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PubMed1980

The relationship between alkylation of specific DNA bases and induction of sister chromatid exchange.

The mechanism of induction of sister chromatid exchange (SCE) was investigated by treating Chinese hamster V-79 cells with two ethylating and two methylating mutagens at doses, taken from linear response curves, that produced 30 SCE/cell. Concentrations of the DNA alkylation products were measured or calculated at 11 DNA base sites and at the phosphodiester bond. Ethyl methanesulfonate, N-methyl- and N-ethyl-N-nitrosourea produced comparable concentrations (3.3 to 3.5 micromol product/mol DNA phosphate) of O6-alkylguanine. Hence, alkylation at O6 of guanine appears relevant to SCE induction for these mutagens. Since alkylation at O6 of guanine has been positively correlated with mutagenesis in V-79 cells, these findings support the suggestion that SCE and mutagenesis can result from a common DNA lesion. Methyl methanesulfonate (MMS) produced very little O6-methylguanine, but did produce 3-methylthymine and 3-methyladenine, either of which might account for the MMS-induced SCE. Thus, for a series of mutagens, induction of SCE does not necessarily result from a single specific DNA lesion. Therefore, SCE can be considered a qualitative indicator of potential mutagenic events.

Alkylating Agents↗
PubMed1982

Differential response of familial polyposis fibroblasts to two bifunctional alkylating agents.

Skin fibroblasts from three subjects with familial polyposis coli, a hereditary disorder predisposing to colon cancer, exhibited an unexpected insensitivity to mitomycin C (MMC). Sister chromatid exchanges (SCEs) were either reduced or unchanged after exposure to MMC, while another bi-functional alkylating agent, diepoxybutane (DEB), induced an increase in SCE level at a low, equitoxic dosage in parallel experiments. Increased sensitivity to MMC as assayed by colony formation was, however, shown by one of the subjects with polyposis. These cells with the familial polyposis genotype may have a deficiency in the DNA repair system handling MMC-induced alkylation damage yet remain capable of repairing damage induced by DEB alkylation. This deficiency may contribute to the increased frequency of neoplastic transformation known to occur in these cells in vivo.

Alkylating Agents↗
PubMed1986

Expression in mammalian cells of a truncated Escherichia coli gene coding for O6-alkylguanine alkyltransferase reduces the toxic effects of alkylating agents.

The lesion O6-alkylguanine (O6-AG) is produced in cellular DNA following exposure to monofunctional alkylating agents and its miscoding and mutagenic properties have been demonstrated in specific in vitro systems. In order to examine whether this lesion could be responsible for any of the biological effects of alkylating agents in mammalian cells, we have constructed a plasmid containing the O6-AG alkyltransferase (ATase) region of the gene from Escherichia coli, the product of which normally repairs both O6-AG and alkylphosphotriesters in DNA. We have transfected the construction into Chinese hamster fibroblasts which are deficient in endogenous ATase activity and selected a clone that expresses the truncated repair gene. We demonstrate that this protein is functional, acts on damage in host cell DNA and protects the cells from the toxic effects of those alkylating agents that react extensively at oxygen atom positions.

Alkylating Agents↗
PubMed2006

The biological impact of sequence-specific DNA alkylation by pyrrole-imidazole polyamides.

We have developed a series of novel DNA alkylating polyamides possessing indole linkers. Investigations using high-resolution gel electrophoresis revealed that the indole linked Py-Im polyamide alkylated at A of a targeted nine base pair matching sequence. Evaluation in human cancer cell lines revealed that the indole linked Py-Im polyamides have strong cytotoxicities. Furthermore, we showed that alkylation of the template strand of the coding region by these polyamides causes effective gene silencing.

Alkylation↗
PubMed2006

Antitumor activity of sequence-specific alkylating agents: pyrolle-imidazole CBI conjugates with indole linker.

DNA-targeting agents, including cisplatin, bleomycin and mitomycin C, are used routinely in cancer treatments. However, these drugs are extremely toxic, attacking normal cells and causing severe side effects. One important question to consider in designing anticancer agents is whether the introduction of sequence selectivity to DNA-targeting agents can improve their efficacy as anticancer agents. In the present study, the growth inhibition activities of an indole-seco 1,2,9,9a-tetrahydrocyclopropa[1,2-c]benz[1,2-e]indol-4-one (CBI) (1) and five conjugates with hairpin pyrrole-imidazole polyamides (2-6), which have different sequence specificities for DNA alkylation, were compared using 10 different cell lines. The average values of -log GI50 (50% growth inhibition concentration) for compounds 1-6 against the 10 cell lines were 8.33, 8.56, 8.29, 8.04, 8.23 and 8.83, showing that all of these compounds strongly inhibit cell growth. Interestingly, each alkylating agent caused significantly different growth inhibition patterns with each cell line. In particular, the correlation coefficients between the -log GI50 of compound 1 and its conjugates 2-6 showed extremely low values (R<0). These results suggest that differences in the sequence specificity of DNA alkylation lead to marked differences in biological activity. Comparison of the correlation coefficients between compounds 6 and 7, with the same sequence specificity as 6, and MS-247, with sequence specificity different from 6, when used against a panel of 37 human cancer cell lines further confirmed the above hypothesis.

Adenine↗
PubMed1989

Induction of the alkylation-inducible aidB gene of Escherichia coli by anaerobiosis.

Induction of the adaptive response to alkylation damage results in the expression of four genes arranged in three transcriptional units: the ada-alkB operon and the alkA and aidB genes. Adaptive-response induction requires the ada gene product and occurs when cells are treated with methylating agents. In previous studies we noted that aidB, but not alkA or ada-alkB, was induced in the absence of alkylation damage as cells were grown to stationary phase. In this note we present evidence that aidB is induced by anaerobiosis. Thus, aidB is subject to dual regulation by ada-dependent alkylation induction and ada-independent anaerobic induction.

Alkylating Agents↗
PubMed2003

Two lead drug designs based on chloramphenicol as the parent structure, which express alkylation activity with potential for clinical applications.

Chloramphenicol is a bacteriostatic antibiotic which acts primarily as an inhibitor of bacterial protein synthesis. Modification of chloramphenicol structure was accomplished by replacing the two hydroxyl groups (-OH) with a chlorine atom for LEAD-1 or a chloroethyl ether group (-OCH(2)CH(2)Cl) for LEAD-2. The resulting daughter compounds expressed significant alkylating activity at physiological temperature of 37 degrees C and at pH 7.4. Alkylation activity was evaluated after reaction with guanosine 5'-diphosphate (GDP), L-serine, L-glutamic acid, and p-chloroaniline. The partition coefficient (log P) was determined for chloramphenicol, LEAD-1 and LEAD-2 to be 0.854, 3.409 and 3.10, respectively. The molecular dipole of chloramphenicol, LEAD-1 and LEAD-2 was calculated to be 5.804, 3.961 and 4.097 Debye, respectively. All three structures showed zero violations of the Rule of 5, which indicates good bioavailability. Values for polar surface area (TPSA) indicate an intestinal absorption of 51% and 35% for LEAD-1 and LEAD-2, respectively. The reduction to only one proton donor in LEAD-1 and LEAD-2 structures (chloramphenicol having three proton donors) indicates an improvement of membrane penetration compared to chloramphenicol. (13)C NMR analysis of molecular structures was accomplished and fast atom bombardment mass spectrometry analysis of a reaction mixture showed LEAD-1 alkylation of guanosine 5'-diphosphate.

Alkylating Agents↗
PubMed1986

Mitochondrial glutathione transferases. The alkylation of mitochondrial membrane yields a catalyst with glutathione-transferase-like properties.

The pretreatment of rat liver mitochondria with alkylating agents (N-ethylmaleimide, iodoacetamide or vinyl pyridine) increased the activity of mitochondrial glutathione transferase (GST) by 100-250%. Further experiments provided evidence that mitochondrial membranes contain an enzymatically inactive protein which can be alkylated through its sulfhydryl groups. By alkylation, this protein achieves catalytic properties similar to those of native GST.

Alkylating Agents↗
PubMedDate not supplied

Glutathione depletion induced by c-Myc downregulation triggers apoptosis on treatment with alkylating agents.

Here we investigate the mechanism(s) involved in the c-Myc-dependent drug response of melanoma cells. By using three M14-derived c-Myc low-expressing clones, we demonstrate that alkylating agents, cisplatin and melphalan, trigger apoptosis in the c-Myc antisense transfectants, but not in the parental line. On the contrary, topoisomerase inhibitors, adriamycin and camptothecin, induce apoptosis to the same extent regardless of c-Myc expression. Because we previously demonstrated that c-Myc downregulation decreases glutathione (GSH) content, we evaluated the role of GSH in the apoptosis induced by the different drugs. In control cells treated with one of the alkylating agents or the others, GSH depletion achieved by L-buthionine-sulfoximine preincubation opens the apoptotic pathway. The apoptosis proceeded through early Bax relocalization, cytochrome c release, and concomitant caspase-9 activation, whereas reactive oxygen species production and alteration of mitochondria membrane potential were late events. That GSH was determining in the c-Myc-dependent drug-induced apoptosis was demonstrated by altering the intracellular GSH content of the c-Myc low-expressing cells up to the level of controls. Indeed, GSH ethyl ester-mediated increase of GSH abrogated apoptosis induced by cisplatin and melphalan by inhibition of Bax/cytochrome c redistribution. The relationship among c-Myc, GSH content, and the response to alkylating agent has been also evaluated in the M14 Myc overexpressing clones as well as in the melanoma JR8 c-Myc antisense transfectants. All together, these results demonstrate that GSH plays a key role in governing c-Myc-dependent drug-induced apoptosis.

Alkylating Agents↗
PubMed2000

DNA alkylation studies of combined treatment with methylnitrosourea and ethylmethanesulfonate in mice.

The interaction of the DNA-alkylating model compounds, ethylmethanesulfonate (EMS) and methylnitrosourea (MNU), was studied in pregnant NMRI mice by measuring DNA adduction in vivo. Previously, large-scale dose-response studies on teratogenicity as well as on DNA modification were performed using these substances. In addition, the risk of low doses in mice was estimated by comparative use of several approaches including molecular dosimetry. The risk was further analysed by combination experiments on teratogenesis with EMS and MNU. This paper describes a further approach with regard to an interaction of these compounds: the formation of DNA adducts was determined using a combined treatment regimen of [(14)C]-labelled MNU and EMS. The mutual influence of EMS and MNU on the DNA alkylation rates was found to be moderate. The dramatic increase in the teratogenic outcome following combined treatment found in previous studies was obviously not due to a massive interaction regarding the initial DNA alkylation rates. It may be explained, however, by the concept of toxic equivalency. Teratogenesis Carcinog. Mutagen. 20:27-34, 2000.

Alkylating Agents↗
PubMed1976

The problem of oncostatic specificity of cyclophosphamide (NSC-26271): Studies on reactions that control the alkylating and cytotoxic activity.

The relatively high oncostatic specificity of cyclophosphamide (CP) in vivo is shown to be due to the cytotoxic specificity of 4-hydroxycyclophosphamide (4-hydroxy-CP), the first product of metabolic activation of CP in the liver. This specificity can be evaluated not only in vivo by measuring the therapeutic index, but also in vitro by determining its cytotoxicity against Yoshida ascites tumor cells. Evidence is given that 4-hydroxy-CP is not an alkylating agent itself, but attains this property only by release of an alkylating N,N-(2-chloroethyl)phosphorodiamic acid moiety and acrolein. The energetic source for this rate-limiting toxication results from the resonance stabilization of the released acrolein. Reactions at the cryptoaldehyde group of 4-hydroxy-CP, which reduce or prevent the resonance stabilization of the 3-carbon unit to be released, lead to a deactivation of the primary metabolite of CP thus reducing or even preventing toxication, and hence influencing both the alkylating and cytotoxic activities of the molecule. Accordingly, it could be demonstrated by the reaction of 4-hydroxy-CP with thiols yielding 4-(S-R)-mercapto CP derivatives that the toxication of 4-hydroxy-CP can be controlled under physiologic conditions of pH and temperature. In the case of free protein sulfhydryl groups, this reaction also leads to fixation onto a macromolecule of the CP metabolite. On the basis of these peculiar reactivities of the oxazaphosphorine ring of 4-hydroxy-CP and of the partial reaction kinetics involved during toxication or deactivation, the significance of these findings to the problem of CP specificity is discussed.

Alkylating Agents↗
PubMedDate not supplied

[Selective modification of polyadenyl fragments of mRNA from Krebs-2 ascites carcinoma cells by an alkylating derivative of nonathymidilyluridine].

It is shown that alkylating reagent 2',3'-O-[4-(N-2-chloroethyl-N-methylamino)]-benzylidene nonathymidilyluridine penetrates into the Krebs-2 ascite carcinoma cells and efficiently alkylates their polymers. Nearly 30% of the reagent penetrated into the cell is consumed by nucleic acids. In conditions providing stability of the complementary complexes the modification extent of poly(A) fragments is two orders of magnitude greater than that of other nucleic acids fractions. No destruction of the oligonucleotide moiety of the reagent occurs in the course of intracellular alkylation.

Alkylating Agents↗
PubMed1995

DNA alkylation by minor groove-binding N1-alkoxyalkyl-bis-benzimidazoles.

Two groups of isomeric N1-alkoxyalkyl-bis-benzimidazoles differing in the orientation of the N-alkoxyalkyl group (R) with respect to the DNA minor groove have been examined as to their reaction with DNA. Agarose gel mobility shift assay demonstrates that the 'R-inward' isomers alkylate and cause thermally induced strand breakage, in contrast to a much weaker reaction of the 'R-outward' isomers. Complementary studies using high-resolution polyacrylamide gel electrophoresis confirmed relatively weak but definite alkylation-induced thermal cleavage at all available G base sites, but at selected A sites. The alkylation reaction is interpreted in terms of an SN2 displacement of the alkoxy group by nucleophiles within the groove, in contrast to the complete lack of such nucleophilic displacement of these drugs in bulk solution. Reaction with all available Gs is interpreted in terms of nucleophilic strength within the DNA minor groove whereas, in contrast, reaction at the A residues appears to be determined primarily by initial molecular recognition of a DNA site by the drug, followed by an SN2 displacement. The relative cytotoxic potencies of these drugs against KB human tumor cells may be explained on the basis of this mechanism.

Alkylation↗
PubMed1997

Cooperative alkylation by duocarmycin A-distamycin A heterodimer.

Duo A (Duo) normally alkylates adenine N3 at the 3' end of A + T-rich sequence in DNA. The addition of another minor groove binder, dist A (Dist), dramatically modulate the site of DNA alkylation by Duo with great acceleration of the reaction rate. In order to examine the mode of alkylation, the kinetics of the reaction under various conditions were examined. Based on the simulation of experimental data, a new reaction pathway was proposed.

Alkylating Agents↗
PubMed1997

Hematopoietic toxicity and cell cycle perturbations induced by new DNA minor groove-alkylating agents.

Some new alkylating agents which bind to the minor groove of DNA and have sequence-specific patterns of alkylation have shown anti-neoplastic activity in pre-clinical systems. Two of them, carzelesin and tallimustine, are now in phase II. Considering the severe dose-limiting bone marrow toxicity of both these drugs in clinical use, it was of interest to investigate the mechanism of their myelotoxicity in a detailed pre-clinical study and compare it with a conventional alkylating agent, such as melphalan. The origin and progression of the myelotoxicity of carzelesin, tallimustine and melphalan were investigated comparatively in mice, combining data on bone marrow and peripheral blood cellularity with data on the proliferative activity of bone marrow cells, obtained by in vivo administration of bromodeoxyuridine. Significant differences were found between the hematopoietic response to the 3 drugs, though all caused severe leukopenia. Carzelesin induced a short-term increase in myeloid proliferative activity, which prevented the high leukocytopenia on day 3 observed with the other drugs. However, when this effect was exhausted, a second nadir was seen in peripheral blood, with a new wave of cell proliferation of all lineages in the bone marrow. Reconstruction of the lymphoid lineage was slow for all 3 drugs but particularly difficult with high-dose tallimustine. In general, the hematopoietic system response to tallimustine was dampened, with no overshoots, suggesting either lasting effects or extensive cytotoxicity from the early to late precursors of all lineages.

Animals↗
PubMed1999

Comparison of DNA damage in plants as measured by single cell gel electrophoresis and somatic leaf mutations induced by monofunctional alkylating agents.

The use of single cell gel electrophoresis (SCGE) has recently been applied to plant systems. We optimized the experimental conditions for SCGE analysis using nuclei isolated from different tissues of intact plants. Concentration-response curves of genomic DNA migration were analyzed in intact plants treated with the monofunctional alkylating agents ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), N-ethyl-N-nitrosourea (ENU), and N-methyl-N-nitrosourea (MNU). These data were used to calibrate SCGE tail moment values to induced somatic mutation in plant leaves. We used a genotoxicity index to compare genomic DNA damage and the induction of somatic mutation in the leaf tissues. The rank order of the genotoxic potency of these alkylating agents assayed by SCGE was MNU >> MMS > ENU > EMS. The rank order for the mutagenic potency of these agents was MNU >> ENU congruent with MMS > EMS. The data demonstrate the utility of SCGE analysis in plant systems. The use of SCGE will permit a larger range of plants for use as in situ environmental monitors. Also, this approach may be used to search for crop plant germplasm accessions with enhanced genomic stability. We investigated whether the intragenomic distributions of DNA damage induced by these alkylating agents were uniform and random. When a plot of the ratio of the %tail DNA and tail length versus the concentration of the test mutagen was generated, the induced SCGE data deviated from a random distribution of genomic DNA damage.

Alkylating Agents↗
PubMed1998

Effects of O6-benzylguanine on growth and differentiation of P19 embryonic carcinoma cells treated with alkylating agents.

The purpose of this study was to evaluate the impact of modulating the repair of O6-alkylguanine adducts on the developmental toxicity of alkylating agents. Alkylating agents that have been shown to induce developmental toxicity following either in vitro or in vivo exposure were chosen for this investigation, and include methylnitrosourea (MNU), ethylnitrosourea (ENU), methyl methanesulfonate (MMS), and ethyl methanesulfonate (EMS). P19 cells are pluripotent murine embryonic carcinoma cells that can be induced by all trans retinoic acid (RA) to differentiate into cells that are biochemically and morphologically very similar to cells of the central nervous system. These cells are useful for studying the ability of chemicals to affect neuronal viability and differentiation. Neuronally differentiating P19 cells were pretreated with O6-benzylguanine (O6-Bg), a potent and specific inhibitor of the O6-alkylguanine-DNA-alkyltransferase (AT) protein that repairs lesions at the O6-position of guanine. In previous studies using micromass rat embryo midbrain cells, O6-Bg greatly potentiated the ability of MNU but not ENU to inhibit differentiation, and did not significantly alter the effects of either MNU or ENU on viability. In the P19 cells, we found that AT inhibition potentiated the effects of MMS, MNU, and EMS to inhibit both viability and differentiation. Additionally, AT inhibition had a much greater effect on toxicity of the methylating agents, as compared to the ethylating agents. These results suggest that O6-alkylguanine adducts can inhibit both viability and differentiation in P19 cells treated with alkylating agents.

Alkylating Agents↗