Anti-CHMP5 single chain variable fragment antibody retrovirus infection induces programmed cell death of AML leukemic cells in vitro

Aim： Over-expressed CriMP5 was found to act as oncogene that probably participated in leukemogenesis. In this study, we constructed the CriMP5 single chain variable fragment antibody （CHMP5-scFv） retrovirus and studied the changes of programmed cell death （PCD） of AML leukemic cells after infection by the retrovirus. Methods The anti-CriMP5 KC14 hybridoma cell line was constructed to generate monoclonal antibody of CriMP5. The protein expression of CriMP5 was studied using immunofluorescence analysis, pMIG-CHMP5 scFv antibody expressible retroviral vector was constructed to prepare CHMP5-scFv retrovirus. AML leukemic U937 cells were infected with the retrovirus, and programmed cell death was studied using confocal microscope, FCM and Western blot. Results： We obtained a monoclonal antibody of CriMP5, and found the expression of CriMP5 was up-regulated in the leukemic cells. After U937 cells were infected with CHMP5-scFv retrovirus, CriMP5 protein was neutralized. Moreover, the infection resulted in a significant increase in apoptosis and necrosis of U937 cells. In U937 cells infected with CHMP5-scFv retrovirus, apoptosis-inducing factor （AIF）-mediated caspase-independent necrotic PCD was activated, but autophagic programmed cell death was not observed. Neither the intrinsic nor extrinsic apoptotic PCD pathway was activated. The granzyme B/perforin-mediated caspase-dependent apoptotic PCD pathway was not activated. Conclusion： CHMP5-scFv retrovirus can neutralize the abnormally high levels of the CriMP5 protein in the cytosol of AML leukemic U937 cells, thereby inducing the programmed cell death of the leukemic cells via AIF-mediated caspase-independent necrosis and apoptosis.