Radiation Research

Published by: Radiation Research Society

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Radiation Research 170(5):628-637. 2008
doi: 10.1667/RR1276.1

Multiparameter Analysis of CHO AL Mutant Populations Sorted on CD59 Expression after Gamma Irradiation

Carley D. Rossa and Michael H. Fox1ab

aCell and Molecular Biology Graduate Program

bDepartment of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, Colorado

1Address for correspondence: Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, CO 80523-1618;

Abstract

Ross, C. D. and Fox, M. H. Multiparameter Analysis of CHO AL Mutant Populations Sorted on CD59 Expression after Gamma Irradiation. Radiat. Res. 170, 628–637 (2008).

The flow cytometry mutation assay is based on detecting mutations in the CD59 gene on human chromosome 11 in CHO AL cells with flow cytometry, but the kinetics of mutant expression and the histogram region for mutant selection have not been studied in detail. CHO AL 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.

Received: November 7, 2007; Accepted: May 1, 2008



REFERENCES

Ross, C. D., C. U. Lim, and M. H. Fox. Assay to measure CD59 mutations in CHO AL cells using flow cytometry. Cytometry A 66:85–90.2005). PubMed
French, C. T., C. D. Ross, S. B. Keysar, D. D. Joshi, C. U. Lim, and M. H. Fox. Comparison of the mutagenic potential of 17 physical and chemical agents analyzed by the flow cytometry mutation assay. Mutat. Res 602:14–25.2006). PubMed
Liber, H. L., K. M. Call, and J. B. Little. Molecular and biochemical analyses of spontaneous and x-ray-induced mutants in human lymphoblastoid cells. Mutat. Res 178:143–153.1987). PubMed, CSA
Liber, H. L. and W. G. Thilly. Mutation assay at the thymidine kinase locus in diploid human lymphoblasts. Mutat. Res 94:467–485.1982. PubMed, CSA
Moore, M. M., D. Clive, B. E. Howard, A. G. Batson, and N. T. Turner. In situ analysis of trifluorothymidine-resistant (TFTr) mutants of L5178Y/TK+/− mouse lymphoma cells. Mutat. Res 151:147–159.1985. PubMed, CSA
Mitchell, A. D., A. E. Auletta, D. Clive, P. E. Kirby, M. M. Moore, and B. C. Myhr. The L5178Y/tk+/− mouse lymphoma specific gene and chromosomal mutation assay: A phase III report of the U.S. Environmental Protection Agency Gene-Tox Program. Mutat. Res 394:177–303.1997). PubMed, CSA
Amundson, S. A. and H. L. Liber. A comparison of induced mutation at homologous alleles of the tk locus in human cells. Mutat. Res 247:19–27.1991). PubMed, CSA
el-Tarras, A., J. S. Dubins, J. Warner, C. Hofman, and R. R. Cobb. Molecular analysis of the TK locus in L5178Y large and small colony mouse lymphoma cell mutants induced by hycanthone methanesulfonate. Mutat. Res 332:89–95.1995). PubMed, CSA
Ross, C. D., C. T. French, S. B. Keysar, and M. H. Fox. Mutant spectra of irradiated CHO AL cells determined with multiple markers analyzed by flow cytometry. Mutat. Res 624:61–70.2007). PubMed
Wedemeyer, N., B. Greve, D. Uthe, T. Potter, D. Denklau, E. Severin, U. Hacker-Klom, W. Kohnlein, and W. Gohde. Frequency of CD59 mutations induced in human-hamster hybrid AL cells by low-dose X-irradiation. Mutat. Res 473:73–84.2001). PubMed, CSA
Waldren, C., L. Correll, M. A. Sognier, and T. T. Puck. Measurement of low levels of x-ray mutagenesis in relation to human disease. Proc. Natl. Acad. Sci. USA 83:4839–4843.1986). CrossRef, PubMed, CSA
Zhou, H., A. Xu, J. A. Gillispie, C. A. Waldren, and T. K. Hei. Quantification of CD59− mutants in human-hamster hybrid (AL) cells by flow cytometry. Mutat. Res 594:113–119.2006). PubMed
Waldren, C., C. Jones, and T. T. Puck. Measurement of mutagenesis in mammalian cells. Proc. Natl. Acad. Sci. USA 76:1358–1362.1979. CrossRef, PubMed
Kraemer, S. M., D. B. Vannais, A. Kronenberg, A. Ueno, and C. A. Waldren. 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). CrossRef, PubMed, CSA
Ross, C. D., C. T. French, S. B. Keysar, and M. H. Fox. Mutant spectra of irradiated CHO AL cells determined with multiple markers analyzed by flow cytometry. Mutat. Res 624:61–70.2007). PubMed
Moore, M. M. and C. L. Doerr. Comparison of chromosome aberration frequency and small-colony TK-deficient mutant frequency in L5178Y/TK+/−–3.7.2C mouse lymphoma cells. Mutagenesis 5:609–614.1990). CrossRef, PubMed
Applegate, M. L., M. M. Moore, C. B. Broder, A. Burrell, G. Juhn, K. L. Kasweck, P. F. Lin, A. Wadhams, and J. C. Hozier. Molecular dissection of mutations at the heterozygous thymidine kinase locus in mouse lymphoma cells. Proc. Natl. Acad. Sci. USA 87:51–55.1990. CrossRef, PubMed, CSA
Kraemer, S. M. and C. A. Waldren. Chromosomal mutations and chromosome loss measured in a new human-hamster hybrid cell line, ALC: studies with colcemid, ultraviolet irradiation, and 137Cs gamma-rays. Mutat. Res 379:151–166.1997). PubMed, CSA
Ueno, A. M., D. B. Vannais, D. L. Gustafson, J. C. Wong, and C. A. Waldren. A low, adaptive dose of gamma-rays reduced the number and altered the spectrum of S1-mutants in human-hamster hybrid AL cells. Mutat. Res 358:161–169.1996). PubMed, CSA
Applegate, M. L., M. M. Moore, C. B. Broder, A. Burrell, G. Juhn, K. L. Kasweck, P. F. Lin, A. Wadhams, and J. C. Hozier. Molecular dissection of mutations at the heterozygous thymidine kinase locus in mouse lymphoma cells. Proc. Natl. Acad. Sci. USA 87:51–55.1990. CrossRef, PubMed, CSA
Evans, H. H. The prevalence of multilocus lesions in radiation-induced mutants. Radiat. Res 137:131–144.1994). CrossRef, PubMed, CSA
McGuinness, S. M., M. L. Shibuya, A. M. Ueno, D. B. Vannais, and C. A. Waldren. Mutant quantity and quality in mammalian cells (AL) exposed to cesium-137 gamma radiation: Effect of caffeine. Radiat. Res 142:247–255.1995). CrossRef, PubMed, CSA
Rudd, P. M. and R. A. Dwek. Glycosylation: heterogeneity and the 3D structure of proteins. Crit. Rev. Biochem. Mol. Biol 32:1–100.1997. CrossRef, PubMed, CSA
Rudd, P. M., B. P. Morgan, M. R. Wormald, D. J. Harvey, C. W. van den Berg, S. J. Davis, M. A. Ferguson, and R. A. Dwek. The glycosylation of the complement regulatory protein, human erythrocyte CD59. J. Biol. Chem 272:7229–7244.1997). CrossRef, PubMed, CSA
Fivaz, M., F. Vilbois, S. Thurnheer, C. Pasquali, L. Abrami, P. E. Bickel, R. G. Parton, and F. G. van der Goot. Differential sorting and fate of endocytosed GPI-anchored proteins. EMBO J 21:3989–4000.2002. CrossRef, PubMed, CSA
Horikawa, M., F. Suzuki, and S. Ban. Mammalian cell mutagenesis: comparison of the sensitivity in assay systems of mutations induced by radiations and chemicals. Mutat. Res 38:337. 1976). PubMed

FIG. 1. Flow cytometry histograms of CD59 expression in CHO AL cells after a dose of 4 Gy. The CD59 mutant region is set at 1% of the mean positive peak; the intermediate region is between the positive and negative peaks. The histograms are scaled to emphasize the small population of mutants. The mutant peak shifts from the intermediate region into the CD59 mutant region by day 6. Histograms such as these were analyzed in Fig. 2

FIG. 2. CHO AL mutant yield induced by γ radiation and CD59 expression measured at various times after irradiation. CD59 expression was measured after doses of 0–4 Gy. These results are from three replicates in one experiment. The error bars represent the standard deviations

FIG. 3. Flow cytometry histogram of CD59 expression 6 days after exposure to 4 Gy of γ radiation. The mutant peak and intermediate regions were gated into Regions 1–6. Sorting was done with an EPICS V cell sorter

FIG. 4. Panel A: Flow cytometry histograms of CD59 expression 8 days after sorting from Regions 1–6 of Fig. 3 (14 days postirradiation). Panel B: At least 1 × 104 cells from the entire lower populations in panel A were sorted, grown an additional 6 days (20 days postirradiation), and analyzed for CD59 expression. The dashed lines indicate the region that is approximately 1% of the CD59+ peak. The data are from one representative experiment of three that were done

FIG. 5. Relative growth of cells from sorted regions in comparison to control. After sorting the cells from regions shown in Fig. 3, cells were counted at various times as indicated on the x axis. Control (♦), Region 1 (), Region 2 (○), Region 3 (█), Region 4 (□), Region 5 (). The line is a linear regression for the control cells

FIG. 6. Survival of irradiated cells sorted from regions shown in Fig. 3. Numbers on the x axis correspond to the region. Control cells (Region 0) were sorted from the CD59-positive peak. Three hundred cells were sorted from each region, and colony survival was determined after a 7-day growth period. Surviving fraction is calculated relative to the plating efficiency from sorted control cells. Experiments were done in triplicate with proper controls. Error bars are the SEM from three independent experiments

FIG. 7. Flow cytometry histogram of CD59 expression 6 days after irradiation with 4 Gy. Region A corresponds approximately to Regions 1–3 in Fig. 3. Cells were sorted from each region and analyzed simultaneously for CD44 and CD90 expression. Cells were analyzed on a CyAn flow cytometer

FIG. 8. Bivariate flow cytometry histograms of populations sorted from regions shown in Fig. 7, then grown for an additional 18 days before analysis. All populations were gated on CD59− populations so that all cells analyzed were CD59− and were then analyzed for CD44 and CD90 expression. Quantification of the numbers of cells with various phenotypes is given in Table 2

table

TABLE 1 Expression of Various CD Markers 6 Days after Irradiation with 4 Gy Measured by Flow Cytometry on Cells Gated from Regions Shown in Figure 7

table

TABLE 2 Cells Sorted from CD59 Regions A–D in Figure 7, Grown 18 Additional Days, then Analyzed by Multiparameter Flow Cytometry as Shown in Figure 8

table

TABLE 3 Comparison of CD Marker Expression Measured by Flow Cytometry on Day 6 and Day 24 after Irradiation with 4 Gy

Cited by

Stephen B. Keysar, Michael H. Fox. (2009) Kinetics of CHO A L mutant expression after treatment with γ radiation, EMS, and asbestos. Cytometry Part A 75A:5, 412-419
Online publication date: 1-Jun-2009.
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