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.
BACKGROUND.The human prostate carcinoma cell line, LNCaP, proliferates under stimulation by a limited number of mitogenic signals, which include members of the growth factor and steroid hormone families. Androgens and epidermal growth factor (EGF) are among the LNCaP cell mitogens. We tested the hypothesis that these mitogens stimulate LNCaP cell proliferation at least in part through the induction of cyclin D 1 , a protein requisite for cell cycle progression, which is expressed in the G 1 phase of the cell cycle. METHODS. LNCaP cells were grown in serum-free medium with 10 ng/ml or 100 ng/ml EGF, 0.1 nM or 1.0 nM mibolerone (a potent androgen agonist), or vehicle (distilled water or 0.01% ethanol). Expression of cyclin D, mRNA, and protein were assessed by Northern and Western blot analyses. Transcription regulation was assessed by nuclear runoff assay. RESULTS. Western analyses demonstrated that EGF stimulated cyclin D 1 protein expression 4-fold over 12 hr. Northern analyses showed a 4-fold increase in mRNA expression, peaking within 4 hr of EGF stimulation. There were no effects on cyclin D 1 protein or mRNA expression with mibolerone treatments. We further explored the mechanism of cyclin D 1 induction. LNCaP cells stimulated for 1 hr with EGF demonstrated a 2-fold increase in cyclin D 1 message, as assayed by nuclear runoff transcription assay. In addition, we demonstrated the involvement of the protein kinase C pathway in mediating the EGF induction of cyclin D 1 . CONCLUSIONS. We conclude that one of the mechanisms by which growth factors such as EGF may stimulate prostate cell proliferation is through the direct induction of cyclin proteins, which are necessary for entry of cells into mitosis.
Insulin-like growth factor I (IGF-I) seems to play an important role in prostate cell growth and its actions may be modified by IGF-binding proteins (IGFBPs) secreted by prostate epithelial cells. The IGFBP system was studied in two human prostate carcinoma cell lines, PC3 and LNCaP. Androgen receptor-negative PC3 cells secrete IGFBP-3, IGFBP-4, and IGFBP-5, as determined by immunoprecipitation of the serum-free conditioned medium with specific IGFBP antibodies. Androgen receptor-positive LNCaP cells secrete IGFBP-2 and IGFBP-3. At neutral pH, there was little or no effect of a 24-h, 37 C cell-free incubation of PC3 and LNCaP conditioned media on IGFBP. On the other hand, when media was incubated at pH 3 for 24 h, [125I]IGFBP-3 hydrolysis and the virtual elimination of endogenous IGFBP detected by Western ligand blotting were observed. This loss was not due to the acid treatment, per se, since IGFBPs remained intact if the incubation at pH 3 was carried out at 4 C. The acid-activated IGFBP protease in LNCaP and PC3 cell-conditioned media was identified as cathepsin D based on acidic pH optimum and immunoblotting. Furthermore, immunoadsorption of cathepsin D from the media attenuated the acid-activated IGFBP hydrolysis [125I]IGF-I binding to prostate cancer cells was reduced in the presence of LNCaP conditioned media that had been incubated at neutral pH for 24 h (i.e. containing intact IGFBP) but not by acid-incubated conditioned media (i.e. cathepsin D-mediated hydrolyzed IGFBP). These data indicate that prostate carcinoma cells secrete specific IGFBPs, as well as a general IGFBP protease, cathepsin D. In the proper environment, cathepsin D is capable of hydrolyzing all endogenous IGFBP and, thus, modifying IGF-I action in prostatic cells.
Elevation of intracellular calcium levels in the presence of normal androgen levels has been implicated in apoptotic prostate cell death.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.