Abstract

The effects of treatment of mice with O6-benzylguanine (O6-BeG) on the levels of O6-alkylguanine-DNA alkyltransferase (ATase) in the hematopoietic compartment and on the in vivo sensitivity of hematopoietic progenitor cells to the toxic and clastogenic effects of the antitumor agents 1,3-bis(2-chloroethyl)-nitrosourea (BCNU) and temozolomide were studied. When the overall effects of BCNU alone or with O6-BeG pretreatment were compared, dose potentiating factors of 4.17 for marrow cellularity, 4.57 for granulocyte macrophage-colony forming cells (GM-CFC) and 8.25 for colony forming unit-spleen (CFU-S) in O6-BeG pretreated versus nonpretreated animals were observed. A similar trend of dose potentiation was observed for temozolomide, although it was of lower magnitude: 1.20 for marrow cellularity, 1.63 for GM-CFC, and 1.68 for CFU-S. When the clastogenic effects of BCNU and temozolomide were examined in the mouse bone marrow micronucleus assay, a significantly (P < .05 to .001) higher frequency of micronuclei formation was observed in mice that received O6-BeG pretreatment compared with mice that received no pretreatment. These data suggest that the use of O6-BeG as a tumor-sensitizing agent before treatment of patients with O6-alkylating agents may lead to more severe hematological toxicity and possibly to an increased incidence of secondary leukemias as a result of elevated mutation frequencies in these patients.

ALKYLATING CHEMOTHERAPEUTIC agents comprising the chloroethylnitrosoureas (eg, BCNU, CCNU, fotemustine) and related methylating agents (eg, temozolomide, streptozotocin, procarbazine) remain one of the most important classes of antineoplastic compounds in clinical practice. The clinical usefulness of these drugs (hereafter called the O6-alkylating agents) is dependent on the extent and persistence of specific alkylation lesions at key DNA sites, damage at the O6-position of guanine being one of the major toxic and promutagenic events.1 Such damage is repaired by the O6-alkylguanine-DNA-alkyltransferase (ATase) protein, which transfers the alkyl group to its active site cysteine residue in a stoichiometric and autoinactivating process.2 This can limit the therapeutic efficacy of chloroethylating and methylating agents, but other cellular mechanisms such as detoxification by glutathione can also contribute to intrinsic or therapy-induced resistance.3,4 It has been shown that a number of human tumors are inherently resistant to the cytotoxic effects of alkylating agents due to higher level of ATase expression5,6 so that therapeutic strategies for depleting tumor ATase are gaining importance and agents that directly inhibit this DNA repair protein are being considered. O6-benzylguanine (O6-BeG) is one such agent: this irreversibly inactivates ATase by acting as an alternative substrate. O6-BeG has been shown to increase tumor cell sensitivity to alkylating agents both in vitro7-9 and in vivo10,11 and based on such studies, it is currently undergoing clinical trial.

Therapy involving the nitrosoureas and related agents is often complicated by acute dose-limiting cytotoxicity to hematopoietic and other tissues such as liver, lung, and gastrointestinal tract. One major problem that could arise as a result of ATase depletion and tumor sensitization by O6-BeG is the potentiation of toxicity to normal tissues. Recently, we have shown that O6-BeG can enhance the toxicity of temozolomide in primary human bone marrow cells in vitro.12 In the present study, we have examined the effect of O6-BeG pretreatment on the toxic and clastogenic effects of temozolomide and BCNU on murine hematopoietic cells in vivo.

MATERIALS AND METHODS

Chemicals

Temozolomide was obtained from the Cancer Research Campaign Formulation Unit, Department of Pharmaceutical Sciences, University of Strathclyde, Glasgow, Scotland. BCNU (carmustine) was purchased from Bristol-Myers Pharmaceuticals, Hounslow, Middlesex, England. O6-BeG was a generous gift from Dr R.S. McElhinney and Prof B. McMurry, Trinity College, Dublin, Ireland. Temozolomide and BCNU were dissolved in DMSO (30 mg/mL) and ethanol (25 mg/mL), respectively and both were diluted appropriately in phosphate-buffered saline (PBS). O6-BeG was homogeneously suspended in corn oil. All the solutions were freshly prepared immediately before use.

Mice

Male and female B6D2F1 mice aged 9 to 11 weeks and weighing between 24 g and 32 g were obtained from the Paterson Institute's breeding colony. The mice were housed three to a cage with wooden chip bedding and given commercial food pellets and water ad libitum. All experimental procedures were performed under United Kingdom Home Office Licence in accordance with the Animals (Scientific Procedures) Act (1986).

Cytotoxicity to Bone Marrow Cells

Groups of three male mice were treated with a single dose of either corn oil (vehicle control) or O6-BeG (30 mg/kg body weight) in corn oil intraperitoneally (IP) 2 hours before the administration of temozolomide or BCNU. Animals were killed by cervical dislocation 48 hours after the last injection and femur cell suspensions were prepared in Iscove's modified Dulbecco's (IMDM) medium. Nucleated cells in the marrow were counted using a Sysmex Microcellcounter (TOA Medical Electronics Co, Ltd, Kobe, Japan).

Granulocyte-Macrophage Colony-Forming Cell (GM-CFC) Assay

The method used was as described by Heyworth and Spooncer.13 Briefly, an aliquot of the femoral bone marrow suspension was added to IMDM supplemented with 20% fetal calf serum, 10% bovine serum albumin (BSA) (100 mg/mL), penicillin (at a final concentration of 0.2 mg/mL), streptomycin (at a final concentration of 0.033 mg/mL) and 5% murine lung conditioned medium as a source of colony-stimulating factor. Agar Noble (Difco, Detroit, MI) was added to 0.33% and aliquots of 1 mL were plated in 35 mm dishes and incubated at 37°C in humidified air plus 5% CO2 for 7 days. Colonies of at least 50 cells were then counted, and the number of GM-CFC per femur were calculated.

Colony-Forming Unit-Spleen (CFU-S) Assay

CFU-S were assayed in potentially lethally irradiated (15.25 Gy 60Co γ-rays; dose rate of 0.95 Gy/h) mice as previously described by Lord.14 Groups of 10 irradiated recipient female mice were intravenously injected with 0.2 mL of appropriately diluted femoral bone marrow suspension. Twelve days later mice were killed, spleens were removed and fixed, colonies were counted and the number of CFU-S per femur was calculated. Irradiation-only control groups of mice that did not receive any injected cells showed zero endogenous colony growth in all experiments.

Micronucleus Assay

Groups of four male B2D6F1 mice were treated with a single dose of either corn oil or O6-BeG (30 mg/kg) in corn oil IP. Two hours after this treatment, mice received a single dose of 0, 0.25, or 0.50 mg/kg BCNU or 0, 5, 10, or 15 mg/kg temozolomide IP and were killed 24 hours later. Femurs were removed and cells flushed out with fetal calf serum. The slides were prepared from the pellet, allowed to air dry, and fixed in methanol for 15 minutes. Slides were then stained with acridine orange as described by Tinwell and Ashby15 and scored blindly by two separate investigators for micronucleated polychromatic erythrocytes (MPE) among 2,000 polychromatic erythrocytes.

ATase Assay

Groups of four male B2D6F1 mice were treated with a single dose of O6-BeG (30 mg/kg IP) and killed at 2, 4, 8, 12, 16, 20, 24, 30, 36, 48, or 72 hours posttreatment. Bone marrow from both the femurs were flushed with 0.55 mL of Buffer I (50 mmol/L Tris-HCl, pH 8.3, 1 mmol/L EDTA, 3 mmol/L dithiothreitol containing 5 μg/mL leupeptin) and a piece of liver (≈0.1 g) was removed and immediately frozen in liquid nitrogen and stored at −70°C until further analysis. The ATase activity was measured in individual samples (four samples per time point) using [3H]-MNU–treated calf thymus DNA substrate. Briefly tissue samples were sonicated (two 10-second bursts at 10 μm peak to peak), and phenylmethylsulfonyl fluoride was added to a final concentration of 87 μg/mL. The tissue homogenate was centrifuged for 10 minutes at 18,000g at 4°C and aliquots of the supernatant were incubated for 1 hour at 37°C with the [3H]-DNA substrate above (≈20,000 cpm). Protein was precipitated by the addition of perchloric acid to a final concentration of 1 mol/L and DNA was hydrolyzed at 75°C for 50 minutes. The precipitated protein was washed with 1 mol/L perchloric acid, the resultant protein pellet resuspended in 10 mmol/L sodium hydroxide, and the amount of radioactivity in the pellet determined by liquid scintillation counting. Specific activities of ATase were calculated from the amount (fmoles) of [3H]-methyl groups transferred per unit amount of total protein under protein-limiting conditions. The protein concentrations were determined using BSA as standard by the method of Bradford.16 ATase activity was expressed in terms of methyl groups (fmol) transferred from DNA to protein (mg).

Statistics

The dose response curves of bone marrow cellularity, GM-CFC, and CFU-S for temozolomide and BCNU in the absence and presence of O6-BeG were compared by analysis of variance using MINITAB statistical software (MINITAB Inc, Pennsylvania). The unpaired Student's t-test was used to compare the bone marrow micronucleus frequencies in mice treated with either BCNU or temozolomide in the absence and presence of O6-BeG.

RESULTS

General Toxicity

Administration of a single IP dose of O6-BeG in corn oil, alone or in combination with either temozolomide or BCNU at any of the doses used did not produce any mortality or obvious morbidity in treated mice during the 48-hour test period.

Effect of O6-BeG on ATase Levels in Liver and Bone Marrow

The base line levels of ATase in liver and bone marrow before treatment with O6-BeG were 131 ± 5 and 102 ± 0.85 fmol/mg total protein, respectively. The ATase inactivation and the kinetics of recovery in mouse liver and bone marrow cells were studied after administration of 30 mg/kg O6-BeG (Fig 1). Exposure to O6-BeG reduced ATase activity in liver to undetectable levels (<2 fmol/mg) at the earliest time point studied and the level remained undetectable over the first 8 hours posttreatment. However, detectable levels of ATase (16% of untreated control; 16.6 ± 0.49 fmol/mg) were found in the bone marrow cells over this same time period. Thus, depletion of ATase in liver was greater in absolute terms than in bone marrow. There was a subsequent rise in ATase activity both in bone marrow and in liver from 12 hours onward with bone marrow ATase levels reaching 91% of untreated control levels at 48 hours and liver ATase levels reaching 79% of control values at 72 hours post-O6–BeG treatment.

Fig. 1.

Inactivation and regeneration kinetics of ATase after O6-BeG treatment in liver (•) and bone marrow (▪). Male B6D2F1 mice received 30 mg/kg O6-BeG and were killed at different time intervals. Data points represent mean ± SE of four animals.

Fig. 1.

Inactivation and regeneration kinetics of ATase after O6-BeG treatment in liver (•) and bone marrow (▪). Male B6D2F1 mice received 30 mg/kg O6-BeG and were killed at different time intervals. Data points represent mean ± SE of four animals.

Potentiation of Cytotoxicity in Bone Marrow

Treatment of mice with 30 mg/kg O6-BeG alone did not alter the bone marrow cellularity (corn oil control = 2.13 ± 0.152 × 107, O6-BeG = 2.10 ± 0.210 × 107 cells per femur; mean ± standard deviation (SD) of eight independent observations). Treatment with temozolomide alone decreased marrow cellularity in a dose-dependent manner and pretreatment of mice with O6-BeG potentiated this cytotoxicity as reflected by a significant shift in the dose response curve (Figs 2 and 3). The dose required to reduce marrow cellularity to 50% of control levels (toxic dose 50%, TD50 , Table 1) was calculated to be 89 mg/kg and 74 mg/kg for temozolomide in the absence and presence of O6-BeG, respectively, which is equivalent to a 1.20-fold potentiation of toxicity. The effects of O6-BeG pretreatment on BCNU toxicity were even more dramatic, with the TD50 for BCNU being 20 mg/kg and 4.8 mg/kg in the absence and presence of O6-BeG, respectively (P < .001), equivalent to a 4.17-fold potentiation of toxicity.

Fig. 2.

Potentiation of temozolomide-induced cytotoxicity by pretreatment with O6-BeG. Male B6D2F1 mice were pretreated with either corn oil or O6-BeG (30 mg/kg) in corn oil and 2 hours later treated with increasing doses of temozolomide. Each data point represents the mean ± SE of at least three independent experiments. Marrows from animals were pooled in each experiment. The data were tested with analysis of variance using MINITAB statistical software. Bone marrow cellularity and numbers of GM-CFC and CFU-S following treatment with O6-BeG plus temozolomide (▪) were significantly (P < .001) different from temozolomide alone (•).

Fig. 2.

Potentiation of temozolomide-induced cytotoxicity by pretreatment with O6-BeG. Male B6D2F1 mice were pretreated with either corn oil or O6-BeG (30 mg/kg) in corn oil and 2 hours later treated with increasing doses of temozolomide. Each data point represents the mean ± SE of at least three independent experiments. Marrows from animals were pooled in each experiment. The data were tested with analysis of variance using MINITAB statistical software. Bone marrow cellularity and numbers of GM-CFC and CFU-S following treatment with O6-BeG plus temozolomide (▪) were significantly (P < .001) different from temozolomide alone (•).

Fig. 3.

Potentiation of BCNU-induced cytotoxicity by pretreatment with O6-BeG. Male B6D2F1 mice were pretreated with either corn oil or O6-BeG (30 mg/kg) in corn oil and 2 hours later treated with increasing doses of BCNU. Each data point represents the mean ± SE of at least three independent experiments. Marrows from three animals were pooled in each experiment. The data were analyzed with analysis of variance using MINITAB statistical software. Bone marrow cellularity and numbers of GM-CFC and CFU-S following the treatment with O6-BeG plus BCNU (▪) were significantly (P < .001) different from BCNU alone (•).

Fig. 3.

Potentiation of BCNU-induced cytotoxicity by pretreatment with O6-BeG. Male B6D2F1 mice were pretreated with either corn oil or O6-BeG (30 mg/kg) in corn oil and 2 hours later treated with increasing doses of BCNU. Each data point represents the mean ± SE of at least three independent experiments. Marrows from three animals were pooled in each experiment. The data were analyzed with analysis of variance using MINITAB statistical software. Bone marrow cellularity and numbers of GM-CFC and CFU-S following the treatment with O6-BeG plus BCNU (▪) were significantly (P < .001) different from BCNU alone (•).

Table 1.

Potentiation of Temozolomide and BCNU Toxicity to Mouse Bone Marrow by O6-Benzylguanine

 Toxic Dose50 (mg/kg) 
 Temozolomide BCNU     
 −BG +BG DPF* −BG +BG DPF*     
Cellularity 89.0 74.0 1.20 20.0 4.80 4.17 
GM-CFU 68.9 42.2 1.63 20.0 4.38 4.57 
CFU-S 88.4 52.6 1.68 13.2 1.60 8.25 
 Toxic Dose50 (mg/kg) 
 Temozolomide BCNU     
 −BG +BG DPF* −BG +BG DPF*     
Cellularity 89.0 74.0 1.20 20.0 4.80 4.17 
GM-CFU 68.9 42.2 1.63 20.0 4.38 4.57 
CFU-S 88.4 52.6 1.68 13.2 1.60 8.25 
*

DPF = TD50 (drug)/TD50 (O6-BeG + drug).

Potentiation of Cytotoxicity to Hematopoietic Progenitor Cells

GM-CFC.Exposure of mice to O6-BeG had no significant effect on the number of femoral GM-CFC (corn oil control = 40,200 ± 2,860; O6-BeG = 48,200 ± 6,880 per femur, mean ± SD of six independent observations). Treatment with temozolomide or BCNU alone reduced the number of GM-CFU in a dose-dependent manner and this was potentiated by O6-BeG pretreatment (Figs 2 and 3). TD50 was calculated as 68.9 mg/kg for temozolomide alone and 42.2 mg/kg for O6-BeG plus temozolomide, equivalent to a 1.63-fold potentiation of toxicity. As with marrow cellularity, the effects on BCNU-induced toxicity were more pronounced than those with temozolomide, with TD50 values of 20 mg/kg for BCNU alone and 4.38 mg/kg with pretreatment with O6-BeG, a 4.57-fold potentiation of toxicity.

CFU-S.In the same experiments, O6-BeG treatment alone did not alter the number of CFU-S in mice (corn oil control = 2,330 ± 200; O6-BeG = 2,310 ± 270 per femur, mean ± SD of six independent observations). Treatment of mice with increasing doses of temozolomide or BCNU significantly depleted the number of CFU-S in a dose-dependent manner (Figs 2 and 3). Potentiation of toxicity by O6-BeG was observed with temozolomide as well as with BCNU. However, the effect with BCNU was more pronounced than with that of temozolomide as evidenced by the TD50 values. TD50 values for temozolomide in the absence and presence of O6-BeG were 88.4 mg/kg and 52.6 mg/kg, respectively, whereas corresponding figures for BCNU were 13.2 mg/kg and 1.60 mg/kg. These data indicate 1.68-fold potentiation of temozolomide toxicity and a dramatic 8.25-fold potentiation of BCNU toxicity to CFU-S following O6-BeG pretreatment (Table 1).

Potentiation of Micronucleus Formation in the Polychromatic Erythrocytes of the Bone Marrow

Micronucleus formation in bone marrow was used as a monitor of clastogenicity after treating mice with low doses of temozolomide or BCNU. A dose-dependent increase in the frequency of micronucleus formation was observed when animals were treated with temozolomide or BCNU alone, while O6-BeG on its own did not alter the frequency of micronucleus formation when compared with vehicle controls. However, O6-BeG pretreatment significantly (P < .05 to .001) increased the frequency of micronuclei observed in the bone marrow of animals that subsequently received temozolomide or BCNU (Fig 4).

Fig. 4.

Effect of O6-benzylguanine pretreatment on the induction of micronuclei in mouse bone marrow following the administration of various doses of temozolomide (A) and BCNU (B). Results are presented as mean ± SE from four mice per group. The level of significance in the presence and absence of O6-BeG is expressed as *P < .05 and ***P < .001 as assessed by unpaired Student's t-test. (□), Control; (▨), O6-BeG.

Fig. 4.

Effect of O6-benzylguanine pretreatment on the induction of micronuclei in mouse bone marrow following the administration of various doses of temozolomide (A) and BCNU (B). Results are presented as mean ± SE from four mice per group. The level of significance in the presence and absence of O6-BeG is expressed as *P < .05 and ***P < .001 as assessed by unpaired Student's t-test. (□), Control; (▨), O6-BeG.

DISCUSSION

There is compelling evidence that alkylation at the O6-position of guanine in DNA is one of the major lesions responsible for the cytotoxicity, mutagenicity, clastogenicity, and carcinogenicity of alkylating agents1 and that the DNA repair protein, ATase, plays a pivotal role in protection against these effects.2 Indeed, the pronounced sensitivity of the hematopoietic compartment to the cytotoxic effects of the O6-alkylating agents is almost certainly related to the finding that rat, mouse, and human bone marrow exhibit low levels of ATase compared with many other organs.17 The levels of ATase detected in bone marrow do presumably confer some protection of the hematopoietic tissue from the effects of O6-alkylating agents. Thus, pretreatment of animals or patients systemically with ATase inhibitors (to sensitize the tumor cells) might be predicted to deplete repair activity in the normal cells and as a consequence increase the toxicity to the normal tissues. Given that treatment with O6-alkylating agents (like most chemotherapeutic agents) results in significant myelosuppression, the use of O6-BeG may greatly exacerbate this effect. In this study, we have examined the effects of O6-BeG pretreatment on the cytotoxic and clastogenic effects of O6-alkylating agents in vivo.

We first examined the extent of ATase depletion and the kinetics of recovery of activity in mice following O6-BeG treatment. As expected, ATase activity in liver decreased to undetectable levels within 2 hours of treatment of animals with O6-BeG. ATase activity in bone marrow also decreased over the same time period, but only to 16% of levels in untreated mice at the dose of O6-BeG tested. The residual ATase activity seen in bone marrow following O6-BeG treatment of mice may reflect the presence of an inactivator resistant population of cells in this tissue. In addition to hematopoietic cells, bone marrow contains a number of other cell types, including fibroblasts and adipocytes, which collectively make up the bone marrow stroma. An O6-BeG resistant cell population might be derived from either the hematopoietic or stromal elements of the bone marrow. Alternatively, differences in the partitioning of O6-BeG between liver and bone marrow, or in the rate of recovery of ATase levels in these tissues might account for this preliminary observation.

Of perhaps more importance than absolute depletion levels is the rate of recovery of ATase in both liver and bone marrow of O6-BeG–treated mice. In such animals, ATase began to rise by 12 hours posttreatment. The recovery of activity, and particularly the timing of the recovery, has important implications for the effects of O6-BeG pretreatment on the cytotoxicity of the agents used in this study and this will be discussed in greater detail below.

We then went on to determine the effects of O6-BeG pretreatment on the sensitivity of the hematopoietic compartment to alkylating agents in vivo. The overall effect on the hematopoietic system was studied by monitoring marrow cellularity, while the effects on particular progenitor populations within the bone marrow were studied using in vitro and in vivo colony-forming assays. These colony-forming (GM-CFC and CFU-S) assays measure the levels of particular (experimentally defined) subpopulations of the hematopoietic tissue at the time of bone marrow harvest. Temozolomide or BCNU treatment of mice led to a dose-dependent depletion of marrow cellularity, GM-CFC, and CFU-S. However, pretreatment with O6-BeG substantially increased the sensitivity of these populations to the cytotoxic effects of temozolomide and BCNU. Quantitatively this enhanced toxicity was expressed as the dose potentiating factor (DPF ) = TD50 (temozolomide or BCNU alone)/TD50 (O6-BeG + temozolomide or BCNU) as summarized in Table 1. The O6-BeG DPF for temozolomide was between 1.20 and 1.68 indicating that O6-BeG pretreatment marginally increased the temozolomide sensitivity of all hematopoietic cells. The general trend was that sensitization varied with the cell population assayed and the more primitive the cell population examined, the more marked was the sensitization effect of O6-BeG: thus sensitization followed the order of CFU-S > GM-CFC > marrow cellularity. A similar trend in the potentiation of toxicity was seen with BCNU, although in this case, the extent of sensitization, as reflected in the DPF, was considerably greater than that seen with temozolomide (eg, 8.25 v 1.68 for sensitization of CFU-S to BCNU and temozolomide, respectively, see Table 1). The greatest drop in progenitor concentration occurred in the day 12 CFU-S compartment, suggesting a greater sensitization of these cells (as compared with GM-CFC) to alkylating agents by O6-BeG. It should be stressed, however, that the apparent differences in sensitization of individual populations of progenitors noted here may reflect modest temporal differences in the initial kill and subsequent short-term recovery of these cell populations. Further work will be necessary to address this point. The observed trend is consistent, however, with the proliferative activity of bone marrow progenitor cells: the effect on cellularity is least pronounced because of the large number of postmitotic cells (both hematopoietic and stromal) present in the bone marrow. More pronounced potentiation of temozolomide toxicity by O6-BeG is seen in the proliferating cell compartment (GM-CFC) and the cells that will be recruited into proliferative activity (CFU-S) following cytoreductive therapy.

The difference in the levels of toxicity seen with temozolomide and BCNU is likely to be due to the nature of the DNA lesion produced. Temozolomide undergoes rapid chemical degradation at physiological pH to 5-(3-methyl-triazeno) imidazole-4-carboxamide (MTIC),18 which reacts with DNA bases leading to methylation at several sites including O6-guanine, N3-adenine, and N7-guanine.7 BCNU is known to form O6-chloroethylguanine, which gives rise to the cyclic intermediate N1, O6-ethanoguanine and this subsequently forms a DNA interstrand cross-link with the cytosine of the opposite strand, an event that may be fatal for the cell.19,20 ATase prevents the formation of cross-links by removing O6-chloroethylguanine groups from the DNA,21 but does not remove the cross-link itself. In those animals treated with BCNU alone, ATase would start repairing the O6-chloroethylguanine adducts before cross-links had formed, a process with a half-life of around 7.6 hours.22 In the ATase recovery experiment shown in Fig 1, recovery of activity was not seen until around 12 hours after O6-BeG pretreatment. Thus, in mice treated with BCNU following O6-BeG pretreatment, the inactivation of ATase probably prevented cross-link precursor repair during this crucial 7.6-hour period, thereby maximizing cross-link formation and manifesting greater BCNU-induced cytotoxicity.

Comparatively less is known about cell killing by O6-methylguanine (O6-meG). However, it seems likely that the abnormal O6-meG: thymine base pair that would be generated after the replication of DNA containing O6-meG is the substrate for a mismatch repair system, which cleaves the newly synthesized DNA strand. Since the excision repair process then restores the mismatch, the cleavage substrate is perpetuated and effectively results in a permanent cytotoxic strand break.23 Recently, however, it has been observed that O6-meG can cause DNA chain termination in in vitro assays and if this occurs in vivo, it may represent an important mode of cytotoxicity for this class of agent.24 In either case, it is likely that the time before commitment of cells to death following formation of O6-meG is longer than that following the formation of O6-chloroethylguanine lesions. Therefore, the window of opportunity for the repair of methyl lesions is probably wider, allowing some repair of potentially lethal O6-meG lesions if the regeneration of the ATase protein occurs before DNA replication.

Extrapolation of the in vivo data presented here, taken together with our earlier study reporting a potentiating effect of O6-BeG on the in vitro toxicity of temozolomide in the human granulocyte/macrophage lineage, suggests that enhanced toxicity to hematopoietic progenitor cells is likely to occur in patients receiving O6-BeG before O6-alkylating agent chemotherapy. Given this, at least two further avenues of investigation seem important in further understanding the likely effect of O6-BeG/nitrosourea combinations on hematopoietic progenitors. The first of these concerns the rate of recovery of hematopoiesis following treatment. Earlier studies with MNU25 have indicated that recovery of hematopoeisis (as measured by marrow GM-CFC numbers) occurs over a 7- to 9-day period with an approximate twofold overshoot of total CFU-GM around day 11, following a single dose of agent. It will be of great interest and importance to further our investigations by comparing the kinetics of hematopoietic recovery in mice following treatment with BCNU or temozolomide plus or minus O6-BeG pretreatment. None the least of reasons for this would be that several doses of O6-BeG might be necessary to keep the tumor ATase levels low for an extended period to inhibit regeneration of ATase and thus reduce the survival of tumor cells after chemotherapy. In this case, multiple treatment of patients with combinations of O6-BeG and nitrosoureas might be required, causing further exacerbation of the toxicity in bone marrow. Clearly, careful consideration and monitoring of the effects of such regimens on normal sensitive tissues will be vital.

A second consideration may be whether or not a differential effect of O6-BeG on the sensitization of tumor and normal tissue might occur, leading to an increased therapeutic index. Were this to be the case, it might be possible to use low levels of chemotherapeutic agent and hence cause less collateral damage in bone marrow, while still achieving significant tumor kill. In a number of xenograft studies reported, increased tumor killing without significant mortality in mice has certainly been seen.11,26-28 In terms of collateral toxicity, there are reports of qualitative changes in normal tissue pathology in animals receiving O6-BeG pretreatment.29 However, most studies of collateral toxicity have concentrated on mortality in mice and clearly, for human studies, this would be an inappropriate expression of dose-limiting toxicity. The studies reported here are the first quantitative analysis of in vivo bone marrow damage and it will be important to extend our studies further with long-term and extensive xenograft experiments to fully assess the clinical potential of a combination of O6-BeG with a range of clinically active alkylating agents. Furthermore, the observations of tissue pathology in mouse models, even in the absence of mortality, raises the issue of the potential for longer term disease as a consequence of treatment.

To assess potential effects of O6-BeG on the clastogenicity of alkylating agents, we exploited the mouse bone marrow micronucleus test. This test is sensitive to all known human carcinogens including leukemogens,30,31 although it is not in itself directly predictive of leukemogenicity. In the present study, analysis of polychromatic erythrocytes from the bone marrow of mice exposed to various doses of temozolomide or BCNU clearly showed that the number of clastogenic events detected was significantly higher in mice that had received O6-BeG pretreatment than in those that had not. This is in agreement with our earlier report showing that overexpression of ATase can protect cells against the clastogenic effects of alkylating agents,32 indicating that O6-alkylguanine is a clastogenic lesion. O6-alkylguanine is also a mutagenic lesion, giving rise to G → A point mutations, as is well-established, for example, in the methylating agent induced activation of the ras oncogene.33 It is also important to note that the effects seen here were achieved at very low (nontoxic) doses of alkylating agent, far below those normally acheived in patients undergoing chemotherapy. Extrapolation of all of these data to the clinical situation raises the possibility that increased mutations may occur in the hematopoietic cells of patients receiving O6-BeG before chemotherapy, and this could lead to an increased incidence of iatrogenic leukemias.

We and others have recently shown that retroviral transduction of bone marrow with vectors encoding human ATase leads to protection of hematopoietic cells against the toxic effects of nitrosoureas,34-37 and a number of investigators are pursuing ATase gene therapy as a means of protecting the hematopoietic compartment from collateral damage during antitumor chemotherapy. However, ATase produced as a result of retroviral transduction would still be sensitive to O6-BeG pretreatment and its activity would be depleted, leading to renewed sensitivity of the hematopoietic system to alkylating agents. One recent report detailed the ability of singly mutated forms of ATase to confer a moderate level of O6-BeG resistance and protect cells from nitrosoureas.38 We have recently shown that a double mutated form of ATase, which is completely insensitive to inactivation by O6-BeG, is able to fully protect cells expressing this mutated protein from a combination of O6-BeG and nitrosoureas, which proves toxic to cells expressing even large amounts of the wild type protein.39 Based on such observations, our proposed gene therapy strategy will involve the use of autologous bone marrow transplantation to reconstitute an O6-alkylating agent resistant bone marrow in which the constitutive expression of the transduced mutant ATase gene capable of expressing a protein resistant to O6-BeG should protect hematopoietic cells from chemotherapy-induced toxicity, while allowing sensitization of tumor tissues to chemotherapy.40,41 

ACKNOWLEDGMENT

The authors thank Lorna Woolford for expert technical assistance, Dr Steve Roberts for advice on statistical analysis, and Dr R.S. McElhinney and Prof B. McMurry for the generous gift of O6-benzylguanine.

Supported by grants from the Medical Research Council and Cancer Research Campaign (UK). T.M.D. is a Gibb Research Fellow of the Cancer Research Campaign (UK).

Address reprint requests to Leslie J. Fairbairn, PhD, Cancer Research Campaign Department of Experimental Haematology, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester M20 4BX, UK.

REFERENCES

REFERENCES
1
Margison GP, O'Connor PJ: Biological consequences of reactions with DNA: Role of specific lesions, in Cooper CS, Grover PL (eds): Handbook of Experimental Pharmacology 94/1. Berlin, Germany, Springer-Verlag, 1990, p 547
2
Pegg
AE
Byers
TL
Repair of DNA containing O6-alkylguanine.
FASEB J
6
1992
2302
3
Waxman
DJ
Glutathione S-transferase: Role in alkylating agent resistance and possible target for modulation chemotherapy: A review.
Cancer Res
50
1990
6449
4
Epstein
RJ
Drug-induced DNA damage and tumor chemosensitivity.
J Clin Oncol
8
1990
2062
5
Brent
TP
Houghton
PJ
Houghton
JA
O6-Alkylguanine-DNA alkyltransferase activity correlated with the therapeutic response of human rhabdomyosarcoma xenografts to 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea.
Proc Natl Acad Sci USA
82
1985
2985
6
Waldstein
E
Cao
E
Miller
M
Extracts of chronic lymphocytic leukemia lymphocytes have a high level of DNA repair activity for O6-methylguanine.
Proc Natl Acad Sci USA
79
1982
4786
7
Baer
JC
Freeman
AA
Newlands
ES
Watson
AJ
Rafferty
JA
Margison
GP
Depletion of O6-alkylguanine-DNA alkyltransferase correlates with potentiation of temozolomide and CCNU toxicity in human tumour cells.
Br J Cancer
67
1993
1299
8
Dolan
ME
Mitchell
RB
Mummert
C
Moschel
RC
Pegg
AE
Effect of O6-benzylguanine analogues on sensitivity of human tumour cells to the cytotoxic effects of alkylating agents.
Cancer Res
51
1991
3367
9
Wedge
SR
Porteous
JK
May
BL
Newlands
ES
Potentiation of temozolomide and BCNU cytotoxicity by O6-benzylguanine: A comparative study in vitro.
Br J Cancer
73
1996
482
10
Michell
RB
Moschel
RC
Dolan
ME
Effect of O6-benzylguanine on the sensitivity of human tumor xenografts to 1,3-bis(2-chloroethyl)-1-nitrosourea and on DNA interstrand cross-link formation.
Cancer Res
52
1992
1171
11
Gerson
SL
Zborowska
E
Norton
K
Gordon
NH
Willson
JKV
Synergistic efficacy of O6-benzylguanine and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in a human colon cancer xenograft completely resistant to BCNU alone.
Biochem Pharmacol
45
1993
483
12
Fairbairn
LJ
Watson
AJ
Rafferty
JA
Elder
RH
Margison
GP
O6-benzylguanine increases the sensitivity of human primary bone marrow cells to the cytotoxic effects of temozolomide.
Exp Hematol
23
1995
112
13
Heyworth C, Spooncer E: In vitro clonal assays for murine multipotential and lineage restricted myeloid progenitar cells, in Testa NG, Molineux G (eds): Haemopoiesis: A Practical Approach. Oxford, UK, Oxford, 1992, p 37
14
Lord B: In vivo assays for multipotential and marrow repopulating cells, in Testa NG, Molineux G (eds): Haemopoiesis: A Practical Approach. Oxford, UK, Oxford, 1992, p 1
15
Tinwell
H
Ashby
J
Comparison of acridine orange and Giemsa stains in several mouse bone marrow micronucleus assay - including a triple dose study.
Mutagenesis
4
1989
476
16
Bradford
MM
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principles of protein-dye binding.
Anal Biochem
72
1976
248
17
Gerson
SL
Trey
JE
Miller
K
Berger
NA
Comparison of O6-alkylguanine-DNA alkyltransferase activity based on cellular DNA content in human, rat and mouse tissues.
Carcinogenesis
7
1986
745
18
Stevens
MFG
Hickman
JA
Langdon
SP
Chubb
D
Vickers
L
Stone
R
Baig
G
Goddard
C
Gibson
NW
Slack
JA
Newton
C
Lunt
E
Fizames
C
Lavelle
F
Antitumour activity and pharmacokinetics in mice of 8-carbamoyl-3-methylimidazo [5,1-d]1,2,3,5-tetrazin-4 (3H) one (CCRG) 81045; M&B 39831) a novel drug with potential as an alternative to dacarbazine.
Cancer Res
47
1987
5846
19
Kohn
KW
Interstrand cross-linking by 1,3-bis(2-chloroethyl)-1-nitrosourea and other 1-(2-haloethyl)-1-nitrosoureas.
Cancer Res
37
1977
1450
20
Tong
WP
Kirk
MC
Ludlum
DB
Formation of the crosslink 1-(N3-deoxycytidyl)-2-(N1-deoxyguanosinyl)-ethane in DNA treated with N,N-bis(2-chloroethyl)-N-nitrosourea.
Cancer Res
42
1982
3102
21
Robins
P
Harris
AL
Goldsmith
I
Lindahl
T
Cross linking of DNA induced by chloroethylnitrosourea is prevented by O6-alkylguanine-DNA methyltransferase.
Nucleic Acids Res
11
1983
7743
22
Gonzaga
PE
Potter
PM
Niu
T
Yu
D
Ludlum
DB
Rafferty
JA
Margison
GP
Brent
TP
Identification of the cross-link between human O6-methylguanine-DNA methyltransferase and chloroethylnitrosourea-treated DNA.
Cancer Res
52
1992
6052
23
Karran
P
Bignami
M
Self-destruction and tolerence of mammalian cells to alkylation damage.
Nucleic Acids Res
20
1992
2933
24
Voigt
JM
Topal
MD
O6-methylguanine-induced replication blocks.
Carcinogenesis
16
1995
1775
25
Gerson
SL
Trey
JE
Miller
K
Benjamin
E
Repair of O6-alkylguanine during DNA synthesis in murine bone marrow hematopoietic precursors.
Cancer Res
47
1987
89
26
Friedman
HS
Dolan
ME
Moschel
RC
Pegg
AE
Felker
GM
Rich
J
Bigner
DD
Schold
SC Jr
Enhancement of nitrosourea activity in medulloblastoma and glioblastoma multiforme.
J Natl Cancer Inst
84
1992
1926
27
Dolan
ME
Pegg
AE
Moschel
RC
Grindey
GB
Effect of O6-benzylguanine on the sensitivity of human colon tumour xenografts to 1,3-bis(2-chloroethly)-1-nitrosourea (BCNU).
Biochem Pharmacol
46
1993
285
28
Dolan
ME
Pegg
AE
Moschel
RC
Vishnuvajjala
BR
Flora
KP
Grever
MR
Friedman
HS
Biodistribution of O6-benzylguanine and its effectiveness against human brain tumor xenografts when given in polyethylene glycol or cremophor-EL.
Cancer Chemother Pharmacol
35
1994
121
29
Dolan
ME
Pegg
AE
Biser
ND
Moschel
RC
English
HF
Effect of O6-benzylguanine on the response to 1,3-bis(2-chloroethly)-1-nitrosourea in the Dunning R3327G model of prostatic cancer.
Cancer Chemother Pharmacol
32
1993
221
30
Shelby
MD
Zeiger
E
Activity of human carcinogens in the Salmonella and rodent bone marrow cytogenetic tests.
Mutat Res
234
1990
257
31
Tinwell
H
Ashby
J
Comparative activity of human carcinogens and NTP rodent carcinogens in the mouse bone marrow micronucleus assay: An integrative approach to genetic toxicity data assessment.
Environ Health Perspect
102
1994
758
32
White
GRM
Ockey
CH
Brennand
Margison GP
Chinese hamster cells harbouring the Escherichia coli O6-alkylguanine alkyltransferase gene are less susceptible to sister chromatid exchange induction and chromosome damage by methylating agents.
Carcinogenesis
7
1986
2077
33
Sukumar
S
Notario
V
Martin-Zanca
D
Barbacid
M
Induction of mammary carcinomas in rats by nitroso-methylurea involves malignant activation of H-ras-1 locus by single point mutations.
Nature
306
1983
658
34
Jelinek
J
Fairbairn
LJ
Dexter
TM
Rafferty
JA
Stocking
C
Ostertag
W
Margison
GP
Retrovirus-mediated expression of the human O6-alkylguanine-DNA-alkyltransferase gene in murine long-term primary bone marrow cultures protects haemopoietic cells against the toxic effects of N-methy-N-nitrosourea.
Blood
87
1996
2025
35
Allay
JA
Dumenco
LL
Koe
ON
Liu
L
Gerson
SL
Retroviral transduction of the human alkyltransferase cDNA provides nitrosourea resistance to haematopoietic cells.
Blood
85
1995
3342
36
Moritz
T
Mackay
W
Glassner
BG
Williams
DA
Samson
L
Retrovirus-mediated expression of a cDNA repair protein in bone marrow protects haemopoietic cells from nitrosourea-induced toxicity in vitro and in vivo.
Cancer Res
55
1995
2608
37
Harris
LC
Marathi
UK
Edwards
CC
Houghton
PJ
Srivastava
DK
Vanin
EF
Sorrentino
BP
Brent
TP
Retroviral transfer of a bacterial alkyltransferase gene into murine bone marrow protects against chloroethylnitrosourea cytotoxicity.
Clin Can Res
1
1995
1359
38
Hickson
I
Fairbairn
LJ
Chinnasamy
N
Dexter
TM
Margison
GP
Rafferty
JA
Protection of mammalian cells against chloroethylating agent toxicity by an O6-benzylguanine-resistant mutant of human O6-alkylguanine-DNA-alkyltransferase.
Gene Therapy
3
1996
868
39
Loktionova
NA Pegg AE
Point mutations in human O6-alkylguanine-DNA-alkyltransferase prevent the sensitisation by O6- benzylguanine to killing by N,N′-bis(2-chloroethyl)-N-nitrosourea.
Cancer Res
56
1996
1578
40
Baum
C
Margison
GP
Eckert
H-G
Fairbairn
LJ
Ostertag
W
Rafferty
JA
Gene transfer to augment the therapeutic index of anti-cancer chemotherapy.
Gene Therapy
3
1996
1
41
Rafferty JA, Hickson I, Chinnasamy N, Lashford LS, Margison GP, Dexter TM, Fairbairn LJ: Chemoprotection of normal tissues by transfer of drug resistance genes. Cancer Metastasis Rev (in press)