Oxazolidinones Inhibit Cellular Proliferation via Inhibition of Mitochondrial Protein Synthesis

The oxazolidinones are a relatively new structural class ofantibacterial agents that act by inhibiting bacterial proteinsynthesis. The oxazolidinones inhibit mitochondrial proteinsynthesis, as shown by [³⁵S]methionine incorporation into intactrat heart mitochondria. Treatment of K562 human erythroleukemiacells with the oxazolidinone eperezolid resulted in a time-and concentration-dependent inhibition of cell proliferation.The cells remained viable, but an increase in doubling timewas observed with eperezolid treatment. Inhibition was reversible,since washing and refeeding of cells in the absence of compoundresulted in a resumption of growth. The growth-inhibitory effectof the oxazolidinones did not appear to be cell type specific,and inhibition of CHO and HEK cells also was demonstrated. Treatmentof cells resulted in a decrease in mitochondrial cytochromeoxidase subunit I levels, consistent with an inhibition of mitochondrialprotein synthesis. Eperezolid caused no growth inhibition ofrho zero ( ${rho}$ ⁰) cells, which contain no mitochondrial DNA; however,the growth of the parent 143B cells was inhibited. These resultsprovide a direct demonstration that the inhibitory effect ofeperezolid in mammalian cells is the result of mitochondrialprotein synthesis inhibition.

INTRODUCTION

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Introduction

The oxazolidinone linezolid is a new antibacterial approvedfor marketing in 2000 that inhibits bacterial protein synthesis(5, 20, 41). It represents a new structural class of antibiotics,with activity against several gram-positive organisms, includingseveral resistant strains. Linezolid has been shown to be effectivein treating nosocomial pneumonia caused by methicillin-susceptibleand -resistant Staphylococcus aureus or multidrug-resistantStreptococcus pneumonia and skin and soft tissue infectionscaused by methicillin-susceptible and -resistant Staphylococcusaureus, Streptococcus pyogenes, and Streptococcus agalactiae.It is also effective against community-acquired pneumonia causedby methicillin-susceptible S. aureus, multidrug-resistant S.pneumoniae, and vancomycin-resistant Enterococcus faecium infections(15, 31).

The oxazolidinones inhibit bacterial protein synthesis, althoughthe exact details concerning the mechanism(s) of inhibitionare still emerging. Early results demonstrated that the oxazolidinoneeperezolid binds to 50S but not 30S ribosomal subunits. Furthermore,binding was inhibited by chloramphenicol and lincomycin (27).Cross-linking studies have been carried out to identify thesites of oxazolidinone binding. Using ribosomes from Escherichiacoli, a number of nucleotide residues in domain V of 23S rRNAwere identified, as well as residue A864 of 16S rRNA (28). Colcaet al. carried out cross-linking experiments using intact Staphylococcusaureus and showed that tRNA, two ribosomal proteins, and nucleotideA2602 of 23S rRNA all were labeled by the cross-linker (10).

The results from mapping oxazolidinone resistance mutationsagree with the cross-linking studies. Linezolid-resistant mutantsof Halobacterium halobium were isolated and shown to containsingle point mutations in the central loop of domain V of 23SrRNA (24). Likewise, Escherichia coli oxazolidinone-resistantmutants contained G2032A and G2447A mutations, which also arein domain V (4, 47). Introduction of the G2032A mutation backinto linezolid-sensitive Escherichia coli conferred resistanceto the oxazolidinone. Domain V of 23S rRNA is part of the peptidyltransferase center of the bacterial ribosome, and biochemicalexperiments demonstrate that the oxazolidinones affect stepsin protein synthesis. Work by Eustice et al. showed that anearly step in synthesis is inhibited at or before the initiationstep (13). Linezolid inhibits the formation of the tRNA Met-mRNA-70Scomplex but not the synthesis of N-formylmethionyl-tRNA (fMet-tRNA)(2). Elongation factor G-dependent translocation of fMet-tRNAfrom the A site to the P site is inhibited, and the oxazolidinonesalso inhibit peptide synthesis, as shown by in vitro assaysusing fMet and puromycin (2, 4, 7, 34, 45). Taken together,all of the available data from mutation, resistance, and biochemicalexperiments are consistent with the hypothesis that the primarybinding site of the oxazolidinones is located within the ribosomalpeptidyl transferase center.

In contrast to the current knowledge of oxazolidinone actionon bacteria, much less is understood about oxazolidinone effectson mammalian cells. Clinically, linezolid therapy results inminimal side effects for most patients. However, reversiblemyelosuppression, characterized by anemia and thrombocytopenia,has been infrequently observed in patients treated with linezolidfor at least 21 days (25). The mechanism of this effect is notknown, but it has been suggested that the myelosuppression mayresult from linezolid inhibition of mitochondrial protein synthesis.In fact, it has been shown that the oxazolidinones inhibit [³⁵S]methionineincorporation into mitochondrial proteins (39).

In the present study we have examined the effect of an oxazolidinone,eperezolid, on the proliferation of a variety of mammalian cells.We show that treatment with eperezolid inhibits proliferation,and further, that the inhibition of proliferation results fromeperezolid-mediated inhibition of mitochondrial protein synthesis.

MATERIALS AND METHODS

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Materials and Methods

Cell culture. The human osteosarcoma-derived cell line 143B (thymidine kinasedeficient) and its mitochondrial DNA (mtDNA)-less derivative,143B206 ${rho}$ ⁰, were cultured in Dulbecco's modified Eagle's (DME)high glucose supplemented with 10% fetal bovine serum, 1 mMsodium pyruvate, and 50 µg/ml uridine as described byKing and Attardi (23). The cells were a gift from G. Attardiand A. Chomyn (California Institute of Technology). Chinesehamster ovary (CHO) cells were grown in alpha minimal essentialmedium supplemented with 10% fetal bovine serum and 4 mM L-glutamine.Human embryonic kidney (HEK) cells were cultured in DME high-glucosemedium supplemented with 10% fetal bovine serum, 4 mM L-glutamine,and 1 mM sodium pyruvate. Human erythroleukemia K562 cells wereobtained from the American Type Culture Collection and grownin RPMI 1640 medium containing 25 mM HEPES, 1.5 mM Glutamax,and 10% fetal bovine serum. Cells were incubated at 37°C,5% CO₂ in the presence or absence of compound. Compound stockswere prepared in 100% dimethyl sulfoxide (DMSO), and the finalDMSO concentration in the assay was 0.5%. Following incubation,quantitation of proliferation was carried out by cell countingusing a Coulter counter.

K562 cell mitochondrial preparation. K562 cells were incubated with compound or vehicle for 5 days,and cells were collected, washed once with phosphate-bufferedsaline (PBS), resuspended in 1 ml of TKM buffer (10 mM TrisHCl, pH 7.5, 10 mM KCl, and 0.15 mM NaCl), and homogenized witha hand-held homogenizer (Ultra-Turrax; IKA Labotechnik) at setting3 for 15 s. An aliquot (300 µl) of TKM buffer containing1 M sucrose was added, and the samples were centrifuged at 1,000x g for 5 min. The supernatant fraction was respun at 5,000x g for 20 min. The mitochondrial pellet was resuspended inTKM buffer containing 0.25 M sucrose in a 100-µl volume.Mitochondrial protein content was measured using the bicinchoninicacid protein assay kit (Pierce).

Western blot analysis. Mitochondrial proteins (10 µg) were electrophoresed on10% Criterion gels (Bio-Rad) and transferred to polyvinylidenedifluoride membranes (Bio-Rad). After blocking overnight at4°C with 1% bovine serum albumin (BSA) and 1% dry milk inTris-buffered saline (Bio-Rad) containing 0.1% Tween 20, themembranes were incubated with 1 µg/ml cytochrome c oxidasesubunit 1 (Cox-1) monoclonal antibody (Molecular Probes A-6403)for 3 h, followed by chemiluminescent detection (Amersham ECLplus) according to the manufacturer's instructions. Membraneswere stripped by RE-blot Plus strong solution (Chemicon 2504)and incubated with 1 µg/ml Tom-20 polyclonal antibody(Gift from B. Wattenberg, University of Louisville), followedby chemiluminescent detection. The Cox-1 bands and Tom-20 bandsin the X-ray films were scanned by a Personal Densitometer SL(Molecular Dynamics) and quantitated by Quantity One (Bio-Rad).

Rat heart mitochondria preparation. The procedure for mitochondria preparation and the protein synthesisassay was adapted from that described by McKee et al. (29).All experiments involving animals were carried out in compliancewith national legislation and subject to local ethical review.Briefly, male Sprague-Dawley rats ( $~$ 250 g) were anesthetizedby inhalation of isoflurane and injected with heparin IV, andthe heart was removed and rinsed in ice-cold MSE buffer (220mM mannitol, 70 mM sucrose, 50 mM HEPES, 2 mM EGTA, pH 7.2)until free of blood. Hearts were minced in MSE buffer containing10 mg/ml Nagarse (Sigma), washed once, and homogenized in MSE-Nagarsebuffer using a Brinkman polytron. Protease inhibitor (Complete;Boehringer) then was added to the homogenate. The homogenatewas centrifuged at 900 x g for 3 min, and the resulting supernatantwas centrifuged at 10,000 x g for 10 min. The pellet was resuspendedin 1.0 ml of ice-cold MSE buffer at a ratio of 20 ml/g ventriculartissue and centrifuged at a speed of 900 x g for 3 min at 4°C.The resulting supernatant was centrifuged at 10,000 x g for10 min at 4°C. Pellets were resuspended in MSE buffer andprotein was determined (Bio-Rad).

Mitochondrial protein synthesis (MPS) assay. Mitochondrial protein synthesis was measured in a 50-µltotal volume containing 90 mM KCl, 4 mM MgSO₄, 2.5 mM KH₂PO₄,50 mM HEPES, pH 7.2, 0.25 mM amino acids without methionine,20 mM glutamate, 0.5 mM malate, 2 mM ADP, 1 mg/ml BSA (SigmaA3059), 0.1 mg/ml cycloheximide (Sigma D-335715), 1 µMcold methionine, [³⁵S]methionine (40 µCi/ml), and mitochondrialprotein (1 mg/ml) in the presence or absence of compound. Thefinal concentration of DMSO was 1% in the assay. Incubationwas carried out at 30°C for 90 min followed by additionof 200 µl 3% sulfosalicylic acid in 0.1 mM EDTA to precipitateproteins. Proteins were harvested onto filtermats (Wallac 1205--404)using a Tomtec harvester followed by counting in a Wallac BetaPlatescintillation counter. The 50% inhibitory concentrations (IC₅₀)were determined by GraphPad Prism software, using a sigmodialdose response equation with a fixed bottom.

RESULTS

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Results

Eperezolid and linezolid (Fig. 1) are oxazolidinones that areactive against gram-positive bacteria, and mechanistic studieshave established that they inhibit bacterial protein synthesis(41). Their IC₅₀ values for inhibiting [¹⁴C]leucine incorporationin the membrane-permeable E. coli strain UC6782 are approximately1 µM, while their MICs at which 90% of the isolates testedare inhibited (MIC₉₀) against gram-positive bacteria such asStaphylococcus aureus is 4 µg/ml (12 µM) (41). Thesecompounds were tested in an in vitro mitochondrial protein synthesisassay. Isolated rat heart mitochondria were incubated with linezolidor eperezolid in the presence of [³⁵S]methionine, and acid-precipitableradioactivity was measured following a 90-min incubation (Fig.2). Both compounds inhibited synthesis, with IC₅₀ values of9.5 ± 1.5 µM (n = 2) and 16 ± 2 µM(n = 2) for eperezolid and linezolid, respectively. The valuefor linezolid is similar to that reported by Sciotti et al.(39).

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FIG. 1. Structures of oxazolidinones linezolid and eperezolid.

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FIG. 2. Linezolid and eperezolid inhibit rat heart mitochondrial protein synthesis. Mitochondrial protein synthesis was carried out in isolated rat heart mitochondria as described in Materials and Methods in the presence or absence of varying concentrations of linezolid ${blacksquare}$ or eperezolid ${blacktriangledown}$ . Results shown are the average of duplicate determinations in one experiment; error bars show the range.

Given the inhibition of mitochondrial protein synthesis andthe role of mitochondrial proteins in oxidative phosphorylationand energy metabolism, we investigated whether the compoundsaffect the growth of mammalian cells. Incubation of K562 humanleukemia cells with varying concentrations of eperezolid resultedin a time- and concentration-dependent inhibition of cell number(Fig. 3). There was a 5% to 10% difference in cell number forthe treated and control cultures at the 24-h time point. Themaximal inhibitory effect on proliferation at each time pointwas achieved with a concentration of 100 µM. After 96h of incubation with 100 µM compound, cell growth wassignificantly slowed. All the cells in the culture were viablethroughout the time course, based on trypan blue exclusion (datanot shown).

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FIG. 3. Eperezolid inhibits the growth of human K562 cells. K562 cells were incubated in the presence or absence of varying concentrations of eperezolid, and cell counts were monitored as described in Materials and Methods. A. Cells were treated with the following: ${blacksquare}$ , vehicle (0.5% DMSO); ${blacktriangleup}$ , 1.7 µM; ${blacktriangledown}$ , 5 µM; ${blacklozenge}$ , 15 µM; •, 45 µM; ${square}$ , 135 µM; or ${Delta}$ , 400 µM eperezolid. B. K562 cells were incubated with varying concentrations of eperezolid, and cell proliferation was monitored by cell counts after 120 h.

Fluorescence-activated cell sorter analysis demonstrated thatthe cells were not undergoing cell cycle arrest (data not shown).The percentage of cells in each phase of the cell cycle wasapproximately the same between control and treated cells. Rather,the major effect of the compound was to slow the growth by increasingthe doubling time of the treated cultures. The time course datawas used to plot the concentration dependency curve of growthinhibition (Fig. 3B). The IC₅₀ for growth inhibition by eperezolidin K562 cells for a 5-day exposure was 12 µM.

The inhibition of proliferation by eperezolid was reversed uponwithdrawal of compound from the growth media (Fig. 4). Cellswere grown in either 15 or 30 µM compound for 72 h followedby removal of the medium, washing, and refeeding the cells withmedium containing either vehicle (0.5% DMSO) or 15 or 30 µMeperezolid. Cells then were cultured for an additional 5 days.At the 5-day time point the cell numbers of cultures incubatedin 15 µM compound and then washed was essentially thesame as the untreated cultures. Reversibility also was observedfor cells treated with 30 µM compound, although the cellnumber did not return completely to that of untreated cellsat the 5-day time point.

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FIG. 4. Growth inhibition by eperezolid is reversible. K562 cells were grown in the presence of ${blacksquare}$ , vehicle (0.5% DMSO); ${blacktriangledown}$ , 15 µM eperezolid; or •, 30 µM eperezolid for 72 h. Cells were then washed in RPMI media and replated in media containing ${blacksquare}$ , vehicle (0.5% DMSO); ${blacktriangledown}$ , 15 µM eperezolid; ${blacktriangleup}$ , vehicle (15 µM eperezolid recovery); •, 30 µM eperezolid; or ${blacklozenge}$ , vehicle (30 µM eperezolid recovery) and cultured for an additional 5 days. Results shown are the average of triplicate determinations, and error bars show the standard errors of the means.

The effect of eperezolid on cell growth of several other celllines was investigated. Eperezolid inhibited proliferation ofhuman HEK cells and Chinese hamster ovary (CHO) cells, as wellas mouse BB88 cells (data not shown). Direct comparison of theeffect of the oxazolidinone on the proliferation of differentcell lines is complicated by the differences in culture conditionsand doubling times; however, the cells were scored during thelogarithmic phase of growth. The IC₅₀ value for eperezolid variedsomewhat between the various cell types and was 63 and 20 µMfor CHO and HEK cells, respectively. In all cases, a progressiveslowing of growth due to an increase in cell doubling time wasobserved. These experiments demonstrate that the antiproliferativeeffect of the oxazolidinones was not cell type specific.

The oxazolidinones inhibit mitochondrial protein synthesis inan in vitro system and they inhibit cell proliferation, butthe link, if any, between these two activities has not beeninvestigated. Therefore, the level of mitochondrial proteinsin K562 cells was examined to determine whether oxazolidinonesinhibited mitochondrial protein synthesis in intact cells. K562cells were incubated with varying concentrations of eperezolidfor 5 days under the same conditions as in Fig. 3, and the cellswere harvested and mitochondria were isolated. This mitochondrialfraction was run on Western blots and probed with a monoclonalantibody to subunit 1 of cytochrome oxidase, which is 1 of the13 proteins encoded by the mitochondrial genome (Fig. 5). Tonormalize for mitochondrial protein loading, after exposureto the Cox-1 antibody the blots were stripped and reprobed withan antibody to Tom-20, an outer mitochondrial membrane proteinencoded by the nucleus. Tom-20 protein levels were relativelyconstant across the gel. However, Cox-1 protein expression decreasedwith increasing concentrations of eperezolid, and 25 µMeperezolid resulted in a greater than 90% decrease in Cox-1levels. As a control, cells also were treated with 100 µMchloramphenicol, a known mitochondrial protein synthesis inhibitor.Almost complete inhibition of Cox-1 levels was observed withthis compound, which also inhibited the proliferation of thecells (data not shown). The decreased level of mitochondrialprotein was not specific for Cox-1, since [³⁵S]methionine labelingof mitochondria isolated from oxazolidinone-treated cells showeddecreased incorporation of radioactivity into all 13 mitochondrialproteins (data not shown).

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FIG. 5. Eperezolid decreases mitochondrial Cox-1 protein levels. K562 cells were treated with vehicle (0.5% DMSO), 100 µM chloramphenicol, or varying concentrations of eperezolid for 5 days, and mitochondria were isolated. Aliquots of mitochondrial protein (10 µg) were electrophoresed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes, and probed with an antibody to Cox-1 (top gel). The blot was stripped and reprobed with an antibody to Tom-20 (bottom gel). Lane 1, vehicle; 2, 3.1 µM eperezolid; 3, 6.2 µM eperezolid; 4, 12 µM eperezolid; 5, 25 µM eperezolid; 6, 50 µM eperezolid; 7, 100 µM eperezolid; 8, 100 µM chloramphenicol. The 30-kDa and 40-kDa molecular mass markers are designated on the left.

Rho zero ( ${rho}$ ⁰) cells are derived from a human osteosarcoma parentcell line (143B), which was depleted of mtDNA by culturing andselection in the presence of ethidium bromide. These cells cannotcarry out oxidative phosphorylation, require pyruvate and uridinefor growth, and have glycolysis as their only source of ATP(23). Rho zero and parent 143B cells were treated with variousconcentrations of eperezolid, and the cell number was quantitated(Fig. 6). 143B cells had a doubling time of 17 ± 1.0(n = 4) h, while that of the rho zero cells was longer, 24 ±0.2 (n = 4) h. As expected, eperezolid resulted in a time- andconcentration-dependent decrease in 143B cell numbers. At the72-h time point, the IC₅₀ value was $~$ 20 µM. In contrast,growth of rho zero cells was not inhibited by treatment withany concentration of the two mitochondrial protein synthesisinhibitors, eperezolid or chloramphenicol. As a control, todemonstrate that rho zero cells are not resistant to all typesof growth arrest, cells were treated with etoposide, a topoisomeraseII inhibitor, or with cycloheximide. As expected, both thesecompounds inhibited growth of rho zero cells, as well as the143B cells.

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FIG. 6. Eperezolid inhibits cell proliferation in 143B cells but not in rho zero cells. Parent 143B (A) or rho zero (B) cells were incubated with ${square}$ , vehicle (0.5% DMSO); ${blacksquare}$ , 3 µM eperezolid; ${blacktriangleup}$ , 10 µM eperezolid; ${blacktriangledown}$ , 100 µM eperezolid; •, 1.7 µM etoposide; ${diamond}$ , 1.75 µM cycloheximide; or ${circ}$ , 30 µM chloramphenicol, and cells were counted at 48, 72, 96, and 120 h.

DISCUSSION

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Discussion

The oxazolidinones inhibit bacterial protein synthesis, andresults from resistance mutations and cross-linking experimentsdemonstrate that they interact with nucleotides in domain Vof 23S rRNA, which contains the peptidyl transferase center.Details concerning the structure, mechanism(s), and activitiesof this region are emerging (17, 32). The crystal structuresof the individual 50S and 30S subunits as well as the functional70S complex have been solved (3, 8, 9). Results from cryoelectronmicroscopy (cryo-EM) studies have shown the conformational changesthis region undergoes during peptide bond formation and translocation,including ratchet-like rotations of the 30S and 50S subunits(16). Proper alignment of the two tRNA substrates appears tobe the driving force for peptide bond formation (42). Mutationaland kinetic analysis demonstrated that nucleotides A2602, A2451,U2585, and C2063, which are located in the innermost layer ofthe active site, are critical for the catalysis of peptide release(34, 48).

In contrast to our understanding of the prokaryotic 70S ribosome,less is known about the structural details of the mitochondrial55S and the mammalian 80S ribosomes. cryo-EM visualization ofthe 80S ribosome was first reported in 2004 with a resolutionof 17 to 20 Å, and more recently, that structure has beenrefined to 9.5-Å resolution (18, 43). These structuresdo not provide details of the peptidyl transferase center ofthe 80S ribosome. Biochemical experiments comparing 70S, 55S,and 80S ribosomes in a peptide "fragment reaction" showed thatantibiotics such as chloramphenicol and lincomycin inhibit bacterialprotein synthesis with no significant effect on mammalian 80Sprotein synthesis (12). Although many aspects of protein translationare conserved between eukaryotes and prokaryotes, the specificityof these antibiotics is believed to result from key differencesbetween these ribosomes.

In the case of the mitochondrial ribosome, results from cryoelectronmicroscopy (cryo-EM) along with extensive physical characterizationof the mitoribosome have shown that there are a number of markeddifferences between bacterial and mitochondrial ribosomes (33,40). The mitoribosome is larger in size and consists of 69%protein and 31% RNA compared to the 33% protein and 67% RNAof the bacterial ribosome. Of the approximately 80 ribosomalproteins of the mitoribosome, only half of them have bacterialhomologs, and the homologous proteins are often larger in sizethan their bacterial counterparts. As a result, the mitoribosomecontains several distinct topological differences. For example,the mRNA entry site is composed primarily of mitoribosome-specificproteins, which form a triangular gatelike structure that isnot present in the bacterial ribosome. Also, the cryo-EM mapof the polypeptide tunnel of the mitoribosome suggests thatthere are two possible pathways for proteins to exit (40).

Despite these differences, there are certain critical featuresof the ribosome that are conserved between bacteria and mitochondria(1). Mears et al. carried out comparative sequence analysesbetween rRNA of archaea, bacteria, eukaryotic nuclear, mitochondrial,and chloroplast ribosomes (30). The areas of greatest similaritiesmap to key functional domains, including the mRNA binding site,the peptidyl transferase center, and the subunit interface,all of which contain the highest number of "universally conserved"nucleotides. An important aspect, however, is areas where thereis the least similarity are primarily on the periphery of therRNA three-dimensional structure in regions that may not beessential for protein translation. In addition, the polypeptideexit tunnel is considerably shortened in the mitochondrial ribosomecompared to the bacterial ribosome.

Given the high degree of homology in the peptidyl transferasecenters, it is not surprising that antibiotics that inhibitbacterial protein synthesis also inhibit mitochondrial proteinsynthesis. Denslow and O'Brien demonstrated that chloramphenicolinhibited E. coli 70S and bovine mitochondrial 55S peptidyltransferase activity with IC₅₀ values of 40 and 100 µM,respectively. Apparent dissociation constants were calculatedfrom Scatchard plots of the inhibition data. The results demonstratedthat E. coli ribosomes have a somewhat higher affinity (100µM) for chloramphenicol than mitochondrial ribosomes (29µM) (11). The ability of antibiotics to inhibit mitochondrialprotein synthesis has been used as evidence to support the ideathat key structural and functional features are shared betweenbacteria and mitochondrial ribosomes (11, 21). The crystal structuresof several different antibacterials with the bacterial 50S subunithave been reported and demonstrate that the compounds all interactat or near the sites of peptide bond formation. The crystalstructure of chloramphenicol binding to Deinococcus radioduransis different than that of its binding to Haloarcula marismortui,although it is bound in the peptidyl transferase center in bothstructures (19, 38). The ability of eperezolid to compete forchloramphenicol binding (27) as well as the resistance mappingindicates that the oxazolidinones bind in the peptidyl transferaseregion as well (47).

The oxazolidinones inhibited cell proliferation in a varietyof cell types, suggesting there is no cell type specificityin their growth-inhibitory effect. The levels of the mitochondrialprotein Cox-1 decreased concomitantly with oxazolidinone treatment.However, the most direct demonstration of the role of mitochondrialprotein synthesis inhibition in the antiproliferative effectof the oxazolidinones was demonstrated in the rho zero cellexperiments. Growth of the parent 143B cells was inhibited,but not that of the rho zero cells. The rho zero cells containno mtDNA and synthesize no mitochondrial proteins, and in thesecells the oxazolidinones had no effect on growth.

The role of mitochondrial protein synthesis inhibition in cellproliferation has been established in a number of studies, andthe results with the oxazolidinones are consistent with thoseobservations. Treatment with eperezolid resulted in a time-dependentlengthening of cell doubling time, consistent with a progressiveloss of oxidative phosphorylation and an increasing relianceon glycolysis. A similar observation was reported by Jazayeriet al., who induced a rho zero phenotype in cells by inducibleexpression of a dominant-negative form of DNA polymerase- ${gamma}$ (22).This resulted in a depletion of mitochondrial DNA, loss of mitochondrialproteins and function, and an increase in cell doubling time.Storrie and Attardie treated HeLa cells with the mitochondrialprotein synthesis inhibitor chloramphenicol and observed a slowingof the growth rate, with the cells ultimately entering a stationaryphase. They concluded that this effect on cell growth was theresult of decreased respiratory rate capacity in the cells,as the mitochondrial proteins were no longer being synthesized(44).

We have shown here a marked effect of the oxazolidinone eperezolidon cell growth. The lack of effect of eperezolid on the proliferationof rho zero cells demonstrates that the effect of the compoundis due to mitochondrial protein synthesis inhibition. Treatmentof wild-type cells with eperezolid in effect causes them toapproach a rho zero phenotype, as seen by an increase in thecell doubling time and a loss of mitochondrial proteins. Culturedcells, as used here, have been shown to derive much of theirenergy from glycolysis when grown in high-glucose medium (36).However, elimination of the contribution of oxidative phosphorylationby mitochondrial protein inhibition by eperezolid or by chloramphenicolor dominant-negative DNA polymerase (as outlined above) nonethelessimpacts cell metabolism as demonstrated by the increase in doublingtime.

Deficits in respiratory chain function resulting from mtDNAmutations are known to affect a variety of different tissues.The degree to which a particular cell type or tissue is affectedreflects the specific "threshold capacity" (35). If a giventissue has a high threshold capacity, then a greater loss inmitochondrial proteins can be sustained without a loss in oxidativephosphorylation function. This has been shown for cultured celllines as well as for different tissues (35, 46). For example,the threshold value for complex IV is higher (around 86%) inthe liver, kidney, and brain compared to the value in muscleand heart (around 67%) (37). In the case of the oxazolidinones,erythroid precursor cells may be particularly sensitive to mitochondrialprotein synthesis inhibition, resulting in the anemia observedin some patients with long-term treatment. In fact, the reversibleanemia observed with the mitochondrial protein synthesis inhibitorchloramphenicol or with human immunodeficiency virus reversetranscriptase inhibitors (which inhibit mtDNA synthesis andthus decrease mitochondrial protein levels) also has been attributedto a loss in mitochondrial function in hematopoietic cells (6,14, 26, 49).

ACKNOWLEDGMENTS

The contributions of Gregory N. Cosma, Michael A. Fisher, andDavid P. Blakeman to the mitochondrial protein synthesis assayare gratefully acknowledged.

FOOTNOTES

* Corresponding author. Mailing address: 2800 Plymouth Rd., Ann Arbor, MI 48105. Phone: (734) 622-7343. Fax: (734) 622-7158. E-mail: karen.l.leach{at}pfizer.com.

REFERENCES

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