Genomic DNA from normal human or mouse cells was transfected together with the selectable marker gpt into the simian virus 40-transformed ataxia telangiectasia fibroblast line, AT5BIVA. From a series of experiments involving over 400000 clones selected for the gpt marker, one unambiguously radiation-resistant clone (clone 67) was recovered following selection with repeated cycles of gamma irradiation.

The normal level of radiation resistance of clone 67 has been maintained for at least 11 months in the absence of further selection by radiation. The resistant clone contains one copy of the gpt gene. Its DNA synthesis following gamma-irradiation is inhibited to an extent intermediate between that of ataxia telangiectasia and normal cells.

Three out of four thioguanine-resistant derivatives of clone 67 have either lost or do not express the gpt sequence and show almost the same sensitivity to gamma irradiation as the original AT5BIVA line. This suggests that the radiation resistance of clone 67 may be linked to the gpt sequence and may have arisen as a consequence of the transfection, rather than as the result of an independent mutation to radiation resistance.

Ataxia telangiectasia (AT) is a recessive genetic disease, which in homozygous individuals leads to neurological disorders, immune deficiency and a high incidence of cancers, especially of the lymphatic system (Bridges & Harnden, 1982). Acute sensitivity to ionizing radiation has been observed in affected individuals (e.g. see Gotoff et al. 1967) and in cells cultured from them (Taylor et al. 1975). In order to obtain a better understanding of the nature of the genetic defect we are attempting to clone the normal gene that complements the defect in AT. As the first step we have transfected DNA sequences from normal human cells into simian virus 40 (SV40)- transformed AT fibroblasts.

One-step or two-step DNA transfection has proved a valuable tool in the identification of human oncogenes (Land et al. 1983) and it has been used to transfer a number of mammalian genes, such as those for hypoxanthine phosphoribosyltransferase (HPRT) (de Jonge et al. 1982) and adenine phosphoribosyltransferase (Lowy et al. 1980) to deficient cells. With these genes, a powerful positive selection system was available. In the case of human syndromes such as AT, which are characterized by enhanced radiation sensitivity, such one-step selection procedures are not possible since repair-deficient cells such as the AT cell line used in our study may only be about twofold more sensitive than the wild-type. In order to select for a possible resistant transfectant in such cases, it is necessary to use a procedure of repeated selective enrichment. This prolonged selection, followed by the subsequent study of resistant transfectants, requires the use of an immortalized human cell line as recipient. In this paper we report the isolation and characterization of a radiation-resistant cell line derived from an SV40-transformed (immortal) AT cell line following DNA-mediated gene transfer.

Cell lines

The SV40-transformed AT cell line used as recipient in our experiments, AT5BIVA, was generously provided by Dr L. Toji, Institute for Medical Research, Camden, NJ, USA. Two SV40-transformed normal cell lines were used. GM0637 was obtained from the Camden Cell Repository (New Jersey) and MRC5-V1 (Huschtscha & Holliday, 1983) was obtained through Dr R. Cox, Harwell.

DNA preparations

High molecular weight genomic DNA was prepared from frozen human placenta, mouse embryos or MRC5-V1 cell cultures by lysis in sodium dodecyl sulphate, digestion with RNase and proteinase K followed by successive extraction with phenol, phenol/chloroform/isoamyl alcohol (50:48:2, by vol.) and chloroform/isoamyl alcohol (24:1, v/v). The plasmids pSV2gpt (containing the gpt gene under SV40 control cloned into pBR322 (Mulligan & Berg, 1981) and pL10 (containing the gpt gene cloned into pBR322) were generously provided by Dr P. Berg. They were extracted from bacteria by an alkaline lysis procedure (Ish-Horowicz & Burke, 1981).

Selection for radiation resistance

Since AT5BIVA is only about twofold more sensitive to gamma-irradiation than SV40-trans- formed normal cells (Fig. 1A), we have been obliged to use a procedure of repeated selective enrichment in order to select for potential resistant transfectants. In a typical DNA-mediated gene transfer experiment 2 × 101 to 5 ×101 AT5BIVA cells were seeded onto 100 9-cm plates. Two days later DNA transfection was carried out by the calcium phosphate precipitation method (Graham & van der Eb, 1973; Wigler et al. 1978) using 20 μg of genomic DNA and 10 μg of pSV2gpt DNA. After 16 h the DNA-containing medium was removed and replaced with fresh medium. Twenty- four hours later this medium was in turn replaced with selective “MAX” medium (after Mulligan & Berg, 1981) containing 25 μg ml −1 mycophenolic acid, 10 μgml −1 xanthine, 15 ×gml −1 hypoxanthine, 0 ·2 μgml −1 aminopterin, 5 μgml −1 thymidine, 2 ·3 μgml −1 deoxycytidine, 5 μgml −1 glycine. The basis of this selection protocol is that mycophenolic acid inhibits the de novo synthesis of GMP. Cells are therefore dependent on an exogenous purine source. Xanthine, supplied exogenously, can be used by xanthine-guanine phosphoribosyltransferase (XPRT), the product of the gpt gene, but not by the endogenous hprt gene. Thus only cells harbouring the gpt gene can survive in MAX medium.

Fig. 1.

Gamma-ray survival curves. A. Survival curves of normal cell lines, MRC5-V1 (filled symbols, 3 experiments), and GM0637 (open symbols, 3 experiments), and of ATSBIVA and clone 67 (c67) mean line of data shown in Fig. IB. B. Survival curves of clone 67 (open symbols) after 5 (▫), 8 (▿), 10 (○) or 11 (╛) months in the absence of gamma-ray selection. Equivalent filled symbols show data for AT5BIVA obtained in the same experiments.

Fig. 1.

Gamma-ray survival curves. A. Survival curves of normal cell lines, MRC5-V1 (filled symbols, 3 experiments), and GM0637 (open symbols, 3 experiments), and of ATSBIVA and clone 67 (c67) mean line of data shown in Fig. IB. B. Survival curves of clone 67 (open symbols) after 5 (▫), 8 (▿), 10 (○) or 11 (╛) months in the absence of gamma-ray selection. Equivalent filled symbols show data for AT5BIVA obtained in the same experiments.

About 20 days after transfection, plates contained between 100 and 1000 gpt+ colonies. These were trypsinized, pooled and half the cells frozen down. The other half were transferred to small flasks (approx. 101 cells per 25 cm1 flask) and gamma-irradiated (3 Gy). At 7-10 days later, the flasks were again irradiated with the same dose and after a further 7 –10 days the cycle of trypsinization, pooling and irradiation was repeated. This procedure, which permits approximately 1 % survival of AT cells and 10 % survival of cells with normal radiation-resistance following each cycle of radiation treatment, was continued until either the culture died out, or an apparently radiation-resistant culture emerged. Clone 67 arose from one such experiment using donor DNA from MRC5-V1.

Survival curves

Survival curves following gamma-irradiation from a 1Co source were obtained using techniques described elsewhere (Arlett & Harcourt, 1980).

Assay for xanthine-guanine phosphoribosyltransferase (XPRT) in cell extracts

XPRT activity in sonicated cell extracts was assayed by the starch gel electrophoresis technique for HPRT as described by Harris & Hopkinson (1977). The substrate, [1C]hypoxanthine (2 ·5 μCi ml −1) can be used by both the endogenous HPRT and the exogenous XPRT enzymes (Miller et al. 1972), so that both enzymes can be detected in a single assay.

Southern analysis

A 25 μg sample of genomic DNA was digested with restriction enzymes for 4h at 2units/μg −1 DNA. The DNA was electrophoresed in 0 ·7 % agarose gels and transferred onto nitrocellulose filters. The filters were hybridized at 42 °C, in the presence of 50% formamide, with pLIO DNA 1P-labelled by nick translation to a specific activity of about 3 ×101 disints min −1μg −1. Standard procedures were used (Maniatis et al. 1982).

Selection of thioguanine-resistant derivatives

Clone 67 cells were plated in the presence of 2 ·5ml −1 or 5 μgml −1 6-thioguanine (TG) at a density of 101 cells per dish. After 3 weeks, individual TG-resistant clones were picked and expanded into mass cultures in the presence of 5 μgml −1 TG.

Selection of clone 67

All our experiments have involved cotransfection of AT5BIVA cells with human or mouse genomic DNA (see Table 1) and the plasmid pSVZgpt, which codes for the dominant selectable gpt gene. The cultures were selected first for the presence of the gpt gene, thus eliminating the vast majority of the cell population that had not incorporated any foreign DNA. The frequency of transfer of the gpt gene to these cells was generally greater than 10 −3. The gpt+ transfectants were then allowed to grow to form clones before applying several cycles of radiation selection. Irradiation of clones should provide the same degree of enrichment for resistance as irradiation of individual cells, but the chance of eliminating an entire clone of resistant cells should be minimal with an appropriate choice of dose. Approximately 400 000 gpt+ clones from mycophenolic acid selection have been grown up and screened for radiation resistance. Table 1 provides a summary of the selection experiments performed to date. Clone 67 was isolated from an experiment using MRC5-V1 DNA for transfection, followed by five cycles of approximately 3 Gy 1Co irradiation.

Table 1.

Summary of experiments designed to correct the defect in AT5BIVA cells and select for radiation-resistant derivatives

Summary of experiments designed to correct the defect in AT5BIVA cells and select for radiation-resistant derivatives
Summary of experiments designed to correct the defect in AT5BIVA cells and select for radiation-resistant derivatives

Survival of clone 67

Some 20 flasks were found to contain apparently radiation-resistant clones in these experiments. Clone 67 was the only one to show good growth, stability and clearly enhanced radiation resistance. Clone 67 was found to have gamma-ray sensitivity within the normal range, approximately equal to that of GM0637 and slightly lower than that of MRC5-V1 (Fig. 1). This normal sensitivity was maintained during a 3-month test period in the absence of the gpt+ selection, and for more than 11 months in the absence of further gamma-radiation selection (Fig. IB). It should be noted that SV40 transformation in its own right increases the resistance of fibroblasts to gamma-irradiation. The distinction between AT and wild-type is nevertheless preserved (Green et al. 1985; Murnane et al. 1985).

Southern analysis

The DNA from clone 67 was subjected to digestion with restriction enzymes and Southern analysis. The probe used in these experiments was the plasmid pL10, which is very similar to pSV2gpt but lacks all the SV40 sequences. Digestion of DNA from clone 67 with which has no cutting site in pSV2gpt, followed by hybridization with 1P-labelled pL10 DNA showed a single band of 15 –20 (×101) bases (Fig. 2A, lane2). Digestion with Eco RI, which cuts at a single site inpSV2gpi, revealed a major hybridizing band of about 12 ×101 bases and a minor band of slightly higher molecular weight (Fig. 2B, lane 1). These results indicate that clone 67 contains a single integrated copy of the pSV2gpt plasmid. It is not possible to estimate the amount of exogenous human DNA that has been integrated into the recipient genome. However, analogous experiments using mouse genomic DNA as donor suggest that no more than 500 ×101 bases of exogenous mammalian DNA (and maybe much less; this being the limit of detection in these experiments) is incorporated into the genome of AT5BIVA cells (unpublished observations).

Fig. 2.

gpt sequences in clone 67. DNA from clone 67 or AT5BIVA was digested with Sad or EcoRI. The digests were run on 0 ·7% agarose gels and then transferred to nitrocellulose. A. Lane 1, pSV2gpt linearized with EcoRI (25 pg DNA); lane 2, clone 67 DNA digested with SacI. B. EcoRI digestion of DNA from clone 67 and TG-resistant derivatives. Lane 1, clone 67; lane 2, 1332.1; lane 3, 1332.2; lane 4, 1338.3; lane 5, 1332.5. Numbers at the sides denote sizes (×101 bases) of HindIII-digested lambda DNA fragments, used as molecular weight markers.

Fig. 2.

gpt sequences in clone 67. DNA from clone 67 or AT5BIVA was digested with Sad or EcoRI. The digests were run on 0 ·7% agarose gels and then transferred to nitrocellulose. A. Lane 1, pSV2gpt linearized with EcoRI (25 pg DNA); lane 2, clone 67 DNA digested with SacI. B. EcoRI digestion of DNA from clone 67 and TG-resistant derivatives. Lane 1, clone 67; lane 2, 1332.1; lane 3, 1332.2; lane 4, 1338.3; lane 5, 1332.5. Numbers at the sides denote sizes (×101 bases) of HindIII-digested lambda DNA fragments, used as molecular weight markers.

DNA synthesis following gamma-irradiation

A characteristic of all primary AT fibroblasts studied to date is that there is less inhibition of DNA synthesis by gamma-irradiation in these cells than in normal cells (Houldsworth & Lavin, 1980; Painter & Young, 1980; Bridges & Hamden, 1982). We found that even though the resistance to gamma-irradiation of clone 67 was indistinguishable from that of normal cells (Fig. 1), the inhibition of DNA synthesis was only slightly greater than in AT5BIVA cells (Lehmann et al. 1986). It did not approach the level seen in normal cells. This finding in clone 67 of normal gammaray sensitivity associated with the reduced inhibition of DNA synthesis typical of AT clearly separates these two phenotypes.

Isoenzyme analysis

We have ruled out the possibility that clone 67 is a contaminant, by isoenzyme analysis of the parental line and of clone 67 (Table 2). The probability of finding this identical isoenzyme pattern with these nine enzymes in two independently derived cell lines is 0 ·003 %.

Table 2.

Isoenzyme patterns in AT5BIVA, clone 67 and their TG-resistant derivatives

Isoenzyme patterns in AT5BIVA, clone 67 and their TG-resistant derivatives
Isoenzyme patterns in AT5BIVA, clone 67 and their TG-resistant derivatives

Effect of loss of gpt on radiation sensitivity of clone 67

Clone 67 could have arisen from a spontaneous reversion or second-site mutation to radiation resistance, in which case its radiation resistance would be completely independent of the transfected DNA. In order to investigate this possibility we attempted by selecting in thioguanine (TG) to obtain derivatives of clone 67 that had lost the gpt gene.

Clone 67 contains both the mammalian hypoxanthine phosphoribosyltransferase hprt and the bacterial guanine xanthine phosporibosyltransferase gpt genes and we were therefore surprised to find that TG-resistant derivatives could be isolated with relatively high frequency. In one experiment in which the selective concentration of TG was 2 ·5 μgml −1, TG-resistant clones arose at a frequency of 2 ·5 ×10 −4; in a subsequent experiment using 5 μgml −1 TG, the frequency was about 10-s. Four TG-resistant lines designated 1332.1, 1332.2, 1338.3 and 1332.5 were examined for gpt sequences, for the activity of the gpt gene product XPRT, and for radio-sensitivity. Isoenzyme analysis confirmed that these four lines were indeed derived from clone 67 (Table 2).

The DNA from the TG-resistant derivatives was digested with EcoRI followed by Southern analysis and hybridization with 1P-labelled pL10. Fig. 2B shows that in lines 1332.1 and 1332.2 the pSV2gpt sequences are completely deleted (lanes 2, 3) and line 1338.3 contains rearranged sequences (lane 4). In contrast in line 1332.5 (lane 5) the gpt sequences were indistinguishable from those of line 67 (lane 1).

The activities of the endogenous mammalian HPRT and the bacterial XPRT enzymes have been measured on starch gel electrophoresis. As anticipated, XPRT activity was not detected in the AT5BIVA parental line (Fig. 3A, lane 7), but it was present in line 67 (lane 8). In the TG-resistant derivatives 1332.1 and 1332.2 in which the gpt gene had been deleted there was no activity (lanes 5,6). Some residual activity could be detected in 1338.3 (lane 3). Line 1332.5 had the unusual property of being able to grow in the presence of TG (selection against the gpt gene), in neutral medium, or in MAX (selection for the presence of the gpt gene). In all cases the gpt gene was expressed as demonstrated by the band of XPRT activity in Fig. 3A, lane 4, and Fig. 3B.

Fig. 3.

XPRT activity in various derivatives. XPRT and HPRT were assayed by starch gel electrophoresis using [1C]hypoxanthine. A. Lane 1, mouse LMTK- cells; lane 2, human lymphoblastoid line BRI-8; lanes 3 –6, TG-resistant derivatives of clone 67 (3, 1338.3; 4, 1332.5; 5, 1332.2; 6, 1332.1); lane 7, AT5BIVA; lane 8, clone 67. Extracts prepared from cells grown in non-selective medium. M, mouse; H, human. B. Extracts of line 1332.5 grown in MAX (lane 1) or TG (lane 2).

Fig. 3.

XPRT activity in various derivatives. XPRT and HPRT were assayed by starch gel electrophoresis using [1C]hypoxanthine. A. Lane 1, mouse LMTK- cells; lane 2, human lymphoblastoid line BRI-8; lanes 3 –6, TG-resistant derivatives of clone 67 (3, 1338.3; 4, 1332.5; 5, 1332.2; 6, 1332.1); lane 7, AT5BIVA; lane 8, clone 67. Extracts prepared from cells grown in non-selective medium. M, mouse; H, human. B. Extracts of line 1332.5 grown in MAX (lane 1) or TG (lane 2).

If the gpt gene were linked to the gene responsible for the increased radiation resistance of clone 67, some of these derivatives may also have deleted or altered the expression of linked sequences, and they may thus show loss of radiation resistance. From Fig. 4 it can be seen that this is indeed the case. All four independent derivatives were more sensitive than clone 67 to gamma-irradiation and were almost as sensitive as AT5BIVA.

Fig. 4.

Gamma-ray survival curves of TG-resistant derivatives of clone 67. Survival curves of AT5BIVA and clone 67 (heavy lines). TG-resistant derivatives of clone 67: 1332.1 (▫ – –▫); 1332.2 (▵ - - - ▵); 1338.3 (• - - - - •); 1332.5 (○ ____○). Best fit curves were determined by a NAG library subroutine.

Fig. 4.

Gamma-ray survival curves of TG-resistant derivatives of clone 67. Survival curves of AT5BIVA and clone 67 (heavy lines). TG-resistant derivatives of clone 67: 1332.1 (▫ – –▫); 1332.2 (▵ - - - ▵); 1338.3 (• - - - - •); 1332.5 (○ ____○). Best fit curves were determined by a NAG library subroutine.

We have isolated a radiation-resistant derivative of AT5BIVA (clone 67) following DNA-mediated gene transfer. Four derivatives of this line that have been selected for TG resistance have radiation sensitivity restored to a level close to that of AT5BIVA. In two of these lines the gpt gene has been deleted and in a third line it is rearranged. These findings suggest that the radiation resistance in clone 67 is linked to the gpt gene, which in turn suggests that the radiation resistance has arisen through transfection rather than by mutation during selection. The properties of the fourth derivative, however, weaken this argument. In this derivative radiation resistance is lost despite the fact that the gpt gene is maintained and continues to be expressed. The properties of this cell line are bizarre. It is able to grow in MAX or TG despite the fact that XPRT activity is expressed in both media. Moreover, its radiation sensitivity is also dependent on the growth medium (results not shown). We have no satisfactory explanation for these observations with line 1332.5.

For the rest of this Discussion we will assume that the gpt gene is linked to radiation resistance in line 67 and therefore that the radiation resistance has arisen by transfection, whilst keeping in mind that our evidence for this is not conclusive. The radiation resistance could then have arisen in a number of different ways. First, the wild-type allele of the gene responsible for the radiation sensitivity of AT5BIVA has itself been transferred and is now linked to the gpt gene. If this were the case, however, one would have expected restoration of post-irradiation DNA synthesis to wild-type levels since the two phenotypes are linked in all the AT complementation groups identified to date. Second, another gene complementing the radiation sensitivity but otherwise unrelated to AT may have been transferred. A third possibility is that no other gene has been transferred, but that the pSV2gpt has become integrated in such a manner as to alter expression of an adjacent gene affecting radiation resistance. In the very large number of gpt transfectants that we have examined, radiation resistance is unaffected, so that this is clearly not a general property of the integrated pSV2gpt plasmid.

Experiments attempting to transfer radiation resistance to repair-deficient human cells have been performed in several laboratories (Royer-Pokora & Haseltine, 1984; reviewed by Lehmann, 1985) with remarkably little success. An early promising result (Takano et al. 1982) has not been reproduced despite attempts in numerous laboratories. This contrasts with the successful correction of repair deficiencies in rodent cell lines using human DNA (Rubin et al. 1983; Westerveld et al. 1984; Maclnnes et al. 1984; Thompson et al. 1985). In one case (Westerveld et al. 1984) this has led to the successful cloning of a human repair gene. One difficulty in the present gene-transfer experiments is the lack of an all-or-none selective system. The approach that we have adopted here should select clones with a moderate increase in radiation resistance, but if, as with xeroderma pigmentosum (Royer-Pokora & Haseltine, 1984), there is an appreciable frequency of mutation to radiation resistance the experiments are likely to become uninterpretable. Our failure to find more than one stable resistant clone among 400 000 transfectants suggests that this may not be a problem with the AT line used here. A second problem concerns the amount of genomic DNA incorporated by our recipient. In experiments with good rodent recipient cells (e.g. mouse L cells or 3T3 cells), on average only 10000 clones need to be screened to detect a particular transferred gene of moderate size. If, as appears to be the case, our recipient takes up genomic DNA less efficiently, a correspondingly larger experiment is likely to be required.

Irrespective of the origin of the radiation resistance in clone 67, the separation of radiation sensitivity from the lack of inhibition of DNA synthesis is of considerable interest with respect to the molecular defect in AT, as discussed in detail earlier (Lehmann et al. 1986). Our isolation of a radiation-resistant derivative of AT in which the gpt gene may be linked to a gene influencing radiation sensitivity may provide an opportunity for cloning a gene that affects DNA repair in humans. We are currently cloning sequences linked to the gpt gene from the DNA of line 67.

This work was supported in part by Euratom contract BIO-E-414-81-UK.

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