Chronic Myeloid Leukaemia (CML) is a clonal haemopoietic stem cell (HSC) disorder characterised by the presence of a disease-specific gene, BCR-ABL, which leads to the production of a bcr-abl mRNA transcript. CML is an ideal candidate for gene therapy using ribozymes (Rz), catalytic RNA molecules that cleave and inactivate target RNA in a sequence specific manner. Limited data is available on the activity of ribozymes in human CML cells. In this study, hammerhead ribozyme sequences directed against the b3a2 bcr-abl mRNA sequence (Rz6-Rz10) were cloned into several retroviral vectors. Initial experiments using MSCVHSA based retroviral constructs failed to express the sequences in cell lines. Rz cDNA fragments were then cloned into an LNL6 based retroviral vector (LGL1) encoding a GFP reporter gene and stable LGLRz constructs produced. Using cell sorting, high-titre PA317 producer cell line clones were isolated. Transcriptional silencing of the LGLRz6 producer cell line occurred with prolonged culture, with partial reversal on treatment with the demethylating agent 5' azacytidine. To assess the activity of these constructs in human cells, CD34+ HSC were isolated from newly diagnosed b3a2 Ph+ CML patients. Cells were transduced with either control LGL vector or the LGLRz6 construct. Transduced human cells were sorted based on GFP expression and placed into long-term HSC culture (LTC-IC assays). Using a common cDNA, RT-PCR was performed to detect the expression of both the transgene and bcr-abl in individual colonies derived from the LTCIC assay at various time points, allowing assessment of the effect of transgene expression on bcr-abl expression. LGLRz transgene expression was detectable for up to 6 weeks in culture. Colony RT-PCR results from 3 patients showed that expression of the LGLRz6 construct was associated with decreased bcr-abl expression. It also appeared that the reduced bcr-abl expression decreased the proliferation of Ph+ cells leading to their loss from culture. In summary, these results appear to show an effect of a retroviral vector containing a bcr-abl Rz sequence on human CML HSC. Targeting of bcr-abl remains a valid therapeutic goal in the Imatinib era, particularly if problems related to effective ribozyme delivery and targeting can be overcome.

"October 2002" Bibliography: leaves 159-198. This thesis describes the mapping of the breakpoints of three different chromosome rearrangements, all involving 1p36, in acute myeloid leukaemia (AML) patients, and an investigation into the molecular outcomes of these rearrangements.

Acute lymphoblastic leukaemia is the most common childhood cancer with cure rates of approximately 80%. This success can be attributed to the introduction of risk stratification for patients and employment of intensified treatment regimes for patients with high risk disease. However, the identification of prognostically important leukaemia subtypes, unfortunately, is an labour-intensive process. In addition, despite the success in treating childhood ALL, specific subgroups of patients nevertheless still have poor survival rates. This is particularly true for leukaemias characterised by chromosomal translocations involving the MLL oncogene on chromosome 11q23. By using a novel bioinformatics approach, GeneRave, a set of 12 classifier genes (PTPRK, FOS, ENG, Lgal-S1, TCFL5, LRMP, CTGF, IGJ, MX1, PENK, CD3D and HBG1) was selected from a publicly available U95 Affymetrix microarray dataset. Real time PCR carried out on a blinded cohort of 58 primary ALL samples yielded an accuracy of 86%. The absence of PENK gene expression in the majority of ALL samples tested appears to have decreased the overall accuracy. Nevertheless, the results indicate that this method of classification can be easily and quickly performed and therefore may be useful as an adjunct to routinely used methodology in the diagnostic classification of childhood ALL. Parellal screening of a 34,000 chemical small molecules library identified 30 ???hits??? that exhibited specificity toward leukaemia, and many of which shared structural similarity. The cytotoxic effect of these compounds was further investigated in a panel of 19 cell lines that included 3 MLL-translocated (MV411, THP-1 and PER-485), 7 non-MLL-translocated leukaemias (REH, Jurkat, K562, HL60, Hal-01, UOB-B, NB4), 2 immortalized normal blood cell lines, 4 non-leukaemic tumour cell lines (Calu6, MCF7, BE(2)-C, and HeLa) and 3 normal cell lines (HSF, MCF10a and MRC5). In particular, two compounds were identified, SM6 and SM7, that were highly effective at killing MLL-translocated cell lines in the low micromolar range while having little or no effect on the other cell lines. Treatment of PER-485 cells with SM6 and SM7 showed mark down-regulation of the MLL chimaeric fusion protein together with its down-stream targets. One other more broadly acting anti-leukaemia compounds were also identified.

Understanding the nature and timing of leukaemogenic events during the development of childhood acute lymphoblastic leukaemia (ALL) will enable intervention that could prevent ALL in the future. We hypothesised that 9p21 deletion in childhood ALL may unmask predisposing genetic events that would allow us to determine the "nature" of initiating events in childhood ALL; whereas the inclusion, or exclusion, of random �N� nucleotides in normal immunoglobulin gene rearrangements from the developing fetus and the expression of terminal deoxynucleotidyl transferase (TdT) in fetal lymphocytes may allow us to unmask the developmental window during which the first transforming leukaemic event occurs in a pre-leukaemic B cell. The most frequent genetic abnormality in childhood ALL is deletion of chromosome 9p21, with the minimal region of deletion including the CDKN2-locus, making genes at this locus candidates for a predisposing genetic event in ALL. To determine whether genomic imprinting might be involved in ALL at the 9p21 locus we investigated the imprinting status of the candidate genes CDKN2A, CDKN2B and ARF. No evidence for genomic imprinting of ARF was found in this study. Because no expressed polymorphisms could be identified for CDKN2B, and CDKN2A expression was too low in normal tissues, the imprinting status of these genes could not be evaluated. Furthermore investigations in our laboratory have been unable to find genomic imprinting at any of these genes in mice. However, we have shown variation in allelic expression of ARF, which suggests a role for ARF haploinsufficiency in the onset of childhood ALL. A key feature of early human fetal lymphoid development is the absence of random �N� nucleotides between the rearranged V[H], D[H] and J[H] gene segments. The addition of �N� nucleotides at these junctions requires the enzyme terminal deoxynucleotidyl transferase (TdT). TdT is reported to not be expressed during early fetal lymphopoiesis but has been observed by the end of the first trimester, but data are sparse. The reported absence of N nucleotides in the majority of childhood ALLs suggests an early fetal origin. By defining the window-in-time during which TdT-negative B cell development occurs, we will be able to refine the timing of the origin of the B cells that give rise to ALL. Therefore we have sequenced and analysed the V[H]-DJ[H] and D[H]-J[H] junctions from immunoglobulin rearrangements in developing B cells in normal human fetuses aged from 5.1 to 11 weeks gestation. In this study 73 fetal IgH gene rearrangements were amplified from 21 different fetal liver samples. Only eight of the seventy-three rearrangements (11%) analysed in this study had no �N� nucleotides at the N1 (D[H]-J[H]) junction. This finding contrasts with the 24-28% of fetal rearrangements with no �N� nucleotides that have been reported in the literature. Furthermore, �N� nucleotides were shown to be present in the earliest sample, 5.1 weeks gestation. TdT expression was demonstrated by immunohistochemistry at 7.3 weeks and by RT-PCR at 8.3 weeks. B cell development in the fetal liver was detected as early as 6.5 weeks using flow cytometric analysis. Then, IgH gene rearrangements from 99 cases of childhood ALL were analysed. In total, 134 clone-specific IgH gene rearrangements were examined in this study. No association was found between the number of �N� nucleotides from complete and incomplete rearrangements at either the N1 (D[H]-J[H]) or N2 (V[H]-DJ[H]) junctions. Nor was any association observed between ALLs from children [less than or equal to] 3 years of age and those >3 years of age at diagnosis. These findings indicate that ALL IgH rearrangements do not have the paucity of �N� nucleotides that has been previously reported. The findings in this thesis suggest that there is no TdT-negative timepoint during B cell development and that there is no paucity of �N� nucleotides at the N1 junction in either fetal or childhood ALL IgH gene rearrangements.

Interleukin (IL)-3, IL-5 and granulocyte-macrophage-colony-stimulating factor (GM-CSF) are cytokines that affect the growth, survival and differentiation of many cells within the haematopoietic system. The functions of these factors are mediated by membrane bound receptor complexes that are composed of specific ligand binding subunits (α)and a common signal transducing subunit(hβc). Constitutively activated mutants of hβc have been previously identified that are able to confer factor-independent signalling in a number of haematopoietic cell lines (including FDC-P1 and FDB-1). These activated mutants fall into two classes defined by the location of the mutation and their biochemical and leukaemogenic properties. In particular, the transmembrane mutant, V449E, causes an acute myeloid leukaemia in vivo, whereas the extracellular mutants (FI∆ or I374N) cause chronic myeloproliferative disorders. The work described in this thesis used the activated hβc mutants to uncover novel transcriptional events induced by the GM-CSF/IL-3/IL-5 receptor complex and to define pathways associated with proliferation and differentiation. Large-scale gene expression profiling techniques were used to investigate the genes involved in these biological processes in the murine myelomonocytic cell line FDC-P1, and the bi-potent FDB-1 myeloid cell line, which are responsive to IL-3 and GM-CSF. Membrane arrays were used to identify differences in gene expression between I374N and V449E expressing FDC-P1 cells. This technique revealed that the gene Ptpmt1 was differentially expressed between V449E and I374N, which was subsequently confirmed by Northern blotting. This finding suggested that the phosphatase encoded by Ptpmt1 may be involved in the different outcomes induced by these two hβc mutants. Northern analysis also revealed Ptpmt1, Nab1 and Ddx26b to be regulated in response to human GM-CSF in FDC-P1 cells expressing human GM CSFα and hβc. A large-scale cDNA microarray experiment was also performed to identify genes that are selectively expressed during differentiation of FI∆ expressing FDB-1 cells, compared to proliferating V449E expressing FDB-1 cells over 24 hours. A comprehensive analysis approach was adopted to examine the microarray data and identify differentially expressed genes. Among the genes displaying differential expression were Btg1, S100a9, Cd24, and Ltf found to be differentiation-associated and Bnip3, Cd34, Myc, Nucleophosmin, and Nucleostemin found to be proliferation-associated. Hipk1, Klf6, Sp100, and Sfrs3 were also identified as potential transcriptional regulators during growth and differentiation. Northern analysis was used to confirm differences in expression for these 13 genes between FI∆ and V449E expressing FDB-1 cells. Eleven of the 13 genes examined were confirmed to be differentially expressed between FI∆ and V449E expressing FDB-1 cells over 24 hours. Furthermore, six genes (Btg1, Hipk1, Cd24, Cd34, Klf6 and Nucleostemin) examined over 72 hours revealed differences in gene expression at early (6-12 hours) and late (48-72 hours) time points. Cell surface expression of CD24 protein was also shown to be induced upon FI∆ expression or GM-CSF induced differentiation of FDB-1 cells, consistent with elevated levels of Cd24 mRNA in FI∆ cells over time. Based on their confirmed gene expression differences seen on the microarrays and Northern analysis, four genes (Btg1, Cd24, Klf6 and Nucleostemin) were selected for over-expression analysis in FDC-P1 or FDB-1 cells, in order to gain insights into the function of these genes. Optimisation of the retroviral infection process was performed so that the role of these genes in proliferation and differentiation could be investigated in the FDB-1 model. Such preliminary functional experiments in FDB-1 cells will enable prioritisation of the genes for further analysis of their function in primary cells. Thus, the work in this thesis describes the first use of microarrays to identify gene expression differences between hβc mutants with differential activities affecting myeloid growth and differentiation. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1284103 / Thesis (PhD)-- School of Medicine, 2005

"October 2002" / Bibliography: leaves 159-198. / xiv, 198 leaves : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This thesis describes the mapping of the breakpoints of three different chromosome rearrangements, all involving 1p36, in acute myeloid leukaemia (AML) patients, and an investigation into the molecular outcomes of these rearrangements. / Thesis (Ph.D.)--University of Adelaide, Dept. of Medicine, 2003

Traumatic lumbar punctures (TLPs) in children with acute lymphoblastic leukemia are associated with a poorer prognosis. The objective of this study was to determine risk factors for TLPs using a retrospective cohort. We compared and contrasted three different regression methods for the analysis of repeated-measures data. In the multivariable model using generalized estimating equations, variables significantly associated with TLPs were age < l year or ≥ 10 years; body mass index percentile ≥ 95; platelet counts < 100 x 103/µL; fewer days since previous LP, and a preceding TLP. The same variables, with similar estimates and confidence-intervals, were identified by the random-effects model. In a fixed-effects model where each patient was used as their own control, days since prior LP and the effect of using image-guidance were significant. Random-effects and GEE lead to similar conclusions, whereas fixed-effects discards between-subject comparisons and leads to different estimates and interpretation of results.

Thesis (M.D.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 130-155).

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

Thesis--PlanA (M.S.)--University of Wisconsin--Stout, 2002. / Includes bibliographical references.