PS1333 OVEREXPRESSION OF PROTEIN TYROSINE PHOSPHATASE NON‐RECEPTOR TYPE1 CAUSES STAT5 DEPHOSPHORYLATION, RESULTING IN SUPPRESSION OF CELL GROWTH SIGNAL IN K562 HUMAN LEUKEMIA CELLS

Background: Protein tyrosine phosphatase non‐receptor type 1 (PTPN1), which functions as a negative regulator of intracellular signaling pathways mediated by tyrosine kinases, is involved in multiple physiological and pathological cellular processes, including cell proliferation and metabolic regulation. The PTPN1 gene is located on long arm of chromosome 20, deletion of which is observed in cases of myelodysplastic syndromes (MDS) and myeloproliferative neoplasms (MPN). Previous our study demonstrated that PTPN1 expression was significantly reduced in bone marrow cells of MDS patients with or without del(20q) compared to those of control subjects, suggesting roles of decreased PTPN1 expression in pathogenesis of MDS. Currently, it was reported that depletion of ptpn1 in hematopoietic cells induced MPN in mouse model, and that markedly increased phosphorylation of intracellular molecules including STAT5 was observed in ptpn1‐depleted bone marrow cells. Aims: In the present study, we examined cellular and molecular biological effects of PTPN1 overexpression in human myeloid leukemia cell lines to elucidate biological significance of reduced PLCG1 expression in myeloid malgnancies, including MDS. Methods: We used the Tet‐On advanced inducible gene expression systems (Clontech) for the experiments. First, we established a stable transfectant (named as pTet‐on K562), which constitutionally expressed tetracycline‐controlled transactivator and showed best inducibility to doxycycline (DOX) treatment. Next, to construct inducible expression vector of PTPN1 (named as pTRE‐Tight‐PTPN1), normal human PTPN1 cDNA was cloned into the multi‐cloning site of the pTRE‐Tight vector, which contains binding site for tetracycline‐controlled transactivator in promotor region. Finally, K562 double stable transfectant (named as pTet‐on K562‐PTPN1), in which PTPN1 expression level was increased 5 folds or more at 24 hours after adding DOX (0.5ug/mL) into culture media, was established and used for following analyses. Results: Significant reduced growth was observed in pTet‐on K562‐PTPN1 cells with DOX compared with those without DOX, indicating that PTPN1 negatively regulates growth of K562 leukemia cells. To elucidate molecular mechanisms of growth inhibition by PTPN1, we examined the effect of PTPN1 overexpression on JAK2/STAT5 pathway, because JAK2/STAT5 pathway is important in human myeloid malignancies, and several previous reports showed that PTPN1 functions as negative regulator of cytokine‐ or hormone‐ induced JAK/STAT pathways in hematopoietic and also non‐hematopoietic cells. We examined STAT5 phosphorylation status by immunoblot, showing that PTPN1 overexpression resulted in reduced phosphorylation of STAT5 with no change in amount of STAT5 protein. We examined expression levels of down‐stream molecules of JAK2/STAT5 pathway, including BCL2L1, an anti‐apoptotic protein, and CCND1, a cell cycle regulator. STAT5 functions as a transactivator and induces expression of BCL2L1 and CCND1. Overexpression of PTPN1 resulted in reduced expression of BCL2L1 and CCND1. We also examined the effect of PTPN1 overexpression on GATA1, EPOR, and HBB, which are down‐stream molecules of JAK2 pathway, but not regulated their expression by STAT5. These three molecules are involved in erythroid cell differentiation. Interestingly, expression level of GATA1, EPOR, and HBB was increased by PTPN1 overexpression. Summary/Conclusion: Our present results indicated that PTPN1 negatively regulates JAK2/STAT5 pathway through STAT5 dephosphorylation, resulting in reduced cell proliferation in human leukemia cells.