MOLECULAR CARCINOGENESIS 45:957–967 (2006)

Carcinoma-Associated eIF3i Overexpression Facilitates mTOR-dependent Growth Transformation

Martin Ahlemann,1 Reinhard Zeidler,2 Stephan Lang,1 Brigitte Mack,2 Markus Mu¨ nz,3 and Olivier Gires2,3* 1Department of Otorhinolaryngology, Head and Neck Surgery, University Hospital of Schleswig-Holstein, Campus Luebeck, Luebeck, Germany 2Department of Head and Neck Surgery, Ludwig-Maximilians-University Munich, Munich, Germany 3Clinical Cooperation Group Molecular Oncology, Department of Head and Neck Research, Ludwig-Maximilians-University Munich, Munich, Germany and GSF-National Research Center for Environment and Health, Munich, Germany

Molecular processes controlling mRNA are complex, multilayered, and their deregulation can lead to cancer pathogenesis. Eukaryotic 3 (eIF3) is involved in the initiation process of translation and overexpression of its subunit initiation factor i (eIF3i) has been observed in carcinomas. Nevertheless, the potential role of eIF3i in carcinogenesis is poorly understood. Here, we show that in vitro overexpression of human eIF3i resulted in cell size increase, proliferation enhancement, cell-cycle progression, and anchorage-independent growth. Without external stimuli, eIF3i overexpressing cells arrested in G1/G0 phase, demonstrating the requirement of additional growth signals. Inhibition of the kinase mTOR, a key player in the integration of nutrition and growth signals into protein synthesis, with rapamycin reduced serine phosphorylation of eIF3i and resulted in a loss of anchorage-independent growth. Thus, eIF3i overexpression fosters the integration of growth signals by mTOR into the mRNA translation process, promoting protein synthesis and tumor growth. ß 2006 Wiley-Liss, Inc. Key words: AMIDA; eIF3i; mTOR; PI3K; rapamycin; mRNA translation

INTRODUCTION (AMIDA) technology, we identified eIF3i as a Changes in transcriptional activity have long tumor-associated antigen [10]. In the present study, been regarded as the major regulatory feature of we conducted a molecular characterization and protein expression. However, nutrients and various uncovered an essential role of human eIF3i in extracellular signals additionally determine cell fate carcinoma-associated growth transformation. Con- upon regulation of translation of specific [1,2]. sistently, inhibition of eIF3i expression interfered The eukaryotic initiation factor 3 (eIF3) is a multi- with anchorage-independent growth. Furthermore, protein complex, which plays a central role in phosphorylation of eIF3i at serine residues was found translation initiation upon maintaining the 40S to depend on mTOR kinase activity and is necessary subunit dissociated from the 60S and by for its ability to induce anchorage-independent promoting the association of the 40S subunit with growth. Thus, this study confirms the prominent the initiator Met-tRNA complex [3]. Recently, a role of eIF3 in disease-associated deregulation of novel function of eIF3 as a scaffold for the mTOR/ protein synthesis and sheds light on the contribu- Raptor complex and its substrate S6 kinase 1 (S6K1) tion of its subunit eIF3i. to allow for the timely coordination of protein translation has been demonstrated [4]. These find- ings unravelled a connection between mTOR and eIF3 [5–7]. The eukaryotic translation initiation factor i (eIF3i), also termed eIF3p36, eIF3i, eIF3S2, or *Correspondence to: Department of Head and Neck Surgery, TRIP-1, is one out of at least 12 subunits of the Ludwig-Maximilians-University Munich, Marchioninstr. 15, D-81377 Munich, Germany. human eIF3 complex [8,9]. In a screen of sera from Received 24 May 2006; Revised 13 July 2006; Accepted 14 July head and neck carcinoma patients using the 2006 autoantibody-mediated identification of antigens DOI 10.1002/mc.20269

ß 2006 WILEY-LISS, INC. 958 AHLEMANN ET AL.

RESULTS noma (data not shown). Hence, we conducted a detailed analysis of eIF3i function in vitro.Inordertostudythe eIF3i Overexpression Induces Cell Size Increase, Cell-Cycle effects of eIF3i overexpression, murine NIH3T3 fibro- Progression, and Anchorage-Independent Growth blasts were stably transfected with an expression plasmid eIF3i was recently isolated as a potential tumor- for human eIF3i (pCAG::eIF3i) or a control vector associated antigen and reported to be overexpressed in (pCAG). The expression of eIF3i in the resulting NIH3T3 hepatocellular carcinomas [10,11]. We could confirm an transfectants was quantified by RT-PCR and immuno- overexpression for head and neck squamous cell carci- blotting analysis as shown in Figure 1A. Murine and

Figure 1. (A) Stable overexpression of human eIF3i in murine and pCAG::eIF3i transfectants were serum-starved (22 h; 0.1% FCS), NIH3T3 fibroblasts. pCAG-control and pCAG::eIF3i expression serum was re-added, and cell-cycle distribution assessed upon plasmids were stably transfected in NIH3T3 cells and the expression propidium iodide (PI) staining after 30 min and 10 h. Left panel of eIF3i mRNA (left panel) and protein (right panel) assessed upon RT- shows a representative flow cytometry histogram; right panel shows PCR and immunoblotting, respectively. As a control, GAPDH and the mean percentage with SD of cells in G1/G0-, S-, and G2/M-phase actin levels were assessed in parallel. Shown are the representative from three independent experiments. (D) eIF3i impacts on cell size of results from three independent experiments. (B) eIF3i induces cell NIH3T3 fibroblasts. NIH3T3 transfectants were serum-starved (0.1% proliferation. Stable NIH3T3 transfectants were plated at equal cell FCS; 22h), serum re-added, and cell size assessed upon flow density (1 104 cells/well) and counted over a time period of 5 days. cytometry after 10 h. Shown are the mean cell sizes with SD from Shown are the mean and SD of three independent experiments. three independent experiments (left panel) and one representative (C) Cell-cycle distribution of NIH3T3 transfectants. pCAG-control histogram of the flow cytometric assessement (right panel).

Molecular Carcinogenesis DOI 10.1002/mc CARCINOMA-ASSOCIATED eIF3i OVEREXPRESSION 959 human eIF3i display 98% identity and were indistin- guishable. The overexpression of eIF3i protein levels was 2.5-fold as determined with the Image J software for quantification and after normalization for actin levels. This reflected the in vivo situation closely. Next, cell numbers of NIH3T3 pCAG::eIF3i and pCAG control cells were assessed over a time period of 5 days. For this purpose, transfectants were plated at equal density and counted every day. At day 5, cells overexpressing eIF3i displayed a mean fourfold higher cell number than control cells (Figure 1B). This phenotype was also reflected in the cell-cycle distribution of NIH3T3 trans- fectants. pCAG::eIF3i and pCAG control cells were serum-starved (22 h, 0.1%FCS), serum was re-added, and the distribution of cells in G1/G0-, S-, and G2/M- phase was assessed after 30 min and 10 h (Figure 1C). After 30 min the distribution of pCAG control and pCAG::eIF3i transfectants was comparable: 78% versus 73% of the cells were arrested in the G1/G0-phase, 3% versus 7% in S-phase, and 13% versus 18% in G2/M- phase, respectively. Notably, eIF3i overexpressing cells did not proliferate without the external growth stimuli supplied by the FCS but arrested just as control cells in G1/ G0-phase. In contrast, after 10 h of serum re-addition, significant differences were observed between both cell lines. eIF3i overexpressing transfectants displayed an increased amount of cells in S-phase as compared with pCAG control cells (19% vs. 8%) and in G2/M-phase (31% vs. 17%). Respectively, less pCAG::eIF3i cells remained in the G1/G0-phase as compared with controls (43% vs. 72%). Thus, eIF3i facilitates re-entry into cell cycle following G1/G0 arrest and implementation of growth signals. Figure 2. eIF3i induces anchorage-independent growth in NIH3T3 Size is a critical parameter for cells prone to divide, fibroblasts. (A) pCAG-control and pCAG::eIF3i NIH transfectants (1– 3 104 cells/6-well) were plated in 1.3% methylcellulose on a 0.4% that is, sufficient cell mass must accumulate before agarose layer and colony formation was analyzed microscopically cells can fulfill mitotic division. For this reason, cell after 7–14 days. Shown are representative sections at 100- and 200- fold magnification. (B) Colony formation was quantified from two growth and division are tightly coupled and coregu- independent experiments performed in duplicates. Shown are the lated processes [12–14]. Therefore, we assessed cell size mean and SD. against eIF3i expression. NIH3T3 transfectants were serum-starved over night (0.1% FCS), serum was re- added, and cell size was measured upon flow cytometry proliferation, and growth transformation in carci- after10h.NIH3T3pCAG::eIF3icellswerelargerin noma cells. Thus, eIF3i-positive HeLa (cervix-carci- size after 10 h of serum re-addition as compared with noma) and PCI-1 (head and neck carcinoma) cell pCAG control cells (Figure 1D, P ¼ 0.002). lines were transfected with eIF3i-specific or control Because eIF3i expression levels influenced cell size siRNA. The expression of eIF3i protein was assessed and proliferation, we next asked whether eIF3i also in a time kinetic of 3 days post transfection of the supports growth transformation under anchorage- respective siRNA. Detection of eIF3i protein levels independent conditions. pCAG and pCAG::eIF3i proved a significant decrease of eIF3i in HeLa and cells were plated in methylcellulose and colony PCI-1 cells 3 days post transfection of specific siRNA formation (>20 cells/colony) was recorded after 7– (Figure 3A and data not shown). Next, the effects of 14 days. Control NIH3T3 transfectants generated 2.6 eIF3i repression on anchorage-independent growth colonies per field of view. At identical time points, were assessed in HeLa cells. Control-siRNA and eIF3i- eIF3i transfectants yielded a mean of 21.7 colonies, siRNA treated HeLa cells were plated in methylcellu- thus 8.3 times more than control cells (Figure 2). lose and colony formation (>20 cells/colony) was recorded after 7–14 days. HeLa cells generated per se eIF3i Knock-Down has a Negative Impact on Cell Size, colonies at a mean rate of 21 colonies per field of view Proliferation, and Anchorage-Independent Growth when treated with control-siRNA. Knock-down of In the next line of experiments, we sought to eIF3i resulted in a mean 64% decrease of anchorage- define the impact of endogenous eIF3i on cell size, independent growth (Figure 3B, C). In accordance

Molecular Carcinogenesis DOI 10.1002/mc 960 AHLEMANN ET AL.

Figure 3. Knock-down of endogenous eIF3i impacts on cell size, formation was quantified depending on the expression of eIF3i. proliferation, and anchorage-independent growth. (A) SiRNA- Shown are the mean and SD of two independent experiments mediated knock-down of eIF3i protein expression. HeLa cells performed in duplicates. (D) Cell numbers of control-siRNA and eIF3i- (5 105/6-well) were transfected with control-siRNA or eIF3i-siRNA siRNA treated HeLa cells were assessed at the indicated time points. (100 nM), lysed at the indicated time point, and the protein amounts Shown are the mean and SD of three independent experiments. (E) of eIF3i and actin assessed upon immunoblotting with specific Cell size of control-siRNA and eIF3i-siRNA treated HeLa cells was polyclonal antibodies. (B) HeLa cells were treated as in (A) and measured in a flow cytometer 3 days after treatment. Shown are the subsequently plated in 1.3% methylcellulose on a 0.4% agarose mean cell sizes with SD of three independent experiments (left panel) layer. Colony formation was recorded after 7–14 days. Shown are and a representative flow cytometry histogram (right panel) for representative sections of one experiment out of two. (C) Colony HEK293 (upper panels) and HeLa cells (lower panels). with the effects observed upon overexpression of reduction of adherent vital cell yield as compared eIF3i in murine fibroblasts, knock-down of eIF3i in with control-treated cells (Figure 3D). HeLa cells resulted in a strongly diminished cell yield We next analyzed the role of endogenous eIF3i in after 5 days. eIF3i inhibition induced a 5.5-fold determining the cell size. Human embryonic kidney

Molecular Carcinogenesis DOI 10.1002/mc CARCINOMA-ASSOCIATED eIF3i OVEREXPRESSION 961

(HEK293) and HeLa cells were transfected with kinase inhibition with the specific inhibitors rapa- control- or eIF3i-siRNA and cell size was assessed mycin or LY294002, respectively, on anchorage- upon flow cytometry 3 days later. Cells transfected independent growth was tested for NIH3T3 transfec- with eIF3i-siRNA were reproducibly smaller as tants. pCAG-control and pCAG::eIF3i cells were control-siRNA treated cells (Figure 3E, HEK293 plated in methylcellulose with 2.5 mM LY294002, P ¼ 0,01; HeLa P ¼ 0.06). 1 ng/mL rapamycin, or with vehicle diluent only and colony formation (>20 cells/colony) was recorded eIF3i Phosphorylation Depends on after 7–14 days. Expectedly, treatment of pCAG- PI3K/mTOR Kinase Activity control cells did not show remarkable colony forma- The regulation of cell size, proliferation, ancho- tion, independently of the inhibitors. Treatment of rage-independent growth, and the integration of human eIF3i expressing NIH3T3 fibroblasts with the extracellular nutritional signals are hallmarks remi- PI3-kinase inhibitor LY294002 induced a marked niscent of mTOR [7]. In addition, critical roles for reduction of colony size and number. Inhibition of PI3K and mTOR in carcinogenesis and the regulation mTOR with rapamycin resulted in a complete of protein translation emerge more and more abrogation of colony formation of eIF3i overexpres- [15,16]. Since PI3K, mTOR, and eIF3i seemingly feed sing NIH3T3 cells (Figure 4). into comparable cellular phenotypes, we studied a When initially identified, eIF3i was demonstrated potential connection. The effect of mTOR or PI3- to be constitutively phosphorylated at serine and

Figure 4. Inhibition of PI3-kinase and mTOR reverses eIF3i-mediated colony formation. NIH3T3 fibroblasts stably carrying the pCAG-control or the pCAG::eIF3i vector were treated with the PI3-kinase inhibitor LY294002 (2.5 mM), the mTOR inhibitor rapamycin (1 ng/mL), or with diluent only. Subsequently, equal cell numbers (1–3 104 cells/well) were plated in 1.3% methylcellulose on a 0.4% agarose layer. Colony formation was recorded after 7–14 days. Shown are representative sections of one experiment out of three.

Molecular Carcinogenesis DOI 10.1002/mc 962 AHLEMANN ET AL. threonine residues [17], thus raising the question, encompassing the predicted mTOS motif (FEFE- whether the observed effects of rapamycin might be FEA*). The resulting HA-tagged mutant HA-eIF3i- attributable to changes in eIF3i phosphorylation. DTOS was stably expressed in NIH3T3 cells yielding HA-tagged eIF3i was stably expressed in NIH3T3 two independent bulk cultures. In parallel, HA- murine fibroblasts. Thereafter, cells were cultured in tagged wild-type eIF3i was stably expressed in the presence or absence of 1 ng/mL rapamycin for a NIH3T3 cells. The expression of both, wild-type time period of 3 days and HA-eIF3i immunoprecipi- and DTOS mutants of eIF3i was assessed in an tated with HA-specific antibodies. Next, the serine immunoblot experiment with HA-specific antibo- phosphorylation of eIF3i was assessed with phos- dies and revealed similar (Figure 6B). Hence, it is phoserine-specific antibodies. Rapamycin treatment excluded that phenotypes observed reflect differ- induced a 50% decrease in serine phosphorylation of ences in protein expression levels. The expected shift eIF3i (Figure 5). Thus, eIF3i is phosphorylated in an in molecular weight due to the C-terminal deletion mTOR-dependent manner. Known targets of mTOR was observed. Next, wild-type and mutated eIF31 such as S6 kinase and 4E binding protein 1 possess a were compared for their ability to induce colony consensus motif in the far N- or C-terminus neces- formation in soft agar. Equal cell numbers of NIH3T3 sary for phosphorylation by mTOR [18–20]. This transfectants were plated in methylcellulose without sequence is termed as mTOR signaling (mTOS) motif. further treatment and colony formation (20 cells/ Mutations in any of the five N-terminal amino colony) was recorded after 7–14 days. None of the acids FDIDL within S6 kinase 1 resulted in a dramatic two bulk cultures was capable of growing in an loss in kinase activity reminiscent of rapamycin anchorage-independent fashion, while NIH3T3 cells treatment. Comparison of the amino acids se- overexpressing a wild-type, HA-tagged version of quences of S6-K1, 4E-BP1, and eIF3i suggested the eIF3i formed 27 colonies per sight of view in average presence of a potential consensus mTOS motif in the (Figure 6C), which is similar to transfectants bearing far C-terminus of eIF3i (FEFEF; Figure 6A). In order to the untagged eIF3S3 (see Figure 2 for comparison). delineate the functionality of this potential mTOS Thus, deletion of the predicted mTOS motif within motif, we deleted the last seven amino acids of eIF3i the C-terminus of eIF3i impaired its transforming capacity.

eIF3i Does Not Specifically Localize to the Plasma Membrane in TGFb-RII-Positive Cells eIF3i was initially identified as an interactor of the TGFb receptor II, which is constitutively phosphory- lated at serine and threonine residues by the receptor kinase [17]. However strong doubts arose, first when TRIP-1 was shown to be identical to the component of the eIF3 complex eIF3i [9], and second when Derynk et al. suggested that eIF3i may also function in a receptor-independent manner [21]. In the present study, we have shown the rapamycin- sensitive phosphorylation of eIF3i, strongly suggest- ing an implication of mTOR in posttranslational modification of eIF3i. This finding added even more question marks to the function of eIF3i in TGFb-RII signaling. Thus, to clarify the localization of eIF3i, we constructed N- and C-terminal fusion of the yellow fluorescence protein and eIF3i, termed eYFP- N1::eIF3i and eYFP-C1::eIF3i, respectively. In a first line, the status of TGFb-RII expression was assessed by RT-PCR in different carcinoma cell lines. HeLa cells expressed decent amounts of TGFb-RII mRNA and thus were chosen for further analysis of Figure 5. Inhibition of mTOR results in decreased serine phos- phorylation of eIF3i. (A) NIH3T3 fibroblasts were stably transfected eIF3i localization. Both fusion proteins were transi- with a HA-tagged version of eIF3i. Transfectants were treated with ently expressed in TGFb-RII-positive HeLa cells rapamycin (1 ng/mL) or diluent only and subjected to a HA-specific (Figure 7A-B) and the localization of the fusion immunoprecipitation. Isolated HA-eIF3i was analyzed for serine phosphorylation upon immunoblotting with a phosphoserine- proteins was monitored upon laser scanning con- specific polyclonal antibody. Equal amounts of immunoprecipitated focal microscopy. Neither eYFP-N1::eIF3i nor eYFP- HA-eIF3i were ascertained upon HA-specific immunoblotting. (B) C1::eIF3i fusion proteins did specifically accumulate Serine phosphorylation of eIF3i was quantified from three indepen- dent experiments with the Image J software. Shown are the mean to the plasma membrane, which would have phosphorylations with SD. been the predicted localization of a ligand of the

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Figure 6. Deletion of the mTOS motif results in a non-transform- were expressed. HC marks heavy chains of the antibodies used for ing eIF3i mutant. (A) Sequence comparison of S6 kinase 1, 4E-BP1, immunoprecipitation that are recognized by HRP-conjugated sec- and eIF3i mTOS motif. FEFEF represents a potential mTOS motif in ondary antibodies. (C) Equal cell numbers of the indicated NIH3T3 the C-terminus of eIF3i. (B) Expression of mTOS deletion variants of transfectants (1–3 104 cells/well) were plated in 1.3% methylcel- eIF3i. A mutant of eIF3i lacking the last C-terminal seven amino acids lulose on a 0.4% agarose layer. Colony formation was recorded after including the FEFEF mTOS motif and wild-type eIF3i were stably 7–14 days. Shown are representative sections of one experiment out expressed in NIH3T3 cells. The expression of each eIF3i variant was of three (left panel). Colony formation was quantified with two assessed upon immunoblotting with HA-specific monoclonal anti- distinct HA-eIF3i-DTOS and one eIF3i-HAwt NIH3T3 bulk cultures. bodies following separation of immuno-precipitates of the indicated Shown are mean with SD from three independent experiments. stable transfectants. Equal amounts of HA-eIF3i and HA-eIF3i-DTOS transmembrane receptor TGFb-RII (Figure 7C, left scopy. In accordance with results obtained with YFP- panel). Rather did both fusion proteins homoge- fusions, endogenous eIF3i distributed across the cell neously distribute all over cells including the with a speckled pattern (Figure 7C, right panel). cytoplasm, distinct areas of the nucleus, and Thus, eIF3i did not exclusively map to the site of the plasma membrane. Because the bulky GFP TGFb-RII expression, which at least allows further residue may impact on the correct localization of phosphorylation by cytosolic kinases such as mTOR. eIF3i, we additionally assessed the localization of endogenous eIF3i in HeLa cells. HeLa cells were DISCUSSION stained with an eIF3i-specific polyclonal antibody eIF3i is a WD40-domain containing protein, and fluorescence monitored upon confocal micro- which was initially isolated as an interactor of the

Molecular Carcinogenesis DOI 10.1002/mc 964 AHLEMANN ET AL.

Figure 7. eIF3i localization in TGFb-RII-positive cells. (A) The C-terminal fusions of eIF3i with eYFP were expressed albeit to far expression of TGFb-RII mRNA was analyzed upon RT-PCR in head lower extend as was eYFP alone. (C) Homogenous localization and neck (GHD, ANT, FaDu) and HeLa cells. GHD-1, FaDu, and HeLa of eYFP-fusions and endogenous eIF3i in HeLa cells. HeLa cells expressed similar levels of TGFb-RII mRNA, while ANT cells did not. As (5 105/6-well) were transiently transfected with eYFP-N1::eIF3i a control, GAPDH mRNA amounts were assessed in parallel. Shown is expression plasmid and green fluorescence monitored upon laser one representative experiment from three. (B) Transient expression scanning confocal microscopy. Nucleic DNA was stained with the of wild-type eYFP-N1, eYFP-C1, eYFP-N1::eIF3i, and eYFP-C1::eIF3i Hoechst 33342 dye (blue). Alternatively, endogenous eIF3i was in HeLa cells. Protein expression was assessed 48 h after transfection stained with a specific polyclonal antibody and detected with a with GFP-specific monoclonal antibodies that equally well recognize Alexa-594 secondary antibody. Shown is one representative section the YFP moiety based on high-. Both, N- and of three independent experiments, respectively.

TGFb-receptor type II and termed TRIP-1, for TGFb- eIF3i in cancer was described for 60% of hepatocel- RII interacting protein 1 [17]. Shortly after, Asano lular carcinomas [11] and could be confirmed for et al. [9] described TRIP-1 to be identical to the 36- head and neck squamous cell carcinoma (data kDa subunit of the eukaryotic translation initiation not shown). The dysregulation of several members factor 3 complex. Recently, we demonstrated that of the eIF3 complex in cancer has been published eIF3i elicits a humoral response in vivo in head previously [22]. These and other findings strongly and neck carcinoma patients [10]. Overexpression of suggested for a considerable time a direct link

Molecular Carcinogenesis DOI 10.1002/mc CARCINOMA-ASSOCIATED eIF3i OVEREXPRESSION 965 between carcinogenesis and the regulation of pro- decreased serine phosphorylation of eIF3i and tein translation initiation [23]. Besides regulation of abrogation of colony formation, we conclude that at the transcriptional level, which eIF3i is essential to integrate mTOR signaling in the has long been considered the main regulatory mRNA translation process. This notion is further feature, changes in translation account for alteration corroborated by the presence of a functional con- in specific polypeptide production, improved global sensus mTOS motif in the C-terminus of eIF3i. protein synthesis [2], and may contribute to cellular Mutation of the mTOS motif resulted in a loss-of- transformation (for an overview ‘‘mRNA transla- function mutant of eIF3i. Inhibition of PI3-kinase, tional control in cancer pathogenesis’’, Oncogene which is an upstream regulatory kinase in the PI3K/ Vol. 23, 2004). AKT/mTOR pathway [27,28], showed a similar In order to study potential effects in an in vitro phenotype. system reflecting the observed in vivo situation, we In summary, the tumor-associated antigen eIF3i overexpressed eIF3i in murine fibroblasts. A moder- contributes to the growth-transformed phenotype of ate overexpression of eIF3i in NIH3T3 fibroblasts cancer cells, fostering the integration of growth resulted in improved proliferation, accelerated cell- signals by mTOR into the mRNA translation process, cycle progression, and increased cell size. In line with promoting protein synthesis, and tumor growth. findings by Huang et al., human eIF3i supported growth in an anchorage-independent fashion [11]. MATERIALS AND METHODS With its WD40 protein-protein interaction domains Cell Lines and Flow Cytometry eIF3i is likely to act as a scaffold for the assembly of the eIF3 subunits to form the complex. Accordingly, HeLa human cervix carcinoma cells, PCI-1 head the deletion of TIF34, the yeast homolog of human and neck carcinoma cells (kind gift from Prof. T. eIF3i, leads to the degradation of other subunits of Whiteside), and murine NIH3T3 fibroblasts were the complex, decreasing the rate of protein synthesis cultured in standard DMEM containing 10% fetal [24]. Thus instability of the eIF3 complex decreases calf serum and passaged three times per week. The global protein synthesis. Reverse, an overexpression empty vector control pCAG, pCAG::eIF3i, pCA- of eIF3i would stabilize the entire eIF3 complex and G::eIF3i-HA, pCAG::Ha-eIF3iDTOS cell lines (HeLa promote its activity, leading to a global increase in and or NIH3T3) were established by transfection mRNA translation. Increased protein synthesis using MATra (IBA, Go¨ttingen, Germany) according allows for faster increase in cell size, interrelated to the manufacturer’s protocol. Stable transfectants with the accumulation of critical mass for cell were grown in 10% FCS DMEM standard medium division, and subsequently proliferation, as seen for containing 2 ng/mL puromycin (Sigma, Tauf- eIF3 overexpressing cells. Consistently, we observed kirchen, Germany). For flow cytometry analysis, diminished proliferation and cell size, as an effect of cells were washed twice in PBS and resuspended in the knock-down of eIF3i by siRNA in carcinoma cells. PBS supplemented with 3% (v/v) FCS. Cell size was Increased growth and proliferation gives eIF3i over- thereafter assessed in a FACS-calibur device (BD expressing carcinoma cells a selection advantage in Clontech, Heidelberg, Germany). tumor development, explaining the observed over- Cell-Cycle Analysis expression in vivo. We show here that a discrete imbalance in eIF3i Cell-cycle analysis was performed using propi- protein levels strongly impacts on growth promoting dium iodide (PI) staining with subsequent FACS 5 effects and features common to tumor cells [25]. analysis. 5–10 10 cells/well were cultured as However, eIF3i overexpressing cells did not prolifer- described and adherent cells were detached with ate without external growth stimuli supplied by trypsin (Biochrom, Berlin, Germany). Detached cells FCS but arrested like control cells in G1/G0-phase. were harvested in complete DMEM medium and Hence, eIF3i overexpression per se does not drive cells centrifuged at 500g for 10 min. Pellets were washed into growth and proliferation but rather improves with PBS and fixed with ice-cold 80% ethanol for 1 h the incorporation of growth signals. The findings at 48C. After fixation, cells were washed twice with that rapamycin, a potent inhibitor of mTOR, has PBS and stained with 500 mL of PI solution (50 mg/mL anticarcinogenic properties, gave new insights into in PBS, 2 mM EDTA) containing 10 units of DNase the underlying molecular mechanisms [26]. PI3- free RNase (Roche, Mannheim, Germany). Cells were kinase and mTOR are key players of protein transla- incubated at 48C for 10–15 min in the dark and tion [7,16], which guide the activity of translational analyzed by flow cytometry. regulators such as S6 kinases 1/2 (S6K1/2) and eIF4E. These mTOR-regulated proteins control cell size and Cell Number facilitate G1 cell-cycle progression [5,6], features NIH3T3 and HeLa transfectants were plated at a reminiscent of the present findings on eIF3i (Figures density of 10 000 cells/well in 6-well plates. Cells 1 and 3). Together with the fact, that rapamycin- were harvested by trypsin treatment, stained with treatment of eIF3i NIH3T3 transfectants led to trypan blue, and living cells were counted.

Molecular Carcinogenesis DOI 10.1002/mc 966 AHLEMANN ET AL.

Methylcellulose Assay MATra system (IBA), and eYFP-fusion proteins Colony formation Assay was carried out as expressing cells were analyzed as living cells in a described elsewhere [29]. Shortly, cells were seeded fluorescence laser scanning system (TCS-SP2 scan- in medium containing 1.3% methylcellulose on a ning system and DM IRB inverted microscope, Leica, bottom layer of 0.4% soft agar medium. Inhibitors Solms, Germany). For the detection of endogenous or diluent were added to the methylcellulose med- eIF3i, cells were fixed according to Brock et al. [30], ium as indicated. After 7–14 days colonies were stained with a polyclonal eIF3i-specific antibody (US counted in three randomly chosen fields of view Biological), and analyzed upon confocal laser scan- on an inverted microscope (Axiovert 200; Zeiss, ning microscopy. Oberkochen, Germany). Representative images SiRNA Treatment were captured with a digital camera (Hamamatsu, Cells (4 105 cells/well) were plated in 6-well Herrsching, Germany). plates and allowed to grow for 24 h before transfec- RNA Isolation and RT-PCR tion with 100 pM target-specific siRNA and control siRNA with MATra transfection reagent (IBA). Two RNA was isolated from 1–5 106 cells with the hours posttransfection, cells were trypsinized and NucleoSpin RNA II Kit (Machery-Nagel, Du¨ren, plated into cell culture plates (, 10 cm) for cell Germany) according to the manufacturer’s protocol. counting and FACS analysis. The following siRNA One microgram of total RNA was reverse transcribed sequences were used: (ImProm-IITM Reverse Transcription System, Pro- mega, Mannheim, Germany) into cDNA and PCR * Control: 50-UUAACCCGAUGAAUUCUUC-30 was performed with sequence specific primers (eIF3i- * eIF3i: 50-CUCCACAACUCUUGAACAU-30 fw 50-TGTCGTCTCTGGGACTGTGAAAC-30, eIF3i- bw 50-ACATTCACCAACACCTCTCCAGAC-30). ACKNOWLEDGMENTS Immunoprecipitation Research described in this article was supported by For immunoprecipitation, cells were lysed in TBS/ Philip Morris Incorporated fundings to R.Z. and S.L. 1% triton and protease inhibitors (Roche). Precleared whole cell lysates (400 mL, 1 mg/mL protein) of transfected NIH3T3 cells were incubated with Pro- REFERENCES tein G beads (30 mL, Amersham Biosciences, Freiburg, 1. Dever TE. Gene-specific regulation by general translation factors. Cell 2002;108:545–556. Germany) loaded with anti-HA mouse monoclonal 2. Nilsson J, Sengupta J, Frank J, Nissen P. Regulation of antibody (Roche) at 48C over-night. Protein G eukaryotic translation by the RACK1 protein: A platform for beads were collected by centrifugation, and the signalling molecules on the ribosome. EMBO Rep 2004;5: pellets were washed five times in cold lysis buffer. 1137–1141. Immunoprecipitates were eluted, incubating beads 3. Gebauer F, Hentze MW. 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Molecular Carcinogenesis DOI 10.1002/mc