Medical Biology - Theses
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ItemFunctional characterisation of Bcl-G( 2011)Apoptosis is a controlled and precise form of cell death necessary for maintaining tissue homeostasis and normal development. Perturbations of this cell death process contribute to a wide range of diseases such as cancer and autoimmunity. The Bcl-2 family regulates the intrinsic pathway of apoptosis and members share homology at one or more of the four different Bcl-2 Homology (BH) domains. They can be subdivided into the pro-survival proteins, the multi-domain pro-apoptotic proteins and the BH3-only proteins. The physiological functions and mechanism of action of the main players of the Bcl-2-regulated apoptotic pathway have been studied extensively using mouse models. However, the roles played by less prominent Bcl-2 family members are not as well characterised. In this thesis, we described the characterisation of Bcl-G, an evolutionarily conserved novel Bcl-2 family member implicated in cancer. Human BCL-G produces two major isoforms, BCL-GL and BCL-GS. BCL-GS only contains a BH3 domain while BCL-GL also contains a BH2 domain. While BCL-GL exhibited little killing ability, in vitro over-expression studies suggested that BCL-GS kills cells by binding BCL-XL. The mouse Bcl-G gene only produces one isoform, which contains both the BH2 and BH3 domains. Little is known about its functions and roles in apoptosis. To characterise the tissue distribution and subcellular localisation of mBcl-G, we produced monoclonal antibodies specific for this protein. Bcl-G was found to be predominantly cytoplasmic when over-expressed in HeLa cells and is present in a wide range of mouse tissues including spleen, thymus, lung, intestine and testis. Anti-Bcl-G immunohistochemistry revealed that it is expressed highly by some dendritic cell (DC) subtypes and certain epithelial cell types including those lining the gastrointestinal tract. To study the functions of mBcl-G and gain clues on its human orthologue, I generated and characterised Bcl-G-deficient mice. Bcl-G-1- mice were born viable, developed normally and had similar numbers of DCs and other immune cell subtypes when compared to their wild-type counterparts. Notably, Bcl-G-deficient cells were normally sensitive to the range of apoptotic stimuli tested. I also analysed the gastrointestinal tract of the Bcl-G-1- mice but did not observe any abnormalities in the gross morphology of the stomach, small intestine and colon. However, Bcl-G loss attenuated the colitis response to acute dextran sodium sulphate (DSS) administration, resulting in reduced immune infiltration and destruction of crypt architecture. The role mBcl-G plays in DSS-induced colitis is unclear and currently under investigation. To gain clues on the processes in which Bcl-G is involved in, I conducted a search for binding partners using two different approaches: co-immunoprecipitation coupled with mass spectrometric analysis (IP-MS) and yeast two-hybrid screening. Importantly, no Bcl-2 family member, whether it is pro-survival or pro-apoptotic, was pulled-down in either screen. In fact, in vitro over-expression studies revealed that Bcl-G’s BH3 domain was incapable of binding Bcl-XL and activating apoptosis when placed in the context of BH3-only protein Bim. These results raise doubts that mBcl-G interacts with the pro-survival proteins and is involved in the Bcl-2-regulated apoptotic pathway. Instead, Trappc6b, a subunit of the Trafficking protein particle (Trapp) complex, was identified as a potential Bcl-G binding partner. Mammalian Trapp is a multi-subunit protein complex involved in intracellular membrane traffic. This finding proposes a novel role of this Bcl-2 family member in protein trafficking and vesicle transport. In a second project, I have studied how BH3-only members activate the essential effectors Bax and Bak. Two models have been proposed, but the issue remains controversial. The indirect activation model suggests that BH3-only proteins simply neutralize all of the pro-survival proteins, whereas the direct activation model proposes that Bim and Bid must activate Bax and Bak by direct binding. As numerous in vitro studies have not resolved this issue, we have investigated Bim's activity in vivo by a genetic approach. Because the BH3 domain determines binding specificity for Bcl-2 relatives, we generated mice having the Bim BH3 domain replaced by that of Bad, Noxa, or Puma. The mutants bound the expected subsets of pro-survival relatives but lost interaction with Bax. Analysis of these mice showed that Bim's pro-apoptotic activity is not solely determined by its ability to engage its pro-survival relatives or solely to its binding to Bax. Thus, initiation of apoptosis in vivo appears to require features of both models.
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ItemThe impact of BH3-only proteins on the response of murine lymphoma to anti-cancer therapy( 2011)Apoptosis, a genetically programmed process for cell killing, is critical for the elimination of damaged or redundant cells during development, homeostasis and defence against pathogens. Defective apoptosis can promote tumour development and impair the response of tumour cells anti-cancer therapy. Apoptosis in response to developmental cues as well as a diverse range of stress stimuli is mediated by the interplay between the pro- and anti-apoptotic members of the Bcl-2 protein family. The BH3-only proteins represent one subset of pro-apoptotic proteins within the Bcl-2 family. The eight mammalian BH3-only proteins that exist are essential for initiation of apoptosis following a death stimulus, and display death stimulus-specific as well as cell type-restricted activity. Some BH3-only proteins, including Puma, Bim and Bid, can bind avidly to all pro-survival Bcl-2-like proteins and therefore have potent apoptotic activity, whilst others, such as Noxa, Bik, Bad and Bmf display more limited binding capabilities and thus more limited killing activity. Conventional cytotoxic chemotherapy drugs, such as cyclophosphamide and etoposide, that are used to treat a variety of cancers, including many haematological malignancies, act by inducing DNA damage in cells, thereby activating the tumour suppressor p53 with consequent triggering of the Bcl-2-regulated apoptotic pathway. The BH3-only protein family members puma and noxa are direct transcriptional targets of p53. Recently, Puma has been shown to play a major, and Noxa a more restricted role in DNA damage-induced p53-mediated apoptosis of normal (non-transformed) cells. Most strikingly, in thymocytes, the combined loss of Puma and Noxa was capable of conferring as much protection from DNA damage-induced killing as loss of p53 itself. How DNA damaging drugs kill tumour cells is currently still poorly defined. To define the essential requirement of the various BH3-only proteins in the response of tumour cells to DNA damage inducing drugs, gene-targeted mice lacking BH3-only proteins were crossed with Eμ-myc transgenic mice to obtain Eµ-myc lymphomas with deficiencies in one or more BH3-only proteins. Analysis of the response of these lymphomas to DNA damaging drugs in vitro and in vivo revealed that Puma was the most critical of the BH3-only proteins in apoptosis induction. Interestingly, combined loss of both p53 activated BH3-only proteins, Noxa and Puma did not result in resistance as severe as that observed with p53 deficiency and these lymphomas displayed no greater drug resistance than lymphomas lacking Puma alone. These observations contrast those reported for primary, non-transformed lymphoid cells, and demonstrate the differences in the requirements for BH3-only proteins for apoptosis induction following DNA damaging drug treatment between Myc-driven tumour cells and non-transformed lymphocytes. As the combined loss of Puma and Noxa could not recapitulate the drug resistance caused by p53 loss, the involvement of other BH3-only proteins was additionally assessed. The induction of another BH3-only protein, Bim, although not known to be a direct p53 target, was also observed following DNA damage in Eµ-myc lymphomas but not in Eµ-myc lymphomas lacking p53. Bim deficiency did not significantly alter the sensitivity of Eµ-myc lymphomas to DNA damaging drugs, however, knockdown of bim levels markedly increased the drug resistance of Eµ-myc lymphomas lacking Noxa and Puma. Remarkably, c-MYC driven lymphoma cells generated from noxa-/-puma-/-bim-/- haematopoietic stem/progenitor cells were as resistant as those lacking p53. These findings demonstrate that the combinatorial action of Puma, Noxa and Bim is critical for the efficacious killing of lymphoma cells by DNA-damaging chemotherapeutic agents and these three BH3-only proteins therefore represent biomarkers for treatment outcome in the clinic. Several BH3-only proteins have been proven to exert tumour suppressive actions in vivo. Loss of the ‘potent’ BH3-only proteins, Bim and Puma that are both capable of binding all pro-survival Bcl-2 family members, has been shown to markedly accelerate Eµ-myc lymphomagenesis, albeit to a lesser extent than p53 deficiency. Interestingly loss of the more selective BH3-only proteins, Bmf and Bad, which are capable of binding only Bcl-2, Bcl-w and Bcl-xL but not Mcl-1 or A1, have also recently been shown to accelerate Eµ-myc-induced lymphoma development. Bik, another selective pro-apoptotic BH3-only protein with similar binding specificity to Bmf and Bad, is widely expressed and has been postulated to function as a tumour suppressor on the basis that its deficiency and loss of function have been reported in many human cancers, including lymphoid malignancies. Surprisingly however, Bik deficiency did not accelerate Eµ-myc induced lymphomagenesis, indicating that in this context, Bik does not function as a tumour suppressor, at least on its own. Moreover, although p53-mediated induction of Bik has been previously documented, loss of Bik did not cause resistance in Eµ-myc lymphomas to DNA damaging drug-induced apoptosis, demonstrating that other BH3-only proteins could adequately compensate for its loss. When ectopically over-expressed, co-operation between Bik and the BH3-only protein Noxa (capable of binding avidly to Mcl-1 and A1) has been described to potently induce apoptosis and this has been attributed to their complementary binding specificities for pro-survival Bcl-2 family proteins. In contrast to these previously published reports, the combined deficiency of Bik and Noxa did not affect onset of Eµ-myc lymphoma development or alter the sensitivity of Eµ-myc lymphomas to killing by DNA damaging drugs. Together, these results suggest that at physiological levels, Bik alone, or even in combination with Noxa, is insufficient to impose a barrier to c-Myc-driven tumorigenesis, and that these two BH3-only proteins on their own do not play a significant role in the p53-mediated DNA damaging drug-induced apoptosis of lymphoma cells. The findings in this thesis highlight the necessity in understanding the crucial requirements of specific BH3-only proteins for tumour suppression, as well as for efficient killing of tumour cells by DNA damaging chemotherapeutic drugs. This knowledge has important implications for predicting treatment success or failure, and ultimately, aid the development of novel anti-cancer agents with improved therapeutic efficacy.