Effects Of Differentiation-inducing Reagents And Gene Transfection Of Heterologous Human DNA Polymerase β On Cell Biological Characteristcs

With study on mechanism of oncogenesis, the important role of maintaining genome stability played by DNA repair enzymes has been considered more and more. The relationship between anomaly of the DNA repair enzymes and oncogenesis has been concerned, which has become a hot spot for research on oncogenesis.Base excision repair enzyme is one of DNA repair enzymes. It can remove 2-6 nucleotide damage and AP sites (apurinic/apyrimidine site) induced by exogenous and endogenous factors. DNA polymerase B (pol B ) is the key enzyme for single base excision repair. The pol B widely exists in both prokaryotes and eukaryotes, and is one with the smallest molecular weight among homologous enzymes, and processes high conservative nature among species. The activity of pol B is very low and independent with the cell cycle change. Besides, the DNA pol B can fill the nick of short patch (one nt) and maintain the genome stability, and it yet can involve DNA replication, recombination and drug resistance in cancer cells. The mutation and deregulation of pol B expression could initiate oncogenesis. Multiple mutations of pol B gene in various kinds of tumor tissues/cell lines were found, overexpression of pol B in mammalian cells could induce genome instability and incidental to oncogenesis. However, the correlation of overexpression and mutation of pol B with the genome stability and the its effect on oncogenesis has been explored primarily and needs further studies. The cell line established with stable transfection of pol B genesis available for study, the cell line establishment of stable transfection with pol B gene was rarely reported and the related research mainly focused on the mouse pol B abroad. The associated report with the cell line establishment of stable transfection with heterologous human pol B genes has not yet been reported.The constructed eukaryotic expression vectors carrying respective wild type and the mutant type DNA polymerase B were transfected into the mammalian cell and their biological effects were observed primarily. It will be a basis for further research on the relationship between the damage- repair from the DNA pol B mutation and the oncogenesis.The deregulation of differentiation and proliferation may result in carcinogenesis. The cell proliferation / differentiation is controlled by multiple genes regulated in multiple steps affected by various factors.Induction of differentiation refers to a reversible process of the malignant cancer cells induced through exogenous and endogenous differentiation reagents towards the normal. Differentiation-induction and its therapy for cancer disease, as a new strategy, had been studied actively in recent years.During the process of differentiation-induction, various kinds of oncogenes and antioncogenes may be altered correspondingly. By using all-trans retinoic acid( ATRA) and dimethyl sulfoxide( DMSO) as differentiation-inducing reagents the DNA polymerase B gene expression and its associated function were explored in human esophageal cancer 109 cells.This research contents can be divided into two parts:1. The effects on the human Eca-109 cell differentiation induced by all-trans retinoic acid(ATRA) and dimethyl sulfoxide(DMSO) and associated with the expression and function of DNA polymerase B .2. Establishment and identification of cell lines stably transfected with heterologous DNA polymerase B gene and the biological effects on NIH3T3 cells.Methods:1. The Eca-109 cell line treated with differentiation reagents and the NIH-3T3cell line stably transfected with pcDNA3.1-pol B -PM3 and pcDNA3.1-wtpol B and their corresponding control cell lines were established respectively. 2. The growth curve of the pol B gene transfected into NIH3T3 cells was drawn by the MTT assay or trypan blue exclusion assay. 3. The Biotin-labeled wtp53 cDNA probe was prepared by random primer method. The Bio-labeled wtpol B antisense RNA probe was prepared by transcription in vitro. The transcription in vitro was carried out by addition of SP6 RNA polymera...