Aberrant Expression And Abnormal Function Of P2X Family Receptors In Leukemia

Nucleotides are new players in intercellular communication and regulate cell function through the interaction with P2 family receptors, which are classified into P2Y and P2X families according to their structures. P2Y family receptors belong to seven-membrane-spanning G protein-coupled receptors, while the P2X receptors including seven distinct subtypes (P2X1-P2X7) are ATP-gated plasma membrane ion channels that mediate transmembrane cation fluxes. P2X7 is distinct from other P2X receptors in terms of its longest C-terminal intracellular domain, pharmacology and ability to form cytolytic pores. Exposure to ATP or the more potent agonist BzATP renders P2X7 permeable to Na+, K+ and Ca2+, while repeated or prolonged application of either agonist induces the formation of a cytolytic pore, which is permeable to larger cations such as fluorescence yellow, positively-charged ethidium and YO-PRO-1.The P2X family receptors are widely distributed in a variety of cell types and involved in diverse biological effects, which suggest that they may participate in both physiological and pathological processes. More and more studies indicated that co-expression of different P2X receptor subtypes might influence the function of a single submit, indicating that it's necessary to know the expression of all seven subtypes of this family on any particular cell type or in any particular process for the better understanding the roles of these receptors. Furthermore, the expression and function of P2X family receptors (mainly P2X7) were focused in hematological malignancies. The abnormal high expression of P2X7 was reported to be related to B-CLL, ALL, AML, MDS, CML. However, the full expression spectrum and significance of P2X family receptors in pediatric hematopoietic malignancies have not been established.P2X7 function is regulated at multiple levels by different mechanisms. Studies on the abnormal function of P2X7 receptor have been point of subject recently, including several polymorphisms leading to dysfunction, different alternative splicing variants, and abnormal membrane location etc. Our previous work demonstrated the lack of P2X7-mediated calcium response in LCL and J6-1 leukemia cell lines upon BzATP stimulation at regular concentration. CD39-associated high ATPase activity contributed to the loss of P2X7 function in LCL-H cells, whereas other mechanism(s) existed in J6-1 cells. Then, the entire coding region of P2X7 was cloned from this cell line and DNA sequencing analysis revealed a substitution of A559 to G559, causing an Asnl87 to Aspl87 substitution (N187D), and we tried to explain the dysfunction of this mutated P2X7 on gene level.Based on the above research background, this paper emphasized on the expression features of P2X family receptors in pediatric acute leukemias and abnormal function of mutated P2X7 (N187D) derived from J6-1 cell line in malignance progression. This paper has completed the work as follows:1. Here we investigated the expression and significance of P2X receptors in BMMC samples from Chinese pediatric acute leukemias. Real-time PCR and Western blot results showed that P2X1,4,5,7 receptors were simultaneously over expressed in leukemias compared with controls, whereas P2X2,3,6 were absent or marginally expressed in both groups. It was worth noting that the co-expression feature of them, especially between P2X4 and P2X7, could be observed and the highest expression of P2X7 was detected in relapsed patients. Moreover, concomitant decrease of P2X4,5,7 expressions was observed at CR stage in a follow-up study. Functional P2X7 was also verified by calcium fluorescence techniques. These results suggested that P2X1,4,5,7 were hematopoiesis-related P2X receptors, and their signaling, especially for P2X7, might play important roles in pediatric leukemias. P2X receptors might co-operatively contribute to the malignant phenotype in human pediatric leukemias.2. We obtained wild type P2X7 gene coding sequences by overlap PCR based on mutated P2X7, stable K562 transfectant clones expressing mP2X7 and wP2X7 (K562-M and K562-W) were obtained. K562-M and K562-W were verified by RT-PCR, Western blot and immunofluorescence methods. Our results showed that the N187D P2X7 needed higher concentration of agonist for its activation leading to Ca2+ influx and ERK phosphorylation, which were not related to high ATPase activity on cell surface and in extracellular space. K562 cells expressing this N187D mutant showed proliferative advantage and reduced pro-apoptosis effects over wild-type P2X7 in vitro and in vivo. Furthermore, elevated angiogenesis and CD206 positive macrophage infiltration were found in tumor tissues formed by K562-M using confocal method. In addition, higher expression of VEGF and MCP1 could be detected in K562-M formed tumor tissues by real time PCR. Our results suggested that this N187D P2X7, representing such mutants hyposensitive to agonist, might be a positive regulator in the progression of hematopoietic malignancies.In summary, our results on P2X expressions in leukemia suggested that they might play important roles in childhood leukemias. The knowledge will help us for better understanding of intercellular communication in leukemias and exploring the potential target for the treatment of leukemias. In addition, we identified a new P2X7 mutant N187D, which was different from previously reported non-functional and gain-functional P2X7 mutant. This new mutant was hyposensitive to its agonist, and the observations on it in the progression of malignancies provide certain inspiration and clues for the understanding of such P2X7 mutations leading to hyposensitive dysfunction in hematologic malignancies or solid tumors.