Elevated Levels of Cyclin D1 Protein in Response to Increased ...

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overexpressed in transfected cells, suggesting that posttranscriptional events may be important in cyclin D1 regulation. The mRNA cap-binding protein ...

MOLECULAR

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Vol. 13, No. 12

CELLULAR BIOLOGY, Dec. 1993, p. 7358-7363

0270-7306/93/127358-06$02.00/0 Copyright © 1993, American Society for Microbiology

Elevated Levels of Cyclin D1 Protein in Response to Increased Expression of Eukaryotic Initiation Factor 4E IGOR B. ROSENWALD,lt ANTHOULA LAZARIS-KARATZAS,2 NAHUM SONENBERG,2 AND EMMET' V. SCHMIDTV*

Massachusetts General Hos ital Cancer Center, Building 149, 13th Street, Charlestown, Massachusetts 02129, and Department of Biochemistry, McGill University, Montreal, Quebec, Canada H3G 1Y62 Received 7 June 1993/Returned for modification 9 July 1993/Accepted 9 September 1993

Cyclin D1 is a Gl-specific cyclin that has been linked to lymphoid, parathyroid, and breast tumors. Recent studies suggested that high protein levels of cyclin D1 are not always produced when cyclin D1 mRNA is overexpressed in transfected cells, suggesting that posttranscriptional events may be important in cyclin D1 regulation. The mRNA cap-binding protein (eukaryotic initiation factor 4E [eIF-4E]) is a potential regulator of several posttranscriptional events, and it can itself induce neoplastic transformation. Consequently, we examined eIF-4E as a potential regulator of cyclin D1. Overexpression of cyclin D1 mRNA in NIH 3T3 cells did not increase cyclin D1 protein. In contrast, overexpression of eIF-4E markedly increased the amount of cyclin D1 protein in NIH 3T3 cells. This increase was specific to cyclin D1 in comparison with the retinoblastoma gene product, c-Myc, actin, and eukaryotic initiation factor 2a. We also examined cyclin D1 protein in cells expressing an estrogen receptor-Myc fusion protein because we previously found that eIF-4E increases after induction of c-myc function. In these cells, increased levels of eIF-4E protein were closely followed by increases in levels of cyclin D1 protein, but the level of cyclin D1 mRNA was not increased. We conclude that increases in cyclin D1 levels may result from increased expression of eIF-4E, and this regulation may be one determinant of cyclin D1 levels in the cell.

pathways are indeed altered in response to overexpression of eIF-4E (13, 18, 19). However, the mechanisms underlying these regulatory events remain unclear. The function of eIF-4E as the mRNA cap-binding protein suggests several potential sites of interaction. The cap structure of eukaryotic mRNAs is added to RNA polymerase II transcripts during the transcription process and functions in multiple biological processes (31). Since eIF-4E binds to the mRNA cap, it has the potential to alter gene expression at levels including translational initiation, mRNA splicing, mRNA 3'-end processing, mRNA nucleocytoplasmic transport, and protection against 5'-exonucleolytic degradation (31). Identification of genes whose expression is specifically altered by eIF-4E would provide important insights for further investigations of the role of eIF-4E in malignant transformation. Numerous studies suggest that the cyclin genes are important regulators of growth and passage through specific points in the cell cycle (6). The mitotic cyclins were initially described in lower eukaryotes as regulators of passage from G2 to M in the cell cycle (27). In contrast, in mammalian cells the G1-S transition is thought to play a more important role in growth regulation, and in these cells the accumulation of unstable proteins is required for cells to pass from the G1 period into S phase (28). The point in late G1 where this accumulation is required is termed the restriction point. A group of recently described G1 cyclins are thought to be important in this transition (35). The expression patterns of the G1 cyclins in mid to late G1 suggest that they may be the unstable proteins that are critical in passage through the restriction point. The importance of specific cyclins in the G1-S transition has been demonstrated by several recent studies (35). Unfortunately, the existence of multiple Gj-phase cyclins and their associated kinases makes it difficult to determine which cyclins are key regulators of this step. Cyclin D1 (cyl 1 or

Cell growth is regulated by complex molecular processes. After growth induction, levels of certain mRNAs increase in the absence of new protein synthesis, defining a group of mRNAs as immediate-early genes in the growth response pathway (17). Several important transcription factors are included among these growth-induced genes. Growth-induced genes may also be important in neoplastic development because several proto-oncogenes are members of this immediate-early gene family. In contrast, the genes which are expressed later in G1 are designated delayed-early genes, and they may be regulated in turn by the immediate-early genes. Members of this class of genes are less well characterized (16). Since new protein synthesis is required for the expression of delayed-early genes, additional posttranscriptional regulatory events may be important in their expression. Transfection studies using eukaryotic initiation factor 4E (eIF-4E) further emphasize the importance of regulation of protein synthesis in cell growth control (32). eIF-4E is an important regulatory component of the protein synthetic machinery because it is present in limiting concentrations in the cell (11). Recent experiments demonstrated the importance of its function because overexpression of eIF-4E transforms cells (18). Acting as the mRNA cap-binding protein, eIF-4E is likely to provide global functions in growth regulation. However, the malignant transformation caused by eIF-4E could be best explained if eIF-4E also selectively increases the expression of specific growth-enhancing genes in contrast to growth inhibitors. Recent studies suggest that specific growth-regulatory * Corresponding author. Electronic mail address: [email protected] Helix.MGH.Harvard.Edu. t Present address: Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA 02139.

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POSTTRANSCRIPTIONAL REGULATION OF CYCLIN Dl

PRAD-1) was identified as a gene that was overexpressed in parathyroid tumors, in breast cancers, and at the bcl-1 breakpoint in certain lymphomas; consequently, cyclin D1 may play an especially important role in growth regulation (15, 26, 37). Furthermore, it complements yeast Cln mutations that prevent passage from G1 to S (21, 38). Interestingly, cyclin D1 has been consistently identified as one important delayed-early gene which is maximally expressed at the time in the cell cycle when protein synthesis is rate limiting (16, 25, 26). Transfection studies with murine cyclin D1 suggested that posttranscriptional processes might be important in regulating its expression (25). We recently found that mRNA for translation initiation factor eIF-4E increases in response to growth induction by c-myc (33). The potential for c-myc to regulate eIF-4E suggests a possible link between the immediate-early growth response and increases in delayed-early proteins like cyclin D1. In assessing the importance of the myc-induced transcriptional increase in eIF-4E during G1, it again seemed likely that eIF-4E might enhance the expression of specific growth-enhancing proteins. To determine whether eIF-4E causes selective increases in one growth-enhancing gene, we examined potential interactions between increased expression of eIF-4E and cyclin D1. MATERIALS AND METHODS Cell culture and transfections. NIH 3T3 cells grown in Dulbecco's modified essential medium (Sigma) containing 10% fetal calf serum were cotransfected with 30 ,ug of pMMTV-PRAD1 (34) and 5 jg of pSVNeo, using standard calcium phosphate precipitation methods (36). Cells were split the following day into several dishes and were incubated in 200 ,ug (active ingredient) of Geneticin (G418; GIBCO). Colonies resistant to G418 were identified and subcloned 2 weeks later. Transfectants were screened for expression of dexamethasone-inducible cyclin D1, using 2 x 10-6 M dexamethasone for 24 h in the medium to induce maximal expression. NIH 3T3 cells expressing translation initiation factor eIF-4E [4E(P2) cells] and eIF-4E containing a serine-to-alanine mutation at codon 53 (Ala cells) were identical to those described previously (18). To arrest cell growth, these cells were cultured in the presence of 0.15 mM hydroxyurea for 24 h before protein lysates were prepared. Cells expressing a genetic construct encoding a protein which contains the estrogen binding domain of the estrogen receptor fused to c-myc were obtained from M. Bishop (7, 8). This fusion decreases the activity of c-myc in the absence of estradiol, and c-myc function is then induced by the addition of estradiol to BALB/c 3T3 cells expressing this construct (myc-er cells). Estrogen stimulation experiments were performed as described previously (7). Normal rat kidney (NRK) cells transfected with the EJ-ras transforming allele of Harvey ras (NRK-ras cells) or a control neomycin resistance plasmid (NRK cells) were obtained from D. Haber. These cells were grown in Dulbecco's modified essential medium supplemented with 10% fetal calf serum. RNA analysis. Total cellular RNA (4) was size fractionated (10 ,ug per lane) on formaldehyde agarose gels, transferred to

Hybond-N nylon matrices, and cross-linked by UV light. Filters were hybridized overnight at 42°C with eIF-4E, actin, tubulin, or cyclin D1 cDNA fragments 32P labeled by the Klenow reaction, using random priming. Western blot (immunoblot) analysis. Cells growing in 100mm-diameter tissue culture dishes were washed with phos-

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FIG. 1. Northern and immunoblot analysis of cyclin D1 in NIH 3T3 transfectants. NIH 3T3 cells were cotransfected with an MMTV-cyclin D1 fusion gene construct and with pSVNeo. G418resistant colonies were screened for increased expression of cyclin D1 that could be induced by dexamethasone. The highest-expressing colony identified was designated cD-10. The blot shown (A) contains 10 pg of total cellular RNA from untreated (-) and dexamethasone-treated (+) NIH 3T3 cells (NIH) and MMTVPRAD-1 transfectants (cD-10). The blots were probed with the PRAD-1 cDNA fragment used in the construct. Equivalent loading of the RNA samples is demonstrated in the ethidium bromidestained gel below each blot. The same cells were cultured under identical conditions, and protein lysates were analyzed for expression of actin, cyclin D1, and translation initiation factor eIF-4E proteins (B). Equivalent loading is demonstrated in the actin probing.

phate-buffered saline and lysed at 4°C by passage through a 20-gauge needle in TNE buffer (50 mM Tris-HCl [pH 8.0], 420 mM NaCl, 0.5% Nonidet P-40, 2 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 1 mg of leupeptin per ml). The lysates were then centrifuged at 10,000 x g for 15 min to remove debris. Protein content of the lysate was determined by using a Bradford assay (Bio-Rad). For each sample, equal amounts (30 to 50 ,ug) of protein lysates were analyzed on a sodium dodecyl sulfate-12% polyacrylamide gel. Proteins were electroblotted onto a nylon membrane (Immobilon PVDF; Millipore), blocked for 1 h in 5% dry skim milk, and then incubated with the indicated antibodies. The antibodies used and their dilutions were as follows: 1:1,500 rabbit polyclonal anti-cyclin D1 (gift of A. Arnold), 1:5,000 monoclonal anti-actin (N-350; Amersham), 1:2,500 polyclonal anti-eIF-4E (19), 1:2,000 monoclonal anti-retinoblastoma (Rb) gene product (Pharmingen), 1:1,500 monoclonal antieIF-2a (gift of E. Henshaw), and 1:2,000 rabbit polyclonal anti-c-Myc (UBI). The secondary antibodies used were those included in an enhanced chemiluminescence detection kit (Amersham) and were chosen according to the species used for the primary antibodies. Exposure times varied according to the antibodies used and ranged from 5 to 60 s. RESULTS Characterization of NIH 3T3 cells transfected with an MMTV-cyclin D1 expression vector. To examine the role of cyclin D1 as a regulator of cell growth, we initially developed an expression vector for cyclin D1 cDNA under the control of mouse mammary tumor virus (MMTV) enhancer and promoter elements. We transfected NIH 3T3 cells with this construct and identified several clones that expressed increased cyclin D1 mRNA of the appropriate size for the transfected construct on Northern (RNA) blots. Several cellular clones expressing this construct were isolated, and we present data from the highest-expressing clone (Fig. 1) to demonstrate that increased cyclin D1 protein was not pro-

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