Ras inactivation of the retinoblastoma pathway by distinct mechanisms in NIH 3T3 fibroblast and RIE-1 epithelial cells.
Although Ras and Raf cause transformation of NIH 3T3 fibroblasts, only Ras causes transformation of RIE-1 intestinal epithelial cells. To determine if the inability of Raf to transform RIE-1 cells is due to a failure to deregulate cell cycle progression, we evaluated the consequences of sustained Ras and Raf activation on steady state levels of cyclin D1, p21(CIP/WAF), and p27(KIP1). Both Ras- and Raf-transformed NIH 3T3 cells showed up-regulated expression of cyclin D1, p21, and p27 protein, increased retinoblastoma (Rb) hyperphosphorylation, and increased activation of E2F-mediated transcription. Similarly, Ras-transformed RIE-1 cells also showed up-regulation of cyclin D1, p21, and hyperphosphorylated Rb. In contrast, Ras-mediated down-regulation of p27 was seen in RIE-1 cells. Conversely, stable expression of activated Raf alone caused only a partial up-regulation of p21 and Rb hyperphosphorylation but no activation of E2F-responsive transcription or down-regulation of p27 in RIE-1 cells. Moreover, we found that the AP-1 site was dispensable for Ras-mediated stimulation of the cyclin-D1 promoter in NIH 3T3 cells but was essential for Ras-mediated stimulation in RIE-1 cells. Thus, up-regulation of p21, rather than the down-regulation seen in previous transient expression studies, is seen with sustained Ras activation. Additionally, p27 may serve a positive (NIH 3T3) or negative (RIE-1) regulatory role in Ras transformation that is cell type-dependent. The involvement of Raf and phosphatidylinositol 3-kinase in mediating Ras changes in cyclin D1, p21, and p27 was also very distinct in NIH 3T3 and RIE-1 cells. Taken together, these results demonstrate the importance of Raf-independent pathways in mediating oncogenic Ras deregulation of cell cycle progression in epithelial cells.
Pruitt
K
K
Department of Pharmacology, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
Pestell
R G
RG
Der
C J
CJ
D016475:3T3 Cells; D000818:Animals; D002453:Cell Cycle; D002471:Cell Transformation, Neoplastic; D019938:Cyclin D1 / Q000096:biosynthesis; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000096:biosynthesis; D011905:Genes, ras / Q000502:physiology*; D007413:Intestinal Mucosa / Q000473:pathology; D051379:Mice; D008840:Microfilament Proteins / Q000032:analysis; D009124:Muscle Proteins; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology; D010766:Phosphorylation; D019908:Proto-Oncogene Proteins c-raf / Q000502:physiology; D016160:Retinoblastoma Protein / Q000378:metabolism*
C493957:Cdkn1a protein, mouse; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D008840:Microfilament Proteins; D009124:Muscle Proteins; D016160:Retinoblastoma Protein; C058992:Tagln protein, mouse; D019938:Cyclin D1; D019869:Phosphatidylinositol 3-Kinases; D019908:Proto-Oncogene Proteins c-raf