|Budget Amount *help
¥2,600,000 (Direct Cost : ¥2,600,000)
Fiscal Year 1999 : ¥900,000 (Direct Cost : ¥900,000)
Fiscal Year 1998 : ¥1,700,000 (Direct Cost : ¥1,700,000)
Glucose 6 phosphate dehydrogenase (G6PD) gene has been strongly expessed and reduced glutathione level was remarkably elevated in the cyclophosphamide (CPA) resistant K562 cells. To assess the possibility that the G6PG itself has an ability to make cells resistant to various cytotoxic drugs, the expression vectors pcDNA3.1(-)/G6PD and pcDNA 3.1(-)/G6PD-R, in which 1.6 kb of G6PD cDNA was forwardly and inversely introduced to the expression vector pcDNA 3.1(-), were constructed. After transfection these vectors to cos7 or K562 cells by elctroporation or transferrifection, cells were selected by G418 to obtain stable tranfectants. When pcDNA 3.1(-)/G6PD-R or pcDNA 3.1(-) were transfected, there was no effect on the G6PD level. When pcDNA3.1(-)/G6PD was transfected, approximately 1.5 fold elevation of mRNA was observed. However, the G6PD activity of pcDNA3.1(-)/G6PD transfected cells was 0.18 μmole/mg protein (control 0.13 μmole/mg protein), reduced glutathione level (GSH) 6.4 U/10^6 cells control 4.5 U/10^6 cells) mRNA and LC90 against 4-hydroperoxyphosphamide, the activated form of CPA, 10 μM (control 5.5 μM), indicating that there was not so much difference in these cells.
The G6PD activity and mRNA level of cells from ten cases with chronic lymphocytic leukemia (CLL), six with treated CLL, two with prolymphocytic leukemia (PLL), three with non-Hodgkin's lymphoma (NHL) and three with refractory cases were almost the same. These results imply that the G6PD gene alone does not contribute to obtaining drug resistance to CPA.