MOLECULAR SENSITIZATION OF P210BCR-ABL POSTIVIE CELLS TO THERAPY--CML

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The P145 product of the cellular A belson (c-abl) gene has been shown to mediate inhibition of cell cycle progression and apoptosis at the Gl/S interface when over-expressed in cells or when cells are exposed to genotoxic stress as radiation therapy and chemotherapy. In contrast, the presence of the abnormal counterpart of P145c-abl, the P210crabl, is associated with the phenotypes of growth factor independent growth, anchorage independent growth, apoptosis rescue and genetic instability in chronic myelogenous leukemia (CML). In order to study the interaction of these two proteins which have structural similarities in the abl domain, but have such opposing effects in the myeloid cells, and to identify the key substrates of bcrabl and c-abl proteins, we have modified a myelogenous leukemia cell line so that the expression of the P210crabl protein is regulated by the extracellular concentration of tetracycline. We have shown that the ration of the P210crabl/p145c-abl proteins, and the growth of these cells, in the absence of IL3, is dependent on the tetracycline concentration. We will use this cell line to test if bcrabl proteins interact with the Stress Activated Protein Kinase (SAP) pathway as does the c-abl, if the substrates or sites of phosphorylation of the c- abl or bcrabl proteins are the same or different, and which substrates or sites of modifications or substrates are associated with the emergence of P210crabl transformation. Since c-abl is known to promote apoptosis in response to genotoxic stress, and the bcrabl is known to rescue from apoptosis, we are proposing to use this cell line to also study if the sensitivity to chemotherapy and interferon therapy is different as the ratio of P210 bcrabl/P145c-abl changes. We will use peptide transcription units which selective interrupt the interaction of P210 bcrabl with it substrates without affecting the P145c-abl kinase to discriminate between effects of P210 bcrabl and P145c-abl, and to reverse the transformed phenotype of the CML cell. We will use the information generated by these studies to design new approaches to the therapy of CML, including the design and testing of peptidomimetic compounds for the inhibition of P210 bcrabl action in P210 bcrabl positive cells.