Increased susceptibility to chemotherapeutic alkylating agents of mice deficient in DNA repair methyltransferase

Akiko Shiraishi1,2, Kunihiko Sakumi1 and Mutsuo Sekiguchi2,3

1 Department of Biochemistry, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582 and
2 Department of Biology and Frontier Research Center, Fukuoka Dental College, 2-15-1 Tamura, Sawara-ku, Fukuoka 814-0193, Japan


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
O6-methylguanine-DNA methyltransferase plays vital roles in preventing induction of mutations and cancer as well as cell death related to alkylating agents. Mice defective in the Mgmt gene, encoding the methyltransferase, were used to evaluate cell death-inducing and tumorigenic activities of therapeutic agents which have alkylation potential. Mgmt–/– mice were considerably more sensitive to dacarbazine, a monofunctional triazene, than were wild-type mice, in terms of survival. When dacarbazine was administered i.p. to 6-week-old mice and survival at 30 days was enumerated, LD50 values of Mgmt–/– and Mgmt+/+ mice were 20 and 450 mg/kg body wt, respectively. Increased sensitivity of Mgmt–/– mice to 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU), a bifunctional nitrosourea, was also noted. On the other hand, there was no difference in survival of Mgmt+/+ and Mgmt–/– mice exposed to cyclophosphamide, a bifunctional nitrogen mustard. It appears that dacarbazine and ACNU produce O6-alkylguanine as a major toxic lesion, while cyclophosphamide yields other types of modifications in DNA which are not subjected to the action of the methyltransferase. Mgmt–/– mice seem to be less refractory to the tumor-inducing effect of dacarbazine than are Mgmt+/+ mice. Thus, the level of O6-methylguanine-DNA methyltransferase activity is an important factor when determining susceptibility to drugs with the potential for alkylation.

Abbreviations: ACNU, 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea; MNU, N-methyl-N-nitrosourea; MTD, maximum tolerated dose.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Alkylating agents exert profound cytotoxic effects on actively growing cells and this is the basic rationale for the use of these agents in cancer chemotherapy (1,2). The biological effects of alkylating agents are attributed to the formation of alkylated bases in cellular DNA. Among various modified bases produced in DNA by alkylating agents, O6-alkylguanine is critical for inducing cell death, as well as mutation (35). This was clearly shown in recent studies with cell lines in which both alleles of the gene for O6-methylguanine-DNA methyltransferase were disrupted by gene targeting (6). The enzyme specifically repairs O6-methylguanine and O4-methylthymine, the latter being produced less frequently as compared with the former, by transferring methyl groups of the bases to the methyltransferase protein itself (7). Lack of the enzyme makes cells hypersensitive to both the cell death-inducing and mutagenic actions of simple alkylating agents, such as N-methyl-N-nitrosourea (MNU).

From gene-targeted embryonic stem cells, mouse lines deficient in O6-methylguanine-DNA methyltransferase were established (8,9). Reflecting the vital role of this enzyme, these mice are sensitive to the lethal and the tumorigenic actions of alkylating agents. A number of thymic lymphomas, as well as lung adenomas, occurred in methyltransferase-deficient mice exposed to a low dose of MNU (10). These mice were also susceptible to dimethylnitrosamine, which induces tumors in liver and lung (11).

Among various types of drugs designed for use in cancer chemotherapy, some have the potential for alkylation. After metabolic activation, these chemicals attack DNA and alkylate their bases, thereby preventing multiplication of rapidly growing tumor cells. These drugs with alkylation capacity can be divided into several groups, according to their preferred sites of action and also abilities to form interstrand crosslinks in DNA. These include the monofunctional triazenes, such as dacarbazine, the bifunctional chloroethylnitrosoureas, such as 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU), and the nitrogen mustard class of compounds, such as cyclophosphamide (2,1214). The base adducts produced by the latter two categories of drugs tend to form interstrand crosslinks, which would block DNA replication.

These facts prompted us to investigate the lethal and tumorigenic actions of chemotherapeutic alkylating agents in mice deficient in DNA repair methyltransferase activity. In the present study we have investigated the actions of dacarbazine, ACNU and cyclophosphamide, representing the three categories of drugs with alkylation capacity, on Mgmt gene knockout mice.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Gene-targeted mice
Development of Mgmt gene-disrupted mice was as described (8). Genotypes of mice were identified by PCR (15). The resulting PCR products were resolved by electrophoresis on 1.5% agarose gels and visualized under UV light after staining with ethidium bromide.

Sensitivity to drugs
Dacarbazine was purchased from Kyowa Co. (Tokyo, Japan). ACNU and cyclophosphamide were obtained from Sankyo Co. (Tokyo, Japan) and Shionogi Co. (Osaka, Japan), respectively. These drugs were dissolved in 0.9% NaCl. Six-week-old mice were given an i.p. injection of 200 µl of the drug solution at defined concentrations. As controls, mice were given an i.p. injection of 200 µl of 0.9% NaCl. Mice were kept for defined periods and survivors were scored.

Peripheral blood analysis
Counts of total peripheral blood platelets were made on tail vein blood, using an automatic hematopoietic cell counter.

Tumor induction
Mice were given i.p. injections of 4 mg/kg body wt dacarbazine daily for five consecutive days at post-natal week 6. Twenty-four weeks after injection, these mice were killed and tissues were examined as described earlier (10).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Susceptibility of Mgmt gene knockout mice to dacarbazine
As a representative of chemotherapeutic alkylating agents, we selected dacarbazine, which is presently in use for treatment of malignant melanoma (13,16). Susceptibility of mice to dacarbazine was examined by applying a fixed dose of the drug to Mgmt+/+ and Mgmt–/– mice. Two types of mice, each consisting of 10 animals (6 weeks old), were given a single i.p. injection of dacarbazine (100 mg/kg body wt). As shown in Figure 1Go, there was a distinct difference in survival of the two types of mice. Death of Mgmt–/– mice occurred as early as 7 days after dacarbazine administration and all mice with this genotype died within 13 days. On the other hand, all of the Mgmt+/+ mice survived for the period of observation (30 days after treatment). As controls, 0.9% NaCl was injected into the two types of mice and all survived for >3 months (data not shown).



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Fig. 1. Survival curves of mice after administration of dacarbazine. Ten each of Mgmt+/+ and Mgmt–/– mice were given dacarbazine (100 mg/kg body wt) i.p. at post-natal week 6. {circ}, Mgmt+/+; •, Mgmt–/–.

 
After dacarbazine treatment, internal organs of Mgmt–/– mice, especially those related to rapid reproduction of cells, such as bone marrow, were severely damaged. In addition to anemia and bleeding, diarrhea and alopecia were remarkable in Mgmt–/– mice treated with dacarbazine. These effects of dacarbazine are similar to those which we observed with Mgmt–/– mice treated with MNU (8).

When Mgmt–/– mice were given a sub-lethal dose of dacarbazine, myelosuppression was evident. Figure 2Go shows changes in number of platelets in peripheral blood after application of 4 mg/kg body wt of the drug for five consecutive days. A dramatic decrease in the number of platelets was observed at an early stage of treatment and this thrombocytopenia continued for >1 week. The change in the number of platelets was nil in Mgmt+/+ mice treated in a similar manner, as was the case for both types of mice receiving 0.9% NaCl. Methyltransferase apparently protects the reproductive capacity of hematopoietic stem cells from alkylation damage.



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Fig. 2. Changes in number of platelets after dacarbazine administration. Two groups of mice were given dacarbazine (4 mg/kg body wt each time) on five consecutive days at post-natal week 6 and other groups of mice (controls) were given 200 µl of 0.9% NaCl at the same times. Arrows indicate the times of drug application. Data are the means of measurements between two and four mice for each point. {circ}, saline-administered Mgmt–/– mice; •, dacarbazine-administered Mgmt–/– mice; {square}, saline-administered Mgmt+/+ mice; {blacksquare}, dacarbazine-administered Mgmt+/+ mice.

 
Tumor formation by dacarbazine
To determine sub-lethal doses of dacarbazine for tumorigenesis experiments, various doses of the drug were given to groups of mice with Mgmt+/+, Mgmt+/– and Mgmt–/– backgrounds. Figure 3Go shows survival curves for the three types of mice, determined on day 30 of treatment. From the curves, the LD50 in Mgmt–/– mice was estimated to be 20 mg/kg body wt while the values for Mgmt+/+ and Mgmt+/– mice were as high as 450 mg/kg. It seems that a single copy of the Mgmt gene is sufficient to provide full repair potency for these mice.



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Fig. 3. Survival of mice given different doses of dacarbazine. Groups of mice with three different Mgmt genotypes (at least 10 for each point) were given different amounts of dacarbazine i.p. on post-natal day 42. Percent survival at day 30 after injection were plotted. {circ}, Mgmt+/+; {triangleup}, Mgmt+/–; •, Mgmt–/–. LD50 values of Mgmt+/+ and Mgmt–/– mice are shown in the figure.

 
Six-week-old mice with the Mgmt+/+ and Mgmt–/– genotypes (~50 each) were given a single i.p. injection of dacarbazine (4 mg/kg body wt) for five consecutive days. The animals were killed 24 weeks after drug administration and their organs examined. As shown in Table IGo, thymic lymphomas were found in two of 52 Mgmt–/– mice, one of which died within 18 weeks after dacarbazine administration. The tumor was ~0.18 g, six times heavier than the normal thymus. No lymphoma was found in Mgmt+/+ mice treated in the same manner. The histology of tumors induced by dacarbazine was similar to that of tumors produced by MNU (10).


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Table 1. Frequencies of tumors in Mgmt +/+ and Mgmt–/– mice with or without dacarbazine administration
 
Lung adenomas were frequent in dacarbazine-treated Mgmt–/– mice, while there were only a few in Mgmt+/+ animals. Since one lung adenoma was found in a control Mgmt+/+ mouse not given dacarbazine administration, tumor incidence observed in the treated Mgmt+/+ mice may be regarded as background. It seems that mice defective in both alleles of the Mgmt gene are more vulnerable to dacarbazine-induced tumorigenesis than are wild-type mice.

The size of testes of male Mgmt–/– mice was considerably reduced after dacarbazine treatment. The mean weight of testes in dacarbazine-treated Mgmt–/– mice was 32.8 mg, whereas the value for mock-treated Mgmt –/– mice was 125.8 mg. There were no differences in Mgmt+/+ mice treated with dacarbazine and untreated Mgmt+/+ mice (128.5 and 114.9 mg, respectively).

Effects of other types of drugs with alkylation potential
In addition to dacarbazine, there are several drugs in clinical use the activities of which are related to the capacity for alkylation of DNA. For our study we used ACNU (2) and cyclophosphamide (12), which belong to the bifunctional nitrosourea and nitrogen mustard groups of drugs, respectively.

Figure 4Go shows survival of methyltransferase-proficient and -deficient mice after treatment with these drugs. ACNU exhibited differential effects on the two types of mice, as observed in the case of dacarbazine treatment. LD50 values for Mgmt+/+ and Mgmt–/– mice were 70 and 14 mg/kg body wt, respectively (Figure 4AGo). This difference in survival between Mgmt+/+ and Mgmt–/– mice with regard to ACNU is less dramatic than that observed with dacarbazine, but is significant.



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Fig. 4. Survival of mice given different doses of chemotherapeutic agents. Groups of mice with different Mgmt genotype (at least 10 per group) were given different amounts of drugs i.p. on post-natal day 42. Percent survival on day 30 after injection was plotted. (A) ACNU. {circ}, Mgmt+/+; {triangleup}, Mgmt+/–; •, Mgmt–/–. (B) Cyclophosphamide. {circ}, Mgmt+/+; •, Mgmt–/–.

 
No significant difference was observed in susceptibility of the two types of mice to cyclophosphamide (Figure 4BGo). Mgmt–/– mice exhibited the same level of resistance as did Mgmt+/+ mice.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
To treat subjects with malignant tumors, various chemicals with alkylation capacity have been developed and some are presently in clinical use. We selected three drugs, dacarbazine, ACNU and cyclophosphamide, which are representatives of monofunctional triazenes, bifunctional chloroethylnitrosoureas and nitrogen mustards, respectively (2,12,13). Figure 5Go shows the structures of these drugs, together with that of MNU, a simple alkylating agent. These three drugs have the potential to alkylate various functional groups of proteins, nucleic acids and other cellular constituents after metabolic activation. Although many groups of various substances in organisms could be modified by these drugs, the most important of these is alkylation of the base moieties of DNA. This type of modification affects DNA metabolism and, in particular, blocks replication. It is expected that rapidly growing cells, including cells in tumor tissues, are preferentially attacked by these agents.



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Fig. 5. Structures of the three therapeutic agents used in the present study and MNU. Methyl or chloroethyl groups circled are transferred to DNA bases to produce alkylated residues.

 
Among various modified bases produced in DNA by alkylating agents, O6-methylguanine has been regarded as critical in cell killing as well as mutation induction. This was clearly shown by recent studies with cell lines and mice defective in O6-methylguanine-DNA methyltransferase (69). Lack of the enzyme makes mice hypersensitive to MNU with respect to both survival and tumor induction. Here we used these gene-targeted animals to evaluate biological activities of chemotherapeutic agents with the potential for alkylation.

Our studies focused on the action of dacarbazine, a triazene type of antitumor drug (13). The LD50 value of dacarbazine in Mgmt–/– mice was ~20 times lower than that for methyltransferase-proficient wild-type mice. Although dacarbazine requires metabolic activation for its action (1719) while MNU acts directly on DNA, their actions in the cell can apparently be ascribed to formation of O6-methylguanine in DNA. The ratio between the LD50 values for dacarbazine in Mgmt+/+ and Mgmt–/– mice is even higher than the ratio for MNU. Thus, dacarbazine is as effective as MNU with regard to cell killing.

In the present tumorigenesis experiments with dacarbazine and previous studies with MNU (10), the age of the mice given the two agents and the observation periods differed slightly. As to the dose, 2.5 mg/kg body wt MNU is half the maximum tolerated dose (MTD) and 20 mg/kg body wt (4 mg each five times) dacarbazine is twice the MTD. At post-natal week 30 Mgmt–/– mice administered MNU had thymic lymphomas and lung adenomas more frequently than did Mgmt–/– mice administered dacarbazine. Dacarbazine administration apparently led to fewer tumors as compared with MNU.

Carcinogenicity of dacarbazine has been tested by giving three i.p. injections per week of 25–50 mg/kg body wt to mice for a 6 month period (20). At the end of the 18 month test period there were large numbers of lung tumors and lymphomas in these treated mice. In our experiment, cumulative exposure of Mgmt–/– mice was 20 mg/kg body wt (estimated as ~110 mg/m2 body surface), about one hundredth of the dose used in former carcinogenicity tests. These low levels of dacarbazine are almost equal to or less than the doses given clinically (21). Mgmt–/– mice exhibited severe myelosuppression and formation of secondary tumors under conditions much the same as those used for chemotherapy. These profound effects of dacarbazine in Mgmt–/– mice raise the question as to whether application of this type of drug may be hazardous for patients, especially those with the Mgmt–/– trait. It is expected that in human populations there are various levels of methyltransferase activity (22,23). Therefore, we consider it important to examine levels of methyltransferase activity prior to application of these drugs to patients. There is the possibility that patients lacking methyltransferase activity might suffer severe myelosuppression and further be at increased risk for secondary tumors.

Similar but less striking effects on methyltransferase-deficient mice were observed with chloroethylnitrosoureas (2,24). We have shown, in the present study, that the LD50 values for ACNU in Mgmt+/+ and Mgmt–/– mice were 70 and 14 mg/kg body wt, while Glassner et al. (9) reported that these values for N,N-bis(2-chloroethyl)-N-nitrosourea were 39 and 14 mg/kg body wt, respectively. These drugs have been used for treatment of subjects with brain tumors, since these drugs pass the blood–brain barrier (25). On application of these drugs chloroethyl groups of nitrosoureas are transferred to a guanine residue of DNA and finally yield a crosslink with a base on the opposite strand, thereby preventing cell proliferation (26). The chloroethyl group at the O6 position of guanine in DNA can be recognized and repaired by O6-methylguanine-DNA methyltransferase (27,28). The finding that Mgmt–/– mice are more sensitive to chloroethylnitrosourea alkylating agents than are wild-type mice implies that relatively large numbers of unpaired chloroethyl groups are present in the DNA, becoming the primary cause of cell death. Cyclophosphamide, on the other hand, alkylates other moieties of DNA, yielding 7-substituted guanine and other alkylated bases (12,14). These modified bases are not recognized by the methyltransferase enzyme and, indeed, there are no differences in sensitivity of Mgmt+/+ and Mgmt–/– mice.


    Notes
 
3 To whom correspondence should be addressed Email: sekim1{at}college.fdcnet.ac.jp Back


    Acknowledgments
 
We thank Drs H. Kawate, Y. Nakabeppu and T. Tsuzuki for discussions and M. Ohara for pertinent advice. This paper was written when one of the authors (M.S.) was the Rothschild-Mayen fellow at the Institut Curie. This work was supported by grants from the Ministry of Education, Science, Sports and Culture of Japan (M.S.) and the Toyota High-Tech Research Grant Program (K.S.).


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received February 7, 2000; revised April 28, 2000; accepted July 7, 2000.