This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Shangary, S. Articles by Johnson, D. E. Search for Related Content PubMed PubMed Citation Articles by Shangary, S. Articles by Johnson, D. E.

Departments of ¹ Medicine, ² Otolaryngology, ³ Molecular Genetics and Biochemistry, and ⁴ Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania

Requests for reprints: Daniel E. Johnson, Division of Hematology/Oncology, University of Pittsburgh, and University of Pittsburgh Cancer Institute, Room 2.18c, Hillman Cancer Center, 5117 Center Avenue, Pittsburgh, PA 15213. Phone: 412-623-3245; Fax: 412-623-7768. E-mail: johnsond{at}pitt.edu

Overexpression of the antiapoptotic proteins Bcl-2 and Bcl-X_L is commonly observed in human malignancies and contributes to chemotherapy and radiation resistance. Bcl-2 and Bcl-X_L inhibit apoptosis by binding to proapoptotic proteins such as Bax, thereby preventing chemotherapy-induced or radiation-induced release of cytochrome c from mitochondria and subsequent activation of the caspase protease cascade. Efforts to inhibit Bcl-2 or Bcl-X_L function in tumor cells have focused on developing agents to inhibit the interactions of these proteins with proapoptotic proteins. Peptides derived from the BH3 domains of proapoptotic proteins have been shown to disrupt the interactions of Bcl-2 and Bcl-X_L with key binding partners in cell-free reactions and to promote cellular apoptosis. However, less is known about the targets of BH3 peptides in intact cells as well as the sequence, length, and conformational requirements for peptide biological activity. In this report, we show that cell-permeable Bax BH3 peptides physically disrupt Bax/Bcl-2 heterodimerization in intact cells and that this disruption correlates with peptide-induced cell death. A point-mutant, control peptide that failed to disrupt intracellular Bax/Bcl-2 interactions also failed to promote apoptosis. To determine important sequence, length, and structural requirements for peptide activity, we generated and systematically analyzed the biological activities of 17 Bax BH3 peptide variants. Peptides were quantitatively examined for their ability to inhibit Bax/Bcl-2 and Bax/Bcl-X_L heterodimerization in vitro and to promote cytochrome c release from mitochondria isolated from Jurkat, HL-60, U937, and PC-3 cells. Our results define 15 amino acids as the minimal length required for Bax BH3 peptide biological activity and show that amino acids COOH terminal to the BH3 core sequence are less critical than those located NH₂ terminal to the core. In addition, circular dichroism spectroscopy revealed that high -helical content generally correlated with, but was not sufficient for, peptide activity. Taken together, these studies provide a basis for future optimization of Bax BH3 peptide as a therapeutic anticancer agent.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

⁵ http://www.cryst.bbk.ac.uk/cdweb/html/home.html

Received 7/13/04; revised 8/18/04; accepted 8/27/04.