A cell surface protein from human acute myelogenous leukemia (AML) cells has been purified. (Al-Rammahy et al., Cancer Immunol. Immunother. 9:181, 1980; Malcolm et al., J. Immunol. 128:2599, 1982). This material was used to immunize rabbits. The resulting antiserum (anti-AML) showed myelogenous leukemia specificity in that it reacted with myelogenous leukemia cell extracts and did not react with cell extracts of normal individuals or patients with non-myelogenous leukemia or other malignant disorders in the enzyme-linked immunosorbent assay (ELISA). Bone marrow and peripheral blood leucocytes (PBL) from either patients with myelogenous leukemia, other disorders or normal individuals were analysed in the fluorescence-activated cell sorter (FACS IV) after labelling with anti-AML, normal rabbit serum (NRS), or antiserum raised to normal human membrane antigens. Of 40 cell samples from patients with AML, 39 reacted strongly with the anti-AML. Similarly, all of 15 specimens from patients with chronic myelogenous leukemia (CML) reacted with the anti-AML. When 42 bone marrow or PBL samples from patients with a variety of lymphoproliferative disorders were examined, only 2 specimens reacted with the antiserum, both from individuals with diagnoses of acute lymphocytic leukemia (ALL). None of the 14 normal bone marrow or PBL donor specimens tested reacted with the anti-AML. It was also found that essentially all samples from patients in clinical remission from AML had high numbers of cells reactive with the anti-AML. When cells from such individuals were labelled and sorted on the FACS IV, it was found that the cell population fluorescing strongly with the anti-AML contained cells of both myeloid and lymphoid origin. The AML antigen was used to produce AML specific monoclonal antibody. Spleens from AML-antigen immunized Balb/c mice were fused to NS-1 myeloma parental cells and a myelogenous leukemia specific monoclonal antibody was selected from the hybrid colonies produced. This monoclonal antibody (MAL-1) as well as the rabbit anti-AML has been used to identify myelogenous leukemia patient samples in the FACS IV. In addition, this monoclonal also demonstrates positive fluorescence binding to HL-60 (a promyelocytic leukemia cell line), while there is no binding to lymphocytic leukemia cell lines, CCRF-SB-ALL-B and CCRF-CEM-ALL-T. The MAL-1 monoclonal has been shown to be specific for myelogenous leukemia cell extracts in the ELISA and has been successfully used as an immunoadsorbent for the isolation of the AML antigen from cell extracts. No equivalent antigen was found when cell extracts from normal cells, lymphocytic leukemia cells and lymphoma cells were similarly absorbed. These findings indicate that both the rabbit anti-AML serum and MAL-1 monoclonal show specificity for an antigen associated with myelogenous leukemia cells. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Thesis (M.S.)--Oregon State University, 2008. / Printout. Includes bibliographical references (leaves 89-99). Also available on the World Wide Web.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

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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.