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.
Vasaturo
F
F
Department of Pathology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814, USA.
Dougherty
G W
GW
Cutler
M L
ML
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