Frequency of CD59 mutations induced in human-hamster hybrid A(L) cells by low-dose X-irradiation

Determination of the genotoxic effects of ionizing radiation, especially at low-doses, is of great importance for risk assessment, e.g. in radiological diagnostics. The human-hamster hybrid A(L) cell line has been shown previously to be a well-suited in vitro model for the study of mutations induced by various mutagens. The A(L) cells contain a standard set of hamster chromosomes and a single human chromosome 11, which confers the expression of the human cell surface protein CD59. Using CD59 specific antibodies, cells mutated in the CD59 gene can be detected and quantified by the loss of the cell surface marker. In contrast to previous studies, prior to irradiation we removed spontaneous mutants by magnetic cell separation (MACS) which allows analysis of radiation-induced mutation events only. We exposed A(L) cells to 100kV X-rays at 0.1 to 5Gy. The proportions of X-irradiation-induced CD59(-) mutants were quantified by flow cytometry after immunofluorescence labeling. Between 0.2 and 5Gy the yield of CD59 mutants was a linear function of dose. The molecular analysis of individual CD59-negative clones induced after exposure of 1, 3 and 5Gy of X-ray revealed a dose-dependent linear increase of large deletions (>6Mbp), whereas, point mutations could be seen only in spontaneous CD59 mutants or after low-dose exposure (< or =1Gy). We conclude that the modified A(L) assay presented here is appropriate for detection and quantification of non-lethal DNA lesions induced by low-dose ionizing radiation.     

Quantification of CD59- mutants in human-hamster hybrid (AL) cells by flow cytometry

Mutation assay is an important approach in evaluating the genotoxic risk of potentially harmful environmental chemicals. The human-hamster hybrid (A(L)) cell mutagenesis system, based on the complement/antibody-mediated cytotoxicity principle, has been used successfully to evaluate the mutagenic potential of a variety of environmental toxicants. The A(L) cells contain a standard set of CHO chromosomes and a single human chromosome 11, which expresses several cell surface proteins including CD59 encoded by the CD59 gene at 11p13.5. A modified mutation assay by flow cytometry was developed to determine the yield of CD59- mutants after either radiation or chemical treatment. After incubation with phycoerythrin-conjugated mouse monoclonal anti-CD59 antibody, the CD59- mutant yields were determined by quantifying the fluorescence of the cells using flow cytometry. This method is faster and eliminates the commonly encountered toxicity problems of the complements with the traditional complement/antibody assay. By comparing the mutant fractions of radiation or chemically treated A(L) cultures using the two methods, we show here that the flow cytometry assay is an excellent substitute in providing an efficient and highly sensitive method in mutant detection for the traditional complement/antibody assay.     

Gamma-ray mutagenesis studies in a new human-hamster hybrid, A(L)CD59(+/-), which has two human chromosomes 11 but is hemizygous for the CD59 gene.

Kraemer, S. M., Vannais, D. B., Kronenberg, A., Ueno, A. and Waldren, C. A. Gamma-Ray Mutagenesis Studies in a New Human-Hamster Hybrid, A(L)CD59(+/-), which has Two Human Chromosomes 11 but is Hemizygous for the CD59 Gene. Radiat. Res. 156, 10-19 (2001).We have developed a human-CHO hybrid cell line, named A(L)CD59(+/-), which has two copies of human chromosome 11 but is hemizygous for the CD59 gene and the CD59 cell surface antigen that it encodes. Our previous studies used the A(L) and A(L)C hybrids that respectively contain one or two sets of CHO chromosomes plus a single copy of human chromosome 11. The CD59 gene at 11p13.5 and the CD59 antigen encoded by it are the principal markers used in our mutagenesis studies. The hybrid A(L)CD59(+/-) contains two copies of human chromosome 11, only one of which carries the CD59 gene. The incidence of CD59 (-) mutants (formerly called S1(-)) induced by (137)Cs gamma rays is about fivefold greater in A(L)CD59(+/-) cells than in A(L) cells. Evidence is presented that this increase in mutant yield is due to the increased induction of certain classes of large chromosomal mutations that are lethal to A(L) cells but are tolerated in the A(L)CD59(+/-) hybrid. In addition, significantly more of the CD59 (-) mutants induced by (137)Cs gamma rays in A(L)CD59(+/-) cells display chromosomal instability than in A(L) cells. On the other hand, the yield of gamma-ray-induced CD59 (-) mutants in A(L)CD59(+/-) cells is half that of the A(L)C hybrid, which also tolerates very large mutations but has only one copy of human chromosome 11. We interpret the difference in mutability as evidence that repair processes involving the homologous chromosomes 11 play a role in determining mutant yields. The A(L)CD59(+/-) hybrid provides a useful new tool for quantifying mutagenesis and shedding light on mechanisms of genetic instability and mutagenesis.     

Mutant spectra of irradiated CHO AL cells determined with multiple markers analyzed by flow cytometry

We have previously developed a sensitive and rapid mammalian cell mutation assay which is based on a Chinese hamster ovary cell line that stably incorporates human chromosome 11 (CHO A(L)) and uses flow cytometry to measure mutations in CD59. We now show that multiparameter flow cytometry may be used to simultaneously analyze irradiated CHO A(L) cells for mutations in five CD genes along chromosome 11 (CD59, CD44, CD90, CD98, CD151) and also a GPI-anchor gene. Using this approach, 19 different mutant clones derived from individual sorted mutant cells were analyzed to determine the mutant spectrum induced by ionizing radiation. All clones analyzed were negative for CD59 expression and PCR confirmed that at least CD59 exon 4 was also absent. As expected, ionizing radiation frequently caused large deletions along chromosome 11. This technology can readily be used to rapidly analyze the mutant yield as well as the spectrum of mutations caused by a variety of genotoxic agents and provide greater insight into the mechanisms of mutagenesis.     

The "pro-drug" RibCys decreases the mutagenicity of high-LET radiation in cultured mammalian cells

We are carrying out studies aimed at reducing the mutagenic effects of high-LET 56Fe ions and 12C ions (56Fe ions, 143 keV/microm; 12C ions, 100 keV/microm) with certain drugs, including RibCys [2-(R,S)-D-ribo-(1',2',3',4'-tetrahydroxybutyl)-thiazolidine-4(R)-carboxylic acid]. RibCys, formed by condensation of L-cysteine with D-ribose, is designed so that the sulfhydryl amino acid L-cysteine is released intracellularly through nonenzymatic ring opening and hydrolysis leading to increased levels of glutathione (GSH). RibCys (4 or 10 mM), which was present during irradiation and for a few hours after, significantly decreased the yield of CD59- mutants induced by radiation in AL human-hamster hybrid cells. RibCys did not affect the clonogenic survival of irradiated cells, nor was it mutagenic itself. These results, together with the minimal side effects reported in mice and pigs, indicate that RibCys may be useful, perhaps even when used prophylactically, in reducing the mutation load created by high-LET radiation in astronauts or other exposed individuals.     

A role for long-lived radicals (LLR) in radiation-induced mutation and persistent chromosomal instability: counteraction by ascorbate and RibCys but not DMSO.

Miazaki, Watanabe, Kumagai and their colleagues reported that induction of HPRT(-) mutants by X-rays in cultured human cells was prevented by ascorbate added 30min after irradiation. They attributed extinction of induced mutation to neutralization by ascorbate of radiation-induced long-lived mutagenic radicals (LLR), found using spectroscopy to have half-lives of minutes or hours. We find that post-irradiation treatment with ascorbate reduces, but does not eliminate, induction of CD59(-) mutants in human-hamster hybrid A(L) cells exposed to high-LET carbon-ions (LET of 100KeV/microm). A(L) cells contain a standard set of Chinese hamster ovary (CHO) chromosomes and a single copy of human chromosome 11 containing the CD59 gene which encodes the CD59 cell surface antigen, a convenient marker for mutation. RibCys [2(R, S)-D-ribo-(1',2',3',4'-tetrahydroxybutyl)thiazolidine-4(R)-carboxylic acid] a 'prodrug' of l-cysteine which also scavenges LLR, had a similar but lesser effect on induced mutation. DMSO, which scavenges classical radicals like H* and OH* but not LLR, also reduced mutation, but only when it was present during irradiation. The lethality of carbon-ions was not altered by ascorbate, RibCys no matter when added. Post-radiation addition of ascorbate and RibCys also affected the quality of CD59(-) mutations induced by carbon-ions. The major change in mutant spectra was a reduction in the prevalence of small, intragenic mutations (mutations not detected by PCR) and in the prevalence of unstable, complicated mutants, which display high levels of persistent chromosomal instability. Thus, ascorbate and RibCys may suppress some kinds of mutations induced by ionizing radiation including those displaying aspects of radiation-induced genomic instability. Countering the effects of both classical radicals and LLR may be important in preventing genetic diseases.     

Increased frequency of spontaneous neoplastic transformation in progeny of bystander cells from cultures exposed to densely ionizing radiation

An increased risk of carcinogenesis caused by exposure to space radiation during prolonged space travel is a limiting factor for human space exploration. Typically, astronauts are exposed to low fluences of ionizing particles that target only a few cells in a tissue at any one time. The propagation of stressful effects from irradiated to neighboring bystander cells and their transmission to progeny cells would be of importance in estimates of the health risks of exposure to space radiation. With relevance to the risk of carcinogenesis, we investigated, in model C3H 10T½ mouse embryo fibroblasts (MEFs), modulation of the spontaneous frequency of neoplastic transformation in the progeny of bystander MEFs that had been in co-culture 10 population doublings earlier with MEFs exposed to moderate doses of densely ionizing iron ions (1 GeV/nucleon) or sparsely ionizing protons (1 GeV). An increase (P<0.05) in neoplastic transformation frequency, likely mediated by intercellular communication through gap junctions, was observed in the progeny of bystander cells that had been in co-culture with cells irradiated with iron ions, but not with protons.     

Comparison of the mutagenic potential of 17 physical and chemical agents analyzed by the flow cytometry mutation assay

Several methods to assess genotoxicity of physical and chemical agents have been developed, most of which depend on growing colonies in selective medium. We recently published a new method for detecting mutations in the CD59 gene in a Chinese hamster ovary cell line that contains a single copy of human chromosome 11 (CHO A_L). The assay is based on detecting the surface expression of CD59 with monoclonal antibodies using flow cytometry. The capabilities of this flow cytometry mutation assay (FCMA) to detect mutations from a wide variety of genotoxic agents are described here. There was a 400-fold separation between CD59⁻ and CD59⁺ populations based on fluorescence intensity. Small numbers of negative cells mixed in with positive cells were detected in a highly linear fashion. Mutation dose response curves over a dose range yielding 80% to 20% survival are shown for ethyl methane sulfonate (EMS), mitomycin C (MMC) and lead acetate. EMS and lead acetate exhibited a threshold in response while MMC had a linear dose response over the full dose range. The mutant fraction was measured over time periods ranging up to 35 days following treatment. The mutant fraction peaked at different times ranging from 6 to 12 days after treatment. An additional 14 chemical and physical agents including point mutagens, heavy metals, ionizing and UV radiation, and DNA intercalators and cross linkers, were analyzed for mutagenic potential after doses giving 80% to 20% survival. The results presented here demonstrate the sensitivity and broad-ranging capability of the FCMA to detect mutations induced by a variety of genotoxic agents.     

Spectrum of complex DNA damages depends on the incident radiation

Ionizing radiation induces bistranded clustered damages--two or more abasic sites, oxidized bases and strand breaks on opposite DNA strands within a few helical turns. Since clusters are refractory to repair and are potential sources of double-strand breaks (DSBs), they are potentially lethal and mutagenic. Although induction of single-strand breaks (SSBs) and isolated lesions has been studied extensively, little is known about the factors affecting induction of clusters other than DSBs. To determine whether the type of incident radiation could affect the yields or spectra of specific clusters, we irradiated genomic T7 DNA, a simple 40-kbp linear, blunt-ended molecule, with ion beams [iron (970 MeV/nucleon), carbon (293 MeV/nucleon), titanium (980 MeV/nucleon), silicon (586 MeV/nucleon), protons (1 GeV/nucleon)] or 100 kVp X rays and then quantified DSBs, Fpg-oxypurine clusters and Nfo-abasic clusters using gel electrophoresis, electronic imaging and number average length analysis. The yields (damages/Mbp Gy(-1)) of all damages decreased with increasing linear energy transfer (LET) of the radiation. The relative frequencies of DSBs compared to abasic and oxybase clusters were higher for the charged particles-including the high-energy, low-LET protons-than for the ionizing photons.     

Comparison of repair of DNA double-strand breaks in identical sequences in primary human fibroblast and immortal hamster-human hybrid cells harboring a single copy of human chromosome 11

We have optimized a pulsed-field gel electrophoresis assay that measures induction and repair of double-strand breaks (DSBs) in specific regions of the genome (L?brich et al., Proc. Natl. Acad. Sci. USA 92, 12050-12054, 1995). The increased sensitivity resulting from these improvements makes it possible to analyze the size distribution of broken DNA molecules immediately after the introduction of DSBs and after repair incubation. This analysis shows that the distribution of broken DNA pieces after exposure to sparsely ionizing radiation is consistent with the distribution expected from randomly induced DSBs. It is apparent from the distribution of rejoined DNA pieces after repair incubation that DNA ends continue to rejoin between 3 and 24 h postirradiation and that some of these rejoining events are in fact misrejoining events, since novel restriction fragments both larger and smaller than the original fragment are generated after repair. This improved assay was also used to study the kinetics of DSB rejoining and the extent of misrejoining in identical DNA sequences in human GM38 cells and human-hamster hybrid A(L) cells containing a single human chromosome 11. Despite the numerous differences between these cells, which include species and tissue of origin, levels of TP53, expression of telomerase, and the presence or absence of a homologous chromosome for the restriction fragments examined, the kinetics of rejoining of radiation-induced DSBs and the extent of misrejoining were similar in the two cell lines when studied in the G(1) phase of the cell cycle. Furthermore, DSBs were removed from the single-copy human chromosome in the hamster A(L) cells with similar kinetics and misrejoining frequency as at a locus on this hybrid's CHO chromosomes.     

Multiparameter analysis of CHO AL mutant populations sorted on CD59 expression after gamma irradiation

The flow cytometry mutation assay is based on detecting mutations in the CD59 gene on human chromosome 11 in CHO A(L) cells with flow cytometry, but the kinetics of mutant expression and the histogram region for mutant selection have not been studied in detail. CHO A(L) cells were analyzed by flow cytometry for CD59 expression at various times after irradiation. The mutant fraction increased to a maximum at day 6 but decreased to near background levels by day 20. Cells were sorted from six different regions on the CD59 histograms after irradiation. The growth rate was similar for cells from all regions, and the surviving fraction was 50% of that for control cells. By 14 days the CD59 expression of cells from regions 2-5 was reduced to that of region 1. Cells were also analyzed for simultaneous expression of CD59, CD44 and CD90 (all on chromosome 11) to roughly characterize the size of the mutations. Triple mutants from the sorted populations were reduced from 41% on day 6 to 8% on day 24. We conclude that the mutant region should be increased to include cells with intermediate CD59 expression; also, the loss of CD59 mutant expression over time could be explained in part by the loss of triple mutants from the population.     

Assay to measure CD59 mutations in CHO A(L) cells using flow cytometry.

BACKGROUND: A sensitive mammalian cell mutation assay was developed previously using a Chinese hamster ovary cell line (CHO A(L)) that stably incorporates human chromosome 11. The assay measures mutations in the CD59 gene on chromosome 11 but it requires the use of rabbit complement and colony growth for mutant selection. We have developed a more rapid flow cytometry-based mutation assay with CHO A(L) cells that uses monoclonal antibodies against CD59 to detect mutants and does not require colony formation. METHODS: CHO A(L) cells were treated with gamma-radiation or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and then allowed to grow for various times for mutant expression. Cells were labeled with monoclonal antibodies against CD59 and analyzed by flow cytometry. RESULTS: Negative and positive populations were separated by over 100-fold. Mixing various proportions of CD59-positive and -negative cells demonstrated that the assay is highly linear (r2 = 0.9999) and sensitive (<0.05% background mutants). The yield of CD59-inducible mutants was linearly related to dose for a clastogen (gamma-radiation) and point mutagen (MNNG). The mutant yield was time and treatment specific. CONCLUSIONS: Mutations induced by genotoxic agents can be rapidly and sensitively measured in CHO A(L) cells using flow cytometry.     

The cytogenetic effects of low doses of accelerated charged particles in human blood lymphocytes in vitro

The aim of the investigation was the study of cytogenetic effects in human blood lymphocytes of low doses of ionizing radiation in vitro. The analysis of unstable chromosome aberrations in human lymphocytes after irradiation by the accelerated ions 12C with the energy 500 MeV/nucleon and LET 10.7 keV/microm was carried out. Blood samples were irradiated on Nuclotron of the High Energy Laboratory of the Joint Institute for Nuclear Research. The doses of irradiation were in the range from 0.05 up to 1.0 Gy. Was shown that the frequency of unstable chromosome aberrations depends from the dose of ionizing radiation and can be described by linear function. At the doses 0.25-0.50 Gy the dose-independent curve was obtained for dicentrics and centric rings. The frequencies of dicentrics and centric rings as markers of the radiation action were slightly different for different donors that could be explained by different radiosensitivity. Using the calibration curve obtained earlier for gamma-rays coefficients of relative biological efficiency of accelerated 12C with the energy 500 MeV/nucleon were defined: they varied from 1.0 at the doses (0.5-1.0 Gy) up to 3.2 at the lower doses (0.05-0.25 Gy).     

Quantitative image analysis of laminin immunoreactivity in skin basement membrane irradiated with 1 GeV/nucleon iron particles

We previously reported that laminin immunoreactivity in mouse mammary epithelium is altered shortly after whole-body irradiation with 0.8 Gy from 600 MeV/nucleon iron ions but is unaffected after exposure to sparsely ionizing radiation. This observation led us to propose that the effect could be due to protein damage from the high ionization density of the ion tracks. If so, we predicted that it would be evident soon after radiation exposure in basement membranes of other tissues and would depend on ion fluence. To test this hypothesis, we used immunofluorescence, confocal laser scanning microscopy, and image segmentation techniques to quantify changes in the basement membrane of mouse skin epidermis. At 1 h after exposure to 1 GeV/nucleon iron ions with doses from 0.03 to 1.6 Gy, neither the visual appearance nor the mean pixel intensity of laminin in the basement membrane of mouse dorsal skin epidermis was altered compared to sham-irradiated tissue. This result does not support the hypothesis that particle traversal directly affects laminin protein integrity. However, the mean pixel intensity of laminin immunoreactivity was significantly decreased in epidermal basement membrane at 48 and 96 h after exposure to 0.8 Gy 1 GeV/nucleon iron ions. We confirmed this effect with two additional antibodies raised against affinity-purified laminin 1 and the E3 fragment of the long-arm of laminin 1. In contrast, collagen type IV, another component of the basement membrane, was unaffected. Our studies demonstrate quantitatively that densely ionizing radiation elicits changes in skin microenvironments distinct from those induced by sparsely ionizing radiation. Such effects may might contribute to the carcinogenic potential of densely ionizing radiation by altering cellular signaling cascades mediated by cell-extracellular matrix interactions.     

Truly incomplete and complex exchanges in prematurely condensed chromosomes of human fibroblasts exposed in vitro to energetic heavy ions

Confluent human fibroblast cells (AG1522) were irradiated with gamma rays, 490 MeV/nucleon silicon ions, or iron ions at either 200 or 500 MeV/nucleon. The cells were allowed to repair at 37 degrees C for 24 h after exposure, and a chemically induced premature chromosome condensation (PCC) technique was used to condense chromosomes in the G2 phase of the cell cycle. Incomplete and complex exchanges were analyzed in the irradiated samples. To verify that chromosomal breaks were truly unrejoined, chromosome aberrations were analyzed using a combination of whole-chromosome specific probes and probes specific for the telomere region of the chromosome. Results showed that the frequency of unrejoined chromosome breaks was higher after irradiation with the heavy ions of high LET, and consequently the ratio of incomplete to complete exchanges increased steadily with LET up to 440 keV/microm, the highest LET included in the present study. For samples exposed to 200 MeV/nucleon iron ions, chromosome aberrations were analyzed using the multicolor FISH (mFISH) technique, which allows identification of both complex and truly incomplete exchanges. Results of the mFISH study showed that 0.7 and 3 Gy iron ions produced similar ratios of complex to simple exchanges and incomplete to complete exchanges; these ratios were higher than those obtained after exposure to 6 Gy gamma rays. After 0.7 Gy of iron ions, most complex aberrations were found to involve three or four chromosomes, which is a likely indication of the maximum number of chromosome domains traversed by a single iron-ion track.     

TAR loop-dependent human immunodeficiency virus trans activation requires factors encoded on human chromosome 12.

The trans-activator response region (TAR) RNA in the human immunodeficiency virus type 1 (HIV-1) and HIV-2 long terminal repeat forms stem-loop secondary structures in which the loop sequence is essential for trans activation. We investigated how the HIV trans-activation mechanism encoded on human chromosome 12 relates to the TAR RNA loop-dependent pathway. DNA transfection experiments showed that trans activation in human-hamster hybrid cells with the single human chromosome 12 and human T-cell lines was highly dependent on the native sequences of the HIV-1 TAR loop and the HIV-2 5' TAR loop. In nonhuman cell lines or hybrid cells without chromosome 12 that supported trans activation, the cellular mechanism was independent of the HIV-1 TAR loop and the response to mutations in the HIV-2 TAR loops differed from that found in human T-cell lines and human-hamster hybrid cells with chromosome 12. Our results suggest that the human chromosome 12 mechanism interacts directly with the TAR RNA loop or indirectly by regulating TAR RNA-binding proteins.     

Confirmation of the localization of the human recombination activating gene 1 (RAG1) to chromosome 11p13

The human recombination activating gene 1 (RAG1) has previously been mapped to chromosomes 14q and 11p. Here we confirm the chromosome 11 assignment by two independent approaches: autoradiographic and fluorescence in situ hybridization to metaphase spreads and analysis of human-hamster somatic cell hybrid DNA by the polymerase chain reaction (PCR) and Southern blotting. Our results unequivocally localize RAG1 to 11p13.     

Identification of two human ferritin H genes on the short arm of chromosome 6

We have found by analyses of human-hamster hybrid cells that two human ferritin H genes lie near the locus of the iron storage disease idiopathic hemochromatosis on chromosome 6p. One of these genes was isolated and shown to be a processed pseudogene. Comparison of its sequence with those of other ferritin H pseudogenes indicates that they may be derived from a functional H gene other than that on chromosome 11.     

Mutation induction and relative biological effectiveness of neutrons in mammalian cells. Experimental observations

The induction of mutation by graded doses of monoenergetic neutrons was examined using the human-hamster hybrid cell system. The AL cells, formed by fusion of human fibroblasts with the gly- A mutant of the Chinese hamster ovary cells, contain the standard set of hamster chromosomes plus a single human chromosome, number 11. These cells contain specific human cell surface antigens that render them sensitive to killing by specific antisera in the presence of complement. Mutant AL cells that have lost the surface markers, however, would survive and give rise to scorable colonies. The cells were irradiated with neutrons produced at the Radiological Research Accelerator Facility of Columbia University. Doses corresponding to low, moderate, and high cytotoxicities and in energies ranging from 0.33 to 14 MeV were used. Neutrons induced a dose-dependent cytotoxicity and mutation frequency in the AL cells. Over the range of doses examined, it was found that the mutagenesis induced by neutrons was energy-dependent and the frequencies were a curvilinear function of dose for both the a1 and a2 antigenic loci examined. In comparison to gamma rays, the relative biological effectiveness (RBE) for cell lethality at the 10% survival level ranged from 5.2 for 0.33 MeV to 1.8 for 14 MeV neutrons. The RBE for mutation induction at the a1 locus, however, ranged from 30 for 0.33 MeV to 4.2 for 14 MeV neutrons at or around the lowest levels of effect examined. Results of the present study demonstrated that neutrons, when measured under conditions which permit detection of a spectrum of gene and chromosomal mutations, in fact, are more efficient mutagens than previously thought.