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CELLULAR AND MOLECULAR

RH1 Induces Cellular Damage in an NAD(P)H:Quinone Oxidoreductase 1-Dependent Manner: Relationship between DNA Cross-linking, Cell Cycle Perturbations, and Apoptosis

Department of Pharmaceutical Sciences, School of Pharmacy and Cancer Center, University of Colorado Health Sciences Center, Denver, Colorado

Received November 24, 2004; accepted January 19, 2005.

	Abstract

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Structure-based development of NAD(P)H:quinone oxidoreductase (NQO1)-directed antitumor quinones resulted in development of RH1 [2,5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone], a methyl-substituted diaziridinyl quinone. We conducted experiments to evaluate the mechanism of RH1-induced cytotoxicity and the inter-relationship between DNA cross-linking, cell cycle changes, and apoptosis using an isogenic cell line pair developed from the human breast cancer cell line MDA-MB-468 differing only in expression of wtNQO1 (NQ16 cells). Statistically significant DNA cross-linking was detected using a modified comet assay in cells with wtNQO1 within 1 h of dosing, whereas in parental cells, only marginal DNA cross-linking was observed and required a concentration up to 50 times higher. Cross-linking in NQ16 cells could be abrogated with 5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]indole-4,7-dione, a mechanism-based inhibitor of NQO1. RH1 prolonged S phase and caused a G₂/M block. Cell cycle changes were observed up to 10-fold lower in RH1 concentrations in NQ16 cells relative to parental cells. Apoptosis was similarly observed morphologically in both cell lines after RH1 treatment but was induced preferentially in NQ16 cells at lower concentrations and earlier time points. Marked cleavage of caspase-3 was observed in NQ16 cells relative to parental cells using lower concentrations of RH1. Temporally, low doses of RH1-induced rapid DNA cross-linking in NQ16 cells followed by induction of apoptosis at times when a G₂/M block was not observed. This suggests that cell cycle arrest is not required for RH1-induced apoptosis and that DNA damage may directly initiate apoptotic events. In summary, RH1-induced preferential DNA cross-linking, cell cycle changes, and apoptosis in an NQO1-dependent manner.

Experiments to evaluate the cellular effects of RH1 were initiated using the human breast adenocarcinoma cell line MDA-MB-468 (MDA468) and the cell line generated from it by transfection with wtNQO1, NQ16. The MDA468 cell line has a point mutation (C609T) in the human NQO1 gene (the NQO1*2/*2 polymorphism) that results in the NQO1*2 protein, which is reported to have no NQO1 activity due to rapid proteasomal degradation (Siegel et al., 2001). The MDA468 and NQ16 cell lines are isogenic apart from expression of wtNQO1 (NQO1*1 protein) in NQ16 cells, and we have previously demonstrated RH1 is an order of magnitude more toxic to NQ16 cells transfected with wtNQO1 than to parental MDA468 cells lacking wtNQO1 (Dehn et al., 2004).

In this work, induction of DNA cross-linking by RH1 was evaluated through the modified comet assay (Ward et al., 1997). In addition to using both cell lines, a highly specific pharmacological inhibitor of NQO1, ES936 (5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]indole-4,7-dione), was used to verify the contribution of NQO1 in activating RH1. Cross-linked DNA can result in the inhibition of cellular division (Rauth et al., 1998), reducing the proliferative potential of cancer cells by interrupting the cell cycle (Balachandran et al., 1999) or inducing cell death. We evaluated the ability of RH1 to disrupt the cell cycle and induce apoptosis under the same conditions we used for investigating the induction of DNA cross-links in both NQO1-deficient MDA468 and NQO1-rich NQ16 cells. This approach has allowed us to perform the first detailed analysis of the mechanism of RH1 cytotoxicity and the inter-relationship among RH1-induced DNA cross-linking, cell cycle changes, and apoptosis.

	Materials and Methods

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Materials. Propidium iodide, Hoechst 33342, acridine orange, and ethidium bromide were purchased from Sigma-Aldrich (St. Louis, MO). RNase was purchased from Worthington Biochemicals (Freehold, NJ). All other chemicals were purchased from Sigma-Aldrich. ES936 and RH1 were synthesized as previously described (Beall et al., 1998; Winski et al., 1998), with stock solutions made up in dimethyl sulfoxide. Cell culture medium and supplements were obtained from Invitrogen (Carlsbad, CA) unless otherwise specified. Fetal bovine serum was purchased from Gemini (Irvine, CA).

Cell Culture. The human breast cancer cell line MDA-MB-468 (MDA468) and its isogenic pair, NQ16 cells, were grown as monolayers in RPMI supplemented with 20% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (complete media) at 37°C in a humidified atmosphere with 5% CO₂. Cells were tested for mycoplasma contamination prior to initiation of experiments.

Treatment with RH1. MDA468 and NQ16 cells were seeded in 60-mm plates at a concentration of 1 x 10⁶ cells/ml. The next day, MDA468 and NQ16 cells were treated with RH1 at 50, 100, and 500 nM or 10, 50, and 100 nM, respectively, in unsupplemented media for 30, 60, or 120 min, after which the dosing medium was aspirated, rinsed with PBS, and then harvested. Additional cells were treated with RH1 at the above doses for 120 min, after which the dosing medium was aspirated, and cells were rinsed with PBS and then incubated in fresh complete medium and harvested at various time points (2 to 48 h later).

Comet Analysis. The modified comet assay was used to detect DNA cross-links as previously described (Dehn et al., 2004). Briefly, control and RH1-treated cells at each time point were irradiated on ice to induce DNA single-strand breaks. Cross-linked DNA is unable to migrate from the head of the comet, and the extent of DNA cross-linking can be indirectly measured by analyzing the relative reduction of DNA migration induced by the irradiation compared with controls. A minimum of 200 comets on each slide (slides were prepared in duplicate for each independent experiment, and three independent experiments were conducted) were visually scored (observer was blind to treatment) as belonging to one of four classes according to the degree of DNA damage observed (Collins et al., 1997; Lebailly et al., 1997; Kamer and Rinkevich, 2002): class 0, intact, well defined nucleus, no DNA damage; class 1, defined nucleus with light tail formation, <25% DNA in the tail; class 2, defined nucleus but weak fluorescence with 25 and 75% DNA in the tail; and class 3, nucleus no longer well defined, and tail consists of more than 75% of the DNA.

The visual classification system was verified independently using Euclid Comet Analysis software (St. Louis, MO) from images captured with a Nikon Coolpix 990 digital camera mounted to a Nikon Eclipse TE300 microscope equipped with epifluorescence capabilities. The total number of comets in classes 0 and 1 (less than 25% DNA in the tail) and in classes 2 and 3 (greater than 25% DNA in the tail) were summed and expressed as a percentage of the total comets counted for each slide. Increases in the percentage of total comets in classes 0 and 1 were indicative of cross-linked DNA.

Cell Cycle Effects. MDA468 and NQ16 cells were treated and dosed as described above. Harvested cells were centrifuged at 1500 rpm for 5 min (4°C) to pellet and were resuspended in PBS to a concentration of 1 x 10⁶ cells/ml, which was again pelleted by centrifugation. The pellet was resuspended in 1 ml of 0.005% propidium iodide, 0.3% saponin, 1 mM EDTA, 1% azide, and 1% fetal bovine serum containing RNase (125 U/ml) and incubated at 4°C in the dark for approximately 12 h. DNA content was determined on a FACScan flow cytometer (BD Biosciences, San Jose, CA) equipped with a 488-nm Ar laser.

Morphologic Assessment of Apoptosis. MDA468 and NQ16 cells were treated and dosed as described above. Hoechst 33342 (1 µg/ml) was added directly to cells in media and allowed to incubate at 37°C for 10 to 15 min. Apoptotic cells lose contact with adjacent cells and lift into the media (Prasad et al., 1999); therefore, the media containing detached cells was transferred to a 15-ml tube and placed on ice. Adherent cells were rinsed in PBS, incubated with 0.5 ml trypsin/EDTA for 2 min at 37°C, and neutralized with 0.5 ml of the reserved media, and the cell suspension was transferred to the existing 15-ml tube on ice containing reserved media and detached cells. Cells were pelleted by centrifugation at 1500 rpm for 5 min, supernatant was aspirated, and pellets were rinsed in ice-cold PBS, recentrifuged at 1500 rpm for 5 min, and resuspended in a volume of 0.5x solution of acridine orange/ethidium bromide (stock solution of 0.3 mg of acridine orange and 1 mg of ethidium bromide per milliliter of PBS) just prior to viewing with fluorescence microscopy. For each treatment, a minimum of 200 cells was scored as viable, necrotic, or apoptotic based on nuclear condensation and membrane integrity.

Caspase-3 Immunoblot Analysis. MDA468 and NQ16 cells were treated and dosed as described above. Detached and adherent cells were centrifuged at 1500 rpm for 5 min to pellet cells. The pellet was washed in ice-cold PBS, recentrifuged, and suspended in 30 µlof caspase lysis buffer (20 mM HEPES, pH 7.5, 10 mM KCl, 1.5 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 250 mM sucrose, and 0.25 mM phenylmethyl sulfonyl fluoride with protease inhibitors; Roche Diagnostics, Indianapolis, IN). The pellet was vortexed and incubated on ice for 30 min, and the probe was sonicated for 5 s and centrifuged at 13,000 rpm for 15 min to pellet debris. All 30 µl of cellular lysate was separated by 12% SDS-polyacrylamide gel electrophoresis (precast minigels) and transferred to a polyvinylidene diflouride membrane. The membrane was immunoblotted for caspase-3 (rabbit anti-caspase-3, 1:1000 dilution; Cell Signaling Technology Inc., Beverly, MA).

Statistical Analysis. Statistical analysis was performed using NCSS (Kaysville, UT) software. Significant differences (p < 0.05) of treated cells compared with time point-matched control cells were determined by one-way analysis of variance using the Tukey-Kramer Multiple Comparison post hoc test analysis and or the Student's paired t tests. Data are presented as mean ± S.E.

	Results

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Cells were treated with various concentrations of RH1 for up to 2 h and washed, and fresh complete media were added to the plate. Cells were harvested at time points up to 48 h later (see Materials and Methods) for assessment of DNA cross-linking, cell cycle changes, and apoptosis.

DNA Cross-Linking. DNA cross-linking was detected by the degree of retardation in the amount of DNA migration into the tail of the comet in treated cells compared with control cells (Tice et al., 2000). Treatment of NQ16 cells with RH1 (50 and 100 nM) for 60 and 120 min resulted in a significant increase (p < 0.05) in cross-linked DNA compared with controls (Fig. 1A). Two hours after RH1 was removed from NQ16 cells, a significant increase (p < 0.05) in cross-linked DNA was observed for all RH1 concentrations used. The maximum amount of DNA cross-linking was observed at this time point for RH1 doses of 50 or 100 nM. Subsequent time points (6, 12, and 24 h) exhibited statistically greater levels of DNA cross-linking than controls, although all were lower than that seen at the 2-h time point. After 48 h in drug-free media, no cross-linked DNA could be detected in drug-treated cells. NQO1 was responsible for DNA cross-linking in the NQ16 cell line, as pretreatment with ES936, an irreversible inhibitor of NQO1 (Winski et al., 2001a) abrogated DNA cross-linking (Fig. 1A, inset). As previously shown, 100 nM ES936 inhibits greater than 95% of NQO1 activity within 30 min (Dehn et al., 2003).

View larger version (36K): [in this window] [in a new window]   Fig. 1. DNA cross-linking after RH1 treatment as assessed by the modified comet assay. Cells were treated with RH1 as described under Materials and Methods. Cross-linking was detected by retardation in the amount of DNA migrating into the tail of the comet after irradiation of treated and control cells. Comets with less than 25% DNA in the tail were considered to have cross-linked DNA. A, percentage of comets with <25% DNA in the tails (indicative of cross-linking) in NQ16 cells after RH1 treatment (10 nM, light gray bars; 50 nM, white bars; 100 nM, cross-hatched bars) or in control cells (black bars). A significant increase (p < 0.05) in the percentage of cells exhibiting cross-linked DNA as compared with controls is shown by *. The graphed insert demonstrates NQO1 is responsible for DNA cross-linking as pretreatment with 100 nM ES936 (), a mechanism-based inhibitor of NQO1, for 30 min prior to RH1 treatment (100 nM) abrogated the DNA cross-linking seen with 100 nM RH1-treated () cells (this is the same data represented by cross-hatched bars in the histogram). B, results of control and RH1 treated (50 nM, white bars; 100 nM, cross-hatched bars; 500 nM, dark gray bars) in MDA468 cells. An approximate 50-fold increase in the concentration of RH1 (500 nM) was required to observe a statistical increase (*, p < 0.05) in cross-linked DNA compared with control cells, which occurred at only a single time point (6 h). The graphed inset demonstrates pretreatment with 100 nM ES936 () prior to treatment with 500 nM RH1 did not result in differences in DNA cross-linking from that of 500 nM RH1 alone () in MDA468 cells at any time point. Data represent mean values of three independent experiments ± S.E.

The MDA468 cell line demonstrated a much different outcome after RH1 treatment (Fig. 1B). An approximate 10-fold increase in the concentration of RH1 (500 nM) induced little DNA cross-linking. A statistical increase (p < 0.05) in DNA cross-linking could only be observed at a single time point (6 h) and was very modest relative to the DNA cross-linking observed in NQ16 cells using lower doses of RH1. ES936 pretreatment had no effect on RH1-induced DNA cross-linking in MDA468 cells at any time point (Fig. 1B, inset). The MDA468 cell line expresses the highest amount of NQO1*2 protein that we have seen in any homozygous mutant cell line (D. L. Dehn, unpublished data). Although this protein is rapidly degraded through the proteasomal pathway (Siegel et al., 2001) and has very low measured activity (Dehn et al., 2004), it may be present in sufficient levels to result in the bioactivation of RH1, an extremely efficient substrate of NQO1, resulting in the DNA cross-linking observed with the highest dose of RH1 used in these experiments (500 nM).

Cell Cycle Effects. In NQ16 cells, although a slight suppression in G₁ (Fig. 2A) was seen for all RH1 doses (10, 50, and 100 nM) after 6 h of incubation in drug-free media, marked changes in the cell cycle were not observed until after 12 h. After 24 h in drug-free media, the two highest RH1 doses had less than 20% of the number of control cells in G₁. A slight increase (up to 1.3-fold) in the number of cells in S phase was seen for all RH1 doses (10, 50, and 100 nM) at 6 h (Fig. 2B). Twelve hours after removal of RH1, almost 55% of the cells that had been treated with 100 nM were in S phase, a 2.4-fold increase over control. Cells that had been treated with 10 or 50 nM RH1 exhibited a similar pattern of increased percentages of cells in S phase but a smaller -fold increase over control (1.3 and 2.2, respectively). At 24 h after treatment, the pattern persisted but was more exaggerated, whereas after 48 h in drug-free media, treated cells were no longer blocked in S phase. By 48 h after drug removal, approximately 70% of the NQ16 cells analyzed that had been treated with RH1 at 50 or 100 nM were in G₂/M (Fig. 2C). NQ16 cells that had been treated with the lowest dose of RH1 displayed a slight increase in the number of cells in G₂/M (2.3-fold) but overall showed a similar cell cycle pattern as controls.

View larger version (19K): [in this window] [in a new window]   Fig. 2. Effects of RH1 treatment on the cell cycle in NQ16 cells as determined by flow cytometry. Cells were treated with RH1 as described under Materials and Methods. Alterations in the cell cycle of NQ16 cells began between 6 and 12 h after RH1 removal. A slight suppression in G1 (A) was seen for cells treated at all RH1 doses (, 10 nM; , 50 nM; , 100 nM) compared with control cells () 6 h after RH1 removal, but marked changes were not observed until 12 h. A concomitant slight increase (up to 1.3-fold) in the percentage of cells in S phase (B) was seen at the 6-h time point. Twelve hours after the removal of RH1, almost 55% of the cells that had been treated with 100 nM were still in S phase, a 2.4-fold increase over control cells. The pattern persisted until the 24-h time point, after which the cells were no longer delayed in S phase but blocked in G2/M (C). Data are representative of results for three independent experiments, with 10,000 cells analyzed per experiment.

In MDA468 cells, higher doses of RH1 were necessary to observe alterations in the cell cycle (Fig. 3, A–C). A dose of 500 nM RH1 was necessary to observe the major alterations that were seen with 50 to 100 nM RH1 in the NQ16 cell line. The two lower doses caused a disturbance in the cell cycle similar to what was seen in the NQ16 line at 10 nM RH1. MDA468 cells that had been treated with 50 or 100 nM RH1 appeared to recover after 48 h in drug-free media. In both NQ16 and MDA468 cells, elevated numbers of cells (relative to control) persisted in G₂/M until at least 72 h (data not shown).

View larger version (18K): [in this window] [in a new window]   Fig. 3. Effects of RH1 treatment on the cell cycle in MDA468 cells. Cells were treated with RH1 as described under Materials and Methods. Higher doses of RH1 (, 50 nM; , 100 nM; , 500 nM) were necessary to observe alterations in the cell cycle of MDA468 cells. A slight suppression in G1 (A) was seen for cells treated at medium and high RH1 doses (, 100 nM; , 500 nM) compared with control cells () 6 h after RH1 removal and a slight increase in the percentage of cells in S phase (B) at the same time point. By 12 h after RH1 removal, all cells treated (regardless of dose) exhibited a suppression in G1 (A) compared with control cells () that was more exaggerated by 24 h but was returning to control levels by 48 h for the two lower RH1 doses. A concomitant increase in the percentage of cells in S phase (B) was seen at the 12-h time point and by 24 h, cells treated at the highest RH1 dose were significantly delayed in S phase. Only cells treated with 500 nM RH1 demonstrated a G2/M block (C) at 48 h after drug removal. Data are representative of results for three independent experiments with 10,000 cells analyzed per experiment.

Morphologic Indicators of Apoptosis. Detached and adherent cells were combined for morphologic assessment of apoptosis (see Materials and Methods). RH1 induced apoptosis in a time- and concentration-dependent manner in NQ16 cells (Fig. 4A). Differences between treated and control cells were observed 6 h after removal of RH1. Thus, apoptosis was detected subsequently to DNA cross-linking (as early as 60 min during treatment) but prior to major cell cycle changes (starting at 12 h after RH1 removal) in NQ16 cells. By 6 h after removal of RH1, all dosed cells exhibited a significantly higher percentage of apoptosis than controls (up to 7% in treated cells compared with 2% in control cells; p < 0.05, n = 3). After 24 h in drug-free media, cells that had been treated with 50 or 100 nM RH1 exhibited a significantly higher percentage of apoptosis (22 to 32%) than those that had been treated with the lowest RH1 dose (10 nM) (6%) or controls (2%). The same pattern persisted 48 h after the removal of RH1. The percentage of apoptosis in cells incubated in drug-free media for 48 h after treatment with 50 or 100 nM RH1 increased to 51 to 56%, whereas the percentage of apoptotic cells after low-dose RH1 treatment increased to 16%; however, only cells treated with RH1 at 50 or 100 nM exhibited significantly higher levels of apoptotic cells than controls.

View larger version (20K): [in this window] [in a new window]   Fig. 4. Apoptosis, as determined by nuclear morphology, after RH1 treatment. Cells were treated with RH1 as described under Materials and Methods. A, percentage of apoptosis in NQ16 cells after RH1 treatment (10 nM, light gray bars; 50 nM, white bars; 100 nM, cross-hatched bars) and control cells (black bars) during (m) or after (h) exposure. Significant differences (p < 0.05) in the percentage of apoptosis between treated (10 to 100 nM) and control cells (as indicated by *) begins 6 h after removal of RH1. B, percentage of apoptosis in MDA468 cells after RH1 treatment (50, 100, and 500 nM, dark gray bars) and in control cells. A significant increase (*, p < 0.05) in the percentage of apoptotic cells was observed in those cells treated at the highest RH1 dose (500 nM) beginning 12 h after RH1 removal. Data represent mean values of three independent experiments ± S.E.

Vehicle-treated MDA468 cells exhibited between 5 to 7% apoptosis and RH1 treatment did not significantly increase apoptosis during the period of incubation (120 min). A statistically significant increase in apoptosis was exhibited in cells 12 h after drug removal (500 nM RH1) compared with controls (7% compared with 5% in control cells) (Fig. 4B), which persisted 24 h after drug removal (19% compared with 7% in control cells). After 48 h in drug-free media, cells treated with 100 and 500 nM RH1 exhibited a significantly higher percentage of apoptosis (19–29%) than controls (7%).

Biochemical Indicators of Apoptosis. RH1-induced caspase-3 cleavage (17- and 12-kDa active subunits) was observed in NQ16 cells after 12 h in drug-free media, whereas a diminution of the 32-kDa procaspase band and an increase in intensity of the subunit bands was observed after 48-h incubation in drug-free media (Fig. 5A). However, morphological analysis was more sensitive in detecting apoptosis than caspase-3 cleavage by immunoblot. Although morphological analysis indicated significantly increased apoptosis in NQ16 cells as early as 6 h after treatment with RH1, the overall percentage of apoptosis was less than 15%, and caspase-3 cleavage was not detected at this time point. In floating cells (which contain a much higher percentage of apoptotic cells than adherent cells), cleavage of caspase 3 could be observed as early as 6 h after treatment (data not shown). In MDA468 cells, caspase-3 cleavage was barely detectable at 5-fold higher RH1 concentrations relative to NQ16 cells. Staurosporine (1 µM), a compound known to cause apoptosis in MDA468 cells in a caspase-dependent manner (Duval et al., 2002), was used as a positive control for caspase-3 activation for both cell lines (data not shown).

View larger version (42K): [in this window] [in a new window]   Fig. 5. Caspase-3 cleavage in NQ16 and MDA468 cells after treatment with RH1 (100 or 500 nM, respectively). A (NQ16 cells) and B (MDA468 cells) represent the immunoblot analysis for caspase-3 using adherent and detached cells at 6, 12, 24, or 48 h after the removal of the RH1 and incubation in drug-free media. Cells were treated with RH1 as described under Materials and Methods.

	Discussion

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RH1 has been shown to be a better substrate for recombinant purified NQO1 than previous NQO1-targeted antitumor quinones such as 2,5-diaziridinyl-3,6-dimethyl-1,4-benzoquinone (MeDZQ), diaziquone, or 3-hydroxymethyl-5-aziridinyl-1-methyl-2-(1H-indole-4,7-dione)-propenol (EO9) (DiFrancesco et al., 2000; Winski et al., 2001b). We have shown RH1 is highly cytotoxic, requiring only nanomolar levels for effective reduction in cell growth in vitro and in xenograft systems in vivo (Dehn et al., 2004). RH1 exhibited an approximate 10-fold higher toxicity in NQ16 cells that contain NQO1*1 protein than in the parental MDA468 cell line. In this study, we performed a detailed examination of the mechanism of cytotoxicity of RH1 in human breast cancer cells. This work is important since RH1 is currently in phase I clinical trials, and the mechanism of cytotoxicity has yet to be defined. Previous studies (Kim et al., 2004a) examining the interaction of RH1 with radiation have demonstrated some cell cycle and apoptotic changes induced by RH1. The present study, however, is the first detailed examination of the mechanism of RH1-induced cytotoxicity and the interrelationship between DNA cross-linking, cell cycle alterations, and the induction of apoptosis. Our results indicate a different sequence of events occurs after treatment with RH1 in cells containing wtNQO1 compared with cells deficient in wtNQO1. RH1 induced DNA cross-linking and subsequent apoptosis and cell cycle changes in NQ16 cells containing wtNQO1. In MDA468 cells, markedly higher doses were required to induce DNA cross-linking and cell cycle changes; only one time point (6 h) demonstrated a significant increase in DNA cross-linking at a dose of 500 nM RH1. In NQ16 cells, doses 50-fold lower generated an earlier and consistently higher degree of DNA cross-linking. At equivalent RH1 doses (50 or 100 nM), the percentage of apoptotic MDA468 cells was one-third to one-fourth that in NQ16 cells after 48 h in drug-free media. Necrosis did not differ between treated and control NQ16 or MDA468 cells at any time point or concentrations (data not shown).

Our data demonstrate RH1 was rapidly activated to an alkylating agent in NQ16 cells. DNA cross-linking was observed within the 1st hour of drug treatment, and maximal cross-linking was seen within 2 h after drug removal, which decreased over 48 h, at which time no difference was seen between control and treated cells, indicating either DNA repair or cell death. Rapid induction of DNA cross-links seems to be the primary mechanism of RH1-induced DNA damage. Single-strand breaks were not observed after treatment of BE cells with RH1 (Winksi et al., 2001b), and we did not observe any single-strand breaks in MDA468/NQ16 cells up to 8 h after treatment (data not shown). Diaziridinylquinones and mitomycin C have previously been demonstrated to induce rapid DNA cross-linking that was subsequently repaired (Ward et al., 1997; Warren et al., 1998). The fact that inhibition of NQO1 in NQ16 cells abrogated DNA cross-linking and little to no cross-linking was observed in MDA468 cells indicates NQO1 is the enzyme responsible for bioreduction of RH1 to a DNA-alkylating species.

The determination of cell cycle by flow cytometry is based on the measurement of relative DNA content. Accumulation of cells in S phase can be interpreted as a reduction in the rate at which cells increase their DNA content reflecting inhibition of DNA synthesis (Dean and Fox, 1984). Bifunctional alkylating agents are known to inhibit the rate of DNA synthesis, resulting in an S phase delay followed by an accumulation of cells in G₂ due to unrepaired DNA cross-links (Roberts et al., 1971; Linfoot et al., 1986). For example, adozelesin caused a spectrum of cell cycle perturbations, including a sequential S phase delay then a G₂ block, in ovarian cancer lines (Nguyen et al., 1992). Nitrogen mustards tend to produce a prolongation in S phase, presumably because of the decreased rate of replicon initiation and fork progression from the DNA-cross links produced (Roberts et al., 1975; Clarkson and Mitchell, 1979). The cell cycle profiles obtained after NQ16 cells were treated with RH1 are consistent with this pattern of a delay in S phase, a G₂/M block, and depletion of cells in G₁. In a recently published study (Kim et al., 2004a) examining the interaction of RH1 and irradiation, RH1 was also found to cause a G₂/M block. Similar effects on the cell cycle were observed in MDA468 cells lacking wtNQO1 but required an RH1 concentration 10 times that required to observe the same effects in NQ16 cells. A G₂/M block was only observed after 48 h in drug-free media in MDA468 cells exposed to 500 nM RH1.

In agreement with preferential RH1 induction of DNA cross-linking and cell cycle changes in cells containing high levels of NQO1, a similar effect was observed in the case of RH1-induced apoptosis. In NQ16 cells, apoptosis was observed earlier and at markedly lower concentrations of RH1 (up to 50-fold depending on the time point) that were necessary to induce apoptosis in MDA468 cells. Tudor et al. (2003) also found a correlation between high NQO1 activity and cytotoxicity accompanied by apoptosis after treatment with aziridinylbenzoquinones.

The measurement of RH1-induced DNA cross-linking, cell cycle changes, and apoptosis under identical conditions allows us to analyze the inter-relationships between these effects. In NQ16 cells, bioreductive activation of RH1 by NQO1 results in immediate DNA cross-linking, which appears to be a primary mechanism of RH1 cytotoxicity in cells containing NQO1*1 protein. Temporal analysis demonstrated that in NQ16 cells, RH1-induced changes in cell cycle and apoptosis occurred subsequent to DNA cross-linking. Significant increases in apoptosis could be detected as early as 6 h after treatment with RH1. Minor perturbations in the cell cycle could also be detected at 6 h; however, S phase delay began at 12 h and cell cycle block in G₂/M was not apparent until 48 h. This suggests that S phase delay and G₂/M arrest are not absolute requirements for apoptosis. It has been reported that cell death resulting from cisplatin-induced DNA damage did not correlate with alterations of the cell cycle; the ratio of antiapoptotic to proapoptotic proteins was thought to be the final determinant of whether a cell became apoptotic (Gonzalez et al., 2001). Shifts in the ratios of pro- and antiapoptotic proteins have been observed in cells after treatment with azirdinylbenozquinones (Tudor et al., 2003). Numerous reports in the literature point to DNA damage as a trigger for apoptosis (Norbury and Zhivotovsky, 2004), which would be consistent with the results of this study. Caspase-2 has been suggested as a signal linking drug-induced DNA damage and mitochondrial dysfunction leading to downstream apoptotic events including caspase-3 activation (Lassus et al., 2002; Dirsch et al., 2004; Robertson et al., 2004). Our data demonstrate that RH1 treatment induced caspase-3 cleavage in NQ16 cells, suggesting a caspase-3-dependent route of RH1-induced apoptosis. Caspase-3 cleavage was barely detectable in MDA468 cells undergoing apoptosis, suggesting that effector pathways leading to RH1-induced apoptosis may be different in these cells. The precise role of apical and effector caspases in RH1-induced apoptosis is deserving of further study.

In summary, our results indicate RH1 can induce cellular toxicity preferentially in cells containing NQO1. In NQ16 cells expressing NQO1*1 protein, low-dose RH1 resulted in early DNA cross-linking and the induction of apoptosis without significant cell cycle alterations and without a G₂/M block. Higher doses of RH1 result in prolonged DNA cross-linking, G₂/M cell cycle arrest, and apoptosis. In MDA468 cells lacking NQO1*1 protein, 10- to 50-fold higher RH1 concentrations were required to induce modest DNA cross-linking, apoptosis, and G₂/M cell cycle arrest. The definition of the mechanisms of antitumor activity of RH1 and the inter-relationships of DNA cross-linking, cell cycle changes, and apoptosis will be useful for the selection of appropriate biomarkers in upcoming clinical trials.

	Footnotes

 
This work was supported by the National Institutes of Health (Grant CA-51210 to D.R.). D.R. discloses a patent interest in RH1.

doi:10.1124/jpet.104.081380.

ABBREVIATIONS: NQO1, NAD(P)H:quinone oxidoreductase; RH1, 2,5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone; ES936, 5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]indole-4,7-dione; PBS, phosphate-buffered saline.

Address correspondence to: Dr. David Ross, University of Colorado Health Sciences Center, Department of Pharmaceutical Sciences, 4200 E. Ninth Ave, Campus Box C238, Denver, CO 80262. E-mail: david.ross{at}uchsc.edu

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