Xeroderma Pigmentosum Cells Research Articles

Complementation of DNA repair defect in xeroderma pigmentosum cells of group C by the transfer of human chromosome 5

Complementation of DNA excision repair defect in xeroderma pigmentosum cells of group C (XP-C) has been achieved by the transfer of human chromosome 5. Individual human chromosomes tagged with a selectable marker were transferred to XP-C cells by microcell fusion from mouse-human hybrid cell lines each bearing a single different human chromosome. Analysis of the chromosome transfer clones revealed that introduction of chromosome 5 into XP-C cells corrected the DNA repair defect as well as UV-sensitive phenotypes, while chromosomes 2, 6, 7, 9, 13, 15, 17, and 21 failed to complement. The introduced chromosome 5 in complemented UVr clones was distinguished from the parental XP-C chromosomes by polymorphism for dinucleotide (CA)n repeats at two loci, D5S117 and D5S209. In addition, an intact marked chromosome 5 was rescued into mouse cells from a complemented UVr clone by microcell fusion. Five subclones of a complemented clone that had lost the marked chromosome 5 exhibited UV-sensitive and repair-deficient phenotypes identical to parental XP-C cells. Concordant loss of the transferred chromosome and reappearance of XP-C phenotype further confirmed the presence of a DNA repair gene on human chromosome 5.

Cellular responses to hematoporphyrin-induced photooxidative damage in fanconi anemia, xeroderma pigmentosum and normal human fibroblasts

Several observations reported in the literature suggest that singlet oxygen ( 1O 2) might play a role in the clastogenic process in Fanconi anemia (FA) cells, and that the antioxidant status of xeroderma pigmentosum (XP) may also be altered. In order to test the ability of FA and XP cells, relative to normal cells, to cope with 1O 2 damage, the effects of photosensitization by hematoporphyrin (HP) have been determined (i) on host cell reactivation (HCR) of damaged infecting herpes simplex virus (HSV) or transfecting SV40 DNA, and (ii) on DNA template capability and clonogenicity of treated cells. Results showed no significant difference among the three types of cells, either for the survival of HP-photosentized HSV, or for the yields of SV40 virus following transfection of cultures with damaged viral DNA. The treatment of cells with HP plus 365-nm light leads to a dose-dependent, homothetic reduction of 18S and 28S ribosomal RNA (rRNA) synthesis, presumably through a mechanism other than the formation of transcription termination sites. After a 24-h post-exposure incubation, the rate of rRNA synthesis was restored to higher than normal levels in all cell lines. Finally, two FA cell lines showed a higher survival to HP photosensitization than two normal cell lines. Another FA cell line and XP-A and XP-C cells were in the range of sensitivity of the two normal strains for this treatment. These results indicate that FA cells possess an antioxidant defense system at least as efficient as that of normal cells for processing 1O 2-induced damage.

Induction of the 72-kD Heat Shock Protein in Xeroderma Pigmentosum Complementation Group A Fibroblasts

In mammalian cells, 72-kD heat shock protein (HSP72) is the major stress-inducible protein that is thought to play a protective role against the various environmental stresses. In order to know the induction mechanism of HSP72, we examined the HSP72 in DNA repair-deficient xeroderma pigmentosum group A fibroblasts (XP2OSSV) and normal fibroblasts (WI38VA13) by the indirect immunofluorescence method using a monoclonal antibody specific for the inducible 72-kD protein. Heat-shock treatment of the same survival fraction (5% survival) induced HSP72 in xeroderma pigmentosum (XP) and normal cells. However, as compared with XP cells, normal cells showed the induction of HSP72 more rapidly and strongly. When XP and normal cells were irradiated with UVC at the same survival dose (10% survival), apparent induction of HSP72 was observed in both cell lines. In the case of UVC irradiation at the same dose (1.0 J/m2), though XP cells showed the induction of HSP72, HSP72 was not induced in normal cells. In both cell lines, heat-shock treatment caused more rapid induction of HSP72 than UV irradiation. These results suggest that the induction mechanism of HSP72 might be different between heat-shock treatment and UV irradiation. In addition, in the case of UV irradiation, the extent of DNA damage after DNA repair or the cell death might be involved in the induction of HSP72.

Expression cloning of a human DNA repair gene involved in xeroderma pigmentosum group C.

Xeroderma pigmentosum (XP) is a rare human autosomal recessive disease characterized by solar sensitivity, high predisposition for developing cancers on areas exposed to sunlight, and, in some cases, neurological abnormalities. XP cells are defective in DNA repair, and complementation of this defect has been used to identify eight genetic groups (A-G and variant). We have developed a simple, highly efficient complementary DNA expression system for use in human cells. Here we use this system to isolate a cDNA clone that restores the ultraviolet sensitivity and unscheduled DNA synthesis of XP-C cells to normal levels. The XP-C complementing clone XPCC encodes a highly hydrophilic protein which is composed of a predicted 823 amino acids and shares limited homology with the product of the yeast DNA repair gene RAD4. The XPCC transcript is undetectable by northern blotting in most XP-C cell lines examined.

A gene that partially complements xeroderma pigmentosum group A cells maps to human chromosome 8.

A gene that partially complements sensitivity of xeroderma pigmentosum cells of group A to UV irradiation has been mapped to human chromosome 8. Isolation of this gene has previously been described. A cDNA clone pEMKR that represents part of this gene was used for mapping. Based upon the nucleotide sequence of pEMKR, a set of oligonucleotide primers were designed for PCR amplification of DNAs from hybrid cell lines. A panel of rodent-human hybrid cell lines representing the total human genome was screened by PCR and Southern blot analysis for chromosomal assignment of this gene. PCR amplification and hybridization occurred only in the case of human and hybrid cell lines that contained human chromosome 8. The pEMKR thus represents a different gene than a DNA repair gene XPAC that has been mapped to human chromosome 9.

UV-induced base substitution mutations in a shuttle vector plasmid propagated in group C xeroderma pigmentosum cells

To assess the contribution to mutagenesis of human DNA repair defects, the UV-irradiated shuttle vector plasmid pZ189 was propagated in fibroblasts derived from a xeroderma pigmentosum (XP) patient in DNA repair complementation group C. In comparison to results with DNA repair-proficient human cells (WI-38 VA13), UV-irradiated pZ189 propagated in the XP-C (XP4PA(SV)) cells showed fewer surviving plasmids and a higher frequency of mutated plasmids. Base sequence analysis of 67 mutated plasmids recovered from the XP-C cells revealed similar classes of point mutations and mutation spectrum, and a higher frequency of G:C to A:T transitions along with a lower frequency of transversions among plasmids with single or tandem mutations compared to plasmids recovered from the normal line. Most single-base substitution mutations (83%) occurred at G:C base pairs in which the 5′-adjacent base of the cytosine was thymine or cytosine. These results indicate that the DNA repair defects in XP-C, in comparison to data previously reported for XP-A, XP-D and XP-F, result in different UV survival and mutation frequency but in similar types of base substitution mutations.

YeastRAD14 and human xeroderma pigmentosum group A DNA-repair genes encode homologous proteins

Xeroderma pigmentosum (XP), a human autosomal recessive disorder, is characterized by extreme sensitivity to sunlight and high incidence of skin cancers. XP cells are defective in the incision step of excision repair of DNA damaged by ultraviolet light. Cell fusion studies have defined seven XP complementation groups, XP-A to XP-G. Similar genetic complexity of excision repair is observed in the yeast Saccharomyces cerevisiae. Mutations in any one of five yeast genes, RAD1, RAD2, RAD3, RAD4, and RAD10, cause a total defect in incision and an extreme sensitivity to ultraviolet light. Here we report the characterization of the yeast RAD14 gene. The available rad14 point mutant is only moderately ultraviolet-sensitive, and it performs a substantial amount of incision of damaged DNA. Our studies with the rad14 deletion (delta) mutation indicate an absolute requirement of RAD14 in incision. RAD14 encodes a highly hydrophilic protein of 247 amino acids containing zinc-finger motifs, and it is similar to the protein encoded by the human XPAC gene that complements XP group A cell lines.

Alteration of a DNA-dependent ATPase activity in xeroderma pigmentosum complementation group C cells.

DNA-dependent ATPase activities in crude extracts prepared from HeLa cells were separated into five peaks by fast protein liquid chromatography Mono Q column chromatography. Similar elution profiles were observed with the extracts from human cells normal in repair and xeroderma pigmentosum cells belonging to complementation groups A through G except for group C. An alteration in elution of one of the five ATPases, designated DNA-dependent ATPase Q1, was observed with a cell line of complementation group C. This alteration was observed with all tested cell lines that belonged to group C. ATPase Q1 in HeLa cell extracts exhibited about 2-fold higher activity with ultraviolet light-irradiated DNA as compared to that with non-irradiated DNA, whereas little difference in the effects of two DNAs was observed with the ATPase activities in the extract from group C cells.

A role for the human single-stranded DNA binding protein HSSB/RPA in an early stage of nucleotide excision repair.

The human single-stranded DNA binding protein (HSSB/RPA) is involved in several processes that maintain the integrity of the genome including DNA replication, homologous recombination, and nucleotide excision repair of damaged DNA. We report studies that analyze the role of HSSB in DNA repair. Specific protein-protein interactions appear to be involved in the repair function of HSSB, since it cannot be replaced by heterologous single-stranded DNA binding proteins. Anti-HSSB antibodies that inhibit the ability of HSSB to stimulate DNA polymerase alpha also inhibit repair synthesis mediated by human cell-free extracts. However, antibodies that neutralize DNA polymerase alpha do not inhibit repair synthesis. Repair is sensitive to aphidicolin, suggesting that DNA polymerase epsilon or delta participates in nucleotide excision repair by cell extracts. HSSB has a role other than generally stimulating synthesis by DNA polymerases, as it does not enhance the residual damage-dependent background synthesis displayed by repair-deficient extracts from xeroderma pigmentosum cells. Significantly, when damaged DNA is incised by the Escherichia coli UvrABC repair enzyme, human cell extracts can carry out repair synthesis even when HSSB has been neutralized with antibodies. This suggests that HSSB functions in an early stage of repair, rather than exclusively in repair synthesis. A model for the role of HSSB in repair is presented.

Correction of xeroderma pigmentosum complementation group D mutant cell phenotypes by chromosome and gene transfer: involvement of the human ERCC2 DNA repair gene.

Cultured cells from individuals afflicted with the genetically heterogeneous autosomal recessive disorder xeroderma pigmentosum (XP) exhibit sensitivity to UV radiation and defective nucleotide excision repair. Complementation of these mutant phenotypes after the introduction of single human chromosomes from repair-proficient cells into XP cells has provided a means of mapping the genes involved in this disease. We now report the phenotypic correction of XP cells from genetic complementation group D (XP-D) by a single human chromosome designated Tneo. Detailed molecular characterization of Tneo revealed a rearranged structure involving human chromosomes 16 and 19, including the excision repair cross-complementing 2 (ERCC2) gene from the previously described human DNA repair gene cluster at 19q13.2-q13.3. Direct transfer of a cosmid bearing the ERCC2 gene conferred UV resistance to XP-D cells.

Pelizaeus-Merzbacher disease: Cellular hypersensitivity to ultraviolet light

We report two connatal cases of Pelizaeus-Merzbacher disease (PMD) with cellular ultraviolet (UV)-hypersensitivity. We studied the UV-sensitivity of cultured fibroblast cells derived from these PMD cases, as compared with UV-sensitive Cockayne syndrome (CS) and xeroderma pigmentosum (XP) cells as positive controls. The ability of the PMD cells to form colonies after UV irradiation was intermediate between those of CS cells and normal controls. There were no differences in both colony-forming ability after x-ray irradiation and unscheduled DNA synthesis (UDS) activity after UV irradiation between the PMD cells and the control cells. These cytological results suggest the possibility that a DNA defect might be involved in PMD.

Striking differences in cellular catalase activity between two DNA repair-deficient diseases: xeroderma pigmentosum and trichothiodystrophy

Xeroderma pigmentosum (XP) and trichothiodystrophy (TTD) are two recessively transmitted human diseases characterized by DNA repair deficiency. While XP is associated with a very high incidence of cancer on skin exposed to sunlight, TTD is not a cancer-prone disease. Therefore, unrepaired UV-induced DNA lesions do not appear to be enough to give rise to tumors. In order to understand the differences between these two syndromes, we measured catalase activity in cellular extracts, UV irradiated or not, and quantified H2O2 production following in vitro UV irradiation. We confirmed on 21 different XP diploid fibroblast lines that catalase activity was decreased on average by a factor of five as compared to controls, while XP heterozygote lines exhibited intermediary responses. All seven TTD lines we tested were deficient in UV-induced lesion repair and exhibited a high level of catalase activity. However, molecular analysis of catalase transcription showed no difference between normal, XP and TTD cell lines. This was confirmed by Western blots where the amount of catalase subunits was identical in all cell lines studied. Finally, UV irradiation induces five and three times more H2O2 production in XP lines compared with TTD or controls respectively. These striking differences between TTD and XP indicate that UV light, directly or indirectly, together with defective oxidative metabolism may increase the initiation and/or the progression steps in the XP environment compared to TTD. This may partly explain the different tumoral phenotype observed between the two diseases.

Evidence for detective repair of cyclobutane pyrimidine dimers with normal repair of other DNA photoproducts in a transcriptionally active gene transfected into Cockayne syndrome cells

Cockayne syndrome (CS) and xeroderma pigmentosum (XP), autosomal recessive diseases with clinical and cellular hypersensitivity to UV radiation, differ in ability to repair UV DNA photoproducts in their overall genome: normal repair in CS, defective repair in XP. In order to characterize a DNA repair defect in an active gene in CS, we measured the capacity of cells from patients with CS and XP to reactive 2 major types of UV-induced DNA damage, photoreactivatable (i.e., cyclobutane pyrimidine dimers) and non-photoreactivable (primarily pyrimidine-(4–6)pyrimidone photoproducts, in the actively transcribing chloramphenicol acetyltransferase (cat) gene of the plasmid expression vector pRSV-cat. Epstein-Barr virus-transformed lymphoblast lines from 4 normal persons and from 3 patients with CS and from two with XP were transiently transfected with the plasmid, and the cat activity in cell extracts was determined. When the cells were transfected with UV-irradiated plasmid, cat expression was abnormally decreased in both the CS and XP cells. When the cyclobutane pyrimidine dimers in the UV-irradiated plasmid were removed by photoreactivation prior to transfection, cat expression in the CS, but not in the XP, lines reached normal levels. These data imply that both XP and CS cells are unable to repair normally the cyclobutane pyrimidine dimer photoproducts which block transcription of cat. However, the CS, but not XP, cells can repair normally the other UV-induced photoproducts which block transcription. The ability of CS, but not XP, cells to repair these non-dimer photoproducts indicates that the active gene repair mechanism treats the cyclobutane pyrimidine dimer differently from the non-dimer photoproducts.

Enhancement of ultraviolet-DNA repair in denV gene transfectants and T4 endonuclease V-liposome recipients.

The phage T4 denV gene, coding for the pyrimidine-dimer specific T4 endonuclease V, was transfected into human repair-proficient fibroblasts, repair-deficient xeroderma pigmentosum fibroblasts, and into wild type CHO hamster cells. Transfectants maintained denV DNA and expressed denV mRNA. Purified T4 endonuclease V encapsulated in liposomes was also used to treat repair-proficient and -deficient human cells. The denV transfected clones and liposome-treated cells showed increased unscheduled DNA synthesis and enhanced removal of pyrimidine dimers compared to controls. Both denV gene transfection and endonuclease V liposome treatment enhanced post-UV survival in xeroderma pigmentosum cells but had no effect on survival in repair-proficient human or hamster cells. The results demonstrate that an exogenous DNA repair enzyme can correct the DNA repair defect in xeroderma pigmentosum cells and enhance DNA repair in normal cells.

High-frequency transformation of human repair-deficient cell lines by an Epstein-Barr virus-based cDNA expression vector

We constructed a human cDNA expression vector by combining an episomal Epstein-Barr virus (EBV) vector with the expression cassette from the transient-expression vector, pCDM8. This new vector, designated pEBS7, exhibited high-level expression of reporter genes in normal and repair-deficient xeroderma pigmentosum cell lines. Reconstruction experiments indicated that marker genes diluted to a frequency of 10(-5) can be rescued on a single transfection dish. Moreover, derivative cell lines that constitutively express the gene encoding EBV nuclear antigen 1 exhibited a tenfold enhancement in the frequency of rescue of marker genes. The feasibility of preparing large-scale directional or nondirectional cDNA libraries in pEBS7 was demonstrated and reconstruction experiments indicated that marker genes could be rescued from either library with equal efficiency. These results establish a high-efficiency system for the isolation of genes by direct phenotypic selection in human mutant cell lines.

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.

Identification of a Specific Protein Factor Defective in Group A Xeroderma Pigmentosum Cells1

A protein factor which corrects the defect in xeroderma pigmentosum cells belonging to complementation group A (XP-A cells) was detected in a cell extract prepared from calf thymus. The activity of this factor was measured as the amount of unscheduled DNA synthesis (UDS) reappearing in UV-irradiated XP-A cells after microinjection of the extract. The native molecular mass of this factor was estimated to be 80 kDa by gel-filtration and 25 kDa by glycerol gradient centrifugation. The activity was, however, recovered at a position corresponding to 43 kDa after renaturation on an SDS-PAGE gel. The isoelectric point was determined to be approximately 7.5 by measuring the activity after renaturation on an IEF gel. These values were obtained with a partially purified sample. A spot corresponding to these values was detected on two-dimensional gel electrophoresis with a highly purified sample recovered from an SDS-PAGE gel. The purified protein stimulated UDS specifically in the XP-A cells and endowed the cells with a normal level of UV-resistance. The XP-A cells injected with the factor also showed a normal level of UDS after treatment with either 4HAQO or psoralen plus UV-A. This factor (XP-A complementing factor; XP-ACF) may be involved in the repair of DNA damage induced by various agents.

Both cross-links and monoadducts induced in DNA by psoralens can lead to sister chromatid exchange formation

The relative importance of DNA-DNA cross-links and bulky monoadducts in sister chromatid exchange (SCE) formation was investigated in three human fibroblast cell lines with different repair capabilities. These cell lines included normal cells, which can repair both classes of lesions; xeroderma pigmentosum (XP) cells, which cannot repair either psoralen-induced cross-links or monoadducts; and an XP revertant that repairs only cross-links and not monoadducts. SCEs were induced by two psoralen derivatives, 4′-hydroxymethyl-4,5′,8-trimethylpsoralen (HMT) and 5-methylisopsoralen (5-MIP). After activation with long-wave ultraviolet light, HMT produces cross-links and monoadducts in DNA, whereas 5-MIP produces only monoadducts. In normal human cells both psoralens induced SCEs, but if cells were allowed to repair for 18 h before bromodeoxyuridine (BrdUrd) was added for SCE analysis, the SCE frequency was significantly reduced. XP cells showed an SCE frequency that remained high regardless of whether SCEs were analyzed immediately after psoralen exposure or 18 h later. In the XP revertant that repairs only cross-links, both psoralens induced a high yield of SCEs when BrdUrd was added immediately after psoralen treatment. When XP revertant cells were allowed 18 h to repair before addition of BrdUrd, the SCEs induced by HMT were greatly reduced, whereas those induced by 5-MIP were only slightly reduced. These observations indicate that both cross-links and monoadducts are lesions in DNA that can lead to SCE formation.

CYCLOBUTANE DIMERS AND (6‐4)PHOTOPRODUCTS IN HUMAN CELLS ARE MENDED WITH THE SAME PATCH SIZES

The size of excision repair patches corresponding to excision of (6-4) pyrimidine-pyrimidone photoproducts and (5-5, 6-6) cyclobutane dimers have been independently determined by using bromodeoxyuridine substitution and density increases in isopycnic gradients of small DNA fragments. The two classes of photoproducts were distinguished by using (a) a xeroderma pigmentosum (XP) revertant cell line that excises (6-4) photoproducts normally, but does not excise cyclobutane dimers from bulk DNA or from an actively transcribed sequence; (b) an XP cell line containing the denV gene of bacteriophage T4, which repairs only cyclobutane dimers by a unique glycosylase mechanism, and (c) normal cells analyzed during time intervals in which cyclobutane dimer repair is the main repair process in action. The patch sizes for the two lesions were similar under all conditions and were estimated to be approximately 30-40 bases. These values are slightly large than corresponding estimates for Escherichia coli and Saccharomyces cerevisiae but close to estimates from in vitro experiments with human cell extracts. The size of 30 bases may consequently be very close to the actual distance between cleavage sites made on either side of a photoproduct during repair.

DNA BREAKS CAUSED BY MONOCHROMATIC 365 nm ULTRAVIOLET‐A RADIATION OR HYDROGEN PEROXIDE and THEIR REPAIR IN HUMAN EPITHELIOID and XERODERMA PIGMENTOSUM CELLS

The induction and repair of DNA single-strand breaks (SSB) assayed by alkaline filter elution was compared in human epithelioid P3 and xeroderma pigmentosum (XP) cells exposed to monochromatic 365-nm UV-A radiation and H2O2. Initial yields of SSB were measured with the cells held at 0.5 degrees C during exposure. The yield from exposure to 365-nm radiation was slightly greater in XP than in P3 cells, whereas H2O2 produced more than three times as many SSB in P3 compared with XP cells. o-Phenanthroline (50 mM) markedly inhibited the yields of SSB induced in XP cells by H2O2, but had no effect on those produced by 365-nm UV-A. These results are consistent with the fact that P3 cells, unlike XP cells, have undetectable levels of catalase. The measured production of trace amounts of H2O2 by the actual 365-nm UV-A exposures was not sufficient to account for the numbers of breaks that were observed. Single-strand breaks produced by both agents were completely repaired after 50 min in P3 cells, as were H2O2-induced SSB in XP cells. However, 25% of the 365-nm UV-A-induced SSB in XP cells remained refractory to repair after 60 min. The results show that SSB produced by these two agents are different and that 365 nm radiation produces most SSB in cells by mechanisms other than by production of H2O2.