Monofunctional Alkylating Agent Methyl Methanesulfonate Research Articles

Late Activation of Stress-activated Protein Kinases/c-Jun N-terminal Kinases Triggered by Cisplatin-induced DNA Damage in Repair-defective Cells

Although stress-activated protein kinases/c-Jun N-terminal kinases (SAPK/JNK) are rapidly activated by genotoxins, the role of DNA damage in this response is not well defined. Here we show that the SEK1/MKK4-mediated dual phosphorylation of SAPK/JNK (Thr-183/Tyr-185) correlates with the level of cisplatin-DNA adducts at late times (16-24 h) after drug treatment in both human and mouse cells. Transfection of platinated plasmid DNA also caused SAPK/JNK activation. A defect in transcription-coupled nucleotide excision repair resting on a mutation in Cockayne syndrome group B protein promoted the late SAPK/JNK activation following cisplatin exposure. Signaling to SAPK/JNK was accompanied by activation of Ataxia telangiectasia mutated- and Rad3-related kinase, replication protein A, and checkpoint kinases as well as by the formation of DNA double strand breaks (DSBs). Ionizing radiation-induced DSBs did not provoke SAPK/JNK activation, and inhibition of transcription also failed to provoke this response. Late activation of SAPK/JNK stimulated by cisplatin-induced DNA lesions was reduced in the absence of specific DNA repair proteins, such as xeroderma pigmentosum protein C, pointing to an essential function of individual repair factors in DNA damage signaling to SAPK/JNK. Collectively, the data indicate that late SAPK/JNK activation is triggered by non-repaired cisplatin adducts in transcribed genes and involves replication-associated events, DSBs, tyrosine kinases, Rho GTPases, and specific repair factors.

On the cytotoxicity of some microbial volatile organic compounds as studied in the human lung cell line A549.

The cytotoxicity of 13 microbial volatile organic compounds (MVOC) was studied using a human lung carcinoma epithelial cell line A549 in a colony formation assay and two colorimetric assays: the microculture tetrazolium assay (MTT assay) and the cellular protein assay (methylene blue-MB assay). For comparison, two known cytotoxic substances: the non-volatile mycotoxin gliotoxin and the mono-functional alkylating agent methyl methanesulfonate (MMS) were studied. Concentration-response curves for each agent were established and the IC50 value (concentration resulting in 50% inhibition of colony growth or absorbance) was estimated. There are differences in toxicity levels between the MVOC tested and gliotoxin and MMS. The most toxic MVOC was 1-decanol which was as effective as MMS in all test systems. 1-decanol was about 10-fold more toxic than the other MVOC. All MVOC tested were more than 1000-fold less toxic than gliotoxin.

Evidence for the involvement of nucleotide excision repair in the removal of abasic sites in yeast.

In eukaryotes, DNA damage induced by ultraviolet light and other agents which distort the helix is removed by nucleotide excision repair (NER) in a fragment approximately 25 to 30 nucleotides long. In humans, a deficiency in NER causes xeroderma pigmentosum (XP), characterized by extreme sensitivity to sunlight and a high incidence of skin cancers. Abasic (AP) sites are formed in DNA as a result of spontaneous base loss and from the action of DNA glycosylases involved in base excision repair. In Saccharomyces cerevisiae, AP sites are removed via the action of two class II AP endonucleases, Apn1 and Apn2. Here, we provide evidence for the involvement of NER in the removal of AP sites and show that NER competes with Apn1 and Apn2 in this repair process. Inactivation of NER in the apn1Delta or apn1Delta apn2Delta strain enhances sensitivity to the monofunctional alkylating agent methyl methanesulfonate and leads to further impairment in the cellular ability to remove AP sites. A deficiency in the repair of AP sites may contribute to the internal cancers and progressive neurodegeneration that occur in XP patients.

DNA Repair Defects and Other Mustakes in Drosophila melanogaster

Preservation of the structural integrity of DNA in any organism is crucial to its health and survival. Such preservation is achieved by an extraordinary cellular arsenal of damage surveillance and repair functions, many of which are now being defined at the gene and protein levels. Mutants hypersensitive to the killing effects of DNA-damaging agents have been instrumental in helping to identify DNA repair-related genes and to elucidate repair mechanisms. In Drosophila melanogaster, such strains are generally referred to as mutagen-sensitive (mus) mutants and currently define more than 30 genetic loci. Whereas most mus mutants have been recovered on the basis of hypersensitivity to the monofunctional alkylating agent methyl methanesulfonate, they nevertheless constitute a phenotypically diverse group, with many mutants having effects beyond mutagen sensitivity. These phenotypes include meiotic dysfunctions, somatic chromosome instabilities, chromatin abnormalities, and cell proliferation defects. Within the last few years numerous mus and other DNA repair-related genes of Drosophila have been molecularly cloned, providing new insights into the functions of these genes. This article outlines strategies for isolating mus mutations and reviews recent advances in the Drosophila DNA repair field, emphasizing mutant analysis and gene cloning.

Isolation and characterization of Chinese hamster cell line resistant to monofunctional alkylating agents.

A clonal derivative of a Chinese hamster Don D-6 cell line resistant to methyl methane sulfonate (MMS) has been isolated following mutagenesis by ethyl methane sulfonate (EMS). The clone, designated as MMSr-1, exhibited high resistance to killing by the monofunctional alkylating agents MMS and EMS. This characteristic had not been acquired by a transient adaptation to the alkylating agents but was found to be a stable heritable trait. MMSr-1 was more sensitive to high-molecular-weight chemicals, such as colchicine and puromycin, than Don D-6. Both MMSr-1 and its parental cells showed the same ability to take up radioactive MMS. The resistance of MMSr-1 appears not to be due to altered uptake of MMS. The resistance was accompanied by low chromosomal aberration and sister chromatid exchange (SCE) induction but not by mutability. Protein synthesis inhibitors such as cycloheximide and puromycin reduced the resistance to the same level as that in Don D-6. SCE induction by MMS in this clone was not antagonized by the protein synthesis inhibitors, whereas mutagenesis was reversed to the normal parental cell level by these inhibitors. Aphidicolin, a DNA-synthesis inhibitor, exhibited no such effects. These results suggest that MMSr-1 might have modified repair capacity, which can be normalized by treatment with the protein-synthesis inhibitors, for lethal DNA damage by monofunctional alkylating agents, and that SCE formation by the alkylating agents is closely correlated with chromosomal aberration and cell lethality.

Methylmethanesulfonate-induced DNA damage and its repair in cultured human fibroblasts: normal rates of induction and removal of alkali-labile sites in xeroderma pigmentosum (group A) cells

Using conventional alkaline sucrose sedimentation analysis, we have compared the initial yield and subsequent enzymatic repair of DNA damage induced in cultured human [normal (GM38 and GM43) and xeroderma pigmentosum (XP12BE)] fibroblasts by the monofunctional alkylating agent methylmethanesulfonate (MMS). Exposure of both cell types to MMS (0-20 mM) resulted in a linear dose-response relationship for the formation of DNA alkali-labile sites (i.e. structurally altered sites that appeared as single-strand interruptions at alkaline pH). The majority (approximately 90%) of the sites detected in the normal cells immediately after chemical treatment (less than or equal to 8 mM) disappeared rapidly, with a half-life of less than or equal to 3 h; the remainder, however, persisted in genomic DNA for at least 72 h. Approximately 40% of the alkali-labile sites induced by 5 mM MMS could be stabilized by methoxyamine, a chemical which is known to react with apurinic/apyrimidinic (AP) sites in DNA so as to prevent alkali-catalyzed beta-elimination; thus this fraction of the alkali-labile sites, which is estimated to constitute approximately 4% of the total genomic injury inflicted by the chemical, may be ascribable to AP sites. XP12BE cells responded normally to MMS exposure as judged by: (i) the rate of initial induction of alkali-labile sites, including those (AP sites) subject to methoxyamine stabilization; (ii) the incidence of alkali-labile sites in cellular DNA at various times (0-72 h) after administration of the alkylating agent; and (iii) the capacity to execute the long-patch mode of excision repair as measured by accumulation of 1-beta-D-arabinofuranosylcytosine-induced strand breaks during post-treatment cell incubation. In addition, we have found that a significant portion of the genetic material in human fibroblasts undergoes degradation upon sustaining MMS damage, as indicated by the appearance of small DNA fragments (sedimenting near the top of alkaline sucrose gradients) in chemically treated cultures incubated for 24 h. Interestingly, the extent of this type of DNA injury proved to be markedly greater in XP12BE than in GM38 cells, and in exponentially growing than in G2-arrested normal cultures.

Cytotoxicity of monofunctional alkylating agents methyl methanesulfonate and methyl- N′-nitro- N-nitrosoguanidine have different mechanisms of toxicity for [formula omitted] cells

N-methyl- N′-nitro- N-nitrosoguanidine (MNNG) and methyl methanesulfonate (MMS) are directly active alkylating agents that methylate cellular macromolecules by SN 1 and SN 2 mechanisms, respectively. These two chemicals produce similar types of alkylation products in DNA and a similar level of total alkylations on a molar basis, but strikingly different proportions of alkylations of ring oxygen atoms of purines and pyrimidines. Because of this attribute, they have been used in combination to attempt to determine which types of alkylation products are responsible for mutation, transformation, and toxicity. Studies have suggested that the mutation rates produced by these and similar chemicals in cells surviving toxicity correlate well with the number of methyl adducts at the O 6 position of guanine, but that cytotoxicity (reduced colony-forming efficiency) does not correlate with any single adduct or with the total level of alkylation of DNA. In this study we have investigated the cytotoxic mechanisms of MNNG and MMS in synchronized 10 T 1 2 cells, using colony-forming ability as a measure of toxicity. Both MNNG and MMS cause dose-dependent reduction in the ability of 10 T 1 2 cells to produce colonies of more than 50 cells after 2 weeks in culture. MNNG is about 100-fold more toxic than MMS on a molar basis. As indicated by the inability of cells to exclude trypan blue, MMS kills a fraction of the population of treated 10 T 1 2 cells after a 30-min exposure; the fraction of cells that excludes trypan blue is correlated with dose of MMS and with colony-forming efficiency. Neither the fraction of cells that is permeable to trypan blue nor the relative colony-forming efficiency is affected by the phase of the cycle when 10 T 1 2 cells are treated with MMS. Furthermore, MMS toxicity for 10 T 1 2 cells is not potentiated by caffeine, MMS treatment does not delay progress of S phase, and cells that survive acute membrane toxicity complete the cell cycle without significant delay. In contrast, MNNG treatment produces toxicity that is maximal when 10 T 1 2 cells are exposed during the S phase and the effect of potentiated by caffeine. MNNG treatment delays DNA replication and this delay is reversed by caffeine. In sharp contrast to 10 T 1 2 cells treated with MMS. MNNG-treated cells are not made permeable to trypan blue, but are blocked in their ability to proliferate. These observations indicate that MNNG and MMS kill 10 T 1 2 cells by drastically different mechanisms, MNNG producing toxicity mainly by preventing chromosome replication and MMS producing toxicity mainly by damaging cell membranes.

Thermal inhibition of repair of methylmethane sulfonate-damaged DNA in chick embryo fibroblasts

Chick embryo fibroblasts were treated with the monofunctional alkylating agent methylmethane sulfonate at various concentrations for 1 h at 42°C, rinsed and then incubated post-treatment at various temperatures at which the kinetics of alkali-labile bond disappearance was followed. Growth experiments showed that these cells grew similarly at temperatures of either 37°C or 42°C. Repair as assessed by removal of alkali-labile bond was also similar for postincubation in the temperature range 37–42°C for damage due to methylmethane sulfonate treatment at concentrations less than 1.5 mM. When the postincubation temperature was raised higher than 42.5–43°C, this type of repair was stopped. The normal internal body temperature of adult chickens is about 41.6°C. Hence the present finding indicates that chick cells are much more severely restricted in DNA repair at temperatures above normal than are mammalian cells, which can function in this respect for several deg. C above 37°C.

Mutagen sensitivity of Drosophila melanogaster I. Isolation and preliminary characterization of a methyl methanesulphonate-sensitive strain

Sensitivity to the monofunctional alkylating agent methyl methanesulfonate (MMS) has been tested as a selection technique to isolate mutant strains which can provide insights into the genetic control of DNA replication, DNA repair and recombination in the complex eucaryote, Drosophila melanogaster. The successful isolation of an X-linked MMS-sensitive strain, mut s , has suggested that mutagen sensitivity is a feasible methodology for the selection of mutant strains of Drosophila which will be useful in the genetic and biochemical analysis of these cellular functions. Preliminary characterization of this mutant strain indicates that: ( A) it is extremely sensitive to killing by MMS; ( B) it is more mutable by MMS than the parent wildtype strain; and ( C) it appears to possess mutator gene activity.

Cross sensitivity to mono- and bifunctional alkylating agents of three radiation-sensitive Saccharomyces mutants.

The inactivation of three radiation-sensitive haploidSaccharomyces mutants, as well as that of corresponding homozygous diploid mutants, following treatment with the bifunctional alkylating agent diepoxybutane (DEB) or the monofunctional alkylating agents methylmethane-sulfonate (MMS) or methylnitrosoguanidine (MNG) was investigated. Sensivity to DEB was correlated with sensitivity to ultraviolet light. The response to MMS or MNG treatment was more complex. The most UV-sensitive haploid and diploid mutants were as resistant as wild type after MMS or MNG treatment. Those mutants with an inter-mediate UV sensitivity were also sensitive after MMS treatment, independent of their state of ploidy. After exposure to MNG, however, a characteristic difference appeared between the sensitivity of the two intermediate sensitive haploids and the corresponding diploids. A model is discussed, interpreting the results obtained as an indication of the existence of at least three different repair mechanisms inSaccharomyces.

The value of hydroxyurea in assessing repair synthesis of DNA in HeLa cells

A simplified assay for quantitating repair replication in HeLa cells was utilized which employed hydroxyurea (HU) for the purpose of selectively suppressing normal, semi-conservative replication. With semiconservative replication inhibited, DNA synthesis by UV-irradiated cells (as measured by the incorporation of [ 3H]thymidine) was shown to be a nearly exclusive product of repair synthesis (> 80%). Thus, the highly selective action of HU affords the opportunity of quantitating repair replication merely by assessing the rate of incorporation of [ 3H]thymidine into DNA. Several classes of chemical compounds were studied (monofunctional alkylating agents, organochlorine insecticides, an organomercurial and N-hydroxy compounds) to ascertain whether they initiated repair synthesis in non-irradiated cells or inhibited the repair process once it had been initiated by UV-irradiation. In those cases where initiation of repair synthesis was observed, attempts were made to confirm the result by isopycnic density gradient analysis in CsCl. Two monofunctional alkylating agents (methylmethanesulfonate (MMS) and ethyl methanesulfonate (EMS) were confirmed to initiate repair synthesis while the other three classes of compounds studied proved to be negative. However, both N-hydroxy compounds tested ( N-hydroxycyclohexylamine (HCHA) and hydroxylamine) gave falsely positive results in the simplified assay. Additional studies indicated that this artifact arose as a consequence of the N-hydroxy compounds interfering with the action of HU; i.e., the ability of HU to inhibit normal replication. None of the compounds tested exerted a marked inhibitory effect upon repair synthesis in UV-irradiated cells.

Alkylation damage and its repair.

The methanesulfonates are efficient mutagenic agents for both eucaryotic and procaryotic organisms. These monofunctional alkylating agents, particularly ethyl methanesulfonate (EMS), have been widely used for the induction of mutations in plants, insects, virus particles, and bacteria. In addition to their mutagenic activity these compounds produce inactivating lesions (Freese and Freese, 1966). In this paper we intend to discuss the following questions: (a) Is damage induced by the monofunctional alkylating agent methyl methanesulfonate (MMS) repaired and, if so, how widespread is the ability of organisms to cope with MMS-induced damage? (b) Which of the several types of damage produced by MMS is recognized by the repair system(s)?