Previous studies have shown that dietary or pharmacological methionine restriction inhibits growth of human prostate cancer cells in vitro or as xenografts in mice. We undertook the present studies to clarify the molecular mechanisms by which methionine restriction inhibits prostate cancer cell growth. We found that PC-3 and DU 145 cells stopped proliferating within six days in growth medium containing homocysteine in place of methionine. In contrast, proliferation of LNCaP cells was only

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... lly inhibited by the absence of methionine. Using flow cytometry, we found that methionine restriction caused PC-3 cells to arrest in all phases of the cell cycle, but predominantly in the G 2 /M phase, whereas LNCaP cells accumulated exclusively in the G 1 phase. Methionine restriction led to accumulation of the cyclin-dependent kinase inhibitors p21 and p27, as determined by Western blot analysis, and inhibited the enzymatic activities of the cyclin-dependent kinases CDK2 and cdc2, as determined by an in vitro kinase assay. However, methionine restriction had little or no effect on CDK2 or cdc2 protein levels. Methionine restriction also induced PC-3 cells to undergo apoptosis, as indicated by the appearance of a typical nucleosomal ladder on gel electrophoresis of genomic DNA. We conclude that methionine restriction has potential as a novel treatment strategy for prostate cancer. Figure 6 . Methionine restriction inhibits activities of cyclin-dependent kinases. LNCaP (A) and PC-3 (B) cells were grown for up to 9 days in methionine-free RPMI medium containing 100 µM homocysteine + 10% fetal calf serum. CDK2 and cdc2 protein levels (top bands) were measured by Western blot analysis. Kinase activities (bottom bands) were measured using an in vitro kinase assay with histone H1 as substrate. Ponceau S (middle bands) indicated equal protein loading.