Hanneke J. M. Kool

Enhanced DDB2 Expression Protects Mice from Carcinogenic Effects of Chronic UV-B Irradiation

UV-damaged DNA-binding protein (UV-DDB) is essential for global genome repair (GGR) of UV-induced cyclobutane pyrimidine dimers (CPD). Unlike human cells, rodent epidermal cells are deficient in GGR of CPDs and express a subunit of UV-DDB, DDB2, at a low level. In this study, we generated mice (K14-DDB2) ectopically expressing mouse DDB2 at elevated levels. Enhanced expression of DDB2 both delayed the onset of squamous cell carcinoma and decreased the number of tumors per mouse in chronically UV-B light-exposed hairless mice. Enhanced expression of DDB2 improved repair of both CPDs and pyrimidine(6-4)pyrimidone photoproducts (6-4PP) in dermal fibroblasts. However, GGR of CPDs in K14-DDB2 mice did not reach the level of efficiency of human cells, suggesting that another repair protein may become rate limiting when DDB2 is abundantly present. To complement these studies, we generated mice in which the DDB2 gene was disrupted. DDB2-/- and DDB2+/- mice were found to be hypersensitive to UV-induced skin carcinogenesis. On the cellular level, we detected a delay in the repair of 6-4PPs in DDB2-/- dermal fibroblasts. Neither the absence nor the enhanced expression of DDB2 affected the levels of UV-induced apoptosis in epidermal keratinocytes or cultured dermal fibroblasts. Our results show an important role for DDB2 in the protection against UV-induced cancer and indicate that this protection is most likely mediated by accelerating the repair of photolesions.

Age-dependent spontaneous mutagenesis in Xpc mice defective in nucleotide excision repair.

DNA damages caused by cellular metabolites and environmental agents induce mutations, that may predispose to cancer. Nucleotide excision repair (NER) is a major cellular defence mechanism acting on a variety of DNA lesions. Here, we show that spontaneous mutant frequencies at the Hprt gene increased 30-fold in T-lymphocytes of 1 year old Xpc−/− mice, possessing only functional transcription-coupled repair (TCR). Hprt mutant frequencies in Xpa−/− and Csb−/− mice that both have a defect in this NER subpathway, remained low during ageing. In contrast to current models, the elevated mutation rate in Xpc−/− mice does not lead to an increased tumour incidence or premature ageing.

Loss of heterozygosity in somatic cells of the mouse: An important step in cancer initiation?

Loss of heterozygosity (LOH) of tumour suppressor genes is a crucial step in the development of sporadic and hereditary cancer. Recently, we and others have developed mouse models in which the frequency and nature of LOH events at an autosomal locus can be elucidated in genetically stable normal somatic cells. In this paper, an overview is presented of recent studies in LOH-detecting mouse models. Molecular mechanisms that lead to LOH and the effects of genetic and environmental variables are discussed. The general finding that LOH of a marker gene occurs frequently in somatic cells of the mouse without deleterious effects on cell viability, suggests that also tumour suppressor genes are lost in similar frequencies. LOH of tumour suppressor genes may thus be an initiating event in cancer development.

Replication Factor C Recruits DNA Polymerase δ to Sites of Nucleotide Excision Repair but Is Not Required for PCNA Recruitment

ABSTRACT Nucleotide excision repair (NER) operates through coordinated assembly of repair factors into pre- and postincision complexes. The postincision step of NER includes gap-filling DNA synthesis and ligation. However, the exact composition of this NER-associated DNA synthesis complex in vivo and the dynamic interactions of the factors involved are not well understood. Using immunofluorescence, chromatin immunoprecipitation, and live-cell protein dynamic studies, we show that replication factor C (RFC) is implicated in postincision NER in mammalian cells. Small interfering RNA-mediated knockdown of RFC impairs upstream removal of UV lesions and abrogates the downstream recruitment of DNA polymerase delta. Unexpectedly, RFC appears dispensable for PCNA recruitment yet is required for the subsequent recruitment of DNA polymerases to PCNA, indicating that RFC is essential to stably load the polymerase clamp to start DNA repair synthesis at 3′ termini. The kinetic studies are consistent with a model in which RFC exchanges dynamically at sites of repair. However, its persistent localization at stalled NER complexes suggests that RFC remains targeted to the repair complex even after loading of PCNA. We speculate that RFC associates with the downstream 5′ phosphate after loading; such interaction would prevent possible signaling events initiated by the RFC-like Rad17 and may assist in unloading of PCNA.

Loss of heterozygosity in somatic cells of the mouse

Loss of heterozygosity (LOH) of tumour suppressor genes is a crucial step in the development of sporadic and hereditary cancer. Recently, we and others have developed mouse models in which the frequency and nature of LOH events at an autosomal locus can be elucidated in genetically stable normal somatic cells. In this paper, an overview is presented of recent studies in LOH-detecting mouse models. Molecular mechanisms that lead to LOH and the effects of genetic and environmental variables are discussed. The general finding that LOH of a marker gene occurs frequently in somatic cells of the mouse without deleterious effects on cell viability, suggests that also tumour suppressor genes are lost in similar frequencies. LOH of tumour suppressor genes may thus be an initiating event in cancer development.

Mutation Research FrontiersLoss of heterozygosity in somatic cells of the mouse: An important step in cancer initiation?

Loss of heterozygosity (LOH) of tumour suppressor genes is a crucial step in the development of sporadic and hereditary cancer. Recently, we and others have developed mouse models in which the frequency and nature of LOH events at an autosomal locus can be elucidated in genetically stable normal somatic cells. In this paper, an overview is presented of recent studies in LOH-detecting mouse models. Molecular mechanisms that lead to LOH and the effects of genetic and environmental variables are discussed. The general finding that LOH of a marker gene occurs frequently in somatic cells of the mouse without deleterious effects on cell viability, suggests that also tumour suppressor genes are lost in similar frequencies. LOH of tumour suppressor genes may thus be an initiating event in cancer development.