The present application proposes to analyze the molecular structure of a number of recurring chromosome abnormalities associated with lymphoid and myeloid neoplasia that share a breakpoint at 11q23, to clone the breakpoint junctions, to search for the genes associated with the breakpoints, and to study their structure and expression. The underlying hypothesis is that genes involved in the regulation of lymphoid and myeloid cell proliferation and differentiation at either 11q23 or at the associated breakpoints sites, are activated to oncogenes by the chromosome rearrangements, and that this is an important step in oncogenesis for lymphoid and myeloid neoplasia. It is also important to determine which are the mechanisms by which these chromosome rearrangements arise, to identify predisposing factors and to devise prophylactic measures to reduce the risk of leukemia and lymphoma. We have mapped the breakpoints of three different translocations: t(9;11)(p22;q23) of monocytic leukemia, t(11;19)(q23;p13) of myeloblastic or lymphoblastic leukemias, and t(4;11)(q21;q23) of lymphoblastic leukemia with cell surface myeloid markers, to a region of about 90 kb within band 11q23. Cloned DNA sequences from inside this region will be used to screen Southern blots, prepared from leukemic cells DNA, to detect rearrangements corresponding to the breakpoint junctions; then, the DNA sequences across the junction will be cloned to search for associated genes. The nucleotide sequence around the junction will provide information about possible mechanisms of origin of the rearrangement. The genomic sequences likely to contain putative oncogenes will be identified by looking for DNA sequences that cross hybridize to unique sequences in other vertebrate species, that identify transcripts in a northern blot, or that lie close to CpG islands. The genes identified will be cloned as CDNAS, their proteins will be expressed in bacterial vectors, and antibodies will be prepared to analyze their expression. The oncogenic nature of these genes, and their effects on the phenotype of hematopoietic cells will be tested by transfecting appropriate constructs into lymphoid or myeloid cell in culture. In order to immortalize these chromosome rearrangements, cell lines will be established from the primary leukemia cells. These and previously established cell lines will be used to study the expression of genes found in association with the breakpoints. A t(1;7)(p34;q34) involving the TCRB gene has been analyzed in the cell line HSB2. The LCK gene coding for a T-cell specific tyrosine-protein- kinase has been found involved at the 1p34 breakpoint. The analysis of this abnormality will be continued, to determine the mechanism of activation of LCK.
| Fair, K; Anderson, M; Bulanova, E et al. (2001) Protein interactions of the MLL PHD fingers modulate MLL target gene regulation in human cells. Mol Cell Biol 21:3589-97 |
