Activation of caspase-8 and Erk-1/-2 in domes regulates cell death induced by confluence in MDCK cells

• Main Authors: Yung-Heng Chang, 張永恆
• Other Authors: Ming-Jer Tang
• Format: Others
• Language: en_US
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/p44fy2

碩士 === 國立成功大學 === 生理學研究所 === 90 === Under normal culture conditions, cells adhere to the culture dish surface, spread out, proliferate and finally cover all areas and reach confluence. During the confluent stage, cell proliferation ceases and differentiation is enhanced. In the meanwhile, cell death occurs during confluent process. To delineate the mechanism of cell death induced by confluent process, we employed Madin-Darby canine kidney (MDCK) cells. We found that approaching confluence, MDCK cells exhibited elevated levels of caspase-2 and enhanced activity of caspase-8. Using various caspase inhibitors to inhibit confluent cell death, we found that only caspase-8 inhibitor, z-IETD-fmk was effective. These results suggest that confluent cell death is mediated by activation of caspase-8. To further delineate whether activated caspase-8 triggered confluent cell death through mitochondria-dependent or -independent pathway, we employed MDCK cell overexpressing Bcl-2. We found that confluence triggered cytochrome c release from mitochondria. Overexpression of Bcl-2 could inhibit cytochrome c release as well as confluent cell death, suggesting the involvement of mitochondria-dependent pathway in confluent cell death. Interestingly, we also found that activity of phospho-Erk (p-Erk) was initially decreased during confluence, but was markedly increased after confluence. Immunofluorescent staining studies showed that p-Erk activity was expressed exclusively on dome-forming cells that underwent apoptosis. Treatment of confluent MDCK cells with PD98059, inhibitor of MEK, enhanced apoptosis and slightly increased activation and activity of caspase-8. These data suggest that elevation of p-Erk activity during confluence may serve to suppress confluent cell death. In summary, activation of caspase-8 is involved in confluent cell death whereas p-Erk prevents MDCK cells from this death process by regulating activation and activity of caspase-8.