The defect in the AT-like hamster cell mutants is complemented by mouse chromosome 9 but not by any of the human chromosomes

X-ray-sensitive Chinese hamster V79 cells mutants, V-C4, V-E5 and V-G8, show an abnormal response to X-ray-induced DNA damage. Like ataxia telangiectasia (AT) cells, they display increased cell killing, chromosomal instability and a diminished inhibition of DNA synthesis following ionizing radiation. To localize the defective hamster gene (XRCC8) on the human genome, human chromosomes were introduced into the AT-like hamster mutants, by microcell mediated chromosome transfer. Although, none of the human chromosomes corrected the defect in these mutants, the defect was corrected by a single mouse chromosome, derived from the A9 microcell donor cell line. In four independent X-ray-resistant microcell hybrid clones of V-E5, the presence of the mouse chromosome was determined by fluorescent in situ hybridization, using a mouse cot-1 probe. By PCR analysis with primers specific for different mouse chromosomes and Southern blot analysis with the mouse Ldlr probe, the mouse chromosome 9, was identified in all four X-ray-resistant hybrid clones. Segregation of the mouse chromosome 9 from these hamster-mouse microcell hybrids led to the loss of the regained X-ray-resistance, confirming that mouse chromosome 9 is responsible for complementation of the defect in V-E5 cells. The assignment of the mouse homolog of the ATM gene to mouse chromosome 9, and the presence of this mouse chromosome only in the radioresistant hamster cell hybrids suggest that the hamster AT-like mutants are homologous to AT, although they are not complemented by human chromosome 11

Dysregulation of Gene Expression in the Artificial Human Trisomy Cells of Chromosome 8 Associated with Transformed Cell Phenotypes

A change in chromosome number, known as aneuploidy, is a common characteristic of cancer. Aneuploidy disrupts gene expression in human cancer cells and immortalized human epithelial cells, but not in normal human cells. However, the relationship between aneuploidy and cancer remains unclear. To study the effects of aneuploidy in normal human cells, we generated artificial cells of human primary fibroblast having three chromosome 8 (trisomy 8 cells) by using microcell-mediated chromosome transfer technique. In addition to decreased proliferation, the trisomy 8 cells lost contact inhibition and reproliferated after exhibiting senescence-like characteristics that are typical of transformed cells. Furthermore, the trisomy 8 cells exhibited chromosome instability, and the overall gene expression profile based on microarray analyses was significantly different from that of diploid human primary fibroblasts. Our data suggest that aneuploidy, even a single chromosome gain, can be introduced into normal human cells and causes, in some cases, a partial cancer phenotype due to a disruption in overall gene expression

Topoisomerase II cleavage activity within the human D11Z1 and DXZ1 alpha-satellite arrays.

Topoisomerase II (Topo II) is a major component of mitotic chromosomes and its unique decatenating activity has been implicated in many aspects of chromosome dynamics including DNA replication, transcription, recombination, chromosome condensation and segregation. Of these, chromosome segregation is the most seriously affected by loss of Topo II, most probably because of residual catenations between sister chromatids. At metaphase, vertebrate chromatids are attached principally through their centromeric regions. Intriguingly, evidence has recently been presented for Topo II cleavage activity within the centromeric alpha-satellite DNA arrays of the human X and Y chromosomes. In this report we extend these observations by mapping distinct sites of Topo II cleavage activity within the alpha-satellite array of human chromosome 11. A single major site of cleavage has been assigned within the centromeric DNA of each of three independently derived, and active, 11 centromeres. Unlike the X and Y centromeres, where cleavage sites mapped close to (within 150 kb of) the short arm edge of the arrays, on chromosome 11, the cleavage sites lie many hundreds of kilobases into each alpha-satellite array. We also demonstrate that catalytically active Topo II is concentrated within the centromere domain through an extended period of G2 and M, with levels declining in G1 and S

Introduction of a Normal Human Chromosome 8 Corrects Abnormal Phenotypes of Werner Syndrome Cells Immortalized by Expressing an hTERT Gene

Werner syndrome (WS) is an autosomal recessive disease characterized by premature aging and caused by mutations of the WRN gene mapped at 8p12. To examine functional complementation of WS phenotypes, we introduced a normal human chromosome 8 into a strain of WS fibroblasts (WS3RGB) immortalized by expressing a human telomerase reverse transcriptase subunit (hTERT) gene. Here, we demonstrate that the abnormal WS phenotypes including cellular sensitivities to 4-nitroquinoline-1-oxide (4NQO) and hydroxy urea (HU), and chromosomal radiosensitivity at G2 phase are corrected by expression of the WRN gene mediated by introducing a chromosome 8. This indicates that those multiple abnormal WS phenotypes are derived from a primary, but not secondary, defect in the WRN gene