Inhibition of Initiation of Protein Synthesis in Mammalian Tissue Culture Cells by L-1-Tosylamido-2-phenylethyl Chloromethyl Ketone

SUMMARY Incorporation of amino acids into proteins in HeLa cells, virus-transformed 3T3 mouse fibroblasts, plasmacytoma cells is inhibited after the addition of L-l-tosyl-amido-Z-phenylethyl chloromethyl ketone, an alkylating agent and chymotrypsin-specific protease inhibitor. Addition of this drug to tissue culture cells at concentrations of 20 to 30 c(g per ml results in an irreversible inhibition of the incorporation of amino acids into cellular proteins, and a rapid and complete breakdown of polyribosomes. several known inhibitors of in protein synthesis, with known mechanisms of action, revealed that an optimal concentration of L-l-tosyl-amido-2-phenylethyl chloromethyl ketone: (a) immediately and selectively inhibits initiation of protein synthesis, (b) does not significantly affect normal elongation rates, and (c) does not promote a premature release of nascent peptides. L-I-Tosylamido-Z-phenylethyl chloromethyl ketone may prove to be a useful tool in investigating the initiation of protein synthesis in eukaryotic cells.

L-I-Tosylamido-Z-phenylethyl chloromethyl ketone may prove to be a useful tool in investigating the initiation of protein synthesis in eukaryotic cells.
Tissue culture cells contain proteolytic enzymes which play a key role in the maturation of several DNA and RNA viruses by specific cleavage of viral precursor proteins (1, 2). Extracts from uninfected cells will convert the isolated picornavirus "polyproteins" (2) into their primary cleavage products (3). This cleavage process can be inhibited by a number of agents in Gvo and in z&o but most effectively by L-l-tosylamido-2phenylethyl chloromethyl ketone, a chymotrypsin-specific protease inhibitor (4). Therefore, TPCKl has recently been used in studies of possible postAranslationa1 cleavage processing in several in vivo systems (3,(5)(6)(7). TPCK also has been used to inhibit proteolytic activities associated with virus-transformed cells and phytohemagglutinin-stimulated and unstimulated lymphocytes (8-10). It had previously been reported that t#reatment of intact cells wit,11 TPCK was accompanied by a reduction in the incorporation of labeled amino acids into proteins (6, 7). In a study designed to investigate the possible role of post-translational cleavage in uninfected HeLa cells, we observed that TPCK is a potent and specific inhibitor of initiation of protein synthesis. Further investigation revealed that protein synthesis in other tissue culture cells, including virus-transformed 3TS mouse fibroblasts and mouse plasmacytoma cells was also inhibited by TPCK t,reatment. This property of TPCK severely rest,ricts the use of this drug in the investigation of the post-translational processing of newly synthesized proteins and in studies examining the role of proteolytic activities in the expression of the transformed state (8,9). However, the results reported here suggest that TPCK may be useful in studying the process of peptide chain initiation in eukaryotic cells. Cell pellets, in both cases, were resuspended at a density of 1.5 X 107 cells per ml in lysis buffer (50 mu Tris-HCl (pH 7.4)-100 m&r KCl-5 mM magnesium acetate) and lysed by the addition of Nonidet P-40 to 0.5y0 at 0" for 10 min.
The cell lysates were cent'rifuged at 16,000 X g Since TPCK has been used as a protease inhibitor in several in uivo studies utilizing serum-supplemented medium, we performed an experiment to examine the effect of serum on the inhibition of amino acid incorporation by TPCK. Fig. 2 shows the effect of TPCK on amino acid incorporation in the mouse plasmacytoma cell  in the absence (A) and presence ; X, 5 pg of TPCK per ml; q !, 10 rg of TPCK per ml; 0, 20 rg of TPCK per ml; A, 30 rg of TPCK per ml.
The incorporation of methionine is also inhibited in the presence of serum, although 15 to 2.0.fold higher concentrations of TPCK are required to obtain an inhibition comparable to that observed in the absence of serum. The inhibition of methionine incorporation after t,he introduction of TFCK is not readily reversible.
As shown in Fig. 3, HeLa cells treated with 30 pg of TPCK per ml for 10 min and then washed extensively with medium lacking TPCK show no subsequent recovery of protein synthesis by 30 min of incubation.
In long t,erm experiments with myeloma cells, in serumsupplemented medium, we observed no recovery in protein synthesis for up to 8 hours after a 20.min treatment with 30 pg of TPCK per ml.
Polyribosome Breakdoum following TPCK Treatment-Analysis, by zonal centrifugation, of cytoplasmic extracts prepared from cells incubated in the presence of 30 pg of TPCK per ml reveals that the observed inhibition of amino acid incorporation is accompanied by a breakdown of polyribosomes. Fig. 4a shows sucrose gradient analysis of polyribosomes from cytoplasmic extracts of HeLa cells treated with 0.3y0 dimethylsulfoxide for 10 min (control) or with 30 pg of TPCK per ml for 4 and 10 min.
It is evident that, by 4 min after TPCK addition, a significant shift in 260 nm absorption from the region corresponding to large polyribosomes to the region corresponding to smaller polyribosomes has occurred. Integration of the area under the absorption profile indicates that 50y0 of the polyribosomes are converted to 80 S ribosomes.
Essentially all of the 260 nm absorbing material is shifted to the monosome region by 10 min after TPCK addition, with no discrete peaks observed migrating ahead of the 80 S peak. Fig. 4b  synthesis, but has no effect on elongation and terminat.ion processes (16). HeLa cell culture suspensions at a density of 4 x lo6 cells per ml were treated with 0.3% dimethylsulfoxide (control), 30 ,ug of TPCK per ml, 100 mu additional NaCl, 100 pg of cycloheximide per ml, or a combination of cycloheximide and TPCK. Fig. 5a shows the characteristic disa,ppearance of polyribosomes following treatment with 30 pg of TPCK per ml. Fig. 5b shows that the addition of NaCl to a fina,i concentration of 210 mM also results in a complete conversion of polyribosomes to 80 S ribosomes.   Cytoplasmic extracts from HeLa cell cultures suspended at a density of 4 X 10" cells per ml, treated as described below, were prepared and fractionated as dcsoribcd in Fig. 4 These results seem to erelude t,he possibilities mentioned above that the observed effect of TPCK on polyribosomes is not directly related t,o the inhibition of protein spnt,hesis.
Although the results of the experiments just described slllow us to conclude that TFCK-induced polyribosome breakdown is a direct result of inhibition of protein synthesis, they do not permit us to make any definite statements with respect to the mechanism of inbibit,ion resulting from TPC:K t,reatment. On the basis of the kinetics of inhibit'ion of amino acid incorporiztion following TPCK addition and the similarity Wween TPCK and NaGinduced polyribosome breakdown, it was thought that TPCK inhibited protein synthesis, most, probably at the level of initiation.
However, the possibility that TPCK might act by prnmot,ing premature release of nascent peptides couId not be excluded. For comparison, Fig. 6B shows the size distribution of labeled polypeptides from cells chased for 12 min at an elevated NaCl concentration and cells chased in the presence of 12 y0 dimethylsulfoxide.
Normal completion of nascent pulse-labeled peptides occurs in cells chased in hypertonic medium.
However, on the basis of the differences in size distribution of labeled peptides from control and dimethylsulfoxide-treated cells, it is evident that premature release of nas- The polgpeptide molecular weight corresponding to each gel fraction was calculated by using the poliovirus peptides as reference.
The percentage of incorporation for each gel fraction was then plotted against the reciprocal of the peptide molecular weight corresponding to that gel fraction. cent polypeptides occurs in cells chased in the presence of 12% dimethylsulfoxide.
These results agree with those of previous studies on the use and mechanism of action of these two protein synthesis inhibitors reported from this laboratory (14,16 well with the times predicted, based on published estimates of the rate of peptide chain synthesis in uninfected HeLa cells (16,17). This fact strongly indicates that the rate of peptide chain elongation is unaffected in TPCK-treated cells. Fig. 8 shows graphically that the percentage of incorporation of methionine into pept.ides, in the pulse labeling experiments just described, is related proportionally to the reciprocal of the moleeular weight of the peptides. This result is predicted for inhibitors that selectively block polypeptide chain initiation. A detailed discussion of this relationship and its use in invcstigating post-translational processing and the specificity of inhibition of peptide chain initiation, will be presented elsewhere.2 The results of Fig. 8  At optimal concentrations of TPCK, inhibition of amino acid incorporation (Fig. 1) and polyribosome breakdown (Fig. 4) is complete wit,hin 5 to 7 min after 1'PCR addition.
Prevention of TPCK-induced polyribvsome breakdown by the addition of cycloheximide (Fig. 5) clearly rules out the possibility that the disappearance of polyribosomes after TPCK treatment is due to increased RNase levels or physical effects following cell lysis. Comparative pulse-chase experiments (Fig. 6) indicate that premature release of nascent pcptides does not occur in TPCX treated cells. Finally, size distribution analysis of peptides pulse-labeled at different t,imes after TPCK addition, and properly chased, clearly shows t)hat peptide chain init)iation is inhibited immediately, but t'hat incorporation of amino acids continues into preinitiated peptides at the normal rate until they are completed (Figs. 7 and 8). TPCK has been used as an inhibitor of proteolytic activities associated with virus-transformed mammalian cells (8, 3) and with mitogen-stimulated lymphocytes (10). Although TPCK is specific in its ability to inhibit proteolytic activities, it is a strong alkylating agent especially reactive with certain sulfhydryl groups (18). It is, therefore, likely that this reagent would have other effects on cell activities.
It has recently been rem ported that TPCK inhibits RNA synthesis (19) and that tosyllysyl chloromethyl ketone, a trypsin-specific protease inhibitor, inhibits DNA synthesis in eukaryotic cells (20). Since TPCK inhibits protein synthesis in a matter of minutes and RNA synthesis only after 30 miqa under the conditions described in this report, we suggest that the effects of this protease inhibitor on the other cellular processes may well be a consequence of t,he inhibition of protein synthesis.
Tosyl-lysyl chloromethyl ketone also affects protein synthesis in HeLa cells under the conditions in this report, but 5 to 10 times higher concentrations are required for a comparable degree of inhibition of amino acid incorporation (data not shown). A reduction in amino acid incorporation and a preferential inhibition in the labeling of small molecular weight peptides following TPCK treatment of virus-infected and uninfected cells has previously been reported (5-7). Since TPCK was reported to have no effect on in vitro protein synthesis in mammalian cell-free extracts (6, 21), it was suggested that the observed relative increase in labeling of large molecular weight peptides, after the addition of TPCK, was a result of inhibition of post-translational processing and not due to inhibition of peptide chain initiation (6). Our data show that TPCK inhibits peptide chain initiation ilz vivo and would, therefore, preferentially inhibit the synthesis of small molecular w-eight peptides very early after addition to the culture medium. Thus, the apparent preferential labeling of large molecular weight peptides in cells treated with TPCK cannot be taken as unequivocal evidence for the existence of post-translational cleavage mechanism. TPCK has been reported by several investigators to inhibit irreversibly peptide chain elongation in bacterial extracts (22,23). However, several observations show that peptide chain initiation and not elongation is affected by TPCK in tissue culture cells. First, inhibition of amino acid incorporation and polyribosome disappearance is completed within 5 to 7 min after addition of an optimal concentration of TPCK. Second, chasing of pulses G. Koch, unpublished data.

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labeled peptides proceeds normally in the presence of TPCK, with no evidence of premature release of nascent peptides.
.Principally, very good agreement was obt,ained between experimental and predicted values for sequential inhibition of peptide labeling with respect to their molecular weights at times after TPCK addition.
The combined data compel us to conclude that TPCK can be used to block the initiation of protein synthesis selectively in a number of tissue culture cells.
Since pcptide chain initiation is a multistep process involving many specific cellular components, one might speculate that TPCK interact,s with certain factors required for in viva peptide chain initiation.
Alternatively, since TPCK effectively inhibits in vivo protein synthesis, but inhibits i?k vitro protein synthesis only to a limited extent+4 it, is suggested that the inhihition of protein synthesis elicited by t&s reagent is membranemediated.
This possibility is currently being investigated. In any event, TPCK, like other inhibitors of initiation ut protein synthesis (14,16,24), may prove to be very useful in studying the mechanism of peptide chain initiation in vivo.