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Transcript
Shubhranshu Debnath Apoptotic cell death signaling in the Human Colon Cancer Cell line (HCT-116) The development of an organism depends upon three basic fundamental cellular processes: proliferation, differentiation and death. Each of these processes is of equal importance in shaping an adult individual. Tumor is defined by uncontrolled proliferation and avoidance of cell death. Hence, most anti-cancer treatments aim to eradicate tumor cells through activation of various cell death processes, including apoptosis. Unfortunately, development of resistance to chemotherapeutic drugs during the course of treatment is a substantial problem in the clinics today. Therefore, a more precise understanding of cell death mechanisms would facilitate development of novel methods for treatment. Programmed cell death, or apoptosis, regulates cell numbers and forms complex organ structures by cell removal. Apoptosis also occurs when a cell is damaged beyond repair. During apoptosis several protein breaker enzymes (proteases) are activated, particularly from the cystine aspartate protease family known as caspases. In apoptosis, caspase-8 and 9 serve as the most upstream activating caspases, respectively in the apoptotic cascade. The activation of caspase-8 as a result of Death Induced Signalling Complex (DISC) formation is a well studied event. If sufficient amounts of active caspase-8 are generated at the DISC to directly process effector caspases then the cells are classified as type I apoptotic cells, while in type II cells, the amount of caspase-8 processed in the DISC is insufficient. Therefore, apoptosis in type II cells is dependent mitochondria, which is also known as the power house of the cell, provides energy to the cell and permiabilization of the outer mitochondrial membrane further damages the cell’s energy production mechanism and thus, intensify the apoptotic process. Our aims for the present project study are to map upstream regulatory events in the apoptotic cell death cascade described and by this means build a more detailed model of apoptotic mechanisms as a response to DNA damage by drug Flurouracil (5-FU) in Human colon carcinoma cells (HCT116). Previous studies show that 5-FU creates cell stress provoked by DNA damage and follow type I cell death. Our results reveal that apoptosis indeed is a death receptor mediated process in this experimental model, by using the gene silencing technique (short interfering RNA- “siRNA”), targeting the DISC regulatory protein Flip (FLICE (Caspase-8) like inhibitor protein) and by creating a stable cell expressing a dominant negative FADD (FAS- associated death domain) protein. Further analyses defined DR5 (Death receptor 5) as the most important death receptor for the signalling cascade studied. Two interesting findings then contributed to following investigations. Firstly, apart from clear plasma membrane localization, DR5 also showed a cytoplasmic punctuate expression pattern. Secondly, upon 5-FU treatment, HCT116 cells are positive for several specific autophagy markers (autophagy- self eating; another cell death mechanism). The present study contains several interesting data which eventually will provide tools to further analyse upstream signalling events in 5-FU induced cell death. More detailed analyses are, however, required to completely map the DR5 mediated apoptotic process in HCT116 cells. Supervisor: Prof. (Dr.) Boris Zhivotovsky Degree project 60 credits in Protein Science – September 2009 to June 2010 Department of Chemistry, Lund University Institution: Division of Toxicology, Institute of Environmental Medicine, Karolinska Institute Shubhranshu Debnath The role of Autophagy for DNA damage-induced Apoptotic signaling in the Human Colon Carcinoma Cell line (HCT-116) The best-characterized apoptotic pathways are the receptor-mediated (extrinsic) and cellular stress-induced (intrinsic) pathways. In which caspase-8 and -9 serve as the most upstream activating caspases, respectively. The activation of caspase-8 as a result of DISC formation is well studied and represents the commencement of the extrinsic apoptotic pathway. In type I cells, sufficient amount of active caspase-8 is generated at the DISC to directly process effector caspases. In type II cells, the amount of caspase-8 processed in the DISC is insufficient. Therefore, apoptosis in type II cells is dependent on the cleavage of the pro-apoptotic Bcl-2 family protein Bid. Conversion of Bid to tBid then leads to oligomerization and activation of Bax/ Bak, succeeded by the opening of specific pores in the outer mitochondrial membrane (OMM) and resulting in the release of cytochrome c and other proteins from the mitochondrial inter membrane space to the cytoplasm. Our aims for the present project are to define upstream regulatory elements in the apoptotic cascade described and by this mean build a more detailed model of apoptotic mechanisms as a response to 5-FU induced DNA-damage in HCT116 cells. Previous findings state that although 5-FU creates cell stress provoked by DNA lesions, it seems that an extrinsic type of apoptosis is activated. Our results revels that apoptosis indeed is death receptor mediated in this experimental model by using siRNA targeting the DISC regulatory protein Flip and by creating a stable cell expressing a dominant negative FADD protein, Further analyses defined DR5 as the most important death receptor for the signalling cascade examined. Two interesting findings then contributed to following investigations. Firstly, apart from clear plasma membrane localization, DR5 also showed a cytoplasmic punctuate expression pattern in immunostainings. Secondly, upon 5-FU treatment, HCT116 cells are positive for several specific autophagy markers. The present investigation contains several interesting data which eventually will provide tools to further analyse upstream signalling events in 5-FU induced cell death. More detailed analyses are, however, required to completely map the DR5 mediated apoptotic process in HCT116 cells. Supervisor: Prof. (Dr.) Boris Zhivotovsky Institution: Division of Toxicology, Institute of Environmental Medicine, Karolinska Institute, Degree project 60 credits in Protein Science – September 2009 to June 2010 Department of Chemistry, Lund University