Functional Analysis of the Beclin-1 Tumor Suppressor Interaction with hVpa34 (Type III PI3' -kinase) in Breast Cancer Cells
Abstract
Macroautophagy plays a pivotal role in type II programmed cell death. This form of cell death entails early accumulation of autophagic vacuoles. Beclin 1 has been implicated in the regulation of macroautophagy. Previous reports indicate that overexpression of Beclin can promote autophagy and inhibit tumorigenesis in cultured breast carcinoma cells, and conversely, that heterozygous disruption of the Beclin gene can promote tumorigenesis in mice. During the first year we have performed immunoprecipitation studies with MCF7 breast carcinoma and U251 glioma cells, and found that Beclin associates with the human class III phosphatidylinositol 3- kinase (PI3K), hVps34, but not with another putative partner, Bcl-2. The lipid product of Vps34, PI(3)P, is required not only for autophagy, but also for assembly of proteins involved in endocytosis and trafficking of enzymes from the trans-Golgi network to the lysosomes. Therefore we set out to determine if the apparent tumor suppressing activity of Beclin is directly related to its role in autophagy, or is instead related to a role in controlling endocytic trafficking of growth factors or cellular nutrients. Retroviral RNAi- mediated gene silencing was initially used to suppress Beclin expression in MCF7 breast carcinoma cells. In these cells, Beclin knockdown (KD) was incomplete (65%). Therefore, we turned to the U-251 cell line, which is more easily infected with retroviral vectors. In these a cells a 95% KD of Beclin was achieved, allowing definitive studies of autophagy and protein trafficking. The results of these studies indicate that Beclin is required for hVps34 to function in autophagy, but is dispensable for hVps34 to function in the trafficking of the lysosomal enzymes or endocytosis of growth factor receptors or fluid phase markers. Beclin and hVps34 appear to exist in a high molecular weight complex in several cell lines including MCF7.
Document Details
- Document Type
- Technical Report
- Publication Date
- Jun 01, 2005
- Accession Number
- ADA437566
Entities
People
- William A. Maltese
Organizations
- University of Toledo Medical Center