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Humans are frequently exposed to acrylamide, a probable human carcinogen found in commonplace sources such as most heated starchy foods or tobacco smoke. Prior evidence has shown that acrylamide causes cancer in rodents, yet epidemiological studies conducted to date are limited and, thus far, have yielded inconclusive data on association of human cancers with acrylamide exposure. In this study, we experimentally identify a novel and unique mutational signature imprinted by acrylamide through the effects of its reactive metabolite glycidamide. We next show that the glycidamide mutational signature is found in a full one-third of approximately 1600 tumor genomes corresponding to 19 human tumor types from 14 organs. The highest enrichment of the glycidamide signature was observed in the cancers of the lung (88% of the interrogated tumors), liver (73%), kidney (>70%), bile duct (57%), cervix (50%), and, to a lesser extent, additional cancer types. Overall, our study reveals an unexpectedly extensive contribution of acrylamide-associated mutagenesis to human cancers.

Progression to malignancy requires that cells overcome senescence and switch to an immortal phenotype. Thus, exploring the genetic and epigenetic changes that occur during senescence/immortalization may help elucidate crucial events that lead to cell transformation. In the present study, we have globally profiled DNA methylation in relation to gene expression in primary, senescent and immortalized mouse embryonic fibroblasts. Using a high-resolution genome-wide mapping technique, followed by extensive locus-specific validation assays, we have identified 24 CpG islands that display significantly higher levels of CpG methylation in immortalized cell lines as compared to primary murine fibroblasts. Several of these hypermethylated CpG islands are associated with genes involved in the MEK-ERK pathway, one of the most frequently disrupted pathways in cancer. Approximately half of the hypermethylated targets are developmental regulators, and bind to the repressive Polycomb group (PcG) proteins, often in the context of bivalent chromatin in mouse embryonic stem cells. Because PcG-associated aberrant DNA methylation is a hallmark of several human malignancies, our methylation data suggest that epigenetic reprogramming of pluripotency genes may initiate cell immortalization. Consistent with methylome alterations, global gene expression analysis reveals that the vast majority of genes dysregulated during cell immortalization belongs to gene families that converge into the MEK-ERK pathway. Additionally, several dysregulated members of the MAP kinase network show concomitant hypermethylation of CpG islands. Unlocking alternative epigenetic routes for cell immortalization will be paramount for understanding crucial events leading to cell transformation. Unlike genetic alterations, epigenetic changes are reversible events, and as such, can be amenable to pharmacological interventions, which makes them appealing targets for cancer therapy when genetic approaches prove inadequate.

Methylated cytosines within CpG dinucleotides (mCpGs) along the DNA-binding domain of the TP53 tumor suppressor gene (exons ~5-8) are the single most significant mutational target in human cancers. The human p53 knock-in (Hupki) mouse model was constructed using gene-targeting technology to create a mouse strain that harbors human wild-type TP53 DNA sequences spanning exons 4-9 in both copies of the mouse p53 gene. To date, however, the methylation status of cytosines within CpGs in the substituted segment of the Hupki mouse genome has not been determined. This lack of information deserves special attention because DNA methylation in mammals, which occurs almost exclusively within CpG dinucleotides, is a dynamic process throughout developmental stages and may vary among different species. Here, we have investigated the status of CpG methylation in the substituted segment of the Hupki mouse genome, and compared it to the methylation profile of the corresponding segment in the human genome using the combined bisulfite-restriction analysis and sodium bisulfite genomic sequencing. We found that all cytosines within CpGs of the TP53 DNA-binding domain, on both the coding and noncoding strands, were heavily methylated in Hupki fibroblasts, as they were in human fibroblasts. This is in keeping with the fully methylated status of TP53 CpGs that is known to prevail in adult human tissues. The remarkably similar patterns of cytosine methylation within CpG dinucleotides in Hupki cells and human cells further validates the suitability of mutagenesis assays in Hupki cells for experimental induction of TP53 mutations that have been observed in human tumors.

Analysis of mutations in the p53 tumor suppressor gene can assist in the diagnosis, prognosis and treatment of cancer. The p53 gene is one of several genes that control DNA repair and monitor the modification process of the human genome. Mutations in this gene have been associated with the development of many types of tumors including hepatocellular and squamous-cell carcinomas. Diagnostic uses of the p53 gene include using it as a biochemical marker in the analysis of cancerous cells. The p53 gene is a useful indicator of disease progression through measurement of such factors as the accumulation of p53 proteins, the number and type of mutations, and the loss of p53 function. Finally, possible p53-based treatments are include its use as a tumor-specific antigen in immune therapy by the production of drugs that would imitate its tumor suppressing function and applications in radiation therapy.

DNA from four human esophageal carcinoma cell lines and 50 primary esophageal carcinomas, taken from various countries for expansion of the cyclin D1 gene, were examined. About 36 of the 50 carcinomas were examined by immunohistochemistry for cyclin D1 and retinoblastoma (RB) protein expression. Approximately 16 tumors and 2 cell lines exhibited a three to ten fold expansion in cyclin D1 gene. During evolution of tumors through reduced RB gene expression or through enhanced cyclin D1 gene expression, the restrictive influence of RB on cell cycle development can be removed.