The role of protein kinase C epsilon in the pathogenesis and treatment resistance of Acute Myeloid Leukaemia

Acute myeloid leukaemia (AML) is a heterogeneous group of haematological malignancies, characterised by the accumulation of abnormally differentiated blast cells in the bone marrow. For most patients, prognosis remains poor due to the prevalence of relapse. Protein kinase C epsilon (PKCε) is a pleiotropic regulator of cell signalling which has demonstrated oncogenic properties and in solid cancers, high PKCε expression is associated with poor outcomes. The aim of this study was to determine the clinical significance and functionality of PKCε in AML. Existing protein expression data showed that PKCε was significantly upregulated in 37% (26/70) of AML patient samples and was associated with reduced CR (p<0.05). To support this, I analysed an independent mRNA dataset where high PKCε expression correlated with significantly reduced overall (p<0.05) and disease-free survival (p<0.05). Together these data suggest that high PKCε expression is associated with poor outcomes in AML. Functionality was investigated by modulating PKCε in normal human haematopoietic cells where PKCε overexpression promoted monocyte differentiation. This finding is inconsistent with a role in leukaemogenesis. Instead, it was hypothesized that PKCε may promote chemoresistance. In two AML cell lines, PKCε overexpression conferred daunorubicin (DNR) resistance (p<0.05) without affecting cytarabine sensitivities. This was accompanied by reduced DNR accumulation, and the upregulation of the selective DNR efflux pump, P-GP. P-GP inhibition restored DNR accumulation and sensitivity, demonstrating P-GP drug efflux as the mechanism of PKCε-mediated DNR resistance. PKC agonist and inhibitor treatments suggested that PKCε modulates P-GP drug efflux through indirect mechanisms, but a correlation between PKCε and P-GP expression and function was supported by analysis of patient samples. However, reducing PKCε expression did not sensitize AML cells to DNR, potentially due to redundancy between PKC isoforms. In conclusion, elevated PKCε expression is a poor prognostic indicator in AML. Functional studies did not support an oncogenic role for PKCε in normal haematopoietic cells or AML cell lines. Nonetheless, PKCε can influence the chemosensitivity of AML cells by promoting P-GP-mediated DNR efflux, providing a mechanism through which PKCε could contribute to poor outcomes in this malignancy

DNA mismatch repair and cellular response to cytarabine :implications for the pathogenesis and treatment of therapy-related acute myeloid leukaemia

PhD ThesisThe DNA mismatch repair (MMR) pathway is responsible for correction of replicative errors, hence is a key factor in maintenance of genomic stability. Paradoxically, functional DNA MMR also mediates the cytotoxicity of certain chemotherapeutic DNA damaging agents. Poor treatment response in therapy-related acute myeloid leukaemia (t-AML) is influenced by a number of factors, one of which might be chemoresistance due to acquired defects in DNA MMR. Using a range of paired MMR proficient and deficient cell lines, investigations herein demonstrate that DNA MMR status mediates response to nucleoside analogues such as cytarabine (Ara-C) used in t-AML chemotherapy. Interestingly, defects of specific MMR components had different and opposing effects on the cytotoxicity of these agents. These findings implicate defects of MMR components as potential prognostic factors in t-AML and suggest assessment of DNA MMR status may be warranted in individual patients when selecting treatment. Cytarabine was mutagenic to DNA at the TK and HPRT loci. Furthermore, the frequency of Ara-C-induced mutation was increased in an MMR-deficient cell line, supporting a role for MMR components in the cellular response to nucleoside analogues, and also suggesting that use of these agents themselves could contribute to the risk of t-AML development. Defective DNA MMR might also contribute to development of relapsed AML, given that genomic instability is demonstrated in some patients at relapse. Genome-wide analysis of DNA copy number aberrations and loss of heterozygosity in a small cohort of matched presentation and relapsed AML samples demonstrated a potential MMR defect in one patient, and also provided some important insights into the clonal origins of relapsed AML. The findings of these investigations together highlight several important considerations for the use of nucleoside analogues in the treatment of t-AML, as well as in other cancers in which dysfunction of the DNA MMR system is implicated.Leukaemia and Lymphoma Researc

The influence of p21WAF1 on cell death pathways in acute lymphoblastic leukaemia

The p53 protein is a primary mediator of apoptosis and growth arrest after exposure to DNA-damaging agents. Previous work has categorised a wild type p53 gene in the majority of childhood acute lymphoblastic leukaemia (ALL) cases, in which instance the p53 protein functions as a modulator of chemotherapy-induced cell death. In contrast, certain p53-induced proteins, such as p21WAF1, can act in an anti-apoptotic manner, and bestow resistance to chemotherapy. Previous studies of the p53 pathway in ALL have utilised cell lines and primary material. In this study a model of ALL was utilised that had previously been developed from a heterogeneous panel of patient biopsies established as xenografts in immune-deficient mice, and are adaptable for short term in vitro culture. A wild-type p53 protein response to etoposide and nutlin-3 exposure was a feature of the whole ALL xenograft panel, irrespective of clinical characteristics and disease biology. While a range of p53 target genes were induced in B-cell precursor (BCP)-ALL and T-ALL xenografts after etoposide exposure, there was negligible induction of p21WAF1 in T- ALL samples. Further work with the histone deacetylase inhibitor vorinostat facilitated p53-independent induction of p21WAF1 in BCP-ALL samples, yet failed to induce p21WAF1 in T- ALL. An association was observed between reduced p21WAF1 expression in the T-ALL samples and decreased histone H3 acetylation in the p21WAF1 promoter together with increased cytosine methylation in the first exon/intron of the p21WAF1 gene. These results suggest that p21WAF1 in T-ALL cells is subject to epigenetic modifications that cause transcriptional silencing. Defective induction of p21WAF1 in T-ALL xenografts was associated with increased sensitivity to the death-inducing effects of drugs, phosphatidylserine (PS) externalisation and caspase-3/-7 activity after drug exposure, indicating that p21WAF1 may exert an anti-apoptotic activity. As proof of principle, p21WAF1 was silenced in Nalm-6 cells by micro-RNA transduction and these cells exhibited increased sensitivity and rapid PS externalisation after drug exposure. A combination of a p21WAF1 inhibitory agent and vorinostat gave some pharmacological evidence to suggest that p21WAF1 inhibition could enhance drug efficacy. Overall, these investigations provide insight into the epigenetic regulation of p21WAF1 and demonstrate an anti-apoptotic role for p21WAF1 in childhood ALL cells

Oncogene and Cancer

This book describes a course of cancer growth starting from normal cells to cancerous form and the genomic instability, the cancer treatment as well as its prevention in form of the invention of a vaccine. Some diseases are also discussed in detail, such as breast cancer, leucaemia, cervical cancer, and glioma. Understanding cancer through its molecular mechanism is needed to reduce the cancer incidence. How to treat cancer more effectively and the problems like drug resistance and metastasis are very clearly illustrated in this publication as well as some research result that could be used to treat the cancer patients in the very near future. The book was divided into six main sections: 1. HER2 Carcinogenesis: Etiology, Treatment and Prevention; 2. DNA Repair Mechanism and Cancer; 3. New Approach to Cancer Mechanism; 4. New Role of Oncogenes and Tumor Suppressor Genes; 5. Non Coding RNA and Micro RNA in Tumorigenesis; 6. Oncogenes for Transcription Factor

DNA mismatch repair and cellular response to cytarabine : implications for the pathogenesis and treatment of therapy-related acute myeloid leukaemia

The DNA mismatch repair (MMR) pathway is responsible for correction of replicative errors, hence is a key factor in maintenance of genomic stability. Paradoxically, functional DNA MMR also mediates the cytotoxicity of certain chemotherapeutic DNA damaging agents. Poor treatment response in therapy-related acute myeloid leukaemia (t-AML) is influenced by a number of factors, one of which might be chemoresistance due to acquired defects in DNA MMR. Using a range of paired MMR proficient and deficient cell lines, investigations herein demonstrate that DNA MMR status mediates response to nucleoside analogues such as cytarabine (Ara-C) used in t-AML chemotherapy. Interestingly, defects of specific MMR components had different and opposing effects on the cytotoxicity of these agents. These findings implicate defects of MMR components as potential prognostic factors in t-AML and suggest assessment of DNA MMR status may be warranted in individual patients when selecting treatment. Cytarabine was mutagenic to DNA at the TK and HPRT loci. Furthermore, the frequency of Ara-C-induced mutation was increased in an MMR-deficient cell line, supporting a role for MMR components in the cellular response to nucleoside analogues, and also suggesting that use of these agents themselves could contribute to the risk of t-AML development. Defective DNA MMR might also contribute to development of relapsed AML, given that genomic instability is demonstrated in some patients at relapse. Genome-wide analysis of DNA copy number aberrations and loss of heterozygosity in a small cohort of matched presentation and relapsed AML samples demonstrated a potential MMR defect in one patient, and also provided some important insights into the clonal origins of relapsed AML. The findings of these investigations together highlight several important considerations for the use of nucleoside analogues in the treatment of t-AML, as well as in other cancers in which dysfunction of the DNA MMR system is implicated.EThOS - Electronic Theses Online ServiceLeukaemia and Lymphoma ResearchGBUnited Kingdo

Étude des mécanismes des voies mitochondriale et lysosomiale dans l'apoptose p53-indépendante induite par les agents chimiothérapeutiques

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal

Programmed cell death

The purpose of this conference to provide a multidisciplinary forum for exchange of state-of-the-art information on the role programmed cell death plays in normal development and homeostasis of many organisms. This volume contains abstracts of papers in the following areas: invertebrate development; immunology/neurology; bcl-2 family; biochemistry; programmed cell death in viruses; oncogenesis; vertebrate development; and diseases

Intestinal model of Inflammation in primary cells

The gastrointestinal tract contains an enormous mucosal surface, which is continuously exposed to antigens hence making it susceptible to an inflammatory response. Such response targets potential pathogens by direct activation of the mucosal immune cells, however, in newborns the continuous inflammation attacks the intestine which leads to induction of necrotising enterocolitis. The present study aims at developing an “in-vitro” intestinal model of inflammation to assist in understanding the complex interplay of pro-inflammatory mediators during the immune response in neonates. Segments (1.5cm length) from the ileum were obtained from SD rat neonates (1-4 days old) and exposed to 0.25% trypsin/EDTA for 30min. Following trituration and subsequent centrifugation for 5min at 450xg, cells were suspended in DMEM-Hepes supplemented with 10% FCS, 2.5% Penicillin/Streptomycin, 2.5% L-Glutamine, and 0.2% Amphotericin B. Cell suspension were transferred to culture flaks and incubated at 37°C. Once confluent, the cell preparation media was replaced by FCS-free media, and treated with 0, 10, 50, and 100'g/ml of LPS. IL-8 and nitric oxide (NO) response were subsequently measured. In separate studies cell proliferation, cell viability, and cell adhesion were analysed. Additionally, the phenotypic properties of the intestinal muscle cells were also investigated via immunocytochemistry. Initial studies demonstrated that LPS treatments induced a significant increase in the release of IL-8 and NO compared to controls. The effect of LPS treatments on cell dynamics demonstrated small changes in cell viability and adhesion, whereas an increase in cell proliferation was observed. Immunocytochemistry studies indicated that LPS treatment caused a decrease in the expression of actin fibers with impaired distribution compared to controls. In the present model key aspects of intestinal inflammation were replicated “in-vitro” including the activation of pro-inflammatory mediators, the loss in enteric innervations and subsequent tissue hyperplasia. Thus, this model may be used as a tool to investigate the anti-inflammatory properties of candidate drugs targeting functional GI diseases