Effect of DNA repair on the cytotoxicity and mutagenicity of polycyclic hydrocarbon derivatives in normal and xeroderma pigmentosum human fibroblasts

The cytotoxicity of the “K-region” epoxides as well as several other reactive metabolites or chemical derivatives of polycyclic hydrocarbons was compared in normally-repairing human diploid skin fibroblasts and in fibroblasts from a classical xeroderma pigmentosum (XP) patient (XP2BE) whose cells have been shown to carry out excision repair of damage induced in DNA by ultraviolet (UV) radiation at a rate approx. 20% that of normal cells. Each compound tested exhibited a 2- to 3-fold greater cytotoxicity in this XP strain than in the normal strain. To determine whether this difference in survival reflected a difference in the capacity of the strains to repair DNA damage caused by such hydrocarbon derivatives, we compared the cytotoxic effect of several “K-region” epoxides in two additional XP strains, each with a different capacity for repair of UV damage. The ration of the slopes of the survival curves for each of the XP strains to that of the normal strain, following exposure to each epoxide, was very similar to that which we had previously determined for their respective UV curves, suggesting that human cells repair damage induced in DNA by exposure to hydrocarbon derivatives with the same system used for UV-induced lesions.To determine whether the deficiency in rate of excision repair in this classical XP strain (XP2BE) causes such cells to be abnormally susceptible to mutations induced by “K-region” epoxides of polycyclic hydrocarbons, we compared them with normal cells for the frequency of induced mutations to 8-azaguanine resistance. The XP cells were two to three times more susceptible to mutations induced by the “K-region” epoxide of benzo(a)pyrene (BP), 7,12-dimethylbenz(a)anthracene (DMBA), and dibenz(a,h)anthracene (DBA). Evidence also was obtained that cells from an XP variant patient are abnormally susceptible to mutations induced by hydrocarbon epoxides and, as is the case following exposure to UV, are abnormally slow in converting low molecular weight DNA, synthesized from a template following exposure to hydrocarbon epoxides, into large-size DNA.     

Comparative studies of host-cell reactivation, cellular capacity and enhanced reactivation of herpes simplex virus in normal, xeroderma pigmentosum and Cockayne syndrome fibroblasts

Host-cell reactivation (HCR) of UV-irradiated herpes simplex virus type 2 (HSV-2), capacity of UV-irradiated cells to support HSV-2 plaque formation and UV-enhanced reactivation (UVER) of UV-irradiated HSV-2 were examined in fibroblasts from 4 patients with Cockayne syndrome (CS), 5 with xeroderma pigmentosum and 5 normals. All UV-survival curves for HSV-2 plaque formation showed 2 components. HCR was similar to normal for the XP variant strain and the 2 CS strains tested, but substantially reduced in the 4 excision-deficient XP strains. The capacity of UV-irradiated fibroblasts to support HSV-2 plaque formation was determined by UV-irradiating fibroblast monolayers with various doses of UV and 48 h later, infecting the monolayers with unirradiated HSV-2. The D37 values for the delayed-capacity curves so obtained were in the range 8.6-12.4 J/m2 for the normal strains, 2.8-3.2 J/m2 for the CS strains, 6.7 J/m2 for an XP variant strain and between 0.3 and 1.5 for the XP excision-deficient strains tested. These results indicate that delayed capacity for HSV-2 plaque formation is a more sensitive assay than HCR in the detection of cellular DNA-repair deficiency for XP and CS. For the examination of UVER, fibroblasts were irradiated with various UV doses and subsequently infected with either unirradiated or UV-irradiated HSV and scored for plaque formation 2 days later. UVER expression was maximum when the delay between UV-irradiation of the cells and HSV infection was 48 h. The magnitude of UVER expression was also found to be dependent on the UV dose to the cells and increased with increasing UV dose to the virus. Using a UV dose to the virus resulting in a plaque survival of about 10(-2) on unirradiated cells, the the maximum UVER factor had a mean value of 1.3 for the normal strains following a dose of 15 J/m2 to the cells. Somewhat higher UVER values were found for all the patient strains tested and resulted from lower UV doses to the cells than for normal strains. Maximum UVER factors for the CS strains ranged from 2.2 to 3.3 at a dose of 5 J/m2 to the cells, for the XP excision-deficient strains; 2.1 to 2.6 at doses of 0.5 to 2.5 J/m2 to the cells and for the XP variant strain tested; 2.5 at UV dose of 10 J/m2 to the cells.     

U.V. enhanced reactivation of U.V.-and gamma-irradiated adenovirus in Cockayne syndrome and Xeroderma pigmentosum fibroblasts

U.V.-enhanced reactivation (UVER) of both U.V.-irradiated and gamma-irradiated human adenovirus type 2 (Ad 2) was examined following the infection of a variety of Cockayne Syndrome (CS) and Xeroderma pigmentosum (XP) fibroblast strains which had been pre-irradiated with U.V. light. U.V.-irradiated or non-irradiated fibroblasts were infected with either non-irradiated or irradiated Ad2, and at 48 hours after infection cells were examined for the presence of viral structural antigens (Vag) using immunofluorescent staining. Normal levels of UVER (i.e. 2-4 fold) of U.V.- and of gamma-irradiated Ad 2 were detected in 2 CS strains (CS IBE and CS 3BE), 2 XP complementation group A strains (XP 12BE and XP 25RO), and 2 XP complementation group D strains (XP 5BE and XP 6BE), although the U.V. doses to these mutant cells which resulted in peak UVER values (0 . 2 Jm-2 for XP 25RO, 0 . 14 Jm-2 for XP 12BE, 0 . 8 Jm-2 for XP 5BE and XP 6BE, and 1 . 6-5 . 0 Jm-2 for CS 1BE and CS 3BE) were considerably lower than those yielding peak UVER in normal strains (10-15 Jm-2). XP variant strains (XP 4BE and XP 115LO), however, showed substantially lower levels of UVER than normal strains.     

DNA excision-repair processes in human cells can eliminate the cytotoxic and mutagenic consequences of ultraviolet irradiation

The ability of DNA excision-repair processes in diploid human fibroblasts to eliminate potentially cytotoxic and mutagenic lesions induced by UV radiation (254 nm) was demonstrated in two ways: (1) Cells with normal rates of excision were compared with cells with an intermediate rate of excision (XP2BE) and cells with an excision rate less than or equal to 1% that of normal (XP12BE) for sensitivity to the killing and mutagenic action of UV radiation. The normal cells proved resistant to doses of UV which reduced the survival of the XP cells to 14% and 0.7%, respectively, and increased the frequency of mutations to 8-azaguanine resistance in the XP cells 5- to 10-fold over background. (2) Cells in confluence were irradiated with cytotoxic and mutagenic doses of UV and allowed to carry out excision repair. After various lengths of time they were replated at lower densities to allow for expression of mutations to 6-thioguanine resistance and/or at cloning densities to assay survival. Normal cells and XP cells with reduced rates of excision repair (from complementation groups C and D) exhibited a gradual increase in survival from an initial level of 15--20% to 100% if held approximately 20 h in confluence. In contrast, XP12BE cells showed no increase from an initial survival of 20% even when held for 7 days. Normal cells irradiated in confluence but prevented from replicating for 7 days exhibited background mutation frequencies, whereas the mutation frequency in XP12BE cells did not change with the time in confluence.     

Ultraviolet mutagenesis of normal and xeroderma pigmentosum variant human fibroblasts

The mutabilities of normal and xeroderma pigmentosum variant (XP4BE) human fibroblasts by ultraviolet light (UV) were compared under conditions of maximum expression of the 6-thioguanine resistance (TGr) phenotype. Selection was with 20 micrograms TG/ml on populations reseeded at various times after irradiation. Approx. 6--12 days (4--8 population doublings), depending on the UV dose, were necessary for complete expression. The induced mutation frequencies were linear functions of the UV dose but the slope of the line for normal cells extrapolated to zero induced mutants at 3 J/m2. The postreplication repair-defective XP4BE cells showed a higher frequency of TGr colonies than normal fibroblasts when compared at equal UV doses or at equitoxic treatments. The induced frequency of TGr colonies was not a linear function of the logarithm of survival for either cell type. Instead, the initial slope decreased to a constant slope for survivals less than about 50%. The UV doses and induced mutation frequencies corresponding to 37% survival of cloning abilities were 6.7 J/m2 and 6.2 X 10(-5), respectively, for normal cells and 3.75 J/m2 and 17.3 X 10(-5) for the XP4BE cells. The lack of an observable increase in the mutant frequency for normal fibroblasts exposed to slightly lethal UV doses suggests that normal postreplication repair of UV-induced lesions is error-free (or nearly so) until a threshold dose is exceeded.     

Roles of DNA interstrand crosslinking and its repair in the induction of sister-chromatid exchange and a higher induction in fanconi's anemia cells

The roles of DNA crosslink and its repair in the induction of sister-chromatid exchanges (SCEs) were studied in normal, xeroderma pigmentosum (XP) complementation group A, and Fanconi's anemia (FA) fibroblasts after treatment with mitomycin C (MC) or decarbamoyl mitomycin C (DMC) for 1 h. FA strains were 5—30-fold more sensitive to MC killing than normal cells, but normally responded to DMC killing. XP group-A cells were twice and only slightly more sensitive to DMC and MC killings, respectively, than normal cells. The induction rate of immediate SCEs by MC was 1.7 times higher, despite a normal SCE rate by DMC, in FA strains than that in normal cells. Alternatively, SCE rates by DMC and MC were 6 times and only 1.3 times higher, respectively, in XP cells than in normal cells. In normal cells, the reduction of MC-induced SCEs as a function of repair time followed a biphasic curve of the first rapid (half-life, 2 h) and the second slow (half-life, 14 h) components. Such components corresponded exactly to the first half-excision and the second slow repair processes of molecular crosslink repair. In MC-induced SCEs, FA17JTO cells exhibited only the slow reduction component without the first rapid component and a higher saturation level in the time-dependent reduction in SCEs. This indicates that SCEs are produced by crosslinks remaining unrepaired for long times (24—48 h) after treatment of FA cells. Conversely, XP group-A cells capable of the first half-excision manifested the first rapid reduction in SCEs, although the second component declined at the slowest rate (half-life, 48 h) owing to a defect in the second mono-adduct repair. The reduction in DMC-induced SCEs followed only the slow component. Thus, these results demonstrate that crosslink can be the lesion leading to SCE, and the MC-induced SCE frequency is higher in FA cells than in normal cells. In the FA20JTO strain, such a repair defect seemed to be less than in FA17JTO cells, judged from the survival and SCE characteristics.     

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.     

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.     

A comparison of the response of unstimulated and stimulated T-lymphocytes and fibroblasts from normal, xeroderma pigmentosum and trichothiodystrophy donors to the lethal action of UV-C.

Unstimulated T-lymphocytes from normal donors are significantly more sensitive to the lethal effects of UV-C than either stimulated T-lymphocytes or fibroblasts as judged by colony-forming ability. Data from other studies suggest that excision repair is more effective in stimulated than unstimulated T-lymphocytes leading to the prediction that these differences in survival should be minimal in cells established from excision defective donors. The prediction was met with XP6BR, a donor of unknown complementation group. For 3 XP's from complementation group D, however, enhanced survival in stimulated T-cells was observed. With cells from an excision-defective TTD who was included in complementation group D of XP both fibroblasts and unstimulated T-lymphocytes were hypersensitive. For a second excision defective TTD patient who was excluded from complementation group D, the unstimulated T-lymphocytes were more resistant than those of normal donors although the fibroblasts were hypersensitive. These results suggest that the in vitro response of stimulated T-lymphocytes or fibroblasts may not reflect the in vivo response of cells, as measured by the response of unstimulated T-lymphocytes.     

Mutational hotspot variability in an ultraviolet-treated shuttle vector plasmid propagated in xeroderma pigmentosum and normal human lymphoblasts and fibroblasts

The mutagenesis shuttle vector, pZ189, was treated with ultraviolet (u.v.) radiation in vitro and passed through a DNA repair-deficient lymphoblastoid cell line derived from a patient with xeroderma pigmentosum complementation group A (XP-A) (XP12BE(EBV)) and a DNA repair-proficient lymphoblastoid cell line (GM606(EBV)). After u.v. treatment, plasmid survival was lower and mutation frequency higher with the XP-A cells mirroring the survival and mutagenesis of the host cells. The nature of the mutations in the suppressor tRNA marker gene was determined by direct sequence analysis. The G.C to A.T transition was the dominant (85%) base substitution mutation with the XP lymphoblasts and was the major (56%) base substitution mutation with the repair-proficient lymphoblasts. We found a G.C to A.T transition mutational hotspot with the XP lymphoblasts not seen in our previous experiments with fibroblasts from the same patient. Comparison of the data presented here with our results with DNA repair-deficient and DNA repair-proficient fibroblasts suggests that hotspot variability is not due to genetic polymorphism or repair capacity of the cells. Instead it appears that cellular factors can influence the probability of mutagenesis of modified DNA at particular sites.     

Herpes simplex virus-infected human fibroblasts are resistant to and inhibit cytotoxic T-lymphocyte activity.

We examined the ability of human anti-herpes simplex virus (HSV) cytotoxic T lymphocytes (CTL) to lyse autologous human fibroblasts infected with HSV. In contrast to HSV-infected human Epstein-Barr virus-transformed B cells (LCL), which were lysed by HLA-restricted anti-HSV CTL, autologous fibroblasts infected with HSV were resistant to lysis. This resistance was not due to a lack of infectivity or production of HSV proteins since greater than 90% of the cells were infected and expressed abundant levels of viral proteins. HSV-infected human fibroblasts were also tested for susceptibility to lysis by alloantigen-specific CTL. Although allogeneic LCL and uninfected allogeneic fibroblasts were killed, human fibroblasts infected with HSV demonstrated a time-dependent resistance to lysis by alloantigen-specific CTL. HSV-infected human fibroblasts were not resistant to all forms of cell-mediated cytotoxicity since they were sensitive to antibody-dependent cellular cytotoxicity. Although one may suspect that the resistance of HSV-infected human fibroblasts to anti-HSV CTL and alloantigen-specific CTL-mediated lysis was due to a lack of major histocompatibility complex expression, Confer et al. (Proc. Natl. Acad. Sci. USA 87:3609-3613, 1990) previously demonstrated that incubation of human natural killer and lymphokine-activated killer cells with monolayers of human fibroblasts infected with HSV "disarmed" the killers in that they were unable to lyse sensitive target cells. We extend their results and show that incubation of anti-HSV CTL or alloantigen-specific CTL with uninfected fibroblasts did not affect their lytic activity, whereas CTL incubated with HSV-infected fibroblasts for 2 to 6 h rendered the CTL incapable of lysing their normally sensitive target cells. Indeed, human fibroblasts infected for merely 2 h with HSV were able to profoundly inhibit the cytotoxic activity of alloantigen-specific CTL. Thus, HSV-infected human fibroblasts are not inherently resistant to lysis by anti-HSV CTL or alloantigen-specific CTL, but rather contact of CTL with HSV-infected fibroblasts resulted in inactivation of the CTL. The inactivation of CTL appears to be HSV specific since incubation of alloantigen-specific CTL in sandwich assays with fibroblasts infected with HSV type 1 (HSV-1) or HSV-2 resulted in inactivation, whereas incubation of CTL with fibroblasts infected with adenovirus or vaccinia virus had no effect. Further, although incubation of alloantigen-specific CTL in sandwich assays with HSV-infected fibroblasts resulted in inhibition of CTL activity, exposure of CTL in Transwell cultures to cell-free supernatant from HSV-infected fibroblasts did not mediate this inhibitory effect.(ABSTRACT TRUNCATED AT 400 WORDS)     

The action of AF 2 on cultured hamster and human cells under aerobic and hypoxic conditions

AF 2 (2-(2-furyl)-3-(5-nitro-furyl)acrylamide) was toxic to Chinese hamster V 79 cells and normal human fibroblasts in aerobic media. However, the toxicity of the drug was increased many times by hypoxia. Similarly, the frequency of AF 2-induced azaguanine- and ouabain-resistant mutants of V 79 cells was much higher in hypoxia than under aerobic conditions. Both hamster V 79 cells and human fibroblasts metabolized AF 2 and other nitrofurans rapidly only under hypoxic conditions. Human fibroblasts were more sensitive to AF 2 both under aerobic conditions and in hypoxia than were V 79 cells under similar conditions. The Chinese hamster cells consistently gave survival curves with marked shoulders while human cells did not. Aerobic cultures of fibroblasts derived from xeroderma pigmentosum (XP) patients were markedly sensitive to AF 2 while fibroblasts from two ataxia telangeictasia patients had normal sensitivity. Under hypoxic conditions the sensitivity of both types of cells was increased but the XP line remained 5--10-fold more sensitive than normal or ataxia cells. These results suggest that the DNA lesions produced by AF 2 may be regarded as similar to those produced by ultraviolet light, at least in terms of their repairability in human cells.     

Structural studies of a membrane-bound acetylcholine receptor from Torpedo californica

We investigated the differential repair of DNA lesions induced by bifunctional mitomycin C, monofunctional decarbamoyl mitomycin C and ultraviolet irradiation in normal human, Xeroderma pigmentosum and Fanconi's anemia cells using assays for the survival of clone-forming ability, alkaline sucrose sedimentation and hydroxyapatite chromatography of DNA. Four FA cell lines exhibited about 5 to 15 times higher sensitivity to MC killing, despite normal resistance to u.v. and DMC, than did normal human cells. The XP cells, however, were highly sensitive to u.v. and DMC killings due to their deficiency in excision repair, but the cells unexpectedly had an almost normal capacity for surviving MC and repairing the MC interstrand cross-links.In experiments to determine the sedimentation velocity of the DNA in alkaline sucrose gradients, normal and XP cells showed evidence for single-strand cutting following MC treatment. The sedimentation velocity of the DNA covalently cross-linked by MC in an FA strain was 2.5 times faster than that of the untreated control, and remained unaltered during post-incubation due to the lack of half-excision⁴ of cross-links. However, FA cells, but not XP cells, had the normal ability to incise DNA with the DMC monoadducts. Hydroxyapatite chromatography revealed the reversibly bihelical property of MC cross-linked DNA after denaturation. Normal and XP cells lost such reversibility during post-MC incubation as the result of cross-link removal with first-order kinetics (half-life = 2 h). The three FA lines studied exhibited two- to eightfold reduced rates of cross-link removal than normal and XP cells, indicating a difference in the repair deficiency of the FA strain. Thus we have been led to conclude that FA cells may have different levels of deficiency in half-excision repair of interstrand cross-links induced by MC, despite having normal mechanisms for repair of u.v.-induced pyrimidine dimers and DMC monoadducts, and vice versa in XP cells.     

The susceptibility of Werner's syndrome and other human skin fibroblasts to SV40-induced transformation and immortalization

In attempts to transform and immortalize human cell cultures, skin fibroblasts from normal donors of different ages, from patients with the premature ageing diseases Werner's syndrome (WS) and progeria (PR), and from donors with the cancer-prone diseases ataxia telangiectasia (AT), Bloom's syndrome (BS) and Fanconi's anaemia (FA), were infected with SV40 virus and their growth monitored thereafter. Lesch-Nyhan (LN) fibroblasts were also infected. SV40-infected cultures from two normal and from WS, AT and LN donors attained a spectrum of transformed properties, high mitotic activity at confluence, presence of T-antigen, anchorage independence and altered morphology. Most of these pretransformed cultures died in the crisis period. However, two cultures from the WS and LN patients survived the crisis period and have now been grown to more than 200 passages. For the LN culture the crisis period was at least 200 days. Both permanent lines retain the properties of pretransformed cells, but differ in their modal chromosome number and ability to grow in methionine-free medium. It can be concluded from these experiments that transformation by SV40 to permanent lines is a rare event in human skin fibroblasts, even when these cells were taken from patients predisposed to form cancers.     

XPC initiation codon mutation in xeroderma pigmentosum patients with and without neurological symptoms

Two unrelated xeroderma pigmentosum (XP) patients, with and without neurological abnormalities, respectively, had identical defects in the XPC DNA nucleotide excision repair (NER) gene. Patient XP21BE, a 27-year-old woman, had developmental delay and early onset of sensorineural hearing loss. In contrast, patient XP329BE, a 13-year-old boy, had a normal neurological examination. Both patients had marked lentiginous hyperpigmentation and multiple skin cancers at an early age. Their cultured fibroblasts showed similar hypersensitivity to killing by UV and reduced repair of DNA photoproducts. Cells from both patients had a homozygous c.2T>G mutation in the XPC gene which changed the ATG initiation codon to arginine (AGG). Both had low levels of XPC message and no detectable XPC protein on Western blotting. There was no functional XPC activity in both as revealed by the failure of localization of XPC and other NER proteins at the sites of UV-induced DNA damage in a sensitive in vivo immunofluorescence assay. XPC cDNA containing the initiation codon mutation was functionally inactive in a post-UV host cell reactivation (HCR) assay. Microsatellite markers flanking the XPC gene showed only a small region of identity ( approximately 30kBP), indicating that the patients were not closely related. Thus, the initiation codon mutation resulted in DNA repair deficiency in cells from both patients and greatly increased cancer susceptibility. The neurological abnormalities in patient XP21BE may be related to close consanguinity and simultaneous inheritance of other recessive genes or other gene modifying effects rather than the influence of XPC gene itself.     

Host-cell reactivation of UV-irradiated and chemically-treated herpes simplex virus-1 by xeroderma pigmentosum, XP heterozygotes and normal skin fibroblasts

The host-cell reactivation of UV-irradiated and N-acetoxy-2-acetylamino-fluorene-treated herpes simplex virus type 1 strain MP was studied in normal and xeroderma pigmentosum human skin fibroblasts. Virus treated with either agent demonstrated lower survival in XP cells from complementation groups A, B, C and D than in normal fibroblasts. The relative reactivation ability of XP cells from the different genetic complementation groups was found to be the same for both irradiated and chemically treated virus. In addition, the inactivation kinetics for virus treated with either agent in the XP variant were comparable to that seen in normal skin fibroblasts. The addition of 2 or 4 mmoles caffeine to the post-infection assay medium had no effect on the inactivation kinetics of virus treated by either agent in the XP variant or in XP cells from the different genetic complementation groups. Treatment of the virus with nitrogen mustard resulted in equivalent survival in normal and XP genetic complementation group D cells. No apparent defect was observed in the ability of XP heterozygous skin fibroblasts to repair virus damaged with up to 100 μg N-acetoxy-2-acetylaminofluorene per ml. These findings indicate that the repair of UV-irradiated and N-acetoxy-2-acetylaminofluorene-treated virus is accomplished by the same pathway or different pathways sharing a common intermediate step and that the excision defect of XP cells plays little if any role in the reactivation of nitrogen mustard treated virus.     

Extent of excision repair before DNA synthesis determines the mutagenic but not the lethal effect of UV radiation

Excision repair-proficient diploid fibroblasts from normal persons (NF) and repair-deficient cells from a xeroderma pigmentosum patient (XP12BE, group A) were grown to confluence and allowed to enter the G₀ state. Autoradiography studies of cells released from G₀ after 72 h and replated at lower densities (3?9 × 10³ cells/cm²) in fresh medium containing 15% fetal bovine serum showed that semiconservative DNA synthesis (S phase) began ～24 h after the replating. To determine whether the time available for DNA excision repair between ultraviolet irradiation (254 nm) and the onset of DNA synthesis was critical in determining the cytotoxic and/or mutagenic effect of UV in human fibroblasts, we released cultures of NF or XP12BE cells from G₀, allowed them to reattach at lower densities, irradiated them in early G₁ (～18 h prior to the onset of S) or just prior to S phase, and assayed the frequency of mutations to 6-thioguanine resistance and the survival of colony-forming ability. The XP12BE cells, which are virtually incapable of excising UV-induced DNA lesions, showed approximately the same frequency of mutations and survival regardless of the time of UV irradiation. In NF cells, the slope of the dose response for mutations induced in cells irradiated just prior to S was about 7-fold steeper than that of cells irradiated 18 h earlier. However, the two sets of NF cells showed no significant difference in survival. Neither were there significant differences in the survival of NF cells released from G₀, plated at cloning densities and irradiated as soon as they had attached and flattened out (～20 h prior to S) or 4, 8, 12, 16, 20 or 24 h later. We conclude that the frequency of mutations induced by UV is dependent upon the number of unexcised lesions remaining at the time of semi-conservative DNA replication. However, the amount of time available for excision of potentially cytotoxic lesions is not determined primarily by the period between irradiation and the onset of S phase.     

The rate of removal of pyrimidine dimers in quiescent cultures of normal human and xeroderma pigmentosum cells

The rate of removal of pyrimidine dimers from DNA of UV (254 nm)-irradiated (1 J/m2) normal and xeroderma pigmentosum (XP) cells maintained in culture as nondividing populations was determined. Several normal and XP strains from complementation groups A, C and D were studied. The excision rates and survival ability of nondividing cells were examined to determine if an abnormal sensitivity was associated with a decreased rate of dimer excision. The results show that all normal strains studied excise pyrimidine dimers at the same rate, with the rate curve characterized by two components. All 'excision-deficient' XP strains excise dimers at a slower-than-normal rate, with the rate curves also characterized by two components. The rate constants for the first components of all of the XP strains (group A, C and D) are the same, one tenth of the normal rate constant, except for XP8LO (group A). XP8LO has a first-component rate constant similar to that of normal strains and a second component rate constant similar to that of other group A strains (XP12BE, XP25RO). Thus, the slower rate of dimer excision in XP8LO is due to a defect in the mechanism responsible for the second component of the excision-rate curve. In general, an abnormal sensitivity of nondividing cells to UV is associated with a reduced dimer-excision rate. A notable exception to this is the group C strain XP1BE which has an initial repair rate similar to that of group A XP12BE but is considerably more resistant when survival is measured.