The Ras and Raf oncogene products are potent agents in neoplastic transformation. Activation of the downstream mitogen-activated protein kinase (MAPK)–extracellular signal-regulated kinase (ERK) cascade by these oncoproteins results in the up-regulation of immediate-early genes through the ability of activated ERK to phosphorylate and modulate the activity of transcription factors, thereby increasing AP1 activity and cyclin D1 accumulation (1
). While the ras
oncogene products were initially isolated from tumors, their role in cell transformation has been mostly studied in established fibroblastic cell lines that are actively dividing upon stimulation by growth factors present in serum-containing media. In such cells, the MAPK-ERK pathway is required for cell proliferation (50
). However, strong activation of the Ras-MAPK pathway was recently found to arrest the cell cycle in NIH 3T3 cells (55
) and to cause senescence of normal human fibroblasts (74
), leading to the notion that the effects of this pathway on key regulators of the cell cycle, including cyclin-dependent kinase inhibitors, depend both on the host cell and the level of ERK activation.
In addition to Raf proteins, other direct Ras effectors have been shown to contribute to the transformation of mammalian cells. These include the catalytic subunit of phosphatidylinositol 3-kinase (PI 3-kinase) (58
) and the family of exchange factors for Ral (30
). Recently, these effectors were shown to contribute to G1
cell cycle progression by cooperating in the induction of E2F activity and cyclin D1 transcription in NIH 3T3 cells (23
). Ras mutants that differ by their ability to bind to and activate these different effectors have been used to evaluate the contribution of each distinct signaling pathway to the transformation of established fibroblast cell lines (38
). These mutants contain a second point mutation in the Ras effector loop, in addition to the V12 activating mutation. Thus, RasS35 mutant binds only to Raf proteins, RasG37 binds only to Ral-GEFs, and RasC40 binds only to the p110 catalytic subunit of PI 3-kinase. Interestingly, each of these mutants poorly transforms NIH 3T3 cells by itself, but they can cooperate to induce a fully transformed phenotype in these cells (38
). While these studies investigated the contribution of the different Ras downstream effectors to the transformation of spontaneously dividing cells, their capacity to induce the proliferation of primary cultures of postmitotic cells has not yet been investigated.
We have shown that primary cultures of differentiating chicken embryonic neuroretina (NR) cells represent a sensitive biological system for the detection of mitogenic signals. Despite the presence of serum growth factors, these cells can be maintained in a nondividing state for several weeks under culture conditions that normally promote the division of primary or established fibroblasts (7
). NR cells are induced to proliferate upon constitutive expression of activated oncogenes, such as v-src
). However, continuous NR cell division not only depends on oncogene expression but also requires the presence of serum and is therefore sustained by two types of signals (24
). Using this model system, we provided the first evidence that an activated raf
oncogene was able to induce cell cycle reentry of postmitotic cells (3
). Similarly, we reported that constitutively activated Ras and MEK, the respective activator and effector of Raf proteins, also promoted NR cell division (12
). In addition, we showed that NR cell division, following infection with retroviruses that do not carry an oncogene, was correlated with the transcriptional activation of mRNAs encoding truncated forms of Raf proteins (17
). This led to the identification of B-raf
, a novel member of the raf
family, as a retrovirally transduced oncogene in NR cells (42
). More recently, we reported that overexpression of full-length B-Raf proteins was sufficient to induce NR cell division in the absence of an activating mutation (51
). Taken together, our previous studies established that constitutive activation of the Raf-MEK-ERK cascade results in cell cycle reentry and sustained division of these postmitotic neuroepithelial cells.
NR cell division results in the down-regulation of QR1
, a retina-specific gene exclusively expressed during late stages of NR development (25
). Transcription of this gene is strictly correlated with growth arrest both in vivo and in cultured cells expressing a v-Src mutant conditionally defective in its mitogenic capacity (25
). This allowed us to identify a quiescence-responsive element in the QR1
) that could serve as sensor for the early detection of mitogenic signals in the NR.
In the present study, we investigated the contribution of the different Ras downstream signaling pathways to NR cell division induced by oncogenic Ras. We show that each of the Ras effector mutants displayed strong mitogenic capacity and repressed the activity of the QR1 promoter. We also show that constitutive activation not only of the Raf-MEK pathway but also of the Ras effectors PI 3-kinase and Rlf, an exchange factor for Ral, results in sustained NR cell proliferation. The mitogenic effect of RasC40–PI 3-kinase appears to be mediated through a Rac-Rho pathway but not to involve Akt (protein kinase B [PKB]). Division induced by RasG37-Rlf appears to be independent of Ral GTPase activation and presumably requires an unidentified mechanism. However, our results also indicate that none of the three Ras downstream pathways is sufficient to induce NR cell division and that this process requires, at least, the cooperation of the Raf-MEK-ERK and Rac-Rho pathways, since cell proliferation was inhibited by dominant negative mutants of both pathways. We finally show that NR cell proliferation resulting from any of the Ras effectors depends on a feedback mechanism, possibly an autocrine or paracrine loop, involving endogenous Ras, since the mitogenic property of all Ras effector mutants was inhibited by the RasN17 dominant negative mutant.