Specific chromosomal translocations are associated with a number of hematologic malignancies. Several of these have been demonstrated to result in the activation of an oncogene. In lymphomas with the t(14;18) translocation, the bcl-2 gene is activated after translocation to the immunoglobulin locus. The goals of this project are to establish the mechanisms of the activation of the bcl-2 gene in human lymphomas and thus to develop a better understanding of the mechanisms of malignant transformation. A. Identification of the molecular mechanisms of transcriptional control of the bcl-2 gene during normal B-cell development and activation. In vivo footprinting and in vitro protein-DNA binding studies will be used to determine how the bcl-2 gene is regulated. B. Identification of the molecular mechanisms of transcriptional control of the translocated bcl-2 gene in t(14;18) lymphomas. Pulsed field electrophoresis will be used to separate the translocated from the normal allele and differences in the binding of transcriptional regulatory factors will be identified by genomic footprinting. C. Identification of differences in methylation at the bcl-2 loci in t(14;18) lymphomas. Methylation of cytosine residues in the regulatory regions of each allele will be studied. D. Effect on bcl-2 expression of mutations identified in the regulatory regions. Sequence information will be acquired, and mutations that occur with a high frequency will be examined for their effects on the binding of regulatory proteins. E. Identification of the mechanisms of transcriptional control of bcl-2 in the 5' translocations found in CLL and comparison to those operative in the t(14;18) lymphomas. In vivo footprinting studies will be performed on both the translocated and normal alleles in CLL cells after separation of the two by electrophoresis. F. Development of a model system to test hypotheses for the mechanisms of bcl-2 activation. A model system will be used to determine whether the results of the in vivo studies can be reproduced in a system that leads to bcl-2 deregulation; this will also be used to determine which elements are required for deregulation.
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