We performed a comprehensive survey of commonly inactivated tumor suppressor genes in esophageal squamous cell carcinoma (ESCC) based on functional reactivation of epigenetically silenced tumor suppressor genes by 5-aza-2'-deoxycytidine and trichostatin A using microarrays containing 12599 genes. Among 58 genes identified by this approach, 44 (76%) harbored dense CpG islands in the promoter regions. Thirteen of twenty-two tested gene promoters were methylated in cell lines, and ten in primary ESCC accompanied by silencing at the mRNA level. Potent growth suppressive activity of three genes including CRIP-1, Apolipoprotein D, and Neuromedin U in ESCC cells was demonstrated by colony focus assays. Pharmacologic reversal of epigenetic silencing is a powerful approach for comprehensive identification of tumor suppressor genes in human cancers.

Breast cancer is the most common cancer among American women, whereas Asian women, who consume a traditional diet high in soy products, have a relatively low incidence. Genistein is a prominent iso¯avonoid in soy products and has been proposed as the agent responsible for lowering the rate of breast cancer in Asian women. We investigated the e ects of genistein on cell growth and apoptosis-related gene expression in breast cancer cells MDA-MB-231. We found upregulation of Bax and p21 WAF1 expressions and downregulation of Bcl-2 and p53 expression in genisteintreated cells. Furthermore, DNA ladder formation, CPP32 activation, and PARP cleavage were observed after treatment with genistein, indicating apoptotic cell deaths. Flow cytometry with 7-amino actinomycin D staining showed that the number of apoptotic cells increased with longer treatment of genistein. From these results, we conclude that genistein inhibits the growth of MDA-MB-231 breast cancer cells, regulates the expression of apoptosis-related genes, and induces apoptosis through a p53-independent pathway. The up-regulation of Bax and p21 WAF1 may be the molecular mechanisms by which genistein induces apoptosis, however, further de®nitive studies are needed. These results suggest that genistein may be a potentially e ective chemopreventive or therapeutic agent against breast cancer.

Prostate cancer is one of the most common cancers in men and it is the second leading cause of cancer related death in men in the United States. Recent dietary and epidemiological studies have suggested the bene®t of dietary intake of fruits and vegetables in lowering the incidence of prostate cancer. A diet rich in fruits and vegetables provides phytochemicals, particularly indole-3-carbinol (I3C), which may be responsible for the prevention of many types of cancer, including hormonerelated cancers such as prostate. Studies to elucidate the role and the molecular mechanism(s) of action of I3C in prostate cancer, however, have not been conducted. In the current study, we investigated whether I3C had any e ect against prostate cancer cells and, if so, attempts were made to identify the potential molecular mechanism(s) by which I3C elicits its biological e ects on prostate cancer cells. Here we report for the ®rst time that I3C inhibits the growth of PC-3 prostate cancer cells. Induction of G1 cell cycle arrest was also observed in PC-3 cells treated with I3C, which may be due to the observed e ects of I3C in the up-regulation of p21 WAF1 and p27 Kip1 CDK inhibitors, followed by their association with cyclin D1 and E and down-regulation of CDK6 protein kinase levels and activity. The induction of p21 WAF1 appears to be transcriptionally upregulated and independent of the p53 responsive element. In addition, I3C inhibited the hyperpohosphorylation of the Retinoblastoma (Rb) protein in PC-3 cells. Induction of apoptosis was also observed in this cell line when treated with I3C, as measured by DNA laddering and poly (ADP-ribose) polymersae (PARP) cleavage. We also found an up-regulation of Bax, and down-regulation of Bcl-2 in I3C-treated cells. These e ects may also be mediated by the down-regulation of NF-kB observed in I3C treated PC-3 cells. From these results, we conclude that I3C inhibits the growth of PC-3 prostate cancer cells by inducing G1 cell cycle arrest leading to apoptosis, and regulates the expression of apoptosisrelated genes. These ®ndings suggest that I3C may be an e ective chemopreventive or therapeutic agent against prostate cancer. Oncogene (2001) 20, 2927 ± 2936.