Kenshi Komatsu

Genetic Mapping Using Microcell-Mediated Chromosome Transfer Suggests a Locus for Nijmegen Breakage Syndrome at Chromosome 8q21-24

Nijmegen breakage syndrome (NBS) is an autosomal recessive disorder characterized by microcephaly, short stature, immunodeficiency, and a high incidence of cancer. Cultured cells from NBS show chromosome instability, an increased sensitivity to radiation-induced cell killing, and an abnormal cell-cycle regulation after irradiation. Hitherto, patients with NBS have been divided into the two complementation groups V1 and V2, on the basis of restoration of radioresistant DNA synthesis, suggesting that each group arises from a different gene. However, the presence of genetic heterogeneity in NBS has been considered to be controversial. To localize the NBS gene, we have performed functional complementation assays using somatic cell fusion between NBS-V1 and NBS-V2 cells, on the basis of hyper-radiosensitivity, and then have performed a genomewide search for the NBS locus, using microcell-mediated chromosome transfer followed by complementation assays based on radiosensitivity. We found that radiation resistance was not restored in the fused NBS-V1 and NBS-V2 cells and that only human chromosome 8 complements the sensitivity to ionizing radiation, in NBS cell lines. In complementation assays performed after the transfer of a reduced chromosome, merely the long arm of chromosome 8 was sufficient for restoring the defect. Our results strongly suggest that NBS is a homogeneous disorder and that the gene for NBS is located at 8q21-24.

Fine localization of the Nijmegen breakage syndrome gene to 8q21: Evidence for a common founder haplotype

Nijmegen breakage syndrome (NBS) is a rare autosomal recessive disorder characterized by microcephaly, a birdlike face, growth retardation, immunodeficiency, lack of secondary sex characteristics in females, and increased incidence of lymphoid cancers. NBS cells display a phenotype similar to that of cells from ataxia-telangiectasia patients, including chromosomal instability, radiation sensitivity, and aberrant cell-cycle-checkpoint control following exposure to ionizing radiation. A recent study reported genetic linkage of NBS to human chromosome 8q21, with strong linkage disequilibrium detected at marker D8S1811 in eastern European NBS families. We collected a geographically diverse group of NBS families and tested them for linkage, using an expanded panel of markers at 8q21. In this article, we report linkage of NBS to 8q21 in 6/7 of these families, with a maximum LOD score of 3.58. Significant linkage disequilibrium was detected for 8/13 markers tested in the 8q21 region, including D8S1811. In order to further localize the gene for NBS, we generated a radiation-hybrid map of markers at 8q21 and constructed haplotypes based on this map. Examination of disease haplotypes segregating in 11 NBS pedigrees revealed recombination events that place the NBS gene between D8S1757 and D8S270. A common founder haplotype was present on 15/18 disease chromosomes from 9/11 NBS families. Inferred (ancestral) recombination events involving this common haplotype suggest that NBS can be localized further, to an interval flanked by markers D8S273 and D8S88.

Restoration of radiation resistance in ataxia telangiectasia cells by the introduction of normal human chromosome 11

In order to identify the human chromosome which carries a mutated gene in cells from a patient with the hereditary disorder ataxia telangiectasia belonging to complementation group D (AT-D), we performed chromosome transfer experiments via microcell fusion. A single, pSV2neo-tagged chromosome, either 11 or 12, derived from normal human fibroblasts was introduced into AT-D cells by microcell fusion, and clones which were resistant to the antibiotic G418 were isolated. All 3 hybrid clones containing an additional copy number of chromosome 11 showed a restoration of the resistance of wild-type cells to killing by X-irradiation, whereas all 3 hybrid clones containing an additional copy number of chromosome 12 remained hyper-radiosensitive, like the parental AT cells. The results indicate that a defective gene of AT-D cells is also located on chromosome 11, since a genetic linkage analysis has previously suggested that a defective gene of its complementation group A is located on this chromosome.

Lack of Correlation between Radiosensitivity and Inhibition of DNA Synthesis in Hybrids (A-T × HeLa)

Hybrid cells obtained from A-T and D98/AH(HeLa) cells showed normal radiation sensitivity to cell killing, but retained radioresistant DNA synthesis similar to parental A-T cells.

Inhibitory effects of Rooibos tea, Aspalathus linealis, on X-ray-induced C3H10T1/2 cell transformation.

Oncogenic transformation of mouse C3H10T1/2 cells induced by X-rays was suppressed in the presence of extract of Rooibos tea, Aspalathus linealis. Transformation was reduced with increased concentration of the extract, so that at an extract concentration of 10%, transformation incidence was similar to the spontaneous level. Suppression was also dependent on treatment time with the extract and was maximal when present during the entire incubation period. In contrast, green tea extract at an equitoxic concentration showed no detectable effect on transformation incidence.

Murine Scid Cells Complement Ataxia-telangiectasia Cells and Show a Normal Post-irradiation Response of DNA Synthesis

The murine severe combined immunodeficient mutation (scid) is characterized by a lack of both B and T cells, due to a defect in lymphoid variable-(diversity)-joining (V(D)J) rearrangement. Scid cells are highly sensitive to both radiation-induced killing and chromosomal aberrations. Significantly reduced D0 and n values were demonstrated in scid cells and were similar to ataxia-telangiectasia (AT) cells (a unique human disease conferring whole body radiosensitivity). However, the kinetics of DNA synthesis after irradiation were different between the two cell types. In contrast with the radioresistant DNA synthesis of AT cells, DNA synthesis of scid cells was markedly inhibited after irradiation. The existence of different mutations was also supported by evidence of complementation in somatic cell hybrids between scid cells and AT cells. Our results indicate that the radiobiological character of scid is similar to AT but is presumably caused by different mechanisms.

Suppression of X-ray-induced chromosome aberrations in ataxia telangiectasia cells by introduction of a normal human chromosome 11

We studied X-ray-induced chromosome aberrations in ataxia telangiectasia (AT) cells containing an introduced chromosome 11 or 12 derived from normal human fibroblasts. We used microcell-mediated chromosome transfer to introduce the normal chromosomes into AT cells belonging to complementation group D. Cells were irradiated with 1 Gy of X-rays in the G2 phase. All 5 hybrid clones with an introduced chromosome 11 showed a reduction in the frequency of chromatid-type aberrations to normal levels, whereas all 4 hybrid clones with an introduced chromosome 12 failed to show this reduction. This finding, taken together with our previous report that chromosome 11 can restore radioresistant cell killing in AT cells, indicates that a defective gene on chromosome 11 in AT cells is responsible for the hypersensitivity to not only cell killing but also chromosome aberrations. Our results suggest that a putative AT gene on chromosome 11 plays an important role in the repair process of radiation-induced DNA damage that leads to chromosome aberrations.

Dose-rate Effects of Neutrons and γ-rays on the Induction of Mutation and Oncogenic Transformation in Plateau-phase Mouse m5S Cells

The dose-rate effect of 252-californium neutrons was investigated using confluent cultures of mouse m5S cells. The relative biological effectiveness (RBE) of neutrons for oncogenic transformation was increased from 3.3 to 5.1 when the dose-rate was reduced from 1.8 to 0.12 cGy/min. Similarly, neutron RBE values for HPRT- mutation were 4.9 and 7.4 at dose-rates of 1.8 and 0.12 cGy/min, respectively. The increases in RBE as dose-rate was reduced were due mainly to diminished transformation- and mutation-induction by gamma-rays (the standard radiation). The yields of neutron-induced oncogenic transformation as well as neutron-induced mutation were constant for both dose rates. Our observation contrasts with reports by others using proliferating cells where both oncogenic transformation and mutation were enhanced with neutron exposure at a reduced dose-rate, the so-called inverse dose-rate effect. Since m5S cells are sensitive to postconfluent inhibition of cell division, this observation could be ascribed to cell growth conditions used in these experiments. The mechanism of the inverse dose-rate effect of neutrons suggests that the enhancement of neutron-induced mutation and oncogenic transformation at a reduced dose-rate is strongly associated with cell proliferation during exposure.

Four novel mutations of the Fanconi anemia group A gene ( FAA ) in Japanese patients

AbstractFanconi anemia (FA) is an autosomal recessive disorder characterized by pancytopenia, predisposition to cancers, and a diverse variety of congenital malformations. At least eight complementation groups, A through H, have been described. Recently, the FA-A gene (FAA) has been isolated, and a large number of distinct mutations reported in ethnically diverse FA-A patients. Here, we report on the mutation analysis of five FA patients by single-strand conformation polymorphism. Out of five patients, at least three were found to have mutations in the FAA gene. The first patient was a compound heterozygote with a 1-bp deletion and a single-base substitution. The second patient had a heterozygous 2-bp deletion, which introduces a premature termination codon, and the third patient had a heterozygous splice donor site mutation in intron 27.

Inhibitory action of (-)-epigallocatechin gallate on radiation-induced mouse oncogenic transformation.

The anticarcinogenic activity of a major component of green tea, (-) epigallocatechin gallate (EGCg) was examined by using the radiation-induced oncogenic transformation in C3H10T1/2 cells. EGCg substantially suppressed the radiation-induced transformation so that the transformation frequency with 15 microM of EGCg was reduced nearly to spontaneous levels. This effect of EGCg was in a dose-dependent manner and significant suppression of transformation was observed even in treatment of cells with 5 microM of EGCg concentration where the cytotoxicity was mild. The inhibitory effect of EGCg was maximal when it was present during the entire incubation period. However, neither treatment prior to nor concurrent with radiation was effective, suggesting that EGCg action is mainly involved in the promotional stage of C3H10T1/2 cell transformation.