Expression Of MDM4S In Acute Myeloid Leukemia And Its Contribution To Complex Karyotype Formation

ObjectiveThe incidence of complex karyotype(CK) is10%to15%in adult patients withacute myeloid leukemia(AML). Moreover, it has become clear that the incidence ofcomplex karyotype increases with age. CK is an independent adverse prognosticfactor in AML, with low complete remission(CR) rate, poor prognosis and lacking ofeffective chemotherapy. We have found15patients with complex karyotype in140denovo AML patients by G/R karyotype analysis, this study was designed to discuss therelationship between the short isoform of mouse double minute4（MDM4-S）andCK-AML. We detected the expression level of full-lenth MDM4(MDM4-FL) andMDM4-S in de novo CK-AML and normal karyotype AML patients(NK-AML). Inorder to explore the possible mechanism for the formation of complex karyotype, weconstructed a lentiviral vector, and transferred the exogenous MDM4S into HepG2cell lines. Cell cycle, cell proliferation and the expression level of P53, P21, BubR1and Securin were detected in HepG2cells stably expressing MDM4S or vectorcontrol.Methods1. To screening CK-AMLpatients from de novoAMLusing karyotype analysisTo summary the characteristics and prognosis of CK-AML patients.Conventional Cytogenetics(G or R banding) was performed at the time of diagnosisof140patients. 2. Gene mutation and MDM4FL,MDM4S expression by means of PCR, RT-PCR,real-time RT-PCR and direct sequencing.To screening mutations of TP53, FLT3-ITD, AML1by direct sequencing of PCRproducts. To detect the expression level of MDM2, MDM4FL and MDM4S by meansof RT-PCR or real-time RT-PCR.3. To construct lentiviral expression vectors of MDM2、MDM4STo design primers containing EcoR Ⅰ andBamH Ⅰ sites, the forward primerincludes initiation codon and the reverse primer includes termination codon. MDM2and MDM4S fragments were amplified by PCR. cDNA templates came from thereverse transcription of K562cell line total RNA. MDM2and MDM4S fragmentswere linked to lentiviral expression vector.For construction of the pCDH1-MDM2or pCDH1-MDM4S plasmid, thepCDH1, MDM2and MDM4S fragments were digested by EcoR Ⅰ and BamH Ⅰ,then linked respectively.293T cells were cotransfected with10μg pCDH1-MDM2orpCDH1-MDM4S and10μg pCDH packaging vectors using calcium phosphatecoprecipitation. Virus was collected48hours and72hours after transfection. HepG2cells in6-well plate were infected. The infection rates were observed by fluorescencemicroscopy.4. Cell cycle and cell proliferation assayCells stably expressing MDM4, MDM4S or vector control were culturedovernight and0.1μg/ml nocodazole was added the following day for18hours. Cellswere stained by PI and cell cycle was detected by flow cytometry(FCM). Cellproliferation was analyzed using the MTT assay. After4h incubation with MTTreagent, cells were lysed with DMSO for10min at37°C and absorbance wasmeasured at570nm. The average percentage is shown for3independent HepG2vector control, MDM2or MDM4S-expressing pools. 5. RT-PCRThe mRNA expression level of TP53, BubR1, Securin, were detected in HepG2cells stably expressing MDM2, MDM4S or vector control by RT-PCR.6. Western-blotThe expression level of P53, P21, BubR1and Securin were detected byWestern-blot.Results1.15CK-AMLpatients were identified from140de novoAMLpatients1.1The characteristics and prognosis of15patients with complex karyotypeIn the entire cohort of15CK-AML, the median age was59years(range,17-80years), and7(46.7%) of which≥60years. The male/female ratio was1.14(8:7).2(13.3%) patients had WBC count greater than100×109/L. There are1patient withM0,4with M2,5with M4and5with M5according to FAB classifications.Karyotype analysis shows monosomy5(-5)(n=2), monosomy7(-7)(n=4),+21(n=9).Of these15patients assessable for therapy response and survival,4achieved CR,2achieved PR.13patients died till now. The median survival time was292days (range66-738days).1.2The karyotype of the15patients are as follows:(1)49,XY,+13,+19,+21(2)52,XXX,+10,+13,+16,+21,+22(3)54,XXX,+8,+11,+15,+16,-17,+19,+20,+21,+22(4)47,XXY,+1,-2,-5,+12,-17,+19,+21,-22(5)56,XXXY,+1,-2,+10,+11,+12,+15,+20,+21,+21,+22(6)49,Y,+1,+5,-7,+8,+13,+21(7)50,XX,+16,+19,+20,+21 (8)51,XX,-7,+13,+15,+21,+21,+21,+22(9)92,XXXX(10)49,XY,+6,add(7)(p22),+8,add(11)(q25),+15(11)49,Y,+1,+5,-7,+8,-11,+13,+22,+t(11;17)(q23;q21)(12)51,XY,-7,+13,+15,+16,+19,+22,+22(13)48,XXY,+1,-2,-5,+12,+18(14)50,XX,+1,-7,+8,+13,+15,+19(15)51,XXX,+10,+13,+15,+162. Gene mutation and expression of de novoAML2.1The results of gene mutationPCR products of TP53and AML1in15CK-AML patients were sequenced andno mutation were found. FISH test showed that2/15cases had lost one copy of TP53.The positive rates of FLT3-ITD were20%and23.2%in CK-AML and NK-AMLrespectively, the difference being not statistically significant(P＞0.05).2.2The results of gene expression levelThe expression ratio of MDM4S/MDM4FL and MDM4-S/MDM4-total werehigher in CK-AML than in NK-AML. The difference were statistically significant(P＜0.05).The difference of mRNA expression levels of MDM2were not statisticallysignificant in CK-AML and NK-AML(P＞0.05). The expression levels of MDM4-Sand MDM4-FL were higher in CK-AML than in NK-AML, the difference beingstatistically significant(P＜0.05).3．To construct lentiviral expression vectors of MDM2,MDM4S and infect HepG2cell lineThe sequences of the pCDH1-MDM2or pCDH1-MDM4S plasmid wereconsistent with the GeneBank. Virus were packaged in293T cells and the titer was determined. HepG2cells were infected with pCDH1-MDM2or pCDH1-MDM4S. Inour test, the positive infection cells were of60%to80%.4. cell cycle and cell proliferation assay4.1cell cycleCells stably expressing MDM2, MDM4S or vector control were culturedovernight and0.1μg/ml nocodazole was added the following day for18hours. Thepercentage of cells in M phase of cell cycle in vector control was51.94%, cells stablyexpressing MDM4, MDM4S were33.35%and37.32%, respectivaly. Compared withthe vector control, there are fewer cells in M phase in stably expressing MDM2,MDM4S, the difference being statistically significant(P＜0.05).The percentage of cells in G0/G1phase of the cell cycle following nocodazoletreatment for0,8,12or18hours. In0h, it was about40%～60%. In8h, thepercentage was decreased sharply. The percentage was gradually increased with theprolonged nocodazole treatment. Following nocodazole treatment for18h, thepercentage of cells in G0/G1phase in stably expressing MDM2, MDM4S were morethan vector control, the difference being statistically significant(P＜0.05).4.2cell proliferation assayThe average percentage were increased in MDM2and MDM4S. Compared withvector control, the difference being statistically significant(P＜0.05).5. Real-time RT-PCR5.1The expression levels of TP53Compared with vector control, the expression level of TP53mRNA in stablyexpressing MDM2, MDM4S cells had not statistically significant(P＞0.05).5.2The expression levels of BubR1and SecurinCompared with control, the expression level of BubR1mRNA in stablyexpressing MDM2, MDM4S cells were decreased, the difference being statistically significant(P＜0.05). The expression of Securin mRNAin stably expressing MDM4Scells were decreased, the difference being statistically significant(P＜0.05).Following nocodazole treatment, the expression level of Securin mRNA increased invector control compared with DMSO treatment cells, the difference had statisticallysignificant(P＜0.05).6. western-blot6.1The expression levels of P53、P21Compared with vector control, the expression level of P53in stably expressingMDM2decreased and had statistically significant(P＜0.05), the expression level ofP53in stably expressing MDM4S had not statistically significant(P＞0.05). Theexpression level of P21was decreased in stably expressing MDM2, MDM4S cells.6.2The expression levels of BubR1and SecurinCompared with vector control, the expression level of BubR1and Securin instably expressing MDM2, MDM4S cells were decreased following nocodazoletreatment, the difference being statistically significant(P＜0.05). Followingnocodazole treatment, the expression level of Securin increased in vector control,compared with DMSO treatment cells, the difference had statistically significant(P＜0.05).Conclusion1. The relative expression level of MDM4S is higher in CK-AML than in normalkaryotype AML.2. Cells stably expressing MDM4-S have a weakened spindle checkpoint.3. Overexpression of MDM4-S, so increasing the MDM4-S/MDM4-FL ratio in cellscan inhibit the activity of P53pathway.4. The mechanism of the complex karyotype are partly due to the inhibition of theP53pathway and the weakness of spindle check point by overexpression MDM4-S.