Study On The Characteristics Of Mutant P2X₇ Receptor And Alternative Splicing Variant From Leukemia Cells

Nucleotides, which bind to P2 family receptors on cell membrane to regulate survival and function of cells, are novel carriers in intercellular communication. According to their structure, P2 receptors are divided into P2X and P2Y subfamilys. P2X receptors contain intrinsic pores that switch conformation from closed to open on ATP binding, allowing ions to flow and P2X₇ receptor is the last identified member of this family. Evidence showed that P2X₇ receptor was widely expressed not only in nervous and muscle systems, but also hematopoietic and immune systems, including monocytes/macrophagocytes, B and T lymphocytes, etc.P2X₇ might play important roles in leukemia. It was reported to be correlated with prognosis of B-CLL. We also showed that it was widely expressed in leukemic cells at higher levels and positive rates, which related to clinical features. In addition, non-functional P2X₇ receptor was found in leukemia cells including Namalva, LCL and J6-1. CD39-associated high ATPase activity contributed to the loss of the P2X₇-mediated calcium response in LCL-H cells, whereas other mechanism(s) existed in J6-1 cells. Hence, cloning of the P2X₇ receptor from J6-1 cells was performed. The results showed that a full length P2X₇ cDNA with a mutation at position 559, and an alternative splicing variant with a 73 bp deletion, were obtained. Based on the above results, we further study the roles of these isoforms on the regulation of P2X7 receptor function in this thesis:1. The P2X₇ receptor from J6-1 cells had a substitution of A₅₅₉→G₅₅₉, causing a substitution of Asn¹⁸⁷→Asp¹⁸⁷ in amino acid sequence. Ramos cells without endogenous P2X receptor were transfected with mutant P2X₇ receptor eukaryotic expressing vector and stably expressing clones (Ramos-P) were obtained. RT-PCR,Western blot and confocal microscopy were used to detect the expression, localization of P2X₇ receptor and the aggregation induced by BzATP. The proliferation was measured by MTT assays and cell cycle was studied by FACS technique. The results showed that both the mutant P2X₇ expressing vector and the control one were successfully constructed. The receptor can be expressed on the membrane and in the cytoplasm of Ramos cells and resulted in the augmented proliferation. To compare with the control cells, apoptosis rate was increased upon higher concentration of the agonist, BzATP (about 300μumol/L), whereas receptor aggregation could be observed upon 100μmol/L BzATP stimulation, respectively.2. We cloned a novel P2X₇ alternative splicing variant with a 73 bp deletion, which might be related to the absence of calcium response in J6-1 cells. To further study its role, we construct five recombinant plasmid vectors with _PIRES vector: the full length vector (_PIRESA), the truncated alternative splicing vector (_PIRESB), the alternative splicing vector (_PIRESC), the full length and the truncated alternative splicing dual expressing vector (_PIRESA+B) , the full length and the alternative splicing dual expressing vector (_PIRESA+C). The DNA sequencing verifies the successful construction of the above vectors.3. The five recombinant plasmid vectors were transfected to Ramos and K562 leukemic cell lines, and stably expressing clones were obtained which lay the foundation of further studies on factional regulation of P2X₇ receptor.This study provide the useful basis for the understanding the roles and functional modifications of P2X7 receptor in leukemias.