Programmed cell death or apoptosis is a cellular suicide process that is necessary to maintain homeostasis in multicellular organisms. In recent years several genes have been cloned that function to either induce or inhibit apoptosis. Many of these genes share homology with the anti-apoptotic oncogene bcl-2. One of these genes, bcl-xL, can function in a similar fashion as bcl-2, yet displays a different pattern of expression. Specifically in lymphocytes Bcl-2 is expressed at high levels regardless of the activation state of the cell while Bcl-xL is expressed only upon cell activation. In addition, upregulation of bcl-x but not bcl-2 occurs in tumor cells following irradiation or induction of multidrug resistance. The objectives of this proposal are to determine the molecular mechanisms by which bcl-x expression is regulated. Specifically to characterize the bcl-x promoter and determine the cis elements which are involved in regulation of gene expression during lymphocyte activation as well induction of drug resistance in tumor cells. A fragment of genomic DNA which extends 2.7 kb 5' of the start methionine has been isolated. This fragment has two putative TATA boxes within 1 kb of the start ATG. The first specific aim is to determine usage of these TATA boxes by primer extension analysis and RNase protection analysis. Functional analysis will be carried out by subcloning promoter fragments into reporter constructs and determining regions of the promoter required for responsiveness to different activation signals. In addition, the cloned fragment contains a GC rich region that displays differential methylation in drug sensitive and drug resistant cell lines.
The final aim of this proposal is to determine the methylation pattern of the bcl-x CpG island by sequencing of sodium bisulfite-treated DNA to reveal methylation sites. The role of methylation of the CpG island on bcl-x expression will be determined by in vitro methylation of reporter constructs. While bcl-x overexpression has not been shown to lead to any specific tumors, it's expression has been demonstrated in several types of tumors including breast, prostrate and the Reed-Sternberg cells of Hodgkin's lymphoma. Understanding the mechanisms by which bcl-x is controlled may result in the development of ways to increase the efficacy of cancer chemotherapy through decreasing the apoptotic threshold of the tumor cell. In addition determination of the methylation state of bcl-x in tumors may be a good indicator of the outcome of cancer chemotherapy of a given tumor.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29CA077837-03
Application #
6173647
Study Section
Allergy and Immunology Study Section (ALY)
Program Officer
Mufson, R Allan
Project Start
1998-04-01
Project End
2003-03-31
Budget Start
2000-06-01
Budget End
2001-03-31
Support Year
3
Fiscal Year
2000
Total Cost
$105,609
Indirect Cost
Name
University of Miami School of Medicine
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
City
Miami
State
FL
Country
United States
Zip Code
33146
McCafferty-Grad, Jennifer; Bahlis, Nizar J; Krett, Nancy et al. (2003) Arsenic trioxide uses caspase-dependent and caspase-independent death pathways in myeloma cells. Mol Cancer Ther 2:1155-64
Parissenti, Amadeo M; Riedel, Heimo (2003) Yeast as a host to screen for modulators and regulatory regions of mammalian protein kinase C isoforms. Methods Mol Biol 233:491-516
Toomey, N L; Deyev, V V; Wood, C et al. (2001) Induction of a TRAIL-mediated suicide program by interferon alpha in primary effusion lymphoma. Oncogene 20:7029-40
Grad, J M; Bahlis, N J; Reis, I et al. (2001) Ascorbic acid enhances arsenic trioxide-induced cytotoxicity in multiple myeloma cells. Blood 98:805-13
Jost, M; Huggett, T M; Kari, C et al. (2001) Epidermal growth factor receptor-dependent control of keratinocyte survival and Bcl-xL expression through a MEK-dependent pathway. J Biol Chem 276:6320-6
Lee, R K; Cai, J P; Deyev, V et al. (1999) Azidothymidine and interferon-alpha induce apoptosis in herpesvirus-associated lymphomas. Cancer Res 59:5514-20