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Studies of chromosome rearrangement and gene fusions involving the human myeloid-lymphiod leukemia gene (MLL).

Nonrandom chromosome exchanges known as chromosome translocations are a hallmark of specific subtypes of leukemia and have been valuable tools in identifying genes associated with malignant transformation of blood cells. MLL is one such gene, identified by its location on chromosome 11 at the breakpoint region in leukemogenic translocations with 40+ unique chromosomal regions. These translocations result in formation of MLL fusion genes which are expressed as oncogenic fusion proteins. MLL translocations are interesting for several reasons. First, MLL is one of the most promiscuous loci in its chromosome rearrangements with numerous translocation partners. In addition, MLL rearrangements are seen in 70-80% of leukemias in children less than 1 year of age and most, if not all of these rearrangements occur in utero. Finally, MLL rearrangements occur in some patients treated for other malignancies, resulting in secondary, therapy-related leukemias. Use of DNA topoisomerase II (topo II) inhibiting drugs correlates with MLL-related secondary leukemias.

Several projects are ongoing to study the mechanism of chromosome breakage and fusion in MLL translocations. Two projects focus on the hypothesis that topo II protein initiates breakage in MLL and in its translocation partner loci. Several in vitro studies have shown that topo II-inhibiting chemotherapy drugs can induce breakage in the translocation breakpoint region of MLL and in one region of an MLL partner gene (AF9). However, other MLL partner genes have not been tested for susceptibility to topo II drugs. We are currently testing AF4, the most common MLL partner gene, for susceptibility to breakage with common topo II inhibitor chemotherapy drugs (see Reinholdt et al.). In a second study, we are attempting to show direct binding of topo II to the MLL translocation breakpoint region. Using an in vivo assay called Chromatin Immunoprecipitation (CHIP), DNA binding proteins are reversibly cross-linked to chromatin in cells. Chromatin is sheared mechanically and immunoprecipitated using antibodies for candidate proteins (such as topo II). Input DNA is compared to the immunoprecipitated fraction for enrichment for specific loci. In our assay regions of the MLL breakpoint will be assayed for enrichment after chromatin immunoprecipitation with topo II antibody. Various culturing conditions (e.g., +/chemotherapy drugs) will be used prior to ChIP analysis. The assay could be applied to MLL partner gene breakpoint regions as well.

In a new phase of MLL study, a novel approach will be taken to modulate expression of MLL fusion genes, in cell lines. RNA interference (RNAi), an endogenous cellular mechanism for silencing specific genes, has recently been identified in many diverse organisms, including mammals. Briefly, RNAi is a conserved mechanism for cleavage of specific RNAs, including mRNAs, by cellular enzyme complexes following detection of the corresponding double-stranded RNA. Today RNAi technology is one of the most powerful and direct ways to silence specific genes for the purpose of studying gene regulation, protein function and protein interactions. In our study we are designing RNAi molecules to target specific MLL fusion mRNAs, (MLL-AF4 or MLL-AF9) which are present in leukemia cell lines. Initial goals of the study are simply to show specific knock-down of MLL fusion mRNAs and MLL fusion proteins. Subsequent analysis will include determining the longevity of the RNAi response, monitoring growth/transformation changes in the cell lines and ultimately determining changes in gene expression profiles following knock down of the MLL fusion proteins.

Heidi J. Super *, Michelle Reinholdt, Jessica Townsend, Stephanie Mueller, and Kyle Pankrantz

Department of Biology, Minor State University, Minot ND, 58707
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Title Annotation:Professional Communications
Author:Super, Heidi J.; Reinholdt, Michelle; Townsend, Jessica; Mueller, Stephanie; Pankrantz, Kyle
Publication:Proceedings of the North Dakota Academy of Science
Date:Apr 1, 2005
Words:570
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