Defective DNA Strand Break Repair Causes Chromosomal Instability and Accelerates Liver Carcinogenesis in Mice

Chromosomal instability is a characteristic feature of hepatocellular carcinoma (HCC) but its origin and role in liver carcinogenesis are undefined. We tested whether a defect in the nonhomologous end-joining (NHEJ) DNA repair gene Ku70 was associated with chromosomal abnormalities and enhanced liver carcinogenesis. Male Ku70 NHEJ-deficient (Ku70-/-), heterozygote (Ku70 +/-), and wild-type (WT) mice were injected with diethylnitrosamine (DEN), a liver carcinogen, at age 15 days. Animals were killed at 3, 6, and 9 months for assessment of tumorigenesis and hepatocellular proliferation. For karyotype analysis, primary liver tumor cell cultures were prepared from HCCs arising in Ku70 mice of all genotypes. Compared to WT littermates, Ku70-/- mice injected with DEN displayed accelerated HCC development. Ku70-/- HCCs harbored clonal increases in numerical and structural aberrations of chromosomes 4, 5, 7, 8, 10, 14, and 19, many of which recapitulated the spectrum of equivalent chromosomal abnormalities observed in human HCC. Ku70-/- HCCs showed high proliferative activity with increased cyclin D1 and proliferating cell nuclear antigen expression, Aurora A kinase activity, enhanced ataxia telangiectasia mutated kinase and ubiquitination, and loss of p53 via proteasomal degradation, features which closely resemble those of human HCC. Conclusion: These findings demonstrate that defects in the NHEJ DNA repair pathway may participate in the disruption of cell cycle checkpoints leading to chromosomal instability and accelerated development of HCC

Antimetastatic gene expression profiles mediated by retinoic acid receptor beta 2 in MDA-MB-435 breast cancer cells

BACKGROUND: The retinoic acid receptor beta 2 (RARβ2) gene modulates proliferation and survival of cultured human breast cancer cells. Previously we showed that ectopic expression of RARβ2 in a mouse xenograft model prevented metastasis, even in the absence of the ligand, all-trans retinoic acid. We investigated both cultured cells and xenograft tumors in order to delineate the gene expression profiles responsible for an antimetastatic phenotype. METHODS: RNA from MDA-MB-435 human breast cancer cells transduced with RARβ2 or empty retroviral vector (LXSN) was analyzed using Agilent Human 1A Oligo microarrays. The one hundred probes with the greatest differential intensity (p < 0.004, jointly) were determined by selecting the top median log ratios from eight-paired microarrays. Validation of differences in expression was done using Northern blot analysis and quantitative RT-PCR (qRT-PCR). We determined expression of selected genes in xenograft tumors. RESULTS: RARβ2 cells exhibit gene profiles with overrepresentation of genes from Xq28 (p = 2 × 10(-8)), a cytogenetic region that contains a large portion of the cancer/testis antigen gene family. Other functions or factors impacted by the presence of exogenous RARβ2 include mediators of the immune response and transcriptional regulatory mechanisms. Thirteen of fifteen (87%) of the genes evaluated in xenograft tumors were consistent with differences we found in the cell cultures (p = 0.007). CONCLUSION: Antimetastatic RARβ2 signalling, direct or indirect, results in an elevation of expression for genes such as tumor-cell antigens (CTAG1 and CTAG2), those involved in innate immune response (e.g., RIG-I/DDX58), and tumor suppressor functions (e.g., TYRP1). Genes whose expression is diminished by RARβ2 signalling include cell adhesion functions (e.g, CD164) nutritional or metabolic processes (e.g., FABP6), and the transcription factor, JUN

Time scale of comet measurement of DNA fragment presence by alkaline integrated comet assay

<p><b>Copyright information:</b></p><p>Taken from "Radiation cataracts: mechanisms involved in their long delayed occurrence but then rapid progression"</p><p></p><p>Molecular Vision 2008;14():274-285.</p><p>Published online 05 Feb 2008</p><p>PMCID:PMC2254966.</p><p></p> This assay integrates the content of migrating DNA fragments at a progressive distance in the gel where the extreme margin is the tip of the comet tail, i.e., integrating the DNA fragment content per linear distance, with the furthest progression through the gel. Means and SEM are shown