DEPLETION OF MAMMALIAN OXYGEN-6-ALKYLGUANINE-DNA ALKYLTRANSFERASE ACTIVITY BY OXYGEN-6-BENZYLGUANINE PROVIDES A MEANS TO EVALUATE THE ROLE OF THIS PROTEIN IN PROTECTION AGAINST CARCINOGENIC AND THERAPEUTIC ALKYLATING-AGENTS
DEPLETION OF MAMMALIAN OXYGEN-6-ALKYLGUANINE-DNA ALKYLTRANSFERASE ACTIVITY BY OXYGEN-6-BENZYLGUANINE PROVIDES A MEANS TO EVALUATE THE ROLE OF THIS PROTEIN IN PROTECTION AGAINST CARCINOGENIC AND THERAPEUTIC ALKYLATING-AGENTS
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DOI：
10.1073/pnas.87.14.5368
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发表时间：
1990-07-01
影响因子：
11.1
通讯作者：
PEGG, AE
中科院分区：
文献类型：
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作者：
DOLAN, ME;MOSCHEL, RC;PEGG, AE
O6-烷基鸟嘌呤-DNA烷基转移酶在接触O6-苯基鸟嘌呤或对氯苯基和对甲基苯类似物后迅速失活，且不可逆。这种失活要比用O6-甲基鸟嘌呤快得多：与2.5µm的O6-苯基鸟嘌呤孵育，10分钟内活性损失超过90%，而同样的还原需要0.2 mM的O6-甲基鸟嘌呤60分钟。O6-苄基鸟嘌呤对体外培养的人结肠癌细胞(HT29)的烷基转移活性有明显的抑制作用。加入O6-苄基鸟嘌呤后15分钟内酶活完全丧失，5微米时效果最好，而O6-甲基鸟嘌呤至少需要100微米作用4小时才能达到最大活性，烷基转移酶仅降低80%。用10微克O6-苄基鸟嘌呤处理HT29细胞2小时后，化疗药物1-(2-氯乙基)-3-环己基-1-亚硝脲(CCNU)或2-氯乙基(甲磺酰基)甲磺酸(Clomesone)对HT29细胞的杀伤作用显著增强。给小鼠灌胃10 mg/kg的O6-苄基鸟嘌呤，可使肝脏和肾脏的烷基转移酶水平降低95%以上。这些结果表明，O6-苄基鸟嘌呤对烷基转移酶的耗竭可能被用来研究DNA修复蛋白在致癌和致突变中的作用，这种治疗可能容易增加氯乙基化药物的化疗效果。
O6-Alkylguanine-DNA alkyltransferase was rapidly and irreversibly inactivated by exposure to O6-benzylguanine or the p-chlorobenzyl and p-methylbenzyl analogues. This inactivation was much more rapid than with O6-methylguanine: incubation with 2.5 .mu.M O6-benzylguanine led to more than a 90% loss of activity within 10 min, whereas 0.2 mM O6-methylguanine for 60 min was required for the same reduction. O6-Benzylguanine was highly effective in depleting the alkyltransferse activity of cultured human colon tumor (HT29) cells. Complete loss of activity was produced within 15 min after addition of O6-benzylguanine to the culture medium and a maximal effect was obtained with 5 .mu.M. In contrast, at least 100 .mu.M O6-methylguanine for 4 hr was needed to get a maximal effect, and this reduced the alkyltransferase by only 80%. Pretreatment of HT29 cells with 10 .mu.M O6-benzylguanine for 2 hr led to a dramatic increase in the cytotoxicity produced by the chemotherapeutic agents 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) or 2-chloroethyl(methylsulfonyl)methanesulfonate (Clomesone). Administration of O6-benzylguanine to mice at a dose of 10 mg/kg reduced alkyltransferase levels by more than 95% in both liver and kidney. These results indicate that depletion of the alkyltransferase by O6-benzylguanine may be used to investigate the role of the DNA repair protein in carcinogenesis and mutagenesis and that this treatment may be vulnerable to increase the chemotherapeutic effectiveness of chloroethylating agents.