Cells cultured from most patients suffering from the sunlight-sensitive hereditary disorder xeroderma pigmentosum are defective in the ability to excise ultraviolet light (UV)-induced pyrimidine dimers from their DNA. There is, however,-one class of these patients whose cells are completely normal in this excision repair process. We have found that these cells have an abnormality in the manner in which DNA is synthesized after UVirradiation. The time taken to convert initially lowmolecular-weight DNA synthesized in UV-irradiated cells into high-molecular-weight DNA similar in size to that in untreated cells is much greater in these variants than in normal cells. Furthermore, this slow conversion of low to high-molecular-weight newly synthesized DNA is drastically inhibited by caffeine, which has no effect in normal cells. Recently, complementation studies have shown that there are several genetically different forms of XP, all deficient in excision-repair (3)(4)(5)(6)(7)(8). In addition there is a further class of XP patients (termed "XP variants" in this communication) which, while exhibiting the usual clinical symptoms, seem to be completely normal in excision-repair of pyrimidine dimers (9-14). All XP lines examined have normal rates of rejoining of DNA single-strand breaks induced by ionizing radiation (14).Despite very low levels of excision-repair in cells from most XP patients (and also in rodent cell lines), they can nevertheless tolerate the production in their DNA of over 105 pyrimi- In this communication we show that fibroblast cultures from three XP variants have normal levels of excision-repair, but are abnormal in postreplication repair. After UV-irradiation, the time taken for the newly synthesized DNA to attain a high molecular weight similar to that in unirradiated controls is much longer than in normal cells. Furthermore, this conversion of low-to high-molecular weight DNA is drastically inhibited by caffeine, which has very little effect in normal human cells. MATERIALS AND METHODSCell Lines used in these experiments were primary fibroblasts from healthy donors and XP patients listed in Table 1.Excision of Pyrimidine Dimers Measured by Loss of UVEndonuclease-Susceptible Sites. This procedure has been described in detail (24). Briefly; fibroblast cells cultured as described in ref. 24 were labeled with [3H]thymidine, UVirradiated, and incubated in the absence of radioactive label for various times. They were then mixed with an equal volume of unirradiated cells whose DNA had been labeled with [14C]-dT. DNA was extracted from the mixed cell population and incubated with or without the UV-specific endonuclease from Micrococcus luteus, which'specifically nicks DNA near pyrimidine dimers (24,25). The DNA products resulting from enzymic attack were centrifuged through 5-20% alkaline sucrose gradients at 40,000 rpm for 135 min at 210 in an SW56 rotor. After centrifugation, fractions were collected, radioactivity was determined, and the weight-average molecular weight of the DNA distributions ...

Ataxia-telangiectasia (AT) is a human autosomal recessive disorder of childhood characterized by: (1) progressive cerebellar ataxia with degeneration of Purkinje cells; (2) hypersensitivity of fibroblasts and lymphocytes to ionizing radiation; (3) a 61-fold and 184-fold increased cancer incidence in white and black patients, respectively; (4) non-random chromosomal rearrangements in lymphocytes; (5) thymic hypoplasia with cellular and humoral (IgA and IgG2) immunodeficiencies; (6) elevated serum level of alphafetoprotein; (7) premature ageing; and (8) endocrine disorders, such as insulin-resistant diabetes mellitus. A DNA processing or repair protein is the suspected common denominator in this pathology. Heterozygotes are generally healthy; however, the sensitivity of their cultured cells to ionizing radiation is intermediate between normal individuals and that of affected homozygotes. Furthermore, heterozygous females are at an increased risk of breast cancer. These findings, when coupled with an estimated carrier frequency of 0.5-5.0%, suggest that (1) as many as one in five women with breast cancer may carry the AT gene and that (2) the increased radiation sensitivity of AT heterozygotes may be causing radiation therapists to reduce the doses of radiation used for treating cancer in all patients. To identify the genetic defect responsible for this multifaceted disorder, and to provide effective carrier detection, we performed a genetic linkage analysis of 31 families with AT-affected members. This has allowed us to localize a gene for AT to chromosomal region 11q22-23.

The purpose of this study is to better understand the roles of the p53 tumor suppressor protein and the product of the p53-regulated gene p21 WAF1 in the response of diploid human dermal ®broblast cultures to 254 nm ultraviolet (UV) light. We report that Li ± Fraumeni syndrome (LFS) ®broblast strains heterozygous for TP53 mutation at either codon 245 or 234 exhibit markedly reduced or no expression of p21 WAF1 following UV irradiation, respectively. These strains also exhibit defective nucleotide excision repair and pronounced inhibition of RNA synthesis following UV exposure, both of which are molecular hallmarks of cells derived from patients with the UV-sensitive syndrome xeroderma pigmentosum. In sharp contrast to xeroderma pigmentosum cells, however, the repair-de®cient LFS cells show abnormal resistance, rather than hypersensitivity, to the killing eect of UV light. We further demonstrate that exposure of normal human ®broblasts to biologically relevant¯uences (415 J/m 2 ) of UV does not induce apoptotic cell death, indicating that UV resistant phenotype displayed by LFS strains is not associated with deregulated apoptosis. In normal ®broblasts, such treatment results in a moderate (*threefold) up-regulation of p53 protein, induction of the p21 WAF1 gene, and a senescence-like growth arrest. On the other hand, exposure to 520 J/m 2 UV results in a striking up-regulation of p53, inhibition of p21 WAF1 expression, and activation of an apoptotic pathway. We conclude that: (i) p21 WAF1-mediated senescence is the principal mode of cell death induced by 415 J/m 2 UV light in normal human ®broblasts; (ii) there is a threshold eect for p53-dependent apoptosis and that, in normal human cells, this threshold level is induced upon expsoure to *20 J/m 2 UV; (iii) the p53 signaling pathway is malfunctional in the TP53 heterozygous LFS strains examined; and (iv) the enhanced resistance to UV-induced cell killing displayed by these LFS strains is a consequence of diminished growth arrest, which is presumably mediated by p21 WAF1 and not abnormalities in an apoptotic pathway.

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