Prostatic Adenocarcinoma Cell Line LNCaP Research Articles

Androgens markedly stimulate the accumulation of neutral lipids in the human prostatic adenocarcinoma cell line LNCaP

Androgens markedly stimulate the accumulation of neutral lipids in the human prostatic adenocarcinoma cell line LNCaP

Androgen regulation of the messenger RNA encoding diazepam-binding inhibitor/acyl-CoA-binding protein in the rat

Our recent finding that diazepam-binding inhibitor/acyl-CoA-binding protein (DBI/ACBP) expression is regulated by androgens in the human prostatic adenocarcinoma cell line LNCaP, prompted us to study whether androgen regulation of DBI/ACBP also occurs in vivo in the prostate and in other organs of the rat. Northern blot analysis demonstrated that DBI/ACBP transcripts were expressed in male accessory sex organs such as ventral prostate, dorsolateral prostate, seminal vesicles and coagulating glands. Castration caused a 1.7- to 2.7-fold reduction in the levels of DBI/ACBP transcripts over a period of 6 days. Readministration of androgens during the last 3 days led to 4.2- to 7.5- fold higher levels of DBI/ACBP transcripts than in untreated castrates. In situ hybridization revealed that in the ventral prostate, DBI/ACBP transcripts were expressed predominantly in epithelial cells, and that the observed effects of androgens were due both to modulation of gene expression per cell and to changes in cell composition. Androgen regulation of DBI/ACBP mRNA expression was also observed in the lacrimal glands, the adrenals, and the submandibular glands, but not in the liver and the kidney. These findings demonstrate that DBI/ACBP is androgen-regulated in vivo in various organs of the rat. In view of the proposed role of DBI/ACBP in the control of multiple biological processes. DBI/ACBP may be one of the target genes by which androgens affect a variety of physiological processes.

A Human Gene Encoding Diazepam-Binding Inhibitor/Acyl-CoA-Binding Protein: Transcription and Hormonal Regulation in the Androgen-Sensitive Human Prostatic Adenocarcinoma Cell Line LNCaP

Diazepam-binding inhibitor (DBI)/acyl-CoA-binding protein (ACBP) is a highly conserved 10-kD polypeptide expressed in various organs and implicated in the regulation of multiple biological processes such as GABAA/benzodiazepine receptor modulation, acyl-CoA metabolism, steroidogenesis, and insulin secretion. To extend our knowledge about the biology of DBI/ACBP and to elucidate the molecular mechanisms responsible for regulating DBI/ACBP gene expression, we have studied the androgen-regulated expression of DBI/ACBP transcripts in the human prostatic adenocarcinoma cell line LNCaP and have cloned and characterized a human gene encoding DBI/ACBP. Northern blotting, reverse transcription-assisted polymerase chain reaction (RT-PCR), ribonuclease protection, and 5' RACE analysis (rapid amplification of cDNA ends) of DBI/ACBP transcripts in LNCaP cells revealed androgen-regulated expression of multiple transcripts originating from multiple transcription start sites and alternative processing. The most abundant type of transcripts (referred to as type 1 transcripts) encodes genuine DBI/ACBP of 86 amino acids, while the minor type (type 2 transcripts) harbors an insertion of 86 bases and might encode an unrelated protein of 67 amino acids. Examination of a cloned DBI/ACBP gene revealed a structural organization of four exons present in all transcripts and one alternatively used exon present only in type 2 transcripts. The promoter region is located in a CpG island and lacks a canonical TATA box. Transient transfection of DBI/ACBP promoter fragments into LNCaP cells demonstrated that a region of 1.1 kb upstream of the translation start site is able to drive high-level expression of luciferase in LNCaP cells in an androgen-regulated fashion. Taken together these data indicate that the isolated human gene encoding DBI/ACBP is functional, has a high degree of structural similarity with the corresponding rat gene, exhibits hallmarks of a typical housekeeping gene, and harbors cis-acting elements that are at least partially responsible for androgen-regulated transcription in LNCaP cells.

PRECLINICAL STUDIES OF GOSSYPOL IN PROSTATE CARCINOMA

Hormone refractory prostate cancer remains an incurable disease and the discovery of newer agents with higher cytotoxic activity is required. Gossypol is a phenolic compound isolated from cottonseed oil which has been shown to have anti-spermatogenic effects. In in vitro studies, gossypol appears to inhibit the growth of rat prostate cancer cell line MAT-LyLu and human prostate adenocarcinoma cell lines PC-3, LNCaP and DU-145. In vive, gossypol appeared to inhibit tumor growth of subcutaneously implanted MAT-LyLu cells in Copenhagen rats. Gossypol may be an active agent for the treatment of hormone refractory metastatic prostate cancer.

Androgen regulation of the messenger RNA encoding diazepam-binding inhibitor/acyl-CoA-binding protein in the human prostatic adenocarcinoma cell line LNCaP

To study the mechanisms by which androgens intervene in the regulation of growth and differentiation of human prostatic epithelial cells, cDNA clones encoding putative prostate-secreted proteins were characterized and tested as potential markers for androgen action. One of the isolated cDNAs expressed diazepam-binding inhibitor/acyl-CoA-binding protein (DBI/ACBP), suggesting that this polypeptide, that has been implicated in a large number of biochemical processes, is expressed and secreted by prostate cells. As demonstrated by Northern blot analysis, the mRNA encoding DBI/ACBP was expressed in prostate tissue and in the three human prostatic adenocarcinoma cell lines tested: LNCaP, PC-3 and DU-145. In androgen-sensitive LNCaP cells, the synthetic androgen R1881 stimulated the DBI/ACBP steady state mRNA levels with half maximal effects at a concentration of 0.2 nM. Increases were a maximal 12 h after addition of the synthetic hormone. DBI/ACBP mRNA levels could also be stimulated by the synthetic androgen mibolerone and by the natural androgens testosterone and dihydrotestosterone. In agreement with the altered steroid specificity of the androgen receptor in LNCaP cells, estradiol and progesterone also exerted a stimulatory effect. Cortisol and the synthetic glucocorticoid dexamethasone were without effect. Androgen stimulation of DBI/ACBP mRNA levels was abolished in the presence of the protein synthesis inhibitor cycloheximide, implying a role for labile or androgen-induced proteins in this androgen stimulation. This is in contrast to the androgen stimulation of the mRNA encoding prostate-specific antigen (PSA), suggesting that different mechanisms are involved in the androgen regulation of these two genes. Although further experiments are required to confirm that DBI/ACBP is secreted by prostatic epithelial cells, these data demonstrate that the mRNA encoding DBI/ACBP is expressed in prostate cells and is affected by androgens in androgen-responsive LNCaP cells.

Antagonism of androgen action in prostate tumor cells by retinoic acid

Recent studies have demonstrated that retinoic acid (RA) can repress the growth of human prostatic epithelial cells. Since the proliferation of prostate cells is highly dependent on androgen stimulation, presumably via its cognate receptor, we investigated the effects of RA on the expression of the androgen receptor and other androgen-regulated genes in the human prostatic adenocarcinoma cell line LNCaP. Using a radioligand binding assay, we found that androgen-binding activity was reduced 30-40% in cells treated with 10(-5) M RA plus 6 nM dihydrotestosterone (DHT), as compared to cells with the androgen alone. Moreover, the reduction of the androgen receptor (AR) was not accompanied by alteration of the ligand-binding affinity. Concomitant changes in the function of AR were manifested by a dramatic reduction in AR-mediated transcription activity in a transfection experiment. Androgen-induced levels of both prostate-specific antigen (PSA) and human glandular kallikrein-1 (hKLK2) mRNAs were significantly repressed by RA in a dose- and time-dependent manner. Consistent with this finding, androgen induction of PSA glycoprotein was also repressed by RA, with maximal inhibition occurring at 10(-5) M. These data suggest that the suppression of proliferation and function of prostatic cells by RA may be via modulatory effects on the AR.

Prostate specific antigen and prostatitis. II. PSA production and release kinetics in vitro.

Elevations in serum prostate specific antigen (PSA) levels in patients with prostatitis are well known, but the pathophysiologic mechanisms involved with this phenomenon are poorly understood. We have recently evaluated the effect of prostatitis on PSA levels in primates. This data demonstrated a rapid rise in PSA, which subsequently fell along the biological decay curve. This suggested a release of sequestered PSA. We evaluated the effect of infection upon PSA production for the prostatic adenocarcinoma cell line LNCaP. The cell line was determined to produce 9.6 +/- 2.7 fg/cell/hr PSA with essentially linear kinetics. No effect was seen with dead bacteria, bacterial supernatant or complement upon the PSA production. Live E. coli had no effect for 4-8 hours at which time the cells sloughed and PSA production ceased. No release of stored PSA was seen. These data do not elucidate the reasons for increased serum PSA levels found with acute bacterial prostatitis, but indicate that it is not a storage phenomenon.

Cytogenetic characterization of several androgen responsive and unresponsive sublines of the human prostatic carcinoma cell line LNCaP.

The cytogenetic evolution of the prostatic adenocarcinoma cell line LNCaP was investigated during long term in vitro culture. Study of five different sublines demonstrated that the original karyotype was well preserved in all sublines, with respect to the chromosome number as well as to the primary markers. All sublines showed additional, subline specific secondary marker chromosomes. Comparison of these markers in androgen responsive and nonresponsive sublines showed rearrangement of the short arm of chromosome 8 in both unresponsive sublines. The breakpoints were in 8p21 and 8p23, respectively, resulting in deletion of the 8p23----pter region in both sublines. In contrast, the hormone responsive sublines did not show any aberrations in chromosome 8. Review of published karyotypes of patients and cell lines seems to support our finding of partial deletion of 8p in androgen unresponsive prostate tumor cells.