(Received for publication, March 28, 1995; and in revised form, June 13, 1995)
From the
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
10 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.
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), ()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.
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.).
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
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.
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.
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.
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.
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
AA8 or 10
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.
Figure 8:
Folinic acid rescue of wild-type AA8 cells
from pyrimethamine cytotoxicity. Parental AA8 cells were plated
(10 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.
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 10
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 (10 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. (
)
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-
-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) .