Mechanism of cell cycle arrest in the ERCC1-deficient mouse model

ERCC1 is a gene essential for the repair process of UV—induced DNA damage known as Nucleotide Excision Repair (NER). Deficiencies in this DNA repair mechanism have been associated with the skin cancer prone human syndrome Xeroderma Pigmentosum (XP) and also with Cockayne's Syndrome (CS) and Trichothiodistrophy (TTD). The main role of RERCC1 is to act as a NER endonuclease but it has also been proposed that it may have a role in recombination. The ERCC1 knock out (k/o) mouse model was first described by McWhir et al. in 1993 . McWhir and co-workers described ERCC 1—deficient mice as being severely runted, dying before weaning at approximately three weeks of age from hepatic failure, having severe aneuploidy and polyploidy in their liver and presenting elevated levels of p53 in this tissue. This k/o model shared no similarities with any of the already known NER deficiency—associated syndromes which led to the theory that a deficiency in the other non—NER ERCC1 function(s) may be responsible for the observed differences. The aim of this project was to gain a better understanding of the precise mechanisms behind this atypical phenotype. Indeed, studies carried out on both liver tissue and primary fibroblast material have resulted in the development of a theory for the ERCC1 k/o phenotype. ERCC1—deficient and wild type primary fibroblasts showed the same response to UV damage in terms of DNA replication arrest after UV. This behaviour, however, differed significantly from that observed in human cells. ERCC1—deficient cells were shown both to induce p53 with lower UV doses than their wild type counterparts and to induce the cyclin—dependent kinase inhibitor, p21, in response to UV in a p53—independent manner. From the liver tissue studies a number of conclusions can be derived which are outlined below. Levels of p21 transcript were also shown to be elevated in ERCC1 deficient livers and this increase of p21 was p53—independent. In order to get a more accurate picture of the cell cycle status in these hepatocytes the DNA synthesis levels as measured by the BrdU incorporation rates and cycling status were studied. Hepatocytes of ERCC1—deficient animals were shown to have reduced levels of DNA synthesis when compared to their wild type counterparts and, through the combination of BrdU, Ki67 and centromeric staining, it was also concluded that the most likely explanation for the aneuploidy and polyploidy we see in these livers is that these cells are arrested in G2. Another point that emerged when morphometric studies were carried out in wild type, ERCC1-/-, p53-/and p53/ERCC1 double null livers, was that binucleation is repressed in the ERCC1 k/o hepatocytes. Although the absence of p53 does not rescue the liver phenotype that is seen in the ERCC1 k/o mice, it results in the bypass of the repression of binucleation , as shown by wild type levels of binucleate hepatocytes in ERCC1/p53 double null livers. When all this data is taken together it is possible to think of a likely explanation for the observed ERCC1 k/o phenotype. Briefly, ERCC 1—deficient livers are subject to 1 McWhir, J. and Selfridge, J. and Harrison, D.J. and Squires, S. and Melton, D.W. (1993) Mice with DNA-repair gene (ERCC1) deficiency have elevated levels of p53, liver nuclear abnormalities and die before weaning. Nature Genetics 5:217-224 endogenous damage that cannot be repaired, this damage results in the activation of two separate pathways, one mediated by p53 and one mediated by p21. Severe, accelerated hepatocyte polyploidisation as a response to the existing endogenous damage results in the observed prematurely aged appearance of the liver. This together with an impairment of liver growth, probably mediated by p2l(as shown by elevated levels of hepatocytes arrested in G2), results in early hepatic failure and the observed premature death. No human syndrome has yet been identified as being caused by a defect in ERCC1, so a subsidiary aim of this project was to see if any human condition could be attributed to an ERRC1 deficiency. Taking as a starting point the prematurely aged appearance of the ERCC1 k/o mice, a rare human premature ageing syndrome called HutchinsonGilford progeria (HGP) was selected as a possible candidate for an ERCC1-related human condition. Primary fibroblasts derived from a HGP patient showed decreased levels of ERCC1 protein, but no evidence for mutations in the ERCC1 gene was found. However, an impaired p53 response to UV—irradiation was observed. Overall, this work has opened the doors for the study of premature ageing in humans in relationship to NER and p53.