Increased methylation of CpG islands and silencing of affected target genes is frequently found in human cancer; however, in vivo the question of causality has only been addressed by loss-of-function studies. To directly evaluate the role and mechanism of de novo methylation in tumor development, we overexpressed the de novo DNA methyltransferases Dnmt3a1 and Dnmt3b1 in Apc Min/+ mice. We found that Dnmt3b1 enhanced the number of colon tumors in Apc Min/+ mice approximately twofold and increased the average size of colonic microadenomas, whereas Dnmt3a1 had no effect. The overexpression of Dnmt3b1 caused loss of imprinting and increased expression of Igf2 as well as methylation and transcriptional silencing of the tumor suppressor genes Sfrp2, Sfrp4, and Sfrp5. Importantly, we found that Dnmt3b1 but not Dnmt3a1 efficiently methylates the same set of genes in tumors and in nontumor tissues, demonstrating that de novo methyltransferases can initiate methylation and silencing of specific genes in phenotypically normal cells. This suggests that DNA methylation patterns in cancer are the result of specific targeting of at least some tumor suppressor genes rather than of random, stochastic methylation followed by clonal selection due to a proliferative advantage caused by tumor suppressor gene silencing.[Keywords: DNA methylation; epigenetics; cancer; Dnmt3b; APC] Supplemental material is available at http://www.genesdev.org. Received July 17, 2007; revised version accepted October 11, 2007. Cancer cells show widespread epigenetic changes when compared with their normal parental tissue, including changes in DNA methylation and chromatin modification (Jones and Baylin 2007). The first epigenetic abnormality reported for human cancer was a global decrease in genomic cytosine methylation (Feinberg and Vogelstein 1983), most often seen in repetitive sequences and intergenic regions. It promotes genetic instability, increases the mobility of transposable elements (Walsh et al. 1998), and induces tumorigenesis in several different mouse models Gaudet et al. 2003;Yamada et al. 2005;Jones and Baylin 2007). Thus, hypomethylation predisposes to genetic damage and increases the risk of tumor development. Conversely, in some tissues global hypomethylation can also inhibit tumor outgrowth (Laird et al. 1995).In addition to global hypomethylation, it was also found that the cancer cell genome frequently contains regions with increased cytosine methylation (Baylin et al. 1986). This regional hypermethylation often affects CpG islands that are associated with promoter regions (Herman and Baylin 2003;Feinberg and Tycko 2004;Jones and Baylin 2007). Regional hypermethylation attracted attention when it was linked to transcriptional silencing of the RB tumor suppressor gene in patients with retinoblastoma tumors (Greger et al. 1989(Greger et al. , 1994. Multiple follow-up studies revealed that in cancer many tumor-relevant genes, in particular tumor suppressor genes, are transcriptionally silenced by hypermethylation. Aberrant DNA met...

Genome-wide DNA hypomethylation and concomitant promoterspecific tumor suppressor gene hypermethylation are among the most common molecular alterations in human neoplasia. Consistent with the notion that both promoter hypermethylation and genome-wide hypomethylation are functionally important in tumorigenesis, genetic and͞or pharmacologic reduction of DNA methylation levels results in suppression or promotion of tumor incidence, respectively, depending on the tumor cell type. For instance, DNA hypomethylation promotes tumors that rely predominantly on loss of heterozygosity (LOH) or chromosomal instability mechanisms, whereas loss of DNA methylation suppresses tumors that rely on epigenetic silencing. Mutational and epigenetic silencing events in Wnt pathway genes have been identified in human colon tumors. We used Apc Min/؉ mice to investigate the effect of hypomethylation on intestinal and liver tumor formation. Intestinal carcinogenesis in Apc Min/؉ mice occurs in two stages, with the formation of microadenomas leading to the development of macroscopic polyps. Using Dnmt1 hypomorphic alleles to reduce genomic methylation, we observed elevated incidence of microadenomas that were associated with LOH at Apc. In contrast, the incidence and growth of macroscopic intestinal tumors in the same animals was strongly suppressed. In contrast to the overall inhibition of intestinal tumorigenesis in hypomethylated Apc Min/؉ mice, hypomethylation caused development of multifocal liver tumors accompanied by Apc LOH. These findings support the notion of a dual role for DNA hypomethylation in suppressing later stages of intestinal tumorigenesis, but promoting early lesions in the colon and liver through an LOH mechanism.APC ͉ Dnmt1 ͉ intestinal cancer ͉ liver cancer ͉ loss of heterozygosity C hanges in the DNA methylation status are among the most common molecular alterations in human neoplasia (1). Although genome-wide DNA hypomethylation was observed in a wide variety of human cancers Ͼ20 years ago (2-4), the functional significance of this alteration is still unclear. In colon carcinogenesis, DNA hypomethylation has been observed in both adenomas and adenocarcinomas (4), suggesting that it is associated with early stages of carcinogenesis. However, similar early-stage lesions also display gene silencing through promoter hypermethylation (1). Ninety percent of colorectal cancers display Wnt pathway activation through either mutation or epigenetic silencing of genes in the canonical Wnt signaling pathway (5). Apc Min/ϩ mice have a mutation in the canonical Wnt pathway and are predisposed to form intestinal tumors (6). In Apc Min/ϩ mice, tumors arise as a result of loss of heterozygosity (LOH) at Apc as well as through LOH-independent mechanisms (7).Several experiments have demonstrated that global DNA hypomethylation induced by hypomorphic Dnmt1 alleles significantly suppresses intestinal tumorigenesis in Apc Min/ϩ mice (8-10). In contrast, global DNA hypomethylation promotes chromosomal instability in ES cells (11) and mic...

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The attachment of kinetochores to spindle microtubules (MTs) is essential for maintaining constant ploidy in eukaryotic cells. Here, biochemical and imaging data is presented demonstrating that the budding yeast CLIP-170 orthologue Bik1is a component of the kinetochore-MT binding interface. Strikingly, Bik1 is not required for viability in haploid cells, but becomes essential in polyploids. The ploidy-specific requirement for BIK1 enabled us to characterize BIK1 without eliminating nonhomologous genes, providing a new approach to circumventing the overlapping function that is a common feature of the cytoskeleton. In polyploid cells, Bik1 is required before anaphase to maintain kinetochore separation and therefore contributes to the force that opposes the elastic recoil of attached sister chromatids. The role of Bik1 in kinetochore separation appears to be independent of the role of Bik1 in regulating MT dynamics. The finding that a protein involved in kinetochore–MT attachment is required for the viability of polyploids has potential implications for cancer therapeutics.

These data show macula-specific increases in mtDNA damage, heteroplasmic mutations, and diminished repair that are associated with aging and AMD severity.

Aberrant gene silencing accompanied by DNA methylation is associated with neoplastic progression in many tumors that also show global loss of DNA methylation. Using conditional inactivation of de novo methyltransferase Dnmt3b in Apc Min/؉ mice, we demonstrate that the loss of Dnmt3b has no impact on microadenoma formation, which is considered the earliest stage of intestinal tumor formation. Nevertheless, we observed a significant decrease in the formation of macroscopic colonic adenomas. Interestingly, many large adenomas showed regions with Dnmt3b inactivation, indicating that Dnmt3b is required for initial outgrowth of macroscopic adenomas but is not required for their maintenance. These results support a role for Dnmt3b in the transition stage between microadenoma formation and macroscopic colonic tumor growth and further suggest that Dnmt3b, and by extension de novo methylation, is not required for maintaining tumor growth after this transition stage has occurred.Altered DNA methylation in the form of global hypomethylation and regional hypermethylation is one of the most consistent epigenetic changes in cancer (18). Global hypomethylation, which is frequently observed at early stages of tumorigenesis in human cancer (10, 11), promotes tumor development in several mouse models and causes chromosomal instability in cultured fibroblasts (9, 12). After the initial observation of DNA hypermethylation within the retinoblastoma tumor suppressor gene (14), dozens of genes have been shown to be hypermethylated and transcriptionally silenced in tumors (2,20). Although the consequences of global hypomethylation and gene-specific hypermethylation have been mechanistically connected to chromosome instability and transcriptional silencing, respectively, the causes of aberrant DNA methylation patterns are currently unknown. DNA methylation is catalyzed by a family of three DNA methyltransferases: Dnmt1, Dnmt3a, and Dnmt3b. Although the three Dnmts partially cooperate to establish and maintain genomic methylation patterns (21), they also have distinctive functions. Dnmt1 has a preference for hemimethylated DNA (1, 15, 38), and indeed a hypomorphic allele of Dnmt1 has been shown to cause global DNA hypomethylation (12). Dnmt1 is therefore considered the major maintenance methyltransferase. Dnmt3a and -3b probably function as de novo DNA methyltransferases because these enzymes were shown to have equal preferences in vitro for unmethylated and hemimethylated DNA (25, 26). Furthermore, de novo methylation of a subset of the CpG sites on stable episomes is detected in human cells overexpressing the murine Dnmt3a or Dnmt3b1 protein (17). Consistent with these notions, inactivation of both Dnmt3a and Dnmt3b by gene targeting blocks de novo DNA methylation in embryonic stem (ES) cells and early embryos, as well as de novo methylation of imprinted genes in germ cells (25,26). These findings support the view that Dnmt3a and Dnmt3b function primarily as de novo methyltransferases during normal development. Nevertheless, the role of...

The protection of RPE cells against oxidative damage is afforded by miR-23a through regulation of Fas, which may be a novel therapeutic target in retinal degenerative diseases.

Exposure to antibiotics during pregnancy can pose a systematic effect on human health. A few biomonitoring studies have demonstrated an extensive exposure of children to antibiotics, but there is still a lack of data for pregnant women. To assess the exposure of pregnant women to antibiotics and potential health risk, we investigated 536 pregnant women aged 16-42 years from two geographically different study sites in Eastern China in 2015. We measured 21 antibiotics of five categories (seven fluoroquinolones, three phenicols, four tetracyclines, three macrolides, and four sulfonamides) in urine using the isotope dilution ultraperformance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry. The hazard index (HI) was calculated on the basis of estimated daily exposure dose and acceptable daily intakes. A total of 16 antibiotics were found in urine, with detection frequencies between 0.2 and 16.0%. Antibiotics were overall detected in 41.6% of urine, and two or more antibiotics were detected in 13.1% of urine. Ciprofloxacin, ofloxacin, and trimethoprim were most frequently detected in urine, with detection frequencies between 10 and 20%. The majority of the antibiotics tested had an estimated daily exposure dose less than 1 μg/kg/day, and 4.3% of pregnant women had a HI value of more than 1. These findings indicated that pregnant women were frequently exposed to antibiotics and some individuals were in the potential risk of adverse microbiological effects induced by antibiotics.