Rad52 partially substitutes for the Rad51 paralog XRCC3 in maintaining chromosomal integrity in vertebrate cells.

Yeast Rad52 DNA-repair mutants exhibit pronounced radiation sensitivity and a defect in homologous re combination (HR), whereas vertebrate cells lacking Rad52 exhibit a nearly normal phenotype. Bio chemical studies show that both yeast Rad52 and Rad55-57 (Rad51 paralogs) stimulate DNA-strand exchange mediated by Rad51. These findings raise the possibility that Rad51 paralogs may compensate for lack of Rad52 in vertebrate cells, explaining the absence of prominent phenotypes for Rad52-deficient cells. To test this hypothesis, using chicken DT40 cells, we generated conditional mutants deficient in both RAD52 and XRCC3, which is one of the five vertebrate RAD51 paralogs. Surprisingly, the rad52 xrcc3 double-mutant cells were non-viable and exhibited extensive chromosomal breaks, whereas rad52 and xrcc3 single mutants grew well. Our data reveal an overlapping (but non-reciprocal) role for Rad52 and XRCC3 in repairing DNA double-strand breaks. The present study shows that Rad52 can play an important role in HR repair by partially substituting for a Rad51 paralog.

Chromosome instability and defective recombinational repair in knockout mutants of the five Rad51 paralogs.

The Rad51 protein, a eukaryotic homologue of Escherichia coli RecA, plays a central role in both mitotic and meiotic homologous DNA recombination (HR) in Saccharomyces cerevisiae and is essential for the proliferation of vertebrate cells. Five vertebrate genes, RAD51B, -C, and -D and XRCC2 and -3, are implicated in HR on the basis of their sequence similarity to Rad51 (Rad51 paralogs). We generated mutants deficient in each of these proteins in the chicken B-lymphocyte DT40 cell line and report here the comparison of four new mutants and their complemented derivatives with our previously reported rad51b mutant. The Rad51 paralog mutations all impair HR, as measured by targeted integration and sister chromatid exchange. Remarkably, the mutant cell lines all exhibit very similar phenotypes: spontaneous chromosomal aberrations, high sensitivity to killing by cross-linking agents (mitomycin C and cisplatin), mild sensitivity to gamma rays, and significantly attenuated Rad51 focus formation during recombinational repair after exposure to gamma rays. Moreover, all mutants show partial correction of resistance to DNA damage by overexpression of human Rad51. We conclude that the Rad51 paralogs participate in repair as a functional unit that facilitates the action of Rad51 in HR.

Enhanced cell killing by overexpression of dominant-negative phosphatidylinositol 3-kinase subunit, Deltap85, following genotoxic stresses.

Phosphatidylinositol 3-kinase (PI3-K) is a heterodimer of a regulatory subunit, p85, and a catalytic subunit, p110. A number of previous reports showed that PI3-K functions in diverse cellular phenomena such as cell proliferation, glucose catabolism, cell adhesion, and vesicle transport. It is also well known that a survival signal from the receptor tyrosine kinases utilizes Akt via PI3-K to protect cells from apoptosis. To examine the role of PI3-K in cellular sensitivity to genotoxic stresses such as cisplatin and ultraviolet (UV), we introduced deletion type p85 (Dp85) into two human glioblastoma cell lines (T98G and A172) and one melanoma cell line (G361). The Deltap85 works in a dominant-negative fashion on PI3-K activity by disrupting its p85 / p110 interaction. In all three transfected cell lines, the overexpression of Deltap85 rendered the cells markedly more sensitive to these DNA-damaging stresses than the cells transfected with the vector alone. Apoptosis was vigorously induced in cells overexpressing Dp85 following the treatment. The present results imply that PI3-K plays a critical role in determining cellular sensitivity to genotoxic stresses in human cancer cells, and that disruption of the p85 / p110 interaction of PI3-K may be a potential molecular target for developing a novel strategy for cancer treatment.

P53 status plays no role in radiosensitizing effects of SN-38, a camptothecin derivative.

PURPOSE: Topoisomerase inhibitors including camptothecin are being studied as potential radiosensitizers. CPT-11 is a derivative of camptothecin and is clinically available. In this study, we investigated the effects of SN-38 (an active metabolite of CPT-11) on four nonirradiated and irradiated murine fibroblast cell lines with different p53 statuses to clarify the role of p53 in the radiosensitizing activity of SN-38. MATERIALS AND METHODS: Four fibroblast cell lines, MT158, MT158/neo, MT158/wtp53 and MT158/mp53 with the same genetic background but with different p53 statuses, were used. Exponentially growing cells were treated with SN-38 (200 nM) and incubated with the drug for 30 min. Cells were then irradiated (0 to 12 Gy) and further incubated with the drug for 2 h. The cell survival rate was determined using a conventional clonogenic assay. The effects of the treatments on the cell cycle were analyzed with a flow cytometric assay. Apoptosis after these treatments was also detected by an annexin V assay. RESULTS: There were no significant differences in sensitivity to radiation or SN-38 treatment among these cell lines. The combined treatment of irradiation and SN-38 showed supraadditive effects in all four cell lines independent of their p53 status. Transient arrest in G2 with a decreased percentage of cells in both the S and G1 phases was observed 8 h after treatment with either SN-38 alone, radiation or their combination, regardless of the p53 status. No significant differences in frequency of apoptosis were observed between treatment and control groups in two cell lines with or without wild-type p53. CONCLUSION: The combination of irradiation and SN-38 treatment showed supraadditive effects in all four cell lines tested, and the p53 status did not play a role in the combination effect.

Enhanced NOR-1 gene expression by exposure of Chinese hamster cells to high-density 50 Hz magnetic fields.

Enhanced expression of neuron derived orphan receptor (NOR-1) gene was observed by exposure of Chinese hamster ovary K1 (CHO-K1) cells to an extremely low frequency magnetic field (ELFMF) of 50 Hz at 400 mT, but not at 5 mT. The enhanced expression, reaching the maximum at 6 h, was transient and reduced to the control level after exposure to 400 mT ELFMF for 24 h. The NOR-1 expression induced by treatment with forskolin and TPA was further enhanced by the simultaneous treatment with 400 mT ELFMF, in which the maximum response was at 3 h. The NOR-1 expression by these treatments was induced more earlier than that by 400 mT ELFMF alone. When cells were treated with an inhibitor of the protein kinase C (calphostin C or crocetin) and Ca2+ entry blockers (nifedipin and dantrolen) during the 400 mT ELFMF exposure, the enhanced NOR-1 expression was not observed. Exposure of CHO-K1 cells to the high-density 400 mT ELFMF may affect the signal transduction in the cells, resulting in the enhanced NOR-1 gene expression.

Systemic arterial supply to normal basilar segments of the left lower lobe: role of continuous thin sliced CT.

A rare congenital anomaly of the left lower pulmonary artery is presented. A 39-year-old woman was admitted to our hospital for evaluation of a splenic mass. Abdominal CT disclosed abnormal vessels in the left lower lobe. Angiography revealed a large systemic artery from the descending aorta supplying the basilar segments of the left lower lobe. Continuous thin sliced CT revealed the anatomical details of this anomaly and showed that the tracheobronchial tree had a normal connection with the lung parenchyma. Although the volume of the left lower lobe was reduced, there were no symptoms related to this lesion. Since the splenic lesion was malignant lymphoma limited to the spleen, only splenectomy was performed. In spite of intensive systemic chemotherapy following the operation, there were no pulmonary complications. Although surgery may be necessary in the future, observation alone has been carried out for more than 3 years because of the lack of symptoms and normal pulmonary function. Continuous thin sliced CT was useful in evaluating this anomaly.