Introduction

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Alkyltriazenes and alkylnitrosoureas demonstrate significant antitumor activity in vivo (Carter et al., 1976; Hill et al., 1989; Church et al., 1990; Mitchel and Dolan, 1993). Substantial evidence suggests that alkylation of DNA at the O⁶ position of guanine is the cytotoxic lesion induced by these agents (Bodell et al., 1985; Tisdale, 1987; Baer et al., 1993; Pegg et al., 1995). Mer+ cells expressing elevated levels of O⁶-alkylguanine transferase, an enzyme capable of repairing the O⁶-alkylguanine lesion, show significant resistance to the action of alkylating agents like mitozolomide, N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU), and temozolomide (Tisdale, 1987; Lee et al., 1991; Chen et al., 1993; Mitchel and Dolan, 1993). Determination of O⁶-alkylguanine transferase levels currently is being investigated as a prognostic tool for brain tumors (Belanich et al., 1996; Mineurs et al., 1996). Patients with tumors containing elevated levels of this enzyme respond poorly to BCNU, dacarbazine, and its second-generation derivative, temozolomide 1 (R = Me) (Belanich et al., 1996a,b).

View larger version (6K): [in this window] [in a new window]   Scheme 1.

Recently, we designed and synthesized a novel class of cyclic compounds, the 1,2,3,5-4-tetrazepinones (PYRZ 2, R = Me; PYRCL 2, R = 2-chloroethyl; NIME 3, QUINCL 4, R = 2-chloroethyl), which, unlike temozolomide and mitozolomide, are weak alkylators (Jean-Claude and Just, 1991, 1998; Jean-Claude, 1992; Jean-Claude et al., 1997). Their weak alkylating ability is believed to result from the resistance of the ureido moiety to hydrolysis. They preferentially decompose by ring-contraction to give metabolites of type 6. In previous studies, we showed that tetrazepinones could induce DNA damage in OVCAR-3 cells and arrest the cell cycle in G2/M (Jean-Claude et al., 1997, 1998).

To determine the possible contribution of O⁶-guanine alkylation to the activity of tetrazepinones, we have investigated their effects on two O⁶-methylguanine-DNA methyl transferase (MGMT)-proficient (SF-188, WiDR) and two MGMT-deficient (SF-126, BE) cell lines. In contrast to temozolomide, mitozolomide, and BCNU, which showed selective cytotoxicity toward the Mer cells, the four tetrazepinones investigated were shown to be almost equiactive in both cell types. Their effect on cell cycle progression was also independent of the cell phenotype. In further experiments, the tetrazepinones were shown to induce a dose-dependent inhibition of MGMT activity in the MGMT-proficient SF-188 cell line.

	Materials and Methods

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Drug Treatment. Temozolomide was supplied by Schering- Plough Research Institute (Kenilworth, NJ). Mitozolomide was a gift from the drug development branch of the National Cancer Institute, and O⁶-benzylguanine was kindly provided by Dr. Robert Mochel of the same department. The tetrazepinones were designed and synthesized in our laboratories (Jean-Claude and Just, 1991, 1998; Jean-Claude and Williams, 1998). In all assays, the drugs were dissolved in dimethyl sulfoxide (DMSO) and diluted in sterile RPMI 1640 medium immediately before treatment of cell cultures. The concentration of DMSO never exceeded 2% (v/v). The cells were treated with the different drugs for 1 h, and treatments were terminated by aspiration of the drug-containing medium and replacement with fresh RPMI 1640 solution.

Cell Culture. The SF-126 and SF-188 cells were obtained from the Brain Tumor Research Center of San Francisco. The WiDR cells were purchased from American Type Culture Collection (Manassas, VA); BE cells were a gift from Dr. Daniel Yarosh (Applied Genetics, Freeport, NY). Cells were maintained as monolayer cultures at 37°C in a humidified atmosphere of 5% CO₂/95% air in RPMI 1640 supplemented with fetal bovine serum (10%), L-glutamine (2 mM), penicillin (50 U/ml), and streptomycin (50 mg/ml). Cells were maintained in logarithmic growth by harvesting with a trypsin-EDTA solution containing 0.5 mg/ml trypsin and 0.2 mg/ml EDTA and replanting before cells reached confluency.

Cytotoxicity Studies. Cell monolayers were incubated with varying amounts of the different drugs for 1 h, and cytotoxicity was evaluated by the sulforhodamine B (SRB) assay 7 days after treatment (Hartley et al., 1988). In cytotoxicity studies involving MGMT depletion, cells were incubated with O⁶-BG (30 µM) for 2 h before the 1-h drug treatment and then grown for 7 days in fresh medium containing O⁶-BG (30 µM).

Flow Cytometry. The effect of 1-h exposure of the different drugs on the cell cycle was evaluated after various recovery times. The cells were harvested by trypsinization at the appropriate times. After fixation in ethanol (70%, v/v), they were stained with an aqueous propidium iodide (PI) solution (100 µl, 100 µg/ml) containing RNase (100 µl, 50 µg/ml) for 30 min at room temperature in the dark. The fluorescence was detected in a spectral range between 580 and 750 nm. Each cytometric analysis was performed on a Becton Dickinson FACScan instrument on 1 to 3 × 10⁵ cells. LYSYS II software was used to estimate cell percentage in each cell cycle phase (Becton Dickinson).

O⁶-Alkylguanine DNA Alkyltransferase Assay. This was carried out as described previously (Baer et al., 1993). Extracts from cells (10⁷) treated with the different drugs for 3 h were incubated with O⁶-[³H]methylguanine containing DNA. The latter was then degraded to acid-soluble materials, and the precipitated protein (containing the methylated MGMT) was collected by centrifugation and counted. The protein content of the cells was determined with a Bio-Rad protein assay kit using bovine serum albumin as a standard.

Statistical Analysis. Statistical significance was determined with the GraphPad Prism software package, using the one-tailed Student's t test.

	Results

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Cytotoxicity Studies. The cell lines used in this study were SF-126 and SF-188 (derived from patients with glioma) and WiDR and BE (human colon tumor cell lines). The IC₅₀ of temozolomide, BCNU, and the four novel tetrazepinones are shown in Table 1. No significant differences were observed between the IC₅₀ values of tetrazepinones in the Mer+ cells and those measured for the Mer cells (one-tailed t test, P < .05). The resistance indices were near 1 for all of the tetrazepinones studied. The IC₅₀ values of temozolomide in Mer+ cells were extremely high (IC₅₀ = 1210.0 µM in SF-188 and 1850.0 µM in WiDR cells), but its activity was 11- to 34-fold higher in the Mer lines (IC₅₀ = 54.1 µM in BE and 114.3 µM in SF-126 cells). Mer cells were approximately 3-fold more sensitive to BCNU than Mer+ cells. Similarly, mitozolomide was 7.5- to 10-fold more potent against the Mer than it was against the Mer+ cells.

O⁶-Alkylguanine DNA Methyltransferase Level. Based on their contrasting levels of MGMT, the Mer+ cell line SF-188 [literature: 370.0 fmol/mg MGMT protein (Bodell et al., 1985); found: 429.0 fmol/mg protein] and the SF-126 line (which expresses barely detectable levels of MGMT) were selected for the determination of the effect of the drugs on MGMT activity. As shown in Fig. 1, all of the tetrazepinones exhibited MGMT inhibitory activity. The strongest inhibition was obtained with PYRCL (IC₅₀ = 18.0 µM), and the inhibitory activity of NIME (IC₅₀ = 272.0 µM) was in the same range as that of temozolomide (IC₅₀ = 215.0 µM). As shown in Fig. 1, the MGMT-inhibitory activity of the tetrazepinones did not correlate with their cytotoxic activity. As an example, PYRCL is a 4-fold-stronger inhibitor of MGMT than PYRZ, but IC₅₀ values for cell survival were in the same range (PYRZ, 31.6 µM; PYRCL, 34.4 µM). Negligible inactivation of MGMT was observed with the bifunctional alkylating agents mitozolomide and BCNU (data not shown).

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Lack of correlation between the cytotoxic activity of the tetrazepinones and their AGT-inhibitory activity.

Flow Cytometry. The differential effects of each agent used in this study on cell cycle progression were examined in the SF-126 and SF-188 cell lines (Figs. 2 and 3). The effects of NIME on the cell cycle were compared with those of the 3-methyltetrazinone temozolomide (Fig. 2, A-F). At the highest concentration (240 µM), NIME induced a significant G₂2-M arrest of almost equal strength in both cell phenotypes with 58.3% of SF-188 and 64.6% of SF-126 cells accumulated in G₂-M 24 h after treatment (Fig. 2, A-C). In contrast, a delayed but strong differential effect appeared in cells treated with temozolomide. As an example, treatment of SF-126 Mer cells with 120 µM temozolomide (Fig. 2E) resulted in minor cell cycle effects 24 h post-treatment; however, a marked perturbation of the cell cycle (59.5% of the cells accumulated in G2/M) was observed 48 h post-treatment. In contrast, under the same conditions, only 28.5% of the SF-188 cells were in G₂-M. Interestingly, while cell cycle arrest induced by temozolomide in SF-126 cells appeared to increase with time (Fig. 2, D-F), the G ₂-M blocks triggered by NIME in both cell phenotypes were observed as early as 24 h post-treatment and decreased by 20.9 to 25.7% at 48 h post-treatment.

View larger version (41K): [in this window] [in a new window]   Fig. 2.   Flow cytometric profiles of SF-126 and SF-188 cell lines treated with 0 µM (A), 120 µM (B), and 240 µM NIME (C) and 0 µM (D), 120 µM (E), and 240 µM (F) temozolomide for 1 h and allowed to recover in drug-free media for either 24 or 48 h. Percentages of cells in each phase of the cell cycle are shown beside each histogram.

View larger version (47K): [in this window] [in a new window]   Fig. 3.   Flow cytometric profiles of SF-126 and SF-188 cell lines treated with the bifunctional drugs for 1 h and allowed to recover in drug-free media for 24 h. Percentages of cells in each phase of the cell cycle are shown beside each histogram.

	Discussion

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Alkylnitrosoureas, 3-alkyl-1-aryltriazenes, and 3-alkylimidazotetrazinones are strong high-nitrogen alkylating agents that are known to alkylate DNA at both O⁶- and N⁷-guanine residues. A significant body of work has accumulated to suggest that O⁶-alkylguanine is the major cytotoxic lesion induced by methylating and chloroethylating agents (Tisdale, 1987; Lee et al., 1991; Chen et al., 1993; Mitchel and Dolan, 1993). Cells expressing high levels of O⁶-alkylguanine transferase, the enzyme capable of repairing the O⁶-alkylguanine lesion by capturing the alkyl group onto its own cysteine residue in an autoinactivating reaction, are resistant to alkyltriazenes, imidazotetrazinones, and nitrosoureas (Lee et al., 1991; Baer et al., 1993; Pegg et al., 1995). Depletion of MGMT in these cells correlates with potentiation of mono- and bifunctional alkylating agents such as temozolomide, mitozolomide, or BCNU (Carter et al., 1976; Gibson et al., 1986; Baer et al., 1993). This differential sensitivity of Mer+ and Mer cells is now well correlated with the O⁶-alkylguanine repair efficiency (Lee et al., 1991; Mitchel and Dolan, 1993; Fairbairn et al., 1995; Belanich et al., 1996a). We have used this model to determine the role of O⁶-alkylation of guanine in the cytotoxic properties of the tetrazepinones.

The results presented here indicate that the tetrazepinones display cytotoxicity profiles that are markedly different from those of their parent triazenes. Likewise, their effects on cell cycle progression differ from those of the most representative member of the previous classes. In this study, strong differential cytotoxicity was observed between the Mer cell lines (SF-126 and BE) and the Mer+ cells (WiDR and SF-188) after exposure to BCNU, mitozolomide, and temozolomide. In contrast, the resistance indices (which are indicative of the relative differential activities of the different drugs against the two cell phenotypes) were near 1 for the tetrazepinones. This indicates that the Mer+ phenotype does not correlate with resistance to tetrazepinones. Furthermore, when the biochemical modulator O⁶-BG was used to inactivate MGMT, significant potentiation of the strong alkylating triazenes, but not the tetrazepinones, was observed in the SF-188 Mer+ cell line. This provided further evidence that the mechanism of action of tetrazepinones does not involve O⁶-alkylguanine and its repair enzyme, MGMT.

All the tetrazepinones tested caused an inhibition of MGMT activity at high concentrations (in the order of potency: PYRCL > PYRZ > QUINCL > temozolomide, NIME > mitozolomide, BCNU). The significant MGMT-inhibitory activity of the tetrazepinones contrasted with their weak alkylating power (Jean-Claude et al., 1995; Jean-Claude et al., 1997). The extent of O⁶-alkylation of guanine is related to the S_N1 character of the reaction mechanism (Ford and Wang, 1993). According to Pearson's hard-soft-acid-base principle (Pearson, 1973), the soft nucleophiles preferentially react with the soft nitrogen (e.g., guanine N⁷) and the harder electrophiles preferentially react with the harder oxygen centers in DNA bases (Ford and Wang, 1993). In both cases the electrophile (e.g., the alkylating species) must be extremely reactive. Therefore, O⁶-methylation or chloroethylation of guanine can be induced only by highly reactive alkylating species such as those generated by alkylnitrosoureas, alkyltriazenes, or their imidazotetrazine prodrugs. Given the weak alkylating capacity of the tetrazepinones (Jean-Claude et al., 1994, 1995, 1997, 1998), it is unlikely that their induced inactivation of MGMT would occur via an O⁶-alkylation of guanine. Hence, we postulate that this may occur by a direct reaction of the tetrazepinones with MGMT enzyme or by mechanisms that can indirectly cause MGMT degradation after cell treatment with the tetrazepinones. Considerable further work is ongoing to verify these hypotheses.

The significance of MGMT inactivation by the tetrazepinones in their cytotoxic activity is, at present, unknown. However, in the present study, a lack of correlation between the IC₅₀ for reduction of cell survival and IC₅₀ for inhibition of MGMT activity was apparent in the high-MGMT-expressing SF-188 cell line (Fig. 1). Despite its significant MGMT-inhibitory activity, the IC₅₀ of PYRCL in the SRB assay was in the same range as those of PYRZ or QUINCL, which are weaker MGMT inhibitors.

A study of the effects of biologically active drugs on the cell cycle often provides insight into their mechanism of action. Differential cell cycle effects were observed in cells treated with temozolomide, mitozolomide, and BCNU. For example, BCNU induced cell cycle arrest in G₂-M in SF-126 cell populations, but exerted a minor effect on the SF-188 cells. Cell cycle arrest in SF-126 cells may be due to a surveillance mechanism that signals the cells to arrest cell cycle progression until the drug-induced DNA lesions (possibly O⁶-alkylguanine and N⁷-alkylguanine lesions) have been repaired. Thus, SF-188 cells that express high levels of MGMT, N³-adenine DNA glycosylase (Matijasevic et al., 1991), and possibly N⁷-guanine base-excision repair enzymes (Bohr et al., 1987; Scicchitano and Hanawalt, 1989) may repair their DNA lesions faster than SF-126 cells and thereby progress faster in the cell cycle after exposure to temozolomide, mitozolomide, or BCNU. In contrast, no significant differential cell cycle arrest was observed for SF-188 and SF-126 cells treated with the tetrazepinones. The absence of differential cell cycle effects may be explained by specific differences in the DNA lesions induced by tetrazepinones. These lesions might not be repaired by enzymes associated with the repair of O⁶- or N⁷-alkylguanine or other alkylated DNA adducts induced by temozolomide, mitozolomide, and BCNU.

In summary, despite their structural similarities to the tetrazinones and nitrosoureas, the tetrazepinones appear to have a mechanism of action different from that previously described for the known high-nitrogen-alkylating agents. Their capacity to 1) inhibit MGMT, 2) arrest cell cycle in G₂-M, and 3) induce significant cell killing in Mer+ cell lines characterize them as novel DNA-reactive agents, which may offer new alternatives to the strong-alkylating, high-nitrogen antitumor agents such as BCNU, mitozolomide, or temozolomide.