The Mechanism Of Communication Between The Abnormal Cells In Leukemia Study

Cellular communication is the foundation of cellular society exists and running. The abnormal intercellular communication is closely related to human diseases. Though classical mechanism of intercellular communication, based on one-directional signal transduction, has been deeply investigated and the research of reverse signal has become one of the hot points in life science. The membrane form of macrophage colony-stimulating factor (mM-CSF) is an alternative splicing variant of this cytokine. As the proteolytic cleavage sites used to release the secreted isoforms have been spliced out, mM-CSF is stably expressed at the cell surface. Our previous research showed that mM-CSF could play juxtacrine and adhesion molecule-like roles. It could transfer two-directional signal after binding its recepteors. Basing on our previous studies on mM-CSF, we try to explore the roles of mM-CSF in reverse signal and the mechanisms of transmission mediated by mM-CSF.In this study, we construct mM-CSF and mM-CSF mutation eukaryotic expression vector pTARGET and retroviruses carrier MSCV-PGK-GFP. Stable and transient transfectant clones expressing mM-CSF and mM-CSF mutation (Namalwa-M and Namalwa-M mutation) were obtained. Namalwa-M and Namalwa-M mutation were verified by RT-PCR and Western blot methods.Using stably transfected clones (Namalwa-M) and transient transfectant clones (HEK293-M), we studied the reverse signal by mM-CSF. The tyrosine phosphorylation level of several proteins with the molecular masses of approximately 40kD was increased by sM-CSFR stimulation. Furthermore, using phospory-kinase arrays, we demonstrated that the phosphorylation level of several protein kinases was changed after sM-CSFR stimulation. The result suggested that ERK/MAPK and Akt signal pathway were involved in this reverse signal by Western blot methods.Using stably transfected Namalwa-M-30 (brachytmema mutation of 30 amino acide located in the intracellular region of mM-CSF) and Namalwa-M 10 (Ser226,Ser235 and Thr246 mutated to Ala) and transient transfectant HEK293-M-30 and HEK293-M10 clones, we studied the role of the intracellular region of mM-CSF in reverse signal. The result suggested demonstrated that there was no reverse signal in above-mentioned cells by the same methods. We demonstrated that the intracellular region of mM-CSF, especially Ser226,Ser2235 and Thr246 located in the intracellular region of mM-CSF play important roles in reverse signal.To further explore the mechanisms of reverse signal mediated by mM-CSF, using stably transfected clones which was expressing one site (Ser226 mutated to Ala) or two sites (Ser235 and Thr246 mutated to Ala) mutation on serine or threonine residues located in the intracellular region of mM-CSF, we studied the different role of serine or threonine residues located in the intracellular region of mM-CSF in reverse signal. The initial result suggested that Ser226 play the important role.In conclusions, we deeply explored the roles of mM-CSF in reverse signal and the mechanisms of transmission mediated by mM-CSF. We demonstrated that mM-CSF can transmit reverse signal and first proved that ERK/MAPK and Akt signal pathway were involved in this reverse signal. We first proved by experiments that the intracellular region of mM-CSF play important roles and Ser226,Ser235 and Thr246 located in the intracellular region of mM-CSF play the different role in reverse signal, especially the important role of the Ser226. RNA editing is an important posttranscriptional process, through which RNA base sequences are altered. Several different forms of RNA editing, catalyzed by two classes of enzymes, occur in humans and the predominant RNA editing in mammals is adenosine-to-inosine (A-I) editing, which is catalyzed by adenosine deaminases acting on RNAs (ADARs). The posttranscriptional RNA editing by the type 1 adenosine deaminase acting on RNAs (ADAR1), expressed as constitutively expressed p110 and IFN-inducible p150 isoforms, is important for both physiological and pathological processes. Their expression and significance in leukemias remain unknown.116 newly diagnosed pediatric acute leukemia patients,20 relapsed patients,16 patients achieving complete remission,26 follow-up patients and 20 control cases were included for the expression of ADAR1 isoforms by real-time PCR and Western blot. The results showed that significant high expression of p110 was detected in leukemias, especially in B-ALL, whereas a slight increase of p150 could be observed. Furthermore, the decrease of p110 expression was observed in B-ALL patients achieving complete remission. Moreover, among prognostic risk groups in ALL, the highest expressions of p110 and p150 were detected in standard-risk group, whereas their lowest expressions were in high-risk group. This observation was further confirmed in comparisons between good and poor prognostic groups based on prognostic related clinical features. These results demonstrated that ADAR1 isoforms showed different expression patterns, suggesting that they might play different roles in pediatric leukemias. Our results will help us for the better understanding of RNA editing, exploring the potential target for the treatment, and making prognostic evaluation in childhood leukemias.