From the Departments of Pharmacology and
§ Therapeutic Radiology and Developmental Therapeutics
Program, Yale Cancer Center, Yale University School of Medicine,
New Haven, Connecticut 06520
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ABSTRACT |
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NADH:cytochrome b5 reductase activates the mitomycins to alkylating intermediates in vitro. To investigate the intracellular role of this enzyme in mitomycin bioactivation, Chinese hamster ovary cell transfectants overexpressing rat NADH:cytochrome b5 reductase were generated. An NADH:cytochrome b5 reductase-transfected clone expressed 9-fold more enzyme than did parental cells; the levels of other mitomycin-activating oxidoreductases were unchanged. Although this enzyme activates the mitomycins in vitro, its overexpression in living cells caused decreases in sensitivity to mitomycin C in air and decreases in sensitivity to porfiromycin under both air and hypoxia. Mitomycin C cytotoxicity under hypoxia was similar to parental cells. Because NADH:cytochrome b5 reductase resides predominantly in the mitochondria of these cells, this enzyme may sequester these drugs in this compartment, thereby decreasing nuclear DNA alkylations and reducing cytotoxicity. A cytosolic form of NADH:cytochrome b5 reductase was generated. Transfectants expressing the cytosolic enzyme were restored to parental line sensitivity to both mitomycin C and porfiromycin in air with marked increases in drug sensitivity under hypoxia. The results implicate NADH:cytochrome b5 reductase in the differential bioactivation of the mitomycins and indicate that the subcellular site of drug activation can have complex effects on drug cytotoxicity.
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INTRODUCTION |
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One of the strategies used in the treatment of cancer attempts to take advantage of drugs that are activated preferentially in the unique environments within solid tumors, minimizing damage to normal tissue. Mitomycin C and porfiromycin (Fig. 1) are prodrugs that can exploit the unique reductive environment in the hypoxic regions of solid tumors, where they are bioactivated to cytotoxic species (1). When triggered by enzymatic bioreduction, these compounds form highly reactive intermediates, which likely kill cells by cross-linking genomic DNA (reviewed in Refs. 2 and 3). Under aerobic conditions, these intermediates redox cycle with molecular oxygen, regenerating the relatively nontoxic parental compound. The combination of radiation, to kill the aerobic tumor cells, and mitomycin C or porfiromycin, used to target the hypoxic tumor cells, has proven to be an effective strategy in the treatment of head and neck cancer, producing significant increases in local control and disease-free survival when compared with radiation alone (4-6).
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We recently demonstrated that human NADPH:cytochrome c
(P-450) reductase (EC 1.6.2.4), when transfected into and overexpressed in a Chinese hamster ovary
(CHO)1 cell line,
CHO-K1/dhfr, can differentially metabolize the
mitomycins, with greater cytotoxicity occurring under hypoxic
conditions (7). In contrast, cells overexpressing rat NAD(P)H
dehydrogenase (DT-diaphorase; EC 1.6.99.2), which has been shown to
activate the mitomycins through a two-electron mechanism, do not
display an aerobic/hypoxic differential sensitivity to the mitomycins,
but rather are sensitized regardless of the degree of oxygenation (8).
This result contradicts two studies utilizing human NAD(P)H
dehydrogenase-transfected NIH 3T3 murine cells (9) or CHO cells (10),
which suggested that this enzyme does not bioactivate these drugs under
aerobic conditions, but agrees with Mikami et al. (11) who
found a 5-10-fold enhancement in aerobic mitomycin C sensitivity of
St-4 gastric carcinoma cells overexpressing transfected human NAD(P)H
dehydrogenase. Likewise, a mouse embryonic stem cell line containing a
homozygous knockout of NAD(P)H dehydrogenase demonstrated increased
aerobic resistance to mitomycin C, suggesting that the mouse enzyme is
capable of activating this drug (12). Experiments exploring the
catalytic differences between the rat and human versions of NAD(P)H
dehydrogenase have determined that the rat enzyme reduces mitomycin C
4.5 times faster than the human enzyme (13), and that changing the
amino acid at position 104 of the human enzyme from glutamine to a
tyrosine residue, which is present in the rat enzyme, can cause the
human enzyme to behave kinetically like the rat enzyme (14).
Other cellular enzymes involved in the bioreductive activation of the mitomycins include NADH:cytochrome b5 reductase (FpD; EC 1.6.2.2) (15, 16), xanthine oxidase (EC 1.1.3.22) (17, 18), and xanthine dehydrogenase (EC 1.1.1.204) (19-21). Because xanthine oxidase and xanthine dehydrogenase are present only at very low levels in tumor tissue, we chose to examine the role of FpD in the bioactivation of the mitomycins in our intact CHO cell system. The expression of the FpD gene involves a combination of transcriptional and translational mechanisms. Two transcripts encoding the three known forms of the rat FpD enzyme are produced from a single gene (22, 23). One transcript produces the myristylated membrane-bound enzyme and is expressed in all tissues. The second transcript, which is produced from an alternate, erythrocyte-specific promoter, encodes a protein which lacks the 7 amino acid myristylation consensus sequence but retains the membrane anchor and an additional 13 noncharged amino acids on the N terminus. A second form of the protein encoded in this transcript produces a soluble isoform which lacks the membrane anchor sequence; this protein is produced through translational initiation at a downstream AUG codon and is the major protein produced from this transcript. In rat liver cells, the myristylated, anchored form of FpD distributes in an approximately equal manner between the endoplasmic reticulum and the mitochondrial outer membrane (24, 25), although the concentration of enzyme measured on either a protein or a phospholipid basis is 11-fold or 4- to 5-fold higher, respectively, in mitochondrial outer membranes than in the endoplasmic reticulum (24). The higher concentration of FpD on the mitochondrial outer membrane may be partially due to a slower rate of degradation of the mitochondrial form of the enzyme compared with its endoplasmic reticulum localized counterpart (26). Studies utilizing a purified rabbit erythrocyte form of FpD have demonstrated that this enzyme can reduce mitomycin C to alkylating species, with greater activation occurring under hypoxia and reduced pH (16).
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EXPERIMENTAL PROCEDURES |
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Materials-- Mitomycin C was contributed by the Bristol-Myers Squibb Company (Wallingford, CT). Porfiromycin was synthesized from mitomycin C by our laboratory. NADH, NADPH, NAD+, chloroquine, rotenone, and Hepes were purchased from Sigma. Glutamine, hypoxanthine, thymidine, geneticin (G418), trypsin, penicillin, and streptomycin were purchased from Life Technologies, Inc. Tissue culture flasks and 60-, 100-, and 150-mm tissue culture dishes were acquired from Corning Costar Corp. (Cambridge, MA). Dicumarol, potassium ferricyanide, and ethanol were obtained from Aldrich. Mercaptoethanol was from Bio-Rad. Na2HPO4·2H2O, dextrose, CaCl2, glycerol, succinic acid, Tris, EDTA, KH2PO4, KCl, and NaCl were obtained from J. T. Baker.
Cell Culture--
The cell line used in this study is a variant
of the CHO-K1 cell line termed CHO-K1/dhfr (27) and was
obtained from the American Type Culture Collection (Rockville, MD).
This cell line is deficient in dihydrofolate reductase. The cells
were maintained in Iscove's modified Dulbecco's medium (Life
Technologies, Inc.) supplemented with 10% fetal bovine serum (Hyclone
Laboratories Inc., Logan, UT), 2 mM glutamine, 0.1 mM hypoxanthine, 0.01 mM thymidine, and
antibiotics (penicillin, 100 units/ml; streptomycin, 100 µg/ml).
Transfected lines were maintained in the identical medium supplemented
with 1 mg/ml G418 to provide for selection of the expression vector.
Cells were grown as monolayers in tissue culture flasks, Petri dishes,
or glass milk dilution bottles at 37 °C under an atmosphere of
95% air, 5% CO2 in a humidified incubator. The doubling
time of CHO-K1/dhfr
cultures is 19 h.
Plasmid Constructions-- The cDNA for the myristylated form of the rat NADH:cytochrome b5 reductase was generously provided by Dr. Nica Borgese of the Consiglio Nazionale delle Ricerche at the University of Milan (Milan, Italy) (22). To allow for the subcloning of the 1348-base pair cDNA into the eukaryotic expression vector pRC/CMV (Invitrogen Corp., San Diego, CA), the cDNA was amplified by the polymerase chain reaction (PCR) utilizing the following oligonucleotides: 1, 5'-CGCGGATCCAAGCTTGGTACCGCCACCATGGGGGCCCAGCTG-3' and 2, 5'-CGCGGATCCAAGCTTGCTAGCCTCTGTCTCTATGTCTGTATCTG-3'. These oligonucleotides incorporate a HindIII restriction site (shown in italics) upstream of the ATG translational initiation codon (shown in bold) and downstream of the termination codon in the 3'-untranslated region (priming from base pairs 1300-1322). To allow for construction of the cytoplasmic form of the reductase, a third oligonucleotide was synthesized: 3, 5'-CGCGGATCCAAGCTTGGTACCGCCACCATGAAGCTGTTTCAGCGCTCC-3'. This oligonucleotide, used in conjunction with oligonucleotide 2 above in a PCR reaction to generate the gene cassette, initiates 70 base pairs within the coding sequence of the reductase gene, resulting in a truncation of 23 amino acids and removal of the membrane binding domain from the amino terminus of the enzyme. The amplified PCR products were extracted with phenol:chloroform, precipitated with 2.5 volumes of ethanol, and resuspended in 50 µl of Tris:EDTA (10 mM Tris-HCl, pH 8.0, 1 mM EDTA). Following digestion of 5 µl of the amplified cDNAs with HindIII (Boehringer Mannheim), the fragments were subcloned into pRC/CMV, and recombinants were screened by restriction analysis to isolate the correct orientation. The plasmids, designated pRC/CMV-FpD (full-length) and pRC/CMV-sol-FpD (truncated), contain the promoter sequences from the immediate early gene of the human cytomegalovirus and the appropriate sequences for polyadenylation and selection (neomycin resistance), and insert stably into the genome of transfected cell lines.
Transfections--
Transfections were performed by the
Ca3(PO4)2-DNA coprecipitation
method essentially as described by Sambrook et al. (28) and
modified by Belcourt et al. (8). Single colonies were
introduced into wells of a 24-well plate using sterile cotton-tipped
applicators (General Medical Corp., Richmond, VA). After expansion, the
isolates were screened for expression of the NADH:cytochrome
b5 reductase cDNA genes. Isolates which had
elevated enzyme activities were cloned by flow cytometric single cell
sorting, and the resulting clones were rescreened. Vector-transfected
control clones were CHO-K1/dhfr cells transfected with
the plasmid without a cDNA insert.
Assays of Enzyme Activity--
Exponentially growing cells
(approximately 5 × 106 total cells) were harvested by
trypsinization, washed in cell culture medium containing 10% fetal
bovine serum to inactivate the trypsin, washed with phosphate buffered
saline (138 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM
KH2PO4, pH 7.4), then resuspended in 2 ml of
the same buffer. Cells were disrupted by sonication using a Branson
sonicator (Branson Ultrasonics Corp., Danbury, CT) with three 10-s
bursts at a setting of 25 with 1-min cooling on ice between each
sonication burst. Cell disruption was confirmed microscopically. NAD(P)H dehydrogenase activity was measured as the
dicumarol-inhibitable reduction of dichlorophenolindophenol, measured
at 600 nm with a Beckman model 25 UV-visible spectrophotometer (Beckman
Instruments Inc., Fullerton, CA) using an extinction coefficient of 21 mM1 cm
1 at 30 °C (29); the
final concentration of dicumarol was 100 µM (30).
NADPH:cytochrome c (P-450) reductase and
rotenone-insensitive NADH:cytochrome c reductase activities
were assayed in cell extracts by monitoring the rate of ferricytochrome
c reduction at 550 nm (extinction coefficient of 21 mM
1 cm
1) at 30 °C (31, 32).
NADH:cytochrome b5 reductase activity was
measured as NADH:ferricyanide reductase at 420 nm (extinction coefficient of 1.02 mM
1 cm
1) at
30 °C essentially as described previously (33), but using a final
concentration of 0.34 mM NADH. Succinate dehydrogenase activity was measured as the succinate-dependent reduction
of dichlorophenolindophenol by a modification of a previously reported method (34) in a 1-ml reaction mixture consisting of 50 mM
potassium phosphate (pH 7.5), 0.04 mM
dichlorophenolindophenol, 1.5 mM KCN, and 3.3 mM succinic acid at 30 °C. Protein concentrations were assayed using the bicinchoninic protein assay reagent (Pierce) (35).
Aerobic/Hypoxic Experiments--
Exponentially growing
monolayers of CHO-K1/dhfr, CHO-FpD-9, CHO-sol-FpD-8, and
CHO-sol-FpD-12 cells were seeded in glass milk dilution bottles at
2 × 105 cells per bottle and were used in
midexponential phase (approximately 3-4 days of growth). Hypoxia was
induced by gassing the cultures with a humidified mixture of 95%
N2, 5% CO2 (<10 ppm O2) at
37 °C for 2 h through a rubber septum fitted with 13-gauge
(inflow) and 18-gauge (outflow) needles. Following induction of
hypoxia, cells were exposed to 1, 2.5, 5, 7.5, 10, 15, or 30 µM mitomycin C or porfiromycin for 1 h; drugs were
injected through the septum without compromising the hypoxia. Cells
under aerobic conditions were treated with the mitomycins in an
identical manner for 1 h in a humidified atmosphere of 95% air,
5% CO2 at 37 °C. Treated cells were washed, harvested
by trypsinization, and assayed for survival by measuring their ability
to form macroscopic colonies (36). Both aerobic and hypoxic vehicle
controls (70% ethanol) were included in each experiment; the surviving
fractions were normalized using these vehicle controls. The plating
efficiencies (colonies/100 plated cells; means ± standard
deviations) for CHO-K1/dhfr
, CHO-FpD-9, CHO-sol-FpD-8,
and CHO-sol-FpD-12 cells were 84 ± 7, 70 ± 4, 55 ± 2, and 52 ± 3, respectively. The surviving fractions for the aerobic
vehicle-treated controls (means ± S.D.) were 1.01 ± 0.04, 0.99 ± 0.02, 1.00 ± 0.02, and 1.00 ± 0.03, while the
surviving fractions for the hypoxic vehicle-treated controls were
somewhat lower, reflecting the toxic effects of the hypoxia: 0.67 ± 0.06, 0.59 ± 0.02, 0.47 ± 0.01, and 0.53 ± 0.03 for CHO-K1/dhfr
, CHO-FpD-9, CHO-sol-FpD-8, and
CHO-sol-FpD-12 cells, respectively.
Subcellular Fractionation--
Exponentially growing
CHO-K1/dhfr parental, CHO-FpD-9, and CHO-sol-FpD-12 cells
were collected and disrupted by sonication as described above, except
that cells were resuspended in 20 ml of Hepes buffer (25 mM
Hepes, 250 mM sucrose, pH 7.4) at a cell concentration of
2 × 107 cells/ml, and sonication was performed at a
setting of 20. The cell sonicates were centrifuged at 12,100 × g for 10 min (mitochondrial/nuclear fraction), then at
105,000 × g for 1 h (microsomal fraction). The
resulting supernatant represented the cytosolic fraction. Each pellet
was washed in Hepes buffer, recentrifuged, then resuspended in 1 ml of
the same buffer. Fractions were assayed for NADH:cytochrome b5 reductase and succinate dehydrogenase enzyme
activities and protein concentration as described above.
Mitomycin C Reduction Rates--
Cell sonicates of
exponentially growing CHO-K1/dhfr parental, CHO-FpD-9,
and CHO-sol-FpD-12 cells were prepared as detailed above for the assays
of enzyme activity. The relative rates of mitomycin C reduction by
NADH-supplemented cell sonicates were determined in a reaction mixture
containing 10 mM potassium phosphate (pH 6.6), 0.05 mM NADH, 8 µM rotenone, 0.3 mM
KCN, 0.1 mM mitomycin C, and 0.2% (v/v) ethanol in a final
volume of 2 ml. The velocity of mitomycin C reduction was expressed as
the disappearance of mitomycin C (quinone moiety) measured at 375 nm
(extinction coefficient of 13.2 mM
1
cm
1) at 30 °C under hypoxia. Hypoxia was accomplished
by stoppering an anaerobic cuvette (Quaracell Products, Inc., Baldwin,
NY) with a rubber septum fitted with 18-gauge (inflow) and 26-gauge
(outflow) needles and pregassing the reaction mixture for 10 min with
humidified nitrogen. Mitomycin C was injected through the septum
without interrupting the hypoxia.
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RESULTS |
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Our in vitro studies using purified FpD indicated that
this enzyme can bioreductively activate mitomycin C (16). After
transfection of an expression vector containing a cDNA encoding the
full length, myristylated, membrane-bound form of the rat FpD enzyme
into CHO-K1/dhfr cells, clones overexpressing FpD enzyme
activity were selected. One such clone, CHO-FpD-9, overexpressed FpD
activity by approximately 9-fold (Table
I). To rule out changes in the other
oxidoreductases implicated in the bioreductive activation of the
mitomycins, including NADPH:cytochrome c (P-450) reductase
and NAD(P)H dehydrogenase, these enzyme activities were also measured
in the transfected cell line and compared with the activities in the
nontransfected parental cell line. No changes were observed in the
other oxidoreductases, indicating that, of the known mitomycin
bioactivating enzymes, only the expression level of FpD was changed
(Table I). The CHO-K1/dhfr
cell line expresses no
xanthine oxidase or xanthine dehydrogenase enzyme activity (7).
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The subcellular localization of FpD was examined in the nontransfected
parental line, CHO-K1/dhfr, and the FpD-transfected cell
line, CHO-FpD-9, by assaying subcellular fractions for FpD enzyme
activity. The majority of the FpD activity in CHO-K1/dhfr
cells co-localized with the mitochondrial enzyme succinate
dehydrogenase, indicating that FpD is found predominantly in the
mitochondria of this CHO cell line, with a significant but smaller
fraction localized in the microsomes (Table
II). This is consistent with the
distribution patterns of this enzyme observed in rat liver cells (37).
In CHO-FpD-9 cells, the distribution pattern of FpD activity was
similar to that of parental cells, with most of the enzyme activity
residing in the mitochondrial fraction (Table II).
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The effect of increasing FpD enzyme activity in the CHO-FpD-9 cell line
on the sensitivity of these cells to mitomycin C and porfiromycin was
measured under aerobic and hypoxic conditions using a clonogenic assay.
Parental cells showed similar sensitivities to mitomycin C under
aerobic and hypoxic conditions, with a small but significant difference
in sensitivity at 10 µM mitomycin C (Fig.
2A). This is a change from
what we previously reported for this cell line (7), where we initially
noted little or no difference in mitomycin C sensitivity at this drug
concentration under aerobic and hypoxic conditions. The acquisition of
additional data on the aerobic/hypoxic drug sensitivities of this cell
line is thought to account for the change from the insignificant
difference reported previously to the small, but significant,
difference reported here. In contrast, porfiromycin was markedly more
cytotoxic to the CHO-K1/dhfr parental cell line under
hypoxia, with relatively little aerobic drug toxicity at concentrations
as high as 30 µM (Fig. 2B). Porfiromycin has
been reported to be much less cytotoxic than mitomycin C to aerobic
cells but essentially equivalent on a molar basis to mitomycin C in
cryptotoxicity to hypoxic EMT6 mouse mammary carcinoma cells (reviewed
in Sartorelli et al. (38)). Our finding that porfiromycin is
significantly more toxic than mitomycin C under hypoxic conditions is
not confined to the CHO-K1/dhfr
cell line, but was also
noted in CHO-HA-1 (39) and CHO-AA8 (40) cells.
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Under oxygenated conditions, the CHO-FpD-9 cell line was significantly
less sensitive to mitomycin C than was the parental cell line despite
the fact that CHO-FpD-9 cells expressed approximately 9-fold more FpD
enzyme activity (Fig. 2C). In contrast, CHO-FpD-9 and
CHO-K1/dhfr parental cells were equally sensitive to
mitomycin C in hypoxia (Fig. 2C). These results were
consistently observed with several independently derived FpD
transfectants (data not shown). The effects of porfiromycin on these
cell lines differed from that of mitomycin C. Porfiromycin, like
mitomycin C, was less cytotoxic to CHO-FpD-9 cells than to parental
cells under oxygenated conditions (Fig. 2D). However, in
contrast to mitomycin C, porfiromycin was less cytotoxic to CHO-FpD-9
cells than to the parental cell line under hypoxic conditions (Fig.
2D).
To investigate whether the subcellular site of activation of the mitomycins is important for their cytotoxic actions, the intracellular location of FpD was altered by constructing and expressing a cytosolic version of the FpD cDNA. The FpD cDNA was modified so as to remove the amino-terminal membrane anchor, creating a gene cassette that produces the soluble form of FpD found in erythrocytes. The membrane-bound and cytosolic forms of the rat FpD enzyme are generated from a single gene through an alternative promoter mechanism (23). Two transfectants were chosen which expressed the cytosolic form of FpD at levels of activity which closely resembled that observed for the full-length FpD transfectant, CHO-FpD-9, expressing the membrane-bound form of FpD. These transfected cell clones, termed CHO-sol-FpD-8 and CHO-sol-FpD-12, expressed 8- and 12-fold more FpD enzyme activity, respectively, than parental cells, with no changes in the levels of the other mitomycin oxidoreductases (Table I). The subcellular localization of FpD enzyme activity was determined for CHO-sol-FpD-12 and was found to be predominantly cytosolic, verifying the successful relocation of the transfected enzyme (Table II). Supporting this conclusion, the measurement of FpD enzyme activity by the rotenone-insensitive NADH:cytochrome c reductase assay, which measures the reduction of cytochrome c via endogenous cytochrome b5, increased in the transfectant expressing the membrane-bound enzyme, CHO-FpD-9, compared with the parental cell line, but did not change in the CHO-sol-FpD-8 and CHO-sol-FpD-12 transfectants expressing the cytosolic form of the enzyme (Table I). In the rotenone-insensitive NADH:cytochrome c reductase assay, the FpD enzyme cannot directly reduce cytochrome c but must first reduce the physiological substrate cytochrome b5, which then transfers its electron to cytochrome c. However, when FpD enzyme activity was measured directly as NADH:ferricyanide reductase, an assay in which the enzyme directly reduces the artificial electron acceptor ferricyanide, an increase in activity was apparent in all three of the transfected cell clones (Table I). These results indicate that the FpD enzyme is in the cytosolic form in CHO-sol-FpD-8 and CHO-sol-FpD-12 and is unable to shunt electrons through the membrane-bound cytochrome b5 in a manner analogous to that seen with the membrane-bound form of FpD expressed in CHO-FpD-9.
Both transfectants expressing the cytosolic form of FpD displayed
parental sensitivity to mitomycin C under aerobic conditions (Fig.
3). This contrasted to the reduced
sensitivity of the transfectant expressing the membrane-bound enzyme,
CHO-FpD-9, to mitomycin C in air, even though all three FpD-transfected
cell lines expressed similar levels of enzyme activity. In hypoxia, the
CHO-sol-FpD-8 and CHO-sol-FpD-12 cell lines were dramatically
sensitized to mitomycin C relative to the parental and CHO-FpD-9 cell
lines (Fig. 4). Likewise, CHO-sol-FpD-8
and CHO-sol-FpD-12 cells exhibited parental sensitivity to porfiromycin
under oxygenated conditions as opposed to the reduced sensitivity of
CHO-FpD-9 cells to this agent (Fig. 5).
Under hypoxic conditions, the CHO-sol-FpD-8 and CHO-sol-FpD-12 cells
were markedly more sensitive to porfiromycin than were the parental
CHO-K1/dhfr cells and the even less sensitive CHO-FpD-9
cells (Fig. 6).
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To determine if differences in mitomycin C metabolism exist between the
membrane-bound and cytosolic forms of the FpD enzyme, the rates of
mitomycin C reduction were measured in cell sonicates of
CHO-K1/dhfr parental, CHO-FpD-9, and CHO-sol-FpD-12
cells. Mitomycin C metabolism in parental cell sonicates was
undetectable. However, cell sonicates from CHO-FpD-9 and CHO-sol-FpD-12
cells exhibited essentially identical rates of mitomycin C reduction,
indicating that the membrane-bound and cytosolic forms of the enzyme
metabolize mitomycin C similarly (Table
III). Collectively, these results
demonstrate that the rat FpD enzyme can enhance the aerobic/hypoxic
differential cytotoxicity of mitomycin C and porfiromycin in intact
cells and that the intracellular site of bioactivation by FpD
influences the cytotoxicity of these drugs.
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DISCUSSION |
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Purified FpD was recently shown to activate
mitomycin C under both aerobic and hypoxic conditions, with slightly
more activation occurring under hypoxia (16). To provide information on
the role of FpD in the bioactivation of the mitomycin antibiotics in
living cells, a full-length rat FpD cDNA was transfected into and
overexpressed in CHO-K1/dhfr cells. A clone, CHO-FpD-9,
which overexpressed the myristylated, membrane-bound form of FpD by
9-fold compared with parental cells, was profiled for the known
mitomycin bioreductive enzymes, including NADPH:cytochrome c
(P-450) reductase and NAD(P)H dehydrogenase, and determined to be
changed only in expression of FpD activity. The subcellular location of
FpD in the transfected cell clone was similar to that of the
parental cell line, with most of the FpD enzyme activity co-localizing
with the mitochondrial enzyme succinate dehydrogenase. This implies
that FpD resides predominantly in the mitochondrial compartment of this
CHO cell line and is consistent with the known localization patterns of
this enzyme in other cell types, including rat liver and MDCK II cells
(24, 25).
The sensitivity of this clone to mitomycin C and porfiromycin yielded
surprising results. Under aerobic conditions, both agents were
significantly less cytotoxic to CHO-FpD-9 cells than to
CHO-K1/dhfr parental cells. Under hypoxia, the CHO-FpD-9
and parental cell lines showed similar mitomycin C sensitivities, while
porfiromycin was less toxic to CHO-FpD-9 cells than to parental cells.
This result suggests that, in living cells, the FpD-catalyzed
bioactivation of the mitomycins and/or the resulting cytotoxicity of
these agents is attenuated by the intracellular environment, because we
demonstrated previously that, in cell-free systems, purified FpD
activates mitomycin C to an intermediate capable of alkylating the
nucleophile 4-(p-nitrobenzyl)pyridine. Alternatively, the
lesion(s) produced by the FpD-generated mitomycin intermediate may not
be cytotoxic at the concentration of drug employed. In fact, the
FpD-transfected cell line is actually less sensitive to the mitomycins
than is the parental line, implying that overexpression of this enzyme protects cells from the cytotoxic effects of the mitomycins.
We demonstrated previously that human NADPH:cytochrome c
(P-450) reductase and rat NAD(P)H dehydrogenase, which are localized to
the endoplasmic reticulum and cytosol, respectively, enhance the
cytotoxicity of mitomycin C and porfiromycin when overexpressed in
CHO-K1/dhfr cells (7, 8). Since FpD is predominantly
situated in the mitochondria in this cell line, we decided to alter the
intracellular location of FpD to ascertain whether the subcellular site
of bioactivation of the mitomycins is important in determining the
cytotoxicity of these drugs. The FpD cDNA was modified to encode a
protein which lacked the N-terminal membrane anchor sequence, producing the cytosolic form of FpD which is expressed exclusively in
erythrocytes. Transfected clones expressing this cytosolic enzyme were
selected which produced similar levels of FpD enzyme activity as
CHO-FpD-9. In contrast to CHO-FpD-9, which expressed the membrane-bound
form of FpD and was less sensitive than the parental line to the
mitomycins in air, the clones expressing the cytosolic enzyme,
CHO-sol-FpD-8 and CHO-sol-FpD-12, were similar to the parental line in
their response to the mitomycins under these conditions of oxygenation. Pronounced increases in sensitivity to the mitomycins under hypoxia were apparent in cells expressing the cytosolic enzyme, with marked increases in the aerobic/hypoxic differential cytotoxicity of both
drugs. Both the full-length and truncated versions of FpD metabolized
mitomycin C at similar rates in cell sonicates, suggesting that the
presence or absence of the membrane binding domain and whether the
enzyme was membrane bound had no effect on the ability of FpD to
metabolize the drug. These results suggest that mitomycin cytotoxicity
can be markedly influenced by the subcellular site of drug activation.
Moreover, we directly implicate the cytosolic form of FpD in the
aerobic/hypoxic differential toxicity of mitomycin C and porfiromycin
in living cells.
The mechanism by which the subcellular location of prodrug
activation influences the ultimate cytotoxicity of the activated drug
is unclear, although several hypotheses can be advanced. Enzymes
capable of bioactivation can be compartmentalized within cellular
organelles and may never interact with a possible substrate. In cases
where an enzyme can interact with a potential prodrug, the activated
drug may not be cytotoxic at the concentration employed because the
activating enzyme is in a region of the cell distal from the drug's
primary site of action; this allows the highly reactive activated
species to interact with oxygen or other macromolecules in a non- or
less cytotoxic event before it is able to diffuse to its target. This
latter hypothesis is most consistent with the data on the mitomycins
presented in this report. Since overexpression of the soluble form of
FpD in CHO-K1/dhfr cells results in a marked enhancement
of the aerobic/hypoxic differential toxicity of the mitomycins, similar
to that seen with NADPH:cytochrome c (P-450) reductase (7),
it can be presumed that the FpD-catalyzed activation of the
mitomycins proceeds through a one-electron reductive mechanism,
producing the oxygen-sensitive semiquinone anion radical
intermediate. In the presence of oxygen, this intermediate redox
cycles, regenerating the parent molecule. Under hypoxia, the
oxygen-quenching of the semiquinone is absent, which allows for the
ensuing cytotoxic alkylations, presumably at the level of nuclear DNA
(41). The semiquinone intermediate is known to be quite reactive in
aqueous solution (reviewed in Franck and Tomasz (42)), which suggests
that, to reach and cross-link nuclear DNA, the mitomycins need to be
activated in a region of the cell where the activated intermediate can
diffuse to the nucleus. Since overexpression of either endoplasmic
reticulum-localized NADPH:cytochrome c (P-450) reductase or
cytosol-localized NAD(P)H dehydrogenase sensitized
CHO-K1/dhfr
cells to the mitomycins, activation of the
mitomycins in these subcellular regions allows the activated
intermediate to reach its target and produce cytotoxic lesions.
Likewise, FpD can enhance the cytotoxicity of the mitomycins when
overexpressed and localized to the cytosol, implying that an
FpD-generated mitomycin intermediate can be lethal when produced in the
cytosolic compartment.
In contrast, when a significant proportion of overexpressed FpD is localized to the outer membranes of the mitochondria, where it occurs normally in non-erythroid cells, this CHO cell line becomes significantly more resistant to mitomycin C under aerobic conditions and more resistant to porfiromycin under both aerobic and hypoxic conditions. The cytotoxicity of mitomycin C under hypoxic conditions was similar to that in parental cells. The decrease in cytotoxicity seen in this transfected cell line compared with parental cells implies that the mitomycins are metabolized by FpD. If the FpD enzyme were compartmentalized such that it never interacted with the mitomycins, no change in mitomycin cytotoxicity should have occurred in the FpD-transfected cell line compared with parental cells. Instead, it appears that the mitomycins are being bioactivated by FpD, but that this drug activation is sequestered in a region from which the activated drug is unable to reach and cross-link nuclear DNA and cause cytotoxicity. This, in effect, protects cells from these drugs because bioactivation in this compartment competes with activation by the endogenous enzymes in the cytosol and endoplasmic reticulum, thereby shunting the drug into a pathway less likely to produce the most highly cytotoxic lesions. This hypothetical protective effect is not seen to the same extent with mitomycin C as with porfiromycin under hypoxia; in hypoxia, the parental and the CHO-FpD-9 cell lines are equally sensitive to mitomycin C, while the CHO-FpD-9 cell line was less sensitive to porfiromycin. We hypothesize that, under hypoxia, more mitomycin C than porfiromycin is activated by the other endogenous bioreductive enzymes because of differences in enzyme/substrate interactions and/or their reactivities with competing nucleophiles and redox cycling pathways.
The fate of the activated mitomycin intermediate in the CHO-FpD-9 cell line is unknown. Under aerobic conditions, redox cycling of the semiquinone with molecular oxygen regenerates the parent quinone. This process may be more likely when the reductive activation of the mitomycins occurs distal from its nuclear target. Recently, mitomycin C was found to damage mitochondrial DNA in EMT6 cells (43) and to disrupt cellular bioenergetics in Balb/c mice (44). This suggests that, following reductive activation of the mitomycins, the activated intermediate can react with mitochondrial macromolecules including mitochondrial DNA, components of the electron transport chain, and/or other mitochondrial proteins thus preventing activated drug from reaching the nucleus. It is also possible that FpD, when bound to the mitochondrial outer membrane, is predominantly in an oxidized state because, upon reduction, it immediately shunts electrons to its redox partner, cytochrome b5. Reduced and activated mitomycin intermediates may be re-oxidized by FpD, thereby restoring the inert prodrug. Soluble FpD, without a redox partner, may be primarily in a reduced state which is able to activate the mitomycins to cytotoxic species. Many enzymes involved in intermediary metabolism can act to reduce or oxidize a single substrate depending on the oxygenation and energy status of the cell (45). These processes may account for the increased resistance of the CHO-FpD-9 cell line compared with its parental counterpart. All of these models suggest that the presence of the endogenous, myristylated form of FpD in its normal subcellular location acts to protect cells from the cytotoxic effects of the mitomycins despite the ability of this enzyme to reductively activate these drugs in vitro. The results imply that the impact of the subcellular location of drug activating enzymes on prodrug cytotoxicity must be considered in assessing the importance of specific enzymes in the bioactivation of drugs in living cells, in designing approaches utilizing gene therapy, and in the use of enzyme modulators to exert optimum therapeutic action.
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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1 The abbreviations used are: CHO, Chinese hamster ovary; FpD, NADH:cytochrome b5 reductase; PCR, polymerase chain reaction; CMV, cytomegalovirus.
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