The Expression And Significance Of PTPROt Gene In Chronic Myelogenous Leukemia

Objective: Chronic myelogenous leukemia (CML) is a clonal myeloproliferative expansion of transformed primitive hematopoietic progenitor cells. CML has the characteristic of Philadelphia chromosome (Ph).Chronic myelogenous leukemia is typified by constitutive activation of the c-abl kinase as a result of its fusion to the breakpoint cluster region (BCR). The fusion protein BCR/ABL is closely related to the pathogenesis of CML. Recently, some scholars noticed the expression of fusion protein BCR/ABL is negatively correlated to the maturity of the CML cells.From chronic phase to blastic phase , CML often accompanied significantly increase of BCR/ABL mRNA . Studies show that the growth and their evolution of many human malignant tumors were caused by the inactivation function of Antioncogenes by genetic point mutation,gene rearrangement,gene loss.The Antioncogenes inactivated play a more important role than the oncogene activated in the transformation process of tumor cells. As other malignant tumors,the occurrence of leukemia is directly related to the abnormal expression of oncogenes and antioncogenes.Antioncogene silence due to hypomethylation of the CpG island in the promoter region of antioncogene is a common phenomenon in human leukemia[1].The studies of antioncogenes on structures and abnormal expressions have important clinical significances in understanding the differentiation characteristics and development of leukemia cells and formulating gene therapy strategies of leukemia. In the studies of the pathogenesis of cancers , tyrosine phosphorylation is an important control mechanism about physiological activities in eukaryotes.It play an important role in communications of cells and endocellular , shape and initiative, regulation of gene transcription, embryonic development, homeostasis and immune function. Abnormal levels of tyrosine phosphorylation plays a key role in the pathogenesis of many inherited and acquired diseases . Protein tyrosine phosphatase (PTPs) and protein tyrosine kinase (PTKs) regulates cellular proteins phosphorylation level. Phosphorylation of PTPs is accompanied by dephosphorylated of PTKs to keep dynamic balance , they are together regulating cell growth, differentiation, signaling, cell cycle regulation, etc. If the balance (or increase PTKs signals or decrease PTPs signals) was breakup,it will promote the phosphorylated tyrosine proteins related to gather up and cause abnormal cellular proliferation, differentiation, result in various malignant diseases. And oncogenesis is connected to the phosphorylation level of cellular proteins .Recent studies show that protein tyrosine phosphatase plays a more prominent role in decision of phosphorylation level of cells [2].Studies on the effects of protein tyrosine phosphatase in the pathogenesis of malignant hematopathy become the hot in recent years. PTPRO (Gleppl,PTP - U2 ) is a new member of protein tyrosine phosphatase (PTP)family, it is a negative control factor in the pathways of JAK/STAT signal transduction. PTPRO locates on 12p12.3 chromosome [3], and it has an important feature that it has loss of the heterozygous(LOH) in a variety of tumor cells such as lung cancer, liver cancer. The truncated isoform of protein-tyrosine phosphatase receptor-type O (PTPROt) is specifically expressed in hematopoietic cells.Some peopele tested the possibility that it could potentially dephosphorylate and inactivate the fusion protein bcr/abl. Ectopic expression of PTPROt in the chronic myelogenous leukemia cell line K562 indeed resulted in hypophosphorylation of bcr/abl and reduced phosphorylation of its downstream targets CrkL and Stat5, confirming that PTPROt could inactivate the function of bcr/abl. Furthermore, the expression of catalytically active PTPROt in K562 cells caused reduced proliferation, delayed transition from G0/G1 to S phase, loss of anchorage independent growth, inhibition of ex vivo tumor growth, and increased their susceptibility to apoptosis, affirming that this tyrosine phosphatase can revert the transformation potential of bcr/abl. The inhibitory action of PTPROt on bcr/abl was also confirmed in a murine myeloid cell line overexpressing bcr/abl. PTPROt expression was suppressed in K562 cells and was relieved upon treatment of the cells with 5-azacytidine, an inhibitor of DNA methyltransferase, with concomitant hypomethylation of the PTPRO CpG island. These data demonstrate that suppression of PTPROt by promoter methylation could contribute to the augmented phosphorylation and constitutive activity of its substrate bcr/abl and provide a potentially significant molecular therapeutic target for bcr/abl-positive leukemia. Based on all these above,my study aims to test the expression level of PTPROt in CML patients and K562 cells treated by 5-azacytidine, observation the changes of cell proliferation and apoptosis in order to research the function and clinical significance of PTPROt gene in the pathogenesis of CML.Methods: The samples were collected from 82 CML patients including 64 chronic-phase(CP) patients and 18 accelerated-phase/blastic-phase(AP/BP) patients , and K562 cells. 20 healthy volunteers as normal controls(NC).We divided the CP patients into initial treatment group(BCR / ABL fusion gene-positive), remission group after treatment(BCR / ABL fusion gene-negative) . K562 cells were divided into untreated and treated group by 5 - azacytidine .The expression of PTPROt in bone marrow of CML patients and peripheral blood of normal controls and K562 cells untreated or treated by 5-azacytidine were measured by realtime-quantity reverse transcription polymerase chain reaction(RQ-PCR). MTT method was used to test cell inhibition rates. FCM methed was used to test cell apoptosis rates.Results:1 The expression levels of PTPROt in CML-CP and CML-AP/BP patients were both obviously lower than that in normal controls,P<0.05,and it has statistical significance.The positive expression rate of PTPROt in the normal controls is 95% (19/20), the relative gene expression levels are between 0 and 0.927, the median is 0.435. The positive expression rate of PTPROt in 64 CML-CP patients is 79.69% (51/64), the relative gene expression levels are between 0 and 0.299, the median is 0.159. The positive expression rate of PTPROt in 18 CML-AP/BP patients is 61.11% (11/18), the relative gene expression levels are between 0 and 0.166, the median is 0.047. The expression levels of PTPROt in CML-AP/BP patients were obviously lower than that in CML-CP patients,P<0.05,and it has statistical significance.2 The expression level of PTPROt in CML patients of initial treatment group and that in CML patients after therapy group were both obviously lower than that in normal controls,P<0.05,and it has statistical significance. The positive expression rate of PTPROt with CML patients of initial treatment group is 79.69%(51/64),the relative gene expression levels are between 0 and 0.299,the median is 0.159.The positive expression rate of PTPROt in CML patients after therapy group is 98.30% (26 / 27),the relative gene expression levels are between 0 and 0.494,the median is 0.272.The expression levels of PTPROt in CML patients after therapy group were obviously higher than that in CML patients of initial treatment group ,P<0.05,and it has statistical significance.3 K562 cells lose the expression of PTPROt .PTPROt mRNA re-expression in K562 cell after 5 - azacytidine treated 5-azacytidine can increase the expression level of PTPROt in K562 cells , and the effect was related to the acting time of 5-azacytidine. And there was significant difference (P<0.05).4 5-azacytidine can inhibit K562 cells , and the effect was related to the concentration and acting time of 5-azacytidine,and presented dosedependent and time-dependent relationship.5 5-azacytidine can induce apoptosis of K562 cells , and the effect was related to the concentration and acting time of 5-azacytidine , and presented dosedependent and time-dependent relationship.Conclusion:1 The expression levels of PTPROt mRNA in CML patients were obviously lower than that in normal controls.The expression levels of PTPROt mRNA in CML-AP/BP patients were obviously lower than that in CML-CP patients.The expression levels of PTPROt in CML patients after therapy group were obviously higher than that in CML patients of initial treatment group . Monitoring changes of expression levels about PTPROt mRNA can be used as a reference indicator to judge the prognosis of CML.2 K562 cells lose the expression of PTPROt .PTPROt mRNA re-expression in K562 cell after 5 - azacytidine treated ; The results suggested that K562 cells lose the expression of PTPROt may be due to gene promoter methylation. 5 - azacytidine demethylation can make PTPROt mRNA re-expression , and presented dosedependent and time-dependent relationship.and thus inhibit tumor cell growth and promote apoptosis.