Identification of molecular mechanism of necroptosis mediated by PI3K / 军事医学

Objective To identify the molecular mechanism of phosphatidylinositol-3-kinase (PI3K) in mediating necroptosis induced by tumor necrosis factor alpha (TNFα).Methods RNA interference mediated by lentivirus short hairpin RNA(shRNA) was used to downregulate p110α in L929 cells and Western blotting was used to determine the knockdown efficiency.In addition,Western blotting was used to detect the phosphorylation level of RIP1,RIP3 and MLKL in L929 cells treated with or without TNFα plus Z-VAD.Duolink assay kit was used to detect the interactions between different proteins or the same proteins.Results p110α knockdown was efficiently mediated by the lentivirus shRNA,which significantly suppressed the phosphorylation of RIP1,RIP3 and MLKL in the absence or presence of TNFα plus Z-VAD stimulation.Moreover,the interactions between p110α and RIP3,but not RIP1,increased in a time-dependent manner during the process of necroptosis,and p110α knockdown significantly inhibited the formation of necrosome and RIP1 homodimer or RIP3 homodimer.Conclusion PI3K mediates the TNFα-induced necroptosis by promoting the activation of RIP1/RIP3/MLKL signaling pathway.Given the increasing direct interactions between p110α and RIP3 during the process of necrosome formation,PI3K may regulate RIP3 kinase activity via direct phosphorylation of RIP3.

Overexpression of CHIP in chronic myeloid leukemia K562 cells induces mitotic abnormality / 中国实验血液学杂志

This study was aimed to investigate the possible influence of a novel E3 ubiquitin ligase CHIP (carboxyl terminus of Hsc70/Hsp70-interacting protein) on biological characteristics of cancer cells. Stable overexpression models in CML K562 cells were established via lipofectamine-mediated wild type CHIP and its TPR or U-box deletion mutants gene transfection. Followed G418 pressure selection, K562-CHIP stable transfected cell clones were obtained by limited dilution. The proliferation status and cell cycle were observed by MTT assay and FACS. The expression of related proteins and morphological changes were detected by Western blot and Wright-Giemsa staining. The results showed that overexpression of wild type CHIP did not inhibit cell proliferation, but slightly increased cell ratio of G(2)/M phase. CHIP gene had no effect on the stability of BCR-ABL kinase protein. HDAC inhibitor FK228-induced BCR-ABL degradation did not enhanced by CHIP. Notably the enlarged cells and abnormal mitotic cells remarkably increased in K562 WT-CHIP cells, indicating that CHIP may involve in the regulation of mitotic process. It is concluded that wild type CHIP induces mitotic abnormity in K562 cells.