The contribution of alkylation to the activity of quinone antitumor agents

Studies have shown that the quinone group can produce tumor cell kill by a mechanism involving active oxygen species. This cytotoxic activity can be correlated with the induction of DNA double strand breaks and is enhanced by the ability of the quinone compound to bind to DNA by alkylation. The cytotoxic activity and the production of DNA damage by model quinone antitumor agents were compared in L5178Y cells, sensitive and resistant to alkylating agents, to assess the contribution of alkylation to the activity of these agents. The resistant L5178Y/HN2 cells were found to be two fold and six fold more resistant to the alkylating quinones, benzoquinone mustard and benzoquinone dimustard, respectively, than parent L5178Y cells. In contrast, the L5178Y/HN2 cells showed no resistance to the nonalkylating quinones, hydrolyzed benzoquinone mustard and bis(dimethylamino)benzoquinone. The alkylating quinones produced approximately two fold less cross-linking in L5178Y/HN2 cells compared with L5178Y sensitive cells. DNA double strand break formation by hydrolyzed benzoquinone mustard and bis(dimethylamino)benzoquinone was not significantly different in sensitive and resistant cells. However, the induction of double strand breaks by the alkylating quinones benzoquinone mustard and benzoquinone dimustard was reduced by 5-fold and 15-fold, respectively, in L5178Y/HN2 cells. These results show that the alkylating activity of the alkylating quinones cannot directly explain all of the enhanced cytotoxic activity of these agents. Furthermore, they provide strong evidence that the enhanced formation of DNA double strand breaks by alkylating quinone agents is directly related to the ability of these agents to bind to DNA. This increased formation of strand breaks may account for the enhanced cytotoxic activity of the alkylating quinones.     

DNA sequence selectivity of guanine-N7 alkylation by nitrogen mustards is preserved in intact cells.

Nitrogen mustard alkylating agents react with isolated DNA in a sequence selective manner, and the substituent attached to the drug reactive group can impose a distinct sequence preference. It is not clear however to what extent the observed DNA sequence preferences are preserved in intact cells. The highly reiterated sequence of human alpha DNA has been used to determine the sites of guanine-N7 alkylation following treatment of cells with three nitrogen mustards, mechlorethamine, uracil mustard and quinacrine mustard, known to react in isolated DNA with distinctly different sequence preferences. Alpha DNA from drug treated cells was extracted, purified, end-labeled, and a 296 base pair, singly end-labelled, fragment isolated. Following the quantitative conversion of alkylation sites to strand breaks the fragments were separated on DNA sequencing gels. Clear differences were observed between the alkylation patterns of the three compounds, and the selectivities were qualitatively similar to those predicted and observed in the same sequence alkylated in vitro. In particular the unique preferences of uracil and quinacrine mustards for 5'-PyGC-3' and 5'-GT/GPu-3' sequences, respectively, were preserved in intact cells suggesting that the pattern of sequence dependent reactivity is not grossly affected by the nuclear milieu.     

DNA sequence selectivity of guanine-N7 alkylation by nitrogen mustards.

Nitrogen mustards alkylate DNA primarily at the N7 position of guanine. Using an approach analogous to that of the Maxam-Gilbert procedure for DNA sequence analysis, we have examined the relative frequencies of alkylation for a number of nitrogen mustards at different guanine-N7 sites on a DNA fragment of known sequence. Most nitrogen mustards were found to have similar patterns of alkylation, with the sites of greatest alkylation being runs of contiguous guanines, and relatively weak alkylation at isolated guanines. Uracil mustard and quinacrine mustard, however, were found to have uniquely enhanced reaction with at least some 5'-PyGCC-3' and 5'-GT-3' sequences, respectively. In addition, quinacrine mustard showed a greater reaction at runs of contiguous guanines than did other nitrogen mustards, whereas uracil mustard showed little preference for these sequences. A comparison of the sequence-dependent variations of molecular electrostatic potential at the N7-position of guanine with the sequence dependent variations of alkylation intensity for mechlorethamine and L-phenylalanine mustard showed a good correlation in some regions of the DNA, but not others. It is concluded that electrostatic interactions may contribute strongly to the reaction rates of cationic compounds such as the reactive aziridinium species of nitrogen mustards, but that other sequence selectivities can be introduced in different nitrogen mustard derivatives.     

Effect of ionic strength and cationic DNA affinity binders on the DNA sequence selective alkylation of guanine N7-positions by nitrogen mustards

Large variations in alkylation intensities exist among guanines in a DNA sequence following treatment with chemotherapeutic alkylating agents such as nitrogen mustards, and the substituent attached to the reactive group can impose a distinct sequence preference for reaction. In order to understand further the structural and electrostatic factors which determine the sequence selectivity of alkylation reactions, the effect of increased ionic strength, the intercalator ethidium bromide, AT-specific minor groove binders distamycin A and netropsin, and the polyamine spermine on guanine N7-alkylation by L-phenylalanine mustard (L-Pam), uracil mustard (UM), and quinacrine mustard (QM) was investigated with a modification of the guanine-specific chemical cleavage technique for DNA sequencing. For L-Pam and UM, increased ionic strength and the cationic DNA affinity binders dose dependently inhibited the alkylation. QM alkylation was less inhibited by salt (100 mM NaCl), ethidium (10 microM), and spermine (10 microM). Distamycin A and netropsin (100 microM) gave an enhancement of overall QM alkylation. More interestingly, the pattern of guanine N7-alkylation was qualitatively altered by ethidium bromide, distamycin A, and netropsin. The result differed with both the nitrogen mustard (L-Pam less than UM less than QM) and the cationic agent used. The effect, which resulted in both enhancement and suppression of alkylation sites, was most striking in the case of netropsin and distamycin A, which differed from each other. DNA footprinting indicated that selective binding to AT sequences in the minor groove of DNA can have long-range effects on the alkylation pattern of DNA in the major groove.     

Use of [8-3H]guanine-labeled deoxyribonucleic acid to study alkylating agent reaction kinetics and stability.

Alkylation at the N7 position of guanine in DNA renders the C8-hydrogen acidic. This serves as the basis for an assay of guanine N7 alkylation using [8-3H]-guanine-labeled DNA. I modified the assay by preparing a high specific activity substrate in vitro and by replacing the distillation step with charcoal adsorption of substrate. Using the appearance of noncharcoal-adsorbable label as a measure of guanine-N7 alkylation I examined the reaction of DNA with dimethyl sulfate and mechlorethamine. The rate of reaction of dimethyl sulfate with the N7 position of guanine in DNA was constant over time, i.e., loss of label from DNA proceeded linearly with time. On the other hand, the rate of reaction of mechlorethamine with DNA increased with time, consistent with the initial formation of the reactive aziridinium ion. The assay can also be used to compare the reaction rates of various alkylating agents with DNA. Thus, the acridine mustards ICR-170 and quinacrine mustard were far more potent alkylating agents than mechlorethamine. Furthermore the assay may be used to determine the alkylating potency and stability of various alkylating agent preparations: while frozen solutions of acridine mustards in organic solvents retained alkylating activity for several months, different commercial preparations of quinacrine mustard had little or no alkylating activity.     

The action of mono- and di-functional sulphur mustards on the ribonucleic acid-containing bacteriophage μ2

Bacteriophage μ2 is inactivated by both mono- and di-functional sulphur mustards at relatively low extents of alkylation. No degradation of alkylated RNA was detected. Cross-linking of RNA to protein was observed with the difunctional agent, but this reaction was only a minor contribution to the inactivation. Analyses of the reaction products in bacteriophage RNA showed that, at the mean lethal doses, more than one mono-alkylation of guanine had occurred but the sum total of other types of RNA alkylation was close to a single event. The results therefore suggest that inactivation results from the mono-alkylation of adenine or cytosine. In experiments with the difunctional agent cross-linking of RNA bases or of RNA to protein also prevented replication, the existence of these reactions accounting for the greater sensitivity of the bacteriophage to this agent.     

In vitro induction of micronuclei by monofunctional methanesulphonic acid esters: possible role of alkylation mechanisms

Six monofunctional alkylating methanesulphonates of widely varying structures were investigated in the in vitro micronucleus assay with Syrian hamster embryo fibroblast cells. The results were compared with the alkylating activities measured in the 4-(nitrobenzyl)pyridine test (NBP-test) and the N-methyl mercaptoimidazole (MMI-test) as measures for S(N)2 reactivity as well as in the triflouoroacetic acid (TFA) solvolysis and the hydrolysis reaction as measures for S(N)1 reactivity in order to provide insights into the role of alkylation mechanisms on induction of micronuclei. Moreover we compared the results of micronucleus assay with those of the Ames tests in strain TA 100 and TA1535 and with those of the SOS chromotest with the strains PQ37, PQ243, PM21 and GC 4798. The potency of methanesulphonates to induce micronuclei depended only to a certain degree, on the total alkylating activity (S(N)1 and S(N)2 reactivity). An inverse, significant correlation between the Ames test and the micronucleus assay was observed and an inverse correlation between the micronucleus assay and the SOS chromotest with the different strains. The results indicate that the primary mechanism leading to induction of micronuclei is not O-alkylation in DNA as it is the case in the Ames test with the hisG46 strains TA1535 and TA100 and not N-alkylation as with the SOS chromotest. There is evidence that protein alkylation, e.g. in the spindle apparatus in mitosis is decisive for induction of micronuclei by alkylating compounds. The structurally voluminous methanesulphonates 2-phenyl ethyl methanesulphonate and 1-phenyl-2-propyl methanesulphonate show a clear higher micronuclei inducing potency than the other tested though the bulky methanesulphonates possess a lower total alkylating activity than the others. This effect can be explained by a higher disturbance during mitosis after alkylation of the spindle apparatus with the structurally more bulky methanesulphonates.     

DNA alkylation by mustard gas in yeast strains of different repair capacity

Reaction of the toxic and mutagenic alkylating agent mustard gas with DNA of the yeast Saccharomyces cerevisiae was analyzed qualitatively and quantitatively. Within the dose range tested (2 X 10(-5)-2 X 10(-3) M) DNA in vivo is alkylated dose-proportionally. DNA alkylation and relative distribution of purine derivatives are not influenced by the cell's sensitivity towards the mutagen. At LD37 (4.4 X 10(-4) M) the wild type contains 44 300 purine derivatives: 9200 3-alkyladenines (20%), 29600 7-alkylguanines (67%) and 5500 diguaninyl derivates (13%) per genome. In sensitive strains the number of derivates per genome at LD37 is reduced according to the dose reduction factor. Alkylation at the position O6 of guanine by mustard gas cannot be shown, the method's limit of detection being 0.3% amongst purine derivates.     

DNA-directed alkylating ligands as potential antitumor agents: sequence specificity of alkylation by intercalating aniline mustards

The sequence preferences for alkylation of a series of novel parasubstituted aniline mustards linked to the DNA-intercalating chromophore 9-aminoacridine by an alkyl chain of variable length were studied by using procedures analogous to Maxam-Gilbert reactions. The compounds alkylate DNA at both guanine and adenine sites. For mustards linked to the acridine by a short alkyl chain through a para O- or S-link group, 5'-GT sequences are the most preferred sites at which N7-guanine alkylation occurs. For analogues with longer chain lengths, the preference of 5'-GT sequences diminishes in favor of N7-adenine alkylation at the complementary 5'-AC sequence. Magnesium ions are shown to selectively inhibit alkylation at the N7 of adenine (in the major groove) by these compounds but not the alkylation at the N3 of adenine (in the minor groove) by the antitumor antibiotic CC-1065. Effects of chromophore variation were also studied by using aniline mustards linked to quinazoline and sterically hindered tert-butyl-9-aminoacridine chromophores. The results demonstrate that in this series of DNA-directed mustards the noncovalent interactions of the carrier chromophores with DNA significantly modify the sequence selectivity of alkylation by the mustard. Relationships between the DNA alkylation patterns of these compounds and their biological activities are discussed.     

Specificity and kinetics of interstrand and intrastrand bifunctional alkylation by nitrogen mustards at a G-G-C sequence.

Previous work showed that melphalan-induced mutations in the aprt gene of CHO cells are primarily transversions and occur preferentially at G-G-C sequences, which are potential sites for various bifunctional alkylations involving guanine N-7. To identify the DNA lesion(s) which may be responsible for these mutations, an end-labeled DNA duplex containing a frequent site of melphalan-induced mutation in the aprt gene was treated with melphalan, mechlorethamine or phosphoramide mustard. The sequence specificity and kinetics of formation of both interstrand and intrastrand crosslinks were determined. All mustards selectively formed two base-staggered interstrand crosslinks between the 5'G and the G opposite C in the 5'G-G-C sequence. Secondary alkylation was much slower for melphalan than for the other mustards and the resulting crosslink was more stable. Mechlorethamine and phosphoramide mustard induced intrastrand crosslinks between the two contiguous Gs in the G-G-C sequence in double-stranded DNA, but melphalan did not. Molecular dynamic simulations provided a structural explanation for this difference, in that the monofunctionally bound intermediates of mechlorethamine and phosphoramide mustard assumed thermodynamically stable conformations with the second arm in a position appropriate for intrastrand crosslink formation, while the corresponding melphalan monoadduct did not.     

Preparation of alkylation agents for bulged DNA microenvironments

A designed molecule with capacity to alkylate DNA bulges has been prepared from readily available starting materials. The spirocyclic template utilized was designed on the basis of established architectures, and equipped with a mustard alkylating group. Preliminary studies confirm alkylation of specific bulged sequences, paving the way for second generation substrates with higher affinity.     

DNA sequence-specific adenine alkylation by the novel antitumor drug tallimustine (FCE 24517), a benzoyl nitrogen mustard derivative of distamycin.

FCE 24517, a novel distamycin derivative possessing potent antitumor activity, is under initial clinical investigation in Europe. In spite of the presence of a benzoyl nitrogen mustard group this compound fails to alkylate the N7 position of guanine, the major site of alkylation by conventional nitrogen mustards. Characterisation of DNA-drug adducts revealed only a very low level of adenine adduct formation. Using a modified Maxam-Gilbert sequencing method the consensus sequence for FCE 24517-adenine adduct formation was found to be 5'-TTTTGA-3'. A single base modification in the hexamer completely abolishes the alkylation of adenine. Using a Taq polymerase stop assay alkylations were confirmed at the A present in the hexamer TTTTGA and, in addition, in one out of three TTTTAA sequences present in the plasmid utilized. The sequence specificity of alkylation by FCE 24517 is therefore the most striking yet observed for an alkylating agent of small molecular weight.     

Cross-resistance ofEscherichia coli B/r tocis-platinum (II) diamminochloride,UV light and alkylating agents

Gradual transfers of the strain Escherichia coli B/r on M9 agar with increasing concentrations of cis-platinum (II) diamminochloride (cis-Pt(II)) yielded a resistant strain SM 405 capable of growing on liquid M9 medium containing 250 muM cis-Pt(II). The parent strain Escherichia coli B/r is completely inhibited in both division and growth at cis-Pt(II) concentrations as low as 30 muM. The resistant mutant has a longer doubling time than the parent strain. No other differences were found between the two strains. To elucidate the nature of the resistance, the effect of cis-Pt(II) on the survival of the two strains was compared with that of nitrogen mustard, UV light and ethyl methanesulphonate (EMS). The resistant strain SM 405 was found to be more hardened against the lethal action of UV light and nitrogen mustard but less so against EMS. It had also a higher ability of a host-cell reactivation of UV-irradiated phage T3. The different resistance of the B/r and SM 405 strains is probably due to a mutation increasing the effectiveness of the excision repair in the latter.     

Inhibition of post-replication repair of alkylated DNA by caffeine in Chinese hamster cells but not HeLa cells

Caffeine has been found to potentiate the lethal effects of sulphur mustard (SM) and N-methyl-N-nitrosourea (MNU) in a line of Chinese hamster cells but not in a line of HeLa cells. The sensitization of SM-treated cells by caffeine was S phase specific, and persisted for up to 24 h after alkylation of asynchronous cell cultures. The sensitization of MNU-treated cells, however, was not S phase specific but persisted for up to 50 h after the initial alkylation. Possible explanations for this difference between these two types of alkylating agent were discussed. Previously, evidence was presented which suggested that the alkylation-induced delay in the time of the peak rate of DNA synthesis in Chinese hamster cells was associated with the operation of post-DNA replication repair mechanism in these cells. Caffeine has now been found to reverse this alkylation-induced delay of DNA synthesis in both SM- and MNU-alkylated Chinese hamster cells. It is therefore proposed that caffeine sensitizes alkylated cells by inhibition of a post-replication DNA repair mechanism. No support was obtained for the alternative possibility that caffeine inhibits alkylation-induced excision repair of damaged DNA. The role of DNA repair in the production of the lethal mutagenic and cytological effects of alkylating agents is discussed.     

Phenyl sulfur mustard derivatives of distamycin A

The design, synthesis, and cytotoxic activity of novel benzoyl and cinnamoyl sulfur mustard derivatives of distamycin A are described and structure activity relationships are discussed. These sulfur mustards are more potent cytotoxics than corresponding nitrogen mustards in spite of the lower alkylating power, while their sulfoxide analogues are substantially inactive. Cinnamoyl sulfur mustard derivative (7) proved to be one of the most active distamycin-derived cytotoxics, about 1000 times more potent than melphalan.     

Gene transfer to suppress bone marrow alkylation sensitivity

Alkylating agents represent a highly cytotoxic class of chemotherapeutic compounds that are extremely effective anti-tumor agents. Unfortunately, alkylating agents damage both malignant and non-malignant tissues. Bone marrow is especially sensitive to damage by alkylating agent chemotherapy, and is a dose-limiting tissue when treating cancer patients. One strategy to overcome bone marrow sensitivity to alkylating agent exposure involves gene transfer of the DNA repair protein O(6)-methylguanine DNA methyltransferase (O(6)MeG DNA MTase) into bone marrow cells. O(6)MeG DNA MTase is of particular interest because it functions to protect against the mutagenic, clastogenic and cytotoxic effects of many chemotherapeutic alkylating agents. By increasing the O(6)MeG DNA MTase repair capacity of bone marrow cells, it is hoped that this tissue will become alkylation resistant, thereby increasing the therapeutic window for the selective destruction of malignant tissue. In this review, the field of O(6)MeG DNA MTase gene transfer into bone marrow cells will be summarized with an emphasis placed on strategies used for suppressing the deleterious side effects of chemotherapeutic alkylating agent treatment.     

radE, a new radiation-sensitive locus in Dictyostelium discoideum

Dictyostelium discoideum strain M28, which has been used widely in genetic studies, was found to carry a radiation-sensitive mutation. This allele, termed rad-100, was recessive in heterozygous diploids and mapped in linkage group III. Complementation analysis and survival studies on strains carrying rad-100 suggested that this allele defines a new radiation-sensitive locus in D. discoideum, and this locus has been designated radE. radE strains were moderately sensitive to ultraviolet light (D10 90 J m-2) and slightly sensitive to 137Cs gamma rays D10 255 krad). radE strains also exhibited increased sensitivity to killing by N-methyl-N'-nitro-N-nitrosoguanidine but not by other alkylating agents such as ethyl methanesulphonate or methyl methanesulphonate. The frequency of spontaneous methanol-resistant (acrA) mutants was approximately the same in cultures of radE and radE+ strains. However, when amoebae of these strains were irradiated with ultraviolet light, the frequency of induced mutants was significantly lower in cultures of the radE strain. Furthermore, when amoebae of wild-type strain NC4 were plated in the presence of caffeine after ultraviolet-irradiation, the survival curves were very similar to the curves obtained for amoebae of radE strains in the presence or in the absence of caffeine. These results suggest that the radE100 mutation and caffeine interfere with an error-prone DNA repair pathway in D. discoideum.     

Physiological modification of alkylating agent induced mutagenesis. II. Influence of the numbers of chromosome replicating forks and gene copies on the frequency of mutations induced in Escherichia coli.

The frequency of reversions induced in Escherichia coli K-12 trpA58 by any of five different monofunctional alkylating agents increased as the growth rate of the organism was raised prior to mutagen treatment. The increase in mutation frequency did not correlate with growth rate-dependent changes in cell area or total cellular protein and DNA. After treatment of cells with N-methyl-N-nitrosourea (MNUA), no growth rate-dependent change was observed in the total DNA alkylation or percentage of O6-methylguanine present in the DNA extracted. The frequency of reversions induced by one mutagen, methyl methanesulphonate (MMS), increased in proportion to the average number of trpA gene copies per cell, whereas the frequency of reversions induced by the other compounds was dependent on the average number of chromosome replicating forks per cell. This difference was attributed to the different ratios of DNA base alkylation products observed, formed after treatment with MMS, an SN2-type reagent, or after treatment with the SN1-type reagents ethyl methanesulphonate (EMS), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), MNUA and N-ethyl-N-nitrosourea (ENUA). Possible reasons for the dependence of mutation frequency on the number of replicating forks per cell are discussed.     

Survival, Deoxyribonucleic Acid Breakdown, and Synthesis in Salmonella typhimurium as Compared with Escherichia coli B Strains

Salmonella typhimurium LT-2 was compared with radioresistant (B/r) and radiosensitive (B(s-2)) strains of Escherichia coli in respect to the survival, deoxyribonucleic acid (DNA) breakdown, and DNA synthesis after X irradiation. It is shown that S. typhimurium LT-2 is about four times more sensitive than E. coli B/r but less sensitive than B(s-2). The DNA breakdown is in S. typhimurium LT-2 lower than the postirradiation breakdown of DNA in both E. coli strains and DNA synthesis proceeds in this bacterium in spite of a much lower survival, as in the radioresistant E. coli B/r.