Chemical-induced Cancer Incidence And Underlying Mechanisms In Fen1 Mutant Mice

A critical observation in sporadic cancers is that not all individuals are equally prone to developing cancer following exposure to a given environmental carcinogen. Epidemiological studies have suggested that the difference in the timing of cancer onset in response to exogenous DNA damage is likely attributable to genetic variations, such as those associated with base excision repair (BER) genes. To test this long-standing hypothesis and elucidate how a genetic variation in the BER gene flap endonuclease 1 (FEN1) results in susceptibility to environment insults and causes cancer, we used the pre-exist mouse model to demonstrate the mechanism. Here, we addressed the questions through several levels, including molecular, cellular and mouse model et.al.1,We established a point-mutations pool of human FEN1 and then optimized the purification protocol for those recombination proteins. We mutated all the amino acids of FEN1 into Alanine by Quick-Change mutagnesis, stored the mutated plasmids and the transformed protein expression strains. Meanwhile, we optimized the protein purification protocol by using FPLC system and obtained extremely-pure FEN1 proteins, which can be used for a bundle of biochemistry assays in the future studies.2,We demonstrated that the E160D mutation impairs the ability of FEN1 to process DNA intermediate structures in long-patch BER;The LP-BER activity is decreased using nuclear extracts or reconstituted purified BER proteins by in vitro biochemistry assays.3,We also observed the phenotypes after chemical induction in cellular level. E160D cells were more sensitive to the base-damaging agents methylnitrosourea (MNU) and hydrogen peroxide (H2O2), leading to DNA strand breaks, chromosomal breakage and chromosome instabilities in response to these DNA insults.4,We further show that E160D mice are more susceptible to exposure to methylnitrosourea and develop lung adenocarcinoma. Thus, our current study demonstrates that a subtle genetic variation (E160D) in BER genes (FEN1) may cause a functional deficiency in repairing base damage, such that individuals carrying the mutation or similar mutations are predisposed to chemical-induced cancer development.