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Originally published In Press as doi:10.1074/jbc.M002235200 on April 25, 2000

J. Biol. Chem., Vol. 275, Issue 30, 22916-22924, July 28, 2000
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Oncogenic Ras-mediated Cell Growth Arrest and Apoptosis are Associated with Increased Ubiquitin-dependent Cyclin D1 Degradation*

Jinyi ShaoDagger , Hongmiao ShengDagger , Raymond N. DuBoisDagger §, and R. Daniel Beauchamp§||

From the Departments of Dagger  Medicine,  Surgery and § Cell Biology, The Vanderbilt-Ingram Cancer Center, Vanderbilt University Medical Center, Nashville, Tennessee 37232

Received for publication, March 14, 2000, and in revised form, April 20, 2000

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The cellular responses to activated Ras vary depending on cell type. Normal cells are often induced into pathways that lead to cell growth arrest, senescence, and/or apoptosis in response to activated Ras expression. These are important protective anti-tumorigenic responses that restrict the propagation of cells bearing activated oncogenes. Here we show that induction of Ha-RasVal-12 in Rat-1 fibroblasts resulted in G1 growth arrest and apoptosis with loss of viable cells that is accompanied by a marked decrease in cyclin D1 levels via increased ubiquitin-proteasome-dependent cyclin D1 turnover. This is in contrast with a rat intestinal epithelial cell line in which induction of Ha-RasVal-12 results in transformation associated with sustained proliferation and increased levels of cyclin D1, that is not accompanied by anoikis or apoptosis. Expression of the cyclin D1 mutant (T286A) that contains an alanine for threonine 286 substitution and is resistant to ubiquitin-proteasome degradation in the Ha-RasVal-12 expressing Rat-1 cells resulted in a sustained transformed phenotype with no accumulation of cells in G1. Inhibition of mitogen-activated protein kinase (MEK1/2) pathway partially reversed the Ras-mediated decrease in cyclin D1. Induction of Ha-RasVal-12 resulted in activation of Akt kinase and inactivation of glycogen-synthase-3beta kinase that are associated with reduction of cyclin D1 protein. These results suggest that Ras-mediated cyclin D1 degradation in Rat-1 cells appears to be partially dependent on activation of mitogen-activated protein kinase pathway and independent of glycogen-synthase-3beta kinase pathway.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The ras mutations are found in a wide variety of human malignancies; with the highest incidences observed in adenocarcinomas of the pancreas (90%), colon (50%), and lung (30%) (1). The cellular responses to activated Ras vary depending on cell type. Normal cells are often induced into pathways that lead to cell growth arrest, senescence, and/or apoptosis in response to activated Ras expression (2-5). These are important protective anti-tumorigenic responses that restrict the propagation of cells bearing activated oncogenes (6, 7). Recent studies have demonstrated that growth arrest and apoptosis after activated Ras expression can result from activation of the tumor suppressor protein p53. This activation occurs through a mechanism that involves transcriptional activation of the p16INK4A locus and the resultant expression of p16INK4A and the product of the alternate reading frame (ARF)1 located within the p16INK4A locus (murine p19ARF, human p14 ARF) (2, 7, 8). ARF can bind to and prevent MDM2-mediated destruction of p53 (9-12). Activation of p53 triggers cell growth arrest and apoptosis, in part through the induction of the cyclin kinase inhibitor p21Waf-1/Cip1 (13). On the other hand, forced expression of oncogenic Ras causes malignant transformation in multiple cell types including murine and rat fibroblasts (14, 15), rat intestinal epithelial cells (16, 17), and mammary epithelial cells (18). Cooperating with SV-40 large T antigen, Ras oncoprotein successfully transforms primary cell cultures (3). SV-40 large T antigen binds to retinoblastoma protein and prevents it from inhibiting E2F-mediated transcriptional activation and thereby enables G1/S transition to proceed. The resultant G1 acceleration contributes to the Ras-mediated transformation.

Expression of the D-type cyclins is dependent on continuous mitogenic stimulation. Progression through the mid to late G1 phase of the mammalian cell cycle is dependent upon the cyclin D1-mediated activation of cyclin-dependent kinase (CDK) 4 (or the related CDK6) (19). The activated cyclin D-dependent kinases phosphorylate and inactivate the retinoblastoma protein, thereby preventing its inhibition of transcription factors (including the E2Fs) that are essential for DNA synthesis. Cyclin D1 overexpression has been reported to cooperate with activated Ha-Ras to induce transformation of primary rat embryo fibroblasts that are not transformed by expression of activated Ha-Ras alone (20). Cyclin D1 is thought to play an important role in the pathogenesis of a variety of neoplastic lesions (21-26). Previous studies suggest that cyclin D1 is regulated by the Ras signaling pathway. Activation of ERK1 and ERK2 increases expression of cyclin D1 (27). Epithelial cells and fibroblasts selected after transformation by Ras exhibit increased levels of cyclin D1, along with a decreased growth factor requirement and accelerated G1 (28-33).

An important mechanism of cyclin D1 regulation is via ubiquitin/proteasome-mediated protein degradation. Phosphorylation of cyclin D1 on threonine 286 is required for its rapid degradation. A cyclin D1 mutant (T286A) containing an alanine for threonine 286 substitution fails to undergo efficient polyubiquitination and is markedly stabilized (t1/2 ~3.5 h) (34). Diehl et al. (35) also recently reported that cyclin D1 is phosphorylated by glycogen-synthase kinase-3beta (GSK-3beta ) specifically on Thr-286 and this triggers rapid cyclin D1 turnover. They also found that activation of Ras may increase the stability of cyclin D1 via phosphatidylinositol 3-kinase and Akt with phosphorylation and inactivation of GSK-3beta .

In this study, we attempted to explore the mechanism responsible for oncogenic Ras induced G1 growth arrest and apoptosis in Rat-1 fibroblasts. Induction of Ha-RasVal-12 in Rat-1 fibroblasts resulted in a marked decrease in cyclin D1 levels via increased ubiquitin-proteasome-dependent cyclin D1 turnover. To determine the role of reduced cyclin D1 in growth arrest of Rat-1 cells, cyclin D1 mutant (T286A) that is resistant to ubiquitin-proteasome degradation was transfected into the Rat-1:iRas cells. Expression of cyclin D1(T286A) resulted in a sustained transformed phenotype with no accumulation of cells in G1. Inhibition of MAP kinase pathway partially inhibited the Ras-mediated down-regulation of cyclin D1. Inactivation of GSK3beta kinase did not reverse the Ras-mediated cyclin D1 turnover. These results suggest that Ras induced G1 arrest in Rat-1 cells may occur through increased ubiquitin-proteasome-mediated cyclin D1 turnover at a GSK3beta -independent manner.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Lines and Stable Transfection-- Rat-1:iRas cell line with an inducible activated Ha-rasVal-12 cDNA was a gift from Dr. Hiroshi of Tokyo University of Technology. The Ha-rasVal-12 cDNA is under the transcriptional control of the Lac operon in an eukaryotic expression system (Stratagene, La Jolla, CA). Rat-1:iRas cells were maintained in DMEM containing 400 µg/ml G418 (Life Technologies, Inc., Gaithersburg, MD), and 150 µg/ml hygromycin B (Calbiochem, SanDiego, CA). IPTG (isopropyl-1-thio-beta -D-galactopyranoside, Life Technologies, Inc., Gaithersburg, MD) at a concentration of 5 mM was used to induce the expression of mutated Ha-Ras. Proteasome inhibitor N-acetyl-Leu-Leu-norleucinal (ALLN) was purchased from Sigma. A rat intestinal epithelial cell line (RIE-1) transfected with the similar Ha-RasVal-12 expression vectors is referred as RIE-iRas cells.

To establish Rat-1:iRas/D1 and Rat1:iRas/D1(T286A) cell lines, stable transfections were performed by using Lipofectin (Life Technologies, Inc., Gaithersburg, MD). An 1.3-kilobase EcoRI-EcoRI fragment containing the open reading frame for mouse cyclin D1, or a Flag-tagged 1 kilobase BamHI-BamHI fragment of cyclin D1(T286A) cDNA (a gift from Dr. C. J. Sherr) were ligated into the eukaryotic expression vector pZeoSV2(+) (InVitrogen, Carlsbad, CA). The resultant pZeoSV2/D1 and pZeoSV2/D1(T286A) vectors were then transfected into the Rat-1:iRas cells and selected in DMEM containing hygromycin, geneticin, and zeocin (250 µg/ml) to generate the Rat-1:iRas/D1 and Rat-1:iRas/D1(T286A) clones.

Northern Analysis-- Total cellular RNA was extracted as described previously (36). RNA samples were separated on formaldehyde-agarose gels and blotted onto nitrocellulose membranes. The blots were hybridized with cDNA probes labeled with [alpha -32P]dCTP by random primer extension (Stratagene, La Jolla, CA). After hybridization and washes, the blots were subjected to autoradiography.

Immunoblotting-- Immunoblot analysis was performed as described previously (36). The anti-pan Ras antibody was purchased from Calbiochem (La Jolla, CA), anti-cyclin D1 antibody was purchased from Upstate Biotechnology (Lake Placid, NY), the anti-Bcl-2 antibody was from Transduction (Lexington, KY), and anti-p53, anti-p16, and anti-Cdk4 antibodies were purchased from Santa Cruz (Santa Cruz, CA). Anti-p21 antibody was purchased from Calbiochem (La Jolla, CA). Phosphorylated Akt and Phosphorylated GSK-3a/b antibodies were purchased from New England BioLabs (Beverly, MA).

DNA Fragmentation Assay-- Cells were lysed in lysis buffer (1% Nonidet P-40 in 20 mM EDTA and 50 mM Tris, pH 7.5). The supernatant containing fragmented DNA was clarified by centrifugation for 5 min at 1600 × g. After the cell lysates were digested with proteinase K (2.5 mg/ml) and RNase A (5 mg/ml) the DNA was separated on 1.6% agarose gel.

Flow Cytometry-- Cells were seeded into 100-mm plates and treated with 5 mM IPTG for the indicated hours. Cells were fixed in 70% ETOH, digested in 1 ml of 0.1% RNase (Sigma), and stained with propidium iodide (Sigma). The DNA was analyzed by a flow cytometer. The cell cycle profile was expressed as percentage of cells in each cell cycle stage.

Metabolic Labeling-- Rat-1:iRas cells were treated with IPTG or vehicle for 24 h. One hour prior to harvesting, culture medium was replaced with methionine-free DMEM supplemented with 10% fetal bovine serum and 300 µCi/ml of [35S]methionine (Amersham Pharmacia Biotech) in the presence or absence of IPTG. Cell lysates were prepared in RIPA buffer (RIPA, 1 × phosphate-buffered saline, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 mg/ml phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 1 mM sodium orthovanadate). An equal amount of protein from each sample was immunoprecipitated with anti-cyclin D1 antibody-conjugated agarose (Upstate Biotechnology, Lake Placid, NY), resolved by SDS-PAGE, and visualized by autoradiography.

Cyclin D1 protein degradation was determined by pulse-chase experiments. The cells were labeled with 300 µCi/ml [35S]methionine for 1 h and then washed three times with DMEM. Fresh DMEM containing 2 mM unlabeled methionine was added into the cultures. Cell lysates were collected at intervals. An equal amount of protein from each sample was immunoprecipitated with anti-cyclin D1 antibody, resolved by SDS-PAGE, and visualized by autoradiography.

Soft Agarose Assay-- 1 × 104 cells were mixed with Sea-plaque-agarose at a final concentration of 0.4% in DMEM, and overlaid onto a 0.8% agarose layer in 35-mm plates. The plates were incubated for 10 days. Colonies were photographed by using an inverted microscope.

GSK3beta Kinase Assay-- For detection of GSK-3beta activity in immune complexes, Rat-1:iRas cells were treated with 5 mM IPTG for the indicated times. Cell lysates were collected in IP buffer (50 mM Tris, pH 7.5, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 0.2% sucrose, 1 mM dithiothreitol, 10 mM beta -glycerophosphate, 1 mM sodium fluoride, 0.1 mM NaVO4, and 1 mM phenylmethylsulfonyl fluoride). GSK-3beta containing complexes were precipitated with a mouse monoclonal antibody directed to GSK-3beta (Transduction Laboratories, Lexington, KY) and mixed with 1 µg of bacterially expressed GST-D1 or GST-D1(T286A) (gifts of Dr. C. J. Sherr) in 20 µl of kinase buffer (50 mM Hepes, pH 8, 10 mM MgCl, 2.5 mM EGTA, 1 mM dithiothreitol, 20 µM ATP, 10 mM beta -glycerophosphate, 1 mM sodium fluoride, 0.1 mM NaVO4, 1 mM phenylmethylsulfonyl fluoride, and 10 µCi of [gamma -32P]ATP). After the mixtures were incubated at 30 °C for 30 min, labeled proteins were separated by SDS-PAGE prior to autoradiography.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Ras Induction of G1 Growth Arrest-- The expression of activated Ha-Ras in Rat-1:iRas cells is markedly induced after treatment with IPTG (5 mM) with a detectable increase by 2 h and a marked increase by 24 h (Fig. 1A). After the induction of Ras, the Rat-1:iRas cells acquired a transformed appearance characterized by growth in overlapping clusters indicating the loss of contact inhibition by 24 h after induction of Ha-Ras expression. The IPTG-treated Rat-1:iRas cells began to detach from the culture surface (anoikis) by 48 h and most of cells were detached and floating by 72 h after IPTG treatment (Fig. 1A), suggesting the induction of cell death. Flow cytometry analysis showed a 42.7% increase in accumulation of cells in the G1 phase of the cell cycle and a 50.9% decrease in the S phase fraction after the Ha-RasVal-12 was expressed in Rat-1:iRas cells for 48 h (Fig. 1B). The floating Rat-1:iRas cells exhibited clumping and fragmentation of chromatin indicating programmed cell death (data not shown). DNA fragmentation assays revealed a time-dependent increase internucleosomal DNA fragmentation after Ha-Ras expression in the Rat-1:iRas cells (Fig. 1C), confirming the induction of apoptosis by activated Ha-Ras. These results suggested that expression of oncogenic Ha-Ras resulted in morphological transformation, but subsequent G1 growth arrest and apoptosis in Rat-1 fibroblasts.


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  		Fig. 1.   The phenotype of Rat-1:iRas cells. A, induction of Ras protein and morphological transformation. Rat-1:iRas cells were treated with IPTG (5 mM) and the levels of Ras protein were determined at the indicated intervals by immunoblotting. The morphology of Rat-1:iRas cells was demonstrated by inverted photomicroscopy (original magnification, ×100). B, flow cytometry. Rat-1:iRas cells were treated with 5 mM IPTG for the indicated hours. Cells were fixed in 70% ETOH and stained with propidium iodide. The DNA was analyzed by flow cytometry. The cell cycle profile was expressed as percentage of cells in each cell cycle phase. C, DNA fragmentation assay. Rat-1:iRas cells were treated with 5 mM IPTG for the indicated times and then lysed in lysis buffer. The supernatant containing fragmented DNA was clarified and separated on 1.6% agarose gel. D, levels of p53, p21Waf-1/Cip1, p16INK4a, Cdk4, and Ras protein in Rat-1:iRas cells at intervals after IPTG treatment. The levels of Cdk4 were only slightly altered by the induction of Ha-Ras in Rat-1:iRas cells and were used as internal loading controls for Western analysis throughout the study.

Recent studies have demonstrated that growth arrest and apoptosis after activated Ras expression can result from activation of the tumor suppressor protein p53, p16INK4a, or p21Waf-1/Cip1 (2, 7, 8, 13). In order to explore the mechanism by which activation of Ha-Ras results in growth arrest in Rat-1 cells we examined the expression of several potential molecular targets of the activated ras oncogene that potentially contribute to the phenotypic response described above. Western analyses show that induction of Ha-RasVal-12 did not result in a significant increase in the levels of p21Waf-1/Cip1, p16INK4a, or p53 in Rat-1 cells (Fig. 1D).

Ras-mediated Down-regulation of Cyclin D1-- Cells that are fully transformed by oncogenic ras often exhibit an increased rate of cell proliferation that is accompanied by an increase in the expression of cyclin D1. In contrast, we found the expression of cyclin D1 protein to be markedly decreased in the Rat-1:iRas cells (Fig. 2A, lower panel) after induction of Ha-Ras. Interestingly, despite the reduction in levels of cyclin D1 protein, induction of Ha-Ras resulted in a modest increase in cyclin D1 mRNA levels during the same interval (Fig. 2A, upper panel).


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  		Fig. 2.   Expression of cyclin D1 in Rat-1:iRas cells. A, expression of cyclin D1. Rat-1:iRas cells were treated with 5 mM IPTG for the indicated hours. Total RNA was isolated for detection of cyclin D1 mRNA. Cellular lysates were collected and the levels of cyclin D1 and Cdk4 were determined by Western analysis. B, forced expression of wild-type cyclin D1 in Rat-1:iRas cells. The eukaryotic expression vector pZeoSV2/D1 was constructed by inserting a mouse cyclin D1 cDNA into pZeoSV2(+). The pZeoSV2/D1 was transfected into Rat-1:iRas cells. Cyclin D1 RNA and protein levels were determined in a clone expressing the transfected gene at intervals after IPTG treatment. C, flow cytometry. Rat-1:iRas/D1 cells were treated with 5 mM IPTG for the indicated hours. The DNA was analyzed by flow cytometry and cell cycle distribution was determined as shown.

These observations raised the question of whether the decrease in cyclin D1 expression was the cause, or simply a consequence, of the G1 arrest and apoptosis that occurred after induction of oncogenic ras. To further investigate the functional significance and regulation of cyclin D1 in cells expressing activated Ha-RasVal-12, we transfected the Rat-1:iRas cells with the mouse wild-type cyclin D1 cDNA to achieve high level constitutive expression of cyclin D1. Several clones of Rat-1:iRas cells were identified that expressed high levels of the transfected cyclin D1 mRNA and protein under basal conditions (referred to as Rat-1:iRas/D1 cells). The regulation of cyclin D1 expression in response to the induction of Ha-Ras is shown for a representative clone (number 14) of the Rat-1:iRas/D1 cells (Fig. 2B). Northern analysis revealed that the endogenous cyclin D1 mRNA (~3.7 kilobases) was increased in Rat-1:iRas-CcnD1 clone number 14. The expression of exogenous cyclin D1 mRNA can be seen as the 1.3-kilobase band in Fig. 2B. The decreased levels of cyclin D1 protein did not correlate with the levels of either endogenously or exogenously expressed cyclin D1 mRNA during the 72 h of observation after IPTG treatment. Despite abundant endogenous and exogenous cyclin D1 mRNA levels, cyclin D1 protein levels decreased by 70-80% from 24 to 72 h after the induction of activated Ha-Ras. Flow cytometry showed 46% of the Rat-1:iRas/D1 cells to be in G1 phase and 44% of cells in S phase prior to the induction of Ras (Fig. 2C). By 48 h after induction of Ha-Ras, the G1 fraction was increased to 67.5% and the cell number in S phase fraction was reduced to 25%. Forced expression of the wild-type mouse cyclin D1 did not delay or prevent the onset of G1 arrest or apoptosis caused by Ha-Ras expression in the Rat-1:iRas/D1 cells, and as with the Rat-1:iRas cells, most of the cells were detached from the culture plate by 72 h after IPTG treatment.

Ras Accelerates Cyclin D1 Protein Turnover-- The decrease in cyclin D1 protein could potentially be due to a decreased rate of synthesis or an accelerated rate of degradation. The Rat-1:iRas/D1 cells were treated with either vehicle or IPTG for 24 h and then were pulse-labeled with [35S]methionine for 1 h. The levels of labeled cyclin D1 protein were reduced by 35-40% by 24 and 48 h after the addition of IPTG (Fig. 3A). A pulse-chase experiment revealed that cyclin D1 protein degradation was accelerated in the Ras-induced Rat-1:iRas/D1 cells (Fig. 3B). The t1/2 of cyclin D1 protein in Rat-1:iRas/D1 cells without Ras induction was 29.7 ± 5.5 min (mean ± S.E. from three separate experiments). The t1/2 of cyclin D1 protein in Ras-induced Rat-1:iRas-cyclin D1 cells was 9.5 ± 3.0 min (mean ± S.E. from three separate experiments). Previous studies have shown that levels of G1 cyclins are largely regulated by their degradation rate via the ubiquitin-proteasome pathway (34, 37-39). To determine whether the rapid degradation of cyclin D1 protein in Ras-induced Rat-1 cells involves the ubiquitin-proteasome pathway the effect of the proteasome inhibitor ALLN on the level of cyclin D1 was investigated (Fig. 3C). Rat-1:iRas cells were treated with vehicle or 5 mM IPTG for either 8 or 24 h prior to ALLN (25 µM) treatment and the levels of cyclin D1 protein were determined at time intervals after the addition of ALLN. The level of cyclin D1 protein was markedly reduced in cells treated with IPTG as compared with untreated cells. Addition of ALLN rapidly restored the levels of cyclin D1 in the IPTG-treated cells. These results suggested that expression of oncogenic ras caused a modest decrease in the rate of cyclin D1 protein synthesis, and a 3-fold increase in the rate of cyclin D1 degradation that involved proteasome function.


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  		Fig. 3.   Cyclin D1 synthesis and degradation in Rat-1:iRas/D1 clone 14. A, cyclin D1 protein synthesis. Rat-1:iRas/D1 (clone 14) cells were treated with 5 mM IPTG (+) or vehicle (-) for 8, 24, or 48 h. The cells were metabolically labeled with [35S]methionine in the presence or absence of IPTG and immunoprecipitated cyclin D1 was resolved by SDS-PAGE and visualized by autoradiography. B, degradation of cyclin D1 protein. Cyclin D1 protein degradation was determined by a pulse-chase experiment. Rat-1:iRas/D1 cells were treated with vehicle (CTR) or 5 mM IPTG for 24 h. The cells were labeled with [35S]methionine for 1 h and then chased by DMEM containing 2 mM unlabeled methionine. Immunoprecipitated cyclin D1 was resolved by SDS-PAGE and visualized by autoradiography. The bottom panel represents the densitometric analysis of the autoradiogram. The results were similar in three separate experiments. C, inhibition of ubiquitin-proteasome pathway. Rat-1:iRas cells were treated with IPTG for 0, 8, or 24 h prior to the treatment with 25 mM ALLN. Cell lysates from the indicated time points were analyzed for the levels of cyclin D1 and Cdk4 protein.

Expression of Cyclin D1(T286A)-- In order to determine whether the reduction of cyclin D1 levels was the consequence or the cause of cell growth arrest and apoptosis after induction of Ha-Ras, we transfected a cyclin D1 mutant (T286A) that is resistant to ubiquitin-proteasomal degradation into the Rat-1:iRas cells (34). Several selected clones expressed the Flag-tagged cyclin D1 at high levels (Fig. 4A). The level of cyclin D1 expressed by the Rat-1:iRas/D1(T286A) cells was significantly greater than the level in the Rat-1:iRas cells in the absence of IPTG treatment. While there was a modest decrease in the cyclin D1 levels in the Rat-1:iRas/D1(T286A) cells by 14-36% between 24 and 72 h after induction of Ha-Ras by IPTG, the level remained more than 3-fold greater than the level in the uninduced Rat-1:iRas cells (Fig. 4B). Pulse-chase experiments revealed that cyclin D1(T286A) protein was very stable (t1/2 ~ 3 h) and induction of Ras did not result in notable alteration of the stability of cyclin D1(T286A) (Fig. 4C). Flow cytometry demonstrated that induction of Ha-Ras in the Rat-1:iRas/D1(T286A) cells did not cause G1 growth arrest (Fig. 5A). The S-phase fraction was 50% prior to induction of Ha-Ras, and 42.4% at 72 h after the induction of Ha-Ras in the Rat-1:iRas/D1(T286A) cells. This is in contrast with the marked decrease in the S-phase fraction in the Rat-1:iRas cells expressing only endogenous wild-type cyclin D1 (as shown in Fig. 1) or exogenous wild-type cyclin D1 (as shown in Fig. 2). The Rat-1:iRas/D1(T286A) cells were able to survive induction of Ha-Ras, grew to high density, and formed foci (Fig. 5B). In addition, the IPTG-treated Rat-1:iRas/D1 cells were incapable of long-term survival or colony formation in soft agarose. Ten days after plating the plates seeded with the Ha-Ras expressing Rat-1:iRas/D1 cells contained only cell debris (Fig. 5C, upper panel), however, all 4 selected clones of the Rat-1:iRas/D1(T286A) cells analyzed formed colonies in soft agarose after induction of Ha-Ras by IPTG treatment (Fig. 5C, lower panel). These results suggest that activation of the Ras signaling pathway triggers increased turnover of cyclin D1 through ubiquitin-proteasome-mediated pathway. Expression of the cyclin D1 mutant (T286A) is sufficient to rescue the Rat-1 fibroblasts from oncogenic Ha-Ras induced growth arrest and cell death.


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  		Fig. 4.   Transfection with cyclin D1(T286A) mutant. A, selection of Rat-1:iRas/D1(T286A) cells. The pZeoSV2D1(T286A) was transfected into Rat-1:iRas cells. The levels of Flag-labeled cyclin D1 were analyzed by Western blotting using an anti-Flag antibody. B, Western analysis for cyclin D1, Flag, Cdk4, and beta -actin in Rat-1:iRas/D1(T286A) clone 8 and in uninduced Rat-1:iRas cells. Rat-1:iRas/D1(T286A) clone 8 was treated with 5 mM IPTG for the indicated intervals. Cell lysates were collected for Western analysis and the levels of cyclin D1, Flag, Cdk4, and beta -actin were compared with the levels in uninduced Rat-1:iRas cells. C, degradation of cyclin D1(T286A). Rat-1:iRas/D1(T286A) cells were treated with vehicle (CTR) or 5 mM IPTG for 24 h. The cells were labeled with [35S]methionine for 1 h and then chased by DMEM containing 2 mM unlabeled methionine. Immunoprecipitated cyclin D1 was resolved by SDS-PAGE and visualized by autoradiography. This experiment was repeated twice.


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  		Fig. 5.   The phenotype of Rat-1:iRas/D1(T286A) cells. A, flow cytometry analysis in Rat-1:iRas/D1(T286A) cells. B, morphology of Rat-1:iRas/D1(T286A) cells. Cells grown on plastic were treated with vehicle or IPTG for 72 h and photographed using an inverted microscope (×100). C, soft agarose assay. Rat-1:iRas/D1 or Rat-1:iRas/D1(T286A) cells were mixed with Sea-plaque-agarose at a final concentration of 0.4% in DMEM medium containing vehicle or 5 mM IPTG and overlaid onto a 0.8% agarose layer in 35-mm plates. Plates were photographed by using an inverted microscope after 10 days.

MAP Kinase and GSK3beta Kinase in Ras-induced Cyclin D1 Turnover-- Activation of extracellular signaling-regulated kinase 1/2 (ERK1/2) up-regulates cyclin D1 (27). GSK-3beta phosphorylates cyclin D1 specifically on Thr-286 and triggers cyclin D1 turnover (35). We observed that rat intestinal epithelial cells transfected with the inducible Ha-RasVal-12 (referred to as RIE-iRas cells) were transformed by IPTG and continued to proliferate without undergoing either anoikis or apoptosis (data not shown). In contrast with what was observed in the Rat-1 cells, induction of Ha-RasVal-12 in RIE-iRas cells resulted not only in sustained proliferation and transformation, but also, increased levels of cyclin D1 protein. Induction of Ha-RasVal-12 increased the levels of active ERK1/2 and elevated levels of cyclin D1 in RIE-iRas cells (Fig. 6A). In contrast, Ras-induced ERK1/2 activity was associated with decreased levels of cyclin D1 in Rat-1:iRas cells (Fig. 6B). Interestingly, inhibition of mitogen-activated protein kinase kinase activity by the treatment with PD-98059 blocked Ras-induced cyclin D1 in RIE-iRas cells and partially restored the reduced cyclin D1 levels in Rat-1:iRas cells, suggesting that the MAP kinase pathway may regulate the levels of cyclin D1 in different directions depending on the cell type.


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  		Fig. 6.   Expression of cyclin D1 and ERK activity. A, the expression of active ERK1/2 and cyclin D1 in RIE-iRas cells. RIE-iRas cells were treated with 5 mM IPTG, with or without PD-98059 (50 µM) for the indicated hours. The levels of active ERK1/2, cyclin D1, and Cdk4 were determined by Western blotting. B, the expression of active ERK1/2 and cyclin D1 in Rat-1:iRas cells. Rat-1:iRas cells were treated with 5 mM IPTG and with or without PD-98059 (50 µM) for the indicated hours. The levels of active ERK1/2, cyclin D1, and Cdk4 were determined by Western blotting.

Expression of mutated Ha-Ras resulted in increased phosphorylation of Akt (protein kinase B) and subsequently increased phosphorylation of GSK-3alpha /beta in both RIE-iRas and Rat-1:iRas cells that were associated with increased levels of cyclin D1 in RIE-iRas cells (Fig. 7A), but with decreased levels of cyclin D1 in Rat-1:iRas cells (Fig. 7B). The GSK-3beta kinase assay revealed that GSK-3beta kinase from uninduced Rat-1:iRas cells efficiently phosphorylated recombinant GST-cyclin D1, but induction of Ha-RasVal-12 almost completely abolished the ability of GSK-3beta to phosphorylate the recombinant cyclin D1 protein (Fig. 7C). The decrease in GSK-3beta activity after induction of Ha-RasVal-12 was accompanied by a decrease in the cyclin D1 level in the Rat-1 cells as shown in Fig. 7B. Lithium inactivates the kinase activity of GSK-3beta (40-42). Treatment with LiCl reduces the phosphorylation of microtubule-associated protein Tau by inhibition of GSK3beta (41) and can induce accumulation of beta -catenin, a known substrate of GSK-3beta , degradation of which requires GSK3beta activity (40). Addition of 20 mM LiCl significantly increased the levels of cyclin D1 protein in parental RIE cells, but did not alter the cyclin D1 levels in parental Rat-1 cells (Fig. 7D). As demonstrated in Fig. 7E, treatment with IPTG for 48 h significantly increased the levels of cyclin D1 in RIE-iRas cells and addition of LiCl further elevated the levels of cyclin D1. IPTG treatment decreased the levels of cyclin D1 protein in Rat-1:iRas cells and treatment with LiCl did not further alter the reduced levels of cyclin D1.


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  		Fig. 7.   The role of GSK3beta kinase in the expression of cyclin D1. Expression of pAkt, pGSKalpha /beta , and cyclin D1 in RIE-iRas (A) and Rat-1:iRas (B) cells. Cells growing in monolayer were treated with 5 mM IPTG for the indicated intervals. The levels of phosphorylated Akt, phosphorylated GSKalpha /beta , and cyclin D1 were determined by Western blotting. C, GSK-3beta kinase activity in Rat-1:iRas cells. Rat-1:iRas cells were treated with 5 mM IPTG for the indicated times. Cell lysates were collected in immunoprecipitation buffer. GSK-3beta -containing complexes were precipitated with a mouse monoclonal antibody directed to GSK-3beta and were incubated with 1 µg of bacterially expressed GST-D1 or GST-D1(T286A) in the presence of [gamma -32P]ATP. After the mixtures were incubated at 30 °C for 30-min labeled proteins were separated by SDS-PAGE prior to autoradiography. Corresponding levels of endogenous cyclin D1 were determined by immunoblotting of the same lysates. D, inhibition of GSK-3beta by LiCl. Rat-1 cells (upper panel) and RIE-1 cells (lower panel) were treated with 20 mM LiCl for the indicated intervals. Levels of cyclin D1, Cdk4, and beta -actin were determined by Western blotting. E, inhibition of GSK-3beta by LiCl in Ras-induced RIE and Rat-1 cells. Except for the first lane (N), RIE-iRas cells or Rat-1:iRas cells were treated with 5 mM IPTG for 48 h prior to LiCl treatment (20 mM). Lane N represents cells growing in the absence of IPTG. Cell lysates were collected at the indicated time points after the addition of LiCl. Levels of cyclin D1 were determined by Western blotting.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Activation of a ras oncogene has different consequences in different cell types. Activated ras may result in oncogenic transformation in some cells, whereas it may cause premature senescence, G1 arrest, and/or apoptosis in others. In this report, we conditionally expressed the Ha-RasVal-12 mutant in rat fibroblasts and in rat intestinal epithelial cells. Although the Rat-1 cells were morphologically transformed by the induction of Ha-RasVal-12 these cells then underwent growth arrest, anoikis, and apoptosis. Induction of Ha-RasVal-12 expression in the Rat-1 cells resulted in a marked reduction in cyclin D1 levels as a result of increased degradation of cyclin D1 protein. Our observations are differ from previously reported observations that ras-mediated transformation results in acceleration of G1 progression and induction of cyclin D1 expression in murine and rat fibroblasts (14, 15, 28). One possible important difference in our results, as compared with previous studies of cells transformed by activated ras, is that inducible expression of activated Ras protein reflects the phenotypic consequences of the whole cell population in response to the acute induction of Ha-RasVal-12. This is in contrast with the selection of clones of cells capable of surviving the constitutive expression of activated Ras after transfection, and it is in contrast with our present observations in the RIE:iRas cells. Ras levels remain low in the Rat-1:iRas cells unless IPTG is present, whereas prior studies of ras-transfected cells have been conducted in cells that were selected to survive constitutive expression of activated Ras oncoprotein. We have also selected clones of Rat-1 cells that survive constitutive expression of Ha-RasVal-12. In contrast with the Rat-1:iRas cells in which acute induction of Ha-RasVal-12 results in cell growth arrest, decreased cyclin D1, and apoptosis, Rat-1 cells selected to survive constitutive expression of Ha-RasVal-12 continue to cycle and maintain the higher levels of cyclin D1 that are typical of other cell types selected to survive transformation by activated Ras (data not shown). Apparently, expression of mutated Ras results in growth arrest and apoptosis in most rat fibroblasts within a population. Only those cells that maintain a high level of cyclin D1 survive complete transformation by oncogenic ras.

Mutational activation of the ras oncogene often results in premature cell senescence (2), G1 arrest (2, 3), and apoptosis (3, 4) in primary cell cultures. The Ras-induced growth arrest, senescence, and apoptosis are often associated with p53, p21Waf-1/Cip1, and p16INK4a induction (2-4). These responses likely reflect a mechanism for protection of cells against the transforming effects of mutational activation of the Ras signaling pathway. We found that p53, p21Waf-1/Cip1, and p16INK4a are expressed in Rat-1 cells, however, levels of these proteins were not significantly regulated by the induction of oncogenic Ras in the Rat-1 cells. This is in contrast with the marked down-regulation of cyclin D1 by Ha-RasVal-12 in the Rat-1 fibroblasts.

The down-regulation of cyclin D1 protein that was observed in the Rat-1:iRas cells between 8 and 24 h preceded the G1 growth arrest that occurred 48 h after the induction of Ha-RasVal-12. G1 cyclins, including cyclin D1, drive cells through G1 by activating CDKs (reviewed in Ref. 43). D-type cyclins are rate-limiting for G1 progression, and growth factor withdrawal results in a rapid decrease in cyclin D1 levels along with G1 cell cycle arrest. Microinjection of cyclin D1 antisense or antibody into fibroblasts during G1 induces cell cycle arrest whereas abrogation of cyclin D1 after the G1/S transition fails to prevent cell cycle progression (28, 44). In the present study, induction of Ha-RasVal-12 in the Rat-1 cells resulted in marked reduction of cyclin D1 protein that was associated with G1 arrest.

Protein degradation is an efficient mechanism for the regulation of cell cycle transitions. Three major cell cycle transitions, including G1/S transition, separation of sister chromatids, and cytokinesis, require the degradation of specific proteins via the ubiquitin-proteasome pathway (reviewed in Ref. 45). G1 cyclins in budding yeast are rapidly degraded throughout the cell cycle (38). The half-life of mammalian cyclin D1 has been previously reported at 20-30 min (46-48). Recent work by Diehl et al. (34, 35) revealed that an important component of cyclin D1 regulation was through alterations in protein stability. These investigators found that cyclin D1 was degraded in an ubiquitin/proteasome-dependent manner, and that the half-life of cyclin D1 was decreased in the absence of growth factor. Phosphorylation of cyclin D1 on threonine 286 is required for its rapid degradation. A cyclin D1 mutant (T286A) containing an alanine for threonine 286 substitution fails to undergo efficient polyubiquitination and is markedly stabilized (t1/2 ~3.5 h) (34).

Diehl et al. (35) also reported that constitutive expression of Ha-RasVal-12 in transformed mouse NIH-3T3 fibroblasts results in increased stability of the cyclin D1 protein. In the present study, we found that activation of the Ras signaling pathway in the Rat-1:iRas fibroblasts had the opposite of the expected effect and that Ras activation accelerated cyclin D1 proteolysis with the concomitant growth arrest of the Rat-1:iRas fibroblasts. The half-life of cyclin D1 in control Rat-1 cells was ~29 min but the stability of cyclin D1 was significantly decreased (t1/2 ~ 9 min) after induction of Ha-RasVal-12. The 35-40% difference of cyclin D1 levels that was observed in the metabolic labeling experiment may also be accounted for by the difference in the protein turnover rate in the Ras-induced as compared with the uninduced Rat-1 cells. Forced expression of wild-type cyclin D1 increased the level of cyclin D1 protein in uninduced Rat-1 cells, but not in the Ras-induced cells despite increased cyclin D1 mRNA levels. Wild-type cyclin D1 expression failed to protect the cells from growth arrest. The stable mutant form of cyclin D1(T286A) was expressed in the Rat-1:iRas(T286A) cells in order to determine whether cyclin D1 levels could be maintained, and to further determine whether this could prevent the cytotoxic effects of Ras activation in the Rat-1 fibroblasts. While growth arrest, anoikis, and apoptosis prevented survival of the Rat-1:iRas cells, expression of stabilized cyclin D1 mutant (T286A) enabled the Rat-1:iRas(T286A) cells to survive induction of Ha-RasVal-12, to grow to high density, and to form foci when growing on plastic, and to grow in an anchorage-independent manner in soft agarose, all consistent with cell transformation. Our results also confirmed that decreased cyclin D1 protein levels were the cause, and not the consequence, of the G1 arrest observed after Ha-RasVal-12 induction in the Rat-1:iRas cells.

GSK-3beta appears to be an important regulator of cyclin D1 protein turnover (35). GSK-3beta phosphorylates cyclin D1 specifically on Thr-286 and triggers rapid cyclin D1 turnover. Since the signaling pathway that sequentially involves Ras, phosphatidylinositol-3-OH, and protein kinase B (Akt), can inhibit the activity of GSK-3beta , activation of Ras may stabilize cyclin D1 via this pathway (35). Similar to previous observations in NIH-3T3 fibroblasts (35), we observed that induction of Ras activates Akt, inactivates GSK-3beta , and increased the level of cyclin D1 in intestinal epithelial (RIE-iRas) cells. In contrast, while activation of Ras in the Rat-1 fibroblasts resulted in activation of Akt and inactivation of GSK-3beta , this did not result in an increase in cyclin D1; in fact, cyclin D1 levels were markedly decreased. Furthermore, inhibition of GSK-3beta activity with lithium chloride increased the level of cyclin D1 in the rat intestinal epithelial cells, but failed to increase the levels of cyclin D1 in the parental and Ras-induced Rat-1 cells. These results suggest that while GSK-3beta -mediated cyclin D1 degradation may important in selected cell types, this mechanism does not appear to be involved in cyclin D1 regulation in the Rat-1 fibroblasts. We conclude from these studies that the ubiquitin/proteosome-mediated degradation of cyclin D1 is important in both cell types, but a protein kinase (or kinases) other than GSK-3beta is likely to regulate this process in the Rat-1 cells. Interestingly, activation of the ERK (MAP kinase) activity appeared to be important for both the increase in cyclin D1 in the RIE:iRas cells and for the decrease in cyclin D1 in the Rat-1:iRas cells, as the effects of activated Ras could be inhibited in both cell types by PD-98059. This suggests that MAP kinase activation is an important signaling component in both cases. The identity of the alternative signaling pathway in addition to MAP kinase that leads to cyclin D1 degradation in response to Ras activation in the Rat-1 cells is not clear at this point and is the focus of ongoing investigation.

In summary, activation of ras often causes cell transformation that is associated with increased expression of cyclin D1. Induction of activated Ha-RasVal-12 in Rat-1 fibroblasts increases cyclin D1 turnover rate via ubiquitin-mediated proteolysis and results in G1 growth arrest, while the opposite effect was observed in rat intestinal epithelial cells. Induction of oncogenic Ras may result in either acceleration of G1 progression or G1 growth arrest depending on the cell type, and both of these effects may be mediated through regulation of cyclin D1 protein levels. The Ras-induced expression of cyclin D1 in intestinal epithelial cells appears to involve the inhibition of GSK-3beta kinase activity, whereas inhibition of GSK-3beta had no effect on cyclin D1 levels in the Rat-1 fibroblasts. While cyclin D1 degradation does appear to be ubiquitin/proteosome mediated, GSK-3beta does not appear to be involved in the regulation of cyclin D1 levels in the Rat-1 fibroblasts.

    ACKNOWLEDGEMENTS

We thank Dr. I. Hiroshi for providing Rat-1:iRas cells. Dr. C. J. Sherr and Dr. J. A. Diehl provided cyclin D1(T286A), GST-cyclin D1, GSK-cyclin D1(T286A) constructs, the protocol for GSK-3beta kinase assay, and helpful discussion for this study. Dr. J. Pietenpol provided critical review and comments on the manuscript.

    FOOTNOTES

* This work was supported by the National Institutes of Health Grants DK-52334, CA-69457 (to R. D. B.), DK-47297 (to R. N. D.), and CA 68485 (to the Vanderbilt Cancer Center).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed: Dept. of Surgery, Vanderbilt University Medical Center, 21st Ave. South, MCN D-5230, Nashville, TN 37232. E-mail: daniel.beauchamp@mcmail.vanderbilt.edu.

Published, JBC Papers in Press, April 25, 2000, DOI 10.1074/jbc.M002235200

    ABBREVIATIONS

The abbreviations used are: ARF, alternate reading frame; Rat-1:iRas, Rat-1 fibroblast transfected with an inducible activated Ha-RasVal-12 cDNA; RIE-iRas, rat intestinal epithelial cells transfected with an inducible activated Ha-RasVal-12 cDNA; IPTG, isopropyl-1-thio-beta -D-galactopyranoside; MAP kinase, mitogen-activated protein kinase; ERK, extracellular signaling-regulated kinase; Akt, protein kinase B; GSK, glycogen-synthase kinase; CDK, cyclin-dependent kinase; DMEM, Dulbecco's modified Eagle's medium; PAGE, polyacrylamide gel electrophoresis; ALLN, N-acetyl-Leu-Leu-norleucinal.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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