With the aim to devise a long-term gene therapy protocol survival, unscheduled DNA synthesis and recovery of RNA for skin cancers in individuals affected by the inherited synthesis, and Western blots. The results show that the autosomal recessive xeroderma pigmentosum, we transrecombinant retroviruses are highly efficient vectors to ferred the human DNA repair XPA, XPB/ERCC3 and XPC transfer and stably express the human DNA repair genes cDNAs, by using the recombinant retroviral vector LXSN, in XP cells and correct the defect of DNA repair of group into primary and immortalized fibroblasts obtained from two A, B and C. With our previous results with XPD/ERCC2, XP-A, one XP-B (associated with Cockayne's syndrome) the present work extends further promising issues for the and two XP-C patients. After transduction, the complete gene therapy strategy for most patients suffering from this correction of DNA repair deficiency and functional cancer-prone syndrome. expression of the transgenes were monitored by UV
Cells from patients with xeroderma pigmentosum complementation group D (XP-D) and most patients with trichothiodystrophy (TTD) are deficient in excision repair of ultraviolet (UV) radiation-induced DNA damage. Although in both syndromes this defect is based on mutations in the same gene, XPD, only XP-D, not TTD, individuals have an increased risk of skin cancer. Since the reduction in DNA repair capacity is similar in XP-D and TTD patients, it cannot account for the difference in skin cancer risk. The features of XP-D and TTD might therefore be attributable to differences in the immune response following UV-irradiation, a factor which is presumed to be important for photocarcinogenesis. Sunlight-induced skin cancer represents the most prevalent malignancy in the Caucasian population, and its incidence is increasing (1). The pathogenesis of photocarcinogenesis is complex and only partially understood. Sunlight is a complete carcinogen, and it is generally accepted that ultraviolet B (UVB; 290-320 nm) radiation-induced DNA mutations constitute the initiation event for the generation of malignant skin cells (2). Studies in animals, however, provide evidence for a second mechanism for the development of clinically apparent skin cancer following UVB radiation exposure. In these studies, UVB radiation at subcarcinogenic doses was found to inhibit the surveillance function of the skin immune system directed against UVB radiation-induced skin tumors (3, 4). The importance of this second mechanism for photocarcinogenesis was demonstrated in tumor transplantation studies in mice, which cannot be carried out in humans. The evidence for a role of UVB radiation-induced immunosuppression in human skin cancer is therefore inevitably circumstantial and includes the observation that immunosuppressed humans who have received renal transplants have an increased frequency of sunlight-induced skin cancers (5-8).Here we take advantage of two human syndromes associated with defects in excision repair of UV-induced DNA lesions, namely xeroderma pigmentosum complementation group D (XP-D) and trichothiodystrophy (TTD) (9-11). Cells derived from XP-D and the majority of TTD patients have a defect in nucleotide excision repair (12, 13), which results from mutations in the same gene (XPD) (refs. 14-16 and unpublished results of B. C. Broughton and A.R.L.). Despite this similarity and a similar frequency of UV-induced mutations (17), only XP-D patients have an increased risk of developing skin cancer (10, 11). Since the increased risk of skin cancer in XP-D, as compared with TTD patients, cannot be explained simply by differences in DNA repair capacity, we hypothesized that XP-D and TTD cells might differ in some aspect of the immune response after UVB radiation.To test this hypothesis, the capacity of UVB radiation to suppress transcriptional expression of the intercellular adhesion molecule 1 (ICAM-1) was assessed in comparative studies employing XP-D and TTD cells. ICAM-1 serves as a ligand for leukocyte function-associated antige...
Nucleotide excision repair (NER)-deficient human cells have been assigned so far to a genetic complementation group by a somatic cell fusion assay and, more recently, by microinjection of cloned DNA repair genes. We describe a new technique, based on the host cell reactivation assay, for the rapid determination of the complementation group of NER-deficient xeroderma pigmentosum (XP), Cockayne's syndrome (CS) and photosensitive trichothiodystrophy (TTD) human cells by cotransfection of a UV-irradiated reporter plasmid with a second vector containing a cloned repair gene. Expression of the reporter gene, either chloramphenicol acetyltransferase (CAT) or luciferase, reflects the DNA repair ability restored by the introduction of the appropriate repair gene. All genetically characterized XP, CS and TTD/XP-D cells tested failed to express the UV-irradiated reporter gene, this reflecting their NER deficiency whereas cotransfection with the repair plasmid expressing a gene specific for the given complementation group increased the enzyme activity to the level reached by normal cells. Selective recovery of both reporter enzyme activities was observed after cotransfection with the XPC gene for the XP17VI cells and with the XPA gene for both XP18VI and XP19VI cells. Using this method, we assigned three new NER-deficient human cells obtained from patients presenting clinical symptoms described as classical XP to either XP group A (XP18VI and XP19VI) and XP group C (XP17VI). Therefore, this technique increases the range of methods now available to determine the complementation group of new NER deficient patients with the advantage, unlike the somatic cell fusion assay or the microinjection procedure, of being simple, rapid, and inexpensive.
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