The Investigation Of Intereaction Between IASPP And Sertad1on Regulation Of Proliferation And Apoptosis Of Leukemic Cells The Investigation Of Value Of N-cadherin And Tie2as Ieukemic Stem Cell Markers

Objective:As the important suprressor of P53, iASPP was found to be overexpressed in leukemia, and functioned as oncogene that inhibited apoptosis of leukemia cells. The amino terminus of iASPP was used as bait in a two-hybrid screen of a cDNA library from human acute leukemia, and Sertadl was identified as one of the proteins that could bind with iASPP. In this study, we investigated the roles of interaction between iASPP and Sertadl in proliferation and apoptosis of leukemic cells.Methods:Co-immunoprecipitation and fluorescence confocal microscopic imaging were used to confirm the intereaction of iASPP and Sertadl, the exact binding domains and the subcellular location. K562cells with high expression of iASPP, Sertadl and both of the genes were established and subcloned. MTT assay was performed to study the proliferation of above K562subclones, flow cytometry for cell cycle and apoptosis analysis, Western blot for P53targeted proteins assay.Results:1. iASPP indeed combined with Sertadl in leukemic cell lines, and the interaction ocuured in the cytoplasm near nuclear membrane. iASPP could intereact with Sertadl through its CA, PB and C terminal domain, except for S domain.2. When Sertadl was overexpressed in K562cell line, the expression of iASPP protein was up-regulated in a time-dependent manner, however, the expression of iASPP protein was not affected after Sertadl expression was down-regulated.3. Overexpression of iASPP in K562cells resulted in the increased cell proliferation, cell cycle arrest in G2/M phase and resistance to apoptosis induced by chemotherapy drugs. While overexpression of iASPP and Sertadl at the same time could slow down the cell proliferation, lead the cell more vulnerable to the chemotherapy durgs. Meanwhile, iASPP and Sertadl protein were found to both relocalize in the cytoplasm mainly.4. Puma, the p53targeted protein, increased gradually after the K562-iASPPhl cells were subjected to the chemotherapy drug VP16in a concentration-and time-dependent manner.Conclusions:P53protein is mutated in K562cell lines, but the mutant still retain some function. iASPP and Sertadl protein could shuttle back and forth between cytoplasm and nucleus in normal condition. But when iASPP was overexpreesed, it mainly retained in nucleus and interacted with p53to exert anti-apoptosis and promote cell proliferation by activation of Puma protein. But when iASPP and Sertadl protein were both overexpressed at the same time, Sertadl could interact with iASPP in the cytoplasm near nuclear membrane, which could block iASPP to enter into nucleus and interact with p53, and inhibited the function of iASPP eventually. Objective:To explore the value of N-cadherin and Tie2, the two promising leukemic stem cell (LSC) markers, in the development of leukemia.Methods:By flow cytometry cell sorting, CD34+CD38+CD123+N-cadherin+(N-cadherin+LSCs) and CD34+CD38’CD123+Tie2+(Tie2+LSCs) cells from bone marrow mononuclear cells (BMMNCs) derived from de novo actue myeloid leukemia(AML) patients were obtained. After NOD/SCID mice were inoculated with different doses of sorted cells, the leukemia development in the mice was observed and the relevant cytogentic markers and surface markers were detected.Results:1. CD34+CD38-proportion varied largely among AML patients with high white blood cell count at diagnosis.2. N-cadherin+N-cadherin+LSCs and Tie2+LSCs population can induce leukemia more rapidly and effectively in NOD/SCDI mice than that of the negative counterparts.3. The blast cells from leukemic mice could also induce leukemia after the second transplantation.4. N-cadherin molecule, with the cooperation of FLT3-ITD mutation, may participate in the process of self-renewal and expansion of the leukemic stem cells.5. Multiple organs were involved in the N-cadherin+LSCs transplanted leukemic mice, but the involved organs in the Tie2+LSCs transplanted leukemic mice were mainly restricted to the bone marrow.Conclusion:N-cadherin and Tie2molecules could participate in the process of self-renewal and expansion of leukemic stem cells, they may become the promising surface markers of leukemic stem cells.