The overall concept of this project is that inhibition of the autoimmune attack mediated by IFNg against pancreatic beta-cells can enable islet recovery and/or better islet transplant survival in type I diabetic patients. The project comprises of two parallel branches. The first branch will study whether the above concept is valid using in vivo animal models. The autoimmune reaction against the beta cells is multifold, however studies of our laboratory found that pro-inflammatory cytokines, interleukin-1b and interferon gamma (IFNg) are major mediators. IFNg deficient mice as well as mice injected with neutralizing anti-IFNg-receptor antibodies were resistant to development of experimentally-induced type I diabetes. Based on this information, the first aim is to evaluate whether INFg neutralization can reverse the progression of manifested diabetes and enhance pancreas recovery.
Uncontrolled proliferation and impaired response to apoptotic signalling are hallmarks of tumorigenesis. Colon carcinoma is the third major cause of cancer-related deaths in Ireland and resistance to drug therapy is a serious problem in its treatment. Thus, restoring the cancer cell’s ability to respond to apoptotic signalling and to ignore proliferative signals are promising strategies for treatment. The cytokine, TNF related apoptosis-inducing ligand (TRAIL) is a very promising anti-cancer agent, as it induces apoptotic death in only cancerous cells, but not in normal, non-transformed cells. However, when TRAIL binds to its two apoptosis-inducing receptors, death receptor 4 (DR4) and DR5, parallel to the apoptotic signal, an inflammatory, pro-survival signal, mediated by nuclear factor-kappa B (NF-kB), is also generated which can counteract the apoptotic signal. This is often the cause of TRAIL resistance of tumours and a significant contributor to metastases. Our previous studies using computational design have identified peptides to prevent the recruitment of TRAF2 to TRADD to the DR4/DR5 receptors and thus prevent DR4/DR5-mediated NF-kB activation. We believe that these TRADD-TRAF2 interaction inhibitors would uncouple the apoptotic signalling from the pro-survival signalling induced by TRAIL in tumour cells without causing toxic side effects in non-cancerous cells. Also, these TRADD-TRAF2 interaction inhibitors used in combination with TRAIL could eliminate/reduce the risk of tumour metastasis induced by NF-kB activation. The objective of this study is to evaluate the potency and specificity of these peptides to block DR4/DR5-mediated NF-kB activation and assess their anti-cancer efficacy in in vivo xenograft models.
Despite the availability of genomics and proteomics and intensive investigation, the ability to predict tumour response to treatments remains limited. Our proteomic analysis of the apoptotic pathway induced by the cytokine and novel anticancer agent, TRAIL, revealed that the expression level of individual proteins only marginally correlates with TRAIL-sensitivity of tumours. On the other hand, expression ratio of these proteins showed strong correlation with TRAIL-sensitivity. This proposal hypothesizes, that rather than the absolute amount, the ratio of the components determines tumour responses. To date, no genomic or proteomic study have examined expression ratios.
In order to assess the predictive power of our ratiometric analysis, we will determine whether the expression ratio of 2 proteins have higher predictive power than the absolute expression of the proteins individually. As a model system, the components of the well-characterised TRAIL cell death machinery will be analysed: TRAIL receptors, caspase-8, c-FLIP, Bcl-2, Bcl-XL and MCl-1. In order to prove the hypothesis, a mechanistic model, i.e. regulated overexpression studies will be used to alter the ratio of predictive protein pairs. Finally, we will apply the ratiometric analysis method to DNA microarrays, i.e. examine the ‘ratiometry’ at the level of the transcriptome. If the hypothesis is proven, it will induce a paradigm shift in the way we analyse proteomic/genomic data and will provide a new insight into cellular mechanics. With this analysis, ratio-fingerprints can be generated to reveal hidden gene/protein interactions which would explain so far unexplainable tumour responses to drugs thus promoting the development of tailor-made therapies.
Physiological or pathological processes that disturb protein folding in the endoplasmic reticulum (ER)activate a set of signalling pathways termed the Unfolded Protein Response (UPR). The initial aim of the UPR is to re-establish homeostasis and normal ER function. However, if the primary stimulus causing protein unfolding in the ER is protracted or excessive, then the stress cannot be resolved and programmed cell death, or apoptosis, ensues. The exact mechanism involved in transition of the UPR from a cell survival to cell death response is not clearly understood. ER stress leading to protein aggregation and cell death is implicated in a wide range of diseases, including neurodegeneration, stroke, bipolar disorder, cardiac disease, cancer, and diabetes. Hypotheses under investigation and main aims: Mutations in the PERK gene, a key mediator of both the UPR and ER stressinduced apoptosis, lead to a decline in pancreatic function and diabetes, in both mice and humans. Mouse embryonic fibroblasts(MEFs) from PERK-/- mice are sensitive to ER stress-induced apoptosis. However, the molecular determinants of the increased sensitivity of PERK-/- cells are not clearly understood. Preliminary data from the laboratory of Dr. Samali show that the expression of BH3 family proteins, such as BMF, PUMA and NOXA is increased in PERK-/- MEFs upon exposure to ER stress as compared to wild type MEFs. Here we propose to determine how the loss of PERK sensitizes the cells specifically to ER stress-induced apoptosis and the relationship between PERK and BH3 family protein expression.
This proposal addresses the question whether inhibition of apoptotic cell death in pancreatic islets by over-expression of Bcl-xL leads to improved islet function in vitro and restoration of normoglycaemia upon transplantation in diabetic animals.