Mutation Research, DNA Repair, 254 (1991) 135-142 © 1991 Elsevier Science Publishers B.V. 0921-8777/91/$03.50 ADONIS 092187779100054F

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MUTDNA 06417

The genetic basis of resistance to ionising radiation damage in cultured mammalian cells John Thacker and R.E. Wilkinson MRC Radiobiology Unit, Chilton, Didcot, Oxon OXl l ORD (Great Britain)

(Received 9 May 1990) (Accepted 16 July 1990)

Keywords: Radioresistance, genetic basis; Complementation groups; Radiosensitive mutants; Hybrids

Summary To test the genetic similarity of independently-isolated hamster cell mutants sensitive to ionising radiation, these were fused in pairs and the hybrids exposed to X-rays. Some mutants (irsl, irs3, xrs-1, XR-1, BLM2) were found to complement all others tested for radiosensitivity in hybrids, and are therefore in separate genetic groups. The mutants irs2 and V-E5, both isolated from V79 cells, did not complement and therefore belong to the same group. Another pair, EM7 and irslSF, formed hybrids with intermediate levels of survival between mutant and wild-type. However, the parental cells fused to irslSF also showed intermediate sensitivity, suggesting a semi-dominant mutant ph¢notype rather than a lack of complementation. Crosses of some of these hamster mutants to the radioserlsitive mouse mutant M10 showed clear complementation (its1 X M10, its2 × M10) but for others the Qomplementation did not greatly exceed the sensitivity of one (irs3 x M10) or both mutants (XR-1 x M10). Taken with our previously-published data, these results show that there are at least 8 genetic groups determining resistance to ionising radiation damage in rodent cells.

In recent years a number of mammalian cell mutants which are hypersensitive to ionising radiation have been isolated (Sato and Hieda, 1979; Thompson et al., 1982; Stamato et al., 1983; Jeggo and Kemp, 1983; Jones et al., 1987; Zdzienicka and Simons, 1987; Robson et al., 1988; Fuller and Painter, 1988). Many of these mutants differ in their degree of radiation sensitivity, and in other attributes, but some are remarkably similar to

Correspondence: Dr. John Thacker, MRC Radiobiology Unit, Chilton, Didcot, Oxon OXll ORD (Great Britain).

each other. Phenotypic characteristics are not, however, a good guide to the genetic similarity of mutants; alleles of the same gene may vary considerably and it is possible that more than one gene controlling sensitivity is mutated in some mutants. Thus, it is important to establish at an early stage in the characterization of mutants displaying a common phenotypic trait (e.g., radiosensitivity) whether or not this trait reflects changes in the same gene. A simple method of establishing genetic identity between any two mutants is to fuse these together and test the radiosensitivity of the resulting hybrid cells: if sensitive then the mutants are

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genetically the same, if resistant then they are genetically dissimilar (but see also below). We reported earlier (Jones et al., 1988) that complementation analyses between 6 radiosensitive mutants of hamster cells (irsl, irs2, irs3, xrs-1, XR-1 and EM7) showed that each fell into a different genetic group. We now report data for a further set of mutants (BLM2, V-E5, irslSF and M10) using the same techniques. Materials and methods

Cell cultures V79 or CHO Chinese hamster cells, and their mutants, were cultured as described previously (Thacker, 1981; Thacker and Stretch, 1985) in Eagle's minimal essential medium (MEM) or aMEM, respectively. Radiation-sensitive mutants were generously supplied as follows: xrs-1 by Dr. P.A. Jeggo; EM7 by Dr. L.H. Thompson; XR-1 by Dr. T.D. Stamato; BLM-2, and parent line CHO(N), by Dr. I.D. Hickson; V-C4, V-E5 and V-G8, and parent line V79(L), by Dr. M. Zdzienicka; irslSF and parent line CHO-AA8 by Dr. R.B. Painter; M10 and parent line L5178Y by Dr. K. Sato. The irsl, irs2, and irs3 mutants, and parent line V79(H), were from the authors' laboratory (Jones et al., 1987). Double-marked sublines of these parent and mutant lines were selected, without mutagen treatment, where necessary; these are designated T O R (thioguanine- and ouabain-resistant). These lines were retested for radiation sensitivity (and in some cases for mitomycin C sensitivity) to ensure that the phenotype was unchanged. Cell fusion and hybrid isolation The double-marker (TOR) selection method was used as described by Thacker (1980) and Jones et al. (1988), following fusion of cells with polyethylene glycol (PEG). Fusions were carried out in monolayer (hamster × hamster) or in suspension (hamster x mouse). Control (self) crosses were usually performed at the same time as experimental crosses to check for reversion of the markers; measurable reversion frequencies were found in two of the fines selected, necessitating the selection of new double-marked lines. It was found that the mouse lines had to be selected in a higher

concentration of ouabain background growth than crosses (1 mM). A number pooled for assessment of otherwise stated.

(2 mM) to eliminate required for hamster of hybrid colonies was radiosensitivity unless

Hybrid cell characterization Chromosome analyses were performed as described previously (Jones et al., 1988). Cells were irradiated as growing cultures with at least 3 doses of 250 kV X-rays and immediately respread at varying densities to assess survival. The cloning efficiencies of hybrids were variable, but usually exceeded 50% for hamster x hamster crosses when cultured for some days after selection; for mouse x hamster crosses, cloning efficiencies were on average about 25% at the time of irradiation. Results

Fusion efficiency and chromosome counts Most hamster X hamster cell fusions yielded large numbers of hybrid cells (frequency >/1 in 103 viable cells), while reversion frequencies were ~< 4 x 10-6 (measured in self-crosses of the component lines). Thus, for most fusions, in excess of 100 hybrid cell clones were pooled for analysis. A few hamster x hamster crosses and most mouse x hamster crosses were less productive, giving up to 10-fold lower hybrid cell frequencies, but in all cases > 10 hybrid cell clones were pooled (unless single clones were deliberately cultured). Counts of chromosome numbers in hybrids and their parents showed that all hamster x hamster hybrids had average numbers within 10% of the sum of the two component lines (with standard errors overlapping this value). However, mouse x hamster hybrids often had greater deviations in chromosome number, commonly by 25-30% above or below the sum of the two component lines. Complementation grouping in hamster x hamster mutant hybrids (0 BLM2. This mutant is very sensitive to bleomycin but has a relatively small increase in sensitivity to ionising radiation as well as to such agents as ethyl methanesulphonate (EMS) and mitomycin C (MMC), compared to wild-type

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hamster cells (Robson et al., 1985). Its sensitivity to radiation, to EMS, and to MMC has similarities to the irs3 mutant, for example, but it was found to complement this mutant and another 5 hamster mutants representing the 6 complementation groups previously established (Fig. 1). Robson et al. (1988) have also found complementation between BLM2 and one of the xrs series of mutants.

(ii) V-C4, V-E5, V-G8. These mutants show a similar moderate increase in radiosensitivity (Zdzienicka et al., 1987) and while this work was in progress were shown to belong in a single complementation group (Zdzienicka et al., 1989). Data are shown for V-E5 to represent this group

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(iii) irslSF. This mutant is derived from the same parent CHO line (AA8) as the EM7 mutant and these mutants have a similar sensitivity to X-rays (Fig. 3). irslSF was crossed to EM7 and to another CHO-derived mutant, XR-1 (Stamato et al. 1983). The irslSF x XR-1 hybrids showed full complementation, but those of irslSF × EM7 did not return to wild-type CHO survival levels (data from two independently-derived hybrid pools are shown in Fig. 3). However, it was also found that the cross irslSF x AA8 did not show wild-type sensitivity, indicating some form of semi-dominance of the irslSF mutant over AA8-derived lines. In contrast, irslSF was found to complement fully the irsl, irs2 and its3 mutants (Fig. 4).

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The genetic basis of resistance to ionising radiation damage in cultured mammalian cells.

To test the genetic similarity of independently-isolated hamster cell mutants sensitive to ionising radiation, these were fused in pairs and the hybri...
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