Ras Inactivation of the Retinoblastoma Pathway by Distinct Mechanisms in NIH 3T3 Fibroblast and RIE-1 Epithelial Cells

Abstract

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 ofRaf 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 upregulation 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.Mutated and constitutively activated forms of Ras are found in 30% of human cancers (1, 2). Therefore, the signaling pathways that mediate oncogenic Ras growth transformation have been studied intensively. Although it is clear that Ras activation of the Raf/MEK 1 /ERK mitogen-activated protein kinase cascade is important for Ras transformation, it has also become clear that Ras transformation also requires the activation of multiple Raf-independent effector pathways (3). Furthermore, the pathways important for Ras transformation also exhibit cell type differences.In addition to Raf, Ras also associates with a spectrum of other downstream effector targets. Among these, the phosphoinositide 3-phosphate lipid kinases (PI3Ks) represent the second best characterized effectors of Ras (4). Activated PI3K, a lipid kinase, facilitates the conversion of phosphatidylinositol 4,5-phosphate to phosphatidylinositol 3,4,5-phosphate. Phosphatidylinositol 3,4,5-phosphate levels are elevated in Rastransformed cells and promote the activation of the Akt/protein kinase B serine/threonine kinase (5, 6). A third class of Ras effectors is a family of guanine nucleotide exchange factors (RalGDS, RGL, and Rlf/RGL2) that serve as activators of the Ral small GTPases ...

Gene Expression Signature of Human Cancer Cell Lines Treated with the Ras Inhibitor Salirasib (S-Farnesylthiosalicylic Acid)

“…Enrichment was highest for E2F and NF-Y, both well-established transcriptional regulators of the cell cycle that are controlled by Ras (19)(20)(21). Evidently, high levels of active Ras and increased activation of E2F and NF-Y are common to many human tumors (19,(21)(22)(23).…”

Pathway- and Expression Level-Dependent Effects of Oncogenic N-Ras: p27Kip1 Mislocalization by the Ral-GEF Pathway and Erk-Mediated Interference with Smad Signaling

“…(i) Ral activation by N-Ras 61K was shown to promote anchorage-independent growth of a human fibrosarcoma cell line, HT1080, by enhancing the degradation of p27 (78); we have formerly shown (38) that p27 is mislocalized in this N-Ras 61K -expressing human cell line and that its nuclear localization is restored upon treatment with a Ras inhibitor. (ii) Oncogenic Ras was shown to enhance the downregulation of p27 in RIE-1 intestinal epithelial cells via a pathway independent of Raf or PI3K activation (57). (iii) It was recently demonstrated that the Ral-GEF pathway, and not the Raf or PI3K pathway, is critical for Ras transformation in human cells (25,37).…”

Inhibitor of apoptosis protein (IAP) survivin is upregulated by oncogenic c-H-Ras

“…It is well established that p21 WAF1 is involved in the assembly of active cyclin/CDK complexes (LaBaer et al, 1997) and inducible by oncogenic Ras (Robles et al, 1998;Pruitt and Der, 2001). Reciprocal expression of both cyclin/CDK-complexes modulators was observed in several transformed human and rodent cell lines (Sherr and Roberts, 1999;Pruitt et al, 2000;Pruitt and Der, 2001).…”