Inducible overexpression of Bak sensitizes HCC-9204 cells to apoptosis induced by doxorubicin
Abstract
"AIM:
To investigate the role of overexpression of Bak in apoptotic pathways and drug susceptibility using doxorubicin and vinorelbine in human HCC-9204 cells.
METHODS:
An inducible system, MT-II regulatory system which allowed controlled expression of protein upon addition of ZnSO4(100 mumol/L) as an external inducer was used. Stable transfection of pMD-Bak gene was performed on HCC-9204 cells. Apoptotic cells were measured by morphological criteria, as well as by TUNEL assay and flow cytometry. The ability of Bak to decrease clonogenic cell survival was studied by colony-forming assays, while decrease in cell viability was assessed by MTT assay.
RESULTS:
Cells overexpressing Bak showed extensive cell death with nucleus fragmentation detected by TUNEL assay. FACS analyses showed that Bak could induce significant G1 accumulation and apoptosis in 19.29% cells 24 h after induction. Bak significantly decreased the clonogenic survival following exposure to adriamycin, but not vinorelbine. Furthermore, the time-course of cell viability rates following exposure of HCC-9204/Bak cells to adriamycin and vinorelbine was in agreement with the above findings. Bak selectively sensitized HCC-9204 cells to death induced by adriamycin while resisted to vinorelbine.
CONCLUSION:
Bak may prolong cell cycle in G1 phase, leading to apoptosis and decrease clonogenic survival of HCC-9204 cells in a drug-specific manner."
Keywords:
To investigate the role of overexpression of Bak in apoptotic pathways and drug susceptibility using doxorubicin and vinorelbine in human HCC-9204 cells.
METHODS:
An inducible system, MT-II regulatory system which allowed controlled expression of protein upon addition of ZnSO4(100 mumol/L) as an external inducer was used. Stable transfection of pMD-Bak gene was performed on HCC-9204 cells. Apoptotic cells were measured by morphological criteria, as well as by TUNEL assay and flow cytometry. The ability of Bak to decrease clonogenic cell survival was studied by colony-forming assays, while decrease in cell viability was assessed by MTT assay.
RESULTS:
Cells overexpressing Bak showed extensive cell death with nucleus fragmentation detected by TUNEL assay. FACS analyses showed that Bak could induce significant G1 accumulation and apoptosis in 19.29% cells 24 h after induction. Bak significantly decreased the clonogenic survival following exposure to adriamycin, but not vinorelbine. Furthermore, the time-course of cell viability rates following exposure of HCC-9204/Bak cells to adriamycin and vinorelbine was in agreement with the above findings. Bak selectively sensitized HCC-9204 cells to death induced by adriamycin while resisted to vinorelbine.
CONCLUSION:
Bak may prolong cell cycle in G1 phase, leading to apoptosis and decrease clonogenic survival of HCC-9204 cells in a drug-specific manner."