Regulation of Differentiation and Proliferation of Marrow Hematogenesis Stem Cells by Notch1 Signaling System from Patients with Aplastic Anemia and Chronic Myelogenous Leukemia.

Notch genes encode evolutionarily conserved transmembrane receptors that regulate cell fate determination. Notch activation promotes proliferation and inhibits differentiation of bone marrow stem cells. Our research was to study the differential expression of Notch1 on bone marrow mononuclear cells (BMMNC) from chronic aplastic anemia (AA) and chronic myelogenous leukemia (CML) patients. We demonstrated that Notch signaling is inactivated in AA patients and activated in CML patients. In CML patients, Notch1 furthers stimulation by its ligand Jagged1, resulting in a strong increase of tumor cell growth. Therefore, we suggest that inactivated Notch signaling plays a pivotal role in the pathogenesis of AA and that activated Notch signaling plays a key role in the pathogenesis of CML. We used the antibody to inactivate Notch1 signaling in mice, and then to detect the hematopoietic state of these mice.

Methods: 5ml marrow were abstracted from chronic AA patient s, CML patients in chronic phase and normal people and the BMMNC was separated from the marrow. RNA was abstracted from these cells. Real Time Quantitative PCR was used to investigate the expression of Notch1 on BMMNCs in 30 AA patients, 20 normal controls and 30 CML patients, and the changes of their expression after chemotherapy. Notch1 protein of these BMMNC was also detected through Western Blots. We injected monoclonal anti-Notch1 and isotonic Na chloride as controls into the tail vein of mice, to detect the hematopoietic state through bone marrow slides and flow cytometry.

Results: Real Time PCR: Expression of Notch1 on BMMNCs from AA patients was lower than that of controls (P<0.05). The expression of Notch1 on BMMNCs from CML patients was higher than that of controls(P<0.05). Notch1 on the BMMNCs from AA patients was expressed higher after chemotherapy (P<0.05). Notch1 on the BMMNCs from CML patients was expressed lower after chemotherapy(P<0.05). Western Blots: Notch1 receptors were highly expressed in BMMNCs of CML patient. BMMNCs of AA patients expressed low amounts of Notch1. Jagged1 induces Notch signaling in cultured cells; We performed RT-PCR analysis of mRNA expression of Hes-1, a member of the hairy enhancer of split family of transcriptional repressors that are direct transcriptional targets of activated Notch. The result was that the expression of Hes-1 of CML was much higher than AA. The growth rate of CML BMMNCs was also much higher than AA BMMNCs. Compared with controls, the hematopoietic state of mice treated with anti-Notch1 had an obvious low hematopoietic ability.

Conclusions: The expression of Notch1 on BMMNCs from AA patients and normal controls was different, which may account for the occurrence of AA. The expression of Notch1 of AA patients was improved after effective chemotherapy, which may be one of the reason of recovery of hematopoietic function. The expression of Notch1 on BMMNCs from CML patients was much higher than that of controls, which decreased after chemotherapy and may be one of the factors accounting for occurrence of CML. BMMNCs cocultured with Jagged-1 expressing cell lines can enhance the growth rate, which indicate that Notch1 can improve proliferation of leukemic cells. The mice treated with anti-Notch1 had lower hematopoietic ability manifesting that Notch1 signaling is essential to maintain normal hematopoietic function.