Reference #: LI-1017-954576
Submit Date: 04/04/2002 14:05:49-0500

P53 and ING1: Enhancing the repair of UV-damaged DNA to suppress skin cancer development

ABSTRACT:
The tumor suppressor, p53, is recognized as a crucial molecule in regulating cellular responses to various DNA-damaging events, including UV irradiation. P53-knockout mice developed significantly more skin tumors with reduced latent period compared to control wild-type mice. One of the important biological functions of p53 is to enhance the repair of UV-damaged DNA. Using the p53 transgenic mouse models, we demonstrated that loss of p53 significantly impairs the normal nucleotide excision repair process in murine keratinocytes. In addition, it appears that p53 enhancement in DNA repair occurs in basal, but not in differentiated keratinocytes. Recent studies indicated that p53 cooperates with the novel tumor suppressor ING1 to exert its biological functions. We hypothesized that p33^ING1 may also be involved in nucleotide excision repair after UV irradiation. We found that overexpression of p33^ING1 significantly enhanced UV-damaged DNA in human melanoma cells and this enhancement requires the presence of functional p53. Furthermore, p33^ING1 physically binds to the p53 downstream target GADD45, which is known to play a role in nucleotide excision repair. Taken together, our data indicate that p53 cooperates with p33^ING1 to enhance the repair of UV-damaged DNA to maintain genomic stability and suppress skin cancer development.

Keywords: p53, ING1, DNA repair, skin cancer

Reference #: FOR-1016-090945
Submit Date: 03/14/2002 01:05:09-0500

Mechanism of p53 and BRCA1 tumor suppressor genes in regulating nucleotide excision repair

ABSTRACT:
The p53 and BRCA1 tumor suppressor genes are involved in multiple aspects of cellular physiology, including DNA repair, and result in cancer when mutated. The p53 gene product is a critical mediator of the cellular response to DNA damage. Its function as a tumor suppressor is due to its role as a transcription factor regulating expression of genes involved in DNA damage response pathways and affecting such biological processes as apoptosis, cell cycle regulation and DNA repair. We have shown that wild-type p53 is required for proficient mammalian nucleotide excision repair (NER) of UV-irradiation induced DNA damage, and that p53 dysfunction or loss results in a DNA repair defective phenotype, specifically affecting the global genomic NER pathway. The dependence of global genomic NER, but not transcription-coupled NER, on p53 function suggests that the mechanism involves those NER enzymes specifically required for GGR. We have identified the DNA damage recognition factors p48 (protein product of the DDB2 gene that is mutated in individuals with xeroderma pigmentosum group E) and XPC as DNA damage inducible in a p53-dependent manner. Both genes contain consensus p53 responsive regulatory elements in either their promoter (p48) or intronic sequence (XPC). Both p48 and XPC localize within minutes of UV-irradiation to sites of DNA damage in vivo, and this does not require the presence of wild-type p53. Furthermore, p53 protein itself does not co-localize to DNA damage sites in cells. Stable ectopic expression of a p48 cDNA results in complementation of the NER defect associated with p53 deficiency in human cells, demonstrating that efficient NER does not require the presence of wild-type p53 protein, but can be restored through expression of damage-inducible, p53-regulated downstream genes. Recent work has also shown that overexpression of the BRCA1 breast cancer susceptibility gene enhances NER and induces expression of p48, XPC and GADD45, all independent of p53. Impaired NER due to loss of BRCA1 and p53 may play a crucial role in the pathogenesis of human cancers in general, and BRCA1-associated breast cancers in particular.

Keywords: p53, BRCA1, nucleotide excision repair, UV-irradiation

Reference #: 095258
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Reference #: SCU-1019-161789
Submit Date: 04/18/2002 15:19:50-0500

Recombination Functions of BRCA1

ABSTRACT:
The hereditary breast and ovarian cancer susceptibility gene, BRCA1, is known to function in the maintenance of genomic integrity. BRCA1 controls homologous recombination and double strand break repair (DSBR) through interactions with the recombination protein, Rad51, the Rad50/NBS1/MRE11 complex and BRCA2. Our previous work showed that wild-type but not mutant alleles of BRCA1 can restore efficient DSBR to a human breast cancer cell line, HCC1937, which lacks endogenous wild-type BRCA1. This indicates that BRCA1 performs a tumor suppressor function in controlling DSBR. Parallel work identified a specific role for BRCA1 in homologous recombination but not in the second major DSBR pathway, non-homologous end-joining. However, we do not yet know how such a function is applied during the somatic cell cycle. We found previously that BRCA1 acts in an S phase-specific DNA damage response pathway, and deduced that BRCA1 may control recombination between sister chromatids in S phase, for example when a DNA polymerase complex stalls on abnormal DNA structure. This idea is reinforced by the finding that BRCA1 is recruited to sites of DNA synthesis in the context of this replication arrest response. In an effort to test this hypothesis, we have developed a new assay for sister chromatid recombination. We are using this to further examine the tumor suppressor functions of BRCA1. This assay may also be of general use in determining the relationship between carcinogen exposure and cancer.

Keywords: breast cancer, BRCA1, recombination

Reference #: DRO-1017-786910
Submit Date: 04/02/2002 16:10:16-0500

Modulation of the DNA damage response in UV-exposed human cells through genetic- vs. functional-inactivation of the p53 tumor suppressor

ABSTRACT:
During periods of genotoxic stress, the p53 tumour suppressor guards against neoplastic transformation through transactivation of target genes, and through protein-protein interactions, that initiate apoptosis, G1 arrest, and nucleotide excision repair (NER). Moreover certain viral gene products, including the human papillomavirus E6 protein (HPV-E6) and the hepatitis B virus X protein (HBx), ostensibly favour carcinogenesis by binding and functionally inactivating p53. However the precise role of p53, and of viral oncoproteins that bind p53, in regulating various protective processes following exposure of cultured human cells to agents that produce bulky (NER-dependent) adducts in DNA, such as UV-induced cyclobutane pyrimidine dimers (CPD), is still not entirely clear. To address this issue, the cellular response to UV-induced DNA damage has been analyzed in the p53-proficient human lymphoblastoid strain TK6 vs. three isogenic p53-deficient derivatives: (i) NH32, carrying a homozygous knockout of p53; (ii) TK6-5E, expressing HPV-E6; and (iii) TK6-HBx, expressing HBx. For each strain, various critical endpoints relevant to the cellular DNA damage response were carefully quantified following UV exposure, including clonogenic survival, apoptosis, G1-S phase progression, and mutagenic specificity at the endogenous hprt locus. In addition, the ligation-mediated PCR technique was employed to quantify the rate of repair for UV-induced CPDs at nucleotide resolution along the transcribed- vs. the nontranscribed strands of different chromosomal gene targets. Using an isogenic human model system, our data provide strong evidence that, following UV exposure, functional p53 is absolutely required for efficent apoptosis and NER (i.e., both global and transcription-coupled subpathways), but plays no role whatsoever in the initiation of G1 arrest.

Keywords: p53 tumour suppressor, UV-induced DNA damage, human papillomavirus E6 protein, hepatitis B virus X protein

Reference #: SAR-1017-132719
Submit Date: 03/26/2002 02:29:29-0500

Activation of Some Signaling Pathways in Skin Tumours from DNA Repair Deficient Xeroderma Pigmentosum Patients

ABSTRACT:
Sun is the most important genotoxic agent involved in skin cancers. The UVB part of the solar spectrum induces DNA lesions at bipyrimidic sites, which can give rise to mutations in the absence of efficient error-free repair. Xeroderma pigmentosum (XP) is a human syndrome transmitted as an autosomal, recessive trait, characterized by early and numerous cutaneous tumours on sun-exposed sites. These patients are defective in Nucleotide Excision Repair which is the major pathway for removing bulky DNA adducts. Cultured XP cells are hypermutable after UV-irradiation. This increased mutation frequency has also been found in key genes, governing signaling pathways implicated in the regulation of cell cycle, in XP skin cancers. A high level of base pair substitutions was detected in the PTCH and the p16 INK4a-ARF locus tumour suppressor genes. Seventy to 80 % of XP skin cancers exhibit one or several mutations in these genes, the type and frequency of mutated genes being different between squamous cell (SCC) or basal cell carcinomas (BCC). The molecular analysis of these mutations reveals specific characteristics which clearly indicate they were produced by unrepaired UV- induced DNA lesions. Most mutations are base pair substitutions, contrary to what is found with the genetically-transmitted deletions found in the PTCH or p16 genes in the Gorlin's and familial melanoma syndromes respectively. These mutations are targeted at pyrimidine-pyrimidine sites which constitute hot spots of UV-induced DNA lesions and correspond essentially to C to T or CC to TT tandem transversions. In XP skin cancers, the CC to TT tandem mutations represent about 60 % of total mutations compared to 12 % in skin tumours from DNA repair-proficient patients. In XP-C patients all base pair substitutions were due to translesion synthesis of unrepaired damage remaining only on the nontranscribed strand of the gene. This result demonstrates the existence of preferential repair in vivo in humans. Activation of these pathways can seriously alter the regulation of cell cycle, particularly the checkpoints activated after genotoxic damage, allowing therefore a damaged cell to replicate and give rise to mutated daughter cells and eventually to originate the carcinogenic process.

Keywords: DNA repair, Ultra-Violet, Skin Cancer, mutagenesis