Thesis (M. Med. Sc.)--University of Queensland, 2001. / Includes bibliographical references.

Thesis (M. Med. Sc.)--University of Queensland, 2002. / Includes bibliographical references.

Thesis (Ph.D.)--George Mason University, 2008. / Vita: p. 175. Thesis director: Ancha Baranova. Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biosciences. Title from PDF t.p. (viewed Jan. 8, 2009). Includes bibliographical references (p. 156-174). Also issued in print.

published_or_final_version / Pathology / Master / Master of Philosophy

A mathematical model of the interaction between chronic lymphocytic leukemia (CLL) and CD4+ (helper) T cells was developed to study the role of T cells in cancer survival. In particular, a system of four nonlinear advection diffusion reaction partial differential equations were used to simulate spatial effects such as chemical diffusion and chemotaxis on CLL survival and proliferation.

B cell chronic lymphocytic leukemia (B-CLL) is a hematologic neoplasm characterised by the proliferation and accumulation of sIgM+/D+ B cells that fail to progress to the final stages of B cell development. The malignant cells in B-CLL also express the pan-T cell antigen CD5, suggesting that CLL is a malignancy of the CD5+ subset of B cells. Additional characteristics of the malignant clone include a low proliferative index, enhanced in vivo survival and constitutive expression of the anti-apoptosis oncoprotein bcl-2. The behaviour of leukemic CD5 B cells in vitro contrasts their arrested in vivo state. That is, despite the majority of cells being arrested in the G0 phase of the cell cycle, the leukemic B cells are not irreversibly frozen as they can be induced to differentiate to Ig-secreting cells under appropriate in vitro conditions. Furthermore, leukemic CD5 B cells rapidly undergo death by apoptosis following in vitro culture. This thesis describes the requirements for in vitro activation of leukemic CD5+ B cells, the characterisation of the events involved in apoptosis of these cells as well as the identification of various growth factors capable of modulating these events. Stimulation of unfractionated peripheral blood lymphocytes (PBLs) from three patients with B-CLL with the phorbol ester PMA and the mitogens PHA and PWM resulted in significant increases in cell proliferation, RNA synthesis and 1gM secretion when compared to unstimulated cell populations. PMA was the most potent inducer of 1gM secretion and this occurred irrespective of the presence of residual T cells. PMA-induced proliferation and RNA synthesis were also independent of T cells. However, in the presence of T cells, these parameters of cellular activation were enhanced during in vitro culture. Thus, the inductive ability of PMA on leukemic CD5 B cells was independent of T cells. In contrast, activation and differentiation of the leukemic CD5 B cells into 1gM-secreting cells following culture with mitogens did not occur in the absence of T cells. Interestingly, co-stimulation of leukemic CD5+ B cells with PMA and anti-Ig induced cellular responses that exceeded those induced by either activator alone. Thus, leukemic CD5+ B cells from patients with B-CLL can be activated in vitro and differentiate in response to stimulation via both T cell-dependent and T cell-independent mechanisms. Apoptotic cell death was characterised in purified leukemic CD5 B cells obtained from six B-CLL patients. All leukemic CD5 B cell populations entered an apoptotic pathway in vitro as evidenced by a reduction in cell size, loss of cell viability and fragmentation of DNA into multimers of -180 base pairs. Following 24 hours of in vitro culture 24.0±16% of DNA was fragmented. After 8 days, the majority of DNA was fragmented, and fewer than 10% of cultured cells were viable. Examination of bcl-2 expression in the malignant B cells by flow cytometry revealed a unimodal pattern of expression in greater than 85% of cells from each B-CLL patient prior to culture. During in vitro culture, bcl-2 expression became bimodal such that the B cells displayed a bcl-2hjgh and bcl-2iow phenotype. The level of expression by the bCl2hjgh cells was similar to that observed prior to in vitro culture, indicating that bcl-2 is down-regulated in apoptosing cells. Interestingly, despite this downregulation, the overall number of cells positive for bcl-2 remained constant. This suggests that the enhanced survival of leukemic CD5+ B cells in vivo is mediated by the sustained expression of bcl-2 and that additional mechanisms exist capable of overriding the protective effect of bcl-2 when bcl-2 is present at reduced levels. Leukemic B cell apoptosis has previously been reported to be delayed or prevented by IL-4, IFN-y and IFN-a. These results were confirmed in this study where it was found that culture of leukemic CD5 B cells with IL-4 or IFN-y enhanced cell viability and delayed apoptosis in 6/6 and 5/6 populations of leukemic B cells, respectively. This function was also found to be shared by IL-2, IL-6, IL-13 and TNF-a as these cytokines enhanced cell viability and delayed apoptosis in some of the cell populations examined at a level similar to that observed for IL-4 and IFN-y. These cytokines may mediate their effect via the expression of bcl2 as culture in the presence of IL-2, IL-4, IL-6, IL-13, IFN-y or TNF-a resulted in a higher percentage of cells displaying the bcl-2high phenotype, compared to unstimulated cells. Taken together, these results suggest that autocrine and/or paracrine growth loops may play a role in the pathogenesis of B-CLL and that cytokines that prevent apoptosis in vitro may be targets for treatment of this B cell malignancy.

B-cell chronic lymphocytic leukemia is the most common leukemia in the adult population of Western developed countries. In 2005, an estimated 9,730 adults in the United States will be diagnosed with B-CLL and an estimated 4,600 deaths will occur. B-CLL is a common heterogeneous malignant disease with variable outcome. B-CLL is divided into two groups based on whether somatic hypermutation is observed in the variable region of the immunoglobulin heavy-chain locus (IgVH). The two distinct groups are named mutated and unmutated. The B-CLL mutated group has a more favorable prognosis than the unmutated group. Gene expression profiling has been used successfully to decipher the biological and clinical diversity of many leukemias and lymphomas. Recently, other small RNAs (microRNAs) have been shown to be important in hematopoiesis. MicroRNAs are small 20-28 nucleotide RNAs that are believed to control many important cellular and developmental processes by posttranscriptional gene silencing, translational repression, and modulating epigenetic events. We are interested in whether microRNA expression correlates with the mutational status of IgVH. This study is significant in the following ways: (1) microRNAs may become surrogate markers for the mutational status of IgVH of B-CLL, which implies a more rapid diagnostic means as compared to the current practice, and (2) microRNAs, in the particular context of B-CLL, may play some significant roles in a gene regulatory network that is further responsible for chromosomal abnormalities found in B-CLL. This thesis presents a study comparing microRNA expression in mutated and unmutated B-CLL groups. Instead of using a genome-wide expression profiling strategy, we selected a specific set of microRNAs based on their chromosome locations and mRNA targets. Specifically, we chose the following eight microRNAs (with their chromosomal abnormalities): mir16-1 (deletion 13), let-7i (trisomy 12), mir196-2 (trisomy 12), mir26a-2 (trisomy 12), mir-34b (deletion 11), mir-125b (deletion 11), mir-181C (trisomy 19), mir-125a (trisomy 19). We used solution hybridization assays to monitor the expression of microRNAs. We successfully characterized the microRNA expression in twelve B-CLL patient samples (eight mutated and four unmutated). Among the eight microRNAs examined, three (mir196-2, mir-125a, mir-125b) are not expressed in the two B-CLL groups, four (mir16-1, mir26a-2, let-7i, mir-34b) have significant differences in expressions over the two groups, and one (mir-181c) has no significant difference in expressions over the two groups.

B-cell chronic lymphocytic leukemia is the most common leukemia in the adult population of Western developed countries. In 2005, an estimated 9,730 adults in the United States will be diagnosed with B-CLL and an estimated 4,600 deaths will occur. B-CLL is a common heterogeneous malignant disease with variable outcome. B-CLL is divided into two groups based on whether somatic hypermutation is observed in the variable region of the immunoglobulin heavy-chain locus (IgVH). The two distinct groups are named mutated and unmutated. The B-CLL mutated group has a more favorable prognosis than the unmutated group. Gene expression profiling has been used successfully to decipher the biological and clinical diversity of many leukemias and lymphomas. Recently, other small RNAs (microRNAs) have been shown to be important in hematopoiesis. MicroRNAs are small 20-28 nucleotide RNAs that are believed to control many important cellular and developmental processes by posttranscriptional gene silencing, translational repression, and modulating epigenetic events. We are interested in whether microRNA expression correlates with the mutational status of IgVH. This study is significant in the following ways: (1) microRNAs may become surrogate markers for the mutational status of IgVH of B-CLL, which implies a more rapid diagnostic means as compared to the current practice, and (2) microRNAs, in the particular context of B-CLL, may play some significant roles in a gene regulatory network that is further responsible for chromosomal abnormalities found in B-CLL. This thesis presents a study comparing microRNA expression in mutated and unmutated B-CLL groups. Instead of using a genome-wide expression profiling strategy, we selected a specific set of microRNAs based on their chromosome locations and mRNA targets. Specifically, we chose the following eight microRNAs (with their chromosomal abnormalities): mir16-1 (deletion 13), let-7i (trisomy 12), mir196-2 (trisomy 12), mir26a-2 (trisomy 12), mir-34b (deletion 11), mir-125b (deletion 11), mir-181C (trisomy 19), mir-125a (trisomy 19). We used solution hybridization assays to monitor the expression of microRNAs. We successfully characterized the microRNA expression in twelve B-CLL patient samples (eight mutated and four unmutated). Among the eight microRNAs examined, three (mir196-2, mir-125a, mir-125b) are not expressed in the two B-CLL groups, four (mir16-1, mir26a-2, let-7i, mir-34b) have significant differences in expressions over the two groups, and one (mir-181c) has no significant difference in expressions over the two groups.

Chronic Lymphocytic Leukaemia (CLL) is a disease of B-lymphocytes that account for significant morbidity and mortality in mostly elderly patients (aged ≥ 70 years). The relative survival of patients with CLL has been shown to decrease with patient age, and this age-related reduction in survival was found to correlate with the levels of two inflammatory cytokine levels in the patients’ plasma. The levels of two inflammatory cytokines, interleukin-6 and -8 (IL-6, IL-8) were found to correlate positively with patient age, and increased levels were associated with lower overall survival. Addition of IL-6 or IL-8 to a co-culture system of CLL cells with bone marrow stromal cells increased the CLL-stromal cell adhesion, and co-culturing increased IL-8 secretion. In a search of a treatment regimen that may be effective and readily tolerated by elderly patients, we examined the combination of fludarabine with valproic acid (VPA), an epileptic that was found to inhibit histone deacetylases (HDACs). The combination was synergistic against human leukaemic cells, including primary CLL cells. In a phase II clinical trial where six elderly patients with relapsed, previously treated CLL were enrolled (half of whom were clinically refractory to fludarabine), the VPAfludarabine combination induced reduction in the peripheral and lymph node tumour loads. Mechanistically, the fludarabine treatment induced disruption of the lysosomes, while VPA induced increase in the level and activity of cathepsin B, a lysosomal protease. The VPA-induced increase in cathepsin B levels was observed in in cell lines (in vitro), primary CLL cells (ex vivo) and in patients treated with VPA (in vivo). Chemical inhibition of cathepsin B was sufficient to dampen the VPA-fludarabine cytotoxicity, and the addition activated cathepsin B to leukaemic cell lysates was sufficient to induce caspase cleavage and reduction in anti-apoptotic protein levels. The VPA-fludarabine combination also lowered phospho-Akt levels and ATM activation, which also contributed to the VPA-fludarabine synergy, and VPA treatment lowered ATM levels and phospho-Akt levels in vivo. In summary, there lies a biological explanation for the poor survival observed with elderly patients, and the VPA-fludarabine may be a useful regimen for these patients.

Chronic Lymphocytic Leukaemia (CLL) is a disease of B-lymphocytes that account for significant morbidity and mortality in mostly elderly patients (aged ≥ 70 years). The relative survival of patients with CLL has been shown to decrease with patient age, and this age-related reduction in survival was found to correlate with the levels of two inflammatory cytokine levels in the patients’ plasma. The levels of two inflammatory cytokines, interleukin-6 and -8 (IL-6, IL-8) were found to correlate positively with patient age, and increased levels were associated with lower overall survival. Addition of IL-6 or IL-8 to a co-culture system of CLL cells with bone marrow stromal cells increased the CLL-stromal cell adhesion, and co-culturing increased IL-8 secretion. In a search of a treatment regimen that may be effective and readily tolerated by elderly patients, we examined the combination of fludarabine with valproic acid (VPA), an epileptic that was found to inhibit histone deacetylases (HDACs). The combination was synergistic against human leukaemic cells, including primary CLL cells. In a phase II clinical trial where six elderly patients with relapsed, previously treated CLL were enrolled (half of whom were clinically refractory to fludarabine), the VPAfludarabine combination induced reduction in the peripheral and lymph node tumour loads. Mechanistically, the fludarabine treatment induced disruption of the lysosomes, while VPA induced increase in the level and activity of cathepsin B, a lysosomal protease. The VPA-induced increase in cathepsin B levels was observed in in cell lines (in vitro), primary CLL cells (ex vivo) and in patients treated with VPA (in vivo). Chemical inhibition of cathepsin B was sufficient to dampen the VPA-fludarabine cytotoxicity, and the addition activated cathepsin B to leukaemic cell lysates was sufficient to induce caspase cleavage and reduction in anti-apoptotic protein levels. The VPA-fludarabine combination also lowered phospho-Akt levels and ATM activation, which also contributed to the VPA-fludarabine synergy, and VPA treatment lowered ATM levels and phospho-Akt levels in vivo. In summary, there lies a biological explanation for the poor survival observed with elderly patients, and the VPA-fludarabine may be a useful regimen for these patients.