Excision repair in xeroderma pigmentosum group C cells is regulated differently in transformed cells and primary fibroblasts

Excision repair in xeroderma pigmentosum group C cells occurs at about 20-30% of normal levels. In confluent fibroblasts a unique characteristic of this low repair is that it is clustered, representing very efficient repair in a small region of the genome. In SV40-transformed fibroblasts and Epstein-Barr virus-transformed lymphocytes of complementation group C, however, excision repair is randomly distributed. This may be a consequence of the high rate of proliferation of both of these cell types, because random repair is also observed in rapidly proliferating group C fibroblasts. The distribution of sites that can be mended in group C cells, therefore, varies according to the transformed and proliferative state of the cells, demonstrating that transformed cells do not always exhibit repair characteristics identical to those of primary fibroblasts.     

Regulation of DNA repair in serum-stimulated xeroderma pigmentosum cells

The regulation of DNA repair during serum stimulation of quiescent cells was examined in normal human cells, in fibroblasts from three xeroderma pigmentosum complementation groups (A, C, and D), in xeroderma pigmentosum variant cells, and in ataxia telangiectasia cells. The regulation of nucleotide excision repair was examined by exposing cells to ultraviolet irradiation at discrete intervals after cell stimulation. Similarly, base excision repair was quantitated after exposure to methylmethane sulfonate. WI-38 normal human diploid fibroblasts, xeroderma pigmentosum variant cells, as well as ataxia telangiectasia cells enhanced their capacity for both nucleotide excision repair and for base excision repair prior to their enhancement of DNA synthesis. Further, in each cell strain, the base excision repair enzyme uracil DNA glycosylase was increased prior to the induction of DNA polymerase using the identical cells to quantitate each activity. In contrast, each of the three xeroderma complementation groups that were examined failed to increase their capacity for nucleotide excision repair above basal levels at any interval examined. This result was observed using either unscheduled DNA synthesis in the presence of 10 mM hydroxyurea or using repair replication in the absence of hydroxyurea to quantitate DNA repair. However, each of the three complementation groups normally regulated the enhancement of base excision repair after methylmethane sulfonate exposure and each induced the uracil DNA glycosylase prior to DNA synthesis. These results suggest that there may be a relationship between the sensitivity of xeroderma pigmentosum cells from each complementation group to specific DNA damaging agents and their inability to regulate nucleotide excision repair during cell stimulation.     

Defective repair of gamma-ray induced DNA damage in xeroderma pigmentosum cells.

We used the bromouracil-photolysis technique to estimate the sizes of the repaired regions in normal human and xeroderma pigmentosum (XP) cells irradiated by gamma-rays aerobically or anoxically. After 1 1/2 hours of incubation, single-strand breaks were repaired and the repaired regions were small--one to two BrUra residues--for cells irradiated aerobically or anoxically. After a 20-hour incubation, the repaired region in normal cells showed a component mimicking U.V.-repair. There were large patches (approximately 30 BrUra residues) in the approximate ratios of one per six chain breaks for aerobic irradiation and one per three chain breaks for anoxic irradiation. XP cells, however, only showed large patches at 20 hours if they had been irradiated aerobically. We could not detect such regions in XP cells irradiated anoxically. These results indicate (1) that some part of ionizing damage mimics excision of U.V. damage in that the repair patches are large and the repair takes an appreciable time; (2) the types of such damage depend on whether the irradiation is done aerobically or anoxically; and (3) XP cells are defective in repairing a component of anoxic damage.     

Excision repair of UV- or benzo[a]pyrene diol epoxide-induced lesions in xeroderma pigmentosum variant cells is 'error free'

It is known that cells from one class of xeroderma pigmentosum (XP) patients, called XP variants, carry out excision repair of UV-induced DNA damage at a normal rate and are only slightly more sensitive than normal cells to the cytotoxic effect of UV radiation, but are much more sensitive to the mutagenic effect of UV. To see if this hypermutability were the result of an 'error-prone', excision repair process, we irradiated fibroblasts derived from an XP variant patient, XP4BE, under conditions that allowed the cells various lengths of time for excision repair before the onset of DNA synthesis (S phase) and assayed the frequency of 6-thioguanine (TG)-resistant mutants. Cells synchronized by release from confluence (G0 state) and irradiated just prior to S phase showed a dose-dependent increase in mutants at very high frequencies; cells irradiated in early G1, approximately 12 h before the onset of S phase, showed frequencies 4 times lower. Cells irradiated in the G0 state and allowed 24 h or 48 h for excision repair before the onset of S phase showed still lower frequencies. A comparison of the relative rates of decrease in mutant frequency with time for excision repair before the onset of S phase in XP variant cells and normal human fibroblasts after a dose of 4 or 6 J/m2 showed that these were equal. However, for every time point, the frequency of mutants induced per dose of UV was significantly higher in the XP variant population than in the normal, suggesting that the XP variant cells have an abnormally error-prone process of replicating DNA on a template containing unexcised lesions or normal cells are by-passing many of such lesions using an error-free process. A similar comparative study in synchronized populations of XP4BE cells and normal cells, using the anti 7,8-diol-9,10-epoxide of benzo[a]pyrene, showed that excision repair prior to the onset of S phase also decreased the frequency of mutants induced in XP variant cells by this agent. But for every dose and time point, the frequencies induced in XP4BE cells and normal cells were identical. Thus, the hypermutability of the XP4BE cells was specific to UV radiation-induced DNA lesions.     

Unique cross-link and monoadduct repair characteristics of a xeroderma pigmentosum revertant cell line

Monoadducts and cross-links formed in DNA of human cells by a psoralen derivative, 4'-hydroxy-methyl-4,5',8-trimethylpsoralen (HMT), have been measured by a new, simple method, based on S1 nuclease digestion of 3H-labeled adducts in DNA, that provides rapid information on the repair of both classes of lesions. Normal human fibroblasts and cells from patients with dyskeratosis congenita and xeroderma pigmentosum (XP) group C were capable of removing both monoadducts and cross-links, whereas XP groups A and D failed to remove either. An XP revertant, isolated from a group A cell line on the basis of an acquired mutagen-induced resistance to ultraviolet light, has the unique property of being capable of removing cross-links but not monoadducts. Consistent with this property, the XP revertant was found to be resistant to cell killing by the cross-linking psoralen derivative, HMT, but as sensitive as its parental cell line to a monofunctional psoralen derivative, 5-methylisopsoralen.     

Mechanisms of impairment of DNA repair in human cells. Interferons stimulated DNA repair in xeroderma pigmentosum cells

DNA repair synthesis and strand break DNA repair induced by 4-nitroquinoline-1-oxide and UV-irradiation in Xeroderma pigmentosum lymphocytes and fibroblasts pretreated by leucocyte interferons were studied. Stimulation of DNA repair synthesis in interferon-pretreated Xeroderma pigmentosum cells, defective in incision, was detected. No such effect was noted for strand break DNA repair. Hence, antimutagenic activity of interferons in human cells is connected with their modificating effect on DNA repair.     

A further definition of characteristics of DNA-excision repair in xeroderma pigmentosum complementation group A strains

The location in the genome of excision repair following exposure to UV (254 nm) of two XP complementation group A strains, XP12BE and XP8LO, that differ considerably in their excision-repair rates, have been determined. Capacity for repair in XP8LO has also been determined. Sites repaired in DNA in a 24-h post-UV period were located relative to the remaining pyrimidine dimers using the M. luteus UV-endonuclease to nick partially repaired DNA and sedimentation in alkaline sucrose to size the resulting DNA. Repair in group A occurs randomly throughout the genome in a manner similar to that observed for normal cells but in contrast to domain-limited repair in group C strains. This observation defines a further similarity of the excision repair detected in group A compared to normal cells that is in addition to the previously reported related characteristics of the respective excision rate curves. A reduced repair capacity in XP8LO relative to normal cells was detected. This strain, which repairs DNA at an initial rate identical to that of normal strains when irradiated with doses of 5 J/m2 or less, repairs DNA at a slower than normal but constant rate at higher doses. This leads to the suggestion that XP8LO is defective in the number of repair enzyme complexes compared to normal cells.     

Unique DNA repair properties of a xeroderma pigmentosum revertant.

A group A xeroderma pigmentosum revertant with normal sensitivity was created by chemical mutagenesis. It repaired (6-4) photoproducts normally but not pyrimidine dimers and had near normal levels of repair replication, sister chromatid exchange, and mutagenesis from UV light. The rate of UV-induced mutation in a shuttle vector, however, was as high as the rate in the parental xeroderma pigmentosum cell line.     

Defective repair replication of DNA in xeroderma pigmentosum. 1968

Most forms of the human hereditary disease xeroderma pigmentation (XP) are due to a defect in nucleotide excision repair of DNA damage in skin cells associated with exposure to sunlight. This discovery by James Cleaver had an important impact on our understanding of nucleotide excision repair in mammals.     

Hypersensitivity of xeroderma pigmentosum cells to dietary carcinogens

Xeroderma pigmentosum patients, in addition to ultraviolet-induced skin cancers, have an increased prevalence of neoplasms occurring in sites shielded from ultraviolet radiation. We postulated that these internal neoplasms might be related to ingestion of dietary carcinogens. As model dietary carcinogens, we studied the tryptophan pyrolysis products, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2). These dietary compounds bind to DNA and are highly mutagenic and carcinogenic. Cytotoxicity of these compounds was examined in cultured lymphoblastoid cell lines from xeroderma pigmentosum patients in complementation groups A, B, C, D and E and the variant form and from normal donors. All xeroderma pigmentosum lymphoblastoid cell lines showed a greater reduction in viable cell concentration than the 2 normal lymphoblastoid cell lines following addition of Trp-P-1 or Trp-P-2 (5 micrograms/ml) to the culture medium. Possible differences in cellular activation of these compounds were overcome by treating the cells with rat-liver microsome-activated Trp-P-2. There was a greater reduction in viable cell concentration in the xeroderma pigmentosum group A and D cells than in the normal lymphoblastoid cell lines after treatment with activated Trp-P-2. These data suggest that the xeroderma pigmentosum DNA-repair system is defective in repairing Trp-P-1 and Trp-P-2 induced DNA damage in addition to being defective in repairing ultraviolet-induced DNA damage. Thus xeroderma pigmentosum patients may be at increased risk of toxicity from some dietary carcinogens.     

Efficient repair of 8-oxo-7,8-dihydrodeoxyguanosine in human and hamster xeroderma pigmentosum D cells

The repair of the endogenous lesion 8-oxo-7,8-dihydrodeoxyguanosine (8-oxodG) was investigated in the nucleotide excision repair mutant xeroderma pigmentosum D (XPD), using human normal or transformed XPD fibroblasts and the Chinese hamster XPD cell line UV5. In vivo repair of 8-oxodG induced by hydrogen peroxide treatment and analyzed by high-performance liquid chromatography/electrochemical detection was normal in the XPD mutant fibroblasts XP15PV and GM434, as compared to normal human fibroblasts GM970, GM5757, and GM6114. Similar results were obtained with the human SV40-transformed XPD mutant cell line GM8207 in comparison to the control cell line GM637. Repair of 8-oxodG was even slightly (2-3-fold) but reproducibly increased in Chinese hamster XPD mutant UV5 cells, as compared to parental AA8 cells. This unexpected effect was reversed by transfection in UV5 cells of a wild-type XPD cDNA and confirmed in in vitro experiments in which a plasmid substrate containing a single 8-oxoG was repaired by UV5 cell extracts. The data show that repair of 8-oxodG is normal in XPD cells, thus indicating that the neurological complications of XPD patients may not be linked to in vivo accumulation of this lesion.     

Complementation of the xeroderma pigmentosum DNA repair defect in cell-free extracts

Soluble extracts from human lymphoid cell lines that perform repair synthesis on covalently closed circular DNA containing pyrimidine dimers or psoralen adducts are described. Short patches of nucleotides are introduced by excision repair of damaged DNA in an ATP-dependent reaction. Extracts from xeroderma pigmentosum cell lines fail to act on damaged circular DNA, but are proficient in repair synthesis of ultraviolet-irradiated DNA containing incisions generated by Micrococcus luteus pyrimidine dimer-DNA glycosylase. Repair is defective in extracts from all xeroderma pigmentosum cell lines investigated, representing the genetic complementation groups A, B, C, D, H, and V. Mixing of cell extracts of group A and C origin leads to reconstitution of the DNA repair activity.     

Retrovirus-mediated DNA repair gene transfer into xeroderma pigmentosum cells: Perspectives for a gene therapy

The rare hereditary disease xeroderma pigmentosum (XP) is clinically characterized by extreme sun sensitivity and an increased predisposition for developing skin cancer. Cultured cells from XP patients exhibit hypersensitivity to ultraviolet (UV) radiation due to the defect in nucleotide excision repair (NER), and other cellular abnormalities. Seven genes identified in the classical XP forms, XPA to XPG, are involved in the NER pathway. In view of developing a strategy of gene therapy for XP, we devised recombinant retrovirus-carrying DNA repair genes for transfer and stable expression of these genes in cells from XP patients. Results showed that these retroviruses are efficient tools for transducing XP fibroblasts and correcting repair-defective cellular phenotypes by recovering normal UV survival, unscheduled DNA synthesis, and RNA synthesis after UV irradiation, and also other cellular abnormalities resulting from NER defects. These results imply that the first step of cellular gene therapy might be accomplished successfully.     

Increase of sister chromatid exchanges in excision repair deficient xeroderma pigmentosum

Summary The distribution of spontaneous sister chromatid exchanges (SCEs) was studied in PHA-stimulated lymphocytes from 15 patients affected by xeroderma pigmentosum (XP). The study of unscheduled DNA synthesis (UDS) in twelve of these patients showed that seven were deficient and five proficient. The number of SCEs in XP patient cells was higher than in those of 19 controls, and the distributions of SCEs per cell were significantly different. However, the results varied when XP patients were considered in relation to their UDS: the group of XP patients with proficient UDS did not differ, whereas the group of XP patients with deficient UDS was very significantly different from controls. The group not tested for UDS was similar to the deficient UDS group. The possible relationship between the increase of SCEs and the type of DNA repair defect is discussed.     

Excision repair in xeroderma pigmentosum group C but not group D is clustered in a small fraction of the total genome

DNA repair in xeroderma pigmentosum complementation groups C and D occurs at a low level. Measurements of pyrimidine dimers remaining in bulk DNA from the whole genome indicated very little excision in either complementation group. The repair sites in group C cells were, however, clustered together in small regions of the genome which appeared to be mended nearly as efficiently as the whole genome is mended in normal cells, while repair in group D cells was randomly distributed. Growth of normal cells in cycloheximide or 3-aminobenzamide neither inhibited repair nor altered the distribution of repair sites. Growth of normal cells in novobiocin or aphidicolin inhibited excision but repair remained randomly distributed. On the basis of these observations, and consideration of other cellular features of group C and D, we suggest that group C may represent a mutation which results in a low level of repair enzymes with normal function. Group D, on the other hand, may represent a mutation resulting in functionally defective repair enzymes.