To study the nature of genes that are induced during the apoptotic death of myeloid pre-cursor cells, the investigators utilized 32Dcl3 cell line, which is derived from normal mouse bone marrow and is non-tumorigenic and diploid. These cells are strictly dependent on IL-3 for growth and apoptosis when deprived of IL-3 from the medium. In the search for genes that are induced during terminal differentiation of 32Dcl3 cells, the investigators identified a novel gene termed AATYK (Apoptosis Associated Tyrosine Kinase), whose expression is dramatically upregulated during IL-3 deprivation. The expression of this gene, which codes for a protein with a tyrosine kinase domain at the N-terminal end and a proline-rich domain at the C-terminal end, is blocked in transformed myeloid cells which are deficient in undergoing apoptosis. The experiments proposed are aimed at understanding the role of AATYK in the apoptosis, differentiation, and transformation of myeloid cells.
The aims are: 1) [a] to test whether other apoptotic stimuli produced by different xenobiotic agents such as calphostin C, methotrexate etoposide, and mitomycin C result in the induction of AATYK, [b] to test whether ectopic over-expression of AATYK renders v-abl and bcr-abl-transformed 32D cells more sensitive to apoptotic death induced by the above xenobiotic agents, and [c] to test whether transgenic expression of AATYK in the v-abl or bcr-abl transformed 32D cells over-rides the block to G-CSF-induced terminal differentiation; 2) to study the effects of transgenic expression of AATYK on 32Dcl3 cell growth, differentiation, and apoptosis and to determine as to how myeloid cell differentiation (in the presence of GCSF) or apoptosis (in the absence of IL-3) is affected when AATYK expression is inhibited using anti-sense vectors; 3) to carry out a detailed biochemical characterization of the protein encoded by AATYK to determine its potential tyrosine kinase activity, post-translational modification patterns, subcellular localization and mechanism of action; and, 4) to carry out a detailed analysis of the promoter/enhancer region of AATYK to examine the sequence elements that play a crucial role in the transcriptional regulation of this gene.
