concentrations of DHT. Second, mannose-6-phosphate, which has been demonstrated to inhibit the activation of latent TGF-beta1, was added in a similar manner to the culture. Results demonstrated that the characteristic bell- shaped growth response following treatment with increasing doses of DHT was converted to a linear dose-response curve as the growth of inhibition seen at the high dose by DHT was abolished. These observations, taken together, indicate that TGF-beta1 mediates at least in part the growth arrest observed at the high concentration of DHT in LNCaP cells.

Profile matching and profile subtraction: application of computer-based image analysis system for two-dimensional electrophoresis gels.

The microcomputer-based image analysis system IB-1000 (developed by Indiana Biotech, Highland, IN) for two-dimensional electrophoresis gels has been described previously (9). It allows the user to compare protein spots between two profiles and identify those spots that are commonly shared in both profiles. This report describes two applications of the system's global comparison routine-profile matching and profile subtraction. This application is able to subtract commonly shared spots from one profile, creating a new profile made up by the unmatched spots in the other profile. These applications can be employed in a large variety of research projects.

Analysis of cell death and cell proliferation in embryonic stages, normal adult, and aging prostates in human and animals.

Homeostasis in the prostate is recognized to be maintained by a complex interplay between the opposing actions of cell proliferation and cell death. Growth regulatory factors that promote or inhibit cell proliferation and promote cellular death have been identified in the prostate. The integration of these forces involves cellular cooperation between the prostatic stroma and epithelium. Hormone-regulated production of growth regulatory factors by one cell type may determine growth stimulation, inhibition, or cell death in a reciprocal cell partner. Imbalance between net cell proliferation and net cell death rates may result in abnormal growth leading to BPH. Additional study of the growth regulatory factors associated with distal vs. proximal epithelial cells and stroma and comparison of growth factor expression by the neonatal, postnatal growing, adult quiescent, and aging prostates will likely provide further insight into the regulation of prostate cell division and death.

Captopril (an inhibitor of angiotensin converting enzyme) inhibits obstructive changes in the neonatal rabbit bladder.

OBJECTIVES: To investigate whether angiotensin II has a role in the regulation of bladder smooth muscle growth and function, we developed a model of bladder neck obstruction (BNO) in the neonatal rabbit and investigated the effect of captopril (angiotensin converting enzyme inhibitor) on the obstructive changes in the developing bladder. METHODS: Partial BNO was induced in a group of 2-day-old rabbits (n = 8) by placing a loose 2-0 silk ligature around the vesicourethral junction. A second group of rabbits subjected to the identical partial BNO procedure (n = 8) was given captopril (1 mg/kg/day). Twelve days postobstruction, bladders from these animals, along with paired controls (n = 8), were harvested and assayed for total protein, DNA, and collagen content. RESULTS: Partial BNO resulted in a 170% increase in wet weight (P < 0.05), 132% increase in protein/deoxyribonucleic acid (DNA) ratio (P < 0.05), 75% increase in total DNA (P < 0.05), and 115% increase in total collagen (P < 0.05). When compared with obstructed animals, captopril administration significantly inhibited the increase in total DNA (P < 0.05) and reduced the amount of total collagen (P = 0.054). Examination of histology specimens demonstrated that captopril inhibited the serosal hyperplasia and collagen deposition associated with obstruction. CONCLUSIONS: These data demonstrate that captopril partially inhibits the changes in the neonatal rabbit bladder associated with obstruction, supporting the hypothesis that angiotensin II is involved in the regulation of bladder smooth muscle growth and collagen production.

Effect of TGF-beta 1, TGF-alpha, and EGF on cell proliferation and cell death in rat ventral prostatic epithelial cells in culture.

A tissue culture system for rat prostatic epithelial cells was developed, and the effect of epidermal growth factor (EGF), transforming growth factor alpha (TGF-alpha), and transforming growth factor beta 1 (TGF-beta 1) on these cells was evaluated. The primary culture was prepared by DNAse/collagenase dissociation of minced ventral prostates. Cells were initially plated in RPMI-1640 medium containing 10% fetal bovine serum to allow the preferential attachment of stromal cells. Twenty-four hours later, the unattached epithelial cells were replated in WAJC-404 medium supplemented with insulin (5 micrograms/ml), transferrin (5 micrograms/ml), and selenious acid (5 ng/ml). Bovine pituitary extract (BPE) (30 micrograms/ml), EGF (10 ng/ml), and TGF-beta 1 (0, 0.1, and 1.0 ng/ml) were added either alone or in combination according to experimental requirements. The rate of cell proliferation was assessed by counting the total cell number and by [3H]thymidine incorporation. Prostatic epithelial cells exhibited a bell-shaped growth curve in a span of 7-8 days, with a growth peak at day 3 or 4 of culture. Treatment of cells with EGF or TGF-alpha resulted in a concentration-dependent increase in cell growth, whereas addition of TGF-beta 1 into the culture resulted in an inhibition of cell proliferation that could be reversed with increasing concentrations of EGF. Cell death was assessed using the terminal deoxynucleotidyl transferase (TdT)-mediated immunoperoxidase-digoxigenin nick end labeling technique and the trypan blue exclusion test. Epithelial cells cultured in media containing EGF had the lowest incidence of cell death. Cells cultured in the absence of EGF demonstrated a marked increase in cells undergoing cell death. The addition of TGF-beta 1 into the EGF-depleted medium caused a further increase of cell death. These results indicated that cell proliferation and cell death in rat prostatic epithelial cells in culture could be modulated by EGF and TGF-beta 1. The former stimulated cell proliferation and prevented cell death, whereas the latter inhibited proliferation in the presence or absence of EGF and induced cell death.

Effect of spermatocele fluid on growth of human prostatic cells in culture.

The present study was carried out to investigate whether testicular fluid derived from a spermatocele contains substance(s) that promote the growth of human prostatic cells in culture. Human spermatocele fluid was centrifuged to sediment spermatozoa. The supernatant was then added to cultures of human prostatic stromal or epithelial cells that were isolated from surgical specimens of benign prostatic hyperplasia. Addition of spermatocele fluid in quantities of 1 microgram/ml of protein resulted in a significant increase in the number of both prostatic stromal and epithelial cells at the end of a 6-day culture period. Human serum at equivalent protein concentrations in the culture medium had no stimulatory effect. At least two separate growth-promoting factors were found in spermatocele fluid, one for stromal cells and one for epithelial cells. The mitogen for stromal cells was heat labile and persisted after treatment with activated charcoal. The factor for epithelial cells was heat stable but was removed by charcoal treatment. These observations are consistent with the concept that the human testis secretes nonandrogenic substances that can promote prostatic growth.

Proteins of the rat prostate. II. Synthesis of new proteins in the ventral lobe during castration-induced regression.

Ventral prostates from adult Sprague-Dawley rats at different days postcastration were cut into one to two mm.3 pieces and incubated in medium containing S35-methionine (100 uCi/ml.) at 37C under 95% oxygen and 5% carbon dioxide for four hours. The incubated tissues were subjected to two-dimensional electrophoresis and radiofluorography. Over 100 spots were developed in the fluorograms. Three groups of spots, representing cytoskeletal proteins, androgen-dependent proteins and castration-induced proteins, were further evaluated by a computer-based densitometer. The level of densitometry absorption is proportional to the amount of radioactivity in each spot. The synthesis of cytoskeletal proteins, such as actin and tropomyosin, were relatively constant throughout the course of prostatic regression. The rate of synthesis of androgen-dependent proteins declined rapidly from a high level of synthesis before castration to a non-detectable level by Day 3 postcastration. However, three proteins, which were either not synthesized (spot G and spot H) or synthesized at a very low level (spot I) before castration, were the major proteins synthesized by the prostate during early stages of its regression. The rate of synthesis of these proteins reached a peak by Day 4 postcastration, declined rapidly and remained at a low level thereafter. The respective molecular weights and isoelectric points for these three proteins were 33 Kd and 7.2 for spot G, 38 Kd and 5.3 for spot H and 64 Kd and 6.0 for spot I. Previous findings showed that prostatic regression in rats was associated with a surge of activities in proteolytic enzymes which peaked five to six days postcastration. The peak of synthesis of three proteins noted in the present study, therefore, preceded the peak of activities of proteolytic enzymes in the regressing prostate by one to two days. Testosterone replacement to animals at the time of castration prevented the synthesis of these proteins in the prostate. Since the synthesis of these three proteins in the ventral prostate is induced by androgen-depletion resulted from castration, they are considered as the castration-induced proteins.

Cell cycle kinetics in rat prostatic epithelia: nuclear migration during G2 phase.

Proliferative events in luminal epithelial cells of the rat ventral prostate were studied in 2 experiments. In experiment 1 a longitudinal survey of the rat prostate was conducted between 22 and 88 days of age. The pattern of temporal changes in the weight of the ventral prostate paralleled that of the testis, an observation consistent with the knowledge that the prostate is an androgen target organ. However, the total number of mitotic cells per prostate showed a biphasic pattern of distribution. The count was high (82 per prostate) in the prostate of 22-day-old rats and it decreased to a low value (25 per prostate) by age 30 days, a period corresponding to the onset of puberty in rats. The total mitotic count per prostate increased gradually and reached a peak (85 per prostate) around age 60 days when it started to decline gradually despite a continuous increase in prostatic weight. In experiment 2 cell cycle parameters of these cells were determined in young adult rats (age 70 days or 250 gm. body weight) by an in vivo 3H-thymidine pulse-chase approach. The apparent duration of various phases of the cell cycle was estimated as G1--11.5 hours, S--6.5 hours, G2--3.0 hours, M--2.0 hours and total cell cycle--23 hours. The fraction of cells in the S phase was 2.1%, which was extrapolated to give rise to a total growth fraction of 7.4%. In addition, it was noted that there was a migration of the nucleus from the base toward the apex of the epithelial cells during the G2 phase of the cell cycle in preparation for cell mitosis. Following mitosis nuclei of the 2 daughter cells returned to the usual basal position. These parameters will be used as basis of our future kinetic studies in the rat prostate.

Acid phosphatase in the rat prostate: comparison with other organs.

Properties of acid phosphatase in the rat ventral prostate were compared with those in seven other organs in adult male rats by the electrophoretic mobility in acrylamide gels, susceptibility to reactions with inhibitors, and by their response to castration and subsequent testosterone replacement. The enzyme in the spleen exhibited the highest values of Km and Vmax. These values in the prostate, the liver, and the kidney showed an intermediate level, while the lowest level was found in the heart, the lung, the adrenals, and the seminal vesicle. Upon electrophoresis, a total of six isoenzyme bands were resolved. Two major zones of activity were noted. They were the slow-moving anodal bands (isoenzymes 1,2, and 3) and the fast-moving cathodal bands (isoenzymes 4,5, and 6). The spleen possessed the highest number of isoenzymes (bands 1,2,4,5 and 6) and the prostate had three (bands 1,2, and 3). The heart contained only one (band 2). Two isoenzymes (bands 1 and 2) were found in remaining organs. Results of the effects of inhibitors showed that NaF inhibited all six isoenzymes, while L(+)tartrate inhibited mainly those in the anodal zone. D(-)tartrate and formaldehyde showed no significant inhibition to any of the isoenzymes. PCMB (para-chloromercuribenzoic acid) was found to be a specific inhibitor for isoenzyme 3. Upon castration in the hosts, the enzyme activity in the prostate, the liver, and the seminal vesicle was significantly reduced. This reduction in enzyme activity involved all isoenzymes in these three organs, but isoenzyme 3 in the prostate disappeared completely after castration. The activities of these isoenzymes were restored by testosterone replacement. These results indicate that acid phosphatase in rat tissues has multiple forms; each has its own electrophoretic mobility in acrylamide gels, sensitivity to inhibitors, and response to hormonal manipulation. Isoenzyme 3 is specific to the prostate and is uniquely sensitive to PCMB inhibition and to androgen stimulation.

Prostatic ductal system in rats: regional variation in morphological and functional activities.

The rat prostate is a complex ductal system with branches and subbranches extending from one end to another. Owing to the relative distance of various regions of the duct from the urethra, the entire length of the ductal system can be arbitrarily divided into three segments, i.e., the proximal, intermediate, and distal segments. The present study was carried out to assess the regional variation in cellular activities in this ductal system. Ventral prostates from adult Sprague-Dawley rats were dissected so that an individual ductal system was mechanically isolated and longitudinally sectioned to reveal various segments. Epithelial cells lining distal segments were tall-columnar type and were actively engaging in mitotic activity. Cells in intermediate segments were also tall-columnar type. However, they were mitotically quiescent, but able to produce secretory proteins. Evidence of programmed cell death was not observed in either of these two segments. Cells in proximal segments, on the other hand, were low-columnar or cuboidal in shape and were stained heavily for cathepsin D, a marker associated with late manifestation of cell death. Following castration in adult rats, there was a reversal in the site of programmed death in cells lining the ductal system. By Day 4 post-castration, distal segments contained many epithelial cells with intense cytoplasmic staining for cathepsin D while proximal segments showed a reduction in number of positively stained cells. By Day 7 post-castration, cells in proximal segments, though atrophied, were devoid of staining for cathepsin D.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of castration-induced cell death in the rat prostate by immunohistochemical localization of cathepsin D.

Activities of cathepsin D (EC 3.4.23.5) were determined in three lobes of the prostate during their involution by both biochemical and immunohistochemical procedures. The activity of cathepsin D in noncastrated rats was 0.9 +/- 0.2 (mean +/- SE) 5.7 +/- 0.6, and 13.1 +/- 0.8 units/mg protein for the ventral, lateral, dorsal lobes, respectively. Following castration, there was a significant increase in enzymatic activity in all three lobes within 2-3 days. In the ventral lobe, the activity peaked in 5 days to 6.2 +/- 0.9 units/mg protein and declined slightly thereafter. In the lateral and dorsal lobes, the activity remained elevated (14-20 units/mg protein) throughout the postcastration period studied. Immunohistochemical staining of cathepsin D was localized in the cytoplasm of prostatic epithelial cells as fine discrete lysosomal granules. These granules were larger and more abundant in the dorsal and lateral lobes than in the ventral lobe and were not detected in prostatic stromal cells and seldom in the luminal fluid. Castration resulted in an immediate increase in the size and number of these granules in the epithelial cells, followed by a sudden further increase in cathepsin D staining in some but not all epithelial cells. Lysosomal granules gradually coalesced in these cells to form large vacuoles that fit the characteristic description of apoptotic bodies. Finally, after day 7 postcastration, collapse and disintegration of the entire glandular structure was noted. Using this procedure to localize cathepsin D as a tool, we were able to follow the morphological events of prostatic cell death during castration-induced involution in the rat at the light microscopic level.

Proteins of the rat prostate. III. Effect of testosterone on protein synthesis by the ventral prostate of castrated rats.

Protein synthetic activities in the ventral prostate were assessed by two-dimensional electrophoresis in either four-day or seven-day castrated rats at different intervals following subcutaneous implantation of testosterone-filled silastic tubings for a period of up to four days. Prostatic tissues were cut into one to two mm. pieces and incubated in tissue culture medium containing S35-methionine (100 microCi/ml.) at 37C under 95% oxygen and 5% carbon dioxide for four hours. The incubated tissues were subjected to two-dimensional electrophoresis and radiofluorography. Analysis of protein spots detected in the fluorograms by computer-assisted densitometry revealed temporal changes in the synthesis of individual proteins by the ventral prostate of castrated rats following androgen treatment. Changes in two groups of proteins were evaluated: castration-induced proteins and androgen-dependent proteins. The level of synthesis of three castration-induced proteins (spots G, H, and I) declined rapidly upon testosterone treatment and reached a non-detectable level for spots G and H and a low level of synthesis for spot I by three days following androgen treatment. Synthesis of androgen-dependent proteins (spots D, E, and F) was activated by testosterone treatment. However, the time interval required to activate the synthesis of these proteins is different. Synthesis of protein spot D (prostatic binding protein) was detected as soon as half hour after the treatment. Synthesis of spots E and F, on the other hand, was not activated until 24 and 48 hours after the treatment, respectively. These changes in patterns of protein synthesis represent the characteristics of cellular responses to testosterone stimulation by the regressed prostate.