Effects of NKG2D haplotypes on the cell-surface expression of NKG2D protein on natural killer and CD8 T cells of peripheral blood among atomic-bomb survivors.

NKG2D is a primary activating receptor that triggers cell-mediated cytotoxicity in NK cells against tumor and virus-infected cells. We previously identified the NKG2D haplotypes in the natural killer gene complex region on chromosome 12p. Two major haplotype alleles, LNK1 and HNK1, were closely related to low and high natural cytotoxic activity phenotypes, respectively. Furthermore, the haplotype of HNK1/HNK1 has revealed a decreased risk of cancer compared with LNK1/LNK1. In the present study, using flow cytometry, we evaluated the functional effects of NKG2D haplotypes and five htSNPs in terms of the cell-surface expression of NKG2D protein on NK and CD8 T cells of peripheral blood among 732 atomic-bomb survivors. NKG2D expression on NK cells showed significant increases, in the order of LNK1/LNK1, LNK1/HNK1 and HNK1/HNK1 haplotypes (p for trend=0.003), or with major homozygous, heterozygous, and minor homozygous genotypes for individual htSNPs (p for trend=0.02-0.003). The same trend was observed for NKG2D expression on CD8 T cells. Our findings indicate that the NKG2D haplotypes are associated with the expression levels of NKG2D protein on NK and CD8 T cells, resulting in inter-individual variations in human cytotoxic response.

SKY and FISH analysis of radiation-induced chromosome aberrations: a comparison of whole and partial genome analysis.

For a retrospective dose estimation of human exposure to ionising radiation, a partial genome analysis is routinely used to quantify radiation-induced chromosome aberrations. For this purpose, fluorescence in situ hybridisation (FISH) with whole chromosome painting probes for selected chromosomes is usually applied covering about 20% of the whole genome. Since genome-wide screening techniques like spectral karyotyping (SKY) and multiplex FISH (mFISH) have been developed the detection of radiation-induced aberrations within the whole genome has now become feasible. To determine the correspondence between partial and whole genome analysis of radiation-induced chromosome aberrations, they were measured comprehensively in this study using in vitro irradiated blood samples from three donors. We were able to demonstrate that comparable results can be detected with both approaches. However, complex aberrations might be misinterpreted by partial genome analysis. We therefore conclude that whole genome analysis by SKY is useful especially in the high dose range to correct aberration data for complex exchange aberrations.

Spontaneous and X-ray-induced chromosomal aberrations in Werner syndrome cells detected by FISH using chromosome-specific painting probes.

Werner syndrome (WS) is a rare autosomal disorder characterized by premature aging exhibiting chromosome instability and predisposition to cancer. Cells derived from WS patients show a variety of constitutionally stable chromosomal aberrations as detected by conventional chromosome banding techniques. We have employed the fluorescence in situ hybridization (FISH) technique using painting probes for 12 different chromosomes to detect stable chromosome exchanges in three WS cell lines and three control cell lines. WS cell lines showed increased frequencies of both stable and unstable chromosome aberrations detected by FISH and Giemsa staining, respectively. One WS lymphoblastoid cell line (KO375) had a 5/12 translocation in all the cells and approximately 60% of the cells had an additional translocated chromosome 12. A high frequency of aneuploid cells was found in all the WS cell lines studied. Though WS cells are known to be chromosomally unstable, unlike other chromosome instability syndromes they are not sensitive to mutagenic agents. We studied the frequencies of X-ray-induced chromosomal aberrations in two WS cell lines and found an approximately 60% increase in the frequencies of fragments and no consistent increase in the frequencies of exchanges.

A radiation hybrid map with 60 loci covering the entire short arm of chromosome 12.

We present a high-resolution radiation hybrid map of the short arm of human chromosome 12 containing 60 loci, including 44 STSs within or closely associated with expressed sequences, 11 highly polymorphic markers, 2 anonymous sequences, 2 subtelomeric sequences, and 1 centromeric sequence. The 60 loci fell into 48 unique retention patterns, providing a comprehensive map covering the entire short arm of chromosome 12 with an average resolution of approximately 800 kb. Twenty-two unique positions were ordered in a 1000:1 framework map with an average resolution of 1.8 Mb. The proposed order is in good agreement with recently published genetic maps, high-resolution FISH maps, and YAC contigs. The noted inconsistencies involved neighboring loci permutations. Our observations further suggest the existence of chromosomal "hot spots" for breakage during irradiation. In three regions an usually high number of breaks was noted between neighboring loci compared to the physical distance derived from existing YAC contigs. Some of these hot spots seem to coincide with known chromosomal aberrations, of which at least two have been involved in the etiology of cancer.

Radiation-induced chromosome aberrations analysed by two-colour fluorescence in situ hybridization with composite whole chromosome-specific DNA probes and a pancentromeric DNA probe.

Fluorescence in situ hybridization with composite whole chromosome-specific DNA probes for human chromosomes 1, 4 and 12 and a degenerate alpha-satellite pancentromeric DNA probe labelled with digoxigenin was used to measure symmetrical translocations and dicentrics induced in vitro by 137Cs gamma-rays (0-6.0 Gy) in peripheral lymphocytes. Despite subtracting our mean background translocation frequency of 0.0016 per cell (11,411 cells scored from 11 individuals) from induced frequencies, about 1.3-1.8-fold more translocations were found than dicentrics at a given dose. Translocation frequencies determined only in stable cells agree well with total translocation frequencies determined also in cells containing additional unstable chromosomal changes. The linear quadratic calibration curve generated for total stable translocations is based on approx. 17,000 cells. The suitability of this curve for biological dosimetry of human radiation exposure can now be evaluated in comparison with dose estimates based on a conventional dicentric dose-response curve.

Radiation-induced chromosome aberrations analysed by fluorescence in situ hybridization with a triple combination of composite whole chromosome-specific DNA probes.

Fluorescence in situ hybridization (FISH) with a combination of three composite whole chromosome-specific DNA probes for human chromosomes 1, 4 and 12 was used to analyse in vitro radiation-induced dicentrics and symmetrical translocations in peripheral lymphocytes. Translocations could be rapidly and efficiently detected by FISH. Their frequencies were 1.8-fold higher than the frequencies for dicentrics at a given dose. The dose-response curves for translocations and dicentrics were linear quadratic with a significant higher quadratic component for translocations. The application of FISH for scoring stable translocations for biological dosimetry of radiation exposures is discussed.

Generation of a panel of somatic cell hybrids containing fragments of human chromosome 12P by X-ray irradiation and cell fusion.

We have employed an irradiation and fusion procedure to generate somatic cell hybrids containing various fragments of the short arm of human chromosome 12 using a 12p-only hybrid (M28) as starting material. For the initial identification of hybrids retaining human DNA, nonradioactive in situ hybridization was performed. Seventeen cell lines appeared to contain detectable amounts of human material. Detailed characterization of these hybrids by Southern blot analysis and chromosomal in situ suppression hybridization (chromosome painting), using hybrid DNAs as probes after Alu element-mediated PCR, resulted in a hybrid panel encompassing the entire chromosome 12p arm. This panel will provide a valuable resource for the rapid isolation of region-specific DNA markers. In addition, this panel may be useful for the characterization of chromosome 12 aberrations in, e.g., human germ cell tumors.