Ectopic expression of Rsu-1 results in elevation of p21CIP and inhibits anchorage-independent growth of MCF7 breast cancer cells.
Signal transduction from tyrosine kinase receptors mediates growth regulation of breast cancer cells in part through the GTPase Ras and downstream kinases. Rsu-1 is a cDNA previously identified as an inhibitor of Ras-induced transformation. An HA-epitope tagged Rsu-1 cDNA was introduced into the MCF7 breast carcinoma cell line. Stable transfectants were selected and used for analysis of Rsu-1 expression on growth control and Ras-dependent kinase pathways. Assessment of biological activity of HA-Rsu-1 transfectants revealed that HA-Rsu-1 clones showed slower anchorage dependent growth rates than control MCF7 cell lines and a significant reduction in anchorage independent growth. Analysis of cell cycle regulatory proteins required for transit through G1 revealed that HA-Rsu-1 transfectant cell lines expressed elevated levels of p21CIP CDK inhibitor. Perturbations in signal transduction pathways which can be activated by Ras were detected in the Ha-Rsu-1 transfectants. Exposure of serum-starved cells to EGF revealed that expression of HA-Rsu-1 increased ERK-2 kinase activation, decreased activation of Jun kinase and inhibited Rho-dependent Rho-alpha kinase (ROK) activity compared to control cells. While serum starvation reduced AKT activity to undetectable levels in HA-Rsu-1 transfectants but not in control MCF7 cells, activation of AKT kinase by serum was unaffected by HA-Rsu-1 expression. Finally, the level of c-myc transcription in HA-Rsu-1 transfectants reached only 60% of the MCF7 control cell line following serum stimulation of starved cells while Fos RNA levels were similar to control cells. These results demonstrate that increased Rsu-1 expression critically altered cell cycle regulation and growth of MCF7 cells as well as signaling pathways in MCF7 cells required for malignant growth.
Department of Pathology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814, USA.
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Vasaturo
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Dougherty
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Cutler
D015152:Blotting, Northern; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000378:metabolism*; D002448:Cell Adhesion; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D009376:Neoplasms, Hormone-Dependent / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D015398:Signal Transduction; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D020741:rho GTP-Binding Proteins / Q000378:metabolism*; D054460:rho-Associated Kinases
D047908:Intracellular Signaling Peptides and Proteins; D014157:Transcription Factors; C076224:RSU1 protein, human; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D020741:rho GTP-Binding Proteins