Hypermethylation of the p16 (Ink4a) promoter in B6C3F1 mouse primary lung adenocarcinomas and mouse lung cell lines.

Primary lung tumors from B6C3F1 mice and mouse lung cell lines were examined to investigate the role of transcriptional silencing of the p16 (Ink4a) tumor suppressor gene by DNA hypermethylation during mouse lung carcinogenesis. Hypermethylation (>/=50% methylation at two or more of the CpG sites examined) of the p16 (Ink4a) promoter region was detected in DNA from 12 of 17 (70%) of the B6C3F1 primary mouse lung adenocarcinomas examined, whereas hypermethylation was not detected in normal B6C3F1, C57BL/6 and C3H/He mouse lung tissues. Immunohistochemistry performed on the B6C3F1 lung adenocarcinomas revealed heterogeneous expression of the p16 protein within and among the tumors. Laser capture microdissection was employed to collect cells from immunostained sections of four tumors displaying areas of relatively high and low p16 expression. The methylation status of the microdissected samples was assessed by sodium bisulfite genomic sequencing. The pattern of p16 expression correlated inversely with the DNA methylation pattern at promoter CpG sites in nine of 11 (82%) of the microdissected areas displaying variable p16 expression. To provide further evidence that hypermethylation is involved in the loss of p16 (Ink4a) gene expression, three mouse lung tumor cell lines (C10, sp6c and CMT64) displaying complete methylation at seven promoter CpG sites and no p16 (Ink4a) expression were treated with the demethylating agent, 5-aza-2'-deoxycytidine. Re-expression of p16 (Ink4a) and partial demethylation of the p16 (Ink4a) promoter were observed in two cell lines (C10 and sp6c) following treatment. These are the first reported studies to provide strong evidence that DNA methylation is a mechanism for p16 inactivation in mouse lung tumors.

Beta-catenin mutations and protein accumulation in all hepatoblastomas examined from B6C3F1 mice treated with anthraquinone or oxazepam.

The molecular pathogenesis of hepatoblastomas in the B6C3F1 mouse is unclear but may involve alterations in the beta-catenin/Wnt signaling pathway as was recently described for chemically induced hepatocellular neoplasms and human liver cancers. The objective of this study was to characterize the mutation frequency and spectrum of beta-catenin mutations and the intracellular localization of beta-catenin protein accumulation in chemically induced hepatoblastomas. In this study, beta-catenin mutations were identified in all 19 anthraquinone-induced hepatoblastomas and all 8 oxazepam-induced hepatoblastomas examined. Although several hepatoblastomas had multiple deletion and/or point mutations, the pattern of mutations in the hepatoblastomas did not differ from that identified in hepatocellular neoplasms. In a majority of the hepatoblastomas (six of seven) examined by immunohistochemical methods, both nuclear and cytoplasmic localization of beta-catenin protein were detected, whereas in hepatocellular adenomas, carcinomas, and normal liver only membrane staining was observed. Our data suggest that beta-catenin mutations and the subsequent translocation of beta-catenin protein from the cell membrane to the cytoplasm and nucleus may be critical steps in providing hepatocellular proliferative lesions with the growth advantage to progress to hepatoblastoma.

Effects of fixation on RNA extraction and amplification from laser capture microdissected tissue.

One of the key end points for understanding the molecular basis of carcinogenesis is the quantitation of gene expression in specific cell populations. Microdissection techniques allow extraction of morphologically distinct cells for molecular analysis. A recent advance in microdissection uses the PixCell laser capture microdissection (LCM) system, which allows for precise removal of pure cell populations from morphologically preserved tissue sections. The objective of this study was to determine the optimal fixation protocol for analyzing RNA from tissue samples using LCM. Optimal fixation must provide acceptable morphology, allow proper laser capture of selected cells, and preserve the integrity of mRNA. We evaluated the effects of both cross-linking and precipitive-type fixatives on frozen and paraffin-embedded mouse liver tissue. For assessment of the quality of the mRNA in LCM samples generated from various fixed tissues, reverse transcription-polymerase chain reaction (RT-PCR)-amplified mouse liver beta2-microglobulin mRNA was detected with ethidium bromide. We also examined mouse glyceraldehyde-3-phosphate-dehydrogenase by using the fluorogenic TaqMan system for real-time quantitative detection of RT-PCR products. Frozen tissues yielded more RT-PCR product than did paraffin-embedded tissues. In both frozen and paraffin-embedded tissues, differences were observed between the fixatives. Precipitive fixatives, such as ethanol and acetone, consistently produced more RT-PCR amplification product than did cross-linking fixatives such as formalin. Optimal fixation protocols for LCM analysis will facilitate the examination of gene expression in specific cell populations, accelerating investigations of the molecular differences responsible for the phenotypic changes observed during carcinogenesis.