The Intracellular Location of NADH:Cytochrome b5 Reductase Modulates the Cytotoxicity of the Mitomycins to Chinese Hamster Ovary Cells*

Michael F. BelcourtDagger , William F. HodnickDagger , Sara RockwellDagger §, and Alan C. SartorelliDagger

From the Departments of Dagger  Pharmacology and § Therapeutic Radiology and Developmental Therapeutics Program, Yale Cancer Center, Yale University School of Medicine, New Haven, Connecticut 06520

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

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.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

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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Fig. 1.   Structures of mitomycin C and porfiromycin.

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).

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

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 mM-1 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.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

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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Table I
Oxidoreductase activities of the CHO-K1/dhfr- parental cell line and NADH:cytochrome b5 reductase-transfected cell lines expressing either a full-length (CHO-FpD-9) or truncated (CHO-sol-FpD-9 and CHO-sol-FpD-12) rat NADH:cytochrome b5 reductase cDNA

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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Table II
Subcellular distribution of NADH:cytochrome b5 reductase and succinate dehydrogenase enzyme activities in CHO-K1/dhfr-, CHO-FpD-9, and CHO-sol-FpD-12 cells

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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Fig. 2.   Survival curves for CHO-K1/dhfr- parental and CHO-FpD-9 cells treated with graded concentrations of mitomycin C (A and C) or porfiromycin (B and D) for 1 h under aerobic (open symbols) or hypoxic (solid symbols) conditions. Surviving fractions were calculated using the plating efficiencies of the aerobic and hypoxic vehicle-treated controls. Points are geometric means of survivals from three to eight independent experiments; S.E.s are shown where larger than the points. open circle , bullet  (solid line), CHO-K1/dhfr- parental cells; square , black-square (dashed line), CHO-FpD-9 cells. Note differences in scale for surviving fraction and drug concentration between the different drugs.

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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Fig. 3.   Survival curves for CHO-K1/dhfr- parental (from Fig. 2), CHO-FpD-9 (from Fig. 2), CHO-sol-FpD-12 (A), and CHO-sol-FpD-8 (B) cells treated with graded concentrations of mitomycin C for 1 h under aerobic conditions. The surviving fractions were calculated using the plating efficiencies of the aerobic vehicle-treated controls. Points are geometric means of survivals from three to eight independent experiments; S.E.s are shown where larger than the points. open circle  (solid line), CHO-K1/dhfr- parental cells; square  (dashed line), CHO-FpD-9 cells; triangle  (dotted line), CHO-sol-FpD-12 cells; down-triangle (dotted line), CHO-sol-FpD-8 cells.


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Fig. 4.   Survival curves for CHO-K1/dhfr- parental (from Fig. 2), CHO-FpD-9 (from Fig. 3), CHO-sol-FpD-12 (A), and CHO-sol-FpD-8 (B) cells treated with graded concentrations of mitomycin C for 1 h under hypoxic conditions. The surviving fractions were calculated using the plating efficiencies of the hypoxic vehicle-treated controls. Points are geometric means of survivals from three to eight independent experiments; S.E.s are shown where larger than the points. bullet  (solid line), CHO-K1/dhfr- parental cells; black-square (dashed line), CHO-FpD-9 cells; black-triangle (dotted line), CHO-sol-FpD-12 cells; black-down-triangle  (dotted line), CHO-sol-FpD-8 cells.


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Fig. 5.   Survival curves for CHO-K1/dhfr- parental (from Fig. 2), CHO-FpD-9 (from Fig. 3), CHO-sol-FpD-12 (A), and CHO-sol-FpD-8 (B) cells treated with graded concentrations of porfiromycin for 1 h under aerobic conditions. The surviving fractions were calculated using the plating efficiencies of the aerobic vehicle-treated controls. Points are geometric means of survivals from three to eight independent experiments; S.E. are shown where larger than the points. open circle  (solid line), CHO-K1/dhfr- parental cells; square  (dashed line), CHO-FpD-9 cells; triangle  (dotted line), CHO-sol-FpD-12 cells; down-triangle (dotted line), CHO-sol-FpD-8 cells.


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Fig. 6.   Survival curves for CHO-K1/dhfr- parental (from Fig. 2), CHO-FpD-9 (from Fig. 3), CHO-sol-FpD-12 (A), and CHO-sol-FpD-8 (B) cells treated with graded concentrations of porfiromycin for 1 h under hypoxic conditions. The surviving fractions were calculated using the plating efficiencies of the hypoxic vehicle-treated controls. Points are geometric means of survivals from three to eight independent experiments; S.E. are shown where larger than the points. bullet  (solid line), CHO-K1/dhfr- parental cells; black-square (dashed line), CHO-FpD-9 cells; black-triangle (dotted line): CHO-sol-FpD-12 cells; black-down-triangle  (dotted line), CHO-sol-FpD-8 cells.

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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Table III
Metabolism of mitomycin C (MC) by sonicates of cell lines expressing either a full-length (CHO-FpD-9) or a truncated (CHO-sol-FpD-12) NADH:cytochrome b5 reductase cDNA

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

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.

    FOOTNOTES

* 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.

To whom correspondence should be addressed. Tel.: 203-785-4533; Fax: 203-737-2045.

1 The abbreviations used are: CHO, Chinese hamster ovary; FpD, NADH:cytochrome b5 reductase; PCR, polymerase chain reaction; CMV, cytomegalovirus.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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