Growth regulation of prostatic stromal cells by prostate-specific antigen.

BACKGROUND: Prostate-specific antigen (PSA) is a serine protease that can cleave insulin-like growth factor-binding protein-3 (IGFBP3), thereby decreasing its affinity for insulin-like growth factor-I (IGF-I). Dissociation of the IGF-I-IGFBP3 complex renders IGF-I available to bind to its receptor and stimulates cellular proliferation. We evaluated the potential for PSA to modulate the effects of IGF-I and IGFBP3 on the proliferation of human benign prostatic hyperplasia (BPH)-derived fibromuscular stromal cells in primary cultures. METHODS: We cultured BPH-derived stromal cells for 48 hours in serum-free RPMI-1640 medium supplemented with 0.2% bovine serum albumin and studied the effects of IGF-I, IGFBP3, PSA, and ZnCl(2) at varying concentrations. Differences in cell growth between control and treated cultures were evaluated by use of Dunnett's test. Concentration-related trends were evaluated by linear regression of log-transformed concentrations of test reagents on BPH-derived stromal cell number responses. Statistical tests were two-sided. RESULTS: We observed a concentration-dependent proliferative response of BPH-derived stromal cells to IGF-I. IGFBP3 inhibited this response in a concentration-dependent fashion. IGFBP3 alone had no effect on stromal cell proliferation. When stromal cells were incubated with PSA alone or with PSA, IGF-I, and IGFBP3, an increase in stromal cell numbers that was dependent on PSA concentration was evident in both instances. Zinc, an endogenous inhibitor of PSA enzymatic activity, was able to attenuate the stimulatory effect of PSA at intraprostatic physiologic concentrations. CONCLUSIONS: These results are consistent with the idea that PSA can modulate in vitro interactions between IGF-I and IGFBP3 and suggest that PSA may play a role in the regulation of human prostatic fibromuscular cell growth.     

Inhibition of tumorigenic potential and prostate-specific antigen expression in LNCaP human prostate cancer cell line by 13-cis-retinoic acid

Prostate-specific antigen (PSA) is a member of the kallikrein family and has been an important biological marker for prostate cancer. The mechanisms regulating PSA expression in prostatic cancer cells are unclear. The present study was designed to elucidate the role of 13-cis-retinoic acid (RA) in regulation of PSA and the tumorigenic potential of the human prostate cancer cell line LNCaP. The growth regulation of LNCaP cells was examined by DNA synthesis and doubling time. The tumorigenic potential of prostate cancer cells was analyzed by soft agar colony-forming assay, in vitro invasion assay, type IV collagenase assay and binding to extracellular matrix assay. The nuclear receptors for retinoic acid (RARα, -β, -γ and RXRα, -β,-γ) as well as PSA mRNA were determined by Northern blot using specific oligonucleotide probes. Our results suggest that 13-cis-RA significantly inhibits PSA secretion and expression both at the mRNA and protein levels compared with untreated cells. Electron microscopic studies suggest that after 13-cis-RA treatment, cells become more differentiated as they contain lumina, lined by plasma membrane and microvilli. Prostate cancer cell growth and tumorigenic potential after 13-cis-RA treatment was significantly decreased compared with controls. Nude mice tumorigenicity studies showed that 13-cis-RA-treated cells produced significantly smaller tumors compared with untreated cell tumors. There was also a significant increase in the expression of RXRa mRNA after 13-cis-RA treatment compared with untreated cells.     

Identification of differentially expressed genes during cyclic adenosine monophosphate-induced neuroendocrine differentiation in the human prostatic adenocarcinoma cell line LNCaP

The LNCaP cell line is a versatile and useful model suitable for the study of human prostate cancer in vitro. It has been determined that the elevation of LNCaP intracellular cyclic adenosine monophosphate (cAMP) levels through the addition of membrane-permeable cAMP analogs, phosphodiesterase inhibitors, adenylate cyclase activators, or components of the cAMP signal-transduction pathway can induce reversible neuroendocrine (NE) differentiation. Elucidation of those genes that are differentially expressed between undifferentiated prostate cancer cells and prostate cancer cells that have been induced to differentiate may present new insights into the molecular mechanisms governing NE differentiation, early detection of prostate cancer, and potential targets for gene therapy. In this study, differential display polymerase chain reaction (PCR) was used to identify 226 differentially expressed PCR products. Twelve of the differential display PCR products were confirmed by Northern blot analysis and were cloned. DNA sequencing and database comparisons were performed. To our knowledge, this is the first report of the use of differential gene expression techniques to analyze gene expression during cAMP-induced NE differentiation in LNCaP cells. Confirmation of NE differentiation reversibility also was accomplished.     

TRAIL-DISC formation is androgen-dependent in the human prostatic carcinoma cell line LNCaP

We and others have previously described that the androgen-responsive human prostatic carcinoma cell line LNCaP is resistant to TRAIL and that TRAIL-mediated apoptosis in LNCaP is PI3K/Akt-dependent. In this study, we found that LNCaP remained resistant to treatment with TRAIL after androgen deprivation even in the presence of the PI3K/Akt pathway inhibitor wortmannin. This resistance was determined by failure to form the TRAIL-DISC and by decreased TRAIL-R1 and TRAIL-R2 levels after androgen deprivation; the capacity of TRAIL to induce DISC formation was completely restored in the presence of DHT. TRAIL and wortmannin together accelerated processing of caspase-8 on the DISC and apparently the release of caspase-8 from the DISC into the cytoplasm. Surprisingly, we found that wortmannin decreased the total amount of TRAIL-R1, but not TRAIL-R2, in the cells as well as the amount of TRAIL-R1 precipitated by TRAIL. Our data suggest that TRAIL-DISC formation and sensitivity to TRAIL treatment are androgen-dependent in LNCaP.     

Effects of soybean isoflavones on cell growth and apoptosis of the human prostatic cancer cell line LNCaP

BACKGROUND: Epidemiological studies have suggested that soybean isoflavones are associated with a lower risk of prostate cancer. However, the mechanisms of prostate cancer prevention by soybean isoflavones have yet to be fully clarified. METHODS: Two soybean isoflavones (genistein and daidzein) and their glucosides (genistin and daidzin) were tested for their effects on cell growth and apoptosis of the LNCaP human prostatic cancer cell line. RESULTS: Among these isoflavones, genistein was found to inhibit the growth of LNCaP most effectively, with an IC50 value of 40 microM. The inhibition of cell growth by genistein was accompanied by the suppression of DNA synthesis and the induction of apoptosis. Expression of prostate-specific antigen (PSA) in LNCaP was also significantly reduced by the treatment with genistein. CONCLUSIONS: The results suggest that genistein might primarily influence human prostate cancer development by reducing tumor growth.      

Tissue concentrations of prostate-specific antigen in prostatic carcinoma and benign prostatic hyperplasia.

Prostate-specific antigen (PSA), as measured in peripheral blood, is currently the most widely used marker for the assessment of tumor burden in the longitudinal study of patients with carcinoma of the prostate (PCA). Studies from other laboratories have led to the conclusion that a given volume of PCA causes a much higher level of PSA in the peripheral circulation of patients than a similar volume of prostate without carcinoma. We have evaluated PSA in the resected tissues immunohistochemically and in extracts of PCA and of prostates resected because of benign prostatic hyperplasia (BPH) with an enzyme-linked immunosorbent assay. Immunohistochemical results were less quantitative than but consistent with the results of the ELISA of tissue extracts. Immunohistochemically, there was considerable heterogeneity in the expression of PSA by both PCA and BPH both within and among prostatic tissues from different patients. While the levels of expression of PSA in these tissues overlap broadly, PSA is expressed at a lower level in PCA than in BPH when PSA is expressed as a function of wet weight of tissue (p = 0.0095), wet weight of tissue/% epithelium (p less than 0.0001), protein extracted from the tissue (p = 0.0039), or protein extracted/% epithelium (p less than 0.0001).     

p53 is involved in tumor necrosis factor-alpha-induced apoptosis in the human prostatic carcinoma cell line LNCaP

The human prostatic carcinoma cell line LNCaP is sensitive to TNF-alpha treatment and expresses wild-type p53. To analyse the possible role of p53 in TNF-alpha-mediated apoptosis, we generated a derivative of LNCaP, LN-56, expressing a dominant-negative element of p53, GSE56. P53 inactivation in LN-56 was associated with an increased resistance to apoptosis induced by TNF-alpha. Surface expression of TNF-alpha receptors was unchanged in LN-56 compared to LNCaP. TNF-alpha treatment resulted in accumulation of p53 in LNCaP and upregulation of p21/WAF1. Activation of caspase-7 and PARP proteolysis were delayed in LN-56 under TNF-alpha treatment. TNF-alpha-induced apoptosis in LNCaP cells was accompanied by caspase-dependent proteolysis of p21/WAF1 and Rb, which was significantly attenuated in LN-56. Cytochrome c release was induced by TNF-alpha treatment in both cell lines, but caspase-9 was not activated. LNCaP and LN-56 were injected s.c. in nude mice and tumors were identified in all LN-56, but not LNCaP, bearing mice indicating that p53 plays an important role in growth control of prostatic neoplasms. Interestingly, accumulation of p53 in TNF-alpha-treated LNCaP cells was decreased in the presence of the caspase inhibitor Z-VAD-FMK, suggesting a new role of activated caspases in acceleration of p53 response. In summary, these results indicate that p53 is involved in TNF-alpha-mediated apoptosis in LNCaP.     

Clinical usefulness of prostate-specific antigen and prostatic acid phosphatase in patients with prostatic cancer.

Serum prostate-specific antigen (PSA) and prostatic acid phosphatase (PAP) levels were measured in 70 patients with benign prostatic hypertrophy (BPH) and in 70 patients with prostatic cancer. PSA was increased above the cutoff level of 10 ng/ml in 13% of patients with BPH and in 87% of patients with prostatic cancer. In contrast, abnormal PAP levels were found in 14 and 76% of patients, respectively. We concluded that, due to its high specificity, PSA is a useful marker in the management of patients with prostatic carcinoma and that it surpasses PAP in this regard.     

LNCaP model of human prostatic carcinoma

The LNCaP cell line was established from a metastatic lesion of human prostatic adenocarcinoma. The LNCaP cells grow readily in vitro (up to 8 x 10(5) cells/sq cm; doubling time, 60 hr), form clones in semisolid media, are highly resistant to human fibroblast interferon, and show an aneuploid (modal number, 76 to 91) human male karyotype with several marker chromosomes. The malignant properties of LNCaP cells are maintained. Athymic nude mice develop tumors at the injection site (volume-doubling time, 86 hr). Functional differentiation is preserved; both cultures and tumor produce acid phosphatase. High-affinity specific androgen receptors are present in the cytosol and nuclear fractions of cells in culture and in tumors. Estrogen receptors are demonstrable in the cytosol. The model is hormonally responsive. In vitro, 5 alpha-dihydrotestosterone modulates cell growth and stimulates acid phosphatase production. In vivo, the frequency of tumor development and the mean time of tumor appearance are significantly different for either sex. Male mice develop tumors earlier and at a greater frequency than do females. Hormonal manipulations show that, regardless of sex, the frequency of tumor development correlates with serum androgen levels. The rate of the tumor growth, however, is independent of the gender of hormonal status of the host.     

Differential diagnostic value of prostate-specific antigen in prostatic hyperplasia

Diagnostic value of prostate specific antigen (PSA) was studied in 163 patients with prostatic hyperplasia. As PSA is not a cancer-specific test, it is recommended to obtain biopsy in patients with prostatic hyperplasia and PSA > 4 ng/ml to diagnose prostatic cancer early. Methods raising PSA specificity for differential diagnosis of prostatic hyperplasia are not absolutely reliable.     

A human gallbladder adenocarcinoma cell line

A cell strain (FU-GBC-1) was established from cancerous ascites of a 68-year-old male patient with well-differentiated adenocarcinoma of the gallbladder. By light and electron microscopy, the cultured cells showed the morphologic features of adenocarcinoma characterized by gland-like structures, intracellular microcystic spaces, and mucous production. Immunoperoxidase stains showed that FU-GBC-1 cells expressed several epithelial tumor antigens including CA 19-9, carcinoembryonic antigen (CEA), and epithelial membrane antigen (EMA). The cell strain has been in continuous culture up to passage 44 for 1 1/2 years, with the population doubling time of 120 hours. The cytogenetic analysis by a G-band technique showed a constant loss of chromosome Y in FU-GBC-1 cells. The modal chromosome number at passage 12 was 82 with a range of 77 to 85. Flow cytometry with an ethidium bromide technique additionally confirmed aneuploid DNA content (4C) in the cultured cells at passage 12 and 35. Inoculation of FU-GBC-1 cells into the dermis of BALB/c nude mice produced transplantable adenocarcinoma identical to the original tumor. Because no continuous cell lines of the well-differentiated type of gallbladder adenocarcinoma have been reported in the literature currently, the newly established cell strain we report may yield a useful system for studying the morphologic and biologic characteristics of gallbladder adenocarcinoma.     

Malignant transformation in a nontumorigenic human prostatic epithelial cell line.

The human prostatic epithelial cell line BPH-1 is normally nontumorigenic in nude mice. The present report demonstrates that this cell line can be permanently transformed by its microenvironment to become tumorigenic. The establishment of a series of tumorigenic sublines based on this parental cell line is described. BPH-1 cells were induced to form tumors either by recombination with human prostatic carcinoma-associated fibroblasts (CAFs) or by exposure to carcinogenic doses of testosterone and estradiol (T+E2) after recombination with rat urogenital sinus mesenchyme. Epithelial cells isolated from these tumors were established as cell strains in culture. When regrafted to nude mouse hosts epithelial cells isolated from CAF- or T+E2-induced tumors were found to be consistently tumorigenic even in the absence of CAF or T+E2. The T+E2-induced cell strains have been designated BPH1(TETD)-A and -B and the CAF-induced strains are designated BPH1(CAFTD)-01 through -08. In vitro, the cells had an epithelial morphology with a less well-defined cobblestone pattern than the parental line. They express SV40 large T antigen, confirming their derivation from the parental BPH-1 line. The BPH1(CAFTD) strains formed colonies in soft agar, whereas the parental BPH-1 cells and the BPH1(TETD) sublines did not. There was no immunocytochemically detectable expression of androgen (AR), alpha-estrogen (ERalpha), or progesterone (PR) receptors by the parental BPH-1 cell line or by any of the tumor-derived cell strains. The cells uniformly coexpressed both basal and luminal cell-type cytokeratins and the basal cell marker p63. When grafted beneath the renal capsule of athymic mouse hosts, all of the tumor-derived cell strains consistently formed tumors. These were predominantly poorly or moderately differentiated squamous or adenosquamous tumors, similar in organization to the primary tumors from which the cell strains were derived. The cell strains continued to express both basal- and luminal-type cytokeratins in vivo. Some of the cell strains also coexpressed vimentin. E-cadherin expression was absent from many of the cells, although patches of cells expressing this marker were seen. The cells continued to express SV40T antigen. These cell strains, which are all derived from a common nontumorigenic progenitor, represent a useful resource for examining genetic and phenotypic changes during carcinogenesis.     

The human prostatic carcinoma cell line LNCaP expresses biologically active, specific receptors for 1 alpha,25-dihydroxyvitamin D3.

The LNCaP prostatic carcinoma cell line was examined for the presence of specific receptors for 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3]. Whole cell binding studies identified approximately 2500 high-affinity (Kd = 1.4 x 10(-9) binding sites per cell. Competition studies revealed that these receptors are specific for the 1 alpha,25(OH)2 metabolite. Binding studies using the synthetic androgen R1881 indicate that separate androgen and vitamin D3 receptors exist in LNCaP cells. The vitamin D3 receptors sediment at approximately 3.5S on linear sucrose gradients. The sedimentation coefficient could be shifted with a monoclonal anti-vitamin D3 receptor antibody (9A7 gamma) but not with a monoclonal antibody to the androgen receptor (AN1-15). The receptor/ligand complex elutes from native DNA cellulose at 0.2 M KCl. Northern blot analysis identified an mRNA of approximately 4.6 kilobases which hybridized with a specific vitamin D3 receptor complementary DNA probe (hVDR). In the absence of androgens, 1 alpha,25(OH)2D3 stimulated growth and prostate-specific antigen production by LNCaP cells in a dose-dependent fashion. Dose-response curves indicated that at physiological concentrations (10(-9) M) 1 alpha,25(OH)2D3 was mitogenic, whereas at higher concentrations (10(-8) M) it promotes differentiation. These studies suggest that 1 alpha,25(OH)2D3 could play an important role in the natural history of and response to hormone therapy by prostatic cancer.     

Synthetic androgens suppress the transformed phenotype in the human prostate carcinoma cell line LNCaP.

Experiments have been designed to investigate hormonal effects on the human prostatic carcinoma cell line LNCaP in the presence of complete foetal calf serum. At physiological concentrations (3.3 x 10(-9)M), several derivatives of 17 alpha-methyl-testosterone led to a significant reduction of cell proliferation, inhibition of colony formation in soft agar, change of morphology, induction of a prostate specific mRNA and down-regulation of c-myc RNA. Two different antiandrogens, hydroxyflutamide and cyproterone acetate, were capable of reversing the effects exerted by the synthetic androgens on growth properties. The proliferation rate of control cells devoid of androgen receptor was not inhibited by synthetic androgens. Our results indicate that the cellular androgen response mechanism of LNCaP cells is intact and that synthetic androgens elicit androgen receptor mediated suppression of the transformed phenotype. Rare cases of remission of prostatic cancer on androgen treatment have been reported. LNCaP cells may be a model of an uncommon class of prostatic cancer which responds favourably to androgen treatment.     

Steroid hormone regulation of prostate-specific antigen gene expression in breast cancer.

We have recently reported that about 30-40% of female breast tumours produce prostate-specific antigen (PSA) and that PSA production is associated with the presence of oestrogen (ER) and progesterone (PR) receptors. We have now developed a tissue culture system to study the regulation of the PSA gene in breast cancer. The breast carcinoma cell line T-47D produces PSA when stimulated by androgens, progestins and glucocorticoids/mineralocorticoids but not oestrogens. PSA mRNA appears approximately 2 h after stimulation; PSA protein appears after 4-8 h. Among 38 compounds tested, only androgens and progestins were able to stimulate PSA production at concentrations below 10(-9) M. Evidence that the progesterone and androgen receptors can regulate the PSA gene independently was provided as follows: (a) the progestin norgestimate, which does not bind to the androgen receptor, up-regulates the PSA gene at concentrations as low as 10(-10) M; (b) triamicinolone acetonide, which does not bind to the androgen receptor (AR) but binds to the PR, acts similarly to norgestimate; (c) the antiandrogen cyproterone acetate, which blocks the androgen receptor but has progestational activity, up-regulates the PSA gene at concentrations as low as 10(-10) M; (d) the antiprogestine mifepristone completely blocks the stimulation of the specific progestin norgestimate. Our tissue culture system identified androgen-progestin agonist activities of 17 alpha-ethinyloestradiol, the antioestrogen RU56, 187 and the antiprogestin mifepristone. Our data suggest that the expression of the PSA gene in the female breast is under the control of androgens and progestins. Our tissue culture system is a highly sensitive in vitro method for evaluating the biological activity of candidate compounds having agonist and antagonist steroid hormone activity.     

Correlation of beta-actin messenger RNA localization with metastatic potential in rat adenocarcinoma cell lines

The actin cytoskeleton is involved in the motility of tumor cells. It has been shown in several cell types that beta-actin mRNA is localized in the protrusions of cells in which actin is actively polymerized, and the ability to localize mRNA is correlated with the efficiency of motility. In this context, we studied the distribution of beta-actin mRNA in two different tumor cell lines and correlated it with their metastatic potential. The two cell lines used were the highly metastatic MTLn3 cells and nonmetastatic MTC cells. Nonmetastatic MTC cells have two different pools of beta-actin mRNA (perinuclear and at the leading edge), whereas highly metastatic MTLn3 cells have only a perinuclear distribution of beta-actin mRNA. These differences in mRNA localization are correlated with profound differences in the polarity and plasticity of cell motility of these cells in culture and the histopathology of primary breast tumors derived from these cells. In particular, MTLn3 cells are unpolarized by all cell shape and motility criteria in culture and in their histopathological organization in primary tumors. By comparison, MTC cells are polarized in all identical measurements. These results suggest that the increased plasticity of cell locomotion and the invasiveness of MTLn3 cells result from the failure of metastatic cells to localize beta-actin mRNA properly, causing them to be less polarized and therefore more flexible in their direction of motility. Thus, differences in the polarized organization of cells in the primary tumor that are correlated with beta-actin mRNA localization may have prognostic value in predicting metastatic potential.     

Detection of tumor-associated antigen in human melanoma cell line supernatants.

Spent tissue culture medium (CDM-S) removed from a single cell line of human malignant melanoma grown in serum-free CDM, contained tumor-associated antigenic activity. Antibodies to CDM-S measured by complement fixation were detected in 44% (31/70) melanoma, 55% (15/27) sarcoma, 63% (24/38) carcinoma and 15% (11/72) normal sera. Delayed cutaneous hypersensitivity reactions (DCHR) were demonstrated in 4/5 melanoma patients at a 500 mug dose, 3/5 at a 100 mug dose and in 1/7 carcinoma patients at the 500 mug dose. One ml of CDM-S was shown to contain antigen equivalent to that obtained from the membranes of 2.9 X 10(7) tissue-cultured melanoma cells. After purification, 84% (16/19) sera from melanoma patients, 66% (12/18) from sarcoma and carcinoma patients and 8% (2/26) from normal controls were positive to the antigen by complement fixation.