Znt. J. Cancer: 65,858-863 (1996) 0 1996 Wiley-Liss, Inc.

Publication of the International Union Against Cancer Publication Ue I‘Union Internationale Contre le Cancer

OVEREXPRESSION OF THE PROTO-ONCOGENE/TRANSLATION FACTOR 4E IN BREAST-CARCINOMA CELL LINES Bruce ANTHONY, Peggy CARTER and Arrigo DE BENEDETTI~ Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, Shreveport, L A , USA.

The expression of the proto-oncogene, translation factor elF-4E, was examined in breast-cell lines: 5 carcinomas and 2 normal. At the protein level, elF-4E was 10 times higher in the carcinoma lines than in normal cells, which is comparable to the level found in breast-cancer biopsies. The elevation appears to be due to increased transcription, since the elF-4E mRNA was correspondingly increased. These results demonstrate that cells isolated from naturally occurring breast carcinomas maintain an elevated expression of the factor. Turnover rates for elF-4E (mRNA and protein) were determined for normal and cancer cells. We found that elF-4E mRNA is relatively stable during an 8-hr incubation with Actinomycin D, but the half-life of the protein is fairly short ( ~ 4 . 5 hr). This suggests that, in proliferat- ing cells, elF-4E may be turning over rapidly, possibly to fine-tune the changes in translation rates which occur during the cell cycle. o 1996 Wiley-Liss, Inc.

The translation factor elF-4E mediates the first and most discriminatory step in the process of protein synthesis: selec- tion and recruitment of mRNA to the ribosomes. During this process, one specific mRNA is selected from the untranslated pool and engaged for translation. Because of the relatively low abundance in elF-4E, the various classes of mRNAs compete with each other for translation initiation, establishing an order of priorities in the spectrum of translated mRNAs. Recently, elF-4E was also identified as a powerful promoter of cell growth and neoplastic transformation, since overexpression of elF-4E causes deregulated cell growth and malignant transfor- mation of rodent and human cells (Lazaris-Karatzas et al., 1990; De Benedetti and Rhoads, 1990; De Benedetti et al., 1994). Furthermore, elF-4E is prominently elevated in some human neoplasms, such as breast carcinomas (Kerekatte et al., 1995), and the mechanism of oncogenesis by elF-4E presents a challenging biological problem. One attractive possibility is that the translation of certain proto-oncogene or growth-factor transcripts becomes preferentially increased by the elevated elF-4E, leading to overexpression of their products. Accord- ingly, a few of these have been identified, e.g., cyclin D1 (Rosenwald et al., 1993a), NF-AT (Barve et al., 1994), orni- thine decarboxylase (Shantz and Pegg, 1994), P23 (Bommer et al., 1995) and c-Myc (De Benedetti et al., 1994). A direct translational effect by elF-4E was recently shown for basic fibroblast growth factor (FGF-2) and Vascular Permeability Factor (VPF), which may have important implications for autocrine stimulation and/or vascularization of the growing tumor (Kevil et al., 1995 and 1996). In fact, we have speculated that increased protein synthesis output, due to elevated elF-4E, may be necessary for cancer cells to achieve adequate proliferation rates (greater than their rate of apoptosis), to promote neo-angiogenesis, and for invasion of the surrounding bed of normal tissue (Kerekatte et al., 1995). Consistent with this, the elevated expression of elF-4E in breast-cancer biop- sies, but not in benign fibroadenomas, suggests that this factor may mark a critical transition in breast-cancer progression.

To begin investigating the reasons and processes leading to the elevation in elF-4E, we set out to examine the expression of elF-4E in several commonly exploited breast-cancer cell lines, primarily MDA435, MDA231 and MCF7; one continuous normal breast cell line (MCFlOA) and one primary breast epithelial cell culture isolated from the milk of a nursing mother. We found that elF-4E was highly elevated in the

breast carcinoma lines, and that this was due to increased transcription of the elF-4E gene.

MATERIAL AND METHODS Cells and tissue culture

The carcinoma cells used in this work were the estrogen- sensitive MCF-7 and estrogen-insensitive MDA-435 and MDA- 231 lines, which were derived from invasive breast carcinomas. These were chosen because they are among the most com- monly used breast-cancer models and have retained their original karyotype. In some experiments we also used SKBR-3 and BT-20 lines, although the karyotype of these cells is highly anomalous. MCF-1OA were originally cloned from a patient suffering from fibrocystic disease, and they are “normal” in most respects, including karyotype and differentiation markers (e.g., milk-fat globule antigen). JA cells used in this study were breast epithelial cells freshly isolated from the milk of a nursing mother. Typically, they were grown in tissue culture for only 4 doublings and then immediately used or frozen. These cells were exfoliates from milk ducts, which were collected weekly over a 1-year period and, therefore, represent a primary epithelial breast-cell culture. All cells were grown in D-MEM with supplemental amino acids and 15% FBS, strep- tomycin, ampicillin and tetracyclin. MCF-1OA and JA cells additionally received insulin (10 pg/ml) and partially purified basic fibroblast growth factor (200 ng/ml), prepared in our laboratory. For experiments, the cells were grown to about 80% confluence, near the end of the exponential phase.

Preparation of cell extracts and determination of protein concentration

Cells were detached in PBS/EDTA, vortexed, and centri- fuged for 5 min at 2,000 rpm ( 5 0 0 ~ g). The cell pellet ( = 2 x loh) was solubilized in RIPA buffer (Harlow and Lane, 1988) with glass beads and vigorously vortexed. Protein concen- tration was determined by the BCA assay (Pierce, Rockford, IL), using dilutions of BSA as a standard. Antibodies, Western blots and immunoprecipitations

Anti-elF4E antiserum was obtained by immunizing a New Zealand rabbit with recombinant human elF-4E, isolated from an overexpressing strain of Escherichia coli (JM109-DE3) by affinity chromatography on m7GTP-Sepharose and prepara- tive SDSIPAGE. The anti-actin (human) monoclonal serum and anti-rabbit IgG conjugated with alkaline phosphatase were purchased from Sigma (St. Louis, MO).

Our standard protocol for Western blots utilized 0.05 mg of total cell protein, separated on 15% polyacrylamide gels, and transferred to Immobilon-P (Millipore, Bedford, MA). The primary antibody was used at a 1500 dilution and incubated overnight at room temperature. The secondary antibody was used at a 1:1,000 dilution for 1 hr. Immunodetection was

‘To whom correspondence and reprint requests should be ad- dressed, at the Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, 1501 Kings Highway, Shreveport, LA 71130-3932, USA. Fax: (318) 675-5180.

Received: October 19, 1995 and in revised form November 28,1995.

859 elF-4E IN BREAST CANCER

RGURE 1 - (a) Western-blot analysis. Cell extracts of the indicated cell lines (described in the text) were prepared, then the proteins were separated by SDS/PAGE and transferred to a membrane. The top panel was probed with elF-4E antiserum and the bottom with actin antiserum, as indicated. Lane 1 contained 30 ng of partially purified, recombinant elF-4E as a marker. Lanes 7 and 8 contain immunoprecipitated elF-4E from the cell extract equivalent of 10 times the amount in lanes 2 and 3. (b) Immunoprecipitations and turnover rates. The loss of labeled elF-4E during a chase was followed in 2 representative cell lines: MDA-231 and MCF-1OA. elF-4E was immunoprecipitated, fractionated on 15% SDS/PAGE, and detected by autoradiography. Time points of the chase are indicated. (c) Determination of decay rates. The elF-4E bands anel b) were excised from the gel, solubilized and counted in aqueous scintillation cocktail. In (c), (0) corresponds to MDA-231 and ( $ ) to MCF-1OA.

performed by staining with bromochloroindoyl phosphate/ nitro blue tetrazolium.

For immunoprecipitations, the cells were incubated for 3 hr in methionine-free medium and 1 mCi/ml [35S]methionine. The culture was then washed 3 times with unlabeled medium to start the chase. Cell extracts were made in RIPA and the specific activity at the beginning of the chase was typically -2 x lo7 cpm/pg(P). Then, 1.5 mg of protein (total cell extract) were reacted with 15 pg of purified elF-4E antiserum for 3 hr on ice, after which 15 p1 of Protein A (Sigma) were added and the incubation was continued for 1 hr. Following a brief centrifugation, the resin was washed 3 times with 0.2 ml RIPA, and the adsorbed proteins were eluted by boiling in 20 p1 of SDS gel-sample buffer containing 50 mM DTT.

Isolation and analysis of RNA Washed cell pellets (lo6) were lysed in 20 volumes of 3%

CETAB (Cetyl-trimethyl-ammonium bromide) in DEPC (di-

ethyl pyrocarbonate)-treated water, vortexed and centrifuged for 3 min at 1,500 x g in a microfuge. RNA pellets were resuspended in 4M guanidinium-HC1 and extracted with phenol/chloroform/isoamyl alcohol (25:24:1) then centrifuged for 3 min at 12,000 x g. Two volumes of cold ethanol were added to the aqueous phase and centrifuged for 8 min at 12,000 x g . RNA was resuspended in 50 p1 of water, then its integrity, purity and concentration were determined spectro- scopically (260/280 nM) and by visual inspection on 1.5% agarose gels.

For Northern blots, 25 pg of total RNA was denatured with glyoxal/DMSO, fractionated on agarose gels and transferred to a ZetaProbe-GT membrane (BioRad, Hercules, CA). Con- ditions for hybridization were as follows: pre-hybridization overnight at 45°C in 6 x SSC, 40% formamide, 0.1% SDS, 0.1% dried milk; hybridization overnight with fresh cocktail and the appropriate 32P-labeled probe (5 x lo5 cpm/ml). The

860 ANTHONY ETAL.

probes were cDNAs labeled by nick-translation using [ C ~ - ~ ~ P ] ~ C T P . The membranes were washed at 65°C for 2 hr in 2 x SSC, 0.1% SDS, 0.05% sodium pyrophosphate, air-dried, and exposed to X-ray film or to a phosphoimager (Molecular Dynamics, Sunnyvale, CA).

For analysis by RT/PCR, equal amounts of RNA ( = 15 pg) were denatured at 75°C for 3 min, then placed on ice. A mixture composed of 2 p1 of random primers (45 pM), 6 p1 of 5 x AMV buffer, 2 pl(20 units) of AMV reverse transcriptase (Promega, Madison, WI), 20 yl dNTPs (1 mM each), and 1 p1 of RNase inhibitor (Promega RNasin) was added to the mRNA and incubated for 2 hr at 42°C. The reaction was stopped by inactivation at 80°C for 3 min, then the mixture was placed on ice. The cDNA was cleaned by gel-filtration through a spin column (Millipore, Ultrafree MC) with a 0.2-ml bed of Biogel P6 (BioRad). Concentrations were determined spectro- scopically (2601280 nM), and all subsequent PCR reactions were performed using equal amounts of RT product. The primers for elF-4E amplification were: 5 ’ -AGATGGCGACT-

cycles wcrc: 94°C for 45 sec, 50°C for 45 sec, and 72°C for 1.5 min. The GAPDH primers were: 5’-CTACTGGCGCATC-

cycles were: 94°C for 45 sec, 58°C for 45 sec, and 72°C for 1.5 min. PCR reactions contained: 30 PI of dNTPs (1 mM each), 20 pCi [CX-~~SI~CTP, 10 p1 of ( lox ) reaction buffer with 25 mM M g Q , 20 ng of RT template, appropriate primers (0.25 pM), and 1 unit of Taq polymerase (Promega) in 0.1 ml final reaction volume. At the indicated cycles, 10-pl samples were removed and electrophoresed on a 1% agarose gel. The products were transferred onto a nylon membrane (BioRad Zeta-Probe GT), dried and exposed to X-ray film.

GTCGAACC-3’; 5‘-CAGCGCCACATACATCAT-3’. PCR

CAAGGCTGT; 5‘-GCCATGAGGTCCACCAC-3’. PCR

RESULTS Analysis by Western blot and imrnunoprectpitation

The expression of elF-4E in MCF-7, MDA-435, MDA231, MCF-1OA and JA cells was determined by SDS/PAGE, followed by Western blotting. Extracts from each cell line were prepared as described above, and 50 pg of each sample were separated on a 15% polyacrylamide gel (Fig. la). Equal protein loading and transfer were further confirmed by prob- ing a parallel blot for p-actin. This analysis showed that the expression of elF-4E was = 10-fold higher in cancer-cell lines than in either MCF-1OA or JA cells. To confirm this, we first immunoprecipitated elF-4E from 500 pg of cell extract (a 10-fold excess) from MCF-1OA and JA cells and then visual- izcd thc irnmunoprecipitated product by Western blotting (lanes 7, 8). This gave a signal comparable to that from the carcinoma lines (lanes 4-6). This elevation in elF-4E is similar to that which we had previously reported for breast-tumor biopsies (8), suggesting that the elevation in elF-4E expression is retained in cultured cancer cells.

It seemed possible that the elevated elF-4E level could be due to slower turnover rates, i.e., stabilization of the protein. Such a situation is encountered, for example, with c-Myc in some plasmacytomas and lymphomas (Cole, 1986). To deter- mine this, we labeled MCF-1OA and MDA231 cells with [35S]methionine for 3 hr. We then washed the cells with unlabeled medium and followed by immunoprecipitation the loss of labeled elF-4E during the chase (Fig. 16). The result was that the rate of elF-4E turnover was similar for the control and the carcinoma cells (Fig. lc), although the initial amount of labeled elF-4E was higher in MDA231 cells, as expected. This implies that the rate of elF-4E decay is not reduced in carcinomas. Examination of the turnover rates indicated that the half-life of elF-4E was approximately 4.5 hr, indicating that elF-4E is not very stable in rapidly dividing cells.

Analysis of elF-4E mRNA To establish that the elevation in elF-4E was also found at

the mRNA level, we isolated RNA from the different cells and analyzed it by Northern blotting (Fig. 2). All the carcinoma cell lines had significantly increased levels of elF-4E mRNA, with the exception of SKBR-3, which had approximately the same level as JA and MCF-1OA. Several autoradiographic exposures of the blot indicated that the level of elF-4E mRNA was elevated roughly 5 to 10-fold in the carcinoma lines in comparison to controls.

In order to obtain a more precise estimate of the level of elF-4E mRNA, we used a quantificative RT/PCR method. First-strand DNA synthesis was generated with random prim- ers, and PCR amplification with either elF-4E or GAPDH specific primers was carried out for the indicatcd number of cycles. We had previously shown that this method yields accurate results (comparable to RNase protection), provided

-28s

LL t 1 8 S aa -

1 2 3 4 5 6 7

FIGURE 2 -Analysis of elF-4E mRNA by Northern blot. The level of elF-4E and GAPDH mRNA was determined for the indicated cell lines, as described in the text. Equal sample loading was also confirmed qualitatively by visual inspection of the 28s and 185 rRNA bands, stained with EtBr.

elF-4E IN BREAST CANCER 861

that samples are taken in the exponential range of amplifica- tion and prior to product saturation (De Benedetti et al., 1991). To better quantify the results [CX-~~S]~CTP was added as a tracer, in the event that different exposures of the films might be necessary. Typical results from these experiments are shown in Figure 3. In JA and MCF-1OA cells, the specific elF-4E PCR product became detectable at cycle 26 and did not quite reach its maximal level at cycle 32. In contrast, elF-4E was clearly detectable at cycle 24 in all 3 cancer-cell lines, and even by cycle 22 in MCF-7. Additionally, the elF-4E product was nearly saturated by cycle 32 for the breast-cancer lines; this is best shown for MCF-7 (compare cycles 30 and 32). All samples gave approximately the same signal (product satura- tion) by cycle 36 (not shown). The blots were analyzed with a densitometer and plotted on a log scale over the number of cycles. This analysis indicated that the level of elF-4E mRNA is 7-, 5-, and 11-fold higher in MDA-435, MDA-231, and MCF-7 cells, respectively, than in control cells. The identity of the elF-4E PCR product was also confirmed by Southern blotting, using a Ddel fragment from elF-4E cDNA as a probe (data not shown). To confirm that the PCR amplification was performed with equivalent amounts of reverse-transcribed samples, we repeated the analysis in panel A with primers specific for GAPDH. In this case, the accumulation of GAPDH product was comparable in all samples and was clearly detect- able by cycle 20 (Fig. 3b), indicating that the representation of GAPDH mRNA is equivalent and that the increase in elF-4E mRNA levels in the cancer lines is specific.

Turnover rates of elF-4E mRNA To establish whether the increased elF-4E mRNA was due

to increased synthesis (transcription) or to slower turnover, we examined its stability during an incubation in the presence of the transcription inhibitor Actinomycin D. For simplicity, we chose a PCR end-point of 26 cycles, which we had previously shown to be within the exponential range of amplification and which gives sufficient signal for each cell line. The decay rates of elF-4E mRNA were determined for MCF-lOA, MDA-435, MCF-7 and MDA-231 (Fig. 4). The t = 0 level of elF-4E mRNA was lower for MCF-IOA cells (as expected), but the elF-4E mRNA appeared to be relatively stable, and no significant differences in turnover rates were observed in any of the cell lines.

DISCUSSION

We recently reported that elF-4E is elevated in breast carcinomas, but not in benign tumors (ie., fibrocystic hyperpla- sia), indicating that the elevation in elF-4E could represent an important and necessary event in cancer progression. Unfortu- nately, tissue from biopsies provides a poor model for studying the changes and mechanism(s) that lead to the elevation in elF-4E. Breast-cancer cell lines offer a more practical model. Before the present study, however, it was not clear that the elevation in elF-4E found in vivo would be retained in cell lines. One possibility was that the elevated elF-4E found in cancer biopsies could be a reversible occurrence, dictated by

FIGURE 3 -Analysis by RT/PCR. The expression of elF-4E panel a ) and GAPDH (panel b) was determined by quantificative RT/PCR, by removing samples at the indicated number of cycles, I ollowed by analysis on agarose gels. After transfer to a membrane, the blots were all exposed to X-ray film for the same amount of time (20 hr).

FIGURE 4 -Turnover rates of elF-4E mRNA. Decay rates of elF-4E mRNA were determined during a course of incubation with Actinomycin D. At the indicated times, RNA samples were prepared and analyzed by RT/PCR at cycle 26 of amplification. The samples from MCF-1OA cells were exposed for 3 times longer to obtain a comparable signal.

862 ANTHONY ETAL.

TABLE I - CELL GENERATION TIMES

Cell name Doubling time (hr)’

MDA-231 31 t 3 MDA-435 MCF-7 MCF-1OA

33 2 5 38 5 7 40 5 10

JA2 36 f 10

‘Cell doubling times were estimated by plating lo4 cells in 25-cm2 flasks with a gridded bottom. During the next 10 days, the cells were counted and the average from 5 squares was determined from triplicate flasks.-*The JA cells were only capable of limited cell division (8-10 doublings) and the results reflect only the first 4 divisions.

the competitive growth conditions in situ. The concern was that breast-tumor cells grown in rich culture medium with plentiful growth factors would no longer need to maintain elevated elF-4E for adequate proliferation. The reverse scenario was also possible, i.e., that “normal” breast cells grown in tissue culture, and potentially immortalized, would up-regulate the expression of elF-4E. Instead, we found that the expression of elF-4E in carcinoma cell lines was similar to that encountered with breast specimens: a 5- to 10-fold elevation in elF-4E. The reason for the elevation in elF-4E is almost certainly transcrip- tional, as its mRNA is also correspondingly elevated. This is also consistent with studies in which the transcriptional regula- tion of elF-4E was examined by run-on assays (Rosenwald et al., 1993b). Furthermore, decreased turnover of elF-4E mRNA in the different lines was ruled out by the Actinomycin D chase, which showed that the elF-4E mRNA does not apprecia- bly decay in 8 hr.

In contrast to the relative stability of elF-4E mRNA, the turnover of the elF-4E protein appears to proceed at a significant rate. This may seem to contrast the reported stability of elF-4E in reticulocytes and peripheral leukocytes (Rychlik et al., 1990). However, these are non-proliferating and essentially terminally differentiated cells, which must nevertheless retain their capacity for protein synthesis. The situation appears to be quite different with dividing cells, and this may have important implications for growth regulation. This work investigates the pattern of elF-4E expression in matched cell lines: normal vs. cancer. Furthermore, all the cells used for this study were derived from a natural source, either normal breast epithelium, or breast biopsies-they were not immortalized or transformed with an introduced onco- gene. Therefore, our finding that the elevation of elF-4E is retained in these carcinoma lines is likely to be relevant to a great number of cases.

We should also stress that the elevated elF-4E level in breast cancer is not a straightforward reflection of increased prolifera- tion rates. In culture, the doubling time of the cells used in this study is quite comparable (see Table I). For MDA-231 and

MDA-435 the generation time is about 30 hr; for MCF-7, MCF-1OA and JA it is about 40 hr. This argues against the notion that the elevation in elF-4E might simply be a marker for increased proliferative rate. Instead, we believe that the elevation of elF-4E is a permanently acquired change reflect- ing the malignant nature of the cells. While this may seem less intuitive, it is consistent with what we know about elF-4E overexpression in model cell lines. The overall spectrum of protein synthesis is not changed by overexpression of elF-4E; only the expression of a few proteins becomes substantially increased (De Benedetti et al., 1994; Kevil et al., 1995). However, when several of these proteins were identified, they turned out to be factors that most directly affect malignancy (Graff et al., 1995; Shants and Pegg, 1994; Kevil et al., 1996). For instance, the large increase in FGF-2 and VPF synthesis could drastically increase neoangiogenesis in vivo, which is essential for tumor vascularization and growth beyond 1 mm.

Most studies thus far, have focused on the activity, rather than the expression, of elF-4E. While the activity of elF-4E is modulated by phosphorylation, which changes either in re- sponse to mitogenic activation or during the cell cycle (Rhoads, 1993; Sonenberg, 1993), its level must also be important for the regulation of cell growth. Significantly, a relatively modest increase in elF-4E (8- to 20-fold) is sufficient for full oncogenic transformation of cultured cells (Lazaris-Karatzas et al., 1990; D e Benedetti et al., 1994). This elevation is typically found in breast carcinomas in vivo (Kerekatte et al., 1995) and in tissue culture (this study). It is not known at present if most cancer-cell lines display an elevation in elF-4E, and whether this is essential for tumor development in vivo. However, we know that in one tumorigenic cell line obtained by transforma- tion with oncogenic Ras (CREF/T24), the level of elF-4E is not elevated in comparison to that of the parental normal cells (Rinker-Schaeffer et al., 1992). Thus, it would seem that the elevation in elF-4E may not be common to all types of malignant cells. However, we should also point out that these cells do not necessarily constitute a contradicting example, since activated Ras drastically up-regulates the phosphoryla- tion of elF-4E (Rinker-Schaeffer et al., 1992; Lazaris-Karatzas et al., 1992). This could bypass the need to upregulate the expression of elF-4E in this aggressive, tumorigenic cell line. The present study also showed that, in the carcinoma line SKBR-3, the level of elF-4E mRNA (and protein, not shown) was not significantly elevated in comparison to that of normal cells. However, this cell line is hypo-tetraploid and underwent substantial genome rearrangements, which may not be typical of most common breast carcinomas, prior to surgical isolation.

ACKNOWLEDGEMENTS

This work was supported by Stiles and BRSG institutional grants (American Cancer Society, through LSUMC), and by LEQSF grant RD-A-43.

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