Biochemical alterations in sex hormone-induced hyperplasia and dysplasia of the dorsolateral prostates of Noble rats.

Simultaneous implantation of intact Noble (Nb) rats with testosterone and 17 beta-estradiol (E2)-filled silastic capsules for 16 weeks caused atypical hyperplasia (dysplasia) and striking enlargement exclusively in the dorsolateral prostates (DLPs) of all animals. The dysplastic lesion may be preneoplastic since long-term administration of these steroids to Nb rats is known to induce a high incidence of adenocarcinoma in the DLP. Treatment of rats with nonaromatizable 5 alpha-dihydrotestosterone (DHT) for 16 weeks caused enlargement but not dysplasia, implicating estrogen as a key factor in the genesis of the proliferative lesion. Compared with controls, the testosterone plus E2 treatment caused a 2.5-fold increase in nuclear type II estrogen binding sites which were confined to the DLP. Neither treatment significantly altered androgen content or levels of androgen receptor in the ventral prostate or DLP. Organ cultures of enlarged DLP containing foci of dysplasia metabolized more [3H]DHT than control tissue, which resulted in increased formation of the 5 alpha-androstane-3 beta, 17 beta-diol (3 beta-androstanediol) metabolite by these explants. Because 3 beta-androstanediol has previously been shown to displace [3H]E2 from cytosolic type I estrogen binding sites, the dysplasia may be caused by hyperstimulation of the DLP by the hormones and their normal metabolites produced in abnormal amounts.

Testosterone-mediated increase in 5 alpha-dihydrotestosterone content, nuclear androgen receptor levels, and cell division in an androgen-independent prostate carcinoma of Noble rats.

An androgen-independent, transplantable prostate carcinoma line (AIT), originally derived from the dorsolateral prostate (DLP) of Noble rat, was implanted into orchiectomized Noble rats and its response to androgen stimulation was studied and compared to that of the regenerating DLP tissue in sexually ablated rats. AIT tumors carried in castrated hosts displayed a high basal level of proliferative activity (mitotic index (MI), 15.0 +/- 0.5) while DLP tissue in untreated castrates exhibited no proliferative activity. Following androgen stimulation by testosterone capsule implantation into host rats, the AIT responded with a marked increase in cell proliferation; MI values doubled to 30.0 +/- 2.9 on Day 5 following androgen stimulation. This androgen-induced increase in MI values was coincident with elevations in nuclear androgen receptor (20-fold increase) and 5 alpha-dihydrotestosterone content (3-fold increase) in the tumor. However, by Day 10 following androgen treatment, indices of cell proliferation in the AIT declined to pre-androgen-stimulated levels (MI, 14.8 +/- 1.9) despite the continued elevations in nuclear androgen receptor and tissue 5 alpha-dihydrotestosterone contents. Parallel changes in MI were also observed in the normal regenerating DLP following androgen stimulation. MI values in this tissue increased from nondetectable levels to 38.1 +/- 4.7 on Day 5 but declined to relatively low levels (4.5 +/- 0.9) by Day 10 following androgen replacement. Taken together these findings led us to conclude that the AIT carried in castrates is capable of responding to testosterone in a manner similar to that observed for androgen-stimulated DLP of sexually ablated rats. Thus, in both the neoplastic and regenerating tissues, the initial response to androgen is characterized by a marked enhancement of cell proliferation which was correlated with an increase in androgen receptor and 5 alpha-dihydrotestosterone content. However, like its tissue of origin, the AIT possesses mechanisms which act to limit androgen-induced cell division despite continued elevations in key parameters of androgen activation.

Characterization of a human, sex steroid-responsive transitional cell carcinoma maintained as a tumor line (R198) in athymic nude mice.

We have established a transplantable tumor line, R198, derived from a papillary (transitional cell) carcinoma of the human urinary bladder. In nude mice, the tumor line exhibits properties attributable to both prostatic and transitional epithelia. In tumor-bearing animals given androgens, the neoplasm has a rapid growth rate, possesses low levels of measurable androgen receptors, produces tartrate-inhibitable acid phosphatase, and forms well-encapsulated, cystic tumors composed of transitional, glandular, and squamous cells. The administration of estrogens or transplantation of the tumor into female mice causes regression of the tumor. In a small percentage of the transplants placed into females or estrogenized animals, selection occurs for tumor cells which can grow under these conditions. The resulting tumors are infiltrating scirrhous carcinomas that closely resemble squamous cell carcinomas of the urinary bladder. These neoplasms grow slowly and do not possess androgen receptors or secretory material. They are composed of a homogeneous population of squamous cells which are locally invasive. The paradox of a bladder tumor with some prostatic characteristics may be explained by the fact that the tumor was derived from the trigone region of the bladder, which embryologically is formed by an admixture of tissue from the wolffian duct and the urogenital sinus. Some trigone-derived neoplasms have characteristics of both bladder and prostate. We hypothesize that sex steroid-sensitive R198, with characteristics of both bladder transitional cells and prostatic epithelia, is a tumor which phenotypically expresses the embryological origins of these tissues. As such, the tumor line will serve as a useful model for studying sex steroid-responsive cells of the urogenital epithelium.

Heterogeneous distribution of filipin-sterol complexes in nuclear membranes.

Filipin, a sterol-specific polyene antibiotic, has been shown by electron microscopy to form complexes in membranes of mouse urinary bladder cells. Following instillation of a glutaraldehyde-filipin-dimethylsulfoxide solution into the bladder lumen, filipin-cholesterol complexes appear as membrane corrugations in thin sections and as 20-25 nm protuberances and depressions on PF and EF faces in freeze-fracture replicas. The complexes are observed in plasmalemma, Golgi membrane, rough endoplasmic reticulum and nuclear membrane of five different cell types (urothelial, endothelial, mesothelial, smooth muscle and fibroblasts). In the present report, we direct particular attention to the localization of numerous filipin-cholesterol complexes present in the nuclear envelopes of these cells. Our results suggest that enrichment of cell membranes with cholesterol occurs at an earlier stage in the flow-differentiation process than previously suspected. In addition, the unequal distribution of complexes in favor of the outer nuclear membrane suggests that it has a higher cholesterol content than the inner membrane.

Cytoplasmic inclusion bodies in superior rectus muscle of the eye.

Cytoplasmic inclusion bodies are found in fibers of superior rectus muscle of the eye. They occur in middle-aged and old individuals dying with various unrelated diseases not affecting muscle and in patients with myotonic dystrophy. With light microscopy they can be observed in fibers with pathologic changes, and with electron microscopy they appear to be associated with crystals of probable viral origin. It is suggested that cytoplasmic inclusion bodies in superior rectus muscle are the result of viral activity and may represent an incidental viral infection of the senescent period.

Gap junctions in the myometrium of hypophysectomized estrogen-treated rats.

Gap junctions, which appear in the myometrium at parturition, are believed to permit the flow of ions between smooth muscle-cell interiors thereby facilitating coordinated contractions. Gap junctions are not observed in thin-sectioned myometrium of immature hypophysectomized rats. Following estrogen administration, numerous gap junctions are present suggesting that this hormone is capable of promoting electrotonic coupling in uterine smooth muscle.

Histochemical studies of epithelial cell glycoconjugates in atrophic, metaplastic, hyperplastic, and neoplastic canine prostate.

The nature and distribution of lectin receptors were studied in normal, atrophic, metaplastic, hyperplastic, and neoplastic epithelium of canine prostate. Results were compared with prostatic epithelium of castrated dogs treated for 2 weeks with estradiol-17 beta 17-cyclopentylpropionate, 5 alpha-adrostane-3 alpha,17 beta-diol dipropionate, or 5 alpha-dihydrotestosterone. Eight biotinylated lectins were used as histochemical probes and avidin-biotin-peroxidase complex served as the visualant. Receptors for Ulex europaeus agglutinin-I were present in atrophic prostatic epithelium. Receptors for U. europaeus agglutinin-I, wheat, germ agglutinin, Dolichos biflorus agglutinin, and soybean agglutinin were present in epithelium that had undergone squamous metaplasia. Binding of peanut agglutinin receptors was present, to a limited extent, in squamous epithelium and was increased after they were unmasked (sialic acid residues cleaved with neuraminidase). In glandular cells of normal canine prostate and in benign prostatic hyperplasia, receptor sites were stained with Ricinus communis agglutinin-I, Concanavalia ensiformis agglutinin wheat germ agglutinin, and U. europaeus agglutinin-I. The basal cells in these tissues did not bind lectins. Prostatic carcinoma cells demonstrated receptors for wheat germ agglutinin and U. europaeus agglutinin-I. Responding and atrophic acini were present in prostates of castrated dogs treated with estradiol-17 beta 17-cyclopentylpropionate. Glandular cells of atrophic acini exhibited lectin receptor profiles similar to counterparts in castrated-untreated dogs. However, glandular cells responding to estrogen exhibited staining of free and cryptic peanut agglutinin receptor sites. Glandular cells of castrated dogs treated with 5 alpha-androstane 3 alpha,17 beta-diol dipropionate and 5 alpha-dihydrotestosterone have a pattern of lectin receptors similar to that found in normal and hyperplastic epithelium. Our studies show significant differences in lectin-binding patterns in the epithelium of atrophic, metaplastic, hyperplastic, and neoplastic canine prostate. They also demonstrate that the species of carbohydrate residues present in the glandular cells can be modified with sex hormones.

Association of asymmetric unit membrane plaque formation in the urinary bladder of adult humans with therapeutic radiation.

Asymmetric unit membrane (AUM) is a component of the luminal membrane of urinary bladder in many species. In normal human adults it is inconspicuous, but it becomes prominent following incidental exposure to therapeutic irradiation.

Immunohistochemical and in situ hybridization studies of androgen receptor expression in a transplantable androgen-independent prostatic carcinoma line (AIT) of Noble rats.

BACKGROUND: In a previous study, we had shown that testosterone (T) administration to castrated Noble (NBL) rats, bearing an androgen-independent prostatic carcinoma line (AIT), caused a dramatic increase in high-affinity nuclear-androgen binding sites in the neoplasms. This increase in nuclear androgen receptors (AR) was accompanied by a transient doubling of the mitotic index in the tumors. EXPERIMENTAL DESIGN: In our current study we used immunohistochemistry and in situ hybridization to investigate the effects of androgen withdrawal and replacement on AR expression at the molecular/cellular level in the AIT. Results from immunohistochemical studies of AR expression in the AIT were compared with those from the prostates of intact, castrated, and castrated T-treated rats. RESULTS: Immunopositive staining for AR was found only in the nuclei of prostatic cells from the glands of intact, castrated or castrated T-treated animals. A few immunopositive tumor cells were present in AITs carried in untreated castrated hosts. In all instances, the reaction product was found in the cytoplasm, but it was also present in the nuclei of some tumor cells. Five days of T administration to castrated AIT-bearing rats caused a dramatic increase in immunopositive tumor cells. Nuclear staining was observed in all positive cells, but the reaction product was also present as well in the cytoplasm of some tumor cells. No differences in AR mRNA expression was detected by in situ hybridization studies of the AITs from castrated and castrated T-treated rats. CONCLUSIONS: The differences in localization of AR between normal prostate and carcinoma cells may reflect alterations in DNA binding domains of the AR protein that occurred with neoplastic transformation. Our in situ findings suggest that unlike the normal prostate, where AR mRNA levels are autoregulated by androgen, AIT cells constitutively express these transcripts. Taken together, our findings suggest that the T-mediated increases in nuclear AR in the AIT, detected previously by binding assay and now by immunohistochemistry, are likely the result of post-transcriptional modifications in the receptor protein.

Comparison of 35S- and digoxigenin-labeled RNA and oligonucleotide probes for in situ hybridization. Expression of mRNA of the seminal vesicle secretion protein II and androgen receptor genes in the rat prostate.

The sensitivity of radiolabeled and digoxigenin-labeled RNA probes and synthetic oligonucleotide probes for the detection of seminal vesicle secretion protein II (SVS II) and androgen receptor (AR) mRNA was compared by in situ hybridization in paraformaldehyde-fixed cryostat sections of the rat prostate. Both genes are expressed in different amounts in the various prostatic lobes and contiguous glands. SVS II or AR RNA probes were either labeled with digoxigenin-11-UTP or [35S]UTP by in vitro transcription. A synthetic SVS II oligonucleotide probe was 3' end-labeled (tailed) with either digoxigenin-11-dUTP or [35S]dATP. Hybridized 35S-labeled probes were detected by autoradiography and digoxigenin-labeled probes by immunohistochemistry using alkaline phosphatase conjugated anti-digoxigenin antibody or gold-labeled antibody followed by protein A-gold and silver enhancement. Digoxigenin-labeled probes provided the same degree of sensitivity as their 35S-labeled counterparts for the detection by in situ hybridization of weakly and strongly expressed mRNA. Using both labeling methods, the SVS II RNA probes were more sensitive than the oligonucleotide probes and background labelling of the 35S-labeled oligonucleotide probe was high. The digoxigenin method produced less background with all probe types, hybridization signals showed higher resolution and results were obtained faster than with radiolabeled probes. The immunogold silver enhancement system provided the fastest detection of digoxigenin-labeled probes with a sensitivity and resolution similar to that provided by alkaline phosphatase anti-digoxigenin immunohistochemistry. It is concluded that digoxigenin probe labeling and detection provides a sensitive, reliable, and efficient alternative to radiolabeled probes for in situ hybridization of mRNA.

Role of canine basal cells in postnatal prostatic development, induction of hyperplasia, and sex hormone-stimulated growth; and the ductal origin of carcinoma.

BACKGROUND: The canine prostate has often been proposed as a model for abnormal growth of the human gland. Hyperplasia of the prostate is common in aging men and has been estimated to be present in 100% of old intact dogs. While prostatic carcinoma is common in older men, it appears to be rare in dogs and unlike the disease in humans, it occurs with relatively high frequency in castrated animals. Since basal cells are thought to be key participants in normal and abnormal growth of the human gland, we used immunohistochemistry to investigate the role that they may play in canine prostatic development, the evolution of hyperplasia and carcinoma, and the effects of sex hormones on these cells. METHODS: Prostate specimens were obtained at autopsy from seven sexually immature dogs, autopsy and biopsy samples from 14 sexually mature intact animals, from four castrates, and from19 dogs with prostatic carcinoma. In addition, we also studied the prostates from two intact dogs treated with 5alpha-dihydrotestosterone (DHT) for 6 months and two castrated dogs that were subsequently treated with 5alpha-androstane-3alpha diol and estradiol-17alpha, as well as specimens from two sexually ablated animals given DHT for 2 weeks. All specimens were immunostained for high molecular weight cytokeratin (HMC), pancytokeratin, androgen receptor (AR), and the proliferative marker KI-67. RESULTS: We find that basal cells are the major proliferative cell type in the neonatal and adult canine prostate and that the expression of HMC staining, which defines these cells, may be regulated by androgens. In the adult gland, ductal basal cells formed a contiguous layer, whereas those lining acini were discontinuous. Populations of both basal cell types were variably AR positive, but while HMC immunostaining was abolished in acinar cells following long-term castration, staining remained in ductal cell counterparts. Paralleling the histological development of hyperplasia, the acinar basal cell population increased with age and were the major cell type that expressed KI-67. In contrast, ductal basal cell populations did not expand in the prostates of older dogs and were seldom positively stained for KI-67. The numbers of HMC and KI-67-stained acinar basal cells were dramatically increased in the prostates of intact dogs treated with DHT when compared with glands of untreated controls. This was not the case with ductal basal cells. Androgens given alone or together with estrogen to castrated dogs induced widespread HMC and KI-67 immunostaining in both populations of basal cells. In addition, our results indicate that the majority of canine prostatic carcinomas likely arise exclusively from ductal epithelium. Only one of the 19 cases of carcinoma contained cells that expressed AR, which suggests that androgens may not be required for the initiation or progression of these cancers. CONCLUSIONS: Our findings indicate that two biologically distinct populations of basal cells may exist in the canine prostate. In this regard, the age-related expansion of proliferating acinar basal cell populations, probably mediated by sex steroids, is a key factor in the pathogenesis of canine prostatic hyperplasia. Additionally, we find that prostatic carcinoma in the dog likely arises from ductal cells. Taken together, these findings may indicate that canine acinar basal cells and ductal epithelium have separate susceptibilities to factors that promote hyperplastic or neoplastic development.

Androgen-supported estrogen-enhanced epithelial proliferation in the prostates of intact Noble rats.

Using a stathmokinetic in vivo metaphase-arrest technique, we studied cell proliferation and histological changes in the ventral (VP) and dorsolateral (DLP) prostate lobes of intact Noble (Nb) rats following a 16 week treatment with testosterone (T) or 5 alpha-dihydrotestosterone (DHT) administered separately or in combination with various estrogens. The combined treatment of rats with T and either estradiol-17 beta, estradiol-17 alpha, or moxestrol induced florid dysplasia and markedly elevated the mitotic index (MI) in affected regions of the DLP. In contrast, joint DHT and estrogen treatment caused only mild proliferative lesions in this lobe. The separate administration of either androgens or estrogens suppressed epithelial proliferation in both the VP and DLP, but they differed in their histological effects on these tissues. Thus DHT or T alone maintained the morphological integrity of VP and DLP, whereas E2-17 beta or moxestrol caused massive atrophy of both lobes. Although dysplastic foci were randomly scattered throughout the DLP, the most dramatic lesions occurred in periurethral ducts. With the exception of joint T and E2-17 alpha treatment, which induced proliferative alterations in the VP, dysplasia was always restricted to the DLP of all animals receiving both androgens and estrogens. Concomitant comparative stathmokinetic studies of the prostates of T-treated castrates suggest that protracted androgen-supported estrogen stimulation of the DLP is necessary to overcome factors that normally limit cell proliferation.

Role of canine basal cells in prostatic post natal development, induction of hyperplasia, sex hormone-stimulated growth; and the ductal origin of carcinoma.

BACKGROUND: The canine prostate has often been proposed as a model for abnormal growth of the human gland. Hyperplasia of the prostate is common in aging men and has been estimated to be present in 100% of old intact dogs. While prostatic carcinoma is common in older men it appears to be rare in dogs and unlike the disease in humans it occurs with relatively high frequency in castrated animals. Since basal cells are thought to be key participants in normal and abnormal growth of the human gland, we used immunohistochemistry to investigate the role that they may play in canine prostatic development, the evolution of hyperplasia and carcinoma, and the effects of sex hormones on these cells. METHODS: Prostate specimens were obtained at autopsy from seven sexually immature dogs, autopsy and biopsy samples from 14 sexually mature intact animals, from four castrates, and from 19 dogs with prostatic carcinoma. In addition, we also studied the prostates from two intact dogs treated with 5 alpha-dihydrotestosterone (DHT) for 6 months and two castrated dogs that were subsequently treated with 5 alpha-androstane-3 alpha diol and estradiol-17 alpha as well as specimens from two sexually ablated animals given DHT for 2 weeks. All specimens were immunostained for high molecular weight cytokeratin (HMC), Pancytokeratin, androgen receptor (AR), and the proliferative marker KI-67. RESULTS: We find that basal cells are the major proliferative cell type in the neonatal and adult canine prostate and that the expression of HMC staining, which defines these cells, may be regulated by androgens. In the adult gland, ductal basal cells formed a contiguous layer whereas those lining acini were discontinuous. Populations of both basal cell types were variably AR positive but while HMC immunostaining was abolished in acinar cells following long-term castration, staining remained in ductal cell counterparts. Paralleling the histological development of hyperplasia, the acinar basal cell population increased with age and were the major cell type that expressed KI-67. In contrast, ductal basal cell populations did not expand in the prostates of older dogs and were seldom positively stained for KI-67. The numbers of HMC and KI-67-stained acinar basal cells were dramatically increased in the prostates of intact dogs treated with DHT when compared with glands of untreated controls. This was not the case with ductal basal cells. Androgens given alone or together with estrogen to castrated dogs induced widespread HMC and KI-67 immunostaining in both populations of basal cells. In addition, our results indicate that the majority of canine prostatic carcinomas likely arise exclusively from ductal epithelium. Only one of the 19 cases of carcinoma contained cells that expressed AR which suggests that androgens may not be required for the initiation or progression of these cancers. CONCLUSIONS: Our findings indicate that two biologically distinct populations of basal cells may exist in the canine prostate. In this regard the age-related expansion of proliferating acinar basal cell populations, probably mediated by sex steroids, is a key factor in the pathogenesis of canine prostatic hyperplasia. Additionally we find that prostatic carcinoma in the dog likely arises from ductal cells. Taken together these findings may indicate that canine acinar basal cells and ductal epithelium have separate susceptibilities to factors that promote hyperplastic or neoplastic development. Prostate 47:149-163, 2001.

Androgen receptor expression in prostatic dysplasia (prostatic intraepithelial neoplasia) in the human prostate: an immunohistochemical and in situ hybridization study.

To evaluate the role of androgens in the pathogenesis of prostatic dysplasia, we compared the localization of androgen receptor (AR) in proliferative and nonproliferative cells in normal and dysplastic acini. Basal cells, the only proliferating cells identified in normal acini, contained AR mRNA but lacked an immunodetectable receptor. Both AR mRNA and immunodetectable receptor were present, however, in secretory and stromal cells. Androgen receptor localization in dysplastic lesions was identical to normal but here the proliferative marker Ki-67 was found in both basal and secretory cells. Our findings suggest that androgens do not directly initiate the division of basal cells, the putative precursors of secretory cells. Instead, the hormone may act through its fully translated receptor to mainly mediate the differentiation of secretory cells. The presence of both AR and Ki-67 in dysplastic secretory cells may indicate an abnormal direct androgen-mediated proliferation in this compartment. This is consistent with previous evidence that secretory cell differentiation is impaired in dysplasia.