The Influence Of ABCA1 On Arsenic Resistance In Mammalian Cell

Objective: The purpose of this research is to study the change of arsenic-resistant ability and the arsenic accumulation in mammalian cell when ATP-binding cassette transporter A1(ABCA1) gene over expressed or knocked down, and presume the possible mechamisms involved in arsenic-resistance function of ABCA1 gene.Methods: Eukaryotic expression vector containing ATP-binding cassette transporter A1 gene was transfected into HeLa cells by Lipofectaonmine 2000. The mRNA level of the ABCA1 gene was examined by real-time PCR and transfectional effectiveness was identified by western blot. After exposed in arsenite for 48 hours, cell survival rate and IC50 were detected by MTT assay. We also examined the arsenic accumulation in HeLa cell by atomic fluorescence spectrophotometry which reflected the effect of arsenic-resistance. At the same time, siRNA fragment of ATP-binding cassette transporter A1 gene was transfected into arsenic-resistance cell ECV-304., the effeciency of interference was examined by western blot. After exposed in arsenite for 48 hours, MTT assay was used to detect the cell survival rate and IC50.Results: The amplification curve is standard in real time PCR and no idio-amplification can be seen. Results of ABCA1 and GAPDH Ct of all the groups shows that the expression level of recombinant plasmid pcDNA3.1/ABCA1 in HeLa cell is 22.6 times(P<0.01) than the control with empty plasmid pcDNA3.1/V5-His.The expression amount between the group with empty plasmid and the group of untransfected HeLa cell showed no obvious distinction(P>0.05).Western blot showed that specific protein straps exist in all the three group of recombinant plasmid,empty plasmid and untransfected groups, they are as the same size as the ABCA1 protein. Express amount of the recombinant plasmid group was higher than the other two groups. It can be proved that recombinant plasmid pcDNA3.1/ABCA1 were successfully transfected into HeLa cells and made ABCA1 protein over expressed in HeLa cells. After been transfected , HeLa cells was exposed in arsenic for 48 hours, we used MTT assay to detect the optical density and calculate cell survival rate and IC50. Results showed that survival rate of those cells in experimental groups were higher than that of control under all concentration of arsenic. The difference of survival rate between two groups has statistical significance(P<0.01). IC50 of the experimental groups was 36.740μmol/l versus 20.721μmol/l for control groups. When ABCA1 protein over expressed in HeLa cells,the results of atomic fluorescence spectrophotometry showed that arsenic accumulation of HeLa cells in experimental groups was lower than that of control groups after incubation with 10μmol/l NaAsO2 for up to 10 h, and arsenic accumulation of experimental groups reached a steady state in 4 h and did not increase significantly thereafter, In contrast, arsenic accumulation of control groups increased throughout the 10h incubation period. After been interfered by siRNA, ABCA1 protein of arsenic-resistance cell ECV-304 in experimental groups was obviously lower than that of control groups. After been interfered, the arsenic-resistance cell ECV-304 was exposed in arsenite for 48 hours, the survival rate of those cells in experimental groups under all concentration of arsenic was lower than that of control groups. The difference of survival rate between two groups had statistical significance.(P<0.01)IC50 of the experimental groups was 7.640μmol/l versus 14.520μmol/l for the control groups.Conclusion: When ABCA1 gene over expressed in HeLa cells, arsenic accumulation in the cells reached a balance in short times, and did not increase significantly, over expressed ABCA1 gene made HeLa cells transfer from arsenic sensitive to arsenic tolerant. While ABCA1 gene was knocked down, arsenic-resistance cell ECV-304 lost its arsenic tolerance. Our data strongly suggests a key role for ABCA1 in resisting arsenic in mammalian cells.