טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentColton Sivan
SubjectRole of Dicer in Mediating Activation-Induced Cell
Death (AICD) in B Cells
DepartmentDepartment of Medicine
Supervisor Professor Doron Melamed


Abstract

B cell development is a multi-step process, that ends in generating mature B cells expressing functionally responsive receptors to a large array of foreign antigens, while being tolerant to self. These changes enable the assembly and expression of repertoire of unique B cell receptors (BCR). Developing B cells that express self-reactive receptors are eliminated by an instructive mechanism for induction of apoptosis, named activation-induced cell death (AICD). This process occurs following BCR ligation in the bone marrow, or FASL-FASR engagement in the periphery.

Our laboratory investigates mechanisms of cell fate decisions. One direction that is studied in the lab is the role of microRNAs (miRNAs) in the determination of B cells development and survival. Our first aim was to determine the role of miRNA biogenesis in AICD. To do so, we applied a shRNA knockdown methodology to reduce the expression of a key regulator of miRNA processing, Dicer, in cell line models for BCR-mediated AICD, DauDi and WHEI-231. The results show that knock-down of Dicer increases sensitivity to apoptosis, proving that miRNAs play a role in BCR mediated AICD. Further biochemical analysis revealed that Dicer knockdown cells had impaired PI3K signaling, proving that miRNAs are major regulators of PI3K pathway for clonal selection of B cell.

Several studies provided an insight that links Dicer to apoptosis. Dicer was found to be a target for activated caspases in stress-mediated apoptosis, ceasing miRNA biogenesis and enhancing the apoptosis process. In agreement with this, our second aim was to test whether Dicer is involved in the process of AICD in B lineage cells. Stimulation of cell line sensitive to BCR mediated AICD, DauDi, have revealed us that Dicer expression was reduced within 14 hours following stimulation, in correlation with increased expression of activated caspase 3 form. Pre-incubation with total caspase inhibitor blocked Dicer degradation, proving that in the process of BCR-mediated AICD the turnover of Dicer is regulated by caspases. Dicer was also found to undergo cleavage during FAS-mediated AICD by activated caspases, in A20 cell line. We next asked whether Dicer’s cleavage facilitates the AICD process, and whether inhibition of it can rescue cells from apoptosis. For that we cloned vector that encodes Dicer wild-type (WT) form or a mutated (mut) form of Dicer, in which the putative caspase 3 cleavage site had been mutated. Unfortunately, we did not observe overexpression of Dicer protein in either of the infected cells and due to this technical limitation, we could not conclude whether Dicer cleavage promotes apoptosis.

In a study preformed on C. elegans, Dicer homologous was found to be cleaved by caspase 3 homologous, resulting in a C-terminal product with DNase activity that promotes apoptosis. In the third part of our study, we aimed to determine whether truncated Dicer might contribute to AICD and demonstrate that Dicer plays an active DNAse role in the process of apoptosis. The putative C-terminal truncated Dicer didn’t seem to induce apoptosis.

Overall, our study had demonstrated miRNAs biogenesis and Dicer role in B cells undergoing AICD.