This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Several types of human acute leukemia show chromosome translocations which fuse the MLL gene to numerous other genes. MLL fusion genes are seen in chemotherapy-related leukemia often after treatment with DNA topoisomerase II (topo II)-inhibiting drugs. Two projects of my lab are based on the hypothesis that DNA topoisomerase II (topo II) plays a role in initiating chromosome translocations in acute leukemia involving MLL. Students are examining the translocation breakpoint of the AF4 gene (the most common MLL translocation partner gene) for susceptibility to breakage with topo II inhibitors. In the study, cell lines or cloned bacteria artificial chromosome DNA have been exposed to chemotherapy drugs and DNA has been examined for breakage via topo II. Students have established control conditions for drug activity and DNA analysis. Students are identifying optimal AF4 breakpoint DNA frgments to test, using Vector NTI DNA analysis software and Genbank database sequences. Other students are using chromatin immunoprecipitation (ChIP) to assay the MLL translocation breakpoint region for topo II binding. We have completed pilot experiments to optimize shearing of DNA, the use of positive control antibodies, and PCR analysis of precipitated, end-product chromatin. Students have established conditions for positive control DNA binding proteins. Students are ready to begin the experiments using topo II antibodies, and MLL-specific primers immediately. In a third project, we have designed short interfering RNAs which correspond to the junction of the MLL-AF4 fusion mRNA in the RS4;11 cell line. Our initial aim is to demonstrate specific knock down of expression of the fusion mRNA using RNA interference. Subsequent studies include monitoring the effect on the cell line (growth/transformation changes), longevity of the RNAi effect, and effect on global gene expression. Preliminary experiments have included optimizing transfection (electroporation) conditions for the cell lines.
Showing the most recent 10 out of 98 publications
