Differentiation (2003) 71:126–134 C Blackwell Verlag 2003

O R I G I N A L A R T I C L E

Derek Walsh ¡ Paula Meleady ¡ Brendan PowerSimon J. Morley ¡ Martin Clynes

Increased levels of the translation initiation factor eIF4E indifferentiating epithelial lung tumor cell lines

Accepted in revised form: 2 January 2003

Abstract Rates of eukaryotic protein synthesis and pro-liferation are dependent upon the availability of eIF4F,the cap-binding translation initiation complex thatguides the ribosome onto the mRNA. One possible rate-limiting factor in eIF4F complex formation is the avail-ability of eIF4E, which interacts specifically with themRNA cap structure. As such, it has a potential role inthe selective translation of growth-related mRNAs, withoverexpression of eIF4E resulting in aberrant cellgrowth and transformation. A number of studies sug-gest that eIF4E may play a role in cellular differentiationas well as proliferation. We have previously reported thatpost-transcriptional regulation is involved in the induc-tion of keratins in epithelial lung tumor cell lines ex-posed to the differentiation-modulating agent, bromo-deoxyuridine (BrdU). Here, we demonstrate that theseBrdU-treated lung cells express elevated levels of eIF4Eprotein and enhanced phosphorylation of eIF4E. Over-expression of eIF4E by cDNA transfection in the poorlydifferentiated, keratin-negative human lung cell line,DLKP, was found to promote a flattened, more epi-thelial appearance to these cells, coupled with the induc-tion of simple keratins (keratins 8 and 18). In contrast,levels of eIF4E expression were found to decrease duringBrdU-induced differentiation of the leukemic cell line,HL-60, suggesting that there are cell-type differences inthe response to BrdU and in the requirement for eIF4Eduring differentiation.

D. Walsh* ¡ P. Meleady* (✉ ) ¡ B. Power ¡ M. ClynesNational Cell and Tissue Culture Centre/National Institute forCellular Biotechnology, Dublin City University, Glasnevin,Dublin 9, IrelandTel: π35 31 700 5700Fax: π35 31 700 5484e-mail: Paula.Meleady/dcu.ie

Simon J. MorleyBiochemistry, School of Biological Sciences, University of Sussex,Falmer, Brighton BN1 9QG, UK

U.S. Copyright Clearance Center Code Statement: 0301–4681/2003/7102–126 $ 15.00/0

Key words eukaryotic initiation factor 4E ¡ epithelialcell differentiation ¡ keratins 8 and 18 ¡5-bromodeoxyuridine

Introduction

The availability of the cap-binding initiation complexeIF4F plays a major role in regulating eukaryotic pro-tein synthesis rates (Morley et al., 1997; Gingras et al.,1999; Raught et al., 2000). Consisting of three majorpolypeptide subunits (eIF4E, eIF4G, and eIF4A), theeIF4F complex guides the ribosome onto the 5ø end ofthe mRNA. eIF4E specifically recognises the 7-methyl-Gppp cap structure at the 5ø end of eukaryotic mRNAsand may be an important rate-limiting factor in trans-lation initiation. The mRNA cap-structure interactswith eIF4E via a hydrophobic pocket in the concave in-ner surface of eIF4E (Marcotrigiano et al., 1997). eIF4Gis a 220 kDa polypeptide that acts as a ‘‘scaffold’’ forthe formation of the eIF4F complex at the 5ø mRNAcap structure and plays a central role in pre-initiationcomplex assembly (Morley et al., 1997; Gingras et al.,1999).

The activity of eIF4E can be regulated by both itsphosphorylation and availability to participate in theinitiation process, mediated by its interaction with theeIF4E binding proteins, 4E-BP1, 4E-BP2, and 4E-BP3(Morley, 1996; Gingras et al., 1999; Raught et al., 2000).In resting mammalian cells, 4E-BPs are hypo-phos-phorylated and bound to eIF4E. Stimulation of cellswith mitogens increases the phosphorylation of theseeIF4E binding proteins, liberating eIF4E to interact

*both these authors contributed equally to this work

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with a conserved hydrophobic region of eIF4G (Gingraset al., 1999; Raught et al., 2000). eIF4E is also a phos-phoprotein, and increased levels of eIF4E phosphoryla-tion and its association with eIF4G have been directlycorrelated with the enhancement of translation whichfollows mitogenic stimulation of mammalian cells (Mor-ley, 1996; Gingras et al., 1999; Raught et al., 2000).Phosphorylation of eIF4E at Ser-209 occurs as part ofthe eIF4F complex (Tauzon et al., 1990), greatly en-hancing and stabilising its association with the capstructure (Minich et al., 1994; Joshi et al., 1995).Multiple signal transduction pathways control the phos-phorylation of eIF4E, mediated via activation of theextracellular-regulated kinases (ERKs) and the p38 fam-ily of mitogen-activated protein (MAP) kinases (Morley,1996; Gingras et al., 1999; Raught et al., 2000). MAPkinase-activated protein kinase, Mnk-1, is able to phos-phorylate eIF4E in vitro at the physiological site (Gingr-as et al., 1999; Raught et al., 2000; Waskiewicz et al.,1997; Wang et al., 1998).

However, there is little work addressing the possiblerole for eIF4E in regulating protein synthesis during cel-lular differentiation. Phosphorylation of eIF4E may in-fluence patterns of differentiation in rat PC12 cellstreated with nerve growth factor (NGF) (Fredericksonet al., 1992), and evidence suggests that the 4E-BPs mayplay a role in regulating eIF4E activity during humanthymocyte maturation (Beretta et al., 1998). Micro-in-jection of mRNA encoding eIF4E into Xenopus laevisembryos has been found to induce mesoderm formation(Klein and Melton, 1994). We have previously describedthe post-transcriptional induction of keratin filamentformation in epithelial lung tumor cell lines exposed tothe differentiating agent, 5ø-bromodeoxyuridine (BrdU)(McBride et al., 1999; Meleady and Clynes, 2001a).One of these cell lines, DLKP, is an extremely poorlydifferentiated carcinoma cell line which does not ex-press keratin proteins (or other epithelial markers suchas epithelial-specific antigen or desmosomal proteins)despite being of epithelial origin (McBride et al.,1998). DLKP cells may represent a stem cell-like cellline of lung epithelial lineage which has the potentialfor both proliferation and differentiation. In the ma-ture lung, no distinct stem cell lineage has so far beenidentified, but at least three different cell types, Clara,Type II pneumocytes and Basal cells, are capable ofnon-terminal division and differentiation. DLKP cellsappear to be at a very early stage of differentiation,shown by the lack of expression of the simple epi-thelial keratins 8 and 18, and can theoretically progresstowards several different phenotypes. BrdU has beenfound to modulate differentiation in a number of dif-ferent cell types (Feyles et al., 1991; Thomas et al.,1993). Here, we show the increased expression andphosphorylation of eIF4E in these systems and suggesta possible role for eIF4E in regulating epithelial celldifferentiation.

Methods

Cell culture

The very poorly differentiated DLKP cell line was established inour laboratory (Law et al., 1992) and expresses many features typi-cal of SCLC-V (variant small cell lung carcinoma) or NSCLC-NE(non-small cell lung carcinoma with neuroendocrine differen-tiation) (McBride et al., 1998). DLKP cells, despite being epithelialin origin, contain no detectable levels of keratin proteins. A549, anadherent human adenocarcinoma cell line, and HL-60, a humanpromyelocytic cell line, were obtained from the American Type Cul-ture Collection. DLKP and A549 cells were cultured in a 1:1 mix-ture of DMEM:Hams F12 (Sigma) supplemented with 5% foetalcalf serum (Sigma) and 2 mM L-glutamine (Life Technologies) insealed tissue culture flasks (Costar) at 37æC. HL-60 cells were cul-tured in RPMI 1640 medium (Life Technologies) supplementedwith 10% foetal calf serum and 2 mM L-glutamine and maintainedin vented flasks (Costar) at 37æC, 5 % CO2. Cells were routinelygrown in antibiotic-free conditions and tested for mycoplasma andalways tested negative.

Immunocytochemical analysis

10 mM BrdU (Sigma) stock solutions were prepared in sterile UHPwater and stored at ª20æC. Cells were seeded at densities of 1¿103 cells per well on sterile multi-well slides (Life Technologies) andincubated for 24 h at 37æC, 5 % CO2. Appropriate dilutions ofBrdU were made in normal growth medium and added to the cellsat a final concentration of 10 mM. Cells were incubated at 37æC, 5% CO2 for 7 days, during which time the medium was replacedevery 3 to 4 days.

Immunocytochemistry was performed using the Vector Red sub-strate system (Vector Laboratories, UK), as described previously(McBride et al., 1999). The primary antibody used was the mouseanti-human monoclonal antibody to eIF4E (Becton Dickinson,clone .87). Staining was visualised under normal light. Low-levelexpression was visualised by excitation of the fluorescent VectorRed substrate through rhodamine filters.

Immunoblot analysis

For both A549 and DLKP, cells were harvested by trypsinisationand washed in ice-cold PBS. Trypsinisation was not necessary forHL-60, which grow in suspension. Equal cell numbers (2¿104 cellsper sample) or equal amounts of total protein (15 mg per sample)were prepared in SDS-PAGE sample buffer (McBride et al., 1999),and proteins were resolved on SDS-polyacrylamide gels. Proteinswere transferred onto nitrocellulose membranes (Amersham Bios-ciences) and blocked for 4 h at room temperature with 5% semi-skimmed milk in TBS/0.5% Tween 20 (Sigma). Membranes wereprobed overnight with the following antibodies: anti-eIF4E(Becton Dickinson, clone .87) or rabbit polyclonal anti-eIF4E(Morley and McKendrick, 1997); rabbit polyclonal anti-eIF4G(Fraser et al., 1999); anti-cytokeratin 8 (Sigma, clone .M-20); oranti-cytokeratin 18 (Sigma, clone .CY-90). Blots were then washed,probed with peroxidase-labelled secondary antibody (Sigma) for 1h and visualised by the enhanced chemiluminescence (ECL)method (Amersham Biosciences) and autoradiography.

Vertical slab iso-electric focusing (V-IEF)

To separate the phosphorylated and non-phosphorylated forms ofeIF4E, a modified vertical slab iso-electric focusing technique wasused, essentially as previously described (Morley and Pain, 1995;Morley and McKendrick, 1997; Fraser et al., 1999). Resolved

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forms of eIF4E were transferred to nitrocellulose and detected byincubation with anti-eIF4E antibody, as described above.

RNA isolation and Northern blot analyses

Cells were grown in 175 cm2 tissue culture flasks (Nunc) andtreated up to 14 days with 10 mM BrdU. Cells were harvested atvarious time points and total RNA was isolated by extraction withTRI REAGENTTM (Sigma) according to manufacturer’s instruc-tions. 20 mg RNA was separated by electrophoresis on 1% agarose-formaldehyde gels in MOPS buffer (Sigma) and transferred ontoHybond-N membranes (Amersham Biosciences). Equal loading ofgels was confirmed by ethidium bromide staining of ribosomalRNA bands on gel. Filters were pre-hybrised for 3 h and hybridisedwith labelled probe at 65 æC for 16–18 h in a buffer containing 0.43M sodium phosphate (pH 7.2), 6% SDS, 1% BSA and 0.02 MEDTA. The eIF4E cDNA probe was 32P-labelled with a randomprimer labelling kit (Promega). Filters were washed once in 2¿SSC/0.1% SDS (5 min), twice in 0.5¿SSC/0.1% SDS (15 min),and twice in 0.1¿SSC/0.1% SDS (15 min) at 65 æC. The humaneIF4E probe was designed in our laboratory by Dr. Noel Daly.

Transfection of cells

DLKP cells were stably transfected with a number of different hu-man eIF4E cDNAs; BK-eIF4E in a BK shuttle vector was a kindgift from Prof. Arrigo DeBenedetti, Louisiana, USA (DeBenedettiand Rhoads, 1990); pcDNA-eIF4E and it’s mutants, pcDNA-eIF4E-S209 and pcDNA-eIF-4E-S209/T210, were kind gifts fromDr. Robert Schneider, New York, USA (Cuesta et al., 2000). ThepcDNA-eIF4E vectors code for a fusion protein between eIF4Eand hemagglutin (HA epitope tag); this protein appears function-ally equivalent to eIF4E (Pyronnet et al., 1999; Cuesta et al., 2000).

On the day prior to transfections, cells were plated from a singlecell suspension into 25 cm2 flasks at 3¿105 cells per flask. On theday of transfection, cDNAs were prepared along with the Lipofec-tinTM (Life Technologies) transfection reagent, according to manu-facturer’s instructions. Cells were incubated with this mixture for 4h in the absence of serum after which the media was supplementedwith 10% FCS, followed by an overnight incubation at 37æC; thefollowing day, cells were re-fed with fresh serum-containing me-dium. Selection with 400 mg/ml geneticin (G418) (Promega) wasinitiated 24 hours later, with the concentration increased to 800 mg/ml over the following 3–4 days. The medium (with selection) waschanged every 4–5 days until large colonies had formed in theflasks (or they had reached about 70% confluency). The G418-resistant cells were then trypsinised and cloned out using limitingdilution. Individual colonies were expanded and the resultant stableclones were routinely maintained in selection medium (i.e. contain-ing 800 mg/ml G418).

Results

Induction of eIF4E protein expression in BrdU-treatedlung epithelial cells

We have previously found that exposure of two humanepithelial lung cancer cell lines (DLKP and A549) toBrdU significantly altered the morphology of the cells,with most of the cells acquiring a stretched, flattened ap-pearance (McBride et al., 1999). Associated with thismorphological change was a significant up-regulation ofa2b1 integrin protein expression, with treated cells adher-

ing significantly faster to ECM proteins (Meleady andClynes, 2001b). Keratins 8, 18, and 19 were also selectivelyinduced in BrdU-treated cells, in a process regulated at thepost-transcriptional level (McBride et al., 1999; Meleadyand Clynes, 2001a).

In order to try to investigate the mechanism of in-creased keratin protein expression levels during BrdU-in-duced differentiation of these cells, we have investigatedwhether BrdU influences the expression of translationfactors in these cells. Total protein cell lysates were pre-pared from untreated cells or from cells treated with BrdUfor various times, and the level of expression of eIF4E wasmonitored by Western blotting. Due to the significant in-crease in cell size when treated with BrdU, Western blot-ting was performed using two scenarios, loading by equalamounts of protein (i.e. 15 mg per sample) and equal cellnumbers (i.e. 2 ¿ 104 cells per sample). There was a sig-nificant increase in eIF4E protein levels after 4 – 6 days oftreatment with BrdU in DLKP cells (Fig. 1A and Fig.1C). Analysis of A549 cells showed that the level of ex-pression of eIF4E also increased over 6 days of BrdUtreatment (Fig. 1B). In contrast, analysis of eIF4G byWestern blotting revealed that expression of eIF4G pro-tein levels showed no significant alteration during BrdU-induced differentiation of DLKP and A549 cells, sug-

Fig. 1 BrdU treatment increases the expression of eIF4E but doesnot influence the expression of eIF4G in DLKP and A549 cells.(A) DLKP cells were grown in the absence or presence of 10 mMBrdU for the times indicated. Extracts were prepared, and equalvolumes corresponding to 2¿104 cells from each culture wereresolved by SDS-PAGE (10% polyacrylamide gels) followed byimmunoblotting with anti-eIF4E (clone .87) and anti-eIF4Gantibodies as described in Methods. (B) A549 cells were grownin the absence or presence of 10 mM BrdU for the times indicatedand equal volumes corresponding to 2¿104 cells from eachculture were resolved by SDS-PAGE and processed as in A. (C)Loading by equal volumes of total protein (i.e. corresponding to15 mg) in DLKP cells revealed a similar increase in eIF4E proteinlevels following exposure to BrdU for 8 days.

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gesting that the effects of BrdU are specific to a subset ofproteins important to the differentiation process. b-actinlevels are increased upon exposure to BrdU (not shown)and so cannot be used as an internal control. Increasedactin levels have been previously found in BrdU-treatedB16 melanoma cells (Gomez et al., 1995). The lack of sig-nificant alteration in eIF4G levels also acts as an internalcontrol for protein loading and excludes the possibility ofnon-specific effects of BrdU increasing levels of proteinsglobally.

Increased phosphorylation of eIF4E in BrdU-treatedlung epithelial cells

Vertical slab iso-electric focussing (V-IEF) was per-formed in order to investigate possible changes in thephosphorylation status of eIF4E in BrdU-treated cells.Treatment of DLKP cells (Fig. 2A) or A549 cells (Fig.2B) with BrdU resulted in increased levels of expressionand enhanced phosphorylation of eIF4E. With both cell

Fig. 2 Exposure to BrdU enhances the phosphorylation of eIF4Ein DLKP and A549 cells but decreases expression of eIF4Eprotein and phosphorylation levels in HL-60 leukemic cells. (A)DLKP cells were grown in the absence or presence of 10 mM BrdUfor the times indicated. Extracts were prepared and equal volumescorresponding to 4 ¿104 cells from each culture were resolved byV-IEF followed by immunoblotting with rabbit anti-eIF4Eantiserum (this anti-serum detects non-phosphorylated andphosphorylated eIF4E), as described in Methods. (B) A549 cellswere grown in the absence or presence of 10 mM BrdU for thetimes indicated and processed exactly as in (A). Rabbitreticulocyte (RR) from in vitro translation extracts serves as amarker for eIF4E. (C) HL-60 cells were grown in the absence orpresence of 10 mM BrdU for the times indicated. Extracts wereprepared and processed exactly as in (A). The phosphorylatedvariants of the protein are indicated on the right (P-4E).

lines, the increased phoshorylation of eIF4E was par-ticularly evident after 2 days of BrdU treatment forDLKP and 5 days of treatment for A549.

eIF4E expression is reduced in BrdU-treated HL-60leukaemic cells

In order to investigate if exposure of cell lines to BrdUresults in a general increase in eIF4E expression, we haveused the non-epithelial, leukaemic cell line, HL-60. InHL-60 cells, BrdU induces myeloid differentiation (Ko-effler et al., 1983; Yen and Forbes, 1990). eIF4E proteinlevels have been found to decrease during phorbol ester-induced myeloid differentiation (Johnston et al., 1998).In contrast to the human epithelial lung cancer cell lines,exposure of HL-60 cells to BrdU resulted in dramaticdown-regulation of eIF4E protein expression, evidentwithin 3 days of treatment (Fig. 2C).

Fig. 3 Exposure to BrdU does not significantly alter the levels ofeIF4E mRNA in DLKP and A549 cells. (A) DLKP and (B)A549 cells were grown in the absence or presence of 10 mM BrdUfor the times indicated. 20 mg of total RNA were loaded per laneand separated by agarose gel electrophoresis, and filters werehybridised with human eIF4E cDNA, as described in Methods.Equal loading of gels was confirmed by ethidium bromidestaining of ribosomal RNA bands on the gels.

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Exposure to BrdU does not significantly alter the levelsof eIF4E mRNA in DLKP and A549 cells

Northern blot analyses of eIF4E mRNA levels in bothDLKP and A549 cells after exposure to 10 mM BrdUfor 7 days indicates no significant change in RNA levelsover the times indicated (Fig. 3). eIF4E has previouslybeen found to be regulated at multiple levels (Raughtand Gingras, 1999).

Overexpression of eIF4E in DLKP lung epithelial cellsinduces keratin 8 and 18 expression

In order to investigate the possible correlation between in-creased eIF4E expression and induction of keratin fila-

Fig. 4 Overexpression of eIF4E alters morphology of DLKPcells. (A) Parental, untransfected DLKP cells were grown in multi-well slides for 48 h; then fixed, permeabilised, and incubated withanti-eIF4E antibody (clone .87). eIF4E was visualised using theVector Red substrate system, as described in Methods. (B) DLKP,stably transfected with the human eIF4E cDNA in a BK shuttlevector (DeBenedetti and Rhoads, 1990), were grown in thepresence of 800 mg/ml G418. Cells were fixed and eIF4E wasvisualised as in (A). Note that increased expression of eIF4Eresults in a flattened, increased volume cell population relativeto untransfected cells. Cells transfected with the empty vectorshowed no change in morphology (data not shown). Bothphotographs were taken at the same magnification (¿20).

ment formation during BrdU-induced differentiation, anumber of different human eIF4E cDNAs were transfect-ed into DLKP cells. As shown in Fig. 4, DLKP cells trans-fected with human eIF4E in a BK shuttle vector (DeBene-detti and Rhoads, 1990) (BK-4E) show increased levels ofeIF4E expression relative to untransfected cells (Fig. 4Bvs. 4A). Subsequent immunoblot analysis showed thatoverexpression of eIF4E in two stable clones BK-4E C1and BK-4E C2 (Fig. 5C) was accompanied by increasedprotein levels of the simple epithelial partner keratins K8(Fig. 5A) and K18 (Fig. 5B). Similarly, DLKP cells trans-fected with a second eIF4E cDNA vector (Cuesta et al.,2000) showed that overexpression of eIF4E in two stableclones, 4E-C1 and 4E-C2, (Fig. 6A; lanes 2 and 3 vs. lane1) was accompanied by increased levels of keratin 8 and18 (Fig. 6B and 6C; lanes 2 and 3 vs. lane 1). In contrast,DLKP cells transfected with eIF4E constructs containingmutations at either Ser209 (clone 4E-S209) or Ser209/Thr210 (clone 4E-S209/T210) did not show increased levelsof keratin 8 or 18 protein (Fig. 6B and 6C; lanes 4 and 5),compared to wild-type transfected eIF4E clones 4E-C1and 4E-C2 (Fig. 6B and 6C, lanes 2 and 3) even thoughthe levels of eIF4E had increased similarly to the wild-type transfected cells (Fig. 6A; lanes 4 and 5 vs. lanes 2and 3). In all four stable clonal cell lines, the levels ofeIF4E protein had increased significantly compared to theuntransfected cell line DLKP (Fig. 6A), which are kera-

Fig. 5 Overexpression of eIF4E in DLKP cells induces expressionof keratin 8 and 18 protein. Extracts from untransfected(DLKP) and two transfected stable clones (BK-4E C1 and BK-4E C2, derived from stable transfection of human eIF4E cDNAinto DLKP cells) were prepared as described in Methods, andequal volumes corresponding to 20 mg of total protein from eachculture were resolved by SDS-PAGE (12% polyacrylamide gels)followed by immunoblotting with antibodies against the followingproteins. Induction of keratin 8 (K8) (A) and keratin 18 (K18) (B)was demonstrated in transfected cells. (C) Overexpression of eIF4Ein transfected cells was confirmed by anti-eIF4E anti-serum.(Note that this anti-serum detects non-phosphorylated andphosphorylated eIF4E that may explain the doublet in the eIF4Econtrol and transfectant clones).

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Fig. 6 Overexpression of eIF4E, and not mutant eIF4E, induceskeratin 8 and 18 protein expression in DLKP cells. Extracts fromDLKP cells stably transfected with pcDNA-eIF4E (two clones,4E-C1 and 4E-C2) and its mutants, pcDNA-eIF4E-Ser209 (clone4E-S209) and pcDNA-eIF4E -Ser209/Thr210 (clone 4E-S209/T210), were prepared as described in Methods. Equal volumes ofprotein corresponding to 20 mg of total protein were resolved bySDS-PAGE (12% polyacrylamide gels) followed byimmunoblotting with antibodies against the following proteins:(A) eIF4E (clone .87), (B) keratin 8, and (C) keratin 18.Induction of keratins 8 and 18 was found only in the wild-typeeIF4E overexpressing DLKP clones (4E-C1 and 4E-C2) and notin mutant eIF4E overexpressing DLKP transfectant clones (4E-S209 and 4E-S209/T210).

tin-negative. This suggests that ‘active’ eIF4E may be re-quired for keratin induction in this cell line model. It alsorules out the possibility of geneticin (G418) selection play-ing a role in this increased keratin expression in transfect-ed cell. Similarly, control empty vector transfectionsshowed no eIF4E overexpression and increased expres-sion of keratin 8 or 18 protein was not detected (notshown).

Discussion

We have used BrdU to study the differentiation of epi-thelial lung cancer cell lines in culture, where BrdU (abromine-substituted thymidine analogue) competes withnaturally occurring thymidine for incorporation intoDNA. The induction or inhibition of differentiation byBrdU depends on the particular cell type, inducing dif-ferentiation in human SCLC (Feyles et al., 1991), HL-60 cells (Koeffler et al., 1983; Yen and Forbes, 1990) andneuroblastoma (Ross et al., 1995), while causing de-dif-ferentiation in human melanomas (Thomas et al., 1993)and chondrocytes (Farrell and Leukens, 1995). It alsoselectively modulates the expression of a class of regula-tory genes, each member of which is important for thedevelopment of a different cell lineage, either throughaltered promoter structure or increased/decreased affin-ity of transcriptional regulatory factors for substitutedDNA. For example, BrdU suppresses the transcriptionof the myogenic determination gene, MyoD1 (Tapscottet al., 1989) and may also modulate a regulatory gene

that affects tyrosinase activity in melanogenesis (Rauthand Davidson, 1993).

Exposure of the human lung epithelial cell linesDLKP and A549 to BrdU resulted in significantly in-creased levels of eIF4E protein but had little or no effecton eIF4G protein levels. Analysis of eIF4E mRNAshowed no significant alterations in the levels of eIF4ERNA following exposure to BrdU. eIF4E has beenfound to be regulated at multiple levels (Raught andGingras, 1999). We are currently investigating the regu-lation of eIF4E mRNA levels and stability of eIF4E pro-tein following exposure to BrdU in our cell-line models.Treatment with BrdU also resulted in an increase inphosphorylated eIF4E in both epithelial lines studied.Initially, cells showed a predominance of the non-phos-phorylated form of eIF4E; however, upon prolonged ex-posure to BrdU, the increase in eIF4E protein levels wasaccompanied by a shift towards the more phosphoryl-ated form of the protein. As these cells differentiate inresponse to BrdU, they also exhibit changes in integrinexpression (Meleady and Clynes, 2001b). Integrins areimportant attachment factor receptors and intra-cellularsignalling factors that can affect activation, for example,of the ERK MAP kinase cascade (Schlaepfer andHunter, 1998). ERK is known to stimulate the activityof Mnk1, possibly resulting in increased cap-binding af-finity of eIF4E through phosphorylation on Ser-209(Waskiewicz et al., 1997). Therefore, increases in botheIF4E protein and phosphorylation levels upon treat-ment with BrdU may influence the expression of keratins8 and 18 at the protein level. However, the role of phos-phorylation of eIF4E in translational control is the sub-ject of much debate at present, with a number of seem-ingly contradictory reports appearing in the literatureover the last couple of years (Scheper and Proud, 2002).Recent reports have suggested that the phosphorylationof eIF4E is not required for de novo protein synthesis inrecovery from cell stress (Morley and Naegele, 2002) butis required, for example, for cellular growth of Droso-phila (Lachance et al., 2002) and maturation of bovineoocytes (Tomek et al., 2002). This suggests that the func-tion of eIF4E phosphorylation is very poorly under-stood at present. Perhaps the role of phosphorylation ofeIF4E may be specific to a cellular process, such asgrowth control, development and differentiation, cellu-lar stress, apopotosis, etc. During foetal development,keratins are the first intermediate filament proteins de-tectable, with the simple epithelial keratins K8 and K18appearing first (Franke et al., 1982). Epithelial cells pos-sess specific keratin profiles characteristic of their celltype, stage of development, disease state, and degree ofdifferentiation. Gene sequence analysis indicates thatthe more specialised keratins, which are expressed atlater stages of development and differentiation, probablyevolved from K8 and K18 (Blumenberg, 1988; Stoler etal., 1988). Induction of keratins 8 and 18 may be indica-

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tive of simple epithelial differentiation in the DLKP cellline.

The 4E-BPs have been shown to play a role in regulat-ing eIF4E activity during human thymocyte maturation(Beretta et al., 1998), suggesting that the availability ofeIF4E may play critical roles in regulating protein syn-thesis during various stages of development. Most re-ports in the literature refer to increased levels of eIF4Ecorrelating with increased growth, and overexpression ofeIF4E by cDNA transfection has been found to causeoncogenic transformation of a number of different celltypes (DeBenedetti and Rhoads, 1990). However, duringcellular differentiation and senescence, cells withdrawfrom the cell cycle but do not abort global protein syn-thesis. Instead, cells adjust the rate, either downward,upward or not at all, depending on species and tissuetype (Efiok et al., 2000). For example, elevated expres-sion of 4E in specific tissues and cell types has beennoted in the primordial germ cells of Drosophila (Her-nandez et al., 1997), oocytes of zebrafish (Fahrenkrug etal., 1999) and during spermatogeneis of C. elegans(Amiri et al., 2001). DLKP cells have been shown to bea stem cell-like cell line derived from the lung (McBrideet al., 1998). It is possible that these cells are beingprimed to differentiate to a number of different lungphenotypes, which may explain the increased levels ofthe translation factor eIF4E after treatment with the dif-ferentiation-modulating agent BrdU and subsequent in-duction of the simple epithelial keratins 8 and 18. In-creased availability of eIF4E at specific points in devel-opment may offer a mechanism for rapid phenotypicchanges during differentiation by recruiting groups ofpoorly competitive or repressed, differentiation-relatedmRNAs to polysomes. Such mRNAs may be producedat earlier stages of development but remain poorly trans-lated until such time as levels of available eIF4E in-crease. Further work is needed to determine whether as-sembly of the eIF4F complex also increases under theseconditions of cell differentiation.

In contrast to the results obtained in these epitheliallung cancer cell lines, levels of eIF4E expression werefound to decrease during BrdU-induced myeloid differ-entiation of the leukaemic cell line, HL-60. However, theinitial loss of phosphorylated eIF4E is far less dramaticthan that of non-phosphorylated eIF4E. Perhaps these,and possibly other cells respond to lower absoluteamounts of eIF4E, increasing the activity of that portionthat remains. This suggests that there are cell-type differ-ences in the response to BrdU and in the requirementfor eIF4E during differentiation. These results are inagreement with those of Johnston et al. (1998), whodemonstrated a decrease in translation rate and eIF4Elevels during phorbol ester-induced myeloid differen-tiation of HL-60. It has been shown that 4E-BP1 and4E-BP2 are independently regulated during differen-tiation of HL-60 cells, reflecting specific effects attribu-table to either the monocytic/ macrophage or the granu-

locytic pathways induced by various differentiatingagents (Grolleau et al., 1999; 2000). Recent studies haveshown the dynamic and asymmetric expression of eIF4Eduring zebrafish embryogenesis and revealed that thisgeneral translation factor may act as a tissue-specifictranslational enhancer (Fahrenkrug et al., 1999; 2000).

Alterations in translational efficacy by BrdU havebeen suggested by studies carried out by Ohtani et al(1997) investigating endothelin receptor expression inhuman melanoma cells during BrdU-induced differen-tiation. While receptor levels on the cell surface werefound to decrease, mRNA levels were shown to remainunchanged. We have previously reported the post-tran-scriptional induction of keratin proteins in BrdU-treatedepithelial lung cancer cell lines (McBride et al., 1999;Meleady and Clynes, 2001a). Here, we show that BrdUis capable of altering expression of eIF4E and thattransfection of DLKP cells with two different eIF4EcDNAs results in the induction of keratin filament for-mation. In contrast, transfection of DLKP cells withmutant eIF4E (eIF4E mutated at Ser209) did not inducekeratin expression, again suggesting that phosphoryla-tion of eIF4E may be required for its activity in thiscellular process, with resultant induction of keratin 8and 18 protein expression. It has been previously re-ported (Cuesta et al., 2000) that these mutants and thedephosphorylation of eIF4E are responsible for inhi-bition of cellular (Cap-dependent) but not adenovirallate mRNA (Cap-independent mRNAs) translation.The presence of keratin 8 and 18 mRNAs in DLKPprior to treatment with BrdU or transfection with eIF4EcDNA suggests that these mRNAs are perhaps unableto compete efficiently for available eIF4E. Alternatively,there may be a general translational block on keratinprotein synthesis in some epithelial cell types (Winterand Schweizer, 1983; Paine et al., 1992; Su et al., 1994)or keratin mRNA translation may be specifically regu-lated by proteins interacting with the 3øUTR of themRNA, in a manner described for developmentallyregulated mRNAs (Stebbins-Boaz et al., 1999). Poorlycompetitive or repressed mRNAs may explain the lackof expression of keratin proteins in DLKP and the dra-matic post-transcriptional induction of filament forma-tion upon exposure to BrdU (McBride et al., 1999). Theincrease in both eIF4E levels and phosphorylation inBrdU-treated DLKP cells may act to increase the avail-ability of eIF4E to poorly competitive or translationallyrepressed, differentiation-related mRNAs. It is alsoworth noting that, from Fig. 4, there appears to be anincrease in nuclear eIF4E staining in the eIF4E-trans-fected cells compared to the parental, untransfectedcells. Recently, it has become evident that eIF4E mayplay a role in the transport of certain mRNAs to thecytoplasm (reviewed Strudwick and Borden, 2002), andin fact, up to 68 % of eIF4E may be present in the nu-cleus (Iborra et al., 2001). Future work will be neededto determine if increased levels of eIF4E in the nucleus

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of DLKP cells is involved in regulating nuclear trans-lation and nuclear export of developmentally-relatedmRNAs such as keratin 8 and 18 in this case.

These data suggest a role for eIF4E in the inductionof differentiation-related keratin synthesis that may rep-resent an important regulatory mechanism during epi-thelial differentiation processes. Further research isnecessary to investigate if this effect of 4E is specific tothe cell lines being studied here and whether such effectscan be observed in primary cells or other established celllines.

Acknowledgements We would like to thank Prof. Arrigo DeBened-etti (Louisiana, USA) for providing the human eIF4E cDNA in aBK shuttle vector and Dr. Robert Schneider (New York, USA) forproviding the pcDNA-eIF4E and mutant pcDNA-eIF4E cDNAs.This work is supported by grants received from Enterprise Ireland,Bioresearch Ireland and Programme for Research in Third LevelInstitutes (PRTLI)/ Higher Education Authority (HEA). Researchin the S.J.M lab is supported by grants from The Wellcome Trust(040800, 050703, 045619, 056778). S. J. Morley is a Senior ResearchFellow of The Wellcome Trust.

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