Study Of Sterigmatocystin-Induced G₂ Phase Checkpoint Adaptation And Its Possible Mechanisms In Immortalized Human Gastric Epithelial Cells（GES-1） In Vitro

Sterigmatocystin(ST), a toxic secondary metabolite mainly produced by Aspergillus nidulans and Aspergillus versicolor. As a contaminant, ST is quite commonly detected in peanuts, corn, rice and grains. According to in vivo as well in vitro studies,ST exhibits potential toxicological, mutagenic and carcinogenic effects and has been classified in Group 2B(possibly carcinogenic) by the International Agency of Research in Cancer. Experiments in vivo showed that ST could lead to lung adenocarcinoma and gastric mucosal hyperplasia of experimental animals and resulted in chromosome aberration of bone marrow cells in rats. Experiments in vitro showed ST also could injury DNA of A549, Het-1A and GES-1cells directly. In addition, ST could cause DNA damage and form DNA adducts. Therefore, many scholars thought effects of ST on DNA damage might be important mechanisms of carcinogenesis.DNA,the basis of genetic material, is stimulated by different harmful factors constantly and cause damage. An important part of the cellular response to DNA damage is checkpoint activation. Cell cycle checkpoint is the speed limit point of cell proliferation cycle. It is now generally believed that the main function of the DNA damage checkpoint is to sense the existence of DNA damage and produce a specific damage signal that is eventually transduced to the downstream effectors to arrest cells in the G1/S-, S- and G2/M-phases of the cell division cycle to give cells enough time to repair damaged DNA. If possible, cells will repair the damaged DNA and reenter the cell cycle. We call this process "recovery". In this process, all of the damage is completely repaired, so that cells can divide without running the risk of transferring damaged gene to their offspring. However, when DNA damage cannot be repaired, cells will undergo apoptosis to eliminate the heavily damaged cells. In this way, genomic integrity can be guaranteed to a maximum extent. The disruption of the DNA damage checkpoint pathway can lead to genome unstable and neoplasia.Note that DNA repair and apoptosis are not the inevitable paths of all damaged DNA. Part of cells can eventually escape a persistent DNA damage-induced arrest and divide without repairing the damage. The process is termed as "adaptation". Checkpoint adaptation is one of the steps that lie between cell cycle arrest and cell death. Although cells of checkpoint adaptation are doomed to die, some researchers also find that a small number of cells actually survive checkpoint adaptation and start proliferating and these cells appear to have rearranged genome. The cells with rearranged genome may be a source of tumor formation.Our previous study showed that ST treatment could induce DNA damage and subsequently lead to cell cycle arrest and apoptosis in normal human gastric epithelial cells(GES-1). However, Can checkpoint adaptation occur after DNA damage induced by ST? It is still unclear the effects and possible consequences of checkpoint adaptation on G2 arrest induced by ST. On the basis of the previous studies, we will explore the development and changes of GES-1 cells induced by ST after G2 arrest. The research is divided into three parts. First, we will explore the effects of ST on cell cycle distribution of GES-1 cells and then further explore whether checkpoint adaptation can occur after a sustained G2 arrest. Furthermore, we will examine whether ST can induce the activation of ATR-Chk1 signaling pathway and explore the role and the possible molecular mechanism of Chk1 in ST-mediated checkpoint adaptation in GES-1 cells. Finally, we will investigate effects of ST on mitosis entry and exit and explore its correlation with PLK1 expression to analyze the role of PLK1 in G2 arrest and subsequently checkpoint adaptation induced by ST.The results of this study will provide a new clue for the study of mechanism in tumorigenesis induced by ST and establish a theoretical basis for the prevention and treatment of gastric cancer.Part Ⅰ Effects of sterigmatocystin treatment on G2 checkpoint in immortalized human gastric epithelial GES-1 cellsObjective: To study the effects of ST on proliferation and G2 arrest of GES-1 cells, and further to explore whether checkpoint adaptation can occur in ST-treated GES-1 cells.Methods:1 Effects of ST at different concentrations(0-48 μM) for 24 h, 48 h, 72 h and 96 h on proliferation of GES-1 cells were tested by MTT.2 Effects of ST on the cell cycle distribution and the expression of mitosis marker protein(p-H3) were detected by FCM.3 Morphology changes in ST-treated GES-1 cells were observed by inverted phase contrast microscope.4 Expression of γH2AX and Changes of G2 phase key factors were detected by Western Blot in cells collected by mechanical shake-off.5 Laser confocal double immunofluorescence staining method was used to detect the expression of Cyclin B1, p-H3 and γH2AX in ST-treated GES-1 cells.Results:1 Effects of ST treatment on proliferation of GES-1 cellsTo explore the cytotoxic effects of ST on GES-1 cells, cells were treated with increasing concentrations(0-48 μM) of ST for 24 h, 48 h, 72 h and 96 h and cell viability were detected by MTT assay. Cell viability in ST-treated groups showed a significant decrease(P<0.05) in dose(r24h=-0.968, r48h=-0.985, r72h=-0.992) and time(r6μM=-0.976, r12μM=-0.980, r24μM=-0.981) dependent manner. The IC50 of ST was 366.18 μM at 48 h and 9.87 μM at 96 h. These data confirmed that ST inhibited growth of GES-1 cells in a dose- and time-dependent manner. According to the above results, we chose 12 μM ST for subsequent experiments.2 Effects of ST on the cell cycle distribution in GES-1cells treated with ST for different timeSimilarly to our previous study, FCM results showed that the proportion of cells in the G2/M phase was significantly increased in ST-treated cells compared with the solvent control group and the percentage of G2/M phase in cells treated with ST for 48 h was increased than cells treated with ST for 24 h(P<0.05). These results further indicated that ST treatment could induce G2/M cell cycle arrest in GES-1 cells.3 Effects of ST on the expression of p-H3(Ser-10) in GES-1cells treated with ST for different timeThe p-H3(Ser-10) is the marker protein of cells in mitotic phase and its positive expression represent the mitotic cells. In order to further determine the exact phase of GES-1 cells treated with ST, we counted cells positive for p-H3. The FCM results showed that the p-H3 expression in the group treated with ST for 24 h was decreased compared with the solvent control group. At 48 h, the p-H3 expression increased sharply(P<0.05). These results indicated that ST treatment could induce G2 arrest in GES-1 cells at first, after a prolonged G2 arrest, part of cells entered M phase.4 Morphology observations of GES-1cells treated with ST for different timeInverted phase contrast microscope showed GES-1 cells were flat and strongly adherent. At 24 h after ST treatment, nearly all cells were similar to normal cells. At 48 h, relative large and rounded cells appeared which had high refraction and were weakly adherent. These rounded cells could be collected by mechanical shake-off, leaving behind the flattened cells.5 Immunophenotypic analyses of large and rounded GES-1cells collected by mechanical shake-offIn order to prove these large and rounded cells treated with ST for 48 h were mitotic cells. A contrast observation of cell cycle distribution was made in these large and rounded cells and cells still tightly adherent after mechanical shake-off. The FCM results revealed that most of the rounded cells collected by mechanical shake-off were in G2/M phase, and the percentage was 74%. The percentage of G2/M phase in adherent cells was 45.7%(P<0.05).The percentage of p-H3 positive cells was 77.1% in cells collected by shake-off, and was 0.534% in adherent cells(P<0.05). We further explored the expression of G2 phase key factors in cells collected by mechanical shake-off.GES-1 cells were treated with DMSO as a solvent control or treated with 12 μM ST for 48 h. Cells treated with ST for 48 h were fractionated by mechanical shake-off as adherent cells and mitotic shake-off cells, Nocodazole as a positive control group. The Western Blot results showed the expression of p-Cdc2 in Shake-off group was lower than ST-treated group and Cyclin B1 expression in Shake-off group was higher than solvent control group(P<0.05). These expression were similar to that of Nocodazole-treated cells(P>0.05).These results indicated that the expression of G2 phase key factors in cells collected by shake-off accorded with cells in mitotic phase. These data confirmed that part of cells passed through G2 checkpoint and entered mitosis after ST treatment.6 Effect of ST on expression of Cyclin B1 in GES-1 cellsCyclin B1 is a kind of proteins related to cell division which translocates from the cytoplasm to the nucleus in early M phase. Confocal microscope showed Cyclin B1 expression was mainly localized in the cytoplasm of cells in solvent control group. Nuclear Cyclin B1 staining was increased in cells treated with ST for 48 h.7 The expression of γH2AX foci in mitotic GES-1 cells positive for p-H3The γH2AX foci were molecular markers for DNA double strand breaks(DSBs). To explore whether GES-1 cells treated with ST came into mitosis with damaged DNA, we detected the expression of γH2AX foci in these cells. Laser confocal double immunofluorescence staining showed many cells positive for p-H3 contained γH2AX foci after ST treatment. Mitotic cells were scored for the number of γH2AX foci, about 100 mitotic cells were scored for each treatment. The results showed: the number of γH2AX foci in cells treated with ST was much more than cells in solvent control group(P<0.05).To further demonstrate the DNA damage in mitotic GES-1 cells treated with ST for 48 h, we collected these mitotic cells by mechanical shake-off.Western Blot results showed the expression of γH2AX was increased dramatically in rounded mitotic cells group than other groups.These results indicated that ST treatment could induce G2 arrest in GES-1 cells at first. However, part of cells came into mitosis phase with damaged DNA after a prolonged G2 arrest. Checkpoint adaptation existed in GES-1 cells treated with ST.Part ⅡRole of Chk1 in sterigmatocystin-induced checkpoint adaptation in GES-1 cellsObjective: Cell cycle checkpoint is a complex signal transduction system. ATR and Chk1 are important components of this system. The ATR-Chk1 signaling pathway is activated in response to DNA damage. Activated Chk1 can pass signals to Cdc25 C phosphatase and Cdc2 to arrest cells in G2 phase. Some studies showed that dephosphorylation of Chk1 played an important role in checkpoint adaptation in tumor cells. The first part of our study suggested G2 checkpoint adaptation occurred in GES-1 cells treated with ST for 48 h. To study the mechanism of checkpoint adaptation, we explored the role of Chk1 in ST-induced G2 checkpoint adaptation in GES-1 cells from the point of signaling pathway.Methods:1 Expression of related gene involved in the ATR-Chk1 signaling pathway was observed by Western Blot and Real-time PCR respectively after ST treatment.2 Effects of Chk1 si RNA on morphology changes of GES-1 cells treated with ST were detected by inverted phase contrast microscope.3 Effects of Chk1 si RNA on the cell cycle distribution and the expression of mitosis marker protein(p-H3) of GES-1 cells treated with ST were detected by FCM.4 Effects of Chk1 si RNA on the expression of p-H3 and nuclear translocation of Cyclin B1 were detected by confocal scanning laser microscope.5 The expression of Chk1 and p-Chk1 of cells collected by shake-off were examined by Western Blot.6 Effects of ST on the G2 phase key factors and p53-p21 signaling pathway were examined by Western Blot and Real-time PCR. Effects of Chk1 si RNA on the G2 phase key factors and p53-p21 signaling pathway were examined by Western Blot.Results:1 Effects of ST on ATR-Chk1 signaling pathway in GES-1 cellsTo explore the effects of ST on ATR-Chk1 signaling pathway, we treated GES-1cells with solvent alone or with 3, 6 or 12 μM ST for 24 h and detected the expressions of releated gene in ATR-Chk1 signaling pathway. Real-time PCR results showed that the expression of ATR and Chk1 increased after ST treatment(P<0.05). The Western Blot results showed p-ATR increased in the ST treatment groups compared with the solvent control group, but there was no significant difference in the average level of ATR protein(P>0.05). Moreover, we investigated Chk1 protein level and phosphorylation of multiple serine residues on Chk1 in response to ST treatment. The expression of Chk1 and p-Chk1 were significantly higher compared with those in the solvent control group(P<0.05). Taken together, these observations indicated that ST-induced DNA damage activated the ATR-Chk1 checkpoint signaling pathway in GES-1 cells.2 Analysis about the crossover between ATR-Chk1 and ATM-Chk2 signaling pathway in GES-1 cellsIn response to different DNA damage, cross-activation often appeared in ATR-Chk1 and ATM-Chk2 signaling pathways. We wanted to explore who mediated Chk1 phosphorylation in GES-1 cells treated with ST.The Western Blot results showed ATR level was decreased by ATR si RNA(P<0.05). However, there was no decrease in the ATM protein expression in response to ATR si RNA suggesting that the effect of ATR si RNA was specific to ATR(P>0.05). The level of p-Chk1(Ser345) decreased remarkably in Cells transfected with ATR si RNA which further indicated that the phosphorylation of Chk1 was dependent on ATR(P<0.05). We used ATM specific inhibitor KU55933 inhibited ST-induced ATM phosphorylation and Chk2 phosphorylation successfully(P<0.05). But Cells treated with KU55933 demonstrated no downregulation in Chk1 phosphorylation, but rather a minor increase was noted, suggesting that ATM did not play a major role in increasing the phosphorylation of Chk1 in ST treatment( P>0.05). This evidence suggested that ST-induced Chk1 phosphorylation was ATR-dependent, but ATM-independent.3 Effects of Chk1 si RNA on cell cycle distribution of GES-1 cells treated with STTo further understand the role of Chk1 activation on the G2/M checkpoint, si RNA against Chk1 was transfected into GES-1 cells. This single transfection resulted in a significant reduction of Chk1 m RNA and protein levels(P<0.05). The FCM analysis confirmed that the strong inhibition of the Chk1 expression did not affect the cell cycle distribution in the absence of ST(P>0.05). However, Following ST treatment, si RNA against Chk1 decreased the G2 population sharply(P<0.05).These results suggested that Chk1 played a major role in cell cycle profile in response to ST treatment.4 Effects of Chk1 si RNA on the entry into mitosis phase of GES-1 cells treated with STGES-1 cells were transfected with Chk1 si RNA or its relative control si RNA and treated with ST at 24 h posttransfection. Inverted phase contrast microscope showed a large number of large and rounded cells appeared which were adherent weakly in Chk1 si RNA + ST group. The cells possessed the morphological characteristics as mitotic cells. Laser confocal immunofluorescence staining and FCM showed the percentage of p-H3 positive cells in Chk1 si RNA + ST group was more than control si RNA + ST group(P<0.05). This evidence illustrated Chk1 si RNA promoted GES-1 cells treated with ST to come into mitosis phase.5 The expression of Chk1 and p-Chk1 of cells collected by shake-offChk1 siRNA experiment revealed that low expression of Chk1 was convenient for GES-1 cells to pass through G2 checkpoint and enter mitosis with damaged DNA. We further explored the expression of Chk1 and p-Chk1 of cells collected by shake-off.GES-1 cells were treated with DMSO as a solvent control or treated with 12 μM ST for 48 h. Cells treated with ST for 48 h were fractionated by mechanical shake-off as adherent cells and mitotic shake-off cells, Nocodazole was the positive control group. The Western Blot results showed the expression of Chk1 and p-Chk1 in Shake-off group was lower than ST-treated group and adherent cells group(P<0.05). These results indicated that cells collected by shake-off expressed low levels of Chk1 and p-Chk1.6 Effects of Chk1 si RNA on G2 phase key factorsThe knockdown of Chk1 promoted the GES-1 cells treated with ST to break through the G2 checkpoint and enter M phase with damaged DNA. Based on these findings, we hypothesized that G2/M checkpoint failure due to a defect of the components among G2 phase key factors. To confirm the hypothesis, we detected the expression of G2 phase key factors. Western blot analysis showed that ST induced an increase in the expression of p-Cdc25 C, p-Cdc2 and Cyclin B1 in GES-1 cells compared with solvent-treated control group(P<0.05). In contrast, with treatment of ST and Chk1 si RNA,the levels of p-Cdc25 C, p-Cdc2 and Cyclin B1 were found to decrease dramatically compared with ST-treated alone cells(P<0.05), suggesting that the Chk1 controlled the p-Cdc25 C, p-Cdc2 and Cyclin B1 to mediate G2/M arrest and the entry into mitosis.7 Effect of ST treatment on nuclear translocation of Cyclin B1 in GES-1 cells transfected with either the control si RNA or Chk1-specific si RNACyclin B1 is a kind of proteins related to cell division which translocates from the cytoplasm to the nucleus in early M phase. Confocal microscope showed many Chk1-depleted cells showed nuclear Cyclin B1 staining, typical for mitotic cells. However, ST-treated alone cells maintained cytoplasmic Cyclin B1 staining. These results confirmed that Chk1 si RNA promoted the thift of Cyclin B1 from the cytoplasm to the nucleus and facilitated the course of mitosis.8 Effect of ST treatment on p53 and p21 proteins of GES-1 cells transfected with either the control si RNA or Chk1-specific si RNAThe p53-p21 signaling pathway plays an important role in the G2 checkpoint; however, it was unclear whether the p53-p21 signaling pathway participated in the checkpoint adaptation triggered by Chk1 si RNA interference.Real-time PCR results showed that the average level of p53 and p21 m RNA were both increased in ST-treated cells relative to the solvent controlcells(P<0.05).Western blot analysis showed that ST induced an increase in theexpression of p53, p-p53(Ser-15), p-p53(Ser-20) and p21 in GES-1 cellscompared with solvent control group(P<0.05).Furthermore, we used si RNA to deplete Chk1 and then subjected the cells to ST treatment for 24 h in GES-1 cells. The Western blotting results showed that Chk1 si RNA effectively down-regulated the expression of p21 in ST-treated GES-1 cells. Moreover, we found that ST-induced high expression of total p53 and phosphorylation of p53 at Ser-20 and Ser-15 were not inhibited in cells transfected with Chk1 si RNA(P>0.05). However, at these concentrations, the checkpoints were partly abrogated by Chk1 knockdown. Therefore, these data indicated that Chk1 participated in the regulation of ST-mediated G2 phase arrest in GES-1 cells through up regulation the expression of p21. But this regulation was not achieved by regulating the expression of p53.PartⅢ Role of PLK1 in sterigmatocystin-induced checkpoint adaptation and the exit of mitosis in GES-1 cellsObjective: PLK1 is a kind of serine/threonine kinases which widely exists in eukaryotic cells. PLK1 regulates multiple processes during mitosis. Researches confirmed that the kinase activity of PLK1 played an important role in checkpoint adaptation. We found that GES-1cells treated with ST came into mitosis with damaged DNA after a prolonged G2 arrest. Whether PLK1 was involved in the process of checkpoint adaptation was still needed to be further researched. DNA damage could inhibit PLK1 and prevent the exit of mitosis. Therefore, we will explore the influence of ST treatment on the exit of mitosis and analyzed the correlation between the influence and PLK1 expression.Methods:1 Effects of ST pretreatment on cell cycle distribution in GES-1 cells synchronized by Nocodazole were detected by FCM.2 Effects of ST pretreatment on the expression of PLK1 and p-PLK1 in GES-1 cells synchronized by Nocodazole were detected by Western Blot.3 Effects of ATR-Chk1 signaling pathway on ST induced inhibition of PLK1 activity and PLK1 phosphorylation at T210 were detected by Western Blot.4 The expression of PLK1 and p-PLK1 of cells collected by shake-off were examined by Western Blot.5 Effects of ST on mitotic exit of GES-1 cells were detected by FCM.6Effects of ST on the expression of PLK1 and Cyclin B1 of GES-1 cells released from M phase.Results:1 Effects of ST pretreatment on cell cycle distribution in GES-1 cells synchronized by Nocodazole and on the expression of PLK1 and p-PLK1GES-1 cells were treated with DMSO as a solvent control or treated with 12 μM ST for 3 h, 8 h and 24 h, followed by Nocodazole treatment for 16 h. FCM results showed Nocodazole-treated cells accumulated in G2M(81.5%) and S(15.06%) phases. However, ST pretreatment inhibited the entry into G2 M phase(P<0.05). Meanwhile, ST pretreatment reduced the percentage of p-H3 positive cells synchronized by Nocodazole(P<0.05). It indicated ST inhibited the entry into G2 M phase of GES-1 cells. Western Blot showed PLK1 was activated in Nocodazole-arrested GES-1 cells, but pretreatment with ST blocked the expression of PLK1 and p-PLK1.2 ATR-Chk1 signaling pathway was involved in ST induced inhibition of PLK1 activity and PLK1 phosphorylation at T210DNA damage inhibited PLK1 phosphorylation and ATR-Chk1 was an important signaling pathway in response to DNA damage. We suspected there might be a relationship between PLK1 and the ATR-Chk1 signaling pathways. Therefore, we used caffeine(an inhibitor of ATM and ATR) and Chk1 si RNA to test if ATR-Chk1 mediated the inhibition of PLK1 phosphorylation.GES-1 cells, were pretreated with caffeine or transfected with Chk1 si RNA, were incubated with solvent control or 12 μM ST for 24 h. Next, Nocodazole was added for 16 h. Western Blot showed ST blocked the expression of PLK1 and p-PLK1.However, caffeine and Chk1 si RNA all could increase the expression of PLK1 and p-PLK1 in cells treated with ST and Nocodazole(P<0.05). This meant ATR-Chk1 signaling pathway reversed the inhibition of PLK1 activity and PLK1 phosphorylation at T210 induced by ST.3 The expression of PLK1 and p-PLK1 of cells collected by shake-offThe Western Blot results showed the expression of PLK1 and p-PLK1 in shake-off group was higher than ST-treated group and adherent cells group group(P<0.05). The results showed PLK1 was expressed and activated in ST-treated GES-1 cells that had undergone G2 checkpoint adaptation.4 Effects of ST pretreatment on mitotic exit in GES-1 cells synchronized by NocodazoleGES-1 cells were treated with DMSO as a solvent control or treated with 12 μM ST for 3 h, 8 h and 2 4 h, followed by Nocodazole treatment for 16 h. Cells were then released in fresh cell culture media, and were harvested and fixed at different time. The results showed cells untreated with ST rapidly exit from mitosis after the removal of Nocodazole. After 8 h, most cells had entered the next G0/G1 phase and PLK1 and Cyclin B1 quickly decreased. Treatment with ST prevented the exit from mitosis, and 8 h after the release a large proportion of the cells remained arrested in G2/M phase(P<0.05) and there was almost no change in the expression of PLK1 and Cyclin B1.5 Cells were synchronized by Nocodazole first, subsequently, were treated with ST for 3h.Explore effects of ST treatment on mitotic exitFCM showed all the cells including untreated and treated with ST both rapidly exited from mitosis after the removal of Nocodazole. There was no difference between them(P>0.05).In conclusion, our data indicated PLK1 phosphorylation and high expression of PLK1 were key reasons to lead to checkpoint adaptation induced by ST treatment.Conclusions:1 ST induces G2 arrest of GES-1 cells, after a prolonged G2 arrest, part of GES-1 cells come into mitosis phase with damaged DNA. G2 checkpoint adaptation occurs in ST-treated GES-1 cells.2 ST activates ATR-Chk1 signaling pathway in GES-1 cells. Chk1 is a key regulator factor of checkpoint adaptation of GES-1 cells treated with ST.Chk1 regulates Cyclin B1 translocation and the expression of G2 phase key regulatory factor and p21 to control the checkpoint adaptation.3 The phosphorylation and high expression of PLK1 are key reasons to lead to checkpoint adaptation induced by ST. ST leads to DNA damage, inhibits mitosis entry and suppresses PLK1 phosphorylation. ATR-Chk1 signaling pathway prevents inhibition of the expression and phosphorylation of PLK1 induced by ST. ST also inhibits mitosis exit, and this inhibition is closely related to the expression of PLK1 and Cyclin B1.The activation of PLK1 is another key reason to result in G2 checkpoint adaptation.4 ST may induce G2 checkpoint adaptation to lead to carcinogenesis.