Complementation of a DNA repair defect in xeroderma pigmentosum cells by transfer of human chromosome 9 (xeroderma pigmentosum complementation group A/microcell fusion/UV irradiation/monochromosomal hybrids)

Complementation of the repair defect in xeroderma pigmentosum cells of complementation group A was achieved by the transfer of human chromosome 9. A set of mouse-human hybrid cell lines, each containing a single Ecogpt-marked human chromosome, was used as a source ofdonor chromosomes. Chromosome transfer to XPTG-1 cells, a hypoxanthine/guanine phosphoribosyltransferase-deficient mutant of simian virus 40-transformed complementation group A cells, was achieved by microcell fusion and selection for Ecogpt. Chromosome-transfer clones of XPTG-1 cells, each containing a different human donor chromosome, were analyzed for complementation of sensitivity to UV irradiation. Among all the clones, increased levels of resistance to UV was observed only in clones containing chromosome 9. Since our recipient cell line XPIG-1 is hypoxanthine/guanine phosphoribosyltransferase deficient, cultivation of Ecogpt+ clones in medium containing 6-thioguanine permits selection of cells for loss of the marker and, by inference, transferred chromosome 9. Clones isolated for growth in 6-thioguanine, which have lost the Ecogpt-marked chromosome, exhibited a UV-sensitive phenotype, confirming the presence of the repair gene(s) for complementation group A on chromosome 9. Xeroderma pigmentosum (XP) is an autosomal-recessive human disease and cells cultured from XP patients exhibit higher sensitivity to UV-radiation and cell transformation than normal cells (1). Nine complementation groups, designated A through I (1-3) and a variant (XP-V) (1) have been identified for the XP condition. Biochemical defects in nine of the XP groups (A-I) have been defined in the nucleotide excision repair pathway that removes pyrimidine dimers (3-5), whereas XP-V is defective in post-replication repair (1). Transient complementation ofXP cells of several groups has been achieved by microinjection of total cell extracts (6-7) and mRNA (8) from normal cells but it has not been possible to define the gene(s) or gene products involved in excision repair synthesis. Another approach toward the study of the repair process has been the isolation of repairdefective mutants of Chinese hamster cells (9). However, none of these mutants has been shown to be related to the XP condition (9). In addition, a human gene isolated by complementation of the CHO mutants (10-12) is also not related to the repair defect in XP cells. Although the UV-sensitive (UV') phenotype of XP cells provides an excellent model for gene isolation by DNA transfection, attempts in this (R.S.A., unpublished results) and other laboratories (22) to rescue the repair gene have been unsuccessful. This may be attributed to inefficient DNA transfer into XP cells, inadequate selection procedures, and/ or a gene of large size. It is also possible that more than one gene is required to complement the repair defect. We, therefore, explored the possibility of transferring intact or large fragments of human chromosomes to XP cells using the method of microcell-mediated chromosome transfer (MMCT). A panel of mouse-human hybrid cell lines, each containing a single Ecogpt-marked human chromosome (13) has been used as a chromosome source to transfer marked human chromosomes individually to hypoxanthine/guanine phosphoribosyltransferase (HGPRT)-deficient XP-A cells. In this paper, we report that human chromosome 9 complements the repair defect in XP-A cells. MATERIALS AND METHODS Cell Lines and Growth Conditions. An HGPRTXP-A cell line, XPTG-1, isolated after mutagenesis of GM4312A cells (Human Genetic Mutant Cell Repository, Camden, NJ) and selection in 6-thioguanine (TG), was used as recipient for MMCT. XPTG-1 cells were also marked with a selectable marker, pSV2neo (14), which facilitates double selection in MMCT experiments. A panel of mouse-human hybrid cell lines, each containing either individual or multiple human chromosomes, were used as microcell donors. All cell lines were routinely cultured at 370C in a 10% C02/90% air atmosphere in Dulbecco's modified Eagle's medium (DMEM) containing 10% (vol/vol) fetal bovine serum. The medium was supplemented with appropriate selective agents such as mycophenolic acid (25 gg/ml) plus xanthine (70 gg/ml) (MX medium) and G418 (400 gg/ml) to isolate chromosome-transfer clones. DMEM containing TG (TG medium) was used to isolate clones for the loss of the Ecogpt-marked human chromosome (13). MMCT. Microcells prepared from donor cell lines as described (13) were fused with the recipient cells in monolayers by using 50% (wt/vol) PEG 1500 (13). After fusion, cells were cultured in DMEM for 48 hr (expression time) and then transferred to MX medium containing G418. Addition of G418 to medium selects against any intact donor cells that might have been present among microcells. Independent chromosome-transfer colonies, isolated individually, were propagated in MX medium for further analysis. Analysis for Complementation. For initial identification of complementation, 106 cells from each chromosome-transfer clone were seeded in the middle of 100-mm tissue culture dishes in triplicate. The cells were rinsed with isotonic phosphate-buffered saline (PBS), UV-irradiated (6 J/m2) at a rate of 0.5 J'm-2 sec-1, and incubated in fresh medium. After 3 days, plates were fixed with absolute methanol, stained with crystal violet, and visually evaluated for cell survival by using an inverted microscope. The clones identified to be UV Abbreviations: XP, xeroderma pigmentosum; TG, 6-thioguanine; S. sensitive; R, resistant; MMCT, microcell-mediated chromosome transfer; HGPRT, hypoxanthine/guanine phosphoribosyltransferase; AK,, adenylate kinase 1. *To whom reprint requests should be addressed. 8872 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Proc. Natl. Acad. Sci. USA 86 (1989) 8873 resistant (UVR) at 6 J/m2 were analyzed for post-UV-irradiation cell survival at doses from 1.5 J/m2 to 12.0 J/m2 by modification of a method described by Cleaver and Thomas (15). Briefly, 106 cells plated in the middle of 100-mm tissue culture dishes in triplicate were cultured for 24 hr in MX medium, rinsed with PBS, UV-irradiated at various doses, and then cultured in MX medium. After 24 hr of further incubation, [3H]thymidine (0.5 uCi/ml; specific activity, 40 Ci/mmol; 1 Ci = 37 GBq) was added to the cultures. After 72 hr of growth in the presence of [3H]thymidine, cells were harvested and lysed in 10 mM Tris HCl/1 mM EDTA (pH 7) containing 0.1% SDS. Relative incorporation of [3H]thymidine into cellular DNA was used as an index for post-irradiation cell survival. Cell survival measured by [3H]thymidine incorporation as described here, was comparable to the data obtained by traditional, but more laborious, post-UV-irradiation colony formation assay (unpublished results). Quantification of unscheduled DNA synthesis was performed by labeling UV-irradiated (10 J/m2) cells with [3H]thymidine followed by autoradiography. Cytogenetic and Biochemical Analysis. Cytogenetic and biochemical analyses were performed to identify the complementing chromosome present in hybrid cells. For cytogenetic identification, metaphase spreads prepared by standard method were stained for G-11 and G-banding to determine the number and identity of the human chromosome (13). Biochemical analysis was performed for the expression of the adenylate kinase 1 (AK1) isozyme by starch gel electrophoresis of cell extracts (16). Southern blot analysis (17) was performed using chromosome 9-specific DNA probes, D9S11 and D9S12, to confirm chromosome identity in hybrid cells and to follow the lineage of complementing chromosome through various cell lines by using Ecogpt as probe.