Autophagy Reduces Micronuclei Formation By Maintaining Redox Homeostasis

Autophagy is a lysosome-dependent degradation pathway,which responds to various types of stress to maintain cell homeostasis.It has been well-documented to play an important role in maintaining genomic stability.Micronuclei,also known as cytoplasmic chromatin fragment,serve as a sensitive indicator of genomic instability that can be used to evaluate the extent of DNA damage.It was reported that autophagy can directly target micronuclei to clear it,but at a very low frequency,only 2-5%.In addition to the direct removal of damaged organelles,proteins and chromatin fragments,autophagy can affect many cellular processes including DNA damage response,regulation of redox homeostasis and cell division,it remains to be determined to what extent each of those processes contributes to the maintenance of genomic stability.Reactive oxygen species(ROS)are natural by-product of normal oxygen metabolism and play an essential role in cell signaling and homeostasis.However,excessive ROS can cause oxidative stress and have toxic effects on important cellular components such as proteins and DNA.Oxidative stress is one of the most significant causes of DNA damage,which further induces the formation of micronuclei.Excessive ROS is also involved in the induction of autophagy,but the reports related to autophagy on the regulation of redox homeostasis are inconsistent.Therefore,as an important process of protecting cells against stress,does autophagy reduce the frequency of micronuclei?And what is the regulatory effect on redox homeostasis?Does this regulation mediate the effect of autophagy on the frequency of micronuclei?Based on the above background and questions,we have designed and carried out a series of work and obtained the following results:1.Genotoxicants induce micronuclei formationThe results of Immunofluorescence assay showed that both hydroxyurea(HU)and X-Ray could effectively induce micronuclei formation.The relationship between micronuclei membrane integrity and y-H2AX examined by immunofluorescence co-staining of LaminB1 and ?-H2AX,we found that compared with MN-?-H2AX(-),the nuclear membrane of MN-?-H2AX(+)tended to be less intact.2.Autophagy reduces micronuclei and ROS of cancer cellsThe autophagy activation efficiency of Rapamycin and Torinl was evaluated by Western Blotting,the results showed that the ratio of LC3 ?/? increased,indicating that autophagy was activated.The frequency of micronuclei was tested by immunofluorescence,and the results showed that autophagy could reduce the spontaneous micronuclei in HT1080 and U2OS cells.We found that the replication stress-induced micronuclei in cancer cells were also significantly reduced by autophagy activators.In addition,Rapamycin could also reduce the frequency of micronuclei induced by X-Ray.The above results showed that autophagy reduced spontaneous and genotoxic stress-induced micronuclei in cancer cells.Flow cytometry analysis of reactive oxygen species revealed that the basal and genotoxic stress-induced ROS level were significantly reduced by autophagy activators.3.The ROS and micronuclei of senescent cells could be reduced by RapamycinHuman umbilical cord mesenchymal stem cells(hUC-MSCs)at different passages were determined for percentage of senescent cells by ?-X-gal staining.The results showed that the percentage of senescent cells at P18 was significantly higher than that at P10.The autophagy efficiency of the two types of cells by Western Blotting and observed abnormal accumulation of LC3-? in senescent MSCs,indicating senescent cells showed blocked autophagy flux.Immunofluorescence assay showed that senescent MSCs had a significant elevation in micronuclei,which could be attenuated by Rapamycin.Correspondingly,the elevated ROS in senescent cells was also reduced by Rapamycin.4.Blockade of autophagy increases micronucleiTreatment of two cancer cell lines and MSCs with autophagy inhibitor chloroquine resulted in an abnormal accumulation of LC3-? and a rise at the autophagy substrate p62 protein level examined by Western Blotting,indicating that autophagy was inhibited.ATG5 knockdown with small interfering RNA was also shown to block the initiation of autophagy.Evaluation of the frequency micronuclei by immunofluorescence showed that both the autophagy inhibitor and ATG5 siRNA could increase the micronuclei formation in cancer cells and MSCs.5.Antioxidants can rescue micronuclei elevation caused by autophagy inhibitionThe results of flow cytometry showed that autophagy inhibition led to a significant elevation in ROS through CQ or ATG5 siRNA in MSCs.However,these results were not observed in the two cancer cell lines.We think it may be due to excessive basal ROS level in the cancer cells.The above results suggested that oxidative stress may mediate the elevation of micronuclei caused by autophagy inhibition in MSCs.The protein level of ?-H2AX was evaluated by Western Blotting.The results showed that the antioxidant N-acetylcysteine(NAC)could reduce the accumulation of y-H2AX caused by autophagy inhibition.The elevation of micronuclei induced by autophagy inhibition could also be reduced by NAC evaluated through Immunofluorescence.6.Autophagy inhibits the activation of CHK1Western Blotting observed the protein level of p-CHK1,and the results showed that autophagy activation could reduce the up-regulation of p-CHK1 induced by replication stress in three cell lines.At the same time,inhibition of autophagy led to a significant upregulation of p-CHK1.This result indicated that autophagy inhibited CHK1 activation,which was not consistent with the explanation in the report that the genomic instability of autophagy deficient cells is mainly due to the impairment of HR function caused by the decrease of p-CHK1.Our results suggest that autophagy does not promote maintain genomic stability by augmenting CHK1 in this experimental system.In summary,this study explored the effect of autophagy on the frequency of micronuclei,clarified the role of autophagy in regulating redox homeostasis We proposed that redox regulation mediated the effect of autophagy on micronuclei frequency under certain conditions.A new explanation was proposed for the key role of autophagy in maintaining genome stability.