Fluorescent-light-induced lethality and DNA repair in normal and xeroderma pigmentosum fibroblasts

Cell survival and induction of endonuclease-sensitive sites in DNA were measured in human fibroblast cells exposed to fluorescent light or germicidal ultraviolet light. Cells from a xeroderma pigmentosum patient were hypersensitive to cell killing by fluorescent light, although less so than for germicidal ultraviolet light. Xeroderma pigmentosum cells were deficient in the removal of fluorescent light-induced endonuclease sites that are probably pyrimidine dimers, and both the xeroderma pigmentosum and normal cells removed these sites with kinetics indistinguishable from those for ultraviolet light-induced sites. A comparison of fluorescent with ultraviolet light data demonstrates that there are markedly fewer pyrimidine dimers per lethal event for fluorescent than for ultraviolet light, suggesting a major role for non-dimer damage in fluorescent light lethality.     

Enhanced expression of procollagenase in ataxia-telangiectasia and xeroderma pigmentosum fibroblasts

Summary Ataxia-telangiectasia and xeroderma pigmentosum are human hereditary diseases in which patients are cancer prone and demonstrate increased sensitivity to DNA damage by ionizing and ultraviolet radiation, respectively. In culture, both ataxia-telangiectasia and xeroderma pigmentosum skin fibroblasts show increased synthesis and secretion of the extracellular matrix proteins fibronectin and collagen. To determine whether these differences in protein production result from fundamental abnormalities in regulation of genes associated with cellular interactions, we compared the effects of trifluoperazine and 12-O-tetradecanoylphorbol-13-acetate on expression of the extracellular matrix-degrading metalloproteinases, procollagenase and prostromelysin, by normal, ataxia-telangiectasia, and xeroderma pigmentosum fibroblasts. After trifluoperazine treatment the overall levels of these metalloproteinases were much greater in three ataxia-telangiectasia cell strains and in cells from xeroderma pigmentosum complementation groups A and D than in normal cells. In contrast, cells from xeroderma pigmentosum complementation group C produced only slightly more procollagenase than normal cells. 12-O-tetradecanoylphorbol-13-acetate also induced higher than normal levels of procollagenase in some ataxia-telangiectasia and xeroderma pigmentosum strains, but less than that induced by trifluoperazine. Because increased extracellular accumulation of matrix-degrading enzymes has long been implicated in metastatic progression, this altered expression of procollagenase and prostromelysin in ataxia-telangiectasia and xeroderma pigmentosum cells could play an important role in the pathogenesis of various tumors in individuals with these genetic diseases. This work was supported by the Office of Health and Environmental Research, U. S. Department of Energy (contract DE-AC03-76-SF01012) (J. A., J. P. M.) and by a Fellowship in Medical Research from the A. P. Giannini/Bank of America Foundation (J. A.). A summary of these results has appeared previously in abstract form (1).     

Normal rate of DNA breakage in xeroderma pigmentosum complementation group E cells treated with 8-methoxypsoralen plus near-ultraviolet radiation

Treatment of normal and xeroderma pigmentosum complementation group E skin fibroblasts with 8-methoxypsoralen plus repeated doses of near-ultraviolet radiation elicited a marked increase in DNA strand breakage during a subsequent incubation. No such induction of breaks was noted with cells from xeroderma pigmentosum groups A and D. The results suggest that the gene product which is deficient in xeroderma pigmentosum group E cells is involved in a critical step of DNA repair of far-ultraviolet photoproducts but not so in the repair of psoralen cross-links.     

The influence of caffeine on the mitomycin C-induced chromosome aberration frequency in normal human and xeroderma pigmentosum cells

The clastogenic effect of mitomycin C (MC) was determined in two normal fibroblast cell lines and two xeroderma pigmentosum (XP) cell lines, a variant and a group A excision-deficient line. The group A xeroderma cell line was substantially more sensitive to MC than either the XP variant or the normal human cells. On caffeine post-treatment potentiation of the MC-induced aberration frequency occurred in all the cell lines. The XP varian cell line exhibited a distinctly higher sensitivity to caffeine than the classical XP or the normal human cell lines.     

Mitomycin C-induced postmitotic fibroblasts retain the capacity to repair pyrimidine photodimers formed after UV-irradiation

The formation and excision of UV-C light-induced cyclobutane-type pyrimidine photodimers were determined in cultures of human skin fibroblasts at time zero and several weeks following treatment with mitomycin C (MMC). Characteristic morphological changes of the fibroblasts and specific shifts in the [35S]methionine polypeptide pattern of total cellular proteins support the notion that MMC accelerates the differentiation pathway from mitotic (MF) to post-mitotic fibroblasts (PMF). No discernible difference could be detected between the fluence-response curves of pyrimidine dimers for untreated and MMC-treated repair-deficient xeroderma pigmentosum cells of group A. Furthermore we investigated the removal of pyrimidine dimers in 3 normal human skin fibroblast strains frequently used in mutation, transformation and aging research. We were able to demonstrate that no significant difference exists in the rate and extent of the excision-repair response to thymine-containing pyrimidine dimers following UV-irradiation shortly after MMC treatment of fibroblasts and in the MMC-induced PMF stage of these cells.     

Detection and repair of a UV-induced photosensitive lesion in the DNA of human cells

Irradiation with UV light results in damage to the DNA of human cells. The most numerous lesions are pyrimidine dimers; however, other lesions are known to occur and may contribute to the overall deleterious effect of UV irradiation. We have observed evidence of a UV-induced lesion other than pyrimidine dimers in the DNA of human cells by measuring DNA strand breaks induced by irradiating with 313-nm light following UV (254-nm) irradiation. These breaks, measured by alkaline sucrose sedimentation, increased linearly with the dose of UV light over the range tested (10-40 J/m2). The breaks cannot be photolytically induced 5 h after a UV dose of 20 J/m2 in normal cells; however, in xeroderma pigmentosum variant cells, the breaks are inducible for up to 24 h after UV irradiation. Xeroderma pigmentosum group A cells in the same 5-h period show an increase in the number of strand breaks seen with 313-nm light photolysis from about 2 to 4 breaks/10(9) dalton DNA. These breaks can then be induced for up to 24 h. These data suggest that, in normal cells, the lesion responsible for this effect is rapidly repaired or altered; whereas, in xeroderma pigmentosum variant cells it seems to remain unchanged. Some change apparently occurs in the DNA of xeroderma pigmentosum group A cells which results in an increase in photolability. These data indicate a deficiency in DNA repair of xeroderma pigmentosum variant cells as well as in xeroderma pigmentosum group A cells.     

Identification and characterization of xpac protein, the gene product of the human XPAC (xeroderma pigmentosum group A complementing) gene

We have cloned human xeroderma pigmentosum group A complementing (XPAC) cDNA that encodes a "zinc finger" protein with a predicted size of 31 kDa. To detect the xpac protein in cells, we raised antibody against a recombinant human xpac protein. Using this antibody, we identified the xpac protein in the nucleus of cells. In normal human cells, 40- and 38-kDa proteins were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A reduced amount of the smaller protein was detected in XP 39OSSV cells, which show low UV sensitivity, and no xpac proteins were detected in XP 2OSSV cells, which show high UV sensitivity. These levels of xpac proteins in xeroderma pigmentosum cells were determinants of heterogeneity of the DNA repair defect in group A xeroderma pigmentosum. Synthesis of the xpac protein did not increase after UV irradiation.     

Proximity of repair patches to persistent pyrimidine dimers in DNA of normal human and xeroderma pigmentosum cells

The proximity of repair patches to persistent pyrimidine dimers in normal human cells and xeroderma pigmentosum group C and D cells was analyzed by sequential digestion of repaired DNA with Micrococcus luteus UV-endonuclease and Escherichia coli DNA polymerase I. Although this enzymatic digestion removed one-third of the pyrimidine dimers, less than 3% of the label associated with repair patches and a similar amount of uniformly labeled DNA were removed. The repair patches therefore appear to be similarly distant from persistent dimers in all cell types, and, in particular, are not adjacent to unexcised dimers in xeroderma pigmentosum group D cells. A previous model that suggested that patches are inserted adjacent to dimers in xeroderma pigmentosum group D cells receives no support from these results.     

Facial resurfacing in xeroderma pigmentosum with monoblock full-thickness skin graft

A case of xeroderma pigmentosum with multiple skin tumors on the face that was treated with total excision and replacement of face skin except the eyelids with a monoblock full-thickness abdominal skin graft is reported. The quality and tumor-free features of the monoblock full-thickness skin graft in xeroderma pigmentosum are indicated. Despite the increased morbidity of the donor region, the radical surgical approach advocated here has improved the prognosis in the case reported.     

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.     

Cutaneous gene therapy: the graft takes

Prospects of ex vivo cutaneous gene therapy rely on stable corrective gene transfer in epidermal stem cells followed by engraftment of corrected cells in patients. In the case of cancer prone genodermatoses, such as xeroderma pigmentosum, cells that received the corrective gene must be selected. However, this step is potentially harmful and can increase risks of immune rejection of grafts. These obstacles have recently been overcome thanks to the labeling of genetically modified stem cells using a small epidermal protein naturally absent in stem cells. This approach was shown to be respectful of the fate of epidermal stem cells that retained full growth and differentiation capacities, as well as their potential to regenerate normal human skin when grafted in a mouse model in the long term. These progresses now open realistic avenues towards ex vivo cutaneous gene therapy of cancer prone genodermatoses such as xeroderma pigmentosum. However, major technical improvements are still necessary to preserve skin appendages which would contribute to aesthetic features and comfort of patients.     

The discovery that xeroderma pigmentosum (XP) results from defective nucleotide excision repair

Most forms of the human hereditary disease xeroderma pigmentosum (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.     

Establishment of cell lines derived from ataxia telangiectasia and xeroderma pigmentosum patients with high radiation sensitivity

Four human fibroblast cell lines, three of which were derived from a patient with ataxia telangiectasia and the other from a patient with xeroderma pigmentosum, were established after transfection with cloned SV40 DNA. These 4 cell lines showed some phenotypes characteristic of neoplastically transformed cells, and had a human karyotype with heteromorphisms identical to those of the parental fibroblasts. Their sensitivity to the cytotoxic effects of gamma-rays or ultraviolet irradiation was as high as those of their parental fibroblasts.     

Complementation of the xeroderma pigmentosum DNA repair synthesis defect with Escherichia coli UvrABC proteins in a cell-free system.

A newly developed cell-free system was used to study DNA repair synthesis carried out by extracts from human cell lines in vitro. Extracts from a normal human lymphoid cell line and from cell lines established from individuals with hereditary dysplastic nevus syndrome perform damage-dependent repair synthesis in plasmid DNA treated with cis- or trans-diamminedichloro-platinum(II) or irradiated with ultraviolet light. Cell extracts of xeroderma pigmentosum origin (complementation groups A, C, D, and G) are deficient in DNA repair synthesis. When damaged plasmid DNA was pretreated with purified Escherichia coli UvrABC proteins, xeroderma pigmentosum cell extracts were able to carry out DNA repair synthesis. The ability of E. coli UvrABC proteins to complement xeroderma pigmentosum cell extracts indicates that the extracts are deficient in incision, but can carry out later steps of repair. Thus the in vitro system provides results that are in agreement with the incision defect found from studies of xeroderma pigmentosum 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.     

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.     

DNA Chain Elongation and Joining in Normal Human and Xeroderma Pigmentosum Cells after Ultraviolet Irradiation

DNA synthesized in human cells after ultraviolet (UV) irradiation is made in segments of lower molecular weight than in unirradiated cells. Within several hours after irradiation these smaller units are both elongated and joined together. This repair process has been observed in normal human fibroblasts, HeLa cells, and fibroblasts derived from three types of xeroderma pigmentosum patients—uncomplicated with respect to neurological problems, complicated (de Sanctis-Cacchione syndrome), and one with the clinical symptoms of xeroderma pigmentosum but with normal repair replication. The ability of human cells to elongate and to join DNA strands despite the presence of pyrimidine dimers enables them to divide without excising the dimers present in their DNA. It may be this mechanism which enables xeroderma pigmentosum cells to tolerate small doses of UV radiation.     

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.     

Defective DNA endonuclease activity on anthramycin treated DNA in xeroderma pigmentosum and mouse melanoma cells

Nine separate DNA endonuclease activities from non-histone chromatin proteins and a corresponding set from the nucleoplasm of normal human and xeroderma pigmentosum, complementation group A, lymphoblastoid and Cloudman mouse melanoma cells, obtained by isoelectric focusing, were tested against circular duplex phage PM2 DNA previously treated with anthramycin. A marked increase in activity against anthramycin treated DNA was found in normal human lymphoblastoid cells in a chromatin fraction with pI 4.6, with lesser increases at pI's 3.9 and 5.4 and a nucleoplasmic fraction at pI 4.6. In the nuclear proteins of xeroderma pigmentosum and mouse melanoma cells, however, no increase in activity against anthramycin DNA could be detected in any fraction.     

Radiation sensitivity of T-lymphocytes grown with recombinant human interleukin-2

gamma-Ray and UV sensitivities of phytohemagglutinin (PHA)-stimulated T-lymphocytes were examined in the presence of the recombinant human interleukin-2 (IL-2). D0 values for the survival curves after gamma-irradiation varied from 0.90 to 1.25 Gy, and were comparable to those reported for human fibroblast cells. By fractionated exposure of gamma-rays, T-lymphocytes were shown to have the repair capacity for the sublethal damage. UV-survival curves yielded D0 of 6.5 J/m2 for T-lymphocytes from normal donors. T-Lymphocytes from a xeroderma pigmentosum patient with extremely low excision repair were markedly hypersensitive to UV (D0, 1.4 J/m2). T-Lymphocytes may be used to detect individuals who are sensitive to radiation or chemicals, and this method takes less time than that using fibroblast cells.