Related Experiment Videos
Absence of the CAAX endoprotease Rce1: effects on cell growth and transformation
Martin O Bergo1, Patricia Ambroziak, Cria Gregory
1Gladstone Institute of Cardiovascular Disease, University of California, San Francisco, California 94141-9100, USA.
Abstract:
After isoprenylation, the Ras proteins and other CAAX proteins undergo two additional enzymatic modifications-endoproteolytic release of the last three amino acids of the protein by the protease Rce1 and methylation of the carboxyl-terminal isoprenylcysteine by the methyltransferase Icmt. This postisoprenylation processing is thought to be important for the association of Ras proteins with membranes. Blocking postisoprenylation processing, by inhibiting Rce1, has been suggested as a potential approach for retarding cell growth and blocking cellular transformation. The objective of this study was to develop a cell culture system for addressing these issues. We generated mice with a conditional Rce1 allele (Rce1(flox)) and produced Rce1(flox/flox) fibroblasts. Cre-mediated excision of Rce1 (thereby producing Rce1(Delta/Delta) fibroblasts) eliminated Ras endoproteolytic processing and methylation and caused a partial mislocalization of truncated K-Ras and H-Ras fusion proteins within cells. Rce1(Delta/Delta) fibroblasts grew more slowly than Rce1(flox/flox) fibroblasts. The excision of Rce1 also reduced Ras-induced transformation, as judged by the growth of colonies in soft agar. The excision of Rce1 from a Rce1(flox/flox) skin carcinoma cell line also significantly retarded the growth of cells, and this effect was exaggerated by cotreatment of the cells with a farnesyltransferase inhibitor. These studies support the idea that interference with postisoprenylation processing retards cell growth, limits Ras-induced transformation, and sensitizes tumor cells to a farnesyltransferase inhibitor.
Insights
Blocking Ras endoproteolytic processing via Rce1 inhibition slows cell growth and limits cancer cell transformation. This study developed a cell system to investigate Rce1
Area of Science:
- Biochemistry
- Cell Biology
- Oncology
Background:
- Ras proteins and other CAAX proteins require post-isoprenylation processing, including endoproteolytic cleavage by Rce1 and methylation by Icmt.
- This processing is crucial for Ras protein membrane association and is a potential target for cancer therapy.
- Previous studies suggested inhibiting Rce1 could impede cell growth and transformation.
Purpose of the Study:
- To establish a cell culture system for studying the effects of blocking post-isoprenylation processing.
- To investigate the role of Rce1 in Ras protein processing, cell growth, and transformation.
- To evaluate the therapeutic potential of Rce1 inhibition in cancer cells.
Main Methods:
- Generation of mice with a conditional Rce1 allele (Rce1(flox)).
- Production of Rce1(flox/flox) and Rce1(Delta/Delta) fibroblasts via Cre-mediated excision.
- Analysis of Ras protein processing, localization, cell growth, and transformation (soft agar assay).
- Assessment of Rce1 excision effects on skin carcinoma cell line growth, with and without farnesyltransferase inhibitor treatment.
Main Results:
- Cre-mediated Rce1 excision in fibroblasts eliminated Ras endoproteolytic processing and methylation.
- Truncated K-Ras and H-Ras fusion proteins showed partial mislocalization in Rce1-deficient cells.
- Rce1(Delta/Delta) fibroblasts exhibited slower growth compared to Rce1(flox/flox) fibroblasts.
- Rce1 excision reduced Ras-induced transformation and significantly retarded skin carcinoma cell growth.
- Combined Rce1 inhibition and farnesyltransferase inhibitor treatment enhanced the anti-cancer effect.
Conclusions:
- Interference with post-isoprenylation processing, specifically Rce1 activity, retards cell growth.
- Blocking Rce1 limits Ras-induced cell transformation, offering a strategy for cancer therapy.
- Rce1 inhibition sensitizes tumor cells to farnesyltransferase inhibitors, suggesting combination therapy potential.