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Volume 270, Number 35, Issue of September 01, pp. 20668-20676, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Alteration of Mitochondrial Gene Expression and Disruption of Respiratory Function by the Lipophilic Antifolate Pyrimethamine in Mammalian Cells (*)

(Received for publication, March 28, 1995; and in revised form, June 13, 1995)

Hannah Sprecher ,  Haim M. Barr ,  Jacob I. Slotky ,  Maty Tzukerman ,  Gera D. Eytan ,  Yehuda G. Assaraf (§)

From the Department of Biology, Technion-Israel Institute of Technology, Haifa 32 000, Israel

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

To clone the mammalian gene(s) associated with a novel lipophilic antifolate resistance provoked by the antiparasitic drug pyrimethamine (Assaraf, Y. G., and Slotky, J. I.(1993) J. Biol. Chem. 268, 4556-4566), differential screening of a cDNA library from pyrimethamine-resistant (Pyr) cells was used. This library was screened with total cDNA from wild-type and Pyr cells. Surprisingly, several differentially overexpressed cDNA clones were isolated from Pyr cells, many of which mapped to the mitochondrial genome. Several lines of evidence establish mitochondria as a new target for the cytotoxic activity of pyrimethamine. (a) At geq10 µM, pyrimethamine inhibited mitochondrial respiration in viable wild-type cells. (b) Electron microscopy revealed degenerated mitochondrial membrane cristae in Pyr cells. (c) Some mitochondrially encoded transcripts were prominently elevated, whereas the normally stable 12 S/16 S rRNA was decreased in Pyr cells. (d) Metabolic pulse-chase labeling suggested an increased turnover rate of mitochondrially synthesized proteins in Pyr cells. (e) The specific activity of the key respiratory enzymatic complex cytochrome c oxidase was reduced by 6-fold in Pyr cells. (f) Consequently, the rate of respiration in intact Pyr cells was reduced by 3-fold. We conclude that pyrimethamine and possibly lipophilic analogues of methotrexate possess a folinic acid nonrescuable toxicity involving disruption of mitochondrial inner membrane structure and respiratory function, thereby establishing a new organellar target for the cytotoxic effect elicited by lipid-soluble antifolates.

INTRODUCTION

Antifolates comprise a large family of chemotherapeutic agents displaying antibacterial, antiparasitic, and antineoplastic activity (1) . The first class of antifolates to have been described for clinical use is represented by methotrexate (MTX), (^1)a hydrophilic folate antagonist that inhibits the target enzyme dihydrofolate reductase. MTX interferes with the biosynthesis of purines, thymidylate, and glycine, thereby leading to inhibition of DNA synthesis and cell death. Aminopterin, a structural homologue of MTX, was first successfully used in the treatment of childhood leukemia(2) . Low-dose MTX is now also considered an efficient anti-inflammatory agent in the treatment of various autoimmune disorders.

The use of MTX as a chemotherapeutic agent has been hampered by the frequent emergence of drug resistance phenomena due to alterations in MTX transport(3, 4) , increased dihydrofolate reductase activity(5) , reduced dihydrofolate reductase affinity for antifolates (6) due to structural alterations in dihydrofolate reductase originating from single amino acid substitutions(7, 8, 9) , and reduced cellular retention of MTX polyglutamates(10, 11, 12) .

Novel lipophilic folate antagonists, capable of accumulating in mammalian cells by diffusion and/or via facilitated diffusion, were consequently designed in an attempt to overcome modes of MTX resistance due to altered transport and impaired intracellular retention(13) . Some of these agents, including pyrimethamine (Pyr) and trimethoprim, are widely used as antiparasitic and antibacterial drugs, respectively(1) . Unfortunately, in various subsequent studies, we demonstrated the development of lipophilic antifolate resistance as a result of dihydrofolate reductase and/or P-glycoprotein overexpression (14, 15) . P-glycoprotein is an integral component of the mammalian plasma membrane that functions as an energy-dependent efflux transporter of multiple hydrophobic cytotoxic agents, thereby leading to multidrug resistance(16, 17) .

In the course of drug resistance studies performed with Chinese hamster ovary (CHO) cells, we have recently described a novel mechanism of resistance to 2,4-diaminopyrimidine (DAP) lipophilic antifolate antibiotics including Pyr(18) ; cross-resistance was extended to trimetrexate (19) and piritrexim(20) , both of which are lipid-soluble analogues of MTX(13) . This lipophilic antifolate resistance was a result neither of qualitative or quantitative alterations in dihydrofolate reductase activity nor of acquisition of the P-glycoprotein-dependent multidrug resistance phenotype (18) . To characterize the biochemical mechanism underlying this lipophilic antifolate resistance, we have used the assay of antifolate competition of fluorescein MTX labeling and flow cytometry(4) . Thus, while fluorescein MTX labeling was competitively displaced from wild-type CHO cells by lipophilic antifolates, it was retained in Pyr-resistant (Pyr) cells even in the presence of high extracellular concentrations of lipophilic antifolates (e.g. DAP). In contrast, MTX was equally potent in displacing fluorescein MTX from wild-type and Pyr cells. These results led us to the conclusion that Pyr cells fail to accumulate DAP and lipophilic analogues of MTX or, alternatively, that DAP are sequestered in subcellular acidic compartments (e.g. lysosomes) in Pyr cells; this putative intravesicular concentration of DAP lipophilic antifolates that behave like hydrophobic weak bases would render them inaccessible to the cytosolic target enzyme dihydrofolate reductase.

Pyr cells retained lipophilic antifolate resistance even after a long-term growth (1200 cell doublings) under nonselective conditions, suggesting that stable genomic changes underlie this lipophilic antifolate resistance phenotype. Toward the elucidation of the mechanism underlying this novel resistance to lipophilic antifolates, we have used differential cDNA library screening. This technique allows for the identification of transcripts present at different levels in paired cell lines (i.e. wild-type and drug-resistant cells). Species of mRNA expressed in similar amounts in both cell types are effectively canceled out, whereas transcripts present at different levels are positively selected. We have therefore used this approach to differentially screen a cDNA library constructed from Pyr cells, using total cDNA derived from Pyr and AA8 cells as probes. Surprisingly, a consistent differential overexpression of a subset of genes encoded by the mitochondrial genome was positively selected in Pyr cells. We further investigated the mitochondrial involvement in the development of resistance to DAP and lipophilic antifolates both at the molecular and physiological levels. Our findings establish that changes in mitochondrial gene expression, structure, and respiratory function are associated with the development of resistance to DAP and lipophilic analogues of MTX. This study therefore establishes mitochondria as a target organelle for the cytotoxic effect elicited by lipophilic antifolates.

EXPERIMENTAL PROCEDURES

Cell Culture

A clonal subline (C11) of CHO wild-type AA8 cells was maintained under monolayer conditions in alpha-minimal essential medium (Biological Industries, Beth Haemek, Israel) containing 5% dialyzed fetal calf serum (Biological Industries), 2 mM glutamine, 100 units/ml penicillin G, and 100 µg/ml streptomycin sulfate (Sigma) at 37 °C in a humidified atmosphere of 5% CO(2). Pyr cells were maintained in the same medium in the presence of 100 µM Pyr. Pyr cells were established using a prolonged multiple step selection to gradually increasing Pyr concentrations initiated at 0.1 µM (the 50% lethal dose for parental AA8 cells) and terminated at 100 µM(18) . Pyr cells displayed a stable (even after 1200 cell doublings of growth under nonselective conditions) 1000-fold resistance to Pyr, and their clonogenic plating efficiency in 130 µM Pyr was 100%. Suspension cultures grown in spinner flasks (Cytostir, Kontes) were maintained in the same medium with the addition of 20 mM HEPES at pH 7.4.

Construction of the cDNA Library

Total RNA was isolated from Pyr cells by acid-guanidinium thiocyanate/phenol/chloroform extraction(21) . Poly(A) RNA was selected from total RNA using a Dynabeads mRNA purification kit (Dynal, Inc.). To synthesize cDNA appropriate for directional cloning, 5 µg of Pyr poly(A) RNA was primed with an oligo(dT) primer containing a 3`-end XhoI site using a commercial cDNA synthesis kit (Stratagene). After ligation of EcoRI linkers to the cDNA, it was digested with XhoI and finally ligated into an EcoRI/XhoI-digested uni-ZAP vector (Stratagene). Ligated DNA was then packaged in vitro using a commercial extract (Stratagene). The cDNA library contained 7.5 10^5 primary plaques.

Differential Screening, Isolation of cDNA Clones, and DNA Sequencing

For screening by differential hybridization, XL1 Blue cells (Stratagene) were transfected with the phages and plated at a density of 5 10^4 plaque-forming units/15-mm Petri dish. Phage DNAs were then blotted onto duplicate filter membranes (Schleicher & Schuell). The blots were hybridized in 50% formamide, 6 SET (0.1 M NaCl, 5 mM EDTA, 0.1 M Tris (pH 7.8)), 0.5% SDS, and 10 µg/ml denatured salmon sperm DNA at 42 °C using 10^6 cpm/ml P-labeled cDNA probe. This P-labeled cDNA probe (10^8 cpm/µg of DNA) was reverse-transcribed using Moloney murine leukemia virus reverse transcriptase (Promega) from poly(A) RNA samples prepared from AA8 and Pyr cells. Plaques expressing differential signals were picked and rescreened two additional rounds. Purified double-stranded cloned cDNAs were sequenced with Sequenase (U. S. Biochemical Corp.) using pBluescript T(3) and T(7) promoter sequences as primers.

Northern Blot Analysis

Poly(A) RNA was fractionated on a 1.5% formaldehyde-agarose gel, blotted onto a GeneScreen Plus nylon membrane (DuPont NEN), and UV-cross-linked. Isolated cDNAs were labeled by random priming (22) using an oligolabeling kit (U. S. Biochemical Corp.) and [alpha-P]dATP (3000 Ci/mmol; DuPont NEN). Blot hybridization and high stringency post-hybridization washes were carried out according to the manufacturers' instructions (DuPont NEN). Linear exposures of the Northern blots were quantitated using a CliniScan 2 scanning densitometer (Helena Laboratories, Beaumont, TX).

Transmission Electron Microscopy

For transmission electron microscopy, exponentially growing cells maintained in growth medium (i.e. Pyr-containing for Pyr cells) under suspension culture conditions were harvested, washed with PBS, and fixed for 2 h in 2.5% glutaraldehyde in PBS. Following an overnight fixation in 1% OsO(4) in PBS, cells were washed, dehydrated, and embedded in an Epon mixture. Thin sections were stained with uranyl acetate (Merck) and examined using a Jeol 100B electron microscope.

Succinate-2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium Reductase Activity Assay

Exponentially growing parental AA8 and Pyr cells were detached by trypsinization and counted. Duplicate cell lysates containing 33-560 µg of protein were gently agitated for 15 min at 37 °C in buffer containing 50 mM potassium phosphate (pH 7.4), 0.1% 2-(p-iodophenyl)-3-(p-nitrophenyl)-3-phenyltetrazolium chloride, 50 mM sodium succinate, and 25 mM sucrose in a total volume of 1 ml. Following incubation, ice-cold trichloroacetic acid was added to a final concentration of 5%, and water-soluble formazan was extracted with 4 ml of ethyl acetate, after which a colorimetric detection at 490 nm was performed using a Gilford 2400 spectrophotometer. Protein determination was performed according to the method of Bradford(23) .

Cytochrome c Oxidase Activity Assay

Cytochrome c oxidase activity was assayed by following the decrease in absorbance at 550 nm during the incubation of cell lysates with 0.1 mM ferrocytochrome c, 10 mM potassium phosphate (pH 7.0) at 37 °C every 15 s.

Purification of Mitochondrial DNA

AA8 and Pyr cells were grown under suspension culture conditions in growth medium containing 20 mM HEPES at pH 7.4. Exponentially growing cells in 3-liter spinner flasks were harvested and counted. Following washing with PBS, 4 10^8 cells were swollen in a hypotonic buffer and disrupted using a Dounce homogenizer, and mitochondria were isolated by differential centrifugation from the post-nuclear fraction as described (24) . Based on known cell equivalents (typically 4 10^8), mitochondrial DNA (mtDNA) was then isolated from the SDS-lysed mitochondria using cesium chloride density gradient centrifugation (24) . Closed circular mtDNA identified as a lower ethidium bromide band was quantitatively collected, precipitated, dialyzed, and quantitated. The purity of the mtDNA was verified by restriction enzyme digestion followed by agarose gel electrophoresis as well as using mitochondrial specific and nuclear specific probes.

Analysis of Mitochondrial Protein Synthesis

Metabolic labeling of mitochondrial proteins was performed according to Chomyn et al.(25) . Samples of 10^6 AA8 or Pyr cells were plated in 5-cm Petri dishes and incubated overnight at 37 °C. After washing the cells with PBS, 5 ml of prewarmed methionine-free growth medium containing either 100 µg/ml cycloheximide or 200 µg/ml emetine was added to each plate. Then, following a 10-min preincubation at 37 °C, L-[S]methionine (specific activity > 1000 Ci/mmol; Amersham Corp.) was added to a final radioactive concentration of 20 µCi/ml, and monolayer cells were labeled for 2 h. In pulse-chase experiments, the [S]methionine-containing medium was replaced by growth medium containing 1 mM nonlabeled L-methionine and further incubated for up to an additional 6 h. Cells were then trypsinized, washed, and lysed with 1% SDS. Samples containing 50 µg of protein were fractionated by electrophoresis on 15-20% exponential polyacrylamide gels containing SDS (Novex).

Lactic Acid Production Assay

Glycolytic activity was estimated in AA8 and Pyr cells by determination of lactate levels in the culture medium. 5 10^4 (AA8) or 1 10^5 (Pyr) cells were plated in 6-cm plates in growth medium. Culture medium aliquots of 1 ml were drawn from individual plates at 12-h intervals and centrifuged, after which the supernatants were frozen until analysis. Parallel cultures were used for the monitoring of cell numbers in each kinetic point. Growth medium samples (0.2 ml) were assayed for lactate levels using a lactate dehydrogenase clinical kit (Boehringer Mannheim) that follows the irreversible production of NADH spectrophotometrically using a COBAS MIRA clinical analyzer.

Oxygen Consumption

Suspension cultures grown in spinner flasks were maintained in growth medium containing 20 mM HEPES at pH 7.4. Pyr cells were maintained in the same medium in the presence of 100 µM Pyr. Exponentially growing cells were harvested by sedimentation, counted, and adjusted to 10^7 cells/ml in serum-lacking growth medium. Respiration rates were measured in a 1-ml oxygen consumption chamber and recorded on a Kipp & Zonen paper recorder.

RESULTS

Differential Screening and Isolation of cDNA Clones

Poly(A) RNA was isolated from Pyr cells, converted into cDNA using reverse transcriptase and oligo(dT) primers, and directionally cloned into a uni-ZAP vector. Approximately 6 10^5 cDNA clones were distributed over 12 Petri dishes. To screen for the cDNA clones differentially expressed in Pyr cells, duplicate filters were hybridized with labeled cDNA prepared from parental AA8 or Pyr cells. Initially, 75 cDNA clones with differential signals were picked up, of which 17 scored positive after two additional rounds of screening. The partial 5`- and 3`-end sequences of these clones were determined and compared with the nucleotide sequence data base of the National Center for Biotechnology Information using Blast E-mail Server, and the results are summarized in Table 1. Surprisingly, nine of these differential cDNA clones turned out to be encoded by a restricted region of the mitochondrial genome (Fig. 1). The eight remaining differential cDNA clones were all nuclear encoded (Table 1): two cDNA clones were found to correspond to ferritin (an intracellular iron-binding protein), two cDNA clones were identified as ribosomal proteins L-35A and L-11, and the four remaining cDNA clones showed no significant homology to sequences present in current data bases.

Figure 1: Alignment of the cloned cDNAs with the mitochondrial transcription map. Light (L) and heavy (H) strands of mammalian mitochondrial DNA are represented as thin and thicksolidbars, respectively. Genes for the various aminoacyl-tRNAs are designated by three-letter abbreviations. The various differential mitochondrial cDNA clones isolated are aligned in respect to the transcription map. ND, NADH-coenzyme Q oxireductase subunits 1-6; CO, cytochrome c oxidase subunits 1-3; ATPase6,8, subunits 6 and 8 of the ATP synthase complex; Cytb, cytochrome b. The various mitochondrial cDNA probes used are also shown.


To examine whether the differential cDNA clones identified were indeed elevated in Pyr-resistant cells, Northern blot analysis was performed with poly(A) mRNA isolated from wild-type AA8 and Pyr cells using the following mitochondrial probes (Fig. 1): 12 S/16 S rRNA (a physiologically stable mitochondrial transcript) and transcripts of the electron transport chain including mitochondrial cytochrome c oxidase I, cytochrome c oxidase III/ATPase 6,8, and NAD dehydrogenase 4/4L. The differentially appearing cDNA of ferritin was also used as a nuclear encoded control. Fig. 2shows that the mRNA levels of the three mitochondrial electron chain transcripts cytochrome c oxidase I, cytochrome c oxidase III/ATPase 6,8, and NAD dehydrogenase 4/4L as well as the nuclear encoded ferritin were significantly elevated in Pyr cells relative to parental AA8 cells. In contrast, reprobing the Northern blots with a 12 S/16 S cDNA sequence, a physiologically stable mitochondrial transcript, revealed a consistent decrease in its RNA levels (Fig. 2). Ethidium bromide staining of the formaldehyde-agarose gels confirmed that the actual amounts of RNA being analyzed were similar in AA8 and Pyr cells (Fig. 2). Scanning densitometric analysis of linear exposures of the Northern blots was performed to quantify the changes in mitochondrially encoded and nuclear encoded transcripts (Table 2). Thus, the mRNA levels of cytochrome c oxidase I, cytochrome c oxidase III/ATPase 6,8 cDNA, and NAD dehydrogenase 4/4L as well as ferritin were elevated in Pyr cells by approximately 3-, 14-, 2-, and 12-fold, respectively (Table 2). In contrast, the normally stable transcript levels of 12 S/16 S rRNA were decreased by 2.5-fold in Pyr cells relative to wild-type cells (Table 2). Of special notice was the finding that although the mature 0.9-kb cytochrome c oxidase III/ATPase 6,8 transcript (26) was only 2-3-fold elevated in Pyr cells, its primary 2-kb transcript strikingly accumulated in these cells ( Fig. 2and Table 2). The lower molecular size transcript corresponded to the mature mRNA of cytochrome c oxidase III and ATPase 6,8(26) . Thus, the differentially increased abundance of the transcripts of the electron transport chain along with the decreased transcript levels of the otherwise stable 12 S/16 S rRNA suggest an altered processing and/or turnover rate of the mitochondrial primary transcripts.

Figure 2: Northern blot analysis of poly(A) RNA from parental AA8 and Pyr cells probed with the cloned cDNAs. Poly(A) RNA (2 µg/lane) isolated from wild-type or Pyr cells was denatured and fractionated by formaldehyde-agarose gel electrophoresis, transferred to GeneScreen Plus, and hybridized with the various mitochondrial cDNA probes specified in Fig. 1. The middlepanels present blots shown in the upperpanels that were reprobed with the specified mitochondrial cDNA sequences. The lower panels provide an ethidium bromide staining of the gels mainly showing the poly(A)-deficient 28 S rRNA band (the minute amounts of which were coisolated with the poly(A) mRNA); this was used to confirm that similar amounts of RNA were being analyzed. Transcript size was estimated using an RNA ladder (Life Technologies, Inc.).


Respiration in Intact Parental AA8 and PyrCells

Based on these data of altered mitochondrial gene expression, we wanted to examine whether or not Pyr has any deleterious effect on the structure and function of mitochondria. Toward this end, respiration rates were measured in intact parental AA8 cells and their Pyr subline by following oxygen consumption polarographically. Given that the cytotoxic effect of hydrophilic and lipophilic antifolates is achieved only after days of exposure required for the depletion of intracellular tetrahydrofolate polyglutamates and that respiration rates were measured here within minutes, any effect detected on whole cell respiration is therefore authentic. Fig. 3A shows that the mean (n = 12-19 independent experiments ± S.D.) respiration rates in parental AA8 and Pyr cells were 28.6 ± 8.6 and 10.3 ± 3.3 ng atoms of oxygen/min, respectively; thus, the respiration rate in Pyr cells suspended in Pyr-containing medium was 3-fold lower than that obtained with their parental AA8 cells. We have therefore examined the effect of Pyr on the respiration rates of AA8 cells that bear wild-type sensitivity to Pyr. Specifically, we determined the degree of coupling of mitochondrial oxidative phosphorylation by comparing the respiration rates in oligomycin-inhibited cells in the presence or absence of the uncoupler 2,4-dinitrophenol (DNP) (Fig. 3B)(27) . Parental AA8 cells were first exposed to oligomycin (an inhibitor of mitochondrial ATP synthesis), which consequently brings about a marked decrease in the rate of respiration (Fig. 3B); the consecutive addition of the uncoupler DNP led to the uncoupling of oxidative phosphorylation, thereby leading to increased rates of respiration (Fig. 3B). Thus, the greater the DNP-induced increase in the uncoupled respiration rates of the oligomycin-inhibited mitochondria, the tighter the coupling and the more active were the enzymatic complexes of oxidative phosphorylation prior to analysis(27) . The addition of 10, 20, and 50 µM Pyr to oligomycin-inhibited AA8 cells caused a dose-dependent inhibition of respiration as evidenced by the decreasing ability of DNP to yield an uncoupling of oxidative phosphorylation (Fig. 3C); these data suggest that at a concentration of geq10 µM, Pyr acts as an inhibitor of mitochondrial respiration in parental AA8 cells. Consistently, when oligomycin-inhibited Pyr cells were treated with 300 µM Pyr (a concentration that yields 2 LD in Pyr cells), respiration was completely blocked as evidenced by the failure of DNP to generate any measurable uncoupling effect of oxidative phosphorylation (Fig. 3D).

Figure 3: Respiration of intact wild-type AA8 and Pyr cells. Exponentially growing AA8 and Pyr cells under suspension conditions were counted, washed, and suspended in medium lacking serum (10^7 cells/ml). Respiration rates of viable wild-type AA8 and Pyr cells were determined polarographically by measuring the time-dependent consumption of oxygen from the cell suspension medium (1 ml) present in the closed chamber in the presence or absence of various mitochondrial inhibitors used at the following concentrations: oligomycin, 7.5 µM; DNP, 1 mM; and rotenone, 3 µM. A, AA8 cells (solid line) and Pyr cells grown in Pyr-containing medium (dashed line). B, AA8 cells consecutively treated (arrows) with the various mitochondrial inhibitors described above. C, oligomycin-treated parental AA8 cells further incubated in the following Pyr concentrations (arrows): 0 µM (a, solid line), 10 µM (b, dashed line), 20 µM (c, dotted line), and 50 µM (d, dashed-dotted line). D, oligomycin-treated Pyr cells further incubated in the absence (a, solid line) or presence (b, dashed line) of 300 µM Pyr.


Activity of Mitochondrial Enzymes

As mitochondrial respiration rates are a reflection of cytochrome c oxidase activity, the specific activities of this enzyme as well as of another mitochondrial enzyme, succinate dehydrogenase, were determined in AA8 and Pyr cells. Despite the increased mRNA levels of cytochrome c oxidase III, the specific activity of this enzymatic complex in Pyr cells was 6-fold lower than that present in parental AA8 cells (Table 3). In contrast, the specific activity of the nuclear encoded mitochondrial enzyme succinate dehydrogenase was essentially identical in AA8 and Pyr cells. Thus, consistent with the 6-fold reduced cytochrome c oxidase activity in Pyr cell extracts was the 3-fold decrease in the respiration rates in intact Pyr cells.

Ultrastructural Studies with AA8 and PyrCells

As these data were suggestive of impaired mitochondrial function in Pyr cells, we examined the fine structure of mitochondria in Pyr-resistant cells (Fig. 4, B and C) as compared with their parental AA8 cells (Fig. 4A). Marked ultrastructural changes were observed in mitochondria from Pyr cells (Fig. 4, B and C). The general organization of the mitochondrial inner membrane cristae into the typical transverse alignment in parental AA8 cells (Fig. 4A) was largely absent in Pyr cells (Fig. 4, B and C). The mitochondria were characterized by highly fractured and degenerated cristae; the mean number of mitochondria (from 12 microtome sections) per 3 µm^2 of cell area in AA8 and Pyr cells was 21 and 45, respectively. Furthermore, numerous intracytoplasmic multilamellar vesicles were identified in Pyr cells that could not be seen in parental AA8 cells (Fig. 4C).

Figure 4: Electron micrographs of parental AA8 and Pyr cells. Wild-type AA8 (A) or Pyr (B and C) cells maintained in growth medium (i.e. containing 100 µM Pyr for Pyr cells) under suspension conditions were harvested, washed, and prepared for transmission electron microscopy as described under ``Experimental Procedures''. N, nucleus; M, mitochondria; V, multilamellar vesicles. Magnification 30,000. The bar shown in the upper-left corner of A denotes 1 µm.


Quantitation and Analysis of Mitochondrial DNA in AA8 and PyrCells

To provide further support to this 2-fold increase in the number of mitochondria in Pyr cells, mitochondria were isolated from a known number of cells, after which cellular mtDNA content was determined by its quantitative purification using cesium chloride density gradient centrifugation. The mean mtDNA content, given as micrograms/10^8 wild-type and Pyr cells, was 11.3 and 36, respectively (Table 3). Thus, transmission electron microscopy as well as quantitation of cellular mtDNA content suggest a 2-3-fold increase in the number of mitochondria/Pyr cell as compared with parental AA8 cells (Fig. 4, A-C).

As the ultrastructural studies demonstrated a degenerated mitochondrial inner membrane structure in Pyr cells, we examined whether the mtDNA from Pyr cells remained structurally intact and unaltered. Thus, mtDNA purified from AA8 and Pyr cells was first digested with various restriction enzymes. Southern blot analysis using a mitochondrial genome-specific probe (cytochrome c oxidase III/ATPase6,8 cDNA) revealed an identical hybridization pattern in AA8 and Pyr cells when comparing the various restriction endonuclease digests (Fig. 5). Hence, no structural alterations could be detected in mtDNA purified from Pyr cells.

Figure 5: Southern blot analysis of purified mitochondrial DNA from wild-type and Pyr cells. Aliquots (1 µg) of mitochondrial DNA purified from wild-type and Pyr cells by cesium chloride density gradient centrifugation were digested with various restriction enzymes. Following fractionation by 0.8% agarose gel electrophoresis and Southern transfer, the blot was probed with a P-oligolabeled cytochrome c oxidase III/ATPase 6,8 cDNA clone.


Mitochondrial Protein Synthesis

Since transmission electron microscopy studies showed alterations in mitochondrial inner membrane structure and, at the same time, enzymatic and respiration studies suggested an impaired mitochondrial oxidative phosphorylation in Pyr cells, we next examined mitochondrial protein synthesis in Pyr cells. We first assessed cytoplasmic protein synthesis of wild-type and Pyr cells. The profile of [S]methionine-labeled total cytosolic proteins from wild-type and Pyr cells was qualitatively and quantitatively indistinguishable (Fig. 6A), thus suggesting that cytoplasmic protein synthesis is intact in Pyr cells. To investigate potential changes in mitochondrially encoded protein synthesis and/or turnover, cell cultures were metabolically pulse-labeled with [S]methionine in the presence of cycloheximide (Fig. 6, B and C) or emetine (Fig. 6, D and E), both of which are potent inhibitors of eukaryotic (i.e. nuclear) but not prokaryotic (i.e. mitochondrial) protein synthesis. Thus, parental AA8 and Pyr cells were labeled with [S]methionine (20 µCi/ml) for 2 h in the presence of 100 µg/ml cycloheximide. Electrophoretic analysis of the Coomassie-stained total cellular radiolabeled proteins from AA8 and Pyr cells showed no qualitative or quantitative changes (Fig. 6B). Furthermore, the [S]methionine-labeled protein profile obtained for both AA8 and Pyr fluorograms (Fig. 6, C and D) closely corresponded to the profile of the mitochondrial translation products of CHO cells described previously(28) . However, the mean (seven independent experiments) intensity of mitochondrially synthesized radiolabeled proteins in Pyr cells was consistently decreased by 2-fold as compared with parental AA8 cells (Fig. 6, C and D). In this respect, the most dramatic change was observed with the low molecular mass (<22 kDa) mitochondrial polypeptides, which were barely detectable in Pyr cells as compared with their parental counterpart (Fig. 6, C and D).

Figure 6: Mitochondrial protein synthesis in wild-type AA8 and Pyr cells. Mitochondrially synthesized proteins were metabolically labeled for 2 h with 20 µCi/ml [S]methionine in the absence (A) or presence of either 100 µg/ml cycloheximide (B and C) or 200 µg/ml emetine (D and E), both of which are inhibitors of cytoplasmic but not mitochondrial protein synthesis (for details, see ``Experimental Procedures''). Samples containing 50 µg of protein derived from cytosolic (A) or total cell lysates (B-E) were fractionated by electrophoresis on exponential 15-20% polyacrylamide gels containing SDS. B, Coomassie Brilliant Blue staining of the gel; C, fluorogram of the gel presented in B showing the radiolabeled mitochondrial proteins synthesized in mitochondria in the presence of cycloheximide; D, fluorogram of mitochondrially synthesized proteins in the presence of emetine; E, fluorogram generated as in D except that following the 2-h labeling with [S]methionine, cells were chased for 6 h in radiolabel-free medium. Rainbow high and low molecular mass markers (Amersham Corp.) are shown for each gel.


To address the possibility of increased turnover rate of mitochondrially synthesized proteins in Pyr cells, we have used a protocol of 2 h of [S]methionine labeling followed by a 6-h chase (longer times involve loss of cellular viability) in radiolabel-free medium. Fig. 6E shows that a further pronounced decrease in the intensity of radiolabeled mitochondrial proteins occurred in Pyr cells relative to parental AA8 cells (Fig. 6, compare E with C and D), thus supporting the suggestion of an increased turnover rate of mitochondrially synthesized polypeptides in Pyr cells.

Production of Lactic Acid in AA8 and PyrCells

It has been shown that a variety of respiration-defective CHO cell mutants are capable of normal growth on glycolysis alone as long as abundant glucose is provided in the culture medium(28) . These studies also showed that several fibroblast wild-type cells can grow in the presence of rotenone, a potent mitochondrial respiration inhibitor, thus indicating that a large fraction of ATP is produced by glycolysis as long as abundant glucose is provided. Thus, to examine whether mitochondrial respiration significantly contributes to the overall ATP production in parental and Pyr cells, we have assessed, by clonogenic assays, the effect of rotenone on these cells in the presence of various glucose concentrations. At 3 µM rotenone in the simultaneous presence of 10 mM glucose, no clonogenic viability could be detected in wild-type AA8 cells, whereas Pyr cells displayed a 3-fold hypersensitivity to rotenone. These data suggest that both wild-type and Pyr cells rely on mitochondrial respiration as a significant source for ATP production.

Based on the data that Pyr cells do rely on mitochondrial ATP production and, at the same time, suffer from a marked impairment of mitochondrial respiration, we hypothesized that a bioenergetic compensatory mechanism involving increased glycolysis may exist in Pyr cells. Thus, glycolytic activity was estimated in AA8 and Pyr cells by determination of lactate levels in the culture medium. A 2-fold elevation of the lactic acid level in the culture medium was observed in Pyr cells as compared with their parental cell line after 36 h of growth (Fig. 7). This finding therefore supports our notion that the glycolytic activity of Pyr cells is increased in the respiration-defective Pyr cells.

Figure 7: Extracellular lactate levels in culture medium of parental AA8 and Pyr cells. At time 0, 5 10^4 AA8 or 10^5 Pyr cells were plated in 6-cm Petri culture dishes in 5 ml of growth medium. Then, at 12-h intervals, 1-ml medium aliquots were drawn from individual cultures (each culture was used only for a single time point) and centrifuged to remove any remaining cells. The supernatant was then collected and assayed for lactate levels using a clinical lactate dehydrogenase assay and an automated COBAS MIRA analyzer (as detailed under ``Experimental Procedures''). Results represent mean values of three independent experiments, whereas errorbars denote the relative deviation obtained for each sample using a lactate-deficient control solution.


To quantitatively assess the bioenergetic deficit that Pyr may introduce to Pyr cells, we have determined, by clonogenic assays, the minimal glucose concentration that is required to support optimal growth of AA8 and Pyr cells in the presence or absence of Pyr. Thus, in the presence of Pyr, Pyr cells required 5 mM glucose for their growth, whereas 25-fold less glucose (i.e. 0.2 mM glucose) sufficed to support an optimal growth of wild-type and Pyr cells cultured in Pyr-free medium. These data show that Pyr cells had a markedly increased requirement for glucose in accord with their defective respiration rates and presumably with a decreased efficiency of conversion of glucose to ATP molecules.

Folinic Acid Rescue of AA8 Cells from Pyr Cytotoxicity

To provide evidence that a component of Pyr cytotoxicity is independent of folic acid metabolism, we have assessed the ability of folinic acid (5-formyltetrahydrofolate, Leucovorin) to protect parental AA8 cells from this drug. 1 µM folinic acid sufficed for the complete protection of AA8 cells from 10 µM Pyr (50 times the LD in AA8 cells) cytotoxicity (Fig. 8). In contrast, increasing Pyr above the latter concentration by 2-, 5-, and 10-fold yielded, respectively, a 10-, 250-, and 50,000-fold increased requirement for folinic acid to achieve a partial reversal of Pyr cytotoxicity (Fig. 8). This dramatic exponential decline in the ability of folinic acid to protect wild-type cells from Pyr cytotoxicity indicates that at high concentrations, Pyr does not act as a folic acid antagonist. Instead, Pyr cytotoxicity possesses a predominant folinic acid-independent component that is consistent with its toxic effects observed on mitochondrial structure and respiratory function.

Figure 8: Folinic acid rescue of wild-type AA8 cells from pyrimethamine cytotoxicity. Parental AA8 cells were plated (10^3 cells/6-cm Petri dish in duplicate cultures) in medium containing 10 µM (squares), 20 µM (circles), 50 µM (triangles), or 100 µM (diamonds) Pyr in the absence or presence of increasing concentrations of folinic acid (calcium Leucovorin). Following 6-14 days of incubation at 37 °C, colonies (>50 cells/colony) were counted, and the plating efficiency representing folinate protection of AA8 cells from Pyr cytotoxicity was calculated based on control cultures that received drug-free growth medium.


DISCUSSION

It has been well established that hydrophilic and lipophilic folic acid antagonists (i.e. antifolates) exert their cytotoxic effect via inhibition of the cytosolic target enzyme dihydrofolate reductase(13, 29) . In contrast, the cytotoxic effect exerted on wild-type AA8 cells by high Pyr concentrations could be poorly mitigated by folinic acid; this folinic acid nonrescuable Pyr cytotoxicity described here is not limited to DAP since piritrexim, an anticancer drug and a lipophilic pyridopyrimidine analogue of MTX(20) , showed a similar pattern of folinate nonrescuable cytotoxicity in cultured normal (30) and malignant (20) human cells. Thus, the present study provides several lines of evidence that establish mitochondria as an important target organelle for the cytotoxic activity elicited by Pyr and possibly other lipophilic antifolate anticancer drugs. (a) At geq10 µM, Pyr proved to be a mitochondrial respiration inhibitor in wild-type AA8 cells. (b) Transmission electron microscopy demonstrated the degeneration of the mitochondrial inner membrane in Pyr cells. (c) Whereas some mitochondrially encoded transcripts were prominently elevated, physiologically stable ones including 12 S and 16 S rRNAs were rather decreased in Pyr cells. (d) Metabolic pulse-chase labeling experiments suggested an increased turnover rate for mitochondrially synthesized proteins in Pyr cells. (e) The specific activity of a key enzymatic complex in the respiratory chain, cytochrome c oxidase, was reduced by 6-fold in Pyr cells as compared with parental AA8 cells. (f) Consequently, the rate of respiration in intact Pyr cells was reduced by 3-fold relative to wild-type AA8 cells.

Taken in toto, these data suggest that Pyr cells, which possess a degenerated mitochondrial inner membrane structure, may face difficulties in the assembly of enzymatic complexes of the electron transport chain. Alternatively, or in addition, Pyr cells may suffer from a decreased import into mitochondria of cytoplasmically synthesized polypeptides required for the biogenesis of the electron transport chain. Either way, this could explain the increased turnover rate of mitochondrially synthesized proteins observed in Pyr cells.

This study suggests that at high concentrations (geq10 µM), Pyr acts as a mitochondrial respiration inhibitor in wild-type cells. Thus, the following data and considerations may provide the underlying basis for Pyr accumulation in mitochondria. First, Pyr possesses an octanol/water copartition coefficient (log P) of 2.69(31) , which points to the hydrophobic nature of this antifolate. Second, Pyr has a pK value of 7.34(31) , which provides the basis for the weak base behavior of Pyr. These two features are therefore useful in predicting that at physiological pH, Pyr will be predominantly found in an uncharged form and will therefore readily traverse biomembranes, among which it could easily reach mitochondrial inner membranes. In this respect, a variety of lipophilic cationic compounds and dyes(32, 33) , including rhodamines(34) , styrylpyridinium dyes(35) , and acridines (36) as well as carbocyanines(37) , specifically accumulate in and are retained by mitochondria due to their lipophilicity and/or due to the mitochondrial transmembrane potential. Obviously, the driving force for Pyr concentration in mitochondria could be the transmembrane potential at the mitochondrial inner membrane with the negative charge inside. This scenario would therefore predict that the local excess of protons at this mitochondrial membrane site could drive the protonation of Pyr and thereby immobilize it irreversibly via a potent hydrophobic interaction of the lipophilic chlorophenyl and ethyl residues with the most hydrophobic inner membrane and crista components including cytochrome c oxidase. This concentration effect of an amphiphilic compound such as Pyr in the site of oxidative phosphorylation could disrupt membrane selectivity and interfere with the activity of enzymes assembled in mitochondrial membranes via a detergent-like activity. (^2)

The significant decrease in the activity of cytochrome c oxidase in Pyr cells along with the increased turnover rate of mitochondrially synthesized polypeptides may result in transcriptional up-regulation of mitochondrial genes in an attempt to compensate for the impaired respiratory function. Indeed, using differential cDNA screening, several mitochondrial DNA-encoded transcripts, the genes of which mapped to a restricted region of the mitochondrial genome, were found to be differentially overexpressed in Pyr-resistant cells. From 17 clones isolated by the differential screening approach, nine were found to correspond to the mitochondrial cytochrome c oxidase III, ATPase 6,8, and NAD dehydrogenase 4/4L genes, with the cytochrome c oxidase III gene being predominantly represented. Since transcription of mitochondrial DNA is driven by a bipartite promoter located in the D-loop region and both strands of the circular molecule are transcribed into polycistronic transcripts, selective activation of a specific cluster of mitochondrial genes cannot simply result from a general stimulation of transcription. Instead, transcriptional up-regulation of a limited region of the mitochondrial DNA could be explained by a variety of other mechanisms including genomic changes such as deletion or amplification and transcriptional attenuation or post-transcriptional regulation mechanisms including variations in mRNA stability, altered transcript processing, or preferential splicing(38) . Thus, we find that whereas several mature mitochondrial DNA-encoded transcripts were elevated in Pyr cells, a dramatic accumulation of the cytochrome c oxidase III/ATPase 6,8 primary transcript was observed. Furthermore, the physiologically stable mitochondrial 12 S/16 S rRNA was decreased in Pyr cells. These data are indeed consistent with an altered processing and/or turnover of mitochondrial DNA-encoded transcripts in Pyr cells. Specific modulation of transcription in discrete regions of the mitochondrial genome has been described in (a) human adenocarcinoma cells treated with trehalose(39) , (b) malignant breast tissues(40) , (c) adrenal cortex cells stimulated with adrenocorticotrophic hormone(41) , and (d) Daudi cells treated with interferon(42) . A large array of mitochondrial transcription-activating factors have also been described in mammalian and other eukaryotic cells(43, 44) , including a specific activator of the cytochrome c oxidase III gene in yeasts (45) . Thus, it is conceivable that the development of resistance to Pyr in hamster cells is associated with up-regulation of a similar activator as an attempt to compensate for the defective mitochondrial respiratory function. Nevertheless, the mtDNA from Pyr cells remained structurally intact as no change could be detected in the restriction pattern when using a mitochondrial DNA-specific probe on Southern blot analysis.

Mitochondria therefore represent a novel cytotoxicity target for lipophilic antifolate antibiotics of the DAP family and possibly for structural lipophilic analogues of MTX including trimetrexate and piritrexim, the anticancer drugs to which Pyr cells display a significant cross-resistance(18) . Other drugs have been shown to display mitochondrial cytotoxicity, including the antiretroviral 2`,3`-dideoxycytidine(46) , the antiparasitic suramin (47) , and 1-beta-D-arabinofuranosylcytosine(48) .

We note a marked increase in the number of multilamellar intracellular vesicles in Pyr cells. The lipophilic weak base properties of Pyr discussed above predict that Pyr will predominantly and irreversibly accumulate in acidic intracellular compartments including lysosomes and endosomes. The increased number of such vesicles observed in Pyr cells could be critical in accumulating Pyr to high concentrations. This increased drug sequestration could be readily followed by an efficient emptying of the intravesicular Pyr load via the physiological endosome-mediated exocytotic pathway. This could serve as an efficient natural mechanism of drug concentration in acidic compartments followed by an efficient ``drug efflux'' via endosome-mediated exocytosis. Hence, this could explain the resistance of Pyr cells to lipophilic antifolates. Such a mechanism for various cationic lipophilic anticancer drugs has been recently reviewed by Simon and Schindler (49) .

FOOTNOTES

*
This study was supported by a research grant from Chemotech Technologies Ltd. (to Y. G. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 972-4-293744; Fax: 972-4-225153.

(^1)
The abbreviations used are: MTX, methotrexate; Pyr, pyrimethamine; CHO, Chinese hamster ovary; DAP, 2,4-diaminopyrimidine(s); PBS, phosphate-buffered saline; mtDNA, mitochondrial DNA; kb, kilobase(s); DNP, 2,4-dinitrophenol.

(^2)
S. Drori and Y. G. Assaraf, unpublished data.

ACKNOWLEDGEMENTS

We thank Drs. Giuseppe Attardi and Youssef Hatefi for excellent discussions and for providing several mitochondrial probes and polyclonal sera. We extend our gratitude to Yaffa Both for expert technical assistance.

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