| Chronic myeloid leukemia（CML）is a clonal bone marrow stem cell disorder in which a proliferation of both mature granulocytes and their precursor myeloid cells occurs.It accounts for 15%of all leukemia cases in adults,The global annual incidence of CML is 1.6-2.0 cases per 100,000people,while epidemiological surveys show an annual incidence of0.39-0.55 per 100,000 in several areas of China.CML patients in China are younger than in the western countries.CML is associated by the presence of the Philadelphia（Ph）chromosome,which represents a specific abnormality of chromosome 22 as a result of a reciprocal translocation between chromosome9 and 22.The result is the juxtaposition of the ABL1 gene belonging to the chromosome 9（region q34）to a part of the BCR gene on chromosome 22（region q11）,creating an elongated chromosome 9 and a truncated chromosome 22（the Ph chromosome）.This chromosomal translocation is thus specifically designated t（9,22）（q34,q11）.This translocation generates an oncogenic and developed BCR-ABL fusion gene,encoding for a BCR-ABL fusion protein of 210 kDa,Since the ABL gene carries a domain that can add phosphate groups to tyrosine residues,the BCR-ABL fusion gene also possesses a strong tyrosine kinase activity.Therefore,tyrosine kinase inhibitors such as Imatinib（IM）,became the first-line treatment against a variety of cancers,including CML.Although IM showed a successful effect in the majority of patients,10-15%of the patients developed a drug-resistance,or showed a disease progression into the CML accelerated phase due to a late and thus less effective treatment.Once the patient goes into the blast phase,the median progression-free survival is 4 months,and the median overall survival is 7 months.Src homology region 2（SH2）domain-containing tyrosine phosphatase-1（SHP-1）,is a gene mainly expressed in hematopoietic cells and it represents a hematopoietic cell malignancy tumor suppressor gene,playing an important role in the negative regulation of growth factor receptor-mediated signal transduction process.previous study showed that SHP-1 is forming a complex with BCR-ABL by immunoprecipitation in an immortalized myeloblast-like cell line 32D and fibroblast 3T3 cell lines,suggesting that SHP-1 may regulate BCR-ABL.A previous study showed a significant decrease of SHP-1expression level in patients with advanced CML compared to its level in the chronic phase,suggesting that SHP-1 may play a role in the onset of the blast crisis of CML.The decreased of SHP-1 expression in a variety of hematologic malignancies may be associated with the methylation of the SHP-1 gene promoter.Epigenetic deregulation is defined as changes in gene expression mediated through mechanisms other than alterations in the DNA sequence itself.Decitabine is the most widely used demethylating agents and reactivates expression of the epigenetically silenced gene by blocking the DNA methyltransferase Dnmt1,resulting in demethylation（lower）On May 2,2006,the Food and Drug Administration（FDA）formally approved DAC for the treatment of MDS patients.Several clinical studies showed that DAC monotherapy possess a substantial effect during all the phases of CML.Therefore,the present study aim to explore the mechanism of action of DAC on restoring the expression of SHP-1 on CML,comparing its effect with the effect of tyrosine kinase inhibitors to provide a new strategy to fight the progression of CML and offer new insights in this disease treatment.In the present study,to study the methylation of SHP-1 in pathogenesis of chronic myeloid leukemia and the mechanism of potential therapeutic effects of decitabine on advanced chronic myeloid leukemia.we will detect four parts in our study:1.Identification of interaction between SHP1 and P210BCR/AB by co-immunoprecipitation.2.DAC conbinated with IM can inhibit the prolifera-tion and indouce apoptosis of K562,3.SHP-1 and BCR/ABL gene expresstion levels in K562 cells and CML patients after decitabine treatment;4.SHP-1gene methylation levels in K562 cells after decitabine treatment.In conclusion,This study investigate the profile of promoter methylation and expression of SHP-1 gene in the progression of chronic myeloid leukemia（CML）,and provide scientific basis decitabine in advanced CML.Part Ⅰ Identification of interaction between SHP-1 and P210BCR/ABL by co-immunoprecipitationObjective:Identification of interaction between SHP-1 and P210BCR/ABL by co-immunoprecipitationMethods:Cell culture of K562 and primary cell culture of mononuclear cells from patients with CML;SHP1 was immunoprecipitated by c-ABL antibody,SHP1 was identified by Western blotting.Results:1.1 The immunoprecipitation showed that SHP-1 protein was expressed in the K562 cells,as well as in the CML-CP and CML-BP mononuclear cells.1.2 The expression of SHP-1 was lower in K562 cells and compared to CML-CP mononuclear cells,and the difference was statistically significant（P<0.05）.The expression of SHP-1 was lower in CML-BP mononuclear cells and compared to CML-CP mononuclear cells,and the difference was statistically significant（P<0.05）.Conclusion:The interaction between SHP-1 and P210BCR/ABL could be identified by co-immunoprecipitation.SHP-1 expression decreased is associ-ated with CML disease progression.Part Ⅱ Decitabine combined with TKIs could inhibit cell proliferation and induce apoptosis in K562 cellObjective:To explore Decitabine combined with TKIs could inhibit cell proliferation and induce apoptosis in K562 cell.Methods:1 culture of K562 cells;2 OD values of K562 cells after 48 h drug treatment were measured by CCK8,and EC50 values were calculated.Nilotinib treatment alone showed an EC50 of 10.55 nM,IM EC50 was 227.5 nM,and DAC EC50 was 6457 nM.3 We culture the K562 cells,group randomly,control group,5μM DAC group,0.1μM IM group,0.1μM IM+5μM DAC,0.01μM Nilotinib+5μM DAC.4 Cell apoptotic rate was examined by flow cytometry（FCM）after 48h.Results:1 The role of single agent and combination on the inhibition rate of K562cells.1.1 DAC caused time and concentration dependent inhibition rate of K562cells.1.2 K562 cells treated with 5μM DAC combined with 0.1μM IM showed a proliferation inhibition that was statistically significant compared to the IM monotherapy treatment groups（P<0.0001）1.3 K562 cells treated with 5μM DAC combined with 1nM Nilotinib showed a proliferation inhibition that was statistically significant compared to the Nilotinib monotherapy treatment groups（P=0.0015）2 The role of single agent or combination on K562 cell apoptosis2.1 Treatment with 5μM DAC for 48h caused 3.3%early apoptotic K562cells.2.2 Treatment with 0.1μM IM for 48h caused 6.7%early apoptoticK562cells.Treatment with 5μM DAC combined with 0.1μM IM caused 28.7%early apoptoticK562 cells.K562 cells treated with 5μM DAC combined with 0.1μM IM showed a early apoptotic that was statistically significant compared to the IM monotherapy treatment groups（P<0.05）2.3 Treatment with 0.01μM Nilotinib for 48h caused 9.3%early apoptotic K562 cells.Treatment with 5μM DAC combined with 0.01μM Nilotinib caused 35.7%early apoptoticK562 cells.K562 cells treated with 5μM DAC combined with 0.01μM Nilotinib showed a early apoptotic that was statistically significant compared to the IMmonotherapy treatment groups（P<0.05）Conclusion:1 DAC caused time and concentration dependent inhibition rate of K562cells.2 K562 cell proliferation inhibition rate was significantly higher in DAC+TKI groups（IM+DAC,and Nilotinib+DAC）than in TKI monotherapy group,revealing that the two-drugs combination effect is highly synergistic.According to Soriano etc.et al.,our results showed a synergistic effect of the drug we used in combination:IM+DAC CI=0.41 that correspond to a synergism;Nilotinib+DAC combined index CI=0.33 correspond to a strong synergism.3 DAC+TKI showed a early apoptotic that was statistically significant compared to the IM monotherapy treatment groups.Part Ⅲ SHP-1 and BCR/ABL gene expresstion levels in K562 cells and CML patients after decitabine treatmentObjective:Effects of decitabine on expression of SHP-1 in Chronic myelogenous leukemiaMethods:K562 cells and Primary cell culture of mononuclear cells from patients with CML treated with 0.1μM IM,or 1nM Nilotinib,or 5μM DAC,or IM+DAC,or Nilotinib+DAC for 48 hours.Relative quantitation detection of SHP-1 expression level by real-time quantitative polymerase chain reaction（RT-qPCR）RT-qPCR quantitative determination of absolute BCR/ABL gene level,Determination of SHP-1 expression in K562 cells,CML-CP,and CML-BP mononuclear cells by immunoprecipitation.Results:1 SHP-1 and BCR/ABL expression in K562 cells after drug treatment.1.1 RT-qPCR results showed no significant difference on SHP-1 gene expression between IM and Nilotinib monotherapy treatment groups and the control group.1.2 DAC group showed a significant difference compared with the control group.1.3 IM+DAC group showed a significant increased expression of SHP-1 gene compared with IM monotherapy group;Nilotinib+DAC group showed a significant increased expression of SHP-1 gene compared with Nilotinib monotherapy.1.4 BCR-ABL and ABL copy number ratio levels decreased in a significant manner in the combined group respect IM monotherapy group.BCR-ABL and ABL copy number ratio levels decreased in a significant manner in the combined group respect Nilotinib monotherapy group.1.5 SHP-1 protein expression in every group.1.6 SHP-1 protein expression significantly increased in IM+DAC group compared with IM monotherapy group（SHP-1 protein expression gray value increased,P<0.01）.1.7 SHP-1 protein expression significantly increased also in Nilotinib+DAC group compared with nilotinib monotherapy group（SHP-1 protein expression gray value increased,P<0.05）2 SHP-1 and BCR-ABL gene and protein expression in patients with CML 48hours after drug treatment2.1 RT-qPCR results showed no significant difference on SHP-1 gene expression between IM monotherapy treatment groups and the control group.2.2 DAC group showed a significant difference compared with the control group.2.3 IM+DAC group showed a significant increased expression of SHP-1 gene compared with IM monotherapy group.2.4 SHP-1 protein expression in every group.2.5 SHP-1 protein expression significantly increased in IM+DAC group compared with IM monotherapy group（SHP-1 protein expression gray value increased,P<0.05）.Conclusion:1 RT-qPCR results showed no significant difference on SHP-1 gene expression between IM monotherapy treatment groups and the control group.2 DAC may be able to recover SHP-1 expression by demethylation,3 SHP-1 mRNA and protein expression increased in the combined group compared with the monotherapy group,while BCR-ABL expression decreased.Part Ⅳ SHP-1 gene methylation levels in K562 cells after decitabine treatmentObjectiv:To investigate the profile of promoter methylation and expression of SHP-1 gene in the progression of chronic myeloid leukemia（CML）Method:SHP-1 gene methylation levels of K562 cells were measured by MSP in control group,IM group,DAC group,and IM+DAC group.Results:1 The results showed the presence of SHP-1 gene methylation in K562control group.;2 The results showed the presence of SHP-1 gene methylation in IM monotherapy group.3 The results showed no methylation in the DAC monotherapy group.4 The results showed showed no methylation in the IM+DAC group.Conclusion:1 the methylation of the SHP-1 gene promoter lead to its inactivation.2 DAC may be able to recover SHP-1 expression by demethylation3 Imatinib does not affect the methylation of SHP-1.Conclusion:1 Immunoprecipitation showed that SHP-1 and BCR-ABL（p210）protein forms a complex in K562 cells and in primary cells from CML,showing therefore an interaction between SHP-1 and BCR-ABL（p210）protein.Therefore,SHP-1 may represent a tyrosine phosphatase which controlled the activity of BCR-ABL（p210）tyrosine kinase.2 SHP-1 expression decreased in K562 cells and CML-BP primary cells respect the expression found in CML-CP,suggesting that it may be related with CML disease progression.3 The results showed the presence of SHP-1 gene methylation in K562control group.4 DAC may be able to recover SHP-1 expression by demethylation5 K562 cells treated with 5μM DAC combined with 0.1μM IM showed a proliferation inhibition that was statistically significant compared to the IM monotherapy treatment groups（P<0.0001）.6 our results suggest that DAC combined with TKI may provide a new strategy for TKI tolerance or progression of CML. |
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