Repair of ultraviolet radiation damage in xeroderma pigmentosum cells belonging to complementation group F

DNA-repair characteristics of xeroderma pigmentosum belonging to complementation group F were investigated. The cells exhibited an intermediate level of repair as measured in terms of (1) disappearance of T4 endonuclease-V-susceptible sites from DNA, (2) formation of ultraviolet-induced strand breaks in DNA, and (3) ultraviolet-induced unscheduled DNA synthesis during post-irradiation incubation. The impaired ability of XP3YO to perform unscheduled DNA synthesis was restored, to half the normal level, by the concomitant treatment with T4 endonuclease V and ultraviolet-inactivated Sendai virus. It is suggested that xeroderma pigmentosum cells of group F may be defective, at least in part, in the incision step of excision repair.     

Preferential repair of nuclear matrix associated DNA in xeroderma pigmentosum complementation group C

The distribution of ultraviolet-induced DNA repair patches in the genome of xeroderma pigmentosum cells of complementation group C was investigated by determining the molecular weight distribution of repair labeled DNA and prelabeled DNA in alkaline sucrose gradients after treatment with the dimerspecific endonuclease V of bacteriophage T4. The results were consistent with the data reported by Mansbridge and Hanawalt (1983) and suggest that DNA-repair synthesis in xeroderma pigmentosum cells of complementation group C occurs in localized regions of the genome. Analysis of the spatial distribution of ultraviolet-induced repair patches in DNA loops attached to the nuclear matrix revealed that in xeroderma pigmentosum cells of complementation group C repair patches are preferentially situated near the attachment sites of DNA loops at the nuclear matrix. In normal human fibroblasts we observed no enrichment of repair-labeled DNA at the nuclear matrix and repair patches appeared to be distributed randomly along the DNA loops. The enrichment of repair-labeled DNA at the nuclear matrix in xeroderma pigmentosum cells of complementation group C may indicate that the residual DNA-repair synthesis in these cells occurs preferentially in transcribing regions of the genome.     

A ninth complementation group in xeroderma pigmentosum, XP I

A new complementation group of excision-deficient xeroderma pigmentosum (XP) is described in 2 patients living in the F.R.G. Dermatological, ophthalmological and neurological symptoms of XP are presented together with DNA repair characteristics such as unscheduled DNA synthesis, colony-forming ability and alkaline elution studied in cultured fibroblasts. The results are compared to normal controls.     

DNA excision in repair proficient and deficient human cells treated with a combination of ultraviolet radiation and acridine mustard (ICR-170) or 4-nitroquinoline 1-oxide

Excision repair was measured in normal human and xeroderma pigmentosum group C fibroblasts treated with ultraviolet radiation and the carcinogens acridine mustard (ICR-170) or 4-nitroquinoline 1-oxide (4NQO) by the techniques of unscheduled synthesis, photolysis of bromodeoxyuridine incorporated into parental DNA during repair, and assays of sites sensitive to ultraviolet (UV)-endonuclease. Doses of ICR-170 and 4NQO, low enough not to inhibit unscheduled DNA synthesis (UDS), caused damage to DNA that was repaired by a long patch type mechanism and the rates of UDS decreased rapidly in the first 12 h after treatment. Repair after a combined action of UV plus ICR-170 or UV plus 4NQO was additive in normal cells and no inhibition of loss of endonuclease sensitive sites was detected. In xeroderma pigmentosum (XP) C cells there was less repair after UV plus ICR-170 than after each treatment separately; whereas there was an additive effect after UV plus 4NQO and no inhibition of loss of endonuclease sensitive sites. The results indicate that in normal human fibroblasts there are different rate limiting steps for removal of chemical and physical damages from DNA and that XP cells have a different repair system for ICR-170, not just a lower level, than normal cells. Possibly the same long patch repair system works on 4NQO damage in both normal and XP cells.     

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.     

Repair of DNA damaged by methylmethane sulfonate in normal human lymphocytes and in xeroderma pigmentosum

Methylmethanesulphonate has been shown to stimulate an intensive unscheduled DNA synthesis in lymphocytes derived from normal donors as well as in those from patients with xeroderma pigmentosum of the classical form. Somewhat less intensive unscheduled DNA synthesis was observed in cells of a patient suffering from xeroderma pigmentosum. In the case of XPII unscheduled DNA synthesis was greatly reduced which supports the peculiarity of this form of xeroderma pigmentosum.     

Repair of UV-endonuclease-susceptible sites in the 7 complementation groups of xeroderma pigmentosum A through G

7 strains of human primary fibroblasts were chosen from the complementation groups A through G of xeroderma pigmentosum; these strains are UV-sensitive and deficient in excision repair of UV damage on the criterion of unscheduled DNA synthesis (UDS). They were compared with normal human fibroblasts and one xeroderma pigmentosum variant with regard to their capacity to remove pyrimidine dimers, induced in their DNA by UV at 253.7 nm. The XP variant showed a normal level of dimer removal, whereas 6 of the other XP strains had a greatly reduced capacity to remove this DNA damage, in agreement with their individual levels of UDS. Strain XP230S (complementation group F), however, only showed a 20% reduction in the removal of dimers, which is much less than expected from the low level of UDS in this strain.     

An immortalized xeroderma pigmentosum, group C, cell line which replicates SV40 shuttle vectors

We have established and characterized an immortalized xeroderma pigmentosum (XP), group C, cell line. Transformation of the human fibroblasts was carried out with a recombinant plasmid, pLAS-wt, containing SV40 DNA encompassing the entire early region with a defective origin of DNA replication. The transformed XP cell line, XP4PA-SVwt, and the normal transformed fibroblasts AS3-SVwt, both express SV40 T antigen together with enhanced levels of the transformation-associated cellular protein, p53. XP4PA-SVwt retains the XP UV-repair defective phenotype as demonstrated by low levels of unscheduled DNA synthesis and by the reduced survival of irradiated SV40 virus. Analysis of cellular DNA shows a single major, stable, integration site of pLAS-wt in the XP4PA-SVwt cells. The T antigen in these cells supports efficiently the replication of SV40 based shuttle vectors and should prove suitable for the introduction, expression and selection of genes related to DNA repair and to the study of mutagenesis using defined molecular probes.     

DNA damage, DNA repair and chromosome aberrations of xeroderma pigmentosum cells and controls following exposure to nitrosation products of methylguanidine

Nitrosation of methylguanidine (MG) led to products that caused DNA fragmentation (shift in sedimentation profiles of velocity centrifugation through alkaline sucrose gradients), a DNA repair synthesis (unscheduled uptake of (³H]TdR), chromosome aberrations and a lethal effect of cultured human fibroblasts. The response of repair-deficient xeroderma pigmentosum cells did not differ from that of controls. The nitrosation of MG must be carried out at a pH level below 3, in order to obtain products that react with cellular DNA. The results show that a DNA repair synthesis of human fibroblasts appear to be a sensitive assay for carcinogenic and mutagenic nitrosation products which may be formed within an organism from non-carcinogenic compounds.     

Cell-cycle defect of DNA repair in progeria skin fibroblasts

We examined the temporal regulation of DNA repair during synchronous cell proliferation in normal and progeroid human fibroblasts. Ultraviolet light-induced (254 nm, 20 J/m2) unscheduled DNA synthesis was measured at 4-h intervals after serum stimulation, for up to 32 h. Normal cells regulated DNA repair in a defined temporal sequence, showing a peak in the induction of DNA repair just before DNA synthesis. Progeroid skin fibroblasts failed to show an increase in nucleotide excision repair before scheduled DNA synthesis, but the background level of DNA repair was not significantly different from that in controls. Regulation of repair in progeroid human fibroblasts appeared similar, but not identical to that previously reported by Gupta and Sirover (1984b) for xeroderma pigmentosum complementation group C. Our results suggest that patients with Hutchinson-Gilford progeria may have a defect in DNA repair; the results offer nominal evidence that the average level of UV-induced DNA is decreased, and that individuals with this disease lack both the normal enhancement of DNA repair before scheduled DNA synthesis and the temporal control of DNA repair.     

DNA repair ability of cultured cells derived from mouse embryos in comparison with human cells

DNA repair in mouse cells derived from embryos of 3 inbred strains were investigated in comparison with that in human cells. The levels of unscheduled DNA synthesis after UV irradiation appeared to change at different passages, but capacities of host-cell reactivation of UV-irradiated herpes simplex virus were always reduced to the same levels as those in xeroderma pigmentosum cells. This implied that mouse cells are reduced in excision-repair capacities and that the apparently high levels of unscheduled DNA synthesis at certain passages are not quantitatively related to high levels of cell survival. Essentially no differences in DNA repair were noted among 3 strains — BALB/c, C3H/He and C57BL/10.     

Human chromosome 15 confers partial complementation of phenotypes to xeroderma pigmentosum group F cells.

Microcell-mediated transfer of a single human chromosome from repair-proficient human cells to genetic complementation group F cells from the hereditary disease xeroderma pigmentosum (XP) results in partial complementation of repair-defective phenotypes. The complementing chromosome was identified by cytogenetic and molecular analysis as human chromosome 15. Transfer of this chromosome to XP-F cells restores approximately 20% of the resistance of wild-type cells to killing by UV radiation or by the UV-mimetic chemical 4-nitroquinoline-1-oxide (4NQO), as well as partial repair synthesis of DNA measured as unscheduled DNA synthesis. Additionally, complemented XP-F cells have an enhanced capacity for reactivation of the plasmid-borne E. coli cat gene following its inactivation by UV radiation. Phenotypic complementation of XP cells by chromosome 15 is specific to genetic complementation group F; no effect on the UV sensitivity of XP-A, XP-C, or XP-D cells was detected. The observation that phenotypic complementation is partial is open to several interpretations and does not allow the definitive conclusion that the XP-F locus is carried on chromosome 15.     

Poly(ADP-ribose)-synthesis and excision repair in light sensitive skin disorders

Several data suggest a relationship of poly(ADP-ribose) (PAR) synthesis to DNA repair and the influence of some trace elements on the semiconservative and unscheduled DNA synthesis (UDS). Previously we found certain alterations in the UV-light induced UDS and in the contents of trace elements in the lymphocytes of patients with light sensitive skin disorders. In the recent study in polymorphic light eruption, cutaneous porphyrias and xeroderma pigmentosum the PAR synthesis and zinc, copper and manganese contents in the chromatin of the lymphocytes (measured by neutron activation analysis) were investigated. UV induced PAR synthesis was generally lower in the cells of polymorphic light eruption and especially in xeroderma pigmentosum with a reduced repair capacity whereas in cutaneous porphyrias no difference was observed. Some correlations occurred between the contents of trace elements studied and UDS as well in each group tested. It seems that PAR investigations throw new light upon our understanding of the pathomechanism of photodermatoses.     

Microinjection of human cell extracts corrects xeroderma pigmentosum defect

Cultured fibroblasts of patients with the DNA repair syndrome xeroderma pigmentosum (XP) were injected with crude cell extracts from various human cells. Injected fibroblasts were then assayed for unscheduled DNA synthesis (UDS) to see whether the injected extract could complement their deficiency in the removal of u.v.-induced thymidine dimers from their DNA. Microinjection of extracts from repair-proficient cells (such as HeLa, placenta) and from cells belonging to XP complementation group C resulted in a temporary correction of the DNA repair defect in XP-A cells but not in cells from complementation groups C, D or F. Extracts prepared from XP-A cells were unable to correct the XP-A repair defect. The UDS of phenotypically corrected XP-A cells is u.v.-specific and can reach the level of normal cells. The XP-A correcting factor was found to be sensitive to the action of proteinase K, suggesting that it is a protein. It is present in normal cells in high amounts, it is stable on storage and can still be detected in the injected cells 8 h after injection. The microinjection assay described in this paper provides a useful tool for the purification of the XP-A (and possibly other) factor(s) involved in DNA repair.     

Microinjection of Escherichia coli UvrA, B, C and D proteins into fibroblasts of xeroderma pigmentosum complementation groups A and C does not result in restoration of UV-induced unscheduled DNA synthesis

The UV-induced unscheduled DNA synthesis (UDS) in cultured human fibroblasts of repair-deficient xeroderma pigmentosum complementation groups A and C was assayed after injection of identical activities of either Uvr excinuclease (UvrA, B, C and D) from Escherichia coli or endonuclease V from phage T4. Under conditions where the T4 enzyme was able to induce repair synthesis in both XP complementation groups in agreement with earlier observations (de Jonge et al., 1985), no effect of the UvrABCD excinuclease could be observed either when the enzymatic complex was injected into the cytoplasm, or when it was delivered directly into the nucleus. In addition, no effect of the E. coli excinuclease was found on the repair ability of normal repair-proficient human fibroblasts. We conclude that the UvrABCD excinuclease may not work on DNA lesions in human chromatin.     

Microinjection of Micrococcus luteus UV-endonuclease restores UV-induced unscheduled DNA synthesis in cells of 9 xeroderma pigmentosum complementation groups

The UV-induced unscheduled DNA synthesis (UDS) in cultured cells of excision-deficient xeroderma pigmentosum (XP) complementation groups A through I was assayed after injection of Micrococcus luteus UV-endonuclease using glass microneedles. In all complementation groups a restoration of the UV-induced UDS, in some cells to the repair-proficient human level, was observed. Another prokaryotic DNA-repair enzyme, T4 endonuclease V, restored the UV-induced UDS in a similar way after microinjection into XP cells. Since both enzymes specifically catalyse only the incision of UV-irradiated DNA, we conclude that this activity is impaired in cells of all 9 excision-deficient XP complemenation groups tested.     

Repair of damage by ultraviolet radiation in xeroderma pigmentosum cell strains of complementation groups E and F

The xeroderma pigmentosum fibroblast strains XP2RO, complementation group E, and XP23OS, group F, were compared with normal human primary fibroblasts with regard to repair of damage induced by 254-nm UV. In XP2RO cells, repair DNA synthesis, measured by autoradiography (unscheduled DNA synthesis = UDS), was about 50% of the value found in normal human cells. In these cells also the removal of UV-induced sites recognized by a specific UV-endonuclease proceeds at a reduced rate. By having BUdR incorporated into the repaired regions, followed by the induction of breaks in these patches by 313-nm UV, it was shown that the reduced repair synthesis is not caused by a shorter length of the repair regions in XP2RO, but is solely due to a reduction in the number of sites removed by excision repair. In XP23OS a discrepancy was observed between the level of UDS, which was about 10% of the normal value, and other repair-dependent properties such as UV survival, host-cell reactivation and removal of UV-endonuclease-susceptible sites, which were less reduced than could be expected from the UDS level. However, when UDS was followed over a longer period than the 2 or 3 h normally used in UDS analysis, it appeared that in XP23OS cells, the rate of UDS remained constant whereas the rate decreased in normal control cells. Consequently, the residual level of UDS varies with the period over which it is studied.     

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.     

Host-Cell Repair of Vaccinia Virus and of Double Stranded RNA of Encephalomyocarditis Virus

IN normal human cells DNA which has been damaged by ultraviolet radiation is repaired by excision of thymidine dimers and by repair replication. Patients suffering from xeroderma pigmentosum have a hereditary defect of the excision step and therefore their cells repair ultraviolet-induced lesions in their DNA less efficiently than do normal cells^1–4. An analogous situation has been well characterized in bacteria⁵.     

Repair of 4-nitroquinoline-1-oxide-induced DNA damage in normal human cells and cells from classical and variant xeroderma pigmentosum

The effect of 4-nitroquinoline-1-oxide (4NQO) upon 3 fibroblast cell lines derived from normal and xeroderma pigmentosum subjects have been compared. Excision-deficient XP cells (XP2BI), complementation group G, are nearly 200-fold more sensitive than normal cells to the lethal effect of 4NQO while XP variants (XP7TA), are 2-fold more sensitive. This cytotoxicity correlates with the levels of unscheduled DNA synthesis performed by the 3 cell lines. 4NQO causes a dose-related inhibition of DNA replication in all cell lines. However, newly replicated DNA synthesised immediately after treatment of cells with 4NQO is slightly smaller in XP7TA variant cells than in normal cells receiving the same dose of 4NQO, but DNA fragments in excision-deficient XP2BI are 50% smaller. It is likely that replicon elongation and joining together of newly replicated DNA fragments is dependent upon the excision of certain 4NQO-induced lesions, possibly normally repaired by a 'short-patch' repair process defective in XP2BI.