The Construction Of Expression Vector Of Fas/RAR Alpha Fusion Gene Containing Retinoic Acid Response Elemennt, And Its Effect Induced By Retinoic Acid On The Apoptosis Of Tumor Cells

Retinoic acid (RA) plays an important role in normal cell growth and tissue development. Because of its activity on acute promyelocytic leukemia (APL) cells RA has been used for clinical differentiative therapy from 1980s. And RA can produce a high remission rate in APL patients by its anti-proliferative action and inducing differentiation. All of these functions are mediated through its specific nuclear receptors, i.e., RARs and RXRs. These activated nuclear receptors, in turn, bind to specific DNA sequences called as retinoic acid response element (RARE) that are located in the regulatory portions of genes and thus modulate gene activity. But now there are occasionally early tolerance to RA, relapse of the disease and retinoic acid syndrome which limit the application of RA during the period of RA treatment. Several studies in vitro indicated that resistance of RA therapy was closely related to RAR gene mutation or translocation which produced fusion proteins such as PML-RAR alpha or PLZF-RAR alpha, as well as up-regulation of metabolic enzyme of RA. So it is necessary to take optimal strategies to overcome the resistance to RA and decrease ill effects in clinical treatment.Apoptosis, or programmed cell death, is essential for organogenesis during development, for proper function of the immune system. And apoptosis is also the main response of cells to chemotherapeutic agents. ATRA combined with chemotherapy can significantly induce apoptosis of malignant cells. It is a good idea to directly trigger cell apoptosis in order to overcome resistance of RA during tumor therapy. Apoptosis is a highly regulated process controlled byIVnumerous genes that determine a proper response to death signals. The relative levels of expression of pro- and anti- apoptosis genes appear to be particularly important. Studies have demonstrated that there are two major cell-intrinsic pathways for inducing apoptosis, one that begins with ligation of cell surface death receptors and another that involves mitochondrial release of cytochrome C. The Fas antigen(APO-l/CD95) is one of the most important death receptors. Ligation of Fas by FasL or a cross-linking antibody results in receptor trimerization followed by binding of the adapter molecule FADD to the cytoplasmic domain (death domain, DD) of the Fas. Then activated FADD mediates the caspases or transcription factors and finally induces cell apoptosis. It is well known that RAR alpha has the similar character of trimerization during binding to RA. So, in this paper we constructed a novel expression vector of fusion gene Fas/RAR alpha containing RARE-TK promoter and reporter gene eGFP controlled by ATRA and observed the effect of apoptosis initiated by fusion protein Fas/RAR alpha after transfection to HL-60 and MCF-7 malignant cells.At present gene therapy targeted at tumor suppressor genes, oncogenes, or central signaling molecules, as well as "suicide gene" is reported in several studies in vivo and in vitro. And cancer has become by far the most important indication for gene therapy in clinical trials. Exogenous fusion gene-Fas/RAR alpha dependent on RA maybe significantly cause apoptosis of tumor cells which have low Fas expression. Application of fusion gene is one kind of important gene therapy methods. It is a key to construct an optimal expression vector of fusion gene and must follow these rules: 1) all of cDNA fragments of fusion gene in the same open-reading frame; 2) active sites of each cDNA independent and respective; 3) a high-copy expression vector or plasmid. The reporter gene enhanced green fluorescent protein (eGFP) has an extensiveapplication of location and trace of novel gene or protein. In this research eGFPvcan trace the Fas/RAR alpha fusion protein and directly reflect changes of fusion protein.In order to verify the model of expression vector induced by RA, at first we constructed a vector engineered to incorporate eGFP under the RARE-TK promoter in this paper. Results showed that the reporter gene was up-regulated induce...