DNA methylation has a role in mediating epigenetic silencing of CpG island genes in cancer and other diseases. Identification of all gene promoters methylated in cancer cells ''the cancer methylome'' would greatly advance our understanding of gene regulatory networks in tumorigenesis. We previously described a new method of identifying methylated tumor suppressor genes based on pharmacologic unmasking of the promoter region and detection of re-expression on microarray analysis. In this study, we modified and greatly improved the selection of candidates based on new promoter structure algorithm and microarray data generated from 20 cancer cell lines of 5 major cancer types. We identified a set of 200 candidate genes that cluster throughout the genome of which 25 were previously reported as harboring cancer-specific promoter methylation. The remaining 175 genes were tested for promoter methylation by bisulfite sequencing or methylation-specific PCR (MSP). Eighty-two of 175 (47%) genes were found to be methylated in cell lines, and 53 of these 82 genes (65%) were methylated in primary tumor tissues. From these 53 genes, cancer-specific methylation was identified in 28 genes (28 of 53; 53%). Furthermore, we tested 8 of the 28 newly identified cancer-specific methylated genes with quantitative MSP in a panel of 300 primary tumors representing 13 types of cancer. We found cancer-specific methylation of at least one gene with high frequency in all cancer types. Identification of a large number of genes with cancer-specific methylation provides new targets for diagnostic and therapeutic intervention, and opens fertile avenues for basic research in tumor biology.

Cervical neoplasia-specific biomarkers, e.g. DNA methylation markers, with high sensitivity and specificity are urgently needed to improve current population-based screening on (pre)malignant cervical neoplasia. We aimed to identify new cervical neoplasia-specific DNA methylation markers and to design and validate a methylation marker panel for triage of high-risk human papillomavirus (hr-HPV) positive patients. First, high-throughput quantitative methylation-specific PCRs (QMSP) on a novel OpenArray TM platform, representing 424 primers of 213 cancer specific methylated genes, were performed on frozen tissue samples from 84 cervical cancer patients and 106 normal cervices. Second, the top 20 discriminating methylation markers were validated by LightCyclerV R MSP on frozen tissue from 27 cervical cancer patients and 20 normal cervices and ROCs and test characteristics were assessed. Three new methylation markers were identified (JAM3, EPB41L3 and TERT), which were subsequently combined with C13ORF18 in our four-gene methylation panel. In a third step, our methylation panel detected in cervical scrapings 94% (70/74) of cervical cancers, while in a fourth step 82% (32/39) cervical intraepithelial neoplasia grade 3 or higher (CIN31) and 65% (44/68) CIN21 were detected, with 21% positive cases for CIN1 (16/75). Finally, hypothetical scenario analysis showed that primary hr-HPV testing combined with our four-gene methylation panel as a triage test resulted in a higher identification of CIN3 and cervical cancers and a higher percentage of correct referrals compared to hr-HPV testing in combination with conventional cytology. In conclusion, our four-gene methylation panel might provide an alternative triage test after primary hr-HPV testing.Infection with high-risk human papillomavirus (hr-HPV) is causally linked to cervical carcinogenesis. 1 Cervical cancer incidence is reduced by cytologic screening, although cytologic morphologic assessment of cervical scrapings is not ideal, because its sensitivity is only $ 55% for CIN2þ. 2 Recently, preventive vaccines against hr-HPV-16 and hr-HPV-18 have been introduced in the Western world, which will reduce the incidence of cervical neoplasia significantly. However, these vaccines do not cover 100% of cervical cancers, while it will take over decades before HPV vaccination affects cervical neoplasia incidence. Therefore, screening needs to be continued. Simultaneously, efficiency of cytologic screening in population-based screening programs will be reduced by vaccination, due to a gradual decline of cervical neoplasia prevalence. Hr-HPV testing of cervical scrapings has been shown to improve sensitivity of cervical screening, 3,4 but is also associated with low specificity, especially in a young screening population. 5 For that reason, other markers are needed especially to improve the positive predictive value for population-based screening of cervical neoplasia.Promoter methylation of tumor suppressor genes has been reported to be an early event in carcinogenesis. 6 Gene pro...

The accumulation of toxic compounds generated by the interaction between reactive oxygen species and polyunsaturated fatty acids of membrane lipids can significantly damage plant cells. A plethora of enzymes act on these reactive carbonyls, reducing their toxicity. Based on the chromosomal localization and on their homology with other stress-induced aldo-keto reductases (AKRs) we have selected three rice AKR genes. The transcription level of OsAKR1 was greatly induced by abscisic acid and various stress treatments; the other two AKR genes tested were moderately stress-inducible. The OsAKR1 recombinant protein exhibited a high nicotinamide adenine dinucleotide phosphate-dependent catalytic activity to reduce toxic aldehydes including glycolysis-derived methylglyoxal (MG) and lipid peroxidation-originated malondialdehyde (MDA). The function of this enzyme in MG detoxification was demonstrated in vivo in E. coli and in transgenic plants overproducing the OsAKR1 protein. Heterologous synthesis of the OsAKR1 enzyme in transgenic tobacco plants resulted in increased tolerance against oxidative stress generated by methylviologen (MV) and improved resistance to high temperature. In these plants lower levels of MDA were detected both following MV and heat treatment due to the activity of the OsAKR1 enzyme. The transgenic tobaccos also exhibited higher AKR activity and accumulated less MG in their leaves than the wild type plants; both in the presence and absence of heat stress. These results support the positive role of OsAKR1 in abiotic stress-related reactive aldehyde detoxification pathways and its use for improvement of stress tolerance in plants.