Impact Of The Transduction Of Ras-MEK1/2-ERK1/2 Signaling Pathway In HL60 Cells After Treatment With PD98059

Introduction95%APL patients have a cytogenetics specificity of t(15;17)(q22;q21), forming the fusion gene of PML-RARα.It is the essential pathogenesis of APL,but not the only one.Recently,a study demonstrated that mice transgened PML-RARαdeveloped into APL soonly when having the mutation of FLT3.This suggested abnormal Ras-MEK1/2-ERK1/2 signaling pathway is important for the happeniss of APL.The Ras-MEK1/2-ERK1/2 signalig pathway is a kind of kinase cascade reaction,and it has been found activated abnormally in many kinds of tumor organizations and cells.PD98059 is a selective pharmacological inhibitor of MEK phosphorylation.The effects of PD98059 have been studied in many cell types,and this inhibitor is known to induce apoptosis and G1 cell cycle arrest of cells,however,the detailed mechanism is unknon.Our study investigated the effects of cellular and molecular mechanism of the Ras-MEK1/2-ERK1/2 signalig pathway,using the acute promyelocytic leulemia cell line HL60 cells.ObjectiveTo explore the suppressive effects of PD98059,the inhibitor of MEK1(extracellular signal-regulated kinase kinase),on the Ras-MEK1/2-ERK1/2 signaling pathway's transduction and on the proliferation of acute promyelocytic leukemia cell line HL60 cells.Materials and MethodsCell cultureHL60 cells were cultured at 37℃,in a 5%CO₂ humidified incubator,and maintained in RPMI 1640 containing 10%fetal bovine serum(FBS).MTT assayThe cells were seeded at 6×10⁴/ml to each well of the 96-well plates after serum-starved for 12h,then added PD98059 immediately.MTT were added to every well after the HL60 cells being cultured with PD98059,then the HL60 cells were cultured for another 4h without lights;The cells were collected and centrifugated,then added DMSO to each well,finally measured the suppressive effects of proliferation of HL60 after treated with PD98059 on microplate reader.Flow cytometryAfter treated with PD98059,1～3×10⁶ HL60 cells were collected,washed twice with ice-cold PBS,and fixed with 70%ethanol at 4℃overnight.The cells were then collected by centrifugation and stained with propidium iodide solution.The fluorescence intensities of 10,000 cells were measured by a flow cytometer with excitation at 488 nm and emission at 620 nm.Semi-quantitative reverse transcription-poly merase chain reactionToal RNA of HL60 cells was isolated using a TRIZOL reagent after treatment with PD98059,.and was diluted to 3mg/ml.With the help of oligo dT and reverse transcriptase,the RNA were converted into cDNA.The resulting PCR products were visualized on 2%agarose gels.The stained image was recorded by an image analyzer, and the band intensity was quantified using densitometric analysis.Statistical analysisAll values are expressed as means±standard deviation,differences were determined using SPSS 15.0RusultSuppressive effects of PD98059 on the proliferation of HL60 cellsThe statistics analysis showed the suppressive effect of 40 or 80μmol/Lof PD98059 on the HL60 cells was the most strong among the different time(P＜0.05).Similarly,the effect of 48h or 72h was the most strong among the different concentrations of PD98059.However,there was no significant deviation between the effect of treated for 24h and that for 48h in 20μmol/L PD98059 treated group.When the cells was treated with PD98059 for 24h,there was still no significant deviation between 20μmol/L group and control group.Effects of PD98059 on HL60 cells apoptosis and cell cycle changsPD98059 induced apoptosis of HL60 cells in vitro,which were dose-dependent and time-dependent(P＜0.05).However,the HL60 cells interphase of G0/G1 phase did not block after treated with PD98059 for 24h,and the G0/G1 phase was significantly arrested after treated with PD98059 for 48h.Changes of mRNA expressions of ERK2,Skp2,p27The mRNA expressions of ERK2 and Skp2 of HL60 cells were reduced and the expressions of p27 were increased after treated with PD98059 for 48h,which were dose-dependent(P＜0.05).DiscussionThe extracellular signal-regulated kinase(ERK) pathway is one of the most important signaling mechanisms involved in the regulation of apoptosis.ERK plays a major role in regulating cell proliferation and differentiation,and protects cells against apoptosis.The Ras-MEK1/2-ERK1/2 pathway is normally regulated by endogenous Raf inhibitors,however,constitutive activation of the ERK pathway has been observed approximately 30%of all human cancers.The present findings suggest that an ERK pathway regulates the levels of Bad,Mcl-1,caspase9,Bcl-2 and cyclin D1,CDK2,p21,p27,thus resulting in prevention of apoptosis.PD98059 is a selective pharmacological inhibitor of MEK1 phosphorylation.The effects of PD98059 have been studied in many cell types,and this inhibitor is known to induce apoptosis and G1 cell cycle arrest.Ras-MEK1/2-ERK1/2 pathways are frequently activated in acute leukemia.In the present study,Dong-Oh Moon found that treatment with PD98059 significantly arrests the G1 phase through up-regulation of cyclin-dependent kinase(CDK) inhibitor,and produces morphological features of apoptosis in U937 cells.PD98059 also decreased the CDK-2,CDK-4,cyclin D1,and cyclin E expression,and increased high levels of the mitotic inhibitors p16,p211,and p27. In our study,the suppressive effect on the proliferation of HL60 cells was enhanced with the increasing doses of PD98059 and the extension of the day,and PD98059 induces a remarkable G0/G1 phase arrest.We have also examined the inhibition effect of ERK pathway on apoptosis in HL60 cells.Hein Schepers demonstrated that N-Ras transformed HL60 cells lack a G0/G1 arrest upon TGF-βtreatment due to absence of p27.The absence of p27 protein was due to elevated levels of the ubiquitin ligase Skp2,which complexes with and targets p27 for degradation.In our study,the quantity of ERK2 and Skp2's mRNA was dow-regulated in HL60 cells after treated with PD98059,on the contrary,the quantity of p27's mRNA was up-regulated.We could demonstrate that Ras-MEK1/2-ERK1/2 signaling pathway could regulate the expressiong of Skp2 and p27,then regulae the process of cell cycle.We also found that PD98059 can't block the HL60 cell's multiplication completely, which suggests that the abnormality of Ras-MEK1/2-ERK1/2 pathways was just one of the mechanisms of APL development.ConclusionPD98059 can block the Ras-MEK1/2-ERK1/2 signaling pathway of HL60 cells,which is probably completed by regulating the expressiong of ERK2,Skp2 and p27,then regulating the process of cell cycle and inducing apoptosis.