Repair-deficient xeroderma pigmentosum cells made UV light resistant by fusion with X-ray-inactivated Chinese hamster cells.

Abstract

Xeroderma pigmentosum (XP) is an inherited human disease caused by a malfunction in the dimer excision step of DNA repair. The disease is manifested as an extreme sensitivity to UV light resulting in the development of skin cancers and, in some cases, neurological disorders. At least nine complementation groups have been identified, indicating that the disease comprises a variety of mutations at several genetic loci (2, 5). The genetic basis of the physiological defect of XP has not yet been characterized. Neither genes nor gene products have been identified, and it is not known whether the deficiency is due to mutations in structural or regulatory aspects of DNA repair.Introduction of isolated DNA into XP cells by standard DNA transfection methods has thus far been unsuccessful in introducing genes that code for UV resistance, presenting a major obstacle to the cloning of genetic information that can complement the deficiency of XP (12,18). In contrast, the introduction of human DNA into repair-deficient hamster cells has resulted in the identification of DNA repair loci not represented in the XP complementation groups (4,16,21,22). We used a method of fusing X-irradiated Chinese hamster ovary (CHO) cells with XP cells so that fragments of CHO chromosomes, which appear to support DNA repair, were transferred into the human cells. This report presents the initial work of establishing stable UV-resistant, repaircompetent hybrid cell lines, which will then be used to clone hamster DNA sequences that correct the repair defect of XP cells. MATERIALS AND METHODSCell lines. Normal human fibroblasts (1508), simian virus 40-transformed normal human fibroblasts (A13-6F and GM637), simian virus 40-transformed XP group A fibroblasts (XP12RO), and Chinese hamster ovary (CHO) cells (AA8) were grown in Eagle minimal essential medium with 10% fetal calf serum.Cell fusion. The cell fusion procedure was similar to that described by Cirullo et al. (1) and was adopted because it * Corresponding author. predisposes hybrids to lose hamster chromosomes rather than human chromosomes. CHO cells were exposed to a lethal dose of X rays (50 Gy, 10 Gy/min), mixed with approximately equal numbers of unirradiated simian virus 40-transformed XP fibroblasts, and dispersed into 100-mm dishes. After 5 h at 37°C, cell fusion was mediated by exposure for 30 s to 40% polyethylene glycol 1000 and 10% dimethyl sulfoxide in minimal essential medium, followed by three rinses with 10% dimethyl sulfoxide in minimal essential medium. Complete culture medium was added after the final rinse, and cells were incubated overnight. To select for hybrids that contained functional CHO DNA repair genes and to eliminate unfused XP cells, we irradiated cells with 13 J of UV light per m2 (254 nm, 1.3 J/m2 per s) once a day for 3 consecutive days. At this dosage, XP12RO cell survival is 5 x 10-3, as determined by colony formation. Cultures were incubated for 2 to 3 weeks until colonies developed, and colonies on each plate were pooled to establish primary hybrid cell l...

Decreased Levels of (6–4) Photoproduct Excision Repair in Hybrid Fish of the Genus Xiphophorus¶

“…Our results differ from those observed in cell fusion experiments in which heterokaryons formed from eukaryotic cells displaying widely diverse repair capacities (14–16) showed enhanced repair relative to the parental cells. Studies by Paterson and Lohman (14) showed that two distinct repair systems ( i.e.…”

Molecular cloning and biological characterization of a human gene, ERCC2, that corrects the nucleotide excision repair defect in CHO UV5 cells.

“…This is due, at least in part, to the reduced amounts of stably integrated DNA in human lines relative to many rodent lines (14,47). Some success with correction of XP cells has been achieved by fusion of X-ray-inactivated CHO cells with XP group A (17), resulting in primary hybrids having -2% hamster DNA. Thus far, cell fusion studies between CHO UV mutants and XP cells have failed to identify any overlap in these complementation groups (40).…”

Unique DNA repair properties of a xeroderma pigmentosum revertant.

“…The sensitivity of XP cells in culture to UV light (254 nm) is 5-to 10-fold greater than nQrmal (1,15), and the cells fail to repair a wide range of radiation-and chemically induced DNA damage (5, 10, 27), including (5-5,6-6)cyclobutane pyrimidine dimers and (6-4) pyrimidine-pyrimidone photoproducts, measured by radioimmunoassay (4, 19-21) and enzymatic methods (7,13,14,44). The relative proportions of cyclobutane dimers to other photoproducts determined from their photoreactivation suggest that (6-4) photoproducts may account for as much as 30% of the total lesions (24), and they are highly mutagenic (2).…”

“…1C), a response characteristic of the mex phenotype of XP12RO (8,31). Normal, XP12RO, and XP129 cells all had similar sensitivity to mitomycin C, with a D37 concentration of 45 nM (Cleaver and Thomas, in press), consistent with the results of Fujiwara et al (11) with normal and XP cells.Cyclobutane pyrimidine dimers in DNA were quantified in purified DNA by digestion with Micrococcus luteus UV endonuclease, as previously described (7,13,14). On the basis of this assay, the XP129 revertant and the parental XP12RO cell line both appear to be defective in the removal of cyclobutane dimers (Table 1), whereas the two normal cell lines removed about 60% of the initial number of dimers in 24 h. When repair of cyclobutane and (6-4)pyrimidinepyrimidone dimers was monitored by radioimmunoassays that specifically detect these lesions in DNA (18,20,21,36), slightly different results were obtained.…”