Molecular mechanisms of the antiproliferative effects of 1(alpha), 25-dihydroxyvitamin D(3) in human prostate cancer cells

Date of Award




Degree Name

Doctor of Philosophy (Ph.D.)


Molecular and Cellular Pharmacology

First Committee Member

Kerry L. Burnstein, Committee Chair


It is now clear that, in addition to its roles in maintaining calcium and phosphorus homeostasis, $1\alpha$,25-dihydroxyvitamin D$\sb{3}$ (1,25 D), the most active metabolite of vitamin D3, is a regulator of cell growth and differentiation. Epidemiologic data suggest that vitamin D deficiency may be a risk factor for prostate cancer mortality. Moreover, 1,25 D inhibits the growth of human prostate cancer cells in vitro and in vivo. However, the magnitude of growth inhibition is variable. Since 1,25 D is being evaluated for its potential chemotherapeutic use in human prostate cancer, it is imperative to understand the molecular basis for the antiproliferative actions and the basis for variability in this response. The majority of 1,25 D effects are mediated by the vitamin D receptor (VDR), a ligand-activated transcription factor and member of the steroid/thyroid hormone receptor superfamily. To understand the different sensitivities of human prostate cancer cell lines to growth inhibition by 1,25 D, we assayed VDR content and VDR transcriptional activity in four human prostate cancer cell lines: LNCaP, ALVA31, PC3 and DU145. PC3 and DU145 cells, which contain the lowest VDR content/transcriptional activity, were relatively insensitive to 1,25 D-mediated growth inhibition. Stable transfection of PC3 with a VDR cDNA (PC3(VDR)) resulted in modest 1,25 D-mediated growth inhibition. However, in LNCaP and ALVA31, VDR content and antiproliferative effects of 1,25 D did not correlate. LNCaP expressed about half the number of functional VDR as ALVA31 yet LNCaP were more profoundly growth inhibited than ALVA31. VDRs in both LNCaP and ALVA31 were transcriptionally active as determined by reporter gene assays. Thus, VDR is necessary but not sufficient for maximal antiproliferative effects of 1,25 D. To determine what cellular mechanisms are involved in 1,25 D-mediated growth inhibition in human prostate cancer cells, we investigated the effect of 1,25 D on apoptosis and on cell cycle distribution. 1,25 D did not promote DNA fragmentation (indicative of apoptosis) in LNCaP, ALVA31, or PC3(VDR) cells, suggesting that apoptosis is not involved in the differential 1,25 D growth sensitivity. 1,25 D caused LNCaP cells to accumulate in the G1/G0 phase of the cell cycle, but had no effect on the cell cycle distribution of ALVA31 or PC3(VDR) cells. To understand the molecular mechanisms for the differential effects of 1,25 D on cell cycle distribution, we studied expression and 1,25 D regulation of genes that are involved in the regulation of G1-S cell cycle progression. 1,25 D treatment of LNCaP resulted in decreased retinoblastoma (Rb) protein phosphorylation, repressed E2F transcriptional activity, increased levels of the cyclin-dependent kinase inhibitors (CKIs) p21$\rm\sp{WAF1,CIP1}$ and p27$\rm\sp{KIP1}$, and a profound reduction of cyclin dependent kinase 2 (CDK2) activity. Effects of 1,25 D appears to be translational or posttranslational as mRNA levels of p21 and p27 were not affected. Moreover, 1,25 D increased the amount of p21 and p27 associated with CDK2 in LNCaP cells. In contrast, neither p21 nor p27 were detected or 1,25 D-inducible in ALVA31 or PC3(VDR) cells. These results suggest that maximal antiproliferative effects of 1,25 D require VDR and a functional Rb pathway, including p21 and p27 expression and 1,25 D regulation of CDK2 activity.


Biology, Cell; Health Sciences, Pharmacology

Link to Full Text