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.     

Interaction of human and chick DNA repair functions in UV-irradiated xeroderma pigmentosum-chick erythrocyte heterokaryons

Fusion of chick erythrocytes with human primary fibroblasts results in the formation of heterokaryons in which the inactive chick nuclei become reactivated. The expression of chick DNA repair functions was investigated by the analysis of the DNA repair capacity after exposure to ultraviolet (UV) irradiation of such heterokaryons obtained after fusion of chick erythrocytes with normal human or xeroderma pigmentosum (XP) cells of complementation groups A, B, C and D. Unscheduled DNA synthesis (UDS) in normal human nuclei in these heterokaryons is suppressed during the first 2–4 days after fusion. The extent and duration of this suppression is positively correlated with the number of chick nuclei in the heterokaryons. Suppression is absent in heterokaryons obtained after fusion of chicken embryonic fibroblasts with XP cells (complementation group A and C).Restoration of DNA repair synthesis is found after fusion in XP nuclei of all complementation groups studied. It occurs rapidly in XP group A nuclei, starting one day after fusion and reaching near normal human levels after 5–8 days. In nuclei of the B, C and D group increased levels of UDS are found 5 days after fusion. At 8 days after fusion the UDS level is about 50% of that found in normal human nuclei. The pattern of UDS observed in the chick nuclei parallels that of the human counterpart in the fusion. A fast complementation pattern is also observed in chick fibroblast-XP group A heterokaryons resulting within 24 h in a UDS level comparable with that in chick fibroblast-normal human heterokaryons. In heterokaryons obtained after fusion of chick fibroblasts with XP group C cells UDS remains at the level of chick cells. These data suggest that reactivation of chick erythrocyte nuclei results in expression of repair functions which are able to complement the defects in the XP complementation groups A, B, C and D.     

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.     

Postreplication repair of DNA in human fibroblasts after UV irradiation or treatment with metabolites of benzo(a)pyrene

We have examined the ability of normal fibroblasts and of excision-deficient xeroderma pigmentosum (XP) and XP variant fibroblasts to perform postreplication DNA repair after increasing doses of either ultraviolet (UV) irradiation or mutagenic benzo(a)pyrene derivatives. XP cells defective in the excision of both UV-induced pyrimidine dimers and guanine adducts induced by treatment with the 7,8-diol-9,10-epoxides of benzo(a)pyrene were partially defective in their ability to synthesize high molecular weight DNA after the induction of both classes of DNA lesions. This defect was more marked in XP variant cells, despite their ability to remove by excision repair both pyrimidine dimers and the diol epoxide-induced lesions to the same degree as observed in normal cells. The benzo(a)pyrene 9,10-oxide had no effect in any of the 3 cell lines. The response of the excision and postreplication DNA repair mechanisms operating in human fibroblasts treated with benzo(a)pyrene 7,8-diol-9,10-epoxides, therefore, appears to resemble closely that seen after the induction of pyrimidine dimers by UV irradiation.     

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.     

Infection of UV-irradiated xeroderma pigmentosum fibroblasts by herpes simplex virus: study of capacity and Weigle reactivation.

The capacity of monolayers of both normal human and xeroderma pigmentosum (XP) filbroblasts to support plaque formation by herpes simplex virus was decreased when the monolayers were ultraviolet (UV) irradiated and infected with virus. Fibroblasts of XP complementation groups A, B, and D were sensitive to UV, being 4-6 fold more sensitive than either fibroblasts of XP complementation group C or fibroblasts from a normal individual. When the monolayers were irradiated 4 days prior to infection, the capacity of normal fibroblasts to support herpes virus growth recovered, whereas the capacity of the XP strains decreased further compared to that measured when infection immediately followed irradiation. Concurrent experiments with UV-irradiated herpes virus showed that the survival of this virus did not increase when infection by irradiated virus immediately followed irradiation of the monolayers. However, if the monolayers were irradiated 4 days prior to infection, the survival of this virus increased by a factor of nearly 2. Such Weigle reactivation (WR) occurred at lower fluences to the XP fibroblasts than to normal fibroblasts, suggesting that WR results from residual cellular DNA damage left after excision repair.     

Complementation analysis of xeroderma pigmentosum variants

DNA repair after UV exposure was studied in multinucleate cells, obtained after fusion of excision-defective and variant xeroderma pigmentosum fibroblasts. Optimal fusion conditions were determined, facilitating the measurement of DNA replication in heterokaryons. In unirradiated multikaryons, entry into the S phase was depressed, when compared with unfused cells. The extent of the depression of S phase entry was dependent on the fusion conditions. In heterokaryons obtained after fusion of XP variant (6 different strains) with excision-defective XP (three cell strains from complementation groups A, C and D) both unscheduled DNA synthesis and postreplication repair after UV irradiation were restored to normal levels. In contrast, complementation was not observed after pairwise fusion of the XP variant cell strains. These results suggest that the XP variants comprise a single complementation group, different from complementation groups A, C and D.     

The influence of caffeine on cell survival in excision-proficient and excision-deficient xeroderma pigmentosum and normal human cell strains following ultraviolet-light irradiation.

A uniform response to UV of four normal cell strains was demonstrated. One excision-proficient xeroderma pigmentosum variant strain (XP7TA) had a wild-type UV response but a second (XP30RO) was more sensitive. An excision-deficient xeroderma pigmentosum strain XP4L0 was substantially more sensitive than wild-type cell strains. A continuous post-irradiation treatment with non-toxic levels of caffeine enhanced the lethal effect of UV light in both xeroderma pigmentosum variant cell strains but not in cells from normal individuals. There was no detectable effect on cells from a xeroderma pigmentosum individual from complementation group A. These results correlate well with observations on the influence of caffeine on post-replication repair in the three classes of cells.     

Transient correction of excision repair defects in fibroblasts of 9 xeroderma pigmentosum complementation groups by microinjection of crude human cell extracts

Crude extracts from human cells were microinjected into the cytoplasm of cultured fibroblasts from 9 excision-deficient xeroderma pigmentosum (XP) complementation groups. The level of UV-induced unscheduled DNA synthesis (UDS) was measured to determine the effect of the extract on the repair capacity of the injected cells. With a sensitive UDS assay procedure a (transient) increase in UV-induced UDS level was found in fibroblasts from all complementation groups after injection of extracts from repair-proficient (HeLa) or complementing XP cells (except in the case of XP-G), but not after introduction of extracts from cells belonging to the same complementation group. This indicates that the phenotypic correction is exerted by complementation-group-specific factors in the extract, a conclusion that is in agreement with the observation that different levels of correction are found for different complementation groups. The XP-G-correcting factor was shown to be sensitive to proteolytic degradation, suggesting that it is a protein like the XP-A factor.     

Cockayne syndrome complementation group B associated with xeroderma pigmentosum phenotype

Two siblings have been reported whose clinical manifestations (cutaneous photosensitivity and central nervous system dysfunction) are strongly reminiscent of the DeSanctis-Cacchione syndrome (DCS) variant of xeroderma pigmentosum (XP), a severe form of XP. Fibroblasts from the siblings showed UV sensitivity, a failure of recovery of RNA synthesis (RRS) after UV irradiation, and a normal level of unscheduled DNA synthesis (UDS), which were, unexpectedly, the biochemical characteristics usually associated with Cockayne syndrome (CS). However, no complementation group assignment in these cells has yet been performed. We here report that these patients can be assigned to CS complementation group B (CSB) by cell fusion complementation analysis. To our knowledge, these are the first patients with defects in the CSB gene to be associated with an XP phenotype. The results imply that the gene product from the CSB gene must interact with the gene products involved in excision repair and associated with XP.     

Inhibition of human excision DNA repair by inorganic arsenic and the co-mutagenic effect in V79 Chinese hamster cells

We investigated the lethal, UV killing-potentiating and repair-inhibiting effects of trivalent arsenic trioxide (As2O3) and pentavalent sodium arsenate (Na2HAsO4) in normal human and xeroderma pigmentosum (XP) fibroblasts. The presence of As2O3 for 24 h after UV irradiation inhibited the thymine dimer excision from the DNA of normal and XP variant cells and thus the subsequent unscheduled DNA synthesis (UDS): excision inhibitions were partial, 30-40%, at a physiological dose of 1 microgram/ml and 100% at a supralethal dose of 5 micrograms/ml. Correspondingly, As2O3 also potentiated the lethal effect of UV on excision-proficient normal and XP variant cells in a concentration-dependent manner, but not on excision-defective XP group A cells. Na2HAsO4 (As5+) was approximately an order of magnitude less effective in preventing all the above repair events than As2O3 (As3+) which is highly affinic to SH-containing proteins. The above results provide the first evidence that arsenic inhibits the excision of pyrimidine dimers. Partially repair-suppressing small doses of As2O3 (0.5 microgram/ml) and Na2HAsO4 (5 micrograms/ml) enhanced co-mutagenically the UV induction of 6-thioguanine-resistant mutations of V79 Chinese hamster cells. Thus, such a repair inhibition may be one of the basic mechanisms for the co-mutagenicity and presumably co-carcinogenicity of arsenic. XP group A and variant strains showed a unique higher sensitivity to As2O3 and Na2HAsO4 killing by a yet unidentified mechanism.     

Genetic recombination of herpes simplex virus, the role of the host cell and UV-irradiation of the virus

Summary Recombination frequencies for two sets of genetic markers of herpes simplex virus were determined in various host cells with and without ultraviolet irradiation of the virus. UV irradiation increased the recombination frequency in all the cell types studied in direct proportion to the unrepaired lethal damage. In human skin fibroblasts derived from a patient with xeroderma pigmentosum (XP) of complementation group A, a given dose of UV stimulated recombination more than that in fibroblasts from normal individuals. On the other hand, UV stimulation of HSV recombination was slightly less than normal in fibroblasts derived from a patient with a variant form XP and from an ataxia telangiectasia patient. Caffeine, an agent known to inhibit repair of UV damage, reduced recombination in most of the cell types studied but did not suppress the UV-induced increase in recombination. These findings suggest that for virus DNA with the same number of unrepaired UV-lesions, each of the tested cell types promoted HSV-recombination to an equivalent extent.     

Repair of closely opposed cyclobutyl pyrimidine dimers in UV-sensitive human diploid fibroblasts

An enzyme-sensitive site assay has been used to examine the fate of closely opposed pyrimidine dimers (bifilar enzyme-sensitive sites) in fibroblasts from individuals afflicted with various genetic disorders that confer increased cellular sensitivity to UV radiation. The disappearance of bifilar enzyme-sensitive sites was found to be normal in cells from individuals with Fanconi's anemia, Cockayne's syndrome, dyskeratosis congenita and the variant form of xeroderma pigmentosum. The rate of bifilar enzyme-sensitive site removal in XP cells assigned to complementation group C was reduced by an amount similar to that observed for the repair of isolated dimers. Our results indicate that the initiation of repair at closely opposed dimers is slow in XP-C cells but normal in all other cells examined.     

Caffeine inhibition of the repair of ultraviolet-irradiated adenovirus in human cells.

Caffeine is shown to block repair of ultraviolet-irradiated adenovirus 2 when the irradiated virus infects normal human fibroblasts from a xeroderma pigmentosum (XP) variant. Such blockage is not observed when the irradiated virus infects XP fibroblasts belonging to XP complementation group A. Thus normal and XP variant cells have a caffeine-sensitive repair process. This may be either excision or an excision dependent repair process because fibroblasts belonging to XP complementation group A are believed to lack the excision repair process.     

Defect in UV-induced unscheduled DNA synthesis in cultured epidermal keratinocytes from xeroderma pigmentosum

DNA repair synthesis in 8 explant-outgrowth cultures of epidermal cells isolated from variant and complementation groups A and E of xeroderma pigmentosum (XP) was examined by measuring unscheduled DNA synthesis (UDS) on autoradiographs. The extents of UDS in XP epidermal cells were compared with those in normal epidermal cells obtained from 26 subjects. In both normal and XP epidermal cells, UDS was induced dose-dependently by radiation at doses of 5-20 J/m2. XP epidermal cells showed various extents of defect in DNA repair depending on the type of XP. In XP-A, the extent of UDS in epidermal cells was very low, being seen in only 3-10% of the normal epidermal cells. But epidermal cells isolated from XP-E and XP-variants exhibited relatively high levels of residual DNA repair; i.e., 69-84% of the control in XP-E and 67-85% in XP-variant. The extents of UDS in XP epidermal cells were almost the same as those in fibroblastic cells isolated from the same specimens.     

Resistance of plateau-phase human normal and xeroderma pigmentosum fibroblasts to the cytotoxic effect of ultraviolet light

Clonogenic survival response to 254-nm ultraviolet light was measured in 2 strains of repair-proficient normal human fibroblasts and 4 strains of xeroderma pigmentosum (XP) fibroblasts belonging to complementation groups A, C, D and variant. In all strains except XPA, cells irradiated in plateau phase and subcultured immediately were much more resistant to the lethal effect of UV than cells irradiated in the exponential phase of growth. Typically, 10-20% of plateau-phase cells were extremely resistant. When the cultures were held in plateau phase for 24 h after irradiation and before subculture, there was a further enhance of survival. By use of a UV-specific endonuclease assay, no difference was found in the number of DNA lesions induced in exponentially growing and plateau cultures by the same dose of UV light. Thus plateau-phase cells appear to be more efficient in their DNA-repair capability than cells in exponential growth. XP group A cells were uniquely found to be deficient in the processes which lead to plateau-phase resistance. Since plateau-phase repair was not lacking in XP groups C, D and variant, it may be related to a DNA-repair process different from that which is responsible for the overall UV sensitivity of these cells.     

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.     

Phenotypic correction of the defect in xeroderma pigmentosum cells after fusion with isolated cytoplasts

The cybridization technique was used to study the role of cytoplasmic and nuclear factors in complementation of the repair defects in xeroderma pigmentosum (XP) cells. Cybrids were prepared by fusion of UV-exposed XP cells with cytoplasts derived from normal human or complementing XP cells. Phenotypic correction of the DNA repair defect measured by unscheduled DNA synthesis (UDS) occurred in these cybrids. The results show that the correcting factors are present in the cytoplasts and can move into the nucleus of the UV-exposed XP cell almost immediately after fusion. The defective repair in the nuclei of XP complementation group A cell strains is corrected with fast kinetics reaching normal UDS levels within 2 h after fusion. In the A-group cybrids the correcting activity decreased with a half-time of about 12 h. Correction of the XP group C defect occurred at a much slower rate, indicating that different factors are involved in the correction of the XP-A and XP-C defects.     

Correction of the ultraviolet light induced DNA-repair defect in xeroderma pigmentosum cells by electroporation of a normal human endonuclease

Cells from patients with xeroderma pigmentosum, complementation group A (XPA), are known to be defective in repair of pyrimidine dimers and other forms of damage produced by 254-nm ultraviolet (UVC) radiation. We have isolated a DNA endonuclease, pI 7.6, from the chromatin of normal human lymphoblastoid cells which recognizes damage produced by UVC light, and have introduced this endonuclease into UVC-irradiated XPA cells in culture to determine whether it can restore their markedly deficient DNA repair-related unscheduled DNA synthesis (UDS). Introduction of the normal endonuclease, which recognizes predominantly pyrimidine dimers, but not the corresponding XPA endonuclease into UVC-irradiated XPA cells restored their levels of UDS to approximately 80% of normal values. Electroporation of both the normal and the XPA endonuclease into normal human cells increases UDS in normal cells to higher than normal values. These results indicate that the normal endonuclease can restore UDS in UVC-irradiated XPA cells. They also indicate that XPA cells have an endonuclease capable of increasing the efficiency of repair of UVC damage in normal cells.     

Defective recovery of semi-conservative DNA synthesis in xeroderma pigmentosum cells following split-dose ultraviolet irradiation.

In normal human fibroblasts we observe an enhancement of the recovery of the rate of semi-conservative DNA synthesis after split-dose UV-irradiation relative to a single total UV dose. The enhanced recovery is totally absent in both a xeroderma pigmentosum variant line and two xeroderma pigmentosum lines belonging to complementation groups A and C.