Xeroderma Pigmentosum Strains Research Articles

Impaired regulation of tumor suppressor p53 caused by mutations in the xeroderma pigmentosum DDB2 gene: mutual regulatory interactions between p48(DDB2) and p53.

Tumor suppressor p53 controls cell cycle progression and apoptosis following DNA damage, thus minimizing carcinogenesis. Mutations in the human DDB2 gene generate the E subgroup of xeroderma pigmentosum (XP-E). We report here that XP-E strains are defective in UV irradiation-induced apoptosis due to severely reduced basal and UV-induced p53 levels. These defects are restored by infection with a p53 cDNA expression construct or with a DDB2 expression construct if and only if it contains intron 4, which includes a nonmutated p53 consensus-binding site. We propose that both before and after UV irradiation, DDB2 directly regulates p53 levels, while DDB2 expression is itself regulated by p53.

Expression of mitochondrial genes and DNA-repair-related nuclear genes is altered in xeroderma pigmentosum fibroblasts.

Differential hybridization was used to detect repair defects in xeroderma pigmentosum (XP) that are not amenable to current analyses. cDNA libraries were constructed from cytoplasmic RNA of normal and XP fibroblast strains (complementation groups A and D) and analyzed for differential gene expression. More than 40,000 lambda gt10 cDNA clones were differentially screened with in vitro transcripts made from cDNA in the pBluescript vector. Six differential clones were detected in the libraries of the XP group A and D strains which caused stronger or weaker signals when probed with transcripts from XP strains than with those from the normal strains. Two clones coded for mitochondrial genes: mitochondrial 16 S rRNA and ATPase 6L. Overexpression of mitochondrial genes in XP may indicate that functions of the ATP-generating system are impaired since such functions are intensified whenever they become insufficient, for example as a consequence of DNA damage. It is tempting to assume that abnormal mitochondria are one of the causes for the neurological malfunctions in XP. Furthermore, densitometric analysis of Northern blots revealed that mRNA of lactate dehydrogenase, chain M, was less abundant in four XP group A strains (extent of reduction: 70%) and in two XP group D strains (extent of reduction: 58%). Enzyme activity was also diminished. In addition, mRNA of the gene for glyceraldehyde-3-phosphate dehydrogenase was less expressed in the same XP group A and D fibroblast strains investigated (reduction in both complementation groups: 50%). Both glycolytic enzymes have nuclear functions apart from their role in sugar metabolism. Lactate dehydrogenase, chain M, is identical to a helix-destabilizing protein; it is closely associated with chromatin and unfolded DNA, suggesting a role in DNA synthesis and transcription. The 37-kDa subunit of glyceraldehyde-3-phosphate dehydrogenase is involved in transcription and was shown to be identical to uracil-DNA glycosylase, a base-excision repair enzyme. We presume that the nuclear functions of these glycolytic enzymes may be thwarted in the XP strains investigated and may account for malfunctions in XP, particularly for neurological disturbances.

Assignment of six patients with xeroderma pigmentosum in Hokkaido area to a variant form.

Six Japanese patients in Hokkaido area were diagnosed as xeroderma pigmentosum (XP) at two University Hospital; one patient, a 29 years old female (XP2AS) at Asahikawa Medical College, and five patients, namely, a 53 years old male (XP1SA), a 45 years old female (XP2SA), a 46 years old female (XP3SA), a 47 years old male (XP4SA) and a 34 years old male (XP5SA) at Hokkaido University School of Medicine. All these XP patients showed mild skin symptoms and had no apparent neurological abnormalities. To identify genetic complementation groups of these patients, DNA repair capacities in skin fibroblasts derived from the patients were analyzed by ultraviolet light (UV)-induced unscheduled DNA synthesis (UDS), and also by colony-forming abilities of UV-irradiated cells incubated with or without caffeine added in post-UV culture medium. The levels of UDS measured by autoradiography in these XP cells irradiated with 20 J/m2 of UV were 67 to 84% of those of normal human fibroblasts. Although one XP cell strain (XP2AS) showed about 2-fold greater sensitivity to killing by UV than normal cells, UV sensitivities of the rest of 5 XP strains were similar or only slightly higher than those of normal cells. However, UV sensitivities of all the 6 XP cell strains markedly increased by addition of 1 mM caffeine in the post-UV incubation medium, a phenomenon characteristic of XP variant cells. We also analyzed post-replication repair in XP1SA cells by sedimentation of newly-synthesized DNA in alkaline sucrose gradient, and found that this strain is defective in this repair system. From these results, we concluded that all the 6 XP strains studied here belong to XP variants.

Metabolic processing of cyclobutyl pyrimidine dimers and (6-4) photoproducts in UV-treated human cells. Evidence for distinct excision-repair pathways.

A new nuclease digestion assay was developed to elucidate the human excision-repair system operating on cyclobutyl pyrimidine dimers and (6-4) photoproducts. We analyzed lesions that accumulated in excised oligonucleotide fragments during incubation of UV-treated cultured fibroblasts. (6-4) photoproducts were removed intact, whereas excised cyclobutyl dimers often contained ruptured interpyrimidine phosphodiester bonds, raising the possibility that the intradimer backbone-cleavage reaction may help promote the bypass of unexcised dimers by the DNA replication or RNA transcription machinery. Cell strains representing eight different inherited forms of the cancer-prone skin disease xeroderma pigmentosum (XP) were generally found to exhibit characteristic abilities to excise the two classes of photolesions, ranging from total deficiency in groups A and G to normal proficiency in the variant. The capacity of any given XP group to act on one class of photoproducts in no way predicted its ability to act on the other. Finally, in those XP strains displaying significant levels of dimer removal, the ratio of intact-versus-modified dimers was normal, implying that rupture of the intradimer backbone linkage occurs independently of subsequent excision-repair reactions. Our data indicate that cyclobutyl dimers and (6-4) photoproducts are processed by distinct nucleotide-excision-repair pathways in human cells.

Enhanced expression of mitochondrial genes in xeroderma pigmentosum fibroblast strains from various complementation groups.

cDNA libraries constructed from cytoplasmic RNA of normal and xeroderma pigmentosum (XP) fibroblast strains were screened for differential gene expression. XP fibroblast strains included one representative of the complementation groups A, C, D, and one XP variant strain. The XP lambda gt10 cDNA libraries were differentially screened with in vitro transcripts made from cDNA in the pBluescript vector using both the same XP strain and the normal fibroblast strain. Eight differential clones were detected in the libraries of the XP group A, D, and C strains, which caused stronger signals when probed with transcripts from XP strains than with those from the normal strain. The cDNA clones were sequenced. Seven of the eight clones detected coded for three mitochondrial genes: subunit I of cytochrome c oxidase (complex IV of the respiratory chain), apocytochrome b (subunit of complex III), and 16-S rRNA. Two clones representing essentially (a) subunit I of cytochrome c oxidase and (b) 16-S rRNA diverged from the sequence of the human mitochondrial genome present in the data-base libraries. Clone a exhibited a transition mutation, clone b reflected a transcript of a mitochondrial genome rearranged in the 16-S rRNA gene, including four nucleotides of the adjacent tRNA(Leu) gene. The apparently enhanced expression of mitochondrial genes in XP cells, together with the changes in DNA sequence, seem to indicate that functions of the ATP-generating system were impaired. This defect may have originated from mutations due to lack of DNA repair. The data can be interpreted in the light of mitochondrial changes that cause human neuromyopathies to occur. In analogy to these diseases the neurological symptoms in XP might be explained by abnormal mitochondria.

The effects of inhibitors of topoisomerase II and quinacrine on ultraviolet-light-induced DNA incision in normal and xeroderma pigmentosum fibroblasts

The aim of our work was to investigate whether DNA topoisomerase II participates in the repair-specific incision of UV-irradiated genomic DNA. Therefore, the influence upon DNA incision of the topoisomerase II inhibitors (nalidixic and oxolinic acid, novobiocin and coumermycin A1) as well as the intercalating agent quinacrine has been measured in normal human fibroblasts using the alkaline elution technique. In addition, inhibition by novobiocin has been determined in fibroblast strains from 11 normal donors and from 16 xeroderma pigmentosum (XP) patients belonging to the complementation groups A, C, D, E, and XP variant. Nalidixic and oxolonic acid did not inhibit endonucleolytic cleavage, whereas novobiocin was a potent inhibitor of DNA incision. It was observed that in normal and in all XP strains 50% inhibition by novobiocin occurred on average in the dose range 315-590 microM. Since inhibition by novobiocin was not paralleled by that with the other topoisomerase II inhibitors nalidixic and oxolinic acid, it must be concluded that reduction of enzyme-catalysed breaks was not due to the participation of topoisomerase II in the incision step, but to the displacement of ATP at the binding site of the DNA-incising enzyme. This enzyme absolutely requires ATP as a cofactor for endonucleolytic cleavage. Quinacrine, however, inhibited DNA incision in normal fibroblasts at a mean Ki of 318 microM. Inhibition by this intercalating agent seems to be caused by structural perturbations in DNA, which render it a poor substrate for endonucleolytic cleavage.

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

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.

Sensitivity of Hyperthermia-Treated Human Cells to Killing by Ultraviolet or γ Radiation

Human xeroderma pigmentosum (XP) or Fanconi anemia (FA) fibroblasts displayed shouldered 45 degrees C heat survival curves not significantly different from normal fibroblasts, a result similar to that previously found for ataxia telangiectasia (AT) cells, indicating heat resistance is not linked to either uv or low-LET ionizing radiation resistance. Hyperthermia (45 degrees C) sensitized normal and XP fibroblasts to killing by gamma radiation but failed to sensitize the cells to the lethal effects of 254 nm uv radiation. Thermal inhibition of repair of ionizing radiation lesions but not uv-induced lesions appears to contribute synergistically to cell death. The thermal enhancement ratio (TER) for the synergistic interaction of hyperthermia (45 degrees C, 30 min) and gamma radiation was significantly lower in one FA and two strains (TER = 1.7-1.8) than that reported previously for three normal strains (TER = 2.5-3.0). These XP and FA strains may be more gamma sensitive than normal human fibroblasts. Since hyperthermia treatment only slightly increases the gamma-radiation sensitivity of ataxia telangiectasia (AT) fibroblasts compared to normal strains, it is possible that the degree of thermal enhancement attainable reflects the genetically inherent ionizing radiation repair capacity of the cells. The data indicate that both repair inhibition and particular lesion types are required for lethal synergism between heat and radiation. We therefore postulate that the transient thermal inhibition of repair results in the conversion of gamma-induced lesions to irrepairable lethal damage, while uv-type damage can remain unaltered during this period.

Xeroderma pigmentosum complementation group F: more assignments and repair characteristics.

Abstract— The specific heterodikaryon complementation method enabled us to assign three patients with mild xeroderma pigmentosum (XP) symptoms (XP25KO, XP27KO, XP28KO) to complementation group F. UV‐induced unscheduled DNA synthesis (UDS) remained unnormalized in the heterodikaryons between either of the above three XP strains and the reference group F XP3YO. All these particular XP strains as well as XP3YO exhibited an equally low level of10–15% UDS by a 3 h [3H]‐thymidine labeling following 10 J/m2 254 nm UV, while they attained 60% UDS of normal at an extended time of 25 h. The present group F strains were 3 and 1.5 times as sensitive to the lethal effect of UV as normal and XP group E cells, respectively, based on the mean lethal dose (Do) comparison. Normal cells had the biphasic time‐UDS kinetics of early rapid and late slow repair. Characteristically, however, all of the present group F strains were defective in only early rapid repair, but normally proficient in slow repair.

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/m 2) 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.

Action spectra for inactivation of normal and xeroderma pigmentosum human skin fibroblasts by ultraviolet radiations.

Abstract—Action spectra for UV‐induced lethality as measured by colony forming ability were determined both for a normal human skin fibroblast strain (lBR) and for an excision deficient xeroderma pigmentosum strain (XP4LO) assigned to complementation group A using 7 monochromatic wavelengths in the range 254‐365 nm. The relative sensitivity of the XP strain compared to the normal skin fibroblasts shows a marked decrease at wavelengths longer than 313 nm. changing from a ratio of about 20 at the shorter wavelengths to just greater than 1.0 at the longer wavelengths. The action spectra thus indicate that the influence on cell inactivation of the DNA repair defect associated with XP cells is decreased and almost reaches zero at longer UV wavelengths. This would occur, for example, if the importance of pyrimidine dimers as the lethal lesion decreased with increasing wavelength. In common with other studies both in bacterial and mammalian cells, our results are consistent with pyrimidine dimers induced in DNA being the major lethal lesion in both cell strains over the wavelength range 254‐313 nm. However, it is indicated that different mechanisms of inactivation operate at wavelengths longer than 313 nm.

The response of a variety of human fibroblast cell strains to the lethal effects of alkylating agents

The sensitivities of fifteen human fibroblast cell strains to the lethal effects of alkylation damage produced by N-methyl-N-nitrosourea (MNU) and N-ethyl-N-nitrosourea (ENU) have been investigated. Nine cell strains were also investigated for their sensitivities to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Included in our survey are representative strains derived from donors with the repair defective syndromes xeroderma pigmentosum (XP) and ataxia-telangiectasia (A-T), as well as strains derived from patients with Cockayne's syndrome, Bloom's syndrome, Huntington's disease and strains derived from individuals with unclassified syndromes. On the basis of our survival data we report that hypersensitivity to MNU is shown by two A-T strains (AT3BI and AT5BI), an XP strain (XP3BR), and strain 46BR derived from a patient with hypogammaglobulinaemia. This sensitivity to methylating agents is also shown by strains 46BR and XP3BR when treated with MNNG, but not for strain AT5BI. Sensitivity to ENU is shown by strain 11961 (derived from a sun-sensitive individual), XP3BR and a single Cockayne's syndrome strain CS697CTO. Of the strains studied only XP3BR was sensitive to both ethylating and methylating agents and only 46BR showed a greater than two-fold increase in sensitivity compared to normal.

Repair of Bleomycin-damaged DNA by human fibroblasts.

The ability of human fibroblasts to repair bleomycin-damaged DNA was examined in vivo. Repair of the specific lesions caused by bleomycin (BLM) was investigated in normal cell strains as well as those isolated from patients with apparent DNA repair defects. The diseases ataxia telangiectasia (AT), Bloom syndrome (BS), Cockayne syndrome (CS), Fanconi anemia (FA), and xeroderma pigmentosum (XP) were those selected for study. The method used for studying the repair of DNA after BLM exposure was alkaline sucrose gradient centrifugation. After exposure to BLM, a fall in the molecular weight of DNA was observed, and after drug removal the DNA reformed rapidly to high molecular weight. The fall in molecular weight upon exposure to BLM was observed in all cells examined with the exception of some XP strains. Prelabeled cells from some XP complementation groups were found to have a higher percentage of low molecular weight DNA on alkaline gradients than did normal cells. This prelabeled low molecular weight DNA disappeared upon exposure to BLM.

The Post-UV Colony-Forming Ability of Normal Fibroblast Strains and of the Xeroderma Pigmentosum Group G Strain

In xeroderma pigmentosum, an inherited disorder of defective DNA repair, post-UV colony-forming ability of fibroblasts from patients in complementation groups A through F correlates with the patients' neurological status. The first xeroderma pigmentosum patient assigned to the recently discovered group G had the neurological abnormalities of XP. We have determined the post-UV colony-forming ability of cultured fibroblasts from this patient and from 5 more control donors. Log-phase fibroblasts were irradiated with 254 nm UV light from a germicidal lamp, trypsinized, and replated at known densities. After 2 to 4 weeks' incubation the cells were fixed, stained and scored for colony formation. The strains' post-UV colony-forming ability curves were obtained by plotting the log of the percent remaining post-UV colony-forming ability as a function of the UV dose. The post-UV colony-forming ability of 2 of the 5 new normal strains was in the previously defined control donor zone, but that of the other 3 extended down to the level of the most resistant xeroderma pigmentosum strain. The post-UV colony-forming ability curve of the group G fibroblasts was not significantly different from the curves of the group D fibroblast strains from patients with clinical histories similar to that of the group G patient.

Variation of DNA repair capacity in progeria cells unrelated to growth conditions

Summary Fibroblasts from two progeria patients were tested for their capacity to repair γ-irradiated DNA. An immunofluorescence host cell reactivation (HCR) assay was employed to compare normal, progeria, and Xeroderma Pigmentosum strains. HCR decreased markedly over a three day period in confluent cultures, indicating that growth conditions can substantially effect the DNA repair observed in HCR of cultured fibroblasts. Using a variety of growth conditions, one progeria strain showed decreased HCR, while a second progeria strain showed normal HCR. These results suggest both that heterogeneity for DNA repair capacity exists among progeria fibroblast strains and that decreased DNA repair capacity is unlikely to be the basic genetic defect in progeria.

Unscheduled DNA synthesis induced by 4-nitroquinoline-1-oxide in xeroderma pigmentosum cells and their complementing heterodikaryons.

Unscheduled DNA synthesis (UDS) induced by 4-nitroquinoline-1-oxide (rNQO) in the complementation groups A to E and variant xeroderma pigmentosum (XP) cells, and various pairs of complementing XP heterokaryons were investigated. The pattern of UDS induced by various concentrations of 4NQO in normal cells was quite different from those in groups A to D XP cells. The patterns of UDS in group E and variant XP cells were indistinguishable from those of normal cells under our experimental conditions. The levels of UDS induced by 5 x 10(-6) M 4NQO were 13% of normal in group A, 9% in group B, 17% in group C, 25% in group D. The heterokaryons obtained by pair-wise fusions between different complementation groups of XP strains showed restored UDS induced by 5 x 10(-6) M 4NQO, while the dikaryons obtained from fusion between the same groups did not.

Repair of DNA damage after exposure to 4-nitroquinoline-1-oxide in heterokaryons derived from xeroderma pigmentosum cells.

Xeroderma pigmentosum (XP) cells are deficient in the repair of damage induced by ultraviolet irradiation. Excision-repair-deficient XP cell strains have been classified into 7 distinct complementation groups, according to results of studies on cell fusion and UV irradiation. XP cells are not only abnormally sensitive to UV, but also to a variety of chemical carcinogens, including 4-nitro-quinoline-1-oxide (4NQO). Complementation analysis with XP strains from 4 different complementation groups with respect to the repair of 4NQO-induced DNA damage revealed that the classification of the strains into complementation groups with respect to 4NQO-induced repair coincides with the classification based on the repair of UV damage.

Herpes Virus Production as a Marker of Repair in Ultra-violet Irradiated Human Skin Cells of Different Origin

Confluent cultures of human skin fibroblasts were irradiated with ultra-violet light 0 to 48 hours before infection with herpes simplex virus type 1 (HSV). The one-cycle viral yield was measured. Different responses were obtained according to the origin of the host cells. (1) Cells from three normal donors showed a dose-dependent recovery of HSV production during the 36--40 hours following U.V. exposure. The recovery was maximal for a dose at which a plateau level of unscheduled DNA synthesis (UDS) was reached (24 Jm-2). (2) In a xeroderma pigmentosum (XP) heterozygote line from a mother of XP children, the level of UDS after irradiation up to 48Jm-2 was normal whereas the extent of recovery of HSV production capacity was lower than that of the normal lines. (3) In strains from two cases of XP children, with a normal UDS (XP variants), the recovery process was slowed down and its extent was lower than in normal or XP heterozygote cells. (4) Excision-deficient XP strains from eight cases of XP children presented either no recovery (two strains having the lowest UDS, less than 2 per cent) or a small recovery, the extent of which was in good agreement with the corresponding level of UDS (between 5 and 30 per cent). Measurement of this recovery seems to be a very sensitive assay for detecting differences in the repair abilities of U.V.-irradiated human skin cells of various origins.

Decreased Repair of Gamma-irradiated Adenovirus inXeroderma PigmentosumFibroblasts

The ability of gamma-irradiated adenovirus to produce viral structural antigens (Vag) was examined in several normal and Xeroderma pigmentosum (XP) fibroblast strains. The fibroblast cultures were infected with either irradiated or nonirradiated adenovirus and at 48 hours after infection, cells were examined for the presence of Vag using immunofluorescent staining. Survival of Vag synthesis for gamma-irradiated adenovirus had a D37 value of 47 +/- 4 x 10(4) rad following the infection of seven normal fibroblast strains. The survival of this viral function was found to be significantly less following infection of the XP strains. D37 values for Vag synthesis expressed as a percentage of that obtained on normal strains were obtained for a representative strain from each of the XP complementation groups: group A, 57 per cent; group B, 61 per cent; group C, 61 per cent, group D, 59 per cent; group E, 73 per cent; and variant, 75 per cent. These results indicate that XP cells have a reduced repair capacity for some type of gamma-ray-induced DNA damage.

Xeroderma pigmentosum neurological abnormalities correlate with colony-forming ability after ultraviolet radiation.

Xeroderma pigmentosum is an autosomal recessive disease in which DNA repair processes are defective. All xeroderma pigmentosum patients develop premature aging of sun-exposed skin, and some develop neurological abnormalities due to premature death of nerve cells. Sensitivity to ultraviolet radiation of 24 xeroderma pigmentosum fibroblast strains was studied in vitro by measuring each strain's ability to divide and form colonies after irradiation. The most sensitive strains were derived from patients who had an early onset of neurological abnormalities; less sensitive strains were from patients with a later onset; and the most resistant strains were from patients without neurological abnormalities. The UV sensitivities of strains from each member of a sibling pair with xeroderma pigmentosum were identical, indicating that UV sensitivity of xeroderma pigmentosum strains is determined by the patient's inherited DNA repair defect. The results suggest that effective DNA repair is required to maintain the functional integrity of the human nervous system by preventing premature death of neurons.