Adult Human Prostate Research Articles

Stepwise immortalization and transformation of adult human prostate epithelial cells by a combination of HPV-18 and v-Ki-ras.

Recent investigations have shown the presence of ras gene mutations and human papillomavirus (HPV) DNA in prostate carcinomas. In the present study, secondary adult human prostatic epithelial cells, upon transfection with a plasmid containing the entire HPV-18 genome, acquired an indefinite life-span in culture but did not undergo malignant conversion. Subsequent infection of these immortalized cells with the Kirsten murine sarcoma virus, which contains an activated Ki-ras oncogene, induced morphological transformation that led to the acquisition of neoplastic properties. These findings demonstrate the malignant transformation of adult human prostate epithelial cells in culture by a combination of viral oncogenes and the successive roles of HPV infection and Ki-ras activation in a multistep process responsible for prostate carcinogenesis.

Immunohistochemical localization of estrogen-specific 17 beta-hydroxysteroid oxidoreductase in the human and mouse prostate.

The estrogen-specific 17 beta-hydroxysteroid oxidoreductase (17 beta-HSOR) enzyme protein was stained immunohistochemically in the newborn and adult human prostate as well as in the mouse prostate. In the newborn human prostate, ductal and urethral epithelia were faintly stained, whereas in the adult human prostate, intense staining for 17 beta-HSOR enzyme antigen could be detected in the epithelium of the collecting ducts and urethral epithelium as well as in the epithelium of the intraprostatic vas deferens and seminal vesicle epithelium. Immunostaining was weak in the prostatic tissues of both newborn and adult prostate. No positive cells were found in stroma. The activity of NADPH-dependent 3H-estrone reductase was detectable in cell-free homogenates prepared from human prostatic tissues. The activities showed a good correlation with immunocytochemical findings. In the mouse, neonatal estrogenization resulted in intensively stained epithelium of the collecting ducts at the age of 14 days. Moreover, when adult control and neonatally estrogenized mice were implanted with 17 beta-estradiol, the metaplastic epithelium of the periurethral collecting ducts of neonatally estrogenized mice was intensively stained with 17 beta-HSOR. These findings suggest that metaplastic epithelium rises from 17 beta-HSOR-positive cells. The similar distributions of 17 beta-HSOR-positive cells confirm the concept of homology in the posterior estrogen-responsive periurethral region (containing the periurethral ducts and periurethral glands) of the mouse and humans. Our findings further suggest that the 17 beta-HSOR-positive cells may have the same origin and hormonal control in both species.

Tumorigenicity of SV40 T antigen immortalized human prostate epithelial cells: Association with decreased epidermal growth factor receptor (EGFR) expression

Our primary objectives were to: 1) develop a system for the study of prostatic tumor evolution; and 2) examine the role of the epidermal growth factor/epidermal growth factor receptor (EGF/EGFR) pathway in prostate tumor progression. Adult human prostate epithelial cells previously immortalized by transfection with the SV40 T antigen gene (P69SV40T) produced tumors in only 2/18 mice with a 6 month latency period. Reinjection of cells recovered from these tumors after 1 or 2 cycles of growth in nude mice produced tumors in 2/4 and 2/3 mice with markedly decreased latent intervals of 12, 25, 25 and 25 days each. The chromosomal complement of each tumor was human, consistently pseudodiploid, and retained the Y chromosome. In both anchorage-independent and adherent cell growth assays, EGF stimulated proliferation by approximately 2-fold in both the parental P69SV40T line and the tumor sublines. The tumor sublines expressed less EGFR protein than the parental line, as assessed by Western immunoblotting and flow cytometric analysis. Immunoprecipitation revealed increased production of the 18 and 25 kDa TGF-alpha precursors parallel to decreases in detectable EGFR. The growth of both the parental P69SV40T line and the tumor sublines was inhibited by a neutralizing antibody to TGF-alpha under serum-free defined conditions. Inclusion of the TGF-alpha neutralizing antibody consistently inhibited the proliferation of the tumor sublines more than P69SV40T in both proliferation and [3H]thymidine incorporation assays. This finding suggests that the increased tumorigenicity and decreased latent interval observed among the human prostate tumor cells is partially due to activation of the TGF-alpha/EGFR autocrine network.

Distinct Androgen 5α-Reduction Pathways in Cultured Fibroblasts and Immortalized Epithelial Cells from Normal Human Adult Prostate

All androgen-sensitive peripheral tissues and cells, including the prostate, are commonly believed to possess the ability to metabolize testosterone. We report on the in vitro metabolism of tritiated testosterone performed in immortalized human adult normal prostatic epithelial cells and in human adult normal prostate fibroblastic cells (stromal cells). These two cell types were incubated separately with increasing testosterone concentrations (1 to 50nM.) for 2 and 4hours, after which the testosterone metabolic profile was analyzed. Data analysis provided evidence, for the first time, of the existence of two different 5a-reduced metabolic pathways. Stromal cells preferred the androsterone pathway via the oxidative androstenedione formation, whereas epithelial cells preferred the reductive 5a-dihydrotestosterone pathway. These two 5a-reduced metabolites were produced in nearly equal quantities regardless of testosterone concentration or time of incubation. Since interactions between epithelial and stromal cells are involved in the development of the prostate, the availability of defined epithelial and stromal cells suitable for in vitro experiments provides a useful tool for the study of the contribution of androgens to these interactions. The model presented in this study would permit a better evaluation of the intraprostatic regulation of androgen metabolism and the contribution of the anti-5a-reductase drugs to the management of benign prostate hyperplasia.

A functional model of adult human prostate epithelium. The role of androgens and stroma in architectural organisation and the maintenance of differentiated secretory function.

A functional model of adult human prostate epithelium is described. This model shows that stromal cells, but not an androgenic stimuli, are required for architectural organisation of prostate epithelium. Within an organised structure, androgenic stimulation is required for the establishment of secretory epithelial cell morphology and associated function. In the absence of stromal cells but in the presence of androgens architectural organisation and secretory function are lost. Epithelial parenchymal units (organoids) from human prostate tissue were isolated, cultured within a three-dimensional collagen matrix, and xenografted subcutaneously into athymic mouse hosts. The grafted gels were rapidly invaded by host fibroblasts. Epithelial organisation initially disappeared but was re-established concurrently with the stromal cell invasion. In intact male hosts, cuboidal and columnar cells that expressed human prostate-specific secretory markers were found. In castrated male and in female hosts epithelial structures were lined with flattened epithelium with no secretory function. This phenomenon could be reversibly replicated by treating intact male hosts with the anti-androgen Flutamide. Gels containing organoids grafted within 0.45 microns Millipore chambers were not invaded by stromal cells and rapidly lost all epithelial organisation and secretory function. When organoids cocultured with human foreskin fibroblasts were grafted within chambers, structural organisation of the epithelium was supported. These results indicate that both heterologous human fibroblasts and mouse stromal cells are capable of permissively supporting adult human prostate epithelial function.

Immunohistochemical detection of c- erbB-2 protein in human benign and neoplastic prostate

Both the polyclonal anti-c- erbB-2 peptide antiserum pAB 60 and the monoclonal anti-c- erbB-2 protein antibody mAB-1 detect the c- erbB-2 protein in human breast adenocarcinomas. We investigated c- erbB-2 expression in adult human benign hyperplastic and neoplastic prostates, using the avidin-biotin complex immunoperoxidase method. Formalin-fixed, paraffin-embedded specimens of benign hyperplastic prostate (13), prostatic adenocarcinoma (22), and prostatic adenocarcinoma lymph node metastases (two) were tested with pAB 60. Ten formalin-fixed, paraffin-embedded specimens of prostate adenocarcinoma, 11 frozen sections of benign hyperplastic specimens, and eight frozen sections of prostate adenocarcinoma were tested with mAB-1. Our results demonstrated consistent detection of c- erbB-2 immunohistochemically in frozen sections of both benign and malignant prostate. Preincubation of pAB 60 with the immunizing peptide blocked subsequent reactivity with prostatic tumor tissue, indicating specificity. However, fixation and processing protocols significantly affected the reactivity of the antigenic determinants detected by these antibodies, as mAB-1 was nonreactive with formalin-fixed, paraffin-embedded prostatic tissues. Differential reactivity of pAB 60 with malignant rather than benign glands was maximized by exposure of the specimen to the antibody at 4°C rather than 22°C. The most frequently observed staining pattern with both antibodies was cytoplasmic. However, mAB-1 produced distinctly membranous staining in two frozen specimens of benign hyperplasia and one specimen of prostate cancer.

Species‐ and organ‐specificity of secretory proteins derived from human prostate and seminal vesicles

Polyclonal antibodies against semenogelin (SG) isolated from human seminal vesicle secretion and acid phosphatase (PAP), beta-microseminoprotein (beta-MSP), and Prostate-Specific Antigen (PSA) derived from human prostatic fluid, as well as a monoclonal antibody against beta-MSP were used for immunocytochemical detection of the respective antigens in different organs from different species. SG immunoreactivity was detected in the epithelium of the pubertal and adult human and in monkey seminal vesicle, ampulla of the vas deferens, and ejaculatory duct. PAP, beta-MSP, and PSA immunoreactivities were detected in the pubertal and adult human prostate and the cranial and caudal monkey prostate. With the exception of a weak PSA immunoreactivity in the proximal portions of the ejaculatory duct, none of the latter antisera reacted with seminal vesicle, ampullary, and ejaculatory duct epithelium. Among the non-primate species studied (dog, bull, rat, guinea pig) only the canine prostatic epithelium displayed a definite immunoreactivity with the PAP antibody and a moderate reaction with the PSA antibody. No immunoreaction was seen in bull and rat seminal vesicle and canine ampulla of the vas deferens with the SG antibody. The same was true for the (ventral) prostate of rat, bull, and dog for beta-MSP. The epithelium of the rat dorsal prostate showed a slight cross-reactivity with the monoclonal antibody against beta-MSP and one polyclonal antibody against PSA. The findings indicate a rather strict species-dependent expression of human seminal proteins which show some similarities in primates, but only marginal relationship to species with different physiology of seminal fluid.

L-Leucyl β-Naphtylamidase Activity and Differentiation of Adult Human Prostate

The histochemical method for the demonstration of the L-leucyl-beta-naphtylamidase (LNAase) activity has been applied to bioptic and autoptic human prostates from very early to adult age to evaluate any correlation between sexual maturity and the appearance of this enzyme. The histochemical results indicate that in the human prostate, the LNAase activity is present at a cellular level in intrauterine life, infancy, and adulthood. This suggests that the appearance of this enzyme is genetically controlled even if different hormonal stimuli play some role in its localization and distribution.

Clonal growth of human prostatic epithelial cells is stimulated by fibroblasts

The interaction of fibroblasts with adult human prostatic epithelial cells was studied in vitro. Stimulation of epithelial cell proliferation, measured by clonal growth assay, was demonstrated when prostatic epithelial cells were grown in coculture with fibroblasts. Epithelial growth in cocultures with fibroblasts was greater than could be obtained in isolated culture in an "optimized," serum-free medium previously described. Fibroblasts were able to compensate for the deletion of several growth factors from this "optimized" medium, including epidermal growth factor and insulin, but were notably unable to replace bovine pituitary extract. Epithelial growth stimulation identical to that achieved in coculture was produced by medium conditioned by fibroblasts. Similar results were obtained by using fibroblasts of adult human prostate origin, adult human skin, human fetal lung fibroblasts (IMR-90), and mouse 3T3 cells. No difference in response was demonstrated between prostatic epithelial cells derived from normal or malignant tissues. Fixed fibroblast monolayers and extracellular matrix prepared from fibroblast cultures failed to stimulate prostatic epithelial growth. These results suggest that a soluble growth factor is secreted by prostatic fibroblasts and other human fibroblasts of nonprostatic origin, as well as by embryonic mesenchymal cells from nonhuman species, which is capable of producing a marked proliferative response in vitro from adult human prostatic epithelium. This proliferative response could not be reproduced by the addition of a variety of known growth factors to prostatic epithelial cell cultures.

Regional distribution of opioidergic nerves in human and canine prostates.

The regional distribution of opioidergic nerves in the juvenile and adult human prostate and in the adult canine prostate has been studied immunohistochemically using well-characterized polyclonal antisera against multiple opioid peptides. Nerves displaying immunoreactivity (ir) for the proenkephalin (PRO-ENK) derivatives met-enkephalin (ME), leuenkephalin (LE), octapeptide, and heptapeptide (ordered in decreasing frequency) were present in the dorsolateral stroma of human prostate. In canine prostate, the situation was similar, but the number of opioid-ir nerve fibers was lower than in human prostate. In both species, staining for the prodynorphin (PRO-DYN) derivatives dynorphin A and alpha-neoendorphin or staining for the pro-opiomelanocortin (POMC)-derived beta-endorphin was not visualized. In addition to their presence in nerve fibers, PRO-ENK-ir opioids (octapeptide) occurred in intrinsic ganglionic cells situated in the capsule. Octapeptide but not LE-ir fibers supplied stromal blood vessels. The periurethral region and tissue adjacent to the ejaculatory ducts appeared to be devoid of opioid-ir innervation. We conclude that endogenous opioids apparently exclusively derived from PRO-ENK may fulfill important comessenger functions in the fine regulation of prostatic stromal tonus and of local vascular perfusion.

Age-dependency and regional distribution of enkephalinergic nerves in human prostate.

The regional distribution of peptidergic nerves in the juvenile and adult human prostate has been studied immunohistochemically using highly specific polyclonal antibodies. Nerves displaying immunoreactivity of Met-enkephalin, Leu-enkephalin, heptapeptide and octapeptide were detected in the dorsolateral stroma closely related to smooth muscle predominantly of juvenile glands. A less strong immunoreactivity was present around stromal vessels and periurethral. Subepithelial peri-acinar nerves showing Leu-enkephalin, Met-enkephalin, octa- and heptapeptide immunoreactivity were found in a few instances. The most prominent immunoreaction was achieved with octapeptide antiserum, followed in decreasing order by Met-enkephalin, Leu-enkephalin and heptapeptide. Their density decreased with age. Antisera against beta-endorphin, alpha-neoendorphin, beta-neoendorphin and dynorphin A gave to positive immunoreaction with prostatic nerves. The regional distribution pattern described is in favor of a postsynaptic peptidergic sympathetic innervation of prostatic stroma acting with classical neurotransmitters.

The Inability of Adrenal Androgens to Stimulate the Adult Human Prostate: An Autopsy Evaluation of Men with Hypogonadotropic Hypogonadism and Panhypopituitarism

Although it is well recognized that testicular androgens provide the major hormonal stimulus to the prostate the role of adrenal androgens is less certain. Recently, it has been suggested that total androgen ablation (adrenal and testicular) is necessary to achieve a complete response to hormonal therapy in men with carcinoma of the prostate. In an effort to elucidate the influence of adrenal androgens on prostatic growth, we reviewed autopsy findings in patients (mean age 65 years, with a range of 57 to 80 years) from 3 distinct groups: group 1 consisted of 4 men with neither adrenal nor testicular function since before puberty (panhypopituitarism), group 2 included 4 men with normal adrenal glands but no testicular function throughout life (hypogonadotropic hypogonadism or prepubertal castration) and group 3 consisted of 8 age-matched controls with normal adrenal glands and normal testes. To assess the degree of androgenic stimulation each prostate was examined by a genitourinary pathologist and was analyzed morphometrically with respect to epithelial cell height, epithelial cell height:nuclear height ratio, acinar area and short axis diameter of randomly selected acini.All prostates in groups 1 and 2 were atrophic histologically, while all prostates in group 3 were enlarged and demonstrated varying degrees of benign prostatic hyperplasia. Morphometric analysis of the 3 groups revealed the same results. For all 4 parameters studied morphometrically there was no statistical difference between groups 1 and 2. However, a statistical difference did exist for these criteria when either group 1 (p less than 0.0001) or group 2 (p less than 0.0001) was compared to group 3. These findings suggest that normal adrenal glands do not have a significant stimulatory effect on the human prostate and that they are not capable of supporting prostatic growth.

Serial culture of adult human prostatic epithelial cells

Techniques for the isolation, establishment, and subculture of normal, benign hyperplastic and malignant epithelial cell cultures from adult human prostates are described. Acini are released from tissues by collagenase digestion, and primary and subcultures are grown in collagen-coated dishes containing medium PFMR-4A supplemented with 1% serum and additional factors. Growth assays can be performed in serum-free medium. Verification of the cultures as prostatic epithelial cells is accomplished by indirect immunofluorescence detection of keratin, prostate-specific antigen, and prostatic acid phosphatase.

Enzyme histochemical profile of the adult human female prostate (paraurethral glands).

Enzyme histochemical profile of the adult human female prostate (paraurethral glands).

A comparative study of the ultrastructure and lack of growth capacity of adult human prostate epithelium mechanically separated from its stroma.

A comparative study of the ultrastructure and lack of growth capacity of adult human prostate epithelium mechanically separated from its stroma.

SERUM "ACID" PHOSPHATASE IN PATIENTS WITH CARCINOMA OF THE PROSTATE GLAND

While investigating the source of acid phosphatases in urine,1Kutscher and Wolbergs in 1935 discovered2that normal human prostate tissue is extremely rich in an acid phosphatase. This observation did not appear at first to have any special significance, since there are many phosphatases and they are widely distributed in animal and plant tissues. Further study of the enzyme, however, led to interesting developments, among them a chemical method for the diagnosis of metastasizing prostate carcinoma, and the introduction by Huggins in 1941 of androgen control, an important new principle in the treatment of prostate carcinoma. PROSTATIC ACID PHOSPHATASE AND PROSTATIC FUNCTION IN MAN Acid phosphatase occurs in and is apparently elaborated by the acinar epithelium of the prostate gland, as Gomori3recently demonstrated by a special histochemical technic. The concentration of the enzyme in adult human prostate tissue is extraordinarily high (500 to 2.500 units activity per gram of fresh tissue), many hundred times that of any phosphatase in kidney,