Retinoic Acid Regulation Of Ifn¦Á Expression Vector And Its Impact On Tumor Cell Apoptosis

Retinoids are derived from vitamin A, which play an essential role in normal cell growth and differentiation. Retinoic acid (RA), particularly all- trans retinoic acid (ATRA), has been regarded as one of the most suitable agents for differentiative therapy because of its activity on acute promyelocytic leukemia(APL) cells at relatively low concentrations,which can be reached in vivo without major toxicities. Indeed, several clinical trials have reported ATRA efficacy in inducing complete remission in APL patients. Retinoids mediate their antiproliferative action, as well as their ability to induce differentiation and apoptosis through their binding and activation of specific nuclear receptors, ie, RARs or 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. Considerable previous studies have demonstrated that retinoids are promising as chemotherapeutic agents for the prevention and treatment of several types of cancer. Nevertheless, the recurrence after RA therapy and/or development of resistance to RA therapy have often been observed in patients. Therefore, it is necessary to take optimal strategies to overcome the clinical resistance to RA. One of the major molecular mechanisms of clinical resistance to RA is up-regulation of metabolic enzyme of RA in cells stimulated with RA. In our previous study, as well as in papers from other groups, evidence was reported that the expression and activity of metabolic enzyme of RA could be inhibited V by interferons (IFNs). Numerous studies have demonstrated differentiation and/or apoptosis induction and growth inhibition in human cancer cells by treatment with retinoids and IFNs. The combination of both compounds, which probably act through different molecular mechanisms, often results in a synergistic amplification. They synergistically inhibited growth and induced differentiation and apoptosis in several types of cancer, including hematological malignancies and solid tumors. IFN a even could restore responsiveness of a subline of HL-60, which was otherwise resistant to the effects of RA. Although the combination of retinoids and lENs has shown promising results in preclinical studies and clinical trials, the major toxicities were not neglectable, and the median duration of response was less than 20 weeks. Thus, it is important and necessary to explore optimal strategies to relieve the side effects and enhance the response. In order to relieve the side effects of combination of RA and LEN a, firstly,the possibility of the expression of exogenous lEN a gene mediated by RA through RARE was investigated in this paper. An IFN a gene eukaryotic expression vector containing four copies of RARE (pRARB4-IFN a ) was constructed with recombinant DNA technique, identificated with restriction endonuclease digest and PCR and DNA sequence analysis, then transfected into the cells of HL-60,Bcap-37 and MCF-7 with liposome DOTAP, the expression of LEN a gene was analyzed in transfected and nontransfected cells stimulated with RA by RT-PCR and ELISA. Secondly, the synergistic effects of RA and the transfection of pRARE4-IFN a on the proliferation and apoptosis of tumor cells and its possible molecular mechanisms were study. After tested that the expression of lEN a in pRARE4-IFN a transfected cells was...