Blocks in differentiation appear to be a major step in tumor progression. The long term objectives of the research plan is to gain a better understanding of the molecular mechanisms that control terminal myeloid differentiation and growth arrest and how these processes can be blocked, thereby contributing to leukemogenesis. The M1 myeloid leukemia cell line, which proliferates autonomously and undergoes terminal differentiation, growth arrest and loss of leukemogenicity when treated with the physiological inducers IL-6 and leukemia inhibitory factor (LIF) is used. The research scheme is to genetically manipulate M1 cells to block induced differentiation, followed by analysis. The research plan encompasses: 1. Studies the role of c-myc suppression in myeloid differentiation; how continuous expression blocks differentiation growth arrest and exit from the cell cycle. Genes directly regulated by c-myc during myeloid growth and differentiation will be cloned using chimeric myc genes. Expression, sequence and functional analysis of the myc regulated genes will be done. The effects of continued expression of c-myc: on the regulation of expression of the cell cycle genes cdc2 and cyclin, the cell cycle regulated phosphorylation of the retinoblastoma protein and the loss of tumorigenicity will be determined. Finally, analyzing proteins in M and M1myc cells by 2-D gel electrophoresis should give an overall picture of how myc blocks the myeloid differentiation genetic program. 2. Studies on the role of c-myc in the regulation of myeloid differentiation prior to its suppression. Premature suppression of c-myc blocks induced differentiation, resulting in cell death. The role of sustained, early c-myc expression during differentiation and where the block in differentiation is will be determined. Premature suppression of c-myc may cause the cells to exit the cell cycle prematurely. Cell cycle analysis by flow cytometry, cyclin and cdc2 expression and RB protein phosphorylation will be analyzed. 3. Studies on c-myb in myeloid differentiation, growth arrest, and blocking differentiation. Continued expression of c-myb blocks M1 differentiation. That c-myb blocks differentiation by activating c-myc will be tested by antisense experiments. When differentiation is blocked will be determined, following the basic plan described for c-myc. 4. The role of Hox-2.4 in blocking terminal myeloid differentiation and growth arrest. The homeobox Hox-2.4 gene appears to impede IL-3 driven terminal myeloid differentiation. M1Hox-2.4 cells constitutively expressing Hox-2.4 will be generated to determine if Hox-2.4 blocks M1 myeloid differentiation, and if so, where the block is, as well as if the loss of tumorigenicity is blocked. Analysis will follow the same scheme as for c-myc and c- myb. In addition, Hox-2.4 genes activated/suppressed in uninduced M1 cells will be cloned and analyzed. Information from these studies should lead to an increased understanding of the regulation of differentiation and how perturbing normal controls can block differentiation and contribute to leukemogenesis, ultimately aiding in diagnosis, prognosis and eventual therapy.
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