| Objective: To explore the role of autophagy in the growth of pre-B acute lymphoblastic leukemia cells and the underlying mechanism.Methods:(1) BM cells of B-ALL patients and normal healthy control were collected and CD34, CD38, CD117, CD45, CD19 and CD10 were measured by Flow Cytometry. CD34+38- and CD19+ B cells were sorted separately and autophagyessential genes were detected by Q-PCR.(2) Human B-ALL 697 cells were transplanted into NOD-SCID mice to built xenograft mouse model, mice were separated into four groups, control, model, preventative and treatment group. Detect differences between no rapamycin administrated and rapamycin administrated mice, including peripheral blood cell counting, appearance liver and spleen, expression of human CD45, CD19 and CD10 markers in the BM cells.(3) Check whether manipulating autophagy would affect in vivo cell cycle of BM cells and LSK cell percentage in the xenograft mice.(4) Image flow cytometry, Western blot and confocal microscopy were used to check the changes of oncoprotein E2A/Pbx1 by rapamycin treatment of in vitro B-ALL 697 cells and in vivo NOD-SCID leukemia mice.(5) With inhibition of autophagy by 3-MA and inhibition of ubiquitination by ubiquitin-proteasome inhibitor MG132, flow cytometry, Western blot were used to check oncoprotein E2A/Pbx1.(6) Check whether E2A/Pbx1 is colocalized with autophagosomes by flow immunofluorescence after the cells are treated with rapamycin or MG-132.Results:(1) It showed higher percentage of hematological stem/progenitor cells in normal control and higher percentage of B lymphocytic cells in B-ALL patients. And it showed low autophagy level in both CD34+38- cells and CD19+ B cells of B-ALL patients.(2) The average survival time was 21.2 ± 3.5, 26.7 ± 5.4 and 30 ± 2.8 days in the model group, preventive group and treatment group, respectively. Activation of autophagy by rapamycin prolonged 5–9 days of the survival time compared with the model group. Peripheral blood cell counting from the animal experiment showed that the white blood cell increased and the platelet decreased significantly in the model group, but rapamycin normalized the white blood cell number and platelet number, particularly in the treatment group. At the time of killing, the liver and spleen in the model group showed enlargement significantly but the enlargement was less significant in the preventive and treatment groups. The liver in the model group also showed significant invasion of cells, which was less significant in the preventive and treatment groups. CD45, CD19 and CD10 markers in the BM cells of the B-ALL mice administrated with or without rapamycin were measured. The results showed that the B-ALL markers expressed highly in the BM cells of model group, while reduced significantly in both rapamycin preventative and treatment groups.(3) The cell cycle in the preventive group did not show a difference from that in the model group, however, cell cycle of the treatment group showed that cell percentage of G0/G1 phase increased and S, G2/M phase decreased significantly compared with that of the model group. This suggests that enhancement of autophagy by treatment with rapamycin after transplantation of the leukemia cells caused cell-cycle arrest of the total BM cells. In the preventive group, but not the treatment group, LSK(including CD34-LSK, CD34+ LSK) cell percentage increased significantly compared with that of model group, suggesting that enhancement of autophagy by preventive administration of rapamycin restores, at least in part, the pool of hematopoietic stem and progenitor cells.(4) Flow-cytometry analysis showed that E2A/Pbx1 protein level downregulated significantly after activation of autophagy by rapamycin or nutrient depletion by HBSS culture medium. Western blot analysis of total cellular protein, cytoplasmic protein and nucleus protein also showed down-regulated E2A/Pbx1 protein level through activation of autophagy by rapamycin and in particular starvation. Western blot analysis of liver proteins from the B-ALL xenograft mouse model indicates that preventive administration with rapamycin partially downregulated E2A/Pbx1 protein level, whereas treatment with rapamycin significantly minimized the E2A/Pbx1 protein level. The Western blot results were confirmed by confocal microscopic analysis, which discloses that E2A/Pbx1-PE was merged with GFP-LC3 in the HBSS and rapamycin treatment groups in the cytoplasm, while nuclear E2A/Pbx1 was relocalized to the cytoplasm upon treatment with HBSS or rapamycin.(5) The results by Western blot analysis showed that similar to the inhibition of basal autophagy with 3-MA that caused accumulation of E2A/Pbx1 protein, inhibition of ubiquitination with MG132 alone or in combination with 3-MA also caused an accumulation of E2A/Pbx1 protein. Furthermore, Western blot analysis with pan-ubiquitin indicates that pan ubiquitination in the B-ALL 697 cells was increased in HBSS starvation. While MG132 alone effectively blocks the degradation of E2A/Pbx1, it also partially blocked the degradation of the oncogenic protein caused by HBSS starvation.(6) Amnis image flow cytometer was used to visually and statistically examine whether this colocalization occurs. Similar to the observation that autophagy activation by amino-acid depletion or rapamycin causes more LC3 or E2A/Pbx1 moving out of the nucleus, and subsequently colocalization in the cytoplasm, starvation by amino-acid depletion with HBSS medium caused colocalization between ubiquitin and E2A/Pbx1, and treatment with MG132 significantly blocked E2A-Pbx1/Ub colocalization.Conclusion:(1) Autophagy is downregulated in pediatric B-ALL cells.(2) Activation of autophagy in vivo by rapamycin or starvation downregulated oncogenic fusion protein E2A/Pbx1.(3) Activation of autophagy in vitro by rapamycin or starvation downregulated oncogenic fusion protein E2A/Pbx1. Our data suggest a collaborative action between autophagy and ubiquitination in the degradation of E2A/Pbx1 in pediatric B-ALL cells. |
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