The Roles Of Transcription Factor Bclafl In DNA Damage Induced Cellular Senescence And Hypoxia Response

Cellular stress responses are reactions when cells undergo different stimuli from inside or outside environment.Stress response plays crucial role for homeostasis maintenance of cells and carcinogenesis prevention.Different cellular stress responses can stimulate different signaling pathways,which regulated by related transcription factors.Since the expression,activity,modification of the crucial transcription factors(TFs)will decide the expression of different genes which finally participate the cell fate decision,the regulators of those TFs attract great attentions.Recently,bunches of researches show Bclafl Is an important protein,which is associated with organ development,tumor genesis,cell apoptosis and so on,but the regulations and functions of Bclafl is limited.Recently,our lab found Bclafl participated in two different stress responses:one is chronic DNA damage therapeutic drug induced cellular senescence stress(TIS),the other is hypoxic stress.Bclafl plays important roles in both pathways and regulates the key TFs:C/EBP? in TIS and HIFla in hypoxia.In the thesis,we report how Bclafl responds to the stimuli and uncover the molecular mechanisms.To address the questions,we used a therapeutic compound,doxorubicin to treat the cancer cells to induce cellular senescence,which we referred as TIS.We identified a DNA damage associated protein,Bclafl was induced in TIS.And Bclafl induction could be inhibited by knockdown of the DDR core kinase ATM or ATR,or the DDR associated NF-?B transducer Nemo by siRNA.Then we found that Bclafl was a transcriptional target of NF-?B family member p65 and c-Rel which could directly bind to the promoter of Bclafl proved by chromatin immunoprecipitation(ChIP)assay and luciferase report assay.The evidence indicated the induction of Bclafl in TIS was relyon the ATM/Nemo/NF-?B pathway.In addition,by using RNA inference against NF-?B,we found C/EBP? was also induced by NF-?B,but the up-regulation was abolished when adding small interference RNA(siRNA)against Bclaf1.Further ChIP assay identified Bclafl could directly bind to the promoter region of C/EBP?,which indicated Bclafl may transcriptionally regulate the expression of C/EBP?.Besides,by using immunoprecipitation we found Bclafl interacted with the C/EBP? ZIP domain and cooperate with C/EBP? to up-regulate IL-8.Furthermore,we show that Bclafl is required for the effectiveness of doxorubicin(Dox)treatment-induced tumor suppression in a HT-29 xenograft tumor model.These finding suggest that Bclafl plays a crucial role in transducing the senescence-inducing signal from NF-?B to C/EBP? during TIS,thus amplifying the signals for the establishment of senescence.Given the recent revelation that Bclafl is involved in tumorigenesis,our data indicate that the responsiveness of Bclafl to NF-?B may determine the effectiveness of therapeutic drugs.In the second part,we found the up-regulation of Bclafl in multiple tumor cells treated in hypoxia.To figure out the function of Bclafl in hypoxia treatment,we used short hairpin RNA to knockdown Bclafl then cultured the cells in hypoxia and found that the dramatical inhibition of cell growth compared with control cells.Further signaling analysis by Western Blot and real-time PCR assay showed that the stability and transcription activity of HIFla under hypoxia were dramatically decreased by Bclafl depletion.In conclusion,our study revealed Bclafl play crucial roles in two different stress responses:In persistent DNA damage induced cellular senescence,Bclafl is induced by NF-?B pathway amplified the signaling by inducing the expression and activity of another key transcription factor C/EBP?,therefore enhances the expression of senescence-associated secretory factors and senescence establishment.In hypoxia stress,the induction of Bclafl is regulated by transcription factor HIF1,while Bclafl contributes to the stability and function of HIF1? in turn.