| Genetic instability is an important factor in the genesis of both cancers and human hereditary diseases. The decreasing of DNA replication fidelity, or damaging of DNA repair system, or the dysfunction of cell cycle DNA damage checkpoint could play an important role in the development of genetic instability..Cells exposed to environmental physical or chemical carcinogens (e.g. ionizing radiation or alklylating agents) also can develop prolonged genetic instability. Previous report from this laboratory proved that alkylating agents, N-methyl-N'-nitro-N- nitrosoguanidine (MNNG) could induce genetic instability in vero cells, which has characteristic manifestation of delayed developed mutagenesis happened at undamaged base sites of DNA (nontargeted mutation). The vero cells were treated with 0.2 u mol/L MNNG for 2.5hr, then a intact shuttle plasmid pZ189 carrying sup.FtRNA gene was tranfected into vero cells after 12hr culture. A higher mutation frequency of the plasmid replicated in vero cells pretreated with MNNG was found, compared with the spontaneous mutation frequency of the plasmid replicated in control cells. Experiments demonstrated that nontargeted mutation had distinct mutation spectrum and showed phase characteristic. Study also indicated that the decaeasing of DNA fidelity might be involved in the genesis of nontargeted mutagenesis, and the change of expression level of DNA polymerase such as polymerase P may be one of the involed mechanisms. Further study revealed that the activating the signaling pathway and modulating of gene expression were very important events in the formation of nontargeted mutation.Structure-specific nuclease FEN-1 (flap endo/exonuclease-1) plays important roles in the DNA replication, DNA repair and the maintenance of the cellular genetic stability. In the process of DNA replication, FEN-1 cleaves the RNA primer attached to an Okazaki fragments during lagging strand synthesis; In DNA repair, it is showed that FEN-1 was involved in the long patch pathway of base excision repair, some pathway of DNA double strand break repair and UV damage repair. In yeast S. cerevisiae, the rad 27 (homolog gene of human FEN-1) mutant showed a strong mutator phenotype, and the majority of the resulting mutation were duplication mutation at Canr and Lys genes. . It was also reported the existence of microsatellite and minsatellite instability in the rad27 mutant. So, FEN-1 is an important gene related with the genesis of genetic instability.In order to study the function of FEN-1 in DNA replication and in the maintenance ofcell genetic stability in human cells, and to search the possible functional relationship in the development of genetic instability between the blocking of FEN-1 gene and nontargeted mutation, in this work a mammalian expression vector expressing antisense FEN-1 gene fragment pMAMneoAmp~FNB~ was constructed after the the Ncol-BamHl fragment of FEN-1 gene was inserted into the mammalian expresion vector pMAMneoAmp- in antisense oritation. After FL cell was transfected with pMAMneoAmp-FNB- and selected by G418, the FL-FEN-1 - Cell, in which the FEN-1 gene expresion was blocked was established. It was verified that there was FEN-1 antisense mRNA fragment expressed in cell line FL-FEN-1 - by a RT-PCR test, which indicates the block of FEN-1 gene expression.With the study of cell growth curve, it was found that the growth of FL-FEN-1 ~ was retarded after dexamethasone and its doubling time TD was 3.03 day, while the TD of FL and FL-M also induced with dexamethasone was 2.03 and 2.22 day respectively, and the TD of the FL-FEN-1"" cell without dexamethanion was 2.38day. The growth of FL-FEN-1 ~ was inhibited at the fourth day of culture, and and was persistently inhibited after then. In a flow cytometer analysis work,, it was also found that the population of S-phase of cell cycle was decreased as compared with the controls, and the cell cycle was delayed in the S-phase DNA synthesis process, and arrested in Gl phase at 12hr release from synchronization. At 24hr release...
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