Growth inhibitory effects of 1alpha, 25-dihydroxyvitamin D(3) are mediated by increased levels of p21 in the prostatic carcinoma cell line ALVA-31.

1alpha, 25-Dihydroxyvitamin D(3) [1alpha, 25-(OH)(2)D(3)] is recognized to have significant antiproliferative effects on certain prostatic carcinoma (PC) cell lines, although the precise mechanisms of action remain in question. We have evaluated the role of the cell cycle-dependent kinase inhibitor p21. In the PC cell lines ALVA-31 and LNCaP, 1alpha, 25-(OH)(2)D(3) inhibits growth and induces both p21 mRNA and protein levels. Growth inhibition of ALVA-31 cells was abolished by stable transfection with a p21 antisense construct. This effect was not attributable to a reduction in functional vitamin D receptors as measured by transcriptional activity with a luciferase-vitamin D response element reporter construct. Therefore, increased p21 expression appears necessary to mediate the antiproliferative effects of this hormone in ALVA-31 cells. Cell lines that are insensitive to the growth inhibitory properties of 1alpha, 25-(OH)(2)D(3) failed to up-regulate p21 expression after hormone treatment; these include sublines of ALVA-31 as well as the cell lines TSU-Pr1 and JCA-1. In the latter two lines, adenovirus-mediated expression of a sense p21 cDNA significantly reduced their proliferation as compared with a control adenoviral construct. This suggests that the signaling pathway downstream of p21 is intact in TSU-Pr1 and JCA-1 cells, although p21 expression appears unregulated by 1alpha, 25-(OH)(2)D(3). We propose a model in which the antiproliferative effect of 1alpha, 25-(OH)(2)D(3) on PC cells is mediated through increased p21 expression. Elucidation of why this effect is absent in select cell lines may provide valuable insight into the variability of responses observed in PC patients treated with vitamin D.

Three-dimensional spheroid cultures of human prostate cancer cell lines.

BACKGROUND: Many of the available human prostate cancer (PC) cell lines have lost androgen sensitivity and no longer secrete prostate-specific proteins after serial culturing in cell monolayers. Three-dimensional spheroid cultures have been found to better mimic the in vivo phenotypes of several nonprostatic cell lines. METHODS: We analyzed seven PC cell lines to determine if spheroid culturing results in greater sensitivity to androgens and 1alpha,25(OH)(2) vitamin D(3) (1,25(OH)(2) D(3)) with regards to their growth, differentiation, and apoptotic potential. RESULTS: Only PC-3 cells showed greater sensitivity to the growth-inhibitory effects of 1, 25(OH)(2) D(3), while ALVA-31 showed a diminished response. The regulation of prostate-specific antigen and prostate-specific acid phosphatase remained unchanged. However, these studies provided several unique findings not observed in cell monolayers. First, three basic spheroid morphologies were observed with varying degrees of intercellular adhesions. Secondly, the cell lines that formed the tightest spheroids consistently grew at the slowest rates, regardless of their growth rate in monolayers. Lastly, 1,25(OH)(2) D(3) treatment of ALVA-31 and PPC-1 spheroids greatly reduced intercellular adhesions, and rendered ALVA-31 spheroids resistant to apoptotic induction by Fas ligand expressed via a recombinant adenoviral construct. CONCLUSIONS: Our results suggest that spheroid cultures of human PC cells may provide unique insights regarding cell adhesion and apoptotic potential that are diminished or absent in monolayer cultures.

Adenovirus-mediated expression of Fas ligand induces apoptosis of human prostate cancer cells.

Several laboratories have reported on the apoptotic potentials of human prostate cancer (PC) cell lines in response to crosslinking of Fas (CD95/APO-1) with agonistic anti-Fas antibodies. We have re-evaluated the apoptotic potentials of seven human PC cell lines using the natural Fas ligand (FasL) in place of agonistic antibody. First, PC cell lines were tested in a standard cytotoxicity assay with a transfected cell line that stably expresses human FasL. Next, we developed an adenoviral expression system employing 293 cells that stably express crmA, a poxvirus inhibitor of apoptosis, to analyze the effects of FasL when expressed internally by the PC cell lines. Our data suggest that the apoptotic potentials of these cell lines were greatly underestimated in previous studies utilizing agonistic anti-Fas antibodies. Lastly, adenoviral-mediated expression of FasL prevented growth and induced regression of two human PC cell lines in immunodeficient mice. These preliminary in vivo results suggest a potential use for adenovirus encoding FasL as a gene therapy for PC.

Fas-mediated apoptosis in seven human prostate cancer cell lines: correlation with tumor stage.

BACKGROUND: Apoptosis, or programmed cell death, can be mediated through an endogenous signaling pathway that emanates from a cell surface receptor known as Fas. Although best recognized for its role in the immune system, recent studies have also suggested a role for Fas in mediating apoptosis in the murine prostate. Little is known, however, regarding the role of Fas-signaling in the human prostate, and if this signaling pathway is abrogated in the development of prostate cancer (PC). METHODS: In the current study, seven human PC cell lines were evaluated for their sensitivities to Fas-mediated apoptosis, using both morphologic and flow cytometric methods. Fas expression by each cell line was quantitated by immunofluorescence, and gene expression of three putative inhibitory molecules was analyzed. RESULTS: The differential sensitivities of the cell lines to Fas-mediated apoptosis were found to correlate with the clinical stage of the parental tumors. Specifically, the three most sensitive cell lines were all derived from primary tumors, while the four most resistant cell lines were derived from distant metastases. Immunofluorescent analyses of the PC cell lines revealed that the observed resistance to apoptosis was not due to reduced expression of membrane-bound Fas. Likewise, this resistance did not correlate with increased gene expression of the inhibitory molecules FAP-1, ICE epsilon, and Ich-1S. CONCLUSIONS: Our results using established PC cell lines support previous studies with prostatic tissue specimens, and suggest that the normal, differentiated prostatic epithelium, as well as locally invasive PCs, have the potential to undergo Fas-mediated apoptosis. Conversely, these studies suggest that metastatic PCs have a reduced apoptotic potential that is mediated by a novel mechanism.

Three synthetic vitamin D analogues induce prostate-specific acid phosphatase and prostate-specific antigen while inhibiting the growth of human prostate cancer cells in a vitamin D receptor-dependent fashion.

Numerous studies have indicated that the secosteroid hormone 1alpha, 25-dihydroxyvitamin D3 protects against the development of clinical prostate cancer (PC). Whether this hormone also has therapeutic potential for patients with advanced PC has not yet been evaluated. Several synthetic vitamin D analogues are now available that have reduced hypercalcemic effects and yet effectively induce differentiation in some cell types. For these reasons, these analogues may be safer and more effective for cancer therapy than the natural hormone. In the current study, 13 such analogues were screened for their abilities to inhibit the growth of PC cell lines. Three of the most consistently effective analogues (Ro 23-7553, Ro 24-5531, and Ro 25-6760) were then chosen for further analysis. Growth studies using clones of the JCA-1 cell line that were transfected with the vitamin D receptor cDNA indicate that the antiproliferative effects of these analogues require vitamin D receptor expression. Furthermore, these three analogues induce the secretion of prostate-specific acid phosphatase and prostate-specific antigen (two markers of the differentiated prostatic phenotype) in the cell line LNCaP. These in vitro studies suggest that Ro 23-7553, Ro 24-5531, and Ro 25-6760 should be further evaluated as therapeutic agents for the treatment of PC.

Vitamin D receptor expression is required for growth modulation by 1 alpha,25-dihydroxyvitamin D3 in the human prostatic carcinoma cell line ALVA-31.

Epidemiological data suggest that vitamin D3, obtained from dietary sources and sunlight exposure, protects against mortality from prostate cancer (PC). In agreement with this, the most active vitamin D metabolite 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2 D3] regulates the growth and differentiation of several human PC cell lines. Both genomic and non-genomic signalling pathways for 1,25(OH)2 D3 have been reported, although the mechanism of action in PC cells has not been defined. We now provide data supporting an active role for the nuclear vitamin D receptor (VDR) in mediating the growth-inhibitory effects of 1,25(OH)2 D3 on these cells. In the VDR-rich cell line ALVA-31, the observed changes in growth by 1,25(OH)2 D3 are preceded by significant changes in VDR mRNA expression. In contrast, the cell line JCA-1, containing few VDRs, fails to show both early changes in VDR gene expression and later changes in growth with 1,25(OH)2 D3. To assess the role of the VDR more directly, transfection studies were pursued. ALVA-31 cells were stably transfected with an antisense VDR cDNA construct in an attempt to reduce VDR expression. Antisense mRNA expression among clones was associated with: (a) reduced or abolished sensitivity to the effects of 1,25(OH)2 D3 on growth; (b) decreased numbers of VDRs per cell, as measured by radiolabelled-ligand binding; and (c) a lack of induction of the VDR-regulated enzyme 24-hydroxylase in response to 1,25(OH)2 D3. From these studies we conclude that the antiproliferative effects of 1,25(OH)2 D3 require expression of the nuclear VDR in this system.

Stable expression of the nuclear vitamin D receptor in the human prostatic carcinoma cell line JCA-1: evidence that the antiproliferative effects of 1 alpha, 25-dihydroxyvitamin D3 are mediated exclusively through the genomic signaling pathway.

The secosteroid hormone 1 alpha, 25-dihydroxyvitamin D3 [1,25-(OH)2D3] has been found to regulate the growth and differentiation of human prostate cancer cells, although the precise mechanisms mediating these effects have not been defined. 1,25-(OH)2D3 is capable of acting through both nongenomic signaling pathways involving a membrane-associated receptor and genomic pathways involving the nuclear vitamin D receptor (VDR). The primary purpose of this study was to directly evaluate the role of the nuclear VDR in mediating the growth inhibitory effects of 1,25-(OH)2D3 on human prostate cancer cells. The cell line JCA-1 was used because it fails to express detectable number of VDRs and is not measurable affected by 1,25-(OH)2D3 in growth studies. These cells were stably transfected with a wild-type VDR complementary DNA construct producing the following results: 1) the expression of high affinity nuclear VDRs, 2) the dose-dependent inhibition of growth by 1,25-(OH)2D3, and 3) a significant increase in 24-hydroxylase up-regulation by 1,25-(OH)2D3 compared to that in controls. These data indicate that nuclear VDR expression is sufficient to mediate the antiproliferative effects of 1,25-(OH)2D3 on prostate cancer cells. In addition, because the stereoisomer 1 beta, 25-dihydroxyvitamin D3 failed to block these antiproliferative effects, we conclude that nongenomic mechanisms of action are not requisite for growth inhibition by 1,25-(OH)2D3.

Vitamin D receptor expression, 24-hydroxylase activity, and inhibition of growth by 1alpha,25-dihydroxyvitamin D3 in seven human prostatic carcinoma cell lines.

Although prostatic cancer is often viewed as an androgen-dependent malignancy, a number of other hormones including 1alpha, 25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] are now recognized to modulate its growth and differentiated phenotype. Seven different continuous human prostatic carcinoma cell lines were examined for the presence of biologically active receptors for 1alpha,25(OH)2D3. All seven lines were found to contain mRNA for the vitamin D receptor using an RNase protection assay. Six of the seven cell lines were found to have high-affinity saturable binding sites for 1alpha,25(OH)2D3. The seventh line was found to contain vitamin D receptors by sucrose gradient analysis. All seven lines were found to express 24-hydroxylase activity by a HPLC assay that measures the conversion of 25-hydroxyvitamin D3 to 24,25-dihydroxyvitamin D3. 24-Hydroxylase activity was up-regulated in all seven cell lines by preincubation with 1alpha,25(OH)2D3. In the presence of fetal bovine serum, the growth of four of the seven cell lines was inhibited. In the majority of cell lines growth inhibition was related not only to the number of receptors per cell, but also in inverse proportion to the 24-hydroxylase activity of each cell line. The ubiquitous presence of vitamin D receptor and 24-hydroxylase activity in human prostatic carcinoma cells suggests new alternatives for the pharmacological treatment of advanced prostatic cancer and implies that chemoprevention strategies could also make use of this endocrine axis.

A serum-free defined medium capable of supporting growth of four established human prostatic carcinoma cell lines.

This paper describes a serum-free defined medium (Gc) that was initially designed to support growth of the human prostatic carcinoma cell line LNCaP. Our studies indicate that this medium formulation is capable of supporting short-term, long-term, and clonal growth of the LNCaP cell line. Component deletion experiments have shown that the three most critical components for LNCaP short-term growth are insulin, triiodothyronine (T3), and fetuin. Additionally, this medium was found to support short-term and clonal growth of three other human prostatic carcinoma cell lines, DU 145, PC-3, and ALVA-31. The availability of such a medium should aid in the distinction of the regulatory factors involved in growth and differentiation of malignant prostatic epithelium.

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.