The Role Of PKA-CREB And P38MAPK-ATF2 Signal Pathway Regulating The Expression Of DNA Polβ In Esophageal Carcinoma

In many carcinoma related genes,oncogene,tumor suppressor genes and DNA repair gene play a major role in cancerogenesis.The DNA repair genes can not correctly to repair DNA damage when they are mutational or their expression are abnormal.DNA polymeraseβ(polβ) is involved in base-excision repair(BER) for DNA maintenance.It also may be involved in replication,recombination,and drug resistance in eukaryotic cells.However,polβlacks any intrinsic 3' to 5' exonuclease activity and is incapable of proofreading,polβcan lead to DNA repair synthesis errors and confers to cells a mutator phenotype,which has been confirmed in some tumor. Our previous studies have shown that polβgene were mutational and its mRNA expression wers increased in Esophageal carcinoma specimens.It is necessary to research deeply on the relation of polβoverexpression and tumorigenesis and it may be novel idea of oncotherapy.Abnomrality of cell signal transduction and regulation is intimately related to the pathogenesis and promotion of tumor.Tumor cells have developed various mechanisms to achieve constitutive activation and overexpression of signal members, to lead to cell malignant transformation,survival,proliferation,resisting apoptosis. polβgene expression is a precisely regulated process in mammalian cells that is controlled,in part,through cis-elements(CRE) in promoters and their cognate DNA-binding factors.It is important to know the pathway of polβexpression and regulate its level and understand its role in tumor.Alternariol(AOH),a secondary metabolite produced by various species of the genera Alternaria,is often found in wheat,grain and fruits.A long-term exposure to low levels of AOH is associated with esophageal cancer.AOH has DNA toxicity.In general,DNA damage,a stimulated signal,induces stress reaction in cells,and increases the expression of DNA repair genes to maintain DNA stabilization.It is unknown that AOH induces DNA polβexpression via some singnal pathway.In this study,we investigated the cytotoxicity and ability of colony formation of AOH on NIH/3T3 Cells.AOH up-regulated DNA polβexpression via the activated PKA-CREB and p38MAPK-ATF2 pathway in NIH3T3 cells.We discussed the relation of overexpression of polβ,the activated pathway of PKA and p38,and cell transformation.In addition we detected the activation status of PKA-CREB and p38MAPK-ATF2 signaling pathway in the EC9706 cells and found that the expression of polβwas dependent on the activation of PKA and p38MAPK pathway. Treatment of EC9706 cells with PKA inhibitor H89 and/or p38 inhibitor SB203580 reduced cell viability and increased apoptosis compared with the untreated control. While combination with cisplatin,the results were more prominent.This may be a target for the development of novel therapeutic strategies.PartⅠAOH affects NIH3T3 cells biological characteristics and activates the signaling pathway of DNA polβexpressionChapter 1 AOH affects NIH3T3 cells biological characteristics and induces the expression of DNA polβMethods1.NIH3T3 cells in logarithmic growth phase were treated at the dosage of 1.0,2.0, 10.0,20.0,50.0μmol/L AOH and treated with DMSO(0.25%) for 24 h,then the effects of AOH on cell proliferations were assessed by morphologic observasion,and cell cycle distributions were detected by flow cytometric assay(FCM).Cell viability was detected by MTT with the same concentrion of AOH treatment for 24h and 48h. Single Cell Gel Electrophoresis(SCGE) assay was used to examine DNA damage induced by AOH for 4h.The ability of AOH-induced transformation of NIH3T3 cells were by clony formation assay.2.The expression of DNA polβevoked by different concentrations of AOH in NIH3T3 cells was investigated with reverse transcription-polymerase chain reaction (RT-PCR),immunocytochemistry and Western Blotting.Results1.The cells treated with AOH presented morlogic changes and the inhibition of cell proliferation occurred(P＜0.05).There were significant comet in the intermediate and high dosage group(ranging from 10.0 to 50.0μmol/L) in the SCGE assay(P＜0.05). In comparison with the control group,the percents of G2/M and S phase cells were increased after treatment of 10.0,20.0,50.0μmol/L AOH for 24 h(P＜0.05).2.The results of RT-PCR,immunocytochemistry and Western Blotting indicated that the expression of polβincreased with treatment of 2.0,10.0,20.0μmol/LAOH for 16h in the NIH3T3 cells,which was a concentration-dependent manner.Chaper 2 AOH activates the PKA-CREB and p38MAPK-ATF2 pathway in NIH3T3 cellsMethods1.NIH3T3 cells were treated by by 2.0,10.0,20.0μmol/LAOH for 1h,then activation of PKA were investigated with immunocytochemistry,immunofluorescence and Western Blotting.The cells were exposed respectively by 20.0μmol/L AOH for 0, 30,60,120min,then activation of PKA were investigated with Western Blotting.In addition,the cells were pretreated with an inhibitor of PKA(H89) for 1h,then exposed to 20.0μmol/L AOH for 1h,and activation of PKA were detected by the same methods.2.NIH3T3 cells were treated by by 2.0,10.0,20.0μmol/LAOH for 2 h,then phosphorylation of CREB were investigated with immunocytochemistry, immunofluorescence and Western Blotting.The cells were exposed respectively by 20.0μmol/L AOH for 0,1,2,4h,then activation of CREB were investigated with Western Blotting.In addition,the cells were pretreated with an inhibitor of PKA(H89) for 1h,then exposed to 20.0μmol/L AOH for 2h,and activation of CREB were detected by the same methods.3.NIH3T3 cells were treated by by 2.0,10.0,20.0μmol/L AOH for 2h,then phosphorylation of p38MAPK and ATF2 were investigated with Western Blotting. The cells were exposed respectively by 20.0μmol/L AOH for 0,1,2,4h,then activation of p38MAPK and ATF2 were investigated with Western Blotting.In addition,the cells were pretreated with an inhibitor of p38(SB203580) for 1h,then exposed to 20.0μmol/L AOH for 2h,and activation of p38MAPK and ATF2 were detected by the same methods.4.Phosphorylation of JNK1/2 in NIH3T3 cells induced by 20.0μmol/L AOH was investigated with Western Blotting.NIH3T3 cells were pretreated with an inhibitor of JNK(SP600125) for 1h,then exposed to 20.0μmol/L AOH for 2h.Phosphorylation of JNK1/2 and ATF2 were investigated with Western Blotting.5.NIH3T3 cells were pretreated with an inhibitor of p38(SB203580) for 1h,then exposed to 20.0μmol/L AOH for 2h.Phosphorylation of CREB was investigated with Western Blotting.Result1.AOH -induced activation of PKA and nuclear translocationIn the results of Western blot,2.0μmol/L AOH-elicited activation of PKA was not obvious,but it was significantly increased elicited by 10.0,20.0μmol/L AOH compared with the control,which was a concentration-dependent manner.The results of immunocytochemistry coincided with Western Blotting.Application of AOH increased nuclear accumulation of PKA in immunofluorescence assay.Activation of PKA was increased in time-dependent manner andhad reached maximal levels by 1h after AOH treatment.The results of immunocytochemistry,immunofluorescence and Western Blotting indicated that H89 blocked the AOH-induced activation of PKA.2.AOH -induced activation of CREB In the results of Western blot,2.0μmol/L AOH-elicited phosphorylated CREB was not obvious,but it was significantly increased elicited by 10.0,20.0μmol/L AOH compared with the control in a concentration-dependent manner.The results of immunocytochemistry and immunofluorescence coincided with Western Blotting.Phosphorylation of CREB was increased in time-dependent manner andhad reached maximal levels by 2h after AOH treatment,which was latter than the time at which the level of p-PKAhad increased. The results of immunocytochemistry,immunofluorescence and Western Blotting indicated that H89 partly blocked the AOH-induced phosphorylation of CREB.3.AOH -induced activation of p38MAPKIn the results of Western blot,AOH-elicited phosphorylated p38 was significantly increased compared with the control(P＜0.05) in a concentrationdependent manner.Phosphorylation of p38 was increased in time-dependent manner and had reached maximal levels by 2h after AOH treatment.The results of Western Blotting indicated that SB203580 blocked the AOH-induced phosphorylation of p38.4.AOH -induced activation of ATF2In the results of Western blot,AOH-elicited phosphorylated ATF2 was significantly increased compared with the control in a concentration- dependent manner.Phosphorylation of ATF2 was increased in time-dependent manner and had reached maximal levels by 2h after AOH treatment,which coincided with the time at which the level of p-p38 kinasehad increased.The results of Western Blotting indicated that SB203580 decreased the AOH-induced phosphorylation of ATF2.5.The effect of AOH on phosphorylation of JNKIn general,the JNK pathways are preferentially activated by genotoxic agents and cytokines,and tend to mediate the stress response.The results of Western Blotting indicated that AOH failed to increase phosphorylation of JNK.Pretreatment of NIH3T3 cells with JNK inhibitor SP600125 failed to decrease AOH-induced ATF-2 phosphorylation at all.These results showed that ATF-2 phosphorylation was catalyzed mainly by p38 kinase but not by JNK.6.Phosphorylation of CREB partly dependent on p38 activationExposure of NIH3T3 cells to AOH resulted in CREB phosphorylation which was significantly decreased after reduction of p38 activity using the specific inhibitor SB203580.Chaper 3 AOH-induced DNA polβexpression is dependent on the activated PKA-CREB and p38MAPK-ATF2 pathway in NIH3T3 cellsMethods1.To study whether AOH-elicited expression of polβwas dependent of PKA-CREB pathway,NIH3T3 cells were pretreated with an inhibitor of PKA(H89) for 1h,then exposed to 20.0μmol/L AOH for 16h.The expression of polβwas investigated with immunocytochemistry and Western Blotting.2.To study whether AOH-elicited expression of polβwas dependent of p38MAPK -ATF2 pathway,NIH3T3 cells were pretreated with an inhibitor of p38(SB203580) for 1h,then exposed to 20.0μmol/L AOH for 16h.The expression of polβwas detected with Western Blotting.3.NIH3T3 cells were pretreated with H89 combination with SB203580 for 1h,then exposed to 20.0μmol/L AOH for 16h.The expression of polβwas investigated with Western Blotting.4.NIH3T3 cells were pretreated with an inhibitor of JNK(SP600125) for 1h,then exposed to 20.0μmol/L AOH.The expression of polβwas investigated with Western Blotting.Results 1.AOH up-regulated polβexpression via the activated PKA-CREB pathway in NIH3T3 cellsThe expression of DNA polβin NIH3T3 cells induced by AOH was significantlyhigher than that in the control group(P＜0.05).However,H89 decreased the AOH-induced DNA polβexpression.The inhibitory effect of H89 was incomplete,however,it indicated that PKA activation was not the only mechanism by which AOH induced DNA polβexpression.2.AOH up-regulated polβexpression via the activated p38-ATF2 pathway in NIH3T3 cellsSB203580 partly blocked the AOH-induced DNA polβexpression which was significantly lower than that in theAOH-induced directly group(P＜0.05).The inhibitory effect of SB203580 was incomplete,however,it indicated that p38 activation was not the only mechanism by which AOH induced DNA polβexpression.3.H89 and SB203580 cooperated to decrease AOH-induced DNA polβexpressionThe Western Blotting results showed H89 combination with SB203580 blocked the AOH-induced DNA polβexpression which was significantly lower than that H89 or SB203580 alone group(P＜0.05).4.The AOH-induced expression of Polβwere independent on JNK pathwayIn general,the JNK pathways are preferentially activated by genotoxic agents and cytokines,and tend to mediate the stress response.Pretreatment of NIH3T3 cells with JNK inhibitor SP600125 failed to decrease AOH-induced polβexpression. PartⅡThe role of activated of PKA-CREB and p38MAPK-ATF2 pathway regulating the expression of DNA polβin EC9706 cellsChapter 1 Activation of PKA-CREB and p38MAPK-ATF2 pathway controls the expression of DNA polβin EC9706 cellsMethods1.The activation status of PKA-CREB signaling pathway in EC9706 cells was investigated with immunocytochemistry and Western Blotting.In addition,cells were treated with an inhibitor of PKA(H89) for 2h,and activation of PKA and CREB were detected by the same methods.2.The activation status of p38MAPK-ATF2 signaling pathway in EC9706 cells was investigated with immunocytochemistry and Western Blotting.In addition,cells were treated with an inhibitor of p38(SB203580) for 2h,and activation of p38 and ATF2 were detected by the same methods.3.EC9706 cells were treated with H89,SB203580 or combinations thereof for 16h, and the expression of polβwas detected with immunocytochemistry and Western Blotting.The CRE-DNA binding activation of CREB or ATF2 was determined by electrophoretic mobility-shift assays(EMSA).Results1.Activated PKA-CREB pathway in EC9706 cellsImmunocytochemistry and Western Blotting showed that EC9706 cellshad activation of PKA and CREB.H89 reduced phosphorylation of both PKA and CREB.2.Activated p38-ATF2 pathway in EC9706 cellsImmunocytochemistry and Western Blotting showed that EC9706 cellshad activation of p38 and ATF2.SB203580 reduced phosphorylation of both p38 and ATF2. 3.Activated PKA-CREB and p38MAPK-ATF2 pathway up-regulated the expression of DNA polβin EC9706 cellsImmunocytochemistry and Western Blotting showed that untreated EC9706 cells had expression of polβ.H89,SB203580 alone or combinations thereof reduced expression of polβcompared with the control group.However,The polβexpression of H89 plus SB203580 group was lower than those of H89 group and SB203580 group alone(P＜0.05).The results indicaed that PKA and p38MAPK acted co-operatively to control DNA polβexpression in EC9706 cells.Chapter 2 The effect of blocking the signal pathway of polβexpression on the biological characteristics and the sensitivity to cisplatin in EC9706 cellsMethods1.EC9706 cells were treated with H89,SB203580 or combinations thereof for 24h in order to block PKA and p38MAPK pathway and decrease the expression of polβ. Then the number of viable cells were determined by WST-8 and the morphologic change of the cells were observed by a inverted microscope,and the cells were stained with FITC-annexin V and PI for analyzing cell apoptosis by flow cytometry in Cell Quest acquisition and analysis programs.2.EC9706 cells were treated with H89 or/and SB203580 combination with cisplatin for 24h in order to block PKA and p38MAPK pathway and decrease the expression of polβ.Then the number of viable cells were determined by WST-8 and the morphologic change of the cells were observed by a inverted microscope.The cells were stained with FITC-annexin V and PI for analyzing cell apoptosis by flow cytometry in Cell Quest acquisition and analysis programs.Results1.The effect of blocking PKA and p38MAPK pathway and decreasing the expression of polβon biological characteristics of EC9706 cells Treatment with H89 or SB203580,EC9706 cells growth were slower compared with the control,and some cells became smaller.When H89 plus SB203580,these changes were more significant.Treatment with H89 or SB203580 for 24h or 48h,EC9706 cells growth were inhibited compared with the control group(P＜0.05).When H89 plus SB203580,these changes were more significant(P＜0.05).H89 or SB203580 alone could increase cell apoptosis(P＜0.05).While H89 combination with SB203580,the results were more prominent(P＜0.05).2.The increasing sensitivity of EC9706 to cisplatin when blocking PKA and p38MAPK pathway and decreasing the expression of polβH89 or SB203580 combination with cisplatin -treated cells showed an decrease in the speed of cell growth,and the number of floating cells increased.While combination with all,the results were more prominent.Treatment with H89 or SB203580 plus cisplatin,the EC9706 cells growth were inhibited drastically compared with cisplatin alone(P＜0.05).While H89 and SB203580 in combination with cisplatin,the results were more prominent(P＜0.05). H89 or SB203580 in combination with cisplatin could increase cell apoptosis (P＜0.05).While H89 and SB203580 in combination with cisplatin,the results were more prominent(P＜0.05).The results likely indicated that H89 and SB203580 enhanced sensitivity of the EC9706 cells to cisplatin.Conclusions1.AOH inhibits NIH3T3 cells proliferation,induces G2/M phase arrest and DNA damage and increases clony formation.AOH also induces the expression of DNA polβ,which may be one of important mechanisms being involved in AOH carcinogenicity.2.The results have shown that AOH-elicited expression of polβare dependent of the activated PKA-CREB and p38MAPK-ATF2 pathway in NIH3T3 cells.3.There is a constitutively activated PKA-CREB and p38MAPK-ATF2 signaling pathway in EC9706 cell lines.They play a cooperation role in the expression of DNA polβ.H89 and SB203580 blocks the two pathway and downregulate the expression of DNA polβ,which causes the changes of biological characteristics of EC9706 cells. H89 and/or SB203580 in combination with cisplatin,can obviously inhibit cell proliferation and induce apoptosis and have synergistic anti-tumor efficacy,which may be become a novel therapeutic strategies of esophageal carcinoma.