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Biomedical subjects

D J Chen

Publications and source records attributed to D J Chen.

At least 73 records · Page 4Linked to original sources
PubMed1996

Disruption of DNA-PK in Ku80 mutant xrs-6 and the implications in DNA double-strand break repair.

The Chinese hamster ovary (CHO) mutant cell line xrs-6C is highly sensitive to radiation and is deficient in DNA double-strand break (DSB) repair. The repair defect of xrs-6C is complemented by the human DSB repair gene designated as XRCC5. This gene was recently identified as Ku80, which encodes the human autoantigen protein Ku p80. Ku80 protein forms heterodimer with the Ku70 subunit to form a complex that possesses a DNA end-binding activity. Ku70/Ku80 heterodimer can recruit the catalytic p350 subunit of the DNA-dependent protein kinase. It is demonstrated here that, while the Ku70 mRNA expression is normal in the xrs-6C mutant, Ku70 protein is undetectable. However, introduction of human Ku80 gene into the mutant lead to increased expression of Ku70 protein and restored Ku70 binding to DNA ends, suggesting that mutation of the Ku80 gene affected the formation of Ku70/Ku80 dimers and the stability of the Ku70 protein. We also demonstrated that, although p350 protein expression in the mutants was unaffected, the capacity of p350 to bind to DNA ends was impaired in the mutants. After introduction of the human Ku80 into the mutant, the association of p350 with DNA end was restored, accompanied by recovery in cell survival and DNA double-strand break repair. The results in this report show that mutation of the Ku80 gene disrupts formation of the Ku70/Ku80 dimer and compromises the ability of Ku protein to recruit the DNA-PK p350 subunit to DNA double-strand breaks, causing a dysfunction of DNA DSB repair in the cell.

Animals↗
PubMed1996

The effect of track structure on cell inactivation and chromosome damage at a constant LET of 120 keV/micrometer.

The influence of track structure on chromosome damage and cell inactivation are being investigated. Plateau-phase normal human fibroblast cultures were irradiated with gamma rays, and He, Ne and Ar ions. Particle velocities were chosen so that all beams had an LET of 120 keV/micrometer. In this constant-LET experimental design, the radial distribution of excitations and ionizations about the particle track is the most significant variable. Using premature chromosome condensation, chromatin breaks were measured at two time points, promptly after irradiation and after a prolonged incubation to allow for repair. These measurements give an indication of both initial chromosomal damage and also residual damage that is either not repaired or is misrepaired. Survival was measured under the same conditions. Results indicate that the RBEs for both cell inactivation and, to a lesser extent, chromosome damage decrease as particle energy increases.

Alpha Particles↗
PubMed1996

Ionizing radiation-induced mutation of human cells with different DNA repair capacities.

We have observed significant differences in the response to ionizing radiation of two closely related human cells lines, and now compare the effects on these lines of both low and intermediate LET radiation. Compared to TK6, WTK1 has an enhanced X-ray survival, and is also more resistant to cell killing by alpha-particles. The hprt locus is more mutable in WTK1 than in TK6 by both X-rays and alpha-particles. WTK1 is also more mutable by alpha-particles than by X-rays at the hprt locus. X-ray-induced mutation at the heterozygous tk locus in WTK1 is about 25 fold higher than in TK6, while alpha-particle-induced mutation is nearly 50 fold higher at this locus. Also, the slowly growing tk- mutants, which comprise the majority of spontaneous and X-ray-induced tk- mutants of TK6, were not induced significantly by alpha-particles. Previously, we showed that TK6 has a reduced capacity for recombination compared with WTK1, and therefore, these results indicate that recombinational repair may contribute to both cell survival and mutation-induction following exposure to ionizing radiation. Such a mechanism may aid cell survival, but could also result in increased deleterious effects such as the unmasking of recessive mutations in cancer suppresser genes.

Alpha Particles↗
PubMed1996

Alpha particle mutagenesis of human lymphoblastoid cell lines.

Despite being derived from the same donor, the human lymphoblastoid cell lines WTK1 and TK6 have markedly different responses to low LET radiation. We originally observed that WTK1 was more resistant to the cytotoxic effects of X-irradiation, but significantly more sensitive to mutation induction at both the TK and HPRT loci. In an effort to better understand these properties, we have examined the effects of alpha-particles on these cells. Relative to TK6, WTK1 has enhanced survival and mutation after both X-ray and alpha-particle exposure. While the HPRT locus was significantly more mutable in WTK1 as a function of alpha-particle versus X-ray dose, the TK locus was only slightly more sensitive to alpha-particle mutagenesis. In addition, the slowly growing TK mutants that constitute the majority of X-ray-induced TK mutants of TK6 were recovered in lower proportions following alpha-particle exposures. This is consistent with the further finding that in both cell lines, loss of heterozygosity occurred in a smaller fraction of alpha-induced TK mutants than X-ray-induced mutants. These results are consistent with our previous model suggesting that WTK1 has an error-prone repair pathway that is either missing or deficient in TK6, and further suggest that this pathway may be involved in the processing of alpha-particle-induced damage.

Alpha Particles↗
PubMed1996

Inverse dose-rate effect for mutation induction by gamma-rays in human lymphoblasts.

In order to define further the effects of differences in recombinational proficiency on cell survival and mutation by ionizing radiation, we exposed the syngenic cell lines TK6 and WTK1 to continuous low dose-rate gamma-irradiation. We previously demonstrated that acute X-ray exposure results in lower survival and lower mutation induction at both the thymidine kinase (tk) and the hypoxanthine-guanine phosphoribosyltransferase (hprt) loci in TK6 cells compared with WTK1 cells. These differences were attributed in part to reduced levels of recombination in the TK6 line relative to WTK1. Using a low dose rate 137Cs irradiator, we exposed asynchronous growing populations of these cells to gamma-rays at 14.3, 6.7 and 2.7 cGy/h. Both cell lines exhibited a dose-rate effect on survival. Compared with acute doses, the low dose-rates also protected against mutation induction at the hrpt locus in WTK1, but protection was inversely related to dose-rate. There was also a slight inverse dose-rate effect in TK6, with mutation induction at the lowest dose-rate exceeding that at acute exposures.

Cell Cycle↗
PubMed1996

Molecular analysis and comparison of radiation-induced large deletions of the HPRT locus in primary human skin fibroblasts.

Genetic alterations in gamma-ray- and alpha-particle-induced HPRT mutants were examined by multiplex polymerase chain reaction (PCR) analysis. A total of 39-63% of gamma-ray-induced and 31-57% of alpha-particle-induced mutants had partial or total deletions of the HPRT gene. The proportion of these deletion events was dependent on radiation dose, and at the resolution limits employed there were no significant differences between the spectra induced by equitoxic doses of alpha particles (0.2-0.4 Gy) and gamma rays (3 Gy). The molecular nature of the deletions was analyzed by the use of sequence tagged site (STS) primers and PCR amplification as a "probe" for specific regions of the human X chromosome within the Xq26 region. These STSs were closely linked and spanned regions approximately 1.7 Mbp from the telomeric side and 1.7 Mbp from the centromeric side of the HPRT gene. These markers include: DXS53, 299R, DXS79, yH3L, 3/19, PR1, PR25, H2, yH3R, 1/44, 1/67, 1/1, DXS86, D8C6, DXS10 and DXS144. STS analyses indicated that the maximum size of total deletions in radiation-induced HPRT mutants can be greater than 2.7 Mbp and deletion size appears to be dependent on radiation dose. There were no apparent differences in the sizes of the deletions induced by alpha particles or gamma rays. On the other hand, deletions containing portions of the HPRT gene were observed to be 800 kbp or less, and the pattern of the partial deletion induced by alpha particles appeared to be different from that induced by gamma rays.

Alpha Particles↗
PubMed1996

Role of copper zinc superoxide dismutase in the short-term treatment of acetic acid-induced colitis in rats.

The present study describe the role of copper zinc superoxide dismutase (CuZnSOD) in the short-term treatment of experimental colitis induced with 8% acetic acid. The colonic mucosa damage index in the group of 10 rats treated intravenously with 30,000 U/kg CuZnSOD was significantly decreased when compared with the control group (10 rats) treated with normal saline (0.4 +/- 0.6 vs 1.5 +/- 0.5 p < 0.01). Assay of SOD in the control group was 0.3 +/- 0.08 and in the SOD treated group, SOD was significantly increased to 0.8 +/- 0.1, glutathione peroxidase was 44.8 +/- 6.3 in the control and 56.4 +/- 9.1 in the treated group (difference not significant). Both myeloperoxidase activity (14.0 +/- 2.5 vs 22.7 +/- 2.5) and lipid peroxidation products (13.8 +/- 2.9 vs 52.9 +/- 9.6) were significantly lower in the colonic mucosa of the SOD treated group in comparison with the control. These results indicate that the anti-inflammatory effects of CuZnSOD were mainly the removal of oxygen free radicals and indirectly the prevention of lipid peroxidation. This study suggests that CuZnSOD may be beneficial in the treatment of patients with ulcerative colitis. However, our experimental data suggest that this treatment will not have strong effects on the control of severe ulcerative colitis.

Acetic Acid↗
PubMed1995

Construction of 110 cosmid markers and a 4.5-Mb YAC contig on human chromosome 8p12-q11.

Microcell hybrids containing various regions of human chromosome 8 were formed by microcell-mediated transfer of neo-tagged chromosome 8 into the cells derived from severe combined immunodeficiency (SCID) mouse. Thus, 110 cosmid markers were isolated from SV40-transformed SCID fibroblast cell line (SCVA) containing a p12-q11.1 region of human chromosome 8 and were assigned to eight regions in 8p12-q11.1, using a microcell-hybrid panel. For positional cloning of a human gene that restores the DNA-repair defect in a mouse with SCID on 8p11.1-q11.1 (SCID region), we constructed a yeast artificial chromosome (YAC) contig of about 4.5 Mb. Overlapping YACs were further aligned by restriction mapping, using rare-cutting restriction endonucleases. The cosmids and YAC contig should facilitate isolation of the SCID gene and other genes, such as the Werner syndrome-responsible gene in or near this region.

Animals↗
PubMed1995

Loss of the catalytic subunit of the DNA-dependent protein kinase in DNA double-strand-break-repair mutant mammalian cells.

The DNA-dependent protein kinase (DNA-PK) consists of three polypeptide components: Ku-70, Ku-80, and an approximately 350-kDa catalytic subunit (p350). The gene encoding the Ku-80 subunit is identical to the x-ray-sensitive group 5 complementing gene XRCC5. Expression of the Ku-80 cDNA rescues both DNA double-strand break (DSB) repair and V(D)J recombination in group 5 mutant cells. The involvement of Ku-80 in these processes suggests that the underlying defect in these mutant cells may be disruption of the DNA-PK holoenzyme. In this report we show that the p350 kinase subunit is deleted in cells derived from the severe combined immunodeficiency mouse and in the Chinese hamster ovary cell line V-3, both of which are defective in DSB repair and V(D)J recombination. A centromeric fragment of human chromosome 8 that complements the scid defect also restores p350 protein expression and rescues in vitro DNA-PK activity. These data suggest the scid gene may encode the p350 protein or regulate its expression and are consistent with a model whereby DNA-PK is a critical component of the DSB-repair pathway.

Animals↗
PubMed1995

The human and mouse homologs of the yeast RAD52 gene: cDNA cloning, sequence analysis, assignment to human chromosome 12p12.2-p13, and mRNA expression in mouse tissues.

The yeast Saccharomyces cerevisiae RAD52 gene is involved in DNA double-strand break repair and mitotic/meiotic recombination. The N-terminal amino acid sequence of yeast S. cerevisiae, Schizosaccharomyces pombe, and Kluyveromyces lactis and chicken is highly conserved. Using the technology of mixed oligonucleotide primed amplification of cDNA (MOPAC), two mouse RAD52 homologous cDNA fragments were amplified and sequenced. Subsequently, we have cloned the cDNA of the human and mouse homologs of yeast RAD52 gene by screening cDNA libraries using the identified mouse cDNA fragments. Sequence analysis of cDNA derived amino acid revealed a highly conserved N-terminus among human, mouse, chicken, and yeast RAD52 genes. The human RAD52 gene was assigned to chromosome 12p12.2-p13 by fluorescence in situ hybridization, R-banding, and DNA analysis of somatic cell hybrids. Unlike chicken RAD52 and mouse RAD51, no significant difference in mouse RAD52 mRNA level was found among mouse heart, brain, spleen, lung, liver, skeletal muscle, kidney, and testis. In addition to an approximately 1.9-kb RAD52 mRNA band that is present in all of the tested tissues, an extra mRNA species of approximately 0.85 kb was detectable in mouse testis.

Amino Acid Sequence↗
PubMed1995

Interspecific complementation between mouse and Chinese hamster cell mutants hypersensitive to ionizing radiation.

Interspecific and intraspecific hybrids were formed between mouse and Chinese hamster cell mutants hypersensitive to ionizing radiation and their radiosensitivities were examined. Chinese hamster cell mutants irs1, irs2 and irs3 and mouse mammary carcinoma cell mutants SX9 and SX10 have been found to belong to five different complementation groups. A radiosensitive mouse lymphoma cell line L5178Y-S has been demonstrated to be different from the X-ray sensitive mouse cell mutants M10 and LX830, both of which are derived from L5178Y cells, in their complementation groups. L5178Y-S is also distinct from SX9 and SX10.

Animals↗
PubMed1995

Non-radioactive mismatch analysis to detect small mutations in human hypoxanthine-guanine phosphoribosyl transferase cDNA.

We have combined a cDNA-driven PCR technique and a non-radioactive chemical-cleavage mismatch method, followed by a direct sequencing for detecting small mutations in the human hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene. HPRT cDNA was synthesized by RT-PCR from 1,000 wild-type or HPRT(-) mutant cells. Wild-type cDNA was hybridized with mutant cDNA to form heteroduplexes. The resultant mismatched bases were modified and cleaved by base-specific chemicals, followed by analysis by denaturing polyacrylamide gel electrophoresis. Cleaved fragments were detected without using radioactive materials. Finally, direct sequencing of the PCR products was performed with a focus on a small limited region indicated by the mismatch analysis (focused sequencing). In this study, three small mutations in exon-3 of HPRT cDNA were detected and characterized completely with this system. As compared with the radioactive method, this system was shown to be very simple and efficient.

Base Sequence↗
PubMed1995

DNA double-strand break repair in two radiation-sensitive mouse mammary carcinoma cell lines.

The capacity of two radiation-sensitive clones (SX9 and SX10) of the mouse mammary carcinoma cell line SR1 to rejoin radiation-induced DNA double-strand breaks (DSBs) was determined by pulsed-field agarose gel electrophoresis. DSBs were produced with equivalent efficiency in all three cell lines. Both the SX9 and SX10 cell lines demonstrated a significantly diminished capacity to rejoin radiation-induced DSBs. The fraction of the original DNA DSB damage remaining in the DNA of 20 Gy-exposed SR1, SX9 and SX10 cells after 6 h of 37 degrees C incubation was estimated to be 14, 82 and 54%, respectively. In addition the SX10 cell line exhibited enhanced cytotoxicity when exposed to the DNA topoisomerase II poison mitoxantrone. The results indicate that both the SX9 and SX10 cell lines are DNA DSB repair mutants.

Animals↗
PubMed1995

Molecular analysis of gamma-ray-induced mutations at the hprt locus in primary human skin fibroblasts by multiplex polymerase chain reaction.

A total of 153 hprt mutants (23 spontaneous, 130 gamma-ray-induced) of primary human skin fibroblasts were isolated and genetic alterations at the locus were studied by multiplex polymerase chain reaction (PCR). The analyses showed that 51% (66/130) of gamma-ray-induced genetic changes were large deletions, whereas the majority of spontaneous mutants (21/23) exhibited point mutations. The spectrum of large genetic alterations appeared to be dependent on dose in gamma-ray-induced (1-4 Gy) mutations; mutants with complete loss of the hprt locus comprised 21 (3/14) or 39% (15/38) of clones isolated after irradiation with 1 or 4 Gy, respectively. The frequency of partial deletions was found to be higher in the mutants isolated from clones irradiated with 2 Gy (38%) than from those irradiated with 4 Gy (8%). Mapping of all intragenic deletion breakpoints exhibited a nonrandom distribution of breakpoints toward the 3' end of the hprt gene.

Base Sequence↗
PubMed1994

Regional assignment of a human DNA repair gene (XRCC5) to 2q35 by X-ray hybrid mapping.

A number of human genes involved in the repair of radiation-induced DNA double-strand breaks have been identified based on the analysis of radiation-sensitive rodent mutants. Recently, one of the DSB repair genes, XRCC5, has been mapped on human chromosome 2 by concordance analysis of somatic cell hybrids and by the microcell-mediated chromosome transfer approach. For the regional mapping of the human XRCC5 gene, we have constructed a panel of X-ray hybrids and a panel of microcell-mediated chromosome 2 hybrids in the mutant background that contain only fragments of human chromosome 2. By using fluorescence in situ hybridization, chromosome banding, and physical mapping of these X-ray hybrids, we have localized the human XRCC5 locus to 2q35. This result is further confirmed by a segregation analysis indicating that the radiation resistant phenotype of a repair-proficient hybrid cosegregates with the human 2q35 chromosome fragment.

Animals↗
PubMed1994

Genomic structure of the mouse apurinic/apyrimidinic endonuclease gene.

A mammalian apurinic/apyrimidinic endonuclease (AP endonuclease) is known to have two distinct functional domains. One domain is responsible for regulating the activity of Fos/Jun proto-oncogene products to bind to DNA at specific recognition sites. The other domain, which is highly conserved from bacteria to mammals, is responsible for repairing DNA damage caused by ionizing radiation, oxidative damage, and alkylating agents. This study reports on the isolation and characterization of the genomic structure of the mouse AP endonuclease gene (Apex). The genomic sequence of the Apex gene was 2.14 kb in length and contained four exons. Exon 1 contained a 0.24-kb untranslated 5' region upstream of the initiation codon. Consensus sequences for two CAAT boxes and a GC box were found upstream of the end of exon 1. A polymorphism was noted in the untranslated region of exon 1 in a comparison of a number of mouse strains. These data indicate that the 5' end of the mouse gene (Apex) differs from the previously isolated human gene (Ape), which has five exons and an untranslated region between exons 1 and 2. Data are also presented that suggest the presence of two pseudogenes in the mouse.

Amino Acid Sequence↗
PubMed1994

Charged-particle mutagenesis II. Mutagenic effects of high energy charged particles in normal human fibroblasts.

The biological effects of high LET charged particles are a subject of great concern with regard to the prediction of radiation risk in space. In this report, mutagenic effects of high LET charged particles are quantitatively measured using primary cultures of human skin fibroblasts, and the spectrum of induced mutations are analyzed. The LET of the charged particles ranged from 25 KeV/micrometer to 975 KeV/micrometer with particle energy (on the cells) between 94-603 MeV/u. The X-chromosome linked hypoxanthine guanine phosphoribosyl transferase (hprt) locus was used as the target gene. Exposure to these high LET charged particles resulted in exponential survival curves; whereas, mutation induction was fitted by a linear model. The Relative Biological Effect (RBE) for cell-killing ranged from 3.73 to 1.25, while that for mutant induction ranged from 5.74 to 0.48. Maximum RBE values were obtained at the LET of 150 keV/micrometer. The inactivation cross-section (alpha i) and the action cross-section for mutant induction (alpha m) ranged from 2.2 to 92.0 micrometer2 and 0.09 to 5.56 x 10(-3) micrometer2, respectively. The maximum values were obtained by 56Fe with an LET of 200 keV/micrometer. The mutagenicity (alpha m/alpha i) ranged from 2.05 to 7.99 x 10(-5) with the maximum value at 150 keV/micrometer. Furthermore, molecular analysis of mutants induced by charged particles indicates that higher LET beams are more likely to cause larger deletions in the hprt locus.

Cell Death↗