35 research outputs found
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Characterization of a TK6-Bcl-xL gly-159-ala Human Lymphoblast Clone
TK6 cells are a well-characterized human B-lymphoblast cell line derived from WIL-2 cells. A derivative of the TK6 cell line that was stably transfected to express a mutated form of the anti-apoptotic protein Bcl-xL (TK6-Bcl-xL gly-159- ala clone #38) is compared with the parent cell line. Four parameters were evaluated for each cell line: growth under normal conditions, plating efficiency, and frequency of spontaneous mutation to 6‑thioguanine resistance (hypoxanthine phosphoribosyl transferase locus) or trifluorothymidine resistance (thymidine kinase locus). We conclude that the mutated Bcl-xL protein did not affect growth under normal conditions, plating efficiency or spontaneous mutation frequencies at the thymidine kinase (TK) locus. Results at the hypoxanthine phosphoribosyl transferase (HPRT) locus were inconclusive. A mutant fraction for TK6‑Bcl-xL gly-159-ala clone #38 cells exposed to 150cGy of 160kVp x-rays was also calculated. Exposure to x-irradiation increased the mutant fraction of TK6‑Bcl-xL gly-159-ala clone #38 cells
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Gene conversion is strongly induced in human cells by double-strand breaks and is modulated by the expression of BCL-XL
Homology-directed repair (HDR) of DNA double-strand breaks (DSBs) is a well-established mechanism that contributes to the maintenance of genomic stability in rodent cells, and it has been assumed that HDR is of similar importance in the repair of DSBs in human cells. However, in addition to promoting genomic stability, some outcomes of homologous recombination can be deleterious, suggesting that factors exist to regulate HDR. We previously demonstrated that overexpression of BCL-2 or BCL-xL enhanced the frequency of x-ray-induced mutations involving the TK1 locus, including loss of heterozygosity (LOH) events presumed to arise by mitotic recombination. The present study was designed to test whether HDR is a prominent DSB repair pathway in human cells, and to directly determine whether ectopic expression of BCL-xL affects HDR. We used the B-lymphoblastoid cell line TK6, which expresses wild-type TP53 and resembles normal lymphocytes in undergoing apoptosis following! genotoxic stress. Using isogenic derivatives of TK6 cells (TK6-neo, TK6-bcl-xL), we find that a DSB in an integrated HDR reporter stimulates gene conversion 40-50-fold in TK6-neo cells, demonstrating that a DSB can be efficiently repaired by gene conversion in human cells. Significantly, DSB-induced gene conversion events are 3- to 4-fold more frequent in BCL-xL overexpressing cells. The results demonstrate that HDR plays an important role in maintaining genomic integrity in human cells and that ectopic expression of BCL-xL enhances HDR of DSBs. To our knowledge, this is the first study to highlight a function for BCL-xL in modulating DSB repair in human cells
Genotoxicity of charged particles of importance in space flight using murine kidney epithelial cells
Ionizing radiation presents significant challenges for human space flight including an increased cancer risk. High-energy heavy ions in the galactic cosmic radiation can produce qualitative and quantitative differences in biological effects when compared with sparsely ionizing radiations. Mutations are induced by charged particle exposure and are integral to the formation and/or progression of human cancers. Most cancer-associated mutations occur on autosomal chromosomes, and most solid cancers occur in epithelial tissues. Here, a combined in vitro/in vivo approach was used to evaluate cell killing and the induction of mutations at a model autosomal locus, Aprt, in mouse kidney epithelium. For in vitro exposures, Aprt heterozygous kidney cells (clones 1a, 4a or 6a) were used from C57BL/6×DBA/2 mice. Additional experiments were performed using whole body irradiation of mice with the same genotype. Both males and females were irradiated in approximately equal numbers. Irradiations were performed at the NASA Space Radiation Laboratories at Brookhaven National Laboratory. For in vitro studies, cells from primary kidney clones were irradiated and seeded at limiting dilution immediately post-irradiation to determine the toxicity of the treatment. The irradiated kidney cells were also seeded in mutation assays within 1 week post-irradiation to determine the Aprt mutant fraction at the earliest time post-exposure. This work was complemented by studies wherein mice were exposed to the same ions with kidneys harvested several months post-irradiation to determine the residual toxicity and the Aprt mutant fraction. Our previous studies focused on sparsely ionizing 1 GeV protons (LET = 0.24 keV/??m) and densely ionizing 1 GeV/amu Fe ions (LET = 151 keV/??m). Our most recent studies have included work with Si ions (240 MeV/amu for in vitro studies, LET = 78 keV/??m; 263 MeV/amu initial energy for in vivo studies to achieve 78 keV/??m near the midline of the animal) and O ions (250 MeV/amu in vitro studies only, LET = 25 keV/??m). Toxicity for the cultured kidney cells in vitro follows this pattern: Fe > Si > O > protons when the results are expressed per unit dose. D0 values were 92 cGy for Fe ions, 103 cGy for Si ions, 192 cGy for O ions and 340 cGy for protons. With regard to the induction of Aprt mutations, Fe ions were more mutagenic than protons. Si ions were also quite mutagenic with evidence for a linear dose???response for Aprt mutations in kidney cells exposed in vitro or in kidneys harvested from mice irradiated several months earlier. These results are consistent with the linear dose???response data obtained previously for Aprt mutation induction following Fe ion exposure in vitro or in vivo, but the results for Si ions differ from the curvilinear dose???response data we recently published following similar exposures to energetic protons. Our most recent studies examined the molecular characteristics of Si ion-induced Aprt mutants following in vitro exposure. A dose of 160 cGy was used to collect 58 Aprt kidney cell mutants. Mutational events were classified as follows based on PCR-based analyses of polymorphic markers along mouse chromosome 8: intragenic events, apparent mitotic recombination, interstitial deletions of Aprt only, multilocus deletions, discontinuous loss of heterozygosity or whole chromosome loss. The results for this group of mutants will be compared against our previous studies on Aprt mutants arising after exposure to sparsely ionizing 1 GeV protons or densely ionizing 1 GeV/amu Fe ions. Additional studies are ongoing to define mutational spectra following Si ion exposure to kidney epithelium in vivo
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Gene conversion is strongly induced in human cells by double-strand breaks and is modulated by the expression of BCL-XL
Homology-directed repair (HDR) of DNA double-strand breaks (DSBs) is a well-established mechanism that contributes to the maintenance of genomic stability in rodent cells, and it has been assumed that HDR is of similar importance in the repair of DSBs in human cells. However, in addition to promoting genomic stability, some outcomes of homologous recombination can be deleterious, suggesting that factors exist to regulate HDR. We previously demonstrated that overexpression of BCL-2 or BCL-xL enhanced the frequency of x-ray-induced mutations involving the TK1 locus, including loss of heterozygosity (LOH) events presumed to arise by mitotic recombination. The present study was designed to test whether HDR is a prominent DSB repair pathway in human cells, and to directly determine whether ectopic expression of BCL-xL affects HDR. We used the B-lymphoblastoid cell line TK6, which expresses wild-type TP53 and resembles normal lymphocytes in undergoing apoptosis following! genotoxic stress. U sing isogenic derivatives of TK6 cells (TK6-neo, TK6-bcl-xL), we find that a DSB in an integrated HDR reporter stimulates gene conversion 40-50-fold in TK6-neo cells, demonstrating that a DSB can be efficiently repaired by gene conversion in human cells. Significantly, DSB-induced gene conversion events are 3- to 4-fold more frequent in BCL-xL overexpressing cells. The results demonstrate that HDR plays an important role in maintaining genomic integrity in human cells and that ectopic expression of BCL-xL enhances HDR of DSBs. To our knowledge, this is the first study to highlight a function for BCL-xL in modulating DSB repair in human cells
Charged particle mutagenesis at low dose and fluence in mouse splenic T cells.
High-energy heavy charged particles (HZE ions) found in the deep space environment can significantly affect human health by inducing mutations and related cancers. To better understand the relation between HZE ion exposure and somatic mutation, we examined cell survival fraction, Aprt mutant frequencies, and the types of mutations detected for mouse splenic T cells exposed in vivo to graded doses of densely ionizing (48)Ti ions (1GeV/amu, LET=107 keV/μm), (56)Fe ions (1GeV/amu, LET=151 keV/μm) ions, or sparsely ionizing protons (1GeV, LET=0.24 keV/μm). The lowest doses for (48)Ti and (56)Fe ions were equivalent to a fluence of approximately 1 or 2 particle traversals per nucleus. In most cases, Aprt mutant frequencies in the irradiated mice were not significantly increased relative to the controls for any of the particles or doses tested at the pre-determined harvest time (3-5 months after irradiation). Despite the lack of increased Aprt mutant frequencies in the irradiated splenocytes, a molecular analysis centered on chromosome 8 revealed the induction of radiation signature mutations (large interstitial deletions and complex mutational patterns), with the highest levels of induction at 2 particles nucleus for the (48)Ti and (56)Fe ions. In total, the results show that densely ionizing HZE ions can induce characteristic mutations in splenic T cells at low fluence, and that at least a subset of radiation-induced mutant cells are stably retained despite the apparent lack of increased mutant frequencies at the time of harvest
Autosomal Mutants of Proton-Exposed Kidney Cells Display Frequent Loss of Heterozygosity on Nonselected Chromosomes
High-energy protons found in the space environment can induce mutations and cancer, which are inextricably linked. We hypothesized that some mutants isolated from proton exposed kidneys arose through a genome-wide incident that causes loss of heterozygosity (LOH)-generating mutations on multiple chromosomes (termed here genomic LOH). To test this hypothesis, we examined 11 pairs of nonselected chromosomes for LOH events in mutant cells isolated from the kidneys of mice exposed to 4 or 5 Gy of 1 GeV protons. The mutant kidney cells were selected for loss of expression of the chromosome 8-encoded Aprt gene. Genomic LOH events were also assessed in Aprt mutants isolated from isogenic cultured kidney epithelial cells exposed to 5 Gy of protons in vitro. Control groups were spontaneous Aprt mutants and clones isolated without selection from the proton-exposed kidneys or cultures. The in vivo results showed significant increases in genomic LOH events in the Aprt mutants from proton-exposed kidneys when compared with spontaneous Aprt mutants and when compared with nonmutant (i.e., nonselected) clones from the proton-exposed kidneys. A bias for LOH events affecting chromosome 14 was observed in the proton-induced Aprt mutants, though LOH for this chromosome did not confer increased radiation resistance. Genomic LOH events were observed in Aprt mutants isolated from proton-exposed cultured kidney cells; however the incidence was fivefold lower than in Aprt mutants isolated from exposed intact kidneys, suggesting a more permissive environment in the intact organ and/or the evolution of kidney clones prior to their isolation from the tissue. We conclude that proton exposure creates a subset of viable cells with LOH events on multiple chromosomes, that these cells form and persist in vivo, and that they can be isolated from an intact tissue by selection for a mutation on a single chromosome