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.     

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.     

Unique DNA repair properties of a xeroderma pigmentosum revertant.

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

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).     

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.     

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.     

Cyclic GMP and cyclic GMP-dependent protein kinase in brown adipose tissue of developing rats.

Human cells (normal and xeroderma pigmentosum variant) irradiated with ultraviolet light and pulse-labelled with [3H]thymidine underwent transient decline and recovery of molecular weights of newly synthesized DNA and rates of [3H]thymidine incorporation. The ability to synthesize normal-sized DNA recovered more rapidly in both cell types than thymidine incorporation. During recovery cells steadily increased in their ability to replicate normal-sized DNA on damaged templates. The molecular weight versus time curves fitted exponential functions with similar rate constants in normal and heterozygous xeroderma pigmentosum cells, but with a slower rate in two xeroderma pigmentosum variant cell lines. Caffeine added during the post-irradiation period eliminated the recovery of molecular weights in xeroderma pigmentosum variant but not in normal cells. The recovery of the ability to synthesize normal-sized DNA represents a combination of a number of cellular regulatory processes, some of which are constitutive, and one of which is altered in the xeroderma pigmentosum variant such that recovery becomes slow and caffeine sensitive.     

The structure of the human centrin 2-xeroderma pigmentosum group C protein complex

Human centrin-2 plays a key role in centrosome function and stimulates nucleotide excision repair by binding to the xeroderma pigmentosum group C protein. To determine the structure of human centrin-2 and to develop an understanding of molecular interactions between centrin and xeroderma pigmentosum group C protein, we characterized the crystal structure of calcium-loaded full-length centrin-2 complexed with a xeroderma pigmentosum group C peptide. Our structure shows that the carboxyl-terminal domain of centrin-2 binds this peptide and two calcium atoms, whereas the amino-terminal lobe is in a closed conformation positioned distantly by an ordered alpha-helical linker. A stretch of the amino-terminal domain unique to centrins appears disordered. Two xeroderma pigmentosum group C peptides both bound to centrin-2 also interact to form an alpha-helical coiled-coil. The interface between centrin-2 and each peptide is predominantly nonpolar, and key hydrophobic residues of XPC have been identified that lead us to propose a novel binding motif for centrin.     

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.     

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.     

UV-light induced sister chromatid exchanges in xeroderma pigmentosum lymphocytes

Summary Cultured lymphocytes from 9 patients with clinically different types of xeroderma pigmentosum were exposed to ultraviolet light at 24 h. An increased rate of sister chromatid exchanges was observed in 6 patients (128–148% increase in three, 34–51% in three), but not in three patients with deSanctis-Cacchione syndrome (xeroderma pigmentosum with mental defect), compared to simultaneously cultured controls. A positive result could be useful as preliminary cytogenetic diagnostic test. The results are interpreted as an expression of UV-light induced chromosomal instability due to impaired DNA repair.     

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.     

Transformation of ultraviolet-irradiated human fibroblasts by simian virus 40 is enhanced by cellular DNA repair functions

Human fibroblasts irradiated with ultraviolet light were either tested for survival (colony formation) or infected with simian virus 40 and examined for transformation (foci formation). For normal cell cultures, the fractions of surviving colonies which were also transformed increased with increasing irradiation dose. In contrast, little increase in the transformation of ultraviolet-irradiated repair-deficient (xeroderma pigmentosum and xeroderma pigmentosum variant) cells was observed. Similar experiments with xeroderma pigmentosum variant cells treated with caffeine following irradiation indicated that, under these conditions, the deficient cells produced more transformants among the survivors of ultraviolet irradiation than did unirradiated cells. These results suggest (1) that DNA repair functions, not DNA damage per se, are required for enhanced viral transformation in normal cells; (2) that functions involved in excision repair and functions needed for replication of ultraviolet-damaged DNA appear necessary for this stimulation; and (3) that blocking DNA replication in ultraviolet-irradiated xeroderma pigmentosum variant cells by caffeine enhances viral transformation.     

Xéroderma pigmentosum: Huit cas d'évolution différente dans deux familles

Eight cases of xeroderma pigmentosum are described-six in family B. and two in family T. The criteria used in making this diagnosis are indicated. The occurrence of epitheliomas and melanoma was observed. In family B. five of the six patients are alive at time of reporting, their ages varying from 40 to 55 years. In family T. the two affected children died at ages 8 and 14 years. The differential diagnosis between xeroderma pigmentosum and other conditions is briefly discussed.     

Apurinic DNA endonuclease activities in repair-deficient human cell lines.

Several autosomal recessive diseases are associated with apparent DNA repair defects in cell culture. It seemed likely that a defect in excision repair reported for ataxia telangiectasia cells might reflect a lack of apurinic endonuclease activity. We report here normal levels of apurinic endonuclease activity in extracts of cell lines derived from patients with ataxia telangiectasia, xeroderma pigmentosum (complementation group D), Cockayne dwarfism, Fanconi anemia and Bloom syndrome.     

Inhibition of protein synthesis does not antagonize induction of UV-induced sister-chromatid exchange in xeroderma pigmentosum cells

Cycloheximide strongly antagonizes the induction of sister-chromatid exchanges by ethyl methanesulfonate or mitomycin C in human skin fibroblast and xeroderma pigmentosum cells (group A). Analogous behavior has been observed in several other species including Chinese hamster and plant cells. This report documents an exception to that pattern: cycloheximide fails to antagonize UV-induced sister chromatid exchange in xeroderma pigmentosum cells, whereas it does in normal human skin fibroblast cells. A genetic defect in these cells is postulated to alter the UV-mediated DNA recombination process.     

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.     

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.     

Genetic Heterogeneity of Xeroderma Pigmentosum demonstrated by Somatic Cell Hybridization

XERODERMA pigmentosum is an autosomal recessive disease characterized by hypersensitivity of the skin to ultraviolet radiation resulting in severe skin lesions. DNA repair replication after ultraviolet irradiation is absent or markedly reduced in cultivated fibroblasts from patients with xeroderma pigmentosum (XP cells) compared with normal cells^1,2. Using the dark repair mechanism in microorganisms as a model, evidence has been presented that XP cells are defective in the incision step of DNA repair^3–5.     

Transformation of DNA repair-deficient human diploid fibroblasts with a simian virus 40 plasmid

Fibroblasts from patients with xeroderma pigmentosum (XP) complementation groups A, C, D, E, and G, as well as Bloom syndrome (BS) and Fanconi anemia (FA) have been transfected with a plasmid, pSV7, containing the early region of Simian virus 40 (SV40). All of the cultures exhibited cytologic changes characteristic of transformed cells and expressed T-antigen. They also contained integrated copies of DNA derived from the vector, and in several cases, extrachromosomally replicated DNA. Not all of the transfected cultures became immortalized. The transformed xeroderma pigmentosum (XP) cultures retained their UV-sensitive phenotype in all but one case. The BS and FA cell lines retained their characteristic phenotype. All of the cultures, except the BS cells, can be readily transfected with the plasmids, pSV2neo and pSV2gpt.