Characterisation of Ercc1 deficiency in the liver and in conditional Ercc1-deficient primary hepatocytes in vitro.
The ERCC1/XPF complex is responsible for incision at the 5' side of the lesion during nucleotide excision repair and is also involved in homologous recombination and interstrand cross-link repair. The aim of the current study was to set up a better model for examination of Ercc1 deficiency in the murine liver and to determine the DNA lesions responsible for the premature polyploidy observed. We used the Cre/lox system with an adenovirus carrying Cre recombinase to conditionally induce Ercc1 deficiency in murine hepatocytes in vitro. Increased levels of apoptosis were apparent in our Ercc1-deficient cultures, both spontaneously and after UV irradiation and oxidative DNA damage. Increased apoptosis was also observed in simple Ercc1-deficient livers and the time course of the development of polyploidy was characterised. Livers from simple Ercc1 knockout mice contained mitochondria with disrupted outer membranes. Lipid accumulation was observed in older Ercc1-deficient hepatocyte cultures and in young Ercc1-deficient and wild-type livers. Lipids disappeared from the wild-type livers with age, but persisted in Ercc1-deficient livers, suggesting that a reduced ability to repair oxidative DNA damage and a malfunction of oxidative pathways could be responsible for the Ercc1-deficient liver phenotype. Real-time RT-PCR was used to determine differences in expression of cell cycle regulation and survival genes between Ercc1-deficient and control livers. Higher mRNA levels of Igfbp2, a possible marker for polyploidy, and p21 were detected in Ercc1-deficient livers. The pro-apoptotic factor, Bax, showed increased levels of mRNA expression in young Ercc1-deficient livers. However, no elevation in the levels of reactive oxygen species, or of malondialdehyde DNA adducts, a product of oxidative DNA damage, were found in Ercc1-deficient liver and no elevated levels of genes involved in the oxidative damage response were seen.
Kirschner
Kristina
K
Sir Alastair Currie Cancer Research UK Laboratories, Molecular Medicine Centre, University of Edinburgh, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, Scotland, UK.
Singh
Rajinder
R
Prost
Sandrine
S
Melton
David W
DW
D000818:Animals; D017209:Apoptosis / Q000235:genetics; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D018736:DNA Adducts / Q000378:metabolism; D004249:DNA Damage / Q000528:radiation effects; D004268:DNA-Binding Proteins / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism; D004720:Endonucleases / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism; D005949:Glucose Oxidase / Q000008:administration & dosage; D022781:Hepatocytes / Q000378:metabolism* / Q000648:ultrastructure; D019426:Integrases / Q000378:metabolism; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008315:Malondialdehyde / Q000378:metabolism; D008563:Membrane Lipids / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D008928:Mitochondria / Q000378:metabolism; D011123:Polyploidy; D017382:Reactive Oxygen Species / Q000378:metabolism; D014466:Ultraviolet Rays
D018736:DNA Adducts; D004268:DNA-Binding Proteins; D008563:Membrane Lipids; D017382:Reactive Oxygen Species; D008315:Malondialdehyde; D005949:Glucose Oxidase; C045073:Cre recombinase; D019426:Integrases; D004720:Endonucleases; C499360:Ercc1 protein, mouse