Leukemia is a hematological neoplasm with abnormal genetic mutation or chromosomal translocation in the myeloblast or lymphoblast, and characterized by accumulation of immature cells and malfunction of lymphocytes and myeloid-derived cells. The prognosis of treatment depends on genetic mutation, chromosomal aberration, disease progression and age of patients. Currently, bone marrow transplantation is a useful therapeutic strategy, but the success in therapy is limited by the bone marrow of donors and life-threatening events such as immune repulsion. Although chemotherapy improves leukemia treatment, long-term chemotherapy usually leads to the production of drug-resistant cancer cells. Thus, the development of new modality in overcoming drug-resistant should be beneficial for in leukemia therapy. In this thesis, Naja nigricollis toxin £^, piceatannol, caffeine, and Bungarus multicinctus protease inhibitor-like protein 1 (PILP-1) are employed to investigate the molecular mechanisms in regulating apoptosis and invasion of leukemic cell lines K562 and U937. Hopefully, the signaling pathways elicited by these treatments may be aid in identifying new targets in treating leukemia. Toxin £^ inducing cell death is found to evoke p38 MAPK-mediated Bcl-2 down-regulation, which facilitates mitochondria dysfunction, ROS generation and cytiochrome c release. Finally, activation of caspases leads to apoptotic death of toxin £^-treated cells. Piceatannol elicits Ca2+/p38£\ MAPK- mediated c-Jun and ATF-2 phosphorylation, leading to up-regulation of Fas/FasL protein expression and autocrine Fas-mediated death pathway activation. Caffeine treatment down-regulates MMP-2/-9 down-regulation via Ca2+/ROS-mediated inactivation of ERK/c-Fos and activation of p38 MAPK/c-Jun pathway. Consequently, caffeine treatment suppresses invasion of leukemia cells. PILP-1-induced ADAM17 down-regulation suppresses Lyn-mediated Akt phosphorylation, resulting in death of PILP-1-treated leukemia cells. Taken together, the results of the present study elucidate the signaling pathways responsible for apoptosis and invasion of leukemia cells. Moreover, these findings might suggest new targets in developing therapeutic strategy in treating leukemia.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2009. / Härtill 5 uppsatser.

Thesis (Ph. D.)--Hong Kong University of Science and Technology, 2004. / Includes bibliographical references (leaves 134-172). Also available in electronic version. Access restricted to campus users.

Thesis (M. Phil.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 92-104). Also available in print.

Ph.D.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

 Acute myeloid leukemia (AML) is a group of heterogeneous diseases characterized by an abnormal increase in myeloblasts. Despite intensive chemotherapy and allogeneic bone marrow transplantation, the treatment outcome of AML remains unsatisfactory, with a cure rate of only about 30%. Therefore, novel therapeutic strategies targeting the pathogenetic pathways of leukemia initiation and progression are needed. Using intracellular phospho-flow analysis with normal bone marrow as reference, we detected an increase in phosphorylated-STAT5 (pSTAT5) in three leukemic cell lines (K562, KG-1 and ML-2) and 15 primary AML samples. Treatment with specific JAK2 inhibitor TG101209 and JAK2/3 inhibitor AG490 significantly reduced pSTAT5 level and leukemia cell growth associated with an increase in apoptosis and decrease in cellular proliferation. The clonogenic activities of these leukemia cell lines were also significantly reduced. Furthermore, treatment with these inhibitors in K562 and KG-1 also significantly reduced the WNT signaling activity, as enumerated by the TOP/FLASH luciferase assay. In addition, genes associated with oncogenic potential and anti-apoptosis were significantly reduced, consistent with the pathogenetic role of JAK-STAT pathway. In summary, the present study highlighted the importance of the JAK2-STAT5 signaling pathway in sustaining AML. The results may open up a new avenue whereby new therapeutic strategies targeting AML can be designed. / published_or_final_version / Medicine / Master / Master of Philosophy

Previously a t(2;14)(p13;q32) translocation was characterized in four unusually aggressive cases of B cell chronic lymphocytic leukemia (B-CLL). A gene located near the 2p13 breakpoint, B cell lymphoma/leukemia 11A (BCL11A), was shown to overexpress 3 isoforms (BCL11A-XL, L and S). Bcl11a knockout mice are severely impaired in B cell development at the early (pro-B) stage. I have further characterized BCL11A, focusing on the most abundant and evolutionarily conserved isoform, BCL11A-XL (XL). I demonstrated that XL resides in the nuclear matrix, is modified by ubiquitination, and is destabilized by B cell antigen receptor ligation. I identified domains within XL required for its localization within nuclear paraspeckles and for its transcriptional repression. While BCL11A-XL represses model promoters in non-B cells, its biologically relevant targets in B lymphocytes were unknown. I have identified and confirmed a number of XL targets which are both up- and down-regulated by XL over-expression in B cell lines. A number of these genes have been implicated in B cell function, including the V(D)J recombination activating (RAG) genes. Both RAG1 and RAG2 transcripts were up-regulated by XL. XL binds to the RAG1 promoter and RAG enhancer (Erag) in vivo as well as in vitro. Unexpectedly, XL repressed RAG1 transcription in non-B cells, indicating that additional B cell-specific factors are required for activation. Overexpression of XL in a V(D)J recombination-competent pre-B cell line markedly induced RAG expression and VDJ recombination. IRF4 and IRF8, transcription factors previously shown to be required for early B cell development, were also induced by BCL11A-XL. I propose that the early B cell progenitor block in Bcl11a knockout mice is, at least in part, a direct result of BCL11A-XL regulation of V(D)J recombination. Further experiments are required to establish how other XL targets promote B cell lineage development and how malignant transformation such as in B-CLL may corrupt BCL11A function.

MicroRNAs (miRNAs) are 20-22 nucleotide non-coding RNAs that can play important roles in developmental transitions by post-transcriptional regulation of mRNA translation and stability. We profiled miRNA expression in mouse thymocytes, mature T cells, and activated T cells, and identified distinctive patterns of miRNA expression during development, maturation, and activation of T cells. The miR-128 and miR-181 miRNA families are expressed at significantly higher levels in thymocytes. Examining the expression levels of these microRNAs in more detail, we observed that the expression pattern of these microRNA families distinguishes cells committed to lymphoid lineages from cells committed to myeloid lineages during normal mouse hematopoiesis. Extending this work to human malignancies, we determine that high miR-128 expression distinguishes lymphoid precursor derived malignancies from myeloid precursor derived malignancies. Little information is available regarding miRNA expression early after CD8 T cell activation. We demonstrate dynamic miRNA expression during early CD8 T cell activation, including the repression of miR-150, miR-181a, miR-26, miR-29 and miR-30, and the induction of miR-155, miR-31, miR-146, and the miR-17-92 cluster. We show that miR-31 is induced by calcium/Calcineurin signaling during acute CD8 T cell activation, and demonstrate elevated miR-31 expression in regulatory and memory T cell populations. We identify miR-31 targets in primary CD8 T cells and propose a model where miR-31 induction primes CD8 T cells for activation by promoting T cell survival, activation, and proliferation. Activation induced miRNA expression patterns are also found in some human malignancies. Chronic lymphocytic leukemia is typically thought to be a disease of resting lymphocytes. However, we demonstrate an activated B cell miRNA expression signature in CLL. Similarities in miRNA expression between activated B cells and CLL cells include high expression of miR-34a, miR-155, and miR-342-3p and low expression of miR-103, miR-181a and miR-181b. Additionally, we show that decreased levels of miR-29c and miR-223 in CLL are negative prognostic markers associated with shorter time to first therapy. These data indicate an activated B cell status for CLL cells and suggest that the expression level of individual miRNAs may predict clinical course in CLL.