The normal physiological response to bacterial infection or wounding with threat of infection, termed inflammation, has been shown to be dysregulated in certain human diseases including (but not limited to): idiopathic pulmonary fibrosis, acute lung injury, arthritis and glomerulonephritis. The earliest arriving and most abundant cell responding to an inflammatory stimulus is the neutrophil granulocyte. It has been shown that under inflammatory conditions neutrophil granulocytes have extended longevity, enhanced responsiveness and upregulated activation parameters. In the setting of non-infective, or prolonged, ineffectuallycleared infective disease where resolution of inflammation does not occur then neutrophil granulocytes may cause tissue damage which is mediated by excessive, misdirected exocytosis of toxic granule contents or by spillage of the same products from necrotic or netotic cell carcasses that have lost membrane integrity. A key process in the resolution of inflammation is the induction of apoptosis in recruited neutrophils following a successful response to an inflammatory stimulus. Cellular signalling from apoptotic cells and from professional phagocytes that have ingested apoptotic cells has been shown to favour resolution of inflammation and restoration of tissue homeostasis. Additionally, the removal of key inflammatory cells in a highly regulated, non-phlogistic fashion robustly assists the resolution process. Cyclin-dependent kinase (CDK) inhibitor drugs are being developed as anti-cancer agents as it is hypothesized that they should interfere with the enhanced cellcycling ability (increased proliferative capacity and extended longevity) which is such a key feature of cancer cell biology. The CDKs that drive the cell cycle are CDKs 1, 2, 4 and 6 and consequently agents were designed to have enhanced specificity for these targets. CDK inhibitor drugs target the ATP-binding domain of CDKs and as a result usually have activity against more than one CDK. The CDK inhibitor drug, R-roscovitine which targets CDKs 2, 5, 7 and 9 was shown to promote neutrophil apoptosis and consequently resolution of inflammation. This thesis aims to investigate the mechanism by which apoptosis is induced in neutrophil granulocytes by CDK inhibitor drugs. The first experimental chapter of this thesis explores in detail the time-course and active concentration range of CDK inhibitor drugs in comparison to known promoters and inhibitors of neutrophil apoptosis. It then dissects the apoptotic machinery which is responsible for the effects of CDK inhibitor drugs before investigating their capacity to promote apoptosis even in the presence of survival mediators relevant to the context of inflammatory disease. Flow-cytometry, light and confocal microscopy as well as western blotting for caspases, mitochondrial dissipation assay, fluorometric caspase assay and the detection of DNA laddering demonstrate that CDK inhibitor drugs promote classical neutrophil apoptosis by the intrinsic pathway and show similar kinetics of apoptosis induction to drugs that inhibit transcription. The second experimental chapter investigates the key neutrophil survival protein and bcl-2 homologue Mcl-1. By flow cytometry, western blotting and RT-PCR it is demonstrated that Mcl-1 is down-regulated at the level of transcription and that this occurs even in the presence of inflammatory mediators that would normally promote neutrophil survival. Additionally, it is shown that pro-apoptotic bcl-2 homologues are affected to a lesser degree suggesting an imbalance of bcl-2 proteins is caused by effects at a transcriptional level mediated by CDK inhibitor drugs. The third experimental chapter identifies CDKs and their binding partner cyclins in neutrophil granulocytes and investigates the impact of CDK inhibitor drugs on CDK protein levels and cellular distribution by differential lysis and western blotting as well as by confocal microscopy. The key transcriptional enzyme RNA polymerase II is also identified and the effect of CDK inhibitor drugs on phosphorylation of this enzyme is documented. Western blotting and confocal microscopy demonstrate the presence of key CDKs 2, 5, 7, 9 and cyclin binding partners of CDKs 7 and 9. It is shown that the phosphorylation of RNA polymerase II mediated by CDKs 7 and 9 is inhibited by CDK inhibitor drugs. This suggests that a key mechanism by which neutrophil apoptosis is induced by CDK inhibitor drugs is the inhibition of transcription of key proteins and suggests that neutrophils require survival proteins for functional longevity. The fourth experimental chapter addresses the production and use of HIV-tat dominant negative CDK 7 and 9 proteins to knockdown CDKs 7 and 9 in neutrophil granulocytes in vitro to provide a molecular biology surrogate for the pharmacological data already presented. The cloning, production, purification and use of HIV-tat dominant negative CDK proteins are described. The final chapter describes the use of a more specific pharmacological inhibitor of CDKs 7 and 9, DRB, in the mouse bleomycin lung injury model. Resolution of inflammation by a compound specifically targeting CDKs 7 and 9 is described. This thesis identifies CDKs 7 and 9 as key targets of CDK inhibitor drugs in neutrophilic inflammation. It shows these drugs acting at the level of transcription to drive neutrophil apoptosis by exploiting the unique dependency of neutrophils on the short-lived survival protein Mcl-1. In so doing the presence of functional and essential transcriptional machinery is identified in neutrophils and the transcriptional profile of resting, stimulated and inhibited neutrophils is delineated. These findings suggest novel approaches to the pharmacological promotion of resolution of inflammation and indicate key new targets for rational drug design. In future, it will be important to further characterize the effects of CDK inhibitor drugs on other cell-types including epithelial cells, fibroblasts and mononuclear cells. This information should prove important to the continued investigation of CDK inhibitor drugs in resolution of inflammation and also to the ongoing experimental trial of these drugs in idiopathic pulmonary fibrosis.

Drugs that alter microtubule dynamics are often used in chemotherapy regimes in combination with other agents in order to treat various cancers. Despite the success over many years, there remain problems in toxicity, resistance and predictability to the drugs. In order to overcome these problems, there is a need to gain an understanding of how these drugs kill cancer cells in cell culture. As microtubule function is particularly important for chromosome movement in mitosis, cells treated with these agents cause a mitotic arrest through activation of the spindle assembly checkpoint. Following induction of a mitotic arrest, cells can escape this arrest (mitotic slippage) or undergo mitotic death, determined in part by the response of the apoptotic network. Levels of an anti-apoptotic protein, Mcl-1, are often lost over time in mitosis. Using time-lapse analysis on a cell line unable to escape the mitotic arrest, this thesis shows that Mcl-1 protein contributes to cell death both in mitosis and the subsequent interphase in response to microtubule toxin, taxol. This suggests that inhibiting Mcl-1 may increase the efficacy of anti-mitotic agents. In addition, mitotic cell lines prone to mitotic slippage were found to have higher levels anti-apoptotic protein, Bcl-xL, in comparison to Mcl-1, indicating one way in which these cells can cope with loss of Mcl-1 during mitosis. Secondly, an evaluation of the contribution of the previously identified interaction between Mcl-1 and mitotic E3 ligase complex, the APC/C-Cdc20, to the rate of mitotic death and mitotic slippage was assessed. Inhibition of APC/C-Cdc20 activity or mutation of a Mcl-1 motif (RxxL) thought to engage with the APC/C-Cdc20 complex did not have a substantial effect on Mcl-1 degradation or mitotic death, thereby questioning the functional significance of this interaction. However, it appears that Mcl-1 protein levels can influence the rate of mitotic slippage and this influence was affected via Mcl-1’s RxxL motif within Mcl-1. This suggests that Mcl-1 protein may delay mitotic slippage via substrate competition for the APC/C-Cdc20 complex with Cyclin B1, whose degradation is required for mitotic exit. Further analysis of this effect showed that this interaction may not be a universal effect. This together with the specific functional effect on mitotic slippage rather than mitotic death, suggests that this is an indirect effect caused by network interference between the components of the death and slippage pathways.

Les glycosides cardiaques (GCs) utilisés en clinique contre les maladies cardiaques sont des inhibiteurs de la pompe sodium-potassium-ATPase. Récemment il a été montré que ces composés ont un effet anti-cancéreux. Nous avons étudié les effets de l’UNBS1450, un GC modifié partiellement extrait de la plante Calotropis procera. Nous avons analysé ses effets sur une protéine responsable de la résistance aux traitements anti-cancéreux : Mcl-1. L’UNBS1450 diminue son expression dans différents types de cancer à des concentrations très faibles. Cette modulation est capitale pour l’induction de la mort cellulaire. De plus, l’UNBS1450 diminue l’expression d’une protéine importante dans la prolifération cellulaire : c-Myc, cette modulation intervient dans les mêmes conditions, que celles qui permettent la diminution de Mcl-1. Nous avons observé que l’UNBS1450 est actif dans les cancers du sein, en bloquant le cycle cellulaire et la prolifération. Dans cette étude, nous avons montré que l’UNBS1450 et les CGs en général, d’une part induisent la mort cellulaire en diminuant l’expression de Mcl-1, et d’autre part affecte la prolifération cellulaire en modulant c-Myc / Cardiac glycosides (CGs) are used in clinics to treat heart diseases. They inhibit the sodium-potassium-ATPase. Recently, anti-cancers activities have been ascribed to these compounds. In this study, we investigated the anti-cancer effects of UNBS1450, a compound partially modified from a CG extracted from the plant Calotropis procera. We analyzed its activities against a protein responsible for the chemoresistance of cancer cells: Mcl-1. We found that UNBS1450 decreases the levels of Mcl-1 in many forms of cancer, at very low concentrations. This modulation is important to induce cancer cell death and is common to other CGs. Furthermore, UNBS1450 also decreases the intracellular levels of another protein important for the cancer cell proliferation: c-Myc. This modulation takes place in the same conditions determining Mcl-1 decrease. Finally, we found UNBS1450 active on breast cancer, by blocking the cell cycle and proliferation of the breast cancer cells. In this study, we have shown that UNBS1450 and CGs in general induce cell death by decreasing the expression of Mcl-1 protein and affect cell proliferation with the concomitant decrease of c-Myc protein

La thématique du laboratoire de l’équipe d’Adam Telerman porte sur la réversion tumorale, un processus rare au cours duquel les cellules cancéreuses perdent leur phénotype malin, et deviennent des cellules dites révertantes. Plusieurs protéines clefs impliquées dans cette transformation ont été mises en évidence, dont TCTP (Translationally Controlled Tumor Protein). La protéine TCTP est également impliquée dans la régulation de l’apoptose en interagissant et en renforçant l’activité anti-apoptotique de Mcl-1 et de Bcl-xl, deux protéines appartenant à la famille des Bcl-2. Ce projet s’attache à comprendre en termes moléculaires le mode d’action de TCTP au cours de l’apoptose. / Adam Telerman’s team research focuses on tumor reversion, a rare process in which cancer cells lose their malignant phenotype, and therefore become revertant. Many key proteins were showed to be involved in this transformation, including TCTP (translationally Controlled Tumor Protein). TCTP protein is also involved in apoptosis regulation by interacting and strengthening the anti-apoptotic activity of Mcl-1 and Bcl-xl, two proteins from Bcl-2 family.

Head and neck cancer is the sixth leading type of cancer with 90 percent of head and neck cancer arising from squamous cell lining on the epithelium of the oral and nasal cavity, pharynx, and salivary gland. Even with tremendous achievements on chemotherapeutic drugs and therapies, the long-term prognosis of patients with advanced head and neck squamous cell carcinoma (HNSCC) has shown little improvement over the last three decades. Cisplatin is one of widely used chemotherapeutic drugs for multiple cancers, including head and neck cancer, but the prolonged use of this drug is limited by its toxicity and by the development of resistance. To overcome these major roadblocks to improved prognosis requires mechanism-based therapeutic strategies to maximize the antitumor effect of drugs while limiting their toxicities. Cisplatin exerts anticancer effects via multiple mechanisms, yet its most prominent mode of action involves the generation of DNA lesions followed by the activation of the DNA damage response and the induction of BCL-2 family-dependent mitochondrial apoptosis. DNA damage activates a tumor suppressor p53 to induce apoptosis. One of its functions is to induce the expression of several pro-apoptotic proteins such as Noxa, which binds to an anti-apoptotic BCL-2 family protein, MCL-1 (myeloid leukemia cell-1) to inactivate its pro-survival function and induce apoptosis. We examined Noxa expression and apoptosis induced by cisplatin in p53-wild-type HN30 and HN31, p53-truncated and inactive HN4 and HN12, and p53-deleted HN22 and HN8 HNSCC cell lines. We found that Noxa was induced in HN30 and HN31 cells and down-regulation of Noxa by shRNA (short-hairpin RNA) decreased apoptosis, indicating Noxa contribution to cisplatin-induced apoptosis. Interestingly, cisplatin treatment induced Noxa and apoptosis even in p53-deleted HN22 and HN8 cells, suggesting the existence of the p53-independent pathways for the induction of Noxa. Based on these observations, we hypothesized that modulation of Noxa/MCL-1 axis could mimic cisplatin-induced cell death. We found that Noxa overexpression induced cell death in all cell lines tested regardless of p53 status. This finding could be applicable as a potential therapeutic strategy to treat head and neck cancer.

<p>Throughout their development in the thymus and during their maintenance and the immunological response in the periphery, T cells rely on the regulation of classical apoptotic pathways to promote cell survival or death. Several proteins of the Bcl-2 family have been shown to be critical in thymocyte and T cell survival and consequently, in T cell function. Among these proteins, the antiapoptotic proteins Bcl-2 and Mcl-1 are critical for promoting T cell survival at multiple stages of the T cell "life cycle." While these proteins have been reported to interact with several of the proapoptotic members of the Bcl-2 family, the specific interactions by which Mcl-1 in particular promotes T cell survival in vivo were not well understood. Further, how different stimuli (for example, cytokine signaling and T cell activation) modulate the specific functions of Mcl-1 had also not been thoroughly explored. </p><p>We utilized mouse models to dissect the roles of Mcl-1 at multiple stages of T cell development and function. We utilized conditional knockout and double knockout strategies to build genetic pathways for Mcl-1 activity during thymocyte development and in peripheral T cells under a variety of conditions. In the thymus, the major role of Mcl-1 is to inhibit the activity of proapoptotic Bak because the loss of Bak, but not the loss of Bax or Bim, rescued the survival of Mcl-1-deficient thymocytes at both the double negative and single positive stages. Further, we concluded that this role is not shared with Bcl-2 because overexpression of Bcl-2 did not rescue DN or SP survival. </p><p>In peripheral T cells, the loss of Bak rescued T cell survival in the presence of IL 7, but not during conditions of cytokine withdrawal. Interestingly, the overexpression of Bcl-2 or the loss of Bim partially rescued the survival of T cells during cytokine withdrawal, indicating that Mcl-1 has dual roles in T cells: cytokine-dependent and cytokine independent. Additionally, we found that cytokines of the common gamma chain family have different effects on the activity of Mcl-1 due to the differential regulation of other proteins of the Bcl-2 family, most notably Bim.</p><p>Finally, we utilized a Bcl-2 reporter mouse model to examine the role of Bcl-2 in the establishment of CD8+ T cell memory to infection. Although it is known that Bcl-2 is dynamically regulated in response to activation, the importance of this regulation in the establishment of T cell memory is not yet clear. We show that a subset of effector T cells within a previously defined memory precursor population retained high Bcl-2 expression at the peak of the immune response. Using adoptive transfer of sorted effector T cells, we provide preliminary evidence that the cells with memory potential lie within a strict range of Bcl-2 expression. These studies indicate that the regulation of Bcl 2 is likely critical in establishing T cell memory and provide a platform for the future study of the factors that influence T cell memory.</p> / Dissertation

The process of autophagy is defined as the degradation of cellular cytoplasmic constituents via a lysosomal pathway. Herein I sought to examine the regulation of autophagy in the placental pathologies preeclampsia (PE) and intrauterine growth restriction (IUGR). I hypothesized that the Bcl-2 family proteins Mcl-1L and MtdL regulate placental autophagy and contribute towards dysregulated autophagy in PE. My results demonstrate that Mcl-1L acts to repress autophagy via a Beclin 1 interaction, while MtdL induces autophagy when it interacts with Mcl-1L. My data indicate that while autophagy is elevated in PE, a pathology characterized by oxidative stress, it is decreased in IUGR, a hypoxic pathology. Treatment with sodium nitroprusside to mimic PE caused a decrease in Mcl-1L and an increase in MtdL levels in response to oxidative stress, thereby inducing autophagy. Overall, my data provide insight into the molecular mechanisms contributing to the pathogenesis of preeclampsia.

The process of autophagy is defined as the degradation of cellular cytoplasmic constituents via a lysosomal pathway. Herein I sought to examine the regulation of autophagy in the placental pathologies preeclampsia (PE) and intrauterine growth restriction (IUGR). I hypothesized that the Bcl-2 family proteins Mcl-1L and MtdL regulate placental autophagy and contribute towards dysregulated autophagy in PE. My results demonstrate that Mcl-1L acts to repress autophagy via a Beclin 1 interaction, while MtdL induces autophagy when it interacts with Mcl-1L. My data indicate that while autophagy is elevated in PE, a pathology characterized by oxidative stress, it is decreased in IUGR, a hypoxic pathology. Treatment with sodium nitroprusside to mimic PE caused a decrease in Mcl-1L and an increase in MtdL levels in response to oxidative stress, thereby inducing autophagy. Overall, my data provide insight into the molecular mechanisms contributing to the pathogenesis of preeclampsia.

One of the main results of Rb-E2F pathway disruption is deregulation of the E2F family of transcription factors, which can lead to inappropriate proliferation, oncogenic transformation, or the induction of apoptosis. Given the potential negative biological effects associated with deregulated E2F activity, it is of great importance to study E2F targets that mediate these effects. In Part I of this manuscript, we identify the RhoBTB2 putative tumor suppressor gene as a direct physiological target of the E2F1 transcription factor. We find that RhoBTB2 is highly upregulated during mitosis due in part to E2F1, and that overexpression of RhoBTB2 increases the S-phase fraction and slows the rate of proliferation. We also find RhoBTB2 similarly upregulated during drug-induced apoptosis due primarily to E2F1 and that knockdown of RhoBTB2 expression via siRNA slows drug-induced apoptosis. Taken together, we describe RhoBTB2 as a novel direct target of E2F1 with roles in cell cycle and apoptosis. In Part II, we independently identify from cancer cell lines two novel variants from the promoter of E2F1 target MCL-1---MCL-1 +6 and +18---as initially published by Moshynska et al (1). In contrast to Moshynska et al., we find the variant promoters identically present in both cancerous and adjacent noncancerous clinical lung samples, suggesting that the variants are germ-line encoded. We also find the variant promoters prevalent in genomic DNA derived from healthy control samples and present at frequencies similar to that observed in cancerous cell lines. In further contrast, we find the activity of the MCL-1 +6 and +18 promoters approximately 50% less than the common MCL-1 +0 promoter---both during normal cellular homeostasis and under conditions that actively induce Mcl-1 transcription. Given our results and those of others, we conclude that the MCL-1 +6 and +18 promoters are likely benign polymorphisms and do no [sic] represent a reliable prognostic marker for CLL as reported by Moshynska et al.

Myeloid Cell Leukemia-1 (Mcl-1) is a widely expressed anti-apoptotic member of the Bcl-2 family that is elevated in a variety of tumour types including breast cancer. Mcl-1 promotes tumour cell survival and drug resistance and was a mechanism of resistance to first generation Bcl-2 family inhibitors. To determine the significance of Mcl-1 in breast cancer, we evaluated the regulation of Mcl-1 by signalling via the epidermal growth factor receptors (EGFRs). EGFR signalling is frequently deregulated in breast cancer and leads to increased proliferation and survival of tumour cells. We aimed to determine whether Mcl-1 is a critical downstream effector of this pathway and therefore an important therapeutic target. We found that Mcl-1 protein and messenger RNA levels were rapidly induced upon stimulation of breast cancer cells with epidermal growth factor. This induction was blocked by inhibitors of the Ras/Raf/Mek/Erk signalling cascade and was dependent upon activation of the transcription factor Elk-1. We found Mcl-1 to be an essential survival protein, as targeted knock-down with small interfering RNA alone was sufficient to induce apoptosis. Mcl-1 may be critical for the survival advantage conferred by EGFR activation, as prevention of its up-regulation by Mek/Erk inhibitors significantly reduced the drug resistance conferred by EGF. Furthermore, we found a correlation between phosphorylated Elk-1 and Mcl-1 protein levels in breast tumour samples. Therefore, we conclude that Mcl-1 is an important downstream effector of survival and drug resistance mediated by elevated EGF signalling, making it an important therapeutic target in breast cancer.