Human O6-alkylguanine-DNA alkyltransferase was rapidly inactivated by low concentrations of O6-benzylguanine, but the alkyltransferase from the Escherichia coli ogt gene was much less sensitive and alkyltransferases from the E. coli ada gene or from yeast were not affected. O6-Benzyl-2'-deoxyguanosine was less potent than the base, but was still an effective inactivator of the human alkyltransferase and had no effect on the microbial proteins. O6-Allylguanine was somewhat less active, but still gave complete inactivation of both the human and Ogt alkyltransferases at 200 microM in 30 min, slightly affected the Ada protein, and had no effect on the yeast alkyltransferase. O4-Benzylthymidine did not inactivate any of the alkyltransferase proteins tested. Inactivation of the human alkyltransferase by O6-benzylguanine led to the formation of S-benzylcysteine in the protein and to the stoichiometric production of guanine. The rate of guanine formation followed second-order kinetics (k = 600 M-1 s-1). Prior inactivation of the alkyltransferase by reaction with a methylated DNA substrate abolished its ability to convert O6-benzylguanine into guanine. These results indicate that O6-benzylguanine inactivates the protein by acting as a substrate for alkyl transfer and by forming S-benzylcysteine at the acceptor site of the protein. The inability of O6-benzylguanine to inactivate the microbial alkyltransferases may be explained by steric constraints at this site.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of inhibition of the capacity to form spermidine and spermine on cell growth were investigated using murine leukaemia L1210 cells and 5'-([(Z)-4-amino-2-butenyl]methylamino)-5'-deoxyadenosine (MDL 73811, AbeAdo), an enzyme-activated irreversible inhibitor of S-adenosyl-L-methionine decarboxylase. Putrescine levels were increased 80-fold, and spermidine and spermine levels were greatly reduced after a 3-day exposure to a maximally inhibitory dose of 10 microM-AbeAdo. Addition of AbeAdo to the culture medium inhibited the growth of L1210 cells measured 3 days later in a dose-dependent manner, but, even at a dose of 10 microM, which was maximally effective, exposure to AbeAdo was not immediately cytostatic. However, the growth rate of L1210 cells chronically exposed to 10 microM-AbeAdo declined steadily until day 12, when the cells stopped growing. L1210 cells exposed to AbeAdo for 12 days could not be rescued from cytostasis by removal of the drug from the culture, but could be rescued by exposure to exogenous spermidine or spermine, indicating that the growth-inhibitory effects of AbeAdo were a result of spermidine and/or spermine depletion. It is suggested that elevated intracellular putrescine in AbeAdo-treated cells sustained limited growth in the absence of physiological levels of spermidine and spermine until certain critical and specific physiological role(s) fulfilled by spermidine (and/or spermine) became deficient resulting in cytostasis. N-(3-Aminopropyl)-1,4-diamino-cis-but-2-ene, a spermidine analogue that is a substrate for deoxyhypusine synthase, was able to mimic the effects of spermidine in reversing AbeAdo-induced cytostasis. Spermidine analogues such as 5,5-dimethylspermidine, which are not substrates for deoxyhypusine synthase, were not active in this way. These results provide evidence that the formation of hypusine in the protein-synthesis initiation factor eIF-5A may be a critical role of spermidine essential for cell growth.

A structural analog, 5'-{[(Z)4-amino-2-butenyl]methylamino}-5'-deoxyadenosine (MDL 73811), of decarboxy S-adenosyl-L-methionine, the product of the reaction catalyzed by S-adenosyl-L-methionine (AdoMet) decarboxylase (DC), was found to inhibit Trypanosoma brucei brucei AdoMet DC. The inhibition was time dependent (T.s, 0.3 min), exhibited pseudo-first-order kinetics (Ki, 1.5 ,uM), and was apparently irreversible.The natural substrate of the reaction, AdoMet, protected the enzyme from inactivation, suggesting that MDL 73811 was directed at the enzyme active site and was probably catalytically activated. Administration of MDL 73811 to T. b. brucei-infected rats resulted in rapid inhibition of AdoMet DC activity, a decrease in spermidine, and an increase in putrescine in the trypanosomes isolated from treated rats. Treatment of T. b. brucei-infected mice with MDL 73811 (20 mg/kg of body weight intraperitoneally twice daily for 4 days) resulted in cures of the trypanosome infections. Additionally, drug-resistant T. brucei rhodesiense infections in mice were cured by either a combination of MDL 73811 (50 mg/kg intraperitoneally three times per day for 5 days) and relatively low oral doses of a-difluoromethylornithine or MDL 73811 (50 mg/kg per day for 7 days) administered alone in implanted miniosmotic pumps. These data suggest that MDL 73811 and, perhaps, other inhibitors of AdoMet DC have potential for therapeutic use in various forms of African trypanosomiasis.

A molecular-genetic approach towards isolating mammalian polyamine-transport genes and their encoded proteins was devised involving the production of Chinese-hamster ovary (CHO) cells expressing a human polyamine-transport protein. CHO cells and a polyamine-transport-deficient CHO mutant cell line (CHOMG) were equally sensitive to the antiproliferative effects of alpha-difluoromethylornithine (DFMO), which blocked endogenous polyamine synthesis. Exposure to exogenous polyamines increased intracellular polyamine levels and reversed this DFMO-induced cytostasis in the CHO cells, but not in the CHOMG cells. CHOMG cells were therefore transfected with human DNA (isolated from HT-29 colon carcinoma cells) and cells expressing the human polyamine-transport system were identified by the ability of these cells to grow in a medium containing DFMO and polyamines. A number of different positive clones were identified and shown to have the capacity for polyamine uptake and an increased sensitivity to the toxic effects of the polyamine analogue methylglyoxal bis(guanylhydrazone). Differences in these properties between the clones are consistent with a multiplicity of polyamine-transport systems. Some clones also showed a change in growth characteristics, which may indicate a relationship between genes involved in the polyamine-transport system and in cell proliferation.