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Markers that reliably identify cancer stem cells (CSC) in ovarian cancer could assist prognosis and improve strategies for therapy. CD133 is a reported marker of ovarian CSC. Aldehyde dehydrogenase (ALDH) activity is a reported CSC marker in several solid tumors but it has not been studied in ovarian CSC. Here we report that dual positivity of CD133 and ALDH defines a compelling marker set in ovarian CSC. All human ovarian tumors and cell lines displayed ALDH activity. ALDH+ cells isolated from ovarian cancer cell lines were chemoresistant and preferentially grew tumors compared to ALDH− cells, validating ALDH as a marker of ovarian CSC in cell lines. Notably, as few as 1000 ALDH+ cells isolated directly from CD133(−) human ovarian tumors were sufficient to generate tumors in immunocompromised mice, whereas 50,000 ALDH− cells were unable to initiate tumors. Using ALDH in combination with CD133 to analyze ovarian cancer cell lines we observed even greater growth in the ALDH+CD133+ cells compared to ALDH+CD133− cells, suggesting a further enrichment of ovarian CSC in ALDH+CD133+ cells. Strikingly, as few as 11 ALDH+CD133+ cells isolated directly from human tumors were sufficient to initiate tumors in mice. Like other CSC, ovarian CSC exhibited increased angiogenic capacity compared to bulk tumor cells. Lastly, the presence of ALDH+CD133+cells in debulked primary tumor specimens correlated with reduced disease-free and overall survival in ovarian cancer patients. Taken together, our findings define ALDH and CD133 as a functionally significant set of markers to identify ovarian CSCs.

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MicroRNAs (miRs) are conserved small non-coding RNAs that negatively regulate gene expression. The miR profiles are markedly altered in cancers and some of them have a causal role in tumorigenesis. Here, we report changes in miR expression profile in hepatocellular carcinomas (HCCs) developed in male Fisher rats-fed folic acid, methionine, and choline-deficient (FMD) diet. Comparison of the miR profile by microarray analysis showed altered expression of some miRs in hepatomas compared to the livers from age-matched rats on the normal diet. family of genes was upregulated, miR-122, an abundant liver-specific miR, was downregulated in the tumors. The decrease in hepatic miR-122 was a tumor-specific event because it did not occur in the rats switched to the folate and methyladequate diet after 36 weeks on deficient diet, which did not lead to hepatocarcinogenesis. miR-122 was also silent in a transplanted rat hepatoma. Extrapolation of this study to human primary HCCs revealed that miR-122 expression was significantly (P = 0.013) reduced in 10 out of 20 tumors compared to the pair-matched control tissues. These findings suggest that the downregulation of miR-122 is associated with hepatocarcinogenesis and could be a potential biomarker for liver cancers. RNAs that block translation by imperfect base pairing to the 3′-untranslated regions (3′-UTR) of specific mRNA and by inducing mRNA degradation. Most miRs are expressed as primary transcripts transcribed by pol II, some miRs in clusters are coordinately expressed, and others are generated from introns. Primary miRs (pri-miRs) have 5′ caps and 3′ poly (A) tails, which is processed to mature miR by specific ribonuclease complexes (for review, see Zeng et al. [2005]). miRs play a key role in regulating diverse cellular processes that include development, differentiation, cell growth, apoptosis, viral infection, and metabolism (for review, see Ambros [2004]). KeywordsLike mRNAs, the majority of miRs are expressed predominantly in a tissue-specific manner whereas some are enriched in certain tissue [Lagos-Quintana et al., 2002]. Recently, much attention has been focused on miRs and cancer since miR genes are located at chromosomal regions, characterized by fragile sites and regions of deletion or amplification [Calin et al., 2004]. Some of these miRs deregulated in cancer function as tumor suppressors or oncogenes (for review, see Hwang and Mendell [2006]).Our laboratory has been studying the transcriptional and epigenetic regulation of gene expression in rodent and human primary hepatocellular carcinomas (HCCs) [Majumder et al., 2002;Ghoshal et al., 2004]. To study the altered regulation of gene expression at different stages of hepatocarcinogeneis, we have used a rat model. In this model, Fisher male rats-fed folate and methyl-deficient (FMD) diets develop preneoplastic nodules after 36 weeks and HCCs after 54 weeks Li, 2006]. Recently, we have used this rat model to identify the genes that are regulated tumor-specifically by epigenetic mechanism ]. This mode...

5-Azacytidine-and 5-aza-deoxycytidine (5-aza-CdR)-mediated reactivation of tumor suppressor genes silenced by promoter methylation has provided an alternate approach in cancer therapy. Despite the importance of epigenetic therapy, the mechanism of action of DNA-hypomethylating agents in vivo has not been completely elucidated. Here we report that among three functional DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B), the maintenance methyltransferase, DNMT1, was rapidly degraded by the proteasomal pathway upon treatment of cells with these drugs.

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The methylation status of the CpG island located within the ribosomal RNA (rRNA) promoter in human hepatocellular carcinomas and pair-matched liver tissues was analyzed by bisulfite genomic sequencing. Significant hypomethylation of methyl-CpGs in the rRNA promoter was observed in the tumor samples compared with matching normal tissues, which was consistent with the relatively high level of rRNA synthesis in rapidly proliferating tumors. To study the effect of CpG methylation on RNA polymerase I (pol I)-transcribed rRNA genes, we constructed pHrD-IRES-Luc (human rRNA promoter-luciferase reporter). In this plasmid, Kozak sequence of the pGL3-basic vector was replaced by the internal ribosome entry site (IRES) of encephalomyocarditis viral genome to optimize pol I-driven reporter gene expression. Transfection of this plasmid into HepG2 (human) cells revealed reduced pol I-driven luciferase activity with an increase in methylation density at the promoter. Markedly reduced luciferase activity in Hepa (mouse) cells compared with HepG2 (human) cells showed that pHrD-IRES-Luc is transcribed by pol I. Site-specific methylation of human rRNA promoter demonstrated that methylation of CpG at the complementary strands located in the promoter (؊9, ؊102, ؊347 with respect to the ؉1 site) inhibited luciferase activity, whereas symmetrical methylation of a CpG in the transcribed region (؉152) did not affect the promoter activity. Immunofluorescence studies showed that the methyl-CpG-binding proteins, MBD1, MBD2, MBD3, and MeCP2, are localized both in the nuclei and nucleoli of HepG2 cells. Transient overexpression of MBD2 suppressed luciferase activity specifically from the methylated rRNA promoter, whereas MBD1 and MBD3 inhibited rRNA promoter activity irrespective of the methylation status. Chromatin immunoprecipitation analysis confirmed predominant association of MBD2 with the endogenous methylated rRNA promoter, which suggests a selective role for MBD2 in the methylationmediated inhibition of ribosomal RNA gene expression.

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