| Introduction:Acute myeloid leukemia (AML) is characterized by the clonal expansion of myeloid hematopoietic cells with uncontrolled proliferation, differentiation arrest and decreased apoptosis. AML is the most common type of leukemia in adults. Despite the fact that improvements in chemotherapy, strengthened supportive treatment and the wild spread use of molecular targeted therapy and hematopoietic stem cell transplantation improve the efficacy of treatment, but the five-year survival rate in patients under the age of 60 years old is only 30-40%, and while in patients over 60 years old is less than 10%. Therefore, it is vital to identify the fundamental pathogenesis of AML, which may lead to the development of novel treatments. CIP2A (Cancerous Inhibitor of PP2A, CIP2A), also called KIAA1524, p90, is a newly identified endogenous inhibitor of protein phosphatase 2A (PP2A). CIP2A interacts directly with the oncogenic transcription factor c-Myc, inhibits PP2A activity toward c-Myc serine 62 (S62), and thereby prevents c-Myc proteolytic degradation. In addition to its function in c-Myc stabilization, CIP2A promotes anchorage independent cell growth and in vivo tumor formation. Thus CIP2A is identified as a human oncoprotein. Although CIP2A is over-expressed in some human solid tumors, such as head and neck squamous cell carcinoma, colon cancer, gastric cancer, and breast cancer, its expression and roles in AML is still unknown. Methods:We collected the bone marrow samples from patients with AML and healthy controls, and then isolated mononuclear cells by centrifugation over Ficoll-Hypaque gradients, extracted total RNAs and protein. CIP2A mRNA and protein expressions were determined in bone marrow mononuclear cells of both patients with AML and healthy controls using reverse transcription polymerase chain reaction (RT-PCR) and Western blot, respectively. We used siRNA to knock-down CIP2A expression in HL60 cells and then examined its potential roles during the pathological progression of AML.1. Cell proliferation study was conducted by drawing cell growth curve, soft agar colony formation assay and serum starvation test.2. Cell differentiation was determined by both microscopic morphology analysis following Wright's staining and immunophenotypic feature of CD11b expression analysed by flow cytometry.3. Cell apoptosis rate was detected by flow cytometry through Annexin?and PI staining.4. After silencing the expression of CIP2A, we detected the changes of c-Myc level and PP2A activity.Results:A total of 151 bone marrow samples from 116 patients with AML [70 patients with newly diagnosed AML,14 patients with relapsed AML and 32 patients with AML in complete remission (AML-CR)] and 35 healthy controls were collected for this study. CIP2A mRNA was presented in 54 of 70 (77.14%) patients with newly diagnosed AML and in 11 of 14 (70.86%) patients with relapsed AML, which was significantly higher than AML-CR specimens (2/32,6.25%) and healthy controls (1/35,2.86%) (P<0.001). However, in terms of CIP2A mRNA expression, the AML-CR group was not statistically significant different from normal controls (P>0.05). Similarly, there was no statistically significant difference between the newly diagnosed and relapsed AML group. In addition, over-expression of CIP2A protein was also verified by Western blot in corresponding specimens. After 72h of serum starvation, CIP2A level was significantly decreased. Knock-down of CIP2A in HL60 cells slowed down cell proliferation, decreased clonogenic activity, promoted cell differentiation and inceased cell apoptosis. Upon the knock-down of CIP2A, the level of c-Myc protein was consequently reduced, and the activity of PP2A recovered dramatically.Conclusions:Our findings suggest that CIP2A is over-expressed in patients with newly diagnosed/relapsed AML, indicating that CIP2A may serve as a novel tumor marker for AML. The high expression of CIP2A in HL60 cells may be related to active cell proliferation, arrest cell differentiation and inhibit cell apoptosis through the inactivation of PP2A and the increase of c-Myc protein. This study may shed light on the molecular function of CIP2A in myeloid leukemogenesis and support the potential development of CIP2A-based targeted therapy for the treatment of AML. Introduction:Chronic myelocytic leukemia (CML) is an acquired clonal hematopoietic stem cell malignant disease, mainly involving myeloid, caused by the BCR/ABL fusion protein which arises from the translocation of chromosomes 9 and 22. Usually CML experiences three phases:chronic phase (CP), accelerated phase (AP) and blastic phase (BP). Once the disease progresses into BP, the survival is usually less than 1 year. Until now, precise mechanisms responsible for transition of CML chronic phase into blast crisis are still unclear. It has been reported that the tumor suppressor PP2A is functionally inactivated in occurrence and blast crisis of CML. So it is believed that inactivation of PP2A is essential for the pathogenesis even blast crisis of CML. CIP2A is a newly identified endogenous inhibitor of PP2A. Therefore the research of CIP2A in CML is very important to disclose possible mechanisms of CML progression and look for new therapy targets. Although CIP2A is over-expressed in some human solid tumors, such as head and neck squamous cell carcinoma, colon cancer, gastric cancer, and breast cancer, its expression and roles in CML is still unknown.Methods:We collected the bone marrow samples from patients with CML and healthy controls, and then isolated mononuclear cells by centrifugation over Ficoll-Hypaque gradients, extracted total RNAs and protein. CIP2A mRNA and protein expressions were determined in bone marrow mononuclear cells of both patients with CML and healthy controls using RT-PCR and Western blot, respectively. The difference between patients with CML-CP and CML-AP/BP was analyzed by QRT-PCR. We used siRNA to knock-down CIP2A expression in K562 cells and then examined its potential roles during the pathological progression of CML.1. Cell proliferation study was conducted by drawing cell growth curve, soft agar colony formation assay and serum starvation test.2. Cell differentiation was determined by microscopic morphology analysis following Wright's staining.3. Cell apoptosis rate was detected by flow cytometry through Annexin V and PI staining. We also detected the expression of c-Myc and the activity of PP2A after knock-down of CIP2A. In addition, we inhibited the expression of BCR/ABL by imatinib mesylate (IM), and detected the variation of CIP2A. On the other hand, we checked out the change of BCR/ABL and SHP-1 after CIP2A was knocked down or up-regulated.Results:Bone marrow samples from 71 patients with CML (58 patients with CML-CP,13 patients with CML-AP/BP) and 35 healthy controls were collected for this study. CIP2A mRNA was presented in 44 of 58 (75.86%) patients with CML-CP and in 10 of 13 (76.93%) patients with CML-AP/BP, which was significantly higher than healthy controls (1/35,2.86%) (P<0.001). Furthermore, in terms of CIP2A mRNA expression, the CML-AP/BP group was significantly higher than CML-CP (P<0.001), and almost four times the CP. In addition, over-expression of CIP2A protein was also verified by Western blot in corresponding specimens. After 72h of serum starvation, the level of CIP2A was significantly decreased. Knock-down of CIP2A in K562 cells slowed down cell proliferation, decreased clonogenic activity, promoted cell apoptosis. Upon scilencing of CIP2A, the level of c-Myc protein was consequently reduced, and the activity of PP2A recovered dramatically. CIP2A mRNA expression in bone marrow mononuclear cells in CML patients was in accordance with the expression of BCR/ABL fusion gene. After the expression BCR/ABL was suppressed by IM, expression of CIP2A also significantly decreased. Followed with down-regulation of CIP2A expression, expression of BCR/ABL also decreased, while the levels of SHP-1 showed a rise trend. On the contrary, up-regulation of CIP2A expression led to the increase of BCR/ABL and decrease of SHP-1. Conclusions:Our findings suggest that CIP2A is over-expressed in patients with CML and the expression of CIP2A mRNA in CML-AP/BP group was significantly higher than that in CML-CP. This shows that CIP2A not only participates in the pathogenesis of CML, but also plays an important role in the progression of CML. The high expression of CIP2A in K562 cells may promote cell proliferation, inhibit cell apoptosis through the inactivation of PP2A and the increase of c-Myc protein. CIP2A and BCR/ABL can regulate each other forming a positive feedback loop. Functional inactivation of PP2A tumour suppressor activity caused by the effect BCR/ABL on CIP2A expression may be one of the possible mechanisms of blast crisis CML. This study may shed light on the molecular function of CIP2A in CML and interfering in CIP2A'expression will be a potential targeted therapy in patients with CML blast crisis. |
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