Impaired Regulation of Tumor Suppressor p53 Caused by Mutations in the Xeroderma Pigmentosum DDB2 Gene: Mutual Regulatory Interactions between p48^DDB2 and p53

Abstract

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.Xeroderma pigmentosum (XP) is a rare hereditary disease with a high incidence of UV light-induced skin cancers (7). Of eight known XP groups, XP-E has unique clinical and cellular phenotypes. For example, XP-E patients suffer from skin cancers without typical xerosis, and cell strains from XP-E patients are not abnormally sensitive to UV and have normal levels of DNA repair synthesis in vivo (26,27). XP-E is caused by mutations in the DDB2 gene (26, 27, 37), which encodes the small subunit, p48 DDB2 , of a heterodimeric damage-specific DNA binding protein (DDB) that also contains p127 DDB1 (10). Expression of the DDB2 gene is perturbed by UV irradiation. In a human normal diploid fibroblast strain, IMR-90, after an initial reduction in DDB activity, there is a two-to fourfold increase in DDB2 mRNA levels 36 h after UV irradiation followed by a peaking of p48 DDB2 protein and DDB activity 48 h after irradiation (27, 37). Since nucleotide excision repair (NER) is all but complete 48 h after UV irradiation (13), and since NER reconstitution studies have shown that DDB is not required for NER in vitro (1, 35), the direct function, if any, of DDB in DNA repair is unclear. In XP-E cell strains, p48 DDB2 protein and DDB activity are undetectable after UV irradiation (except in strain XP82TO), yet DDB2 mRNA levels are abnormally high both before (2-to 4-fold) and after (11-to 37-fold) UV irradiation (27). However, the regulation of DDB2 induction after UV damage is poorly understood.The tumor suppressor protein p53 is important in cellular responses to DNA damage and mutations in the TP53 gene are frequently identified in human cancers (12,41,45). For example, 40 to 60% of skin cancers have mutations in TP53, suggesting that abnormal p53 function is crucial in perhaps a majority of human skin cancers (8,9,16). Activation of p53 in response to DNA damage occurs as a result of a rapid increase in its levels through stabilization of the protein and a number of posttranslational modifications. These changes allow p53 to mediate transcriptional activation of a number of genes (3,30,40). The CDKN1A (WAF1) and BAX genes, whose products trigger cell cycle arrest and apoptosis, respectively, are regulated in this manner (20,41).While the degradation of p53 is known to be regulated by MDM2-mediated ubiquitination and consequent targeting for prote...

Tumor-prone phenotype of the DDB2-deficient mice

“…Therefore, it is possible that, in the MEFs, XPC/HR23B can substitute for the damaged-DNA recognition function of DDB2. In addition, consistent with the previous observations (Itoh et al, 2003(Itoh et al, , 2004, we did not see any difference in the survival between the wild-type and the DDB2-deficient MEFs following UV irradiation (data not shown). Nevertheless, we think that the difference in the CPDpositive cells in the skin sections is consistent with the UV-induced skin cancers observed in the DDB2-deficient mice.…”

“…Therefore, it is believed that the human DDB2 gene is a downstream mediator in the tumor suppression pathways of p53 and BRCA1. Recent studies provided evidence for a feedback loop in which DDB2 plays a role in the stabilization of p53 (Itoh et al, , 2003. The XP-E cells were shown to be somewhat impaired in activating the levels of p53 following UV irradiation, indicating a role of the DDB2 gene in the stabilization of p53 (Itoh et al, 2003).…”

“…Recent studies provided evidence for a feedback loop in which DDB2 plays a role in the stabilization of p53 (Itoh et al, , 2003. The XP-E cells were shown to be somewhat impaired in activating the levels of p53 following UV irradiation, indicating a role of the DDB2 gene in the stabilization of p53 (Itoh et al, 2003). DDB2 has been shown to associate with cullin 4A, an E3 ubiquitin ligase, which is likely to be involved in regulating the protein level of DDB2.…”

“…XP is a rare autosomal recessive disease characterized by sun-sensitivity and a high incidence of skin malignancies, and these phenotypes are believed to arise from a deficiency in the NER pathway of DNA repair. The DDB2 gene has been linked to XP-E because patients belonging to the XP-E group were shown to harbor mutations in the DDB2 gene (Nichols et al, , 2000Chu and Chang, 1988;Itoh et al, 2000Itoh et al, , 2003. Despite these studies indicating an involvement of DDB2 in DNA repair, it is not clear how DDB2 participates nucleotide excision repair (NER) because the NER assays work efficiently on naked DNA without DDB2 (Reardon et al, 1993;Kazantsev et al, 1996).…”

Impaired repair of cyclobutane pyrimidine dimers in human keratinocytes deficient in p53 and p63

“…The importance of NER in the response to solar ultraviolet radiation (UVR) is illustrated by the disease xeroderma pigmentosum in which mutations in certain NER proteins dramatically increase the risk of non-melanoma skin cancer due to impairment of the global genomic repair (GGR) subpathway of NER in which repair in non-transcribed DNA is defective. In many cell types, p53 has been shown to regulate GGR by transcriptionally activating certain genes, including XPC and DDB2 that encode key DNA damage recognition proteins (1)(2)(3). In several cell types, a p53 deficiency impairs GGR of certain DNA lesions, including the most common UVR-induced lesions, cyclobutane pyrimidine dimers (CPDs) (4).…”