A missense mutation, p.V132G, in the X-linked spermine synthase gene (SMS) causes Snyder-Robinson syndrome.

Snyder-Robinson syndrome (SRS, OMIM 309583) is a rare X-linked syndrome characterized by mental retardation, marfanoid habitus, skeletal defects, osteoporosis, and facial asymmetry. Linkage analysis localized the related gene to Xp21.3-p22.12, and a G-to-A transition at point +5 of intron 4 of the spermine synthase gene, which caused truncation of the SMS protein and loss of enzyme activity, was identified in the original family. Here we describe another family with Snyder-Robinson syndrome in two Mexican brothers and a novel mutation (c.496T>G) in the exon 5 of the SMS gene confirming its involvement in this rare X-linked mental retardation syndrome.

Autoradiographic identification of ornithine decarboxylase in mouse kidney by means of alpha-[5-14C]difluoromethylornithine.

alpha-Difluoromethylornithine is an enzyme-activated irreversible inhibitor of ornithine decarboxylase that forms a covalent bond with the enzyme. When alpha-[5-14C]difluoromethylornithine was administered to androgen-treated mice, only ornithine decarboxylase became labeled. Autoradiographic examination of kidney, liver, and brain indicated much more extensive incorporation of labeled difluoromethylornithine into kidney protein than into the protein of the other tissues. Such incorporation was greatly reduced by prior treatment of the mice with cycloheximide. These results correlate with the presence of ornithine decarboxylase activity which is much higher in the kidney than in the other tissues and is lost rapidly when protein synthesis is inhibited. The binding of this drug in vivo, therefore, is useful for determining the distribution of ornithine decarboxylase. The enzyme was predominantly located in the proximal tubule cells of the kidney in androgen-treated mice.

New SMS mutation leads to a striking reduction in spermine synthase protein function and a severe form of Snyder-Robinson X-linked recessive mental retardation syndrome.

We report the identification of a novel mutation at a highly conserved residue within the N-terminal region of spermine synthase (SMS) in a second family with Snyder-Robinson X-linked mental retardation syndrome (OMIM 309583). This missense mutation, p.G56S, greatly reduces SMS activity and leads to severe epilepsy and cognitive impairment. Our findings contribute to a better delineation and expansion of the clinical spectrum of Snyder-Robinson syndrome, support the important role of the N-terminus in the function of the SMS protein, and provide further evidence for the importance of SMS activity in the development of intellectual processing and other aspects of human development.

Spermine and spermidine mediate protection against oxidative damage caused by hydrogen peroxide.

The polyamines spermidine and spermine have been hypothesized to possess different functions in the protection of DNA from reactive oxygen species. The growth and survival of mouse fibroblasts unable to synthesize spermine were compared to their normal counterparts in their native and polyamine-depleted states in response to oxidative stress. The results of these studies suggest that when present at normal or supraphysiological concentrations, either spermidine or spermine can protect cells from reactive oxygen species. However, when polyamine pools are pharmacologically manipulated to produce cells with low levels of predominately spermine or spermidine, spermine appears to be more effective. Importantly, when cells are depleted of both glutathione and endogenous polyamines, they exhibit increased sensitivity to hydrogen peroxide as compared to glutathione depletion alone, suggesting that polyamines not only play a role in protecting cells from oxidative stress but this role is distinct from that played by glutathione.

Crystal structure of the human O(6)-alkylguanine-DNA alkyltransferase.

The mutagenic and carcinogenic effects of simple alkylating agents are mainly due to O(6)-alkylation of guanine in DNA. This lesion results in transition mutations. In both prokaryotic and eukaryotic cells, repair is effected by direct reversal of the damage by a suicide protein, O(6)-alkylguanine-DNA alkyltransferase. The alkyltransferase removes the alkyl group to one of its own cysteine residues. However, this mechanism for preserving genomic integrity limits the effectiveness of certain alkylating anticancer agents. A high level of the alkyltransferase in many tumour cells renders them resistant to such drugs. Here we report the X-ray structure of the human alkyltransferase solved using the technique of multiple wavelength anomalous dispersion. This structure explains the markedly different specificities towards various O(6)-alkyl lesions and inhibitors when compared with the Escherichia coli protein (for which the structure has already been determined). It is also used to interpret the behaviour of certain mutant alkyltransferases to enhance biochemical understanding of the protein. Further examination of the various models proposed for DNA binding is also permitted. This structure may be useful for the design and refinement of drugs as chemoenhancers of alkylating agent chemotherapy.

The crystal structure of human S-adenosylmethionine decarboxylase at 2.25 A resolution reveals a novel fold.

BACKGROUND: S-Adenosylmethionine decarboxylase (AdoMetDC) is a critical regulatory enzyme of the polyamine synthetic pathway, and a well-studied drug target. The AdoMetDC decarboxylation reaction depends upon a pyruvoyl cofactor generated via an intramolecular proenzyme self-cleavage reaction. Both the proenzyme-processing and substrate-decarboxylation reactions are allosterically enhanced by putrescine. Structural elucidation of this enzyme is necessary to fully interpret the existing mutational and inhibitor-binding data, and to suggest further experimental studies. RESULTS: The structure of human AdoMetDC has been determined to 2.25 A resolution using multiwavelength anomalous diffraction (MAD) phasing methods based on 22 selenium-atom positions. The quaternary structure of the mature AdoMetDC is an (alpha beta)2 dimer, where alpha and beta represent the products of the proenzyme self-cleavage reaction. The architecture of each (alpha beta) monomer is a novel four-layer alpha/beta-sandwich fold, comprised of two antiparallel eight-stranded beta sheets flanked by several alpha and 3(10) helices. CONCLUSIONS: The structure and topology of AdoMetDC display internal symmetry, suggesting that this protein may be the product of an ancient gene duplication. The positions of conserved, functionally important residues suggest the location of the active site and a possible binding site for the effector molecule putrescine.

Alkylation of rat liver DNA by dimethylnitrosamine: effect of dosage on O6-methylguanine levels.

Alkylation of liver DNA was studied following administration to Sprague-Dawley rats of doses of dimethylnitrosamine (DMN) varying from 0.25 to 20 mg/kg body weight. Measurements were made of the amounts of O6-methylguanine and 7-methylguanine present in liver DNA at 4 and 24 hours after treatment with the carcinogen. There was a linear relationship between 7-methylguanine levels and dose of the nitrosamine at both of these times. In contrast, the corresponding levels of O6-methylguanine were not directly proportional to dosage but were less than expected, particularly at low doses below 2.5 mg/kg. This discrepancy was significant at 4 hours, but was even more marked at 24 hours. Only doses above 4 mg/kg at the 4-hour time point gave rise to a 0.11 ratio of alkylation of guanine at the O6-position to that at the 7-position. This ratio was that expected for the initial interaction of the alkylating species derived from DMN with DNA. Evidence was obtained to support the hypothesis that these results were due to an enzymatic removal of O6-methylguanine from liver DNA, which occurred much more efficiently at lower initial levels of alkylation. Repeated daily injections of DMN up to 11 days alos gave rise to O6-methylguanine levels that were not proportional to dosage but were relatively greater at higher dose levels. The significance of these findings in the induction of liver cancer by feeding or repeated injection of DMN was explored.

Enhancement of nitrosourea activity in medulloblastoma and glioblastoma multiforme.

BACKGROUND: Although chemotherapy offers promise of increased survival for children with medulloblastoma and glioblastoma multiforme, drug resistance occurs frequently, resulting in tumor progression and death. Resistance to nitrosoureas and methylating agents, which damage DNA, can be mediated by a DNA repair protein, O6-alkylguanine-DNA alkyltransferase (AGAT). Depletion of this protein with alkylguanines or methylating agents, however, restores tumor cell sensitivity to the cytotoxicity of chloroethylnitrosoureas (e.g., carmustine [BCNU]). PURPOSE: This study was designed to determine whether resistance to the activity of nitrosourea (the drug BCNU) in BCNU-resistant human medulloblastoma (D341 Med) and human glioblastoma multiforme (D-456 MG) can be reversed by the methylating agent streptozocin and the O6-substituted guanines O6-methylguanine and O6-benzylguanine. METHODS: Xenografts were grown subcutaneously in athymic BALB/c mice. BCNU was administered as a single intraperitoneal injection at doses of 100 mg/m2, 75 mg/m2, or 38 mg/m2--i.e., 1.0, 0.75, or 0.38, respectively, of the dose lethal to 10% of treated animals (LD10). Mice were treated intraperitoneally with a single dose of O6-benzylguanine or O6-methylguanine (240 mg/m2) or with streptozocin (600 mg/m2) daily for 4 days. Response was assessed by tumor growth delay and tumor regression. AGAT activity in the xenografts was measured at 1 and 6 hours after pretreatment, at the time tumors were excised. RESULTS: Pretreatment with O6-benzylguanine, O6-methylguanine, or streptozocin reduced AGAT activity to 4%, 25%, and 95% of control values, respectively, in D341 Med and 0%, 0%, and 25% of control values, respectively, in D-456 MG 1 hour after injection. After 6 hours, levels changed to 7%, 61%, and 116% of control values in D341 Med and 0%, 79%, and 21% of control values in D-456 MG, respectively. Both D341 Med and D-456 MG xenografts were completely resistant to BCNU at its LD10. Pretreatment with O6-benzylguanine increased BCNU sensitivity in both types of xenograft. In contrast, treatment with BCNU plus O6-methylguanine or streptozocin did not produce growth delays substantially different from those produced by BCNU alone, reflecting the more efficient depletion of AGAT by O6-benzylguanine. Following therapy with BCNU plus O6-benzylguanine at 0.38 LD10, tumor regressions were seen in eight of 10 D341 Med and in all 10 D-456 MG xenografts. CONCLUSION: We recommend comprehensive clinical toxicologic evaluation of combination therapy with O6-benzylguanine plus BCNU, which would allow subsequent design of phase I clinical trials.

Polyamine metabolism and its importance in neoplastic growth and a target for chemotherapy.

The polyamine-biosynthetic pathway represents an inviting target for the development of agents inhibiting carcinogenesis and tumor growth. Polyamines play an essential role in the proliferation and development of mammalian cells. Deranged polyamine metabolism may be an important factor in carcinogenesis. Depletion of polyamines inhibits growth of neoplastic cells in vitro and in animal models. Several different classes of other anticancer agents may under some conditions exert enhanced effects when polyamine levels are depleted. Some suitable inhibitors of polyamine production are currently available and other promising compounds are presently being tested. It should soon prove possible to block polyamine biosynthesis at every step in the pathway. The use of these inhibitors alone and combined either with each other or with other antitumor agents will enable a full examination of the potential of this approach.

A single amino acid change in human O6-alkylguanine-DNA alkyltransferase decreasing sensitivity to inactivation by O6-benzylguanine.

Mammalian O6-alkylguanine-DNA alkyltransferases (AGTs) are readily inactivated by incubation with the pseudosubstrate, O6-benzylguanine, but the equivalent protein from the Escherichia coli ogt gene is much less sensitive and the Saccharomyces cerevisiae and E. coli ada gene product AGTs are completely resistant to this compound. We have expressed the normal human AGT and various point mutations (C145A, W100A, and P140A) in an ada- ogt- strain of E. coli and tested these proteins against DNA substrates containing O6-methylguanine, for inactivation by O6-benzylguanine and for the ability to produce guanine from O6-benzylguanine. The C145A mutation was inactive as expected since this residue forms the methyl acceptor site. Mutants W100A and P140A were fully active against methylated DNA substrates but the P140A mutant was much less sensitive to inactivation by O6-benzylguanine and failed to form significant amounts of [3H]guanine when incubated with O6-benzyl[8-3H]-guanine. The proline at position 140 in mammalian AGTs is replaced by alanine in the Ada and yeast AGTs and by serine in the Ogt AGT. These results suggest that this proline residue affects the configuration of the active site allowing the O6-benzylguanine to enter and react with the mammalian AGT. The production of resistance to O6-benzylguanine by a single base change raises the possibility that such resistance may arise quite readily in cells of tumors treated therapeutically with the combination of O6-benzylguanine and an alkylating agent.

Regulation of O6-methylguanine-DNA methyltransferase levels in rat liver and kidney.

It was found that rat kidney contains a protein similar to that previously described in rat liver which catalyzes the transfer of the methyl group from O6-methylguanine in DNA to a protein-bound cysteine residue. The amount of the renal O6-methylguanine-DNA methyltransferase was increased up to 2.5-fold during renal hypertrophy in response to unilateral nephrectomy or treatment with folic acid. These results indicate that the protein in kidney resembles that in rat liver which is known to be increased in response to a variety of hepatotoxins or to partial hepatectomy. The liver O6-methylguanine-DNA methyltransferase was reduced by hypophysectomy or thyroidectomy and could be increased by treatment with growth hormone or thyroxine. The level in the liver was considerably lower than the adult value in 1-day-old rats and increased to adult values by 14 to 21 days. At no time was the amount in the neonatal rat liver higher than in the adult, indicating that liver cell proliferation alone is not obligatorily coupled with an elevated methyltransferase level. The high sensitivity of neonatal rats to liver carcinogenesis by dimethylnitrosamine may be related to the high rate of cell proliferation and the lower capacity to repair O6-methylguanine.

Induction of spermidine N1-acetyltransferase by dialkylnitrosamines.

Treatment of rats with dimethylnitrosamine (30 mg/kg) or diethylnitrosamine (200 mg/kg) produced a rapid increase in the activity of spermidine N1-acetyltransferase which peaked at values 7-fold greater than did control at 48 hr after exposure. This increase led to a small accumulation of N1-acetylspermidine in the liver but produced a more striking effect on putrescine which increased 30- to 40-fold after 2 days. Most of this increase appeared to be due to the conversion of N1-acetylspermidine into putrescine which is catalyzed by polyamine oxidase. Treatment with the nitrosamines also increased the conversion of spermine into spermidine which replaced the spermidine converted into putrescine. Spermine levels were therefore significantly depressed by treatment with these carcinogens. These results indicate that these hepatocarcinogens bring about an increase in putrescine and in the spermidine/spermine ratio in the liver not only by enhancement of ornithine decarboxylase but also by induction of the spermidine N1-acetyltransferase activity which is the rate-limiting step in the acetylase-oxidase pathway for interconversion of the polyamines.

Alkylation of intracellular and extracellular DNA by dimethylnitrosamine following activation by isolated rat hepatocytes.

Freshly prepared rat hepatocytes isolated by perfusion with collagenase were able to metabolize microM concentrations of dimethylnitrosamine to a methylating agent. The methylation of hepatocyte DNA in this system was complete within 2 hr, and after this time, the content of O6-methylguanine in the DNA declined, showing that the repair system for this product was active in the isolated hepatocytes. When extracellular calf thymus DNA was added to the incubated hepatocytes, this also became methylated. Methylation of this DNA was not due to cell lysis releasing activating enzymes into the medium, showing that the methylating species formed by the hepatocytes from dimethylnitrosamine is sufficiency stable to pass out of the cell in substantial amounts. These results support the possibility that alkylation of liver cells would not be confined to those cells metabolizing dimethylnitrosamine but could be extended to those cells which are in close proximity to the activating cells. These cells could include nonparenchymal cells which are known to be targets for the carcinogenic action of dimethylnitrosamine.

Alkylation of DNA in rat tissues following administration of streptozotocin.

Streptozotocin, an antibiotic widely used for induction of diabetes in experimental animals and for the treatment of pancreatic neoplasms, was shown to be a potent methylating agent reacting with DNA in vitro to form methylated purines. The reaction was similar in extent and relative proportions of methylation products to that produced by N-methyl-N-nitrosourea, the aglycone of streptozotocin. When streptozotocin was administered to rats by i.v. injection, DNA was methylated with the formation of 7-methylguanine, O6-methylguanine, 3-methyladenine, and 7-methyladenine in liver, kidney, intestine, and pancreas. In contrast to N-methyl-N-nitrosourea which produced approximately equal amounts of methylation in DNA of liver, brain, and kidney, streptozotocin caused virtually no methylation in brain DNA; but, both liver and kidney DNA were alkylated to a greater extent than with N-methyl-N-nitrosourea. This methylation of renal DNA may account for the ability of streptozotocin to induce renal tumors. Streptozotocin produced significant methylation of pancreatic DNA which, if concentrated in the beta-cells, may account for their destruction. Pretreatment with nicotinamide reduced the extent of methylation of pancreatic DNA but did not affect the methylation in the liver or kidney. Methylation of beta-cell DNA in the pancreas may lead to the initiation of tumors if the extent of alkylation is not so great that cell death occurs.

Point mutations in human O6-alkylguanine-DNA alkyltransferase prevent the sensitization by O6-benzylguanine to killing by N,N'-bis (2-chloroethyl)-N-nitrosourea.

Chinese hamster ovary (CHO) cells lack O6-alkylguanine-DNA alkyltransferase (AGT) activity and are sensitive to killing by N,N'-bis (2-chloroethyl)-N-nitrosourea (BCNU). Transfection of these cells with a plasmid leading to the production of wild-type human AGT rendered them resistant to BCNU but this resistance could be overcome by treatment with O6-benzylguanine, an AGT inhibitor. Transfection with plasmids expressing mutants of the AGT in which either proline140 is converted to alanine or glycine156 is converted to alanine also gave rise to CHO cells resistant to BCNU, but these mutations rendered the expressed AGT less sensitive to O6-benzylguanine, and O6-benzylguanine was therefore much less effective in restoring sensitivity to BCNU. The G156A mutation provided the greater amount of resistance to O6-benzylguanine, and the CHO cells expressing this mutant AGT were not effectively killed by the O6-benzylguanine plus BCNU combination. CHO cells expressing the mutant AGTs were also much less sensitive than those expressing the control protein with respect to loss of AGT activity and enhancement of killing by BCNU in response to the more potent AGT inhibitor, 2,4-diamino-6-benzyloxy-5-nitrosopyrimidine. Although these results raise the possibility that resistance to therapy with O6-benzylguanine and chloroethylating agents may arise by the selection for tumor cells expressing a mutated AGT, they also provide a means by which the therapeutic effectiveness of agents forming O6-alkylguanine adducts such as BCNU might be enhanced. Expression of the G156A mutant AGT in hematopoietic progenitor cells by gene therapy techniques could be used to increase their AGT activity and provide a form that was resistant to O6-benzylguanine. Such resistance would provide a way to select for cells expressing the inserted gene and would provide an increase in the therapeutic index for treatment of tumors which would have an AGT activity sensitive to O6-benzylguanine.

Ornithine decarboxylase induction in transformation by H-Ras and RhoA.

The objective of these studies has been to develop a better understanding of the regulation of ornithine decarboxylase (ODC) during the neoplastic process, and to determine whether induction of ODC is a necessary component in the action of the ras oncogene. Specifically, we have studied the role of ODC overexpression in signaling pathways mediated by Raf or RhoA. Cells transformed by ras are known to have constitutively high levels of ODC activity that correlate with oncogenic transformation. To determine which pathways downstream of Ras contribute to the regulation of ODC activity, NIH-3T3 cells were transfected with plasmids coding for activated mutants of either H-Ras or RhoA, or oncogenic nu-Raf. There was a good correlation between increasing ODC specific activity and change in morphology from normal to transformed in the nu-Raf, HRas(61L), and RhoA(63L) clones. Increasing ODC activity also correlated positively with the ability to grow in soft agar in both the H-Ras- and RhoA-expressing cells. In stable transfections, coexpression of the ODC dominant negative mutant K69A/C360A with either HRas(61L) or RhoA(63L) both inhibited intracellular ODC activity and caused a reversion of the transformed phenotype, as measured by a dramatic reduction in the ability of these cells to grow in soft agar and form foci on a monolayer. These results suggest strongly that ODC induction is necessary for transformation by oncogenic Ras. In contrast, expression of K69A/ C360A had no effect on the ability of nu-Raf-transformed cells to grow in soft agar, although intracellular ODC levels were inhibited. When grown on a monolayer, these cells also maintained their transformed appearance. Furthermore, expression of the ODC dominant negative mutant did not affect the phosphorylation of mitogen-activated protein kinase in nu-Raf-transformed cells. These experiments provide strong support for the concept that transformation by activated ras is accompanied by an induction of ODC. The results using RhoA(63L) and nu-Raf suggest that this increase in ODC activity is mediated at least in part through a Raf/ mitogen-activated protein kinase independent pathway.

Overproduction of ornithine decarboxylase caused by relief of translational repression is associated with neoplastic transformation.

The mRNAs for two key enzymes in polyamine biosynthesis, ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC), both long 5' untranslated regions (5'UTRs) that could be important in the regulation of enzyme levels by affecting the translation of these mRNAs. In order to test this hypothesis, ODC and AdoMetDC activities were measured in 3T3 cells and in 3T3 cells overexpressing eIF-4E (P2 cells). eIF-4E has been reported to be a limiting factor in the translation of mRNAs with extensive secondary structures in the 5'UTR. AdoMetDC activity was not greatly different in the two cell lines, but ODC activity was much greater in the P2 cells. These results were confirmed by transfecting these cells with plasmids containing a luciferase complementary DNA fused to follow the 5'UTR from ODC or AdoMetDC. The ODC 5'UTR construct produced a higher luciferase activity in the P2 cells. The high level of expression of ODC may be a critical factor in the transformed phenotype of the P2 cells since the ability of these cells to grow in soft agar was blocked by levels of the ODC inhibitor, alpha-difluoromethylornithine, that reduced the ODC activity to values comparable to those of the parent 3T3 cells. These results provide more evidence for a critical role of ODC activity in neoplastic transformation and for the importance of its translational regulation in cell growth and transformation.

Immunohistochemical staining of human spermidine/spermine N1-acetyltransferase superinduced in response to treatment with antitumor polyamine analogues.

A superinduction of the polyamine catabolic enzyme, spermidine/spermine N1-acetyltransferase (SSAT) accompanies the phenotype-specific cytotoxic response to a class of antitumor polyamine analogues in several important human solid tumor models. A highly specific antiserum against the human SSAT protein has been developed. Using this antiserum we demonstrate that polyamine analogue treatment in vitro or in vivo results in an induction of SSAT protein which is uniformly distributed throughout the cytoplasm of treated tumor cells. This new biochemical tool will be useful in the examination of the association of superinduced SSAT activity and cell type-specific cytotoxicity. Additionally, since clinical trials have begun on one of the SSAT-inducing polyamine analogues, this antiserum may be useful as a diagnostic tool in differentiating responsive and nonresponsive tumor cell populations in treated patients.

Use of oligodeoxynucleotides containing O6-alkylguanine for the assay of O6-alkylguanine-DNA-alkyltransferase activity.

A sensitive assay procedure was developed for the measurement of the activity of mammalian O6-alkylguanine-DNA-alkyltransferase. The procedure utilized oligodeoxynucleotides containing O6-methylguanine as substrates for the reaction. The oligodeoxynucleotides were end labeled with 32P by the reaction with polynucleotide kinase and [gamma-32P]ATP and allowed to react with organ or cell extracts containing the alkyltransferase. The unmethylated product which was formed was separated from the substrate by reverse-phase high-pressure liquid chromatography. Since the repair by the alkyltransferase is bimolecular, the second order rate constants for the reaction between the labeled oligomer and repair protein from several different sources were determined. The amount of alkyltransferase present was then calculated from the amount of product formed and the appropriate second order rate constant for the reaction. Excellent agreement was obtained between the alkyltransferase levels determined in this procedure and those measured by conventional assay procedures in a variety of cell lines having both high and low activity. The method also gave results in good agreement with other assay procedures for a number of rat tissues, although a few tissues gave anomalous results owing to a high level of nuclease activity which degraded the substrate. This method should prove useful for the measurement of alkyltransferase activity in samples in which the activity is very low or the amount of material available is limited.

Effect of O6-alkylguanine pretreatment on the sensitivity of human colon tumor cells to the cytotoxic effects of chloroethylating agents.

Two human colon carcinoma cell lines which differ greatly in their content of O6-alkylguanine-DNA alkyltransferase were analyzed for their response to 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) and 2-chloroethyl(methylsulfonyl)methanesulfonate (ClEtSoSo) before and after treatment with O6-alkylguanines. HT29 cells contained about 17 times more alkyltransferase activity than BE cells. The alkyltransferase activity of HT29 cells was reduced by 60-80% by treatment for 24 h with 0.05-0.4 mM O6-methylguanine or O6-n-butylguanine. Such pretreatment prior to exposure to CCNU or ClEtSoSo increased the sensitivity of HT29 cells to these drugs. The exposure to O6-alkylguanines gave a greater enhancement of the toxic effects of ClEtSoSo than of CCNU. There was no significant increase in the toxicity of these agents towards the BE cells which contained much lower levels of the alkyltransferase. When added alone neither O6-methylguanine nor O6-n-butylguanine showed any toxicity towards HT29 or BE cells at the doses used. These results provide strong evidence that the formation of adducts at the O6-position of guanine by these agents contributes significantly to their lethality and that this reaction is more critical for ClEtSoSo than CCNU. The enhancement of the activity of chloroethylating agents by pretreatment with nontoxic doses of O6-alkylguanines may be clinically useful in terms of increasing their therapeutic efficacy towards cells containing high levels of alkyltransferase.