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 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.
Highlights
Mutated and constitutively activated forms of Ras are found in 30% of human cancers [1, 2]
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, p21CIP/WAF, and p27KIP1
We evaluated whether the contribution of the Raf/ERK pathway to Ras deregulation of cell cycle progression differs in NIH 3T3 fibroblasts and RIE-1 epithelial cells
Summary
Mutated and constitutively activated forms of Ras are found in 30% of human cancers [1, 2]. The contribution of PI3K and RalGDS to Ras transformation has been supported by studies with effector domain mutants (E37G and Y40C) of activated HaRasVal-12 that cause the loss of Raf binding and a failure to activate ERKs (6, 9 –11). RasVal-12/Gly-37 is impaired in interaction with PI3K but retains interaction with RalGDS, whereas the RasVal-12/Cys-40 mutant is impaired in interaction with RalGDS but retains interaction with PI3K The ability of both mutants to cause growth transformation of NIH 3T3 cells demonstrates that Ras can cause transformation by Raf-independent pathways. One aspect of Ras signaling clearly important for growth transformation involves regulation of components of the cell cycle machinery that is critical for progression through the G1 phase [12,13,14]. Whether the links between Ras and the cell cycle in epithelial cells, the cell type from which the majority of ras mutation positive cancers arise, will show cell type differences is not clear
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