Study Of Sterigmatocystin-induced DNA Damage And Cell Cycle Arrest In Immortalized And Primary Human Esophageal Epithelial Cells In Vitro

Sterigmatocystin （ST）, the secondary metabolite of Aspergillus verdicolorand Aspergillus nidulans etc, is a carcinogenesis mycotoxin. As a contaminant,ST is quite commonly detected in grains, corn, bread, cheese, spices, coffeebeans, soybeans, pistachio nuts, animal feed and silage. In addition, it is evenfound in indoor environment, such as carpet and building materials. Therefore,ST exposure to human is a public health issue. ST shows differenttoxicological, mutagenic and carcinogenic effects in animals and has beenrecognized as a2B carcinogen （possible human carcinogen） by InternationalAgency for Research on Cancer.Cixian county in China is one of the highest incidence areas ofesophageal cancer in the world. Studies in the past showed that foodstuffsconsumed by the local residents in this area were frequently contaminatedwith many kinds of fungi. Among the contaminating mycotoxins, ST is one ofthe major pollution mycotoxin. Both the detection rate （60.25%） and the meancontent （9.14μg/kg） of ST in Cixian county were significantly higher thanthose in the relative low risk area. Precancerous lesions in the esophagus ofmice could be induced by feeding ST-contaminated foodstuffs, suggesting thatST exposure might be a risk factor for esophageal cancer. Therefore, it ishighly significant to explore the role of ST on human esophageal epithelium.In this study, on the basis of preliminary epidemiological investigationsand animal experiments, we used immortalized human esophageal epithelialcells （Het-1A） and primary human esophageal epithelial cells （EPC） toexplore the toxic effect of ST treatment on esophageal epithelial cells. First,we explore the effect of ST treatment on DNA damage, DNA repairmechanisms, and cell cycle distribution and to evaluate the role of MMR proteins in ST-induced DNA damage and cell cycle arrest in Het-1A cells.Furthermore, we explore the effect of ST treatment on DNA damage and cellcycle distribution in EPC cells, and the reason of different cell cycle arrestinduced by ST in Het-1A cells and EPC cells. Finally, we investigated thechanges of ATM-Chk2and p53-p21signaling pathway to explore the putativemolecular mechanism of ST-induced different cell cycle arrest in Het-1A cellsand EPC cells. Our study may provide some clues on the putative esophagealtoxicity and carcinogenic effects of ST. It is highly significant to improve thefood security of rural residents, especially the residents of high incidence areaof esophageal cancer in China.Part Ⅰ Role of hMLH1in sterigmatocystin-induced DNA damage and G₂phase arrest in human esophageal epithelial Het-1A cellsObjective: To explore the role of hMLH1in sterigmatocystin-induced G₂phase arrest in human esophageal epithelial Het-1A cells in vitroMethods:1Effects of ST on the cell cycle distribution of Het-1A cellswere detected by FCM, Giemsa staining and immunofluoresence staining.2The changes of the G₂phase key factors （Cdc25C, Cdc2and CyclinB1） weredetected both at mRNA and protein level by Western Blot and Real time-PCRrespectively.3Effects of ST on the Cdc2-CyclinB1complex was detected byimmunoprecipitation.4DNA damage induced by ST was observed by alkalinecomet assay in Het-1A cells.5The changes of the DNA repair geneexpression were detected both at mRNA and protein level by Western Blot andReal time-PCR respectively.6Effects of ST treatment on cell cycledistribution and G₂phase key factors of Het-1A cells transfected with eitherthe control siRNA or hMLH1and hMSH2-specific siRNA were detected byFCM and Western Blot.7We examined the extent of DNA damage in hMLH1siRNA-transfected Het-1A cells treated with or without ST using an alkalinecomet assay.Results:1Effects of ST on the cell cycle distribution of Het-1A cellsThe FCM results showed that the proportion of cells in the G₂/M phase was significantly increased and the proportion of cells in G0/G₁and S phasewas significantly decreased in ST-treated cells compared with the solventcontrol group （P<0.05）. Giemsa staining results showed that compared withthe mitotic index （MI） of the solvent control group （8.02±0.59%）, the MI of12and24μM ST treatment cells （5.77±0.23%and5.35±0.28%） wassignificantly lower （P<0.05）. Immunofluorescence results showed that thephospho-histone H3（Ser-10） levels in24μM ST treatment group weresignificantly reduced compared with that of the solvent control group （P<0.05）.These results indicated that ST treatment could induce G₂cell cycle arrest inHet-1A cells.2Effects of ST on G₂phase key regulatory factor in Het-1A cellsWestern Blot results showed that ST treatment could increase theexpression of Cdc25C, Cdc2and CyclinB1proteins. Similarly, ST treatmentcould also increase the phosphorylation level of Cdc25C and Cdc2. Theimmunoprecipitation results showed that compared to the solvent controlgroup, the level of the Cdc2-CyclinB1complex decreased significantly in theST treatment groups. Real-time PCR results showed that ST treatment couldincrease Cdc25C, Cdc2and CyclinB1expression at the mRNA level （P<0.05）.3Effects of ST on DNA damage in Het-1A cellsAn alkaline comet assay was used to examine the status of the cells afterST treatment. Cells treated with6,12or24μM ST showed clearly visiblecomet tails of DNA fragments indicative of broken DNA strands, whereas inthe solvent control group, these changes were not observed. The extent ofdamage was analyzed further by CASP. The results showed that treatmentwith ST caused a significant increase in Tail DNA%, Tail Length and Olivetail moment （P<0.05）. These data confirmed that ST could cause DNAdamage in Het-1A cells.4Effects of ST on DNA repair systems in Het-1A cellsReal-time PCR results showed that the expression of XPC, hMLH1andhMSH2mRNA increased significantly after24μM ST treatment, but no suchchanges were found in XRCC1, RAD51or DNA-PK expression （P<0.05）. These data suggested that ST-induced DNA damage could initiate NER andMMR.To further confirmed tha effect of ST on MMR system, we use theWestern Blot method to observe hMLH1and hMSH2protein expression. Theresults showed that ST treatment significantly increased the expression ofhMLH1and hMSH2proteins （P<0.05）.5The efficiency of hMLH1and hMSH2siRNA interferenceAt48h post transfection, the cells were harvested and processed foranalysis of Western Blot and Real-time PCR. The results showed that thesiRNAs dramatically reduced hMLH1and hMSH2expressions at both themRNA and protein levels. These data indicated that both of the two siRNAsequences could knockdown the expression of hMLH1and hMSH2in Het-1Acells. Therefore, hMLH1and hMSH2siRNA were selected to be used in thesubsequent experiments.6Effects of ST treatment on cell cycle distribution of Het-1A cellstransfected with hMLH1and hMSH2-specific siRNAThe FCM results showed that cell cycle distribution in hMLH1siRNA+DMSO treatment group have no changed compared with the ControlsiRNA+DMSO treatment group （P>0.05）. The proportion of cells in the G₂/Mphase was significantly decreased in hMLH1siRNA+24μM ST treatmentgroup compared with that of Control siRNA+24μM ST treatment group（P<0.05）, but still higher than that in Control siRNA+DMSO treatment group（P<0.05）. It indicated that ST-induced G₂phase arrest was markedlyattenuated in cells transfected with hMLH1siRNA compared with the controlsiRNA group treated with24μM ST. However, silencing hMSH2had noeffect on ST-induced G₂phase arrest. Based on these results, we concludedthat hMLH1, but not hMSH2, of the MMR system is involved in the G₂phasearrest induced by ST in Het-1A cells.7Effects of ST treatment on G₂phase key factors of Het-1A cellstransfected with hMLH1and hMSH2-specific siRNAThe Western Blot results showed that G₂phase key factors in hMLH1 siRNA+DMSO treatment group have no changed compared with the ControlsiRNA+DMSO treatment group （P<0.05）. The expressions of G₂phase keyfactors was significantly decreased in hMLH1siRNA+24μM ST treatmentgroup compared with Control siRNA+24μM ST treatment group （P<0.05）,but still higher than that in Control siRNA+DMSO treatment group （P<0.05）.It indicated that hMLH1-siRNA prevented the ST-induced up-regulation of theCyclin B1, Cdc2/p-Cdc2and Cdc25C/p-Cdc25C proteins.8hMLH1may act as a direct sensor of ST-mediated DNA damageWe examined the extent of DNA damage in hMLH1siRNA-transfectedHet-1A cells treated with or without ST using an alkaline comet assay. Boththe hMLH1siRNA-transfected cells and the control siRNA-transfected cellsexhibited global, but small, DNA tails. Moreover, we found that the level oftotal DNA strand breaks was similar in the hMLH1siRNA-transfected cellsand control siRNA-transfected cells after ST treatment. Analysis by CASPshowed nearly identical values of Tail DNA%, Tail Length and Olive tailmoment between the two groups （P<0.05）. These results suggested thathMLH1did not lead to the formation of secondary lesions, indicating thatDNA damage recognized by hMLH1might lead to cell cycle arrest throughthe general DNA damage sensor model in Het-1A cells after ST treatment.Part Ⅱ Role of SV40LT in sterigmatocystin-induced different cell cyclearrest in Het-1A and EPC cellsObjective: To explore the role of SV40LT in sterigmatocystin-induceddifferent cell cycle arrest in Het-1A and EPC cells.Methods:1DNA damage induced by ST was observed by alkaline cometassay in EPC cells.2Effects of ST on the cell cycle distribution of EPC cellsdetected by FCM.3Effects of ST on the G₁phase key factors were detectedby Western Blot in EPC cells and Het-1A cells.4Effects of ST treatment oncell cycle distribution of Het-1A cells and cell cycle regulatory factorstransfected with either the control siRNA or SV40LT-specific siRNA wasdetected by FCM and Western Blot. Results:1Effects of ST on DNA damage in EPC cellsAn alkaline comet assay was used to examine the status of the cells afterST treatment. Cells treated with24μM ST showed clearly visible comet tailsof DNA fragments indicative of broken DNA strands, whereas in the solventcontrol group, these changes were not observed. The extent of damage wasanalyzed further by CASP. The results showed that treatment with ST causeda significant increase in Tail DNA%, Tail Length and Olive tail moment（P<0.05）. These data confirmed that ST could cause DNA damage in EPCcells.2Effects of ST on the cell cycle distribution of EPC cellsThe FCM results showed that the proportion of cells in the G₁phase wassignificantly increased and the proportion of cells in G₂/M and S phase wassignificantly decreased in ST-treated EPC cells compared with the solventcontrol group （P<0.05）. These results indicated that ST treatment could induceG₁phase arrest in EPC cells.3Effects of ST on G₁phase regulatory factor in EPC cellsWestern Blot results showed that ST treatment could decrease theexpression of Rb/p-Rb and E2F1proteins. Similarly, the expression ofCyclinD1, Cdk4, CyclinE1and Cdk2were reduced after exposure to ST inEPC cells （P<0.05）.4Effects of ST on G₁phase regulatory factor in Het-1A cellsWestern Blot results showed that ST treatment could increase theexpression of Rb/p-Rb and E2F1proteins. Moreover, the expression ofCyclinD1and Cdk4were reduced, while the expression of CyclinE1and Cdk2were increased after exposure to ST in Het-1A cells （P<0.05）.5The efficiency of SV40LT siRNA interferenceAt48h post transfection, the cells were harvested and processed foranalysis of Western Blot and Real-time PCR. The results showed that thesiRNA dramatically reduced SV40LT expression at both the mRNA andprotein levels. These data indicated that the siRNA sequences could knockdown the expression of SV40LT in Het-1A cells. Therefore, SV40LTsiRNA were selected to be used in the subsequent experiments.6Effects of ST treatment on cell cycle distribution of Het-1A cellstransfected with SV40LT-specific siRNAThe FCM results showed that cell cycle distribution in SV40LTsiRNA+DMSO treatment group have no changed compared with the ControlsiRNA+DMSO treatment group （P<0.05）. The proportion of cells in the G₂/Mphase was significantly decreased in SV40LT siRNA+24μM ST treatmentgroup compared with Control siRNA+24μM ST treatment group （P<0.05）,but still higher than that in Control siRNA+DMSO treatment group （P<0.05）.7Effects of ST treatment on G₁phase regulatory factors of Het-1A cellstransfected with SV40LT-specific siRNAThe Western Blot results showed that the expression of Rb/p-Rb andE2F1in SV40LT siRNA+DMSO treatment group have no changed comparedwith the Control siRNA+DMSO treatment group （P<0.05）. The expression ofRb/p-Rb and E2F1were significantly decreased in SV40LT siRNA+24μM STtreatment group compared with Control siRNA+24μM ST treatment group（P<0.05）. It indicated that SV40LT-siRNA reversed the ST-inducedup-regulation of the Rb/p-Rb and E2F1proteins.8Effects of ST treatment on G₂phase regulatory factors of Het-1A cellstransfected with SV40LT-specific siRNAThe Western Blot results showed that the expression of Cdc2/p-Cdc2andCyclinB1in SV40LT siRNA+DMSO treatment group have no changedcompared with the Control siRNA+DMSO treatment group （P<0.05）. Theexpression of Cdc2/p-Cdc2and CyclinB1were significantly decreased inSV40LT siRNA+24μM ST treatment group compared with ControlsiRNA+24μM ST treatment group （P<0.05）. It indicated that SV40LT-siRNAreversed the ST-induced up-regulation of the Cdc2/p-Cdc2and CyclinB1proteins.All the above results showed that knockdown SV40LT antigen in Het-1Acells could partially reversed the effect of ST on the cell cycle distribution and cell cycle regulatory proteins, suggesting that Het-1A cells which has lowerexpression of SV40LT antigen after ST treatment have a tendency to occur G₁phase arrest. It indicated that SV40LT-immortalized Het-1A cells exhibited anabsence of G₁phase arrest.PartⅢ Role of ATM-Chk2and p53-p21signaling pathway insterigmatocystin-induced cell cycle arrest in Het-1A and EPC cellsObjective: To explore the putative molecular mechanism of ST-induceddifferent cell cycle arrest in Het-1A cells and EPC cells.Method:1The expression of related molecules involved in theATM-Chk2and p53-p21signaling pathway was observed by Western Blotafter ST treatment alone.2The activation of ATM-Chk2signaling pathwayand cell cycle distribution were detected by FCM and Western blot afterpretreatment with ATM specific inhibitor-KU55933.3Effects of ST treatmenton cell cycle distribution of Het-1A cells and G₂phase key factors transfectedwith either the control siRNA or p53-specific siRNA were detected by FCMand Western Blot.Results:1Effects of ST on ATM-Chk2and p53-p21signaling pathway in EPCcellsWestern Blot results showed that the expresion of ATM and Chk2proteinhave no significant difference in ST treatment group compared with solventcontrol group in EPC cells （P>0.05）. However, the phosphorylation of ATMand Chk2were significantly increased in ST treatment group as compared tothe solvent control group in EPC cells （P<0.05）. These results suggested thatATM-Chk2signaling pathway could be activated by ST treatment in EPCcells.Western Blot results showed that the expression of p53/p-p53and p21were decreased in ST treatment group compared with solvent control group inEPC cells （P<0.05）. It indicated that p53-p21signaling pathway was notactivated after ST treatment in EPC cells.All the above results showed that ATM-Chk2signaling pathway, but not p53-p21signaling pathway, could be activated after ST treatment in EPC cells.2Effects of ST on ATM-Chk2and p53-p21signaling pathway in Het-1AcellsWestern Blot results showed that the expresion of ATM and Chk2proteinhave no significant difference in ST treatment group compared with solventcontrol group in Het-1A cells （P>0.05）. However, the phosphorylation of ATMand Chk2were significantly increased in ST treatment group as compared tothe solvent control group in Het-1A cells （P<0.05）. These results suggestedthat ATM-Chk2signaling pathway could be activated by ST treatment inHet-1A cells.Western Blot results showed that the expression of p53/p-p53and p21were significantly increased in ST treatment group as compared to the solventcontrol group in Het-1A cells （P<0.05）. It indicated that p53-p21signalingpathway was activated after ST treatment in Het-1A cells.All the above results showed that ATM-Chk2and p53-p21signalingpathway were both activated after ST treatment in Het-1A cells.3Effects of KU55933pretreatment on ATM-Chk2signaling pathway inHet-1A cellsWe further explored the effect of ATM-Chk2signaling pathway after STtreatment with or without the ATM specific inhibitor-KU55933pretreatment.The results showed that the phosphorylation levels of ATM and Chk2inKU55933pretreatment+ST treatment group was decreased significantlycompared with ST treatment group （P<0.05）. It indicated that KU55933canblock ST-induced activation of ATM-Chk2signaling pathway in Het-1A cells.4Effects of ST on cell cycle distribution in Het-1A cells with KU55933pretreatmentFCM analysis showed that the cell cycle distribution has no significantdifferences in KU55933pretreatment+ST treatment group compared with STtreatment group （P<0.05）. The proportion of cells in G₂/M phase increased byST was not affected after KU55933pretreatment, suggesting that theATM-Chk2signaling pathway is not involved in G₂phase arrest induced by ST in Het-1A cells.5The efficiency of p53siRNA interferenceAt48h post transfection, the cells were harvested and processed foranalysis of Western Blot and Real-time PCR. The results showed that thesiRNA dramatically reduced p53expression at both the mRNA and proteinlevels. These data indicated that the siRNA sequences could knockdown theexpression of p53in Het-1A cells. Therefore, p53siRNA were selected to beused in the subsequent experiments.6Effects of ST treatment on p53-p21signaling pathway of Het-1A cellstransfected with p53-specific siRNAWestern Blot results showed that the expression of p53/p-p53and p21was significantly decreased in p53siRNA+24μM ST treatment groupcompared with Control siRNA+24μM ST treatment group （P<0.05）. Itindicated that p53siRNA can block ST-induced activation of p53-p21signaling pathway in Het-1A cells.7Effects of ST treatment on cell cycle distribution of Het-1A cellstransfected with p53-specific siRNAThe FCM results showed that cell cycle distribution in p53siRNA+DMSO treatment group has no changed compared with the ControlsiRNA+DMSO treatment group （P>0.05）. The proportion of cells in the G₂/Mphase was significantly decreased in p53siRNA+24μM ST treatment groupcompared with Control siRNA+24μM ST treatment group （P<0.05）. Itindicated that the ST-induced G₂phase arrest was markedly reversed in cellstransfected with p53siRNA compared with the control siRNA group treatedwith24μM ST.8Effects of ST treatment on G₂phase regulatory factors of Het-1A cellstransfected with p53-specific siRNAWestern Blot results showed that G₂phase regulatory factors in p53siRNA+DMSO treatment group has no significant differences compared withthe Control siRNA+DMSO treatment group （P>0.05）. The expression of G₂phase key factors were significantly decreased in p53siRNA+24μM ST treatment group compared with Control siRNA+24μM ST treatment group（P<0.05）. It indicated that p53-siRNA reversed the ST-induced up-regulationof the CyclinB1, Cdc2/p-Cdc2and Cdc25C/p-Cdc25C proteins.All the above data indicated that ST treatment could activate ATM-Chk2and p53-p21signaling pathway in Het-1A cells. Further studies showed thatthe activation of ATM-Chk2signaling pathway was not involved in G₂phasearrest induced by ST in Het-1A cells. However, the activation of p53-p21signaling pathway might be involved in G₂phase arrest induced by ST inHet-1A cells.Conclusions:1ST could induce DNA damage and triggered G₂phase arrest in Het-1Acells.2hMLH1might act as a direct sensor of ST-mediated DNA damage and beparticipated in the G₂phase arrest induced by ST through up-regulatingG₂/M regulatory proteins.3ST could induce DNA damage and triggered G₁phase arrest in EPC cells4G₁phase arrest of EPC cells might be the general response of humanesophageal epithelial cells to ST-DNA damage, whileSV40LT-immortalized Het-1A cells exhibited an absence of G₁arrest.5ATM-Chk2signaling pathway might be involved in G₁phase arrestinduced by ST in EPC cells rather than participating in G₂phase arrestinduced by ST in Het-1A cells.6p53-p21signaling pathway might be involved in G₂phase arrest inducedby ST through up-regulating G₂/M regulatory proteins.