Clinical characteristics, DNA repair, and complementation groups in xeroderma pigmentosum patients from Egypt.

Xeroderma pigmentosum (XP) has been reported to be unusually frequent among Middle Eastern populations. This report describes the first survey of DNA repair characteristics among Egyptians. Sixteen XP patients were contacted, and biopsies from eight were analyzed for unscheduled DNA synthesis, strand breakage during pyrimidine dimer excision, and complementation groups. The patients were equally distributed between Complementation Groups A and C. Unscheduled synthesis and strand breaks were significantly higher in Group C than in Group A cells. Central nervous system disorders were found in all of the Group A patients and in none of the Group C patients. No clinical symptoms were observed in the heterozygotes. A 2-month-old sib of an XP patient was free of symptoms, but unscheduled synthesis and strand breakage in cultures from this sib were the same as in the related XP homozygote. From the relative frequencies of each complementation group found in various parts of the world, we offer a hypothesis concerning the relative sizes and roles for gene products specified by the alleles or genes corresponding to each complementation group.

Repair of ultraviolet light damage in a variety of human fibroblast cell strains.

Postreplication repair of DNA damage after ultraviolet light irradiation has been examined in a wide variety of human fibroblast strains. The donors were patients with xeroderma pigmentosum (XP) of different complementation groups or other hereditary disorders with indications of radiosensitivity, or with light sensitivity or multiple cancers. The defect in postreplication repair previously found in XP variants (excision-proficient XP's) has now been observed in a total of five XP variants and a less severe defect in postreplication repair has been found in excision-defective XP's in Complementation Groups A, B, C, and D. Complementation Group E and all other cell strains studied showed a response that was not significantly different from that of cells from normal donors. Excision repair was also measured in some of these cell strains and was found to be defective only in XP cells. Ultraviolet cell survival characteristics have been obtained for may of the cell strains. The most sensitive were cells from the excision-deficient XP's and from a sun-sensitive child (11961); the latter had no measurable defect in either excision or postreplication repair. The rest of the survival curves lay in a band limited by normal cell strains on the one hand and the slightly more sensitive excision-proficient XP variant XP30RO. Only in the case of the variants XP30RO and XP7TA were we able to demonstrate any influence of caffeine on cell survival.

Establishment and characterization of a melanoma cell line from a xeroderma pigmentosum patient: activation of N-ras at a potential pyrimidine dimer site.

Patients suffering from the genetic disorder xeroderma pigmentosum (XP) display an extreme sensitivity of their skin to sun (UV) exposure and predisposition to skin cancer due to deficiencies in the excision DNA repair pathway. Here we describe the establishment and characterization of the first tumor cell line derived from an XP patient (belonging to complementation group C). The melanoma cell line designated XP44RO(Mel) has retained its tumorigenic and XP phenotype (UV sensitivity, reduced unscheduled DNA synthesis) and showed karyotypic abnormalities characteristic of melanomas. Transfection of XP44RO(Mel) DNA to NIH3T3 cells and oligonucleotide hybridization revealed that the N-ras oncogene was activated by an A.T to T.A or C.G transversion at the third position of codon 61. This mutation occurs at a dipyrimidine site. It is likely initiated by a UV-induced pyrimidine dimer and is of a type rarely observed in mammalian shuttle vector systems and endogenous genes after UV irradiation.

Activated ras genes in human seminoma: evidence for tumor heterogeneity.

The incidence of mutations in cellular ras genes was determined in human seminoma, a germ cell tumor of the testis, with the aid of specific oligonucleotide probe hybridization. To eliminate the large number of nonneoplastic cells present in seminomas, aneuploid tumor cell nuclei were isolated from the tumor tissue by flow sorting. Mutations were detected in 40% of the seminomas at codons 12 or 61 of either the Ki-ras or the N-ras gene. No correlation was found with histopathological or clinical features. In some seminomas the mutant gene was present in only a fraction of the tumor cell population, suggesting tumor heterogeneity for ras gene mutations. Yet, flow cytometric measurement of nuclear DNA contents and histological examination of tumor tissue did not reveal two different tumor cell populations. We conclude from these observations that ras mutation is probably not the initial genetic event in the development of seminoma.

Induction of sister chromatid exchanges in xeroderma pigmentosum cells after exposure to ultraviolet light.

The role of DNA repair mechanisms in the induction of sister chromatid exchanges (SCE) after exposure to ultraviolet radiation was investigated in xeroderma pigmentosum cells. Cells from different excision-deficient XP strains, representing the 5 complementation groups in XP, A, B, C, D and E, and from excision-proficient XP variant strains were irradiated with low doses of UVR (0-3.5 J/m2). The number of SCE was counted after two cycles in the presence of BUdR. In cells of the complementation groups A, B, C and D the number of SCE was significantly higher than in UV-exposed control cells. The frequencies of SCE in group E cells and in XP varient cells were not different from those in control cells. Treatment with caffeine (0-200 microgram/ml) did not result in a different response of variant cells compared with normal cells. A simple correlation between SCE frequency and residual excision-repair activity was not observed. The response of the excision-repair deficient cells suggest that unrepaired damage, produced by UVR is involved in the production of SCE.

Microinjection of Micrococcus luteus UV-endonuclease restores UV-induced unscheduled DNA synthesis in cells of 9 xeroderma pigmentosum complementation groups.

The UV-induced unscheduled DNA synthesis (UDS) in cultured cells of excision-deficient xeroderma pigmentosum (XP) complementation groups A through I was assayed after injection of Micrococcus luteus UV-endonuclease using glass microneedles. In all complementation groups a restoration of the UV-induced UDS, in some cells to the repair-proficient human level, was observed. Another prokaryotic DNA-repair enzyme, T4 endonuclease V, restored the UV-induced UDS in a similar way after microinjection into XP cells. Since both enzymes specifically catalyse only the incision of UV-irradiated DNA, we conclude that this activity is impaired in cells of all 9 excision-deficient XP complementation groups tested.

A seventh complementation group in excision-deficient xeroderma pigmentosum.

Cells from a xeroderma pigmentosum patient XP2BI who has reached 17 years of age with no keratoses or skin tumours constitute a new, 7th complementation group G. These cells exhibit a low residual level of excision repair, 2% of normal after a UV dose of 5 J/m2 and an impairment of post-replication repair characteristic of excision-defective XPs. They are also sensitive to the lethal effects of UV and defective in host-cell reactivation of UV-irradiated SV40 DNA.

A ninth complementation group in xeroderma pigmentosum, XP I.

A new complementation group of excision-deficient xeroderma pigmentosum (XP) is described in 2 patients living in the F.R.G. Dermatological, ophthalmological and neurological symptoms of XP are presented together with DNA repair characteristics such as unscheduled DNA synthesis, colony-forming ability and alkaline elution studied in cultured fibroblasts. The results are compared to normal controls.

Five complementation groups in xeroderma pigmentosum.

A collaborative study was undertaken to determine the relationship between the three DNA repair complementation groups in xeroderma pigmentosum found at Erasmus University, Rotterdam, and the four groups found at the National Institutes of Health, Bethesda. The results of this study reveal that there are five currently known complementation groups in xeroderma pigmentosum.

Interspecies complementation analysis of xeroderma pigmentosum and UV-sensitive Chinese hamster cells.

Complementation analysis was performed 24 h after fusion of UV-sensitive CHO cells (CHO 12 RO) with XP cells of complementation groups A, B, C, D, F and G. The parental cells are characterized by low levels of unscheduled DNA synthesis (UDS). In all combinations, the UDS levels observed in heterokaryons were higher than those in parental mutant cells, clearly indicating cooperation of human and Chinese hamster repair functions. In heterokaryons of CHO 12 RO with XP-A and XP-C cells, the UDS values reached about the normal human level, whereas in heterokaryons with XP-B, XP-D and XP-F, UDS was restored at a level approaching that in wild-type CHO cells. The results obtained after fusion of CHO cells with two representative cell strains from the XP-G group, XP 2 BI and XP 3 BR, were inconsistent. Fusion with XP 3 BR cells yielded UDS levels ranging from wild-type Chinese hamster to normal human, whereas fusion with XP 2 BI cells resulted in a slight increase in UDS which even after 48 h remained below the level found in wild-type CHO cells. The occurrence of complementation in these interspecies heterokaryons indicates that the genetic defect in the CHO 12 RO cells is different from the defects in the XP complementation groups tested.

Differences in the levels of UV repair and in clinical symptoms in two sibs affected by xeroderma pigmentosum.

UV-repair activity was studied in two sibs affected by XP showing different clinical symptoms. Complementation studies indicated that both patients fit into complementation group A. The levels of UV-induced 3H-thymidine incorporation, in fibroblasts and in lymphocytes, are different in the two patients: residual level of repair DNA synthesis in the sister is higher than in the brother. In one of the cell samples analyzed UDS analysis showed that in the sister a low proportion of cells with normal repair synthesis is present.

Localization of a gene involved in complementation of the defect in xeroderma pigmentosum group A cells on human chromosome 1.

Human, Chinese hamster or Chinese hamster/human hybrid cytoplasts were fused with UV-irradiated xeroderma pigmentosum group A (XP-A) cells. Unscheduled DNA synthesis (UDS) of the XP-A nucleus was measured 0-2 and 2-4 h after seeding of the fused population. Human cytoplasts did correct the defect in the XP-A nucleus immediately after fusion, whereas the chinese hamster cytoplasts did not show this rapid increase in excision repair. The results obtained after fusion of cytoplasts isolated from a panel of 26 Chinese hamster-human hybrids showed that chromosome 1 bears genetic information that is necessary for the rapid correction of the XP-A defect. Furthermore, this genetic information was regionally assigned to 1q42-qter by analysing hybrid cell lines having retained various segments of chromosome 1. Cytoplasts from a Chinese hamster/XP-A hybrid containing chromosome 1 of XP-A origin corrected also the defect with fast kinetics. This result indicate that the correcting factor consists of human and Chinese hamster components. As a consequence, the gene mapped on chromosome 1 may not be the gene which is mutated in XP-A cells.

Prenatal diagnosis of xeroderma pigmentosum (group C) using assays of unscheduled DNA synthesis and postreplication repair.

The analysis of DNA repair processes is described in two pregnancies at risk for xeroderma pigmentosum. In both cases, excision repair (measured by unscheduled DNA synthesis) and postreplication repair were analyzed. An affected and an unaffected fetus were identified within 3 weeks after amniocentesis. The cells from the affected fetus were found to be deficient in excision DNA repair, whereas the PRR patterns were intermediate between those of normal and PRR deficient cells. This indicates the possibility of prenatal diagnosis of PRR deficient XP patients (XP variants).