Androgen-responsive Prostate Carcinoma Cell Line Research Articles

Insulin-like growth factor binding protein-3 is regulated by dihydrotestosterone and stimulates deoxyribonucleic acid synthesis and cell proliferation in LNCaP prostate carcinoma cells.

We have investigated the production and actions of a growth regulatory protein, insulin-like growth factor binding protein (IGFBP)-3, in the androgen-responsive prostate carcinoma cell line LNCaP. Confluent monolayers of cells secreted approximately 0.7 ng/ml IGFBP-3 over 24 h. Dihydrotestosterone (DHT, 10 nM) and 1,25-dihydroxyvitamin D3 (vitamin D, 10 nM) increased IGFBP-3 in media to 149 +/- 15% and 206 +/- 18% of control, respectively, when added separately, and to 453 +/- 28% of control when used in combination. IGFBP-2, secreted at approximately 25-fold higher concentrations than IGFBP-3, was increased 50% by 10 nM DHT, but there was no effect of vitamin D on IGFBP-2 production in the absence or presence of DHT. Cell-associated IGFBP-3, and immunoreactive IGFBP-3 species of 20 kDa and 30 kDa were also increased in response to vitamin D plus DHT. A combination of vitamin D and DHT increased DNA synthesis in LNCaP cells 3-fold, and this was at least partly mediated by endogenous IGFBP-3 because anti-IGFBP-3 IgG, but not nonimmune serum IgG, reduced the stimulatory effect of vitamin D and DHT from 293 +/- 11.6% to 161 +/- 30.7% of control levels (P < 0.0001). Basal and DHT plus vitamin D-stimulated thymidine incorporation was significantly increased by 50 ng/ml human plasma-derived purified IGFBP-3. After 4 days treatment with vitamin D plus DHT, or pure IGFBP-3, LNCaP cell numbers were increased relative to control. These results indicate a role for IGFBP-3 in the proliferation of androgen-responsive prostate carcinoma cells.

Insulin-Like Growth Factor Binding Protein-3 Is Regulated by Dihydrotestosterone and Stimulates Deoxyribonucleic Acid Synthesis and Cell Proliferation in LNCaP Prostate Carcinoma Cells

We have investigated the production and actions of a growth regulatory protein, insulin-like growth factor binding protein (IGFBP)-3, in the androgen-responsive prostate carcinoma cell line LNCaP. Confluent monolayers of cells secreted approximately 0.7 ng/ml IGFBP-3 over 24 h. Dihydrotestosterone (DHT, 10 nm) and 1,25-dihydroxyvitamin D3 (vitamin D, 10 nm) increased IGFBP-3 in media to 149 ± 15% and 206 ± 18% of control, respectively, when added separately, and to 453 ± 28% of control when used in combination. IGFBP-2, secreted at approximately 25-fold higher concentrations than IGFBP-3, was increased 50% by 10 nm DHT, but there was no effect of vitamin D on IGFBP-2 production in the absence or presence of DHT. Cell-associated IGFBP-3, and immunoreactive IGFBP-3 species of 20 kDa and 30 kDa were also increased in response to vitamin D plus DHT. A combination of vitamin D and DHT increased DNA synthesis in LNCaP cells 3-fold, and this was at least partly mediated by endogenous IGFBP-3 because anti-IGFBP-3 IgG, but not nonimmune serum IgG, reduced the stimulatory effect of vitamin D and DHT from 293 ± 11.6% to 161 ± 30.7% of control levels (P < 0.0001). Basal and DHT plus vitamin D-stimulated thymidine incorporation was significantly increased by 50 ng/ml human plasma-derived purified IGFBP-3. After 4 days treatment with vitamin D plus DHT, or pure IGFBP-3, LNCaP cell numbers were increased relative to control. These results indicate a role for IGFBP-3 in the proliferation of androgen-responsive prostate carcinoma cells.

Autocrine effect of androgen on proliferation of an androgen responsive prostatic carcinoma cell line, LNCAP: role of interleukin-6.

LNCaP is an androgen-responsive prostatic carcinoma cell line that exhibits a bell-shaped growth response curve to increasing doses of dihydrotestosterone (DHT) in culture. Although the precise mechanism responsible for this unique growth response to androgen stimulation remains unclear, many studies have suggested that androgen modulates the level of various growth factors. In an early study, we demonstrated that LNCaP proliferation was stimulated by interleukin (IL)-6 in a paracrine manner, because these cells did not express a significant amount of IL-6. In the present study, the role of IL-6 in mediating androgen regulated proliferation in LNCaP cells was investigated. DHT, at increasing doses up to 10(-8) M, resulted in a release of IL-6 from LNCaP cells. This dose-dependent effect of DHT on LNCaP proliferation could be partially inhibited by the addition of antibody against IL-6 into the culture medium. These results indicate that the DHT-induced expression of IL-6 stimulates proliferation of LNCaP cells in culture in an autocrine manner.

Characterization of an early growth response gene, which encodes a zinc finger transcription factor, potentially involved in cell cycle regulation.

Using differential display polymerase chain reaction, early growth response gene alpha (EGR alpha) was first isolated as a 291-base pair 3'-cDNA clone, which was highly expressed in the androgen-independent prostate carcinoma cell lines PC3 and DU145, as compared with the androgen-responsive prostate carcinoma cell line LNCaP. Full length cloning of the EGR alpha coding region revealed that EGR alpha was a new member of an important subfamily of nuclear zinc finger transcription factors (others members e.g. Sp1, EGR-2, and Wilms' tumor gene). Moreover, it was observed that EGR alpha, as with most Sp1 subfamily members, was conserved between mammalian species ranging from human to rabbit. Two hormones important for prostate development and differentiation were found to be potent regulators of EGR alpha mRNA expression. Androgens were observed to induce a down-regulation of EGR alpha mRNA expression (70% in 72 h), while epidermal growth factor induced a rapid transient up-regulation (6-fold in 100 min). The up-regulation was controlled at the transcriptional level and effectively blocked by staurosporine (which suggests the involvement of the protein kinase C pathway). Functional analysis demonstrated that EGR alpha could bind to, and stimulate transcription from, a basic transcription element (BTE) consensus sequence on DNA (BTE is a transcription-modulating sequence in the promoter region of some cytochrome P450 family members). Furthermore, in stage-synchronized prostate cells, EGR alpha mRNA was highly expressed in the early G1 phase of the cell cycle, similar to c-fos mRNA expression. These results indicated that the zinc finger transcription factor EGR alpha seems to play a role in cell cycle regulation.

Effect of Retinoic Acid on the Proliferation and Secretory Activity of Androgen-Responsive Prostatic Carcinoma Cells

We studied the effect of retinoic acid on the growth and secretory activity of the androgen-responsive prostatic carcinoma cell line LNCaP. Our data showed that retinoic acid at 0.01μM. stimulated the proliferation of LNCaP cells but inhibited their growth at 0.1μM. under androgen-free conditions. In the presence of 0.1nM. dihydrotestosterone (DHT), LNCaP cell proliferation was inhibited by 10μM. retinoic acid but not by lower concentrations of retinoic acid. Retinoic acid reduced LNCaP cell growth at concentrations of 0.1μM. in the presence of 10nM. DHT. Retinoic acid (10μM.) also reduced the growth response of LNCaP cells to epidermal growth factor and transforming growth factor alpha and potentiated the inhibitory effect of transforming growth factor beta. In additional studies, retinoic acid induced a dose-dependent increase in prostate specific antigen (PSA) secretion at concentrations of 0.1 to 1μM. Dihydrotestosterone (10nM.) also enhanced the secretion of PSA by LNCaP cells, and this effect was potentiated in a dose-dependent fashion by the addition of retinoic acid at 0.1–10μM. Competitive binding studies showed that retinoic acid did not bind to androgen receptors. Overall, retinoic acid had a biphasic effect on LNCaP proliferation and promoted the secretion of PSA. The biphasic effect of retinoic acid on LNCaP growth should be considered in designing in vivo studies to determine the impact of retinoic acid on solid prostatic tumor growth. In addition, the ability of retinoic acid to increase PSA secretion may complicate the interpretation of serum PSA levels used for diagnostic and prognostic purposes.

Expression of mRNA for epidermal growth factor, transforming growth factor-alpha and their receptor in human prostate tissue and cell lines

Enhanced expression of the epidermal growth factor receptor (EGFR) or its ligands, epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha) can increase signalling via receptor-mediated pathways which may lead to excessive proliferation and cellular transformation. Such autocrine regulation of growth has been demonstrated for prostate cancer cell lines in culture but its role in prostate cancer in vivo has not been established. To assess the potential of such a mechanism, we have examined the pathway components in prostate carcinomas (CaP) in comparison with non-malignant benign prostatic hyperplasias (BPH). In the present study, we investigate the dosage, structure and expression of EGF, TGF-alpha and EGFR genes in a series of 34 human prostate samples and 3 prostate cancer cell lines. All of the samples contained transcripts from each of the genes. The expression of pre-pro-TGF-alpha mRNA and pre-pro-EGF mRNA were significantly higher in CaP (n = 13) than BPH (n = 21) specimens (p < 0.05). The androgen-responsive prostatic carcinoma cell line, LNCaP, expressed high levels of EGF mRNA while the androgen-independent DU145 and PC-3 cell lines expressed high levels of TGF-alpha mRNA and EGFR mRNA. In general, overexpression of these mRNAs was not associated with amplification or detectable gene rearrangement; only DU145 cells demonstrated any alteration in these genes, with apparent amplification of the TGF-alpha gene. Relative to BPH, all prostate carcinomas and cell lines studied had elevated levels of mRNA for one or both mRNA coding for the ligands for EGFR.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of prostatic carcinoma cell proliferation and secretory activity by extracellular matrix and stromal secretions

Previous studies from our laboratory have shown that reconstituted basement membrane and stromal secretory products are important regulators of benign prostatic epithelial cell growth and differentiation. In the present study we evaluated the impact of extracellular matrix (ECM) and soluble stromal secretory products on the proliferation and secretory activity of the androgen-responsive prostatic carcinoma cell line LNCaP. In these studies, dihydrotestosterone (DHT) was a potent mitogen for LNCaP cells cultured on plastic or on type I collagen. The growth response to DHT was greatly attenuated when LNCaP cells were grown on prostatic stromal ECM. Cells grown on stromal ECM also exhibited clustered morphology compared to the monolayer growth observed on plastic and secreted elevated levels of prostate specific antigen (PSA) and prostatic acid phosphatase (PAP). These findings indicate that cultivation of LNCaP on stromal ECM will promote the expression of differentiated functions. In additional studies, stromal cell conditioned medium (SCM) significantly increased PSA/PAP secretion by LNCaP cells in the presence of 10 nM DHT. The enhancement of DHT-induced PSA/PAP secretion by SCM was most pronounced when LNCaP cells were grown on stromal ECM. SCM did not significantly alter LNCaP proliferation. These studies indicate that prostatic stromal ECM and soluble secretory products will promote differentiated function in cultured LNCaP cells. In addition, we show that DHT can act as either a growth or differentiation-promoting stimulus depending on the presence of stromal factors.