Seiji Tachiiri

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

ABSTRACT 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.

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.

Role of NAD‐dependent deacetylases SIRT1 and SIRT2 in radiation and cisplatin‐induced cell death in vertebrate cells

Yeast Sir2 is a nicotinamide adenine dinucleotide (NAD)‐dependent histone deacetylase that plays a central role in transcriptional silencing, chromosomal stability, DNA damage response and aging. In mammals, Sir2‐like genes constitute a seven‐member family whose function is largely unknown. To investigate the role of the Sir2 family in vertebrates, we have disrupted Sir2 homologues SIRT1 and SIRT2 in the p53‐deficient chicken cell line DT40. Both SIRT1−/− and SIRT2−/− cells had mild growth defects. Colony survival assays showed moderate and mild sensitivity to cisplatin in SIRT1−/− and SIRT2−/− cells, respectively, while SIRT1−/−, but not SIRT2−/− cells, were sensitive to ionizing radiation (IR). Cells rendered doubly deficient in SIRT1 and SIRT2 exhibited the same levels of IR and cisplatin sensitivity as SIRT1−/− cells. SIRT1−/− cells appeared to be defective neither in DNA double strand break repair nor in G2/M checkpoints, but were more susceptible to cell death induction following IR than wild‐type cells. Furthermore, both SIRT1‐ and SIRT2‐deficient cells were more sensitive to pro‐apoptotic stimuli including cisplatin and staurosporine. Our results indicate that SIRT1 and SIRT2 regulate stress‐induced cell death pathways in a p53‐independent manner.

IL12RB2 and ABCA1 Genes Are Associated with Susceptibility to Radiation Dermatitis

Purpose: Severe acute radiation dermatitis is observed in approximately 5% to 10% of patients who receive whole-breast radiotherapy. Several factors, including treatment-related and patient-oriented factors, are involved in susceptibility to severe dermatitis. Genetic factors are also thought to be related to a patients susceptibility to severe dermatitis. To elucidate genetic polymorphisms associated with a susceptibility to radiation-induced dermatitis, a large-scale single-nucleotide polymorphism (SNP) analysis using DNA samples from 156 patients with breast cancer was conducted. Experimental Design: Patients were selected from more than 3,000 female patients with early breast cancer who received radiotherapy after undergoing breast-conserving surgery. The dermatitis group was defined as patients who developed dermatitis at a National Cancer Institute Common Toxicity Criteria grade of ≥2. For the SNP analysis, DNA samples from each patient were subjected to the genotyping of 3,144 SNPs covering 494 genes. Results: SNPs that mapped to two genes, ABCA1 and IL12RB2, were associated with radiation-induced dermatitis. In the ABCA1 gene, one of these SNPs was a nonsynonymous coding SNP causing R219K (P = 0.0065). As for the IL12RB2 gene, the strongest association was observed at SNP-K (rs3790568; P = 0.0013). Using polymorphisms of both genes, the probability of severe dermatitis was estimated for each combination of genotypes. These analyses showed that individuals carrying a combination of genotypes accounting for 14.7% of the Japanese population have the highest probability of developing radiation-induced dermatitis. Conclusion: Our results shed light on the mechanisms responsible for radiation-induced dermatitis. These results may also contribute to the individualization of radiotherapy.

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.

Enhanced Cell Killing by Overexpression of Dominant-negative Phosphatidylinositol 3-Kinase Subunit, Δp85, 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 (Δp85) into two human glioblastoma cell lines (T98G and A172) and one melanoma cell line (G361). The Δp85 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 Δp85 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 Δp85 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.

Influence of Radiation Dose Rate and Lung Dose on Interstitial Pneumonitis after Fractionated Total Body Irradiation: Acute Parotitis May Predict Interstitial Pneumonitis

This study evaluated patients for the influence of the dose rate and lung dose of fractionated total body irradiation (TBI) in preparation for allogeneic bone marrow transplantation (BMT) on the subsequent development of interstitial pneumonitis (IP). Sixty-six patients at our institute were treated with TBI followed by BMT. All of the patients received a total TBI dose of 12 Gy given in 6 fractions over 3 days and were divided into 3 groups according to the radiation dose rate and lung dose: group A, lung dose of 8 Gy (n = 18); group B, lung dose of 12 Gy at 8 cGy/min (n = 25); and group C, lung dose of 12 Gy at 19 cGy/min (n = 23). The overall survival rate, the cumulative incidence of relapse, and the cumulative incidence of IP were evaluated in relation to various potential indicators of future IP. There were no significant differences in survival and relapse rates between patient group A and combined groups B and C. Clinically significant IP occurred in 13 patients. The cumulative incidence of IP was significantly higher in patients who developed acute parotitis as indicated by either an elevation in the serum amylase level or parotid pain of grade 1 to 2. There was no difference in IP incidence among groups A, B, and C. There was no significant difference in IP incidence between lung dose values of 8 Gy (with lung shielding) and 12 Gy (without lung shielding) and between dose rate values of 8 cGy/min and 19 cGy/ min, at least when TBI was given in 6 fractions. The presence of acute parotitis during or just after TBI may be a predictor of IP.

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.

Comparison of dose–volume analysis between standard Manchester plan and magnetic resonance image-based plan of intracavitary brachytherapy for uterine cervical cancer

To investigate the feasibility of image-based intracavitary brachytherapy (IBICBT) for uterine cervical cancer, we evaluated the dose–volume histograms (DVHs) for the tumor and organs at risk (OARs) and compared results from the IBICBT plan and the standard Manchester system (Manchester plan) in eight patients as a simulation experiment. We performed magnetic resonance imaging (MRI) and computed tomography (CT) following MRI-adapted applicator insertion, then superimposed MR images on the planning CT images to describe the contours of high-risk clinical target volume (HR CTV) and OARs. The median volume of HR CTV was 29 cm3 (range, 21–61 cm3). Median D90 (HR CTV) and V100 (HR CTV) were 116.1% prescribed doses (PD) (90.0–150.8%) and 96.7% (84.2–100%), respectively, for the Manchester plan. In comparison, we confirmed that the median D90 (HR CTV) was 100% PD in the IBICBT plan for all patients. Mean D2cc (bladder) was 101.8% PD for the Manchester plan and 83.2% PD for the IBICBT plan. Mean D2cc (rectum) was 80.1% PD for the Manchester plan and 64.2% PD for the IBICBT plan. Mean D2cc (sigmoid) was 75% PD for the Manchester plan and 57.5% PD for the IBICBT plan. One patient with a large tumor (HR CTV, 61 cm3) showed lower D90 (HR CTV) with the Manchester plan than with the IBICBT plan. The Manchester plan may represent overtreatment for small tumors but insufficient dose distribution for larger tumors. The IBICBT plan could reduce OAR dosage while maintaining adequate tumor coverage.

A case of metachronous bilateral Breast Cancer with bilateral radiation pneumonitis after breast-conserving therapy

We report a patient with metachronous bilateral breast cancer who has twice developed radiation pneumonitis after breast-conserving therapy for each breast. The patient was a 48-year-old woman, who presented with Stage I right breast cancer. After wide excision of the right breast tumor and dissection of level I axillary lymph nodes, systemic therapy with oral 5-FU and tamoxifen was started. Subsequently, tangential irradiation with a total dose of 50 Gy in 25 fractions was given. Seven months after irradiation, she developed respiratory symptoms and radiation pneumonitis was diagnosed. The symptoms resolved with oral prednisolone. Thirty months after the right breast cancer treatment, Stage I left breast cancer was diagnosed. After wide excision of the left breast tumor and partial removal of the level I axillary lymph nodes, the same oral systemic chemo-hormonal therapy was initiated. Thereafter, tangential irradiation with a total dose of 50 Gy in 25 fractions was given. Four months after irradiation, she developed respiratory symptoms. A chest X-ray showed an area of increased density in the left lung consistent with radiation pneumonitis. The symptoms were mild and they improved spontaneously without medication. Although there is insufficient evidence to justify or withhold whole breast radiation therapy from patients with a history of contralateral breast cancer and radiation pneumonitis, it is essential to discuss the adequacy of whole breast irradiation and the possibility of alternative approaches, such as breast-conserving surgery without irradiation or partial breast irradiation for this rare condition.