ODDD Controlled HSV1-tk Mediated Gene Directed Enzyme Prodrugs Therapy Strategy In Tumor Cells

Human solid tumors contain hypoxic regions that have considerably lower oxygen tension than normal tissues. These impart resistance to radiotherapy and anticancer chemotherapy, as well as predisposing to increased tumor metastases. To date, efforts to overcome the problem of hypoxia have had only limited success. However, the very low levels of oxygen and the presence of necrosis are unique features of solid tumors under normal physiological conditions they do not occur in normal tissues and so are potentially exploitable in cancer therapy. DNA vaccine targeted in hypoxia had been proposed and more than 200 items had been used in clinical research.A key limitation of present day gene therapy of cancer is the lack of specificity of the gene-delivery system. An alternative to direct targeting of tumors is to have the therapeutic gene transcribed or translated by a tumor-specific vector so that expression of a particular protein would be tumor specific. The transcription factor, HIF-1α, is an important mediator of the hypoxic response of tumor cells and controls the up-regulation of a number of factors vital for solid tumor expansion. The up-regulation of HIF-1αoccurs at the level of protein expression via changes in the half-life of protein in response to hypoxia .The ODDD (oxygen dependent degradation domain) of HIF-1αis located in its central region and consists of 200 amino acid residues. The ODDD controls the degradation of HIF-1αby the ubiquitin-proteasome pathway, and the deletion of this entire region is required for DNA binding and transactivation in the absence of hypoxic signals. The ODDD conferred oxygen-dependent stability to a fusion protein and the covalent attachment of Casp3 and ODDD548–603 allowed quick delivery and specific stabilization in a solid tumor.The purpose of our study is to develop a potentially therapeutic DNA vaccine highly specific for solid tumors. we constructed recombinant plasmid vector pEGFP-tk-ODDD and detect whether it would be selectively stabilized in hypoxic tumor cells and had the potency of specific killing effect in tumor and had not caused any obvious side effects.1. Luciferase reporter vectors for verifying the potential of ODDD responsive to hypoxia. Methods: To observe whether the expressions of the targeted gene controled by ODDD were sensitive to decreased cellular O2 concentration, we constructed three recombinant eukaryotic plasmids composed of parts of the ODDD of HIF-1αand EGFP as a luciferase reporter. The cDNA fragments encoding ODDD were amplified by RT-PCR using corresponding oligonucleotides as primers from cDNA prepared from total RNA of SMMC7721cells.The cDNA fragments encoding various lengths of the ODDD of human HIF-1αwere inserted pEGFP-c1 respectively and acquired the plasmids of pEGFP-ODDD401-603(consisting of the residues of HIF-1αfrom 401 to 603), pEGFP-ODDD557-574 , pEGFP-ODDD2(557-574) The reporter vectors then were transected intoφA cells as well as pEGFP-c1 with lipofectamineTM2000. TheφA cells with reporter vector were cultured in normoxic conditions and under hypoxia respectively. Hypoxia was achieved by incubating cells in a humidified environment at 37°C in CO2 incubators maintained at 94% N2, 5% CO2, and 1% O2. Relative EGFP levels of these cells measured by Fluorescence activated cell sorting (FACS) analysis and RT-PCR technique respectivelyResults: The integrity reporter vectors were verified by restriction enzyme digestion and sequencing and aligned to the expected sequences. The EGFP expression levels inφA cells transfected with pEGFP-ODDD401-603 in normoxic conditions were 6.04%, and were much lower than those of hypoxia sample 36.63% detected by FCM method(p<0.05), but there were no difference by RT-PCR method. Another two luciferase reporter vectors pEGFP-ODDD2(557-574)and pEGFP-ODDD557-574 also had a higher EGFP expression in hypoxic cells than in normoxic cells. The plasmid of pEGFP-ODDD2(557-574)had same responsive effect to hypoxia with plasmid of pEGFP-ODDD401-603 in some extent.Conclusion: Luciferase reporter vectors regulated by the region of ODDD were successfully constructed and were specifically expressed in hypoxia which makes it possible to further investigate ODDD potency of specific antitumor under hypoxia. The region of ODDD2(557-574)could be used for next research.2. ODDD controlled HSV1-tk/GCV GDEPT specific killing in tumors in hypoxia Most firmly established among the suicide genes is the enzyme thymidine kinase from the herpes simplex virus (HSV-tk). This enzyme alone is not harmful to cells, and in tk-negative cells it can allow cell survival. HSV-tk is capable of phosphorylating specific nucleoside analogues acyclovir (GCV) to nucleoside monophosphate (MP). The nucleoside MP is then phosphorylated by cellular kinase to nucleoside triphosphate (TP) and incorporated into DNA, leading to inhibition of DNA synthesis and cell death. Some hurdles must be overcome before HSV-tk/GCV GDEPT will become a clinically efficient treatment of cancers. Major improvements are needed in vector design to enhance targeting and delivery of suicide genes. Here we constructed pEGFP-tk-ODDD2(557-574)plasmid vector and examine the potential of ODDD responsive effect to hypoxia and to control the specific killing effects herpes simplex virus thymidine kinase on SMC7721 cells and Hela cells under hypoxia which makes it possible to further investigate their potency of antitumor under hypoxia.Methods: The tk gene were amplified with PCR with the plasmid of pHSV106 as template and were fused into pEGFP-ODDD2(557-574)and pEGFP-c1, acquired gene vaccines of pEGFP-tk and pEGFP-tk-ODDD2(557-574). Transient transfectants with the vaccines were obtained by exposing the SMMC7721 cells and Hela cells to the complexes of DNA with lipofectamineTM2000 for efficient transfection. Following hypoxic exposure for 24h, cells were added to media containing conditions Prodrugs (GCV) were with different concentrations. The viabilities of cells were determined by the method of MTT.Results: The successful construction of recombinant plasmid vector was certificated through enzyme cutting and sequencing. SMMC7721 cells and Hela cells had the same transfection rate of pEGFP-tk-ODDD2 (557-574)with 33.23% and 36.97%(p>0.05).The sensitivity of cells to GCV showed that concentrations of GCV under 50mg·L-1 had no killing effect to tumors without transfection of gene vaccine and could be safe to clinical research. SMMC7721 cells and Hela cells transfected with the plasmid of pEGFP-tk were sensitive to GCV and viabilities of cells at 12.5 mg·L-1 concentration of GCV were 64.21% and 57.41%. Compared with the normoxic sample transfected with the same plasmid pEGFP-tk-ODDD2(557-574)which viabilities were almost 100%, cell survival of SMMC7721 cells and Hela cells under hypoxia were reduced to with 81.25% and 76.55% at GCV concentration of 12.5mg·L-1, the higher the GCV concentration,the greater the difference.Conclusions: HSV-TK/GCV GDEPT had the killing effect of SMMC7721 cells and Hela cells. The plasmid containing the region of ODDD2(557-574)were responded to hypoxia and had the specific killing potency to tumor cells.