MOLECULAR DETERMINANTS OF SENSITIVITY TO IFN IN CML

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Alpha-interferon induces cytogenetic remissions in 15% of CML patients. These remissions are associated with a reduced probability of evolution to the blastic phase of the disease. this proposal is designed to identify molecular mechanisms which may be used to alter the responsiveness of myeloid cells to alpha-interferon and the cellular determinants of sensitivity or resistance. The regulation of the interferon genes, their cellular actions, and the sensitivity to interferon and its effect on interferon-inducible genes are mediated through transcriptional regulatory proteins. Among these are IRF-1 and IRF-2, positive and negative regulatory factors respectively, the ratio of which determines the sensitivity of the cell to activation of the interferon signal transduction pathway. IRF-1 has been shown to bind to the transcriptional enhancers of the interferon genes and the interferon- inducible genes. In addition, ISGF3alpha and ISGF3-gamma are directly involved in the transcriptional activation of interferon-inducible genes by alpha-interferon. In order to develop methods of altering the ratio of IRF-1 and IRF-2 within cells, therefore sensitizing the cells to activation of the interferon signal transduction pathway, we set out to clone and analyze the 5' transcriptional regulatory regions of the promoters of IRF-1 and IRF-2 genes. We have completed the cloning and total sequencing of the IRF-1 gene and its 5' promoter region (Cha, DNA and Cell Biology 11:605-611, 1992). We have functionally characterized the transcriptional promoter of the IRF-1 gene (Sims, In press, Molecular and Cellular Biology, 1992). We have cloned the IRF-2 gene and are currently characterizing its promoter (Cha, 1992). In addition, our studies have identified a cellular factor which alters the electrophoretic mobility of the complexes which form between the ISGF3 and IRF-1 nuclear regulatory proteins and the transcriptional enhancers of interferon-inducible genes (Seong et al, JCI 86:1664-1770, 1990). this is a protein disulfide isomerase (PDI) protein (Johnson et al, JBC 267:14412-14417, 1992) which is elevated in CML cells, and which is associated with altered states of sensitivity to interferon (Howard et al, Blood 76:1117-1130, 1990). finally, we have shown that the duration and magnitude of the cellular response to alpha-interferon can be altered by gamma-interferon pretreatment (Gao et al, Submitted, JBC, 1992). We are proposing to extend these findings by: (1) developing ways of altering the ratio of IRF-1 and IRF-2 within ells to alter their sensitivity to the interferon pathway; (2) functionally characterizing the redox protein which is associated with altered states of alpha- interferon sensitivity; and (3) characterizing how gamma-interferon induces a change in the magnitude and duration of the cellular response to alpha-interferon. These studies will help us to understand the mechanism of the alpha-interferon response, to identify the molecular determinants of the sensitivity and resistance to alpha-interferon, to lay the foundation for new therapeutic strategies to enhance cellular sensitivity to alpha-interferon response, and to circumvent resistance. This may extend the benefits of alpha-interferon to greater numbers of CML patients.