Upon stimulation by CPPD crystals, the expression of both Bim and Bax-alpha decreased after 3 h suggesting a reduced proapoptotic effect of these proteins so that the static expression of the prosurvival proteins Bcl-xl and Mcl-1 might allow for a temporary shift in the balance to a prosurvival state of the cells. Because a sudden (but transient) increase in the phosphorylated form of Bim was observed in CPPD-stimulated neutrophils it is possible that this species might act as a signaling intermediate, resulting in the observed downregulation of Bax-alpha.
influence the effectiveness of radiotherapy, we carried out the present study to determine whether microenvironmental factors of the tumor tissue would influence the cell killing efficiency of suicide gene system. Materials/Methods: Rat gliosarcoma 9L cells, stably transduced with double suicide genes containing two therapeutic genes, were used. The two suicide genes were fused and the 9L cells expressed cytosine deaminase (E.coli CD) and thymidine kinase (HSV-1 TK) stoichiometrically. E.coli CD converts non-toxic prodrug, 5-fluorocytosine (5-FC) into 5-fluorouracil (5-FU), and HSV-1 TK converts anti-viral drug, ganciclovir (GCV) into toxic ganciclovir monophosphate. The following microenvironmental conditions were examined to determine the cell killing efficiency of two suicide genes. Hypoxia of acute and chronic conditions was achieved with oxygen concentrations of less than 10 mmHg. The cells at pH 6.7 (acidic) were compared to cells at pH 7.2 (neutral) with varying times up to 24 hrs. The effect of cellular density from 104 to 106 cells per dish and the effect of growth status of exponential vs plateau phase of the cell cultures were examined. The endpoint for cell killing was clonogenic assay.Results: Compared to oxic condition, acute or chronic hypoxia did not affect the cell killing of either prodrugs, 5-FC or GCV.In the E.coli CD/5-FC gene system, there was no cell survival difference at pH 6.7 or pH 7.2. However, there was more effective cell killing at the pH7.2 than at pH 6.7 with the HSV-1 TK/GCV gene system. With the cells in exponential growth phase, the higher the cell density plated, the higher the killing achieved with either suicide gene system. Compared to exponential growth phase, cytotoxicity of E.coli /5-FC gene system was decreased in plateau phase. But there was no different cell killing by HSV-1 TK/GCV gene therapy system between cells in exponential phase and plateau phase.Conclusions: Tumor cells transduced with double suicide genes were equally sensitive to prodrugs under both oxic and hypoxic conditions. E.coli CD/5-FC system was effective in cell killing equally in acidic and neutral cell culture environment. HSV-1 TK/GCV gene system was more effective at neutral pH. E.coli CD/5-FC was less efficient with plateau phase cell culture. However, combining the two therapeutic genes showed complementary and synergistic cell killing under different microenvironments.
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