Methyl Methanesulfonate Treatment Research Articles

Methyl methanesulfonate induces necroptosis in human lung adenoma A549 cells through the PIG-3-reactive oxygen species pathway.

Methyl methanesulfonate (MMS) is an alkylating agent that can induce cell death through apoptosis and necroptosis. The molecular mechanisms underlying MMS-induced apoptosis have been studied extensively; however, little is known about the mechanism for MMS-induced necroptosis. Therefore, we first established MMS-induced necroptosis model using human lung carcinoma A549 cells. It was found that, within a 24-h period, although MMS at concentrations of 50, 100, 200, 400, and 800μM can induce DNA damage, only at higher concentrations (400 and 800μM) MMS treatment lead to necroptosis in A549 cells, as it could be inhibited by the specific necroptotic inhibitor necrostatin-1, but not the specific apoptotic inhibitor carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-fmk). MMS-induced necroptosis was further confirmed by the induction of the necroptosis biomarkers including the depletion of cellular NADH and ATP and leakage of LDH. This necroptotic cell death was also concurrent with the increased expression of p53, p53-induced gene 3 (PIG-3), high mobility group box-1 protein (HMGB1), and receptor interaction protein kinase (RIP) but not the apoptosis-associated caspase-3 and caspase-9 proteins. Elevated reactive oxygen species (ROS) level was also involved in this process as the specific ROS inhibitor (4-amino-2,4-pyrrolidine-dicarboxylic acid (APDC)) can inhibit the necroptotic cell death. Interestingly, knockdown of PIG-3 expression by small interfering RNA (siRNA) treatment can inhibit the generation of ROS. Taken together, these results suggest that MMS can induce necroptosis in A549 cells, probably through the PIG-3-ROS pathway.

Phenylpropanoid metabolism, hormone biosynthesis and signal transduction-related genes play crucial roles in the resistance of Paulownia fortunei to paulownia witches’ broom phytoplasma infection

Paulownia witches’ broom (PaWB) caused by an obligate biotrophic plant pathogen called phytoplasma, is a devastating disease of paulownia trees over a large part of the world. However, little is known about the molecular mechanisms that underlie phytoplasma pathogenicity in paulownia or about the mode of interactions with host plants. In this study, genome-wide gene expression profiling was used to compare healthy, phytoplasma-infected, and both phytoplasma-infected and 20 mg L−1 methyl methane sulfonate (MMS) treated Paulownia fortunei plants using high-throughput mRNA sequencing analysis. A total of 6571 and 1377 differentially expressed unigenes were identified in the phytoplasma-infected plants versus healthy plants and in 20 mg L−1 MMS-treated plants versus phytoplasma-infected plants, respectively. Expression changes of 16 candidate differentially expressed unigenes were validated by qRT-PCR, indicating significant differences among the three P. fortunei samples. Our analysis showed that dramatic changes occurred in the gene expression profile of P. fortunei after PaWB phytoplasma infection and MMS treatment. The transcription of a large number of genes related to the plant–pathogen interaction, including phenylpropanoid metabolism, hormone biosynthesis and signaling, defense and/or pathogenesis, and signal transduction, were significantly up-regulated in the phytoplasma-infected paulownia and then returned to the levels in the healthy controls after MMS treatment. Our systematic analysis provides comprehensive transcriptomic data about P. fortunei trees infected by PaWB phytoplasma. The findings will help unraveling the molecular mechanisms of plant-phytoplasma interactions and may pave the way for engineering P. fortunei trees with improved properties.

Protective effect of vitamin E on methyl methanesulfonate-induced teratozoospermia in adult Sprague-Dawley rats.

The protective effect of vitamin E (VE, α-tocopherol) on methyl methanesulfonate (MMS)-induced teratozoospermia was investigated in adult rats. Rats (n=6 per group) were divided into three groups: i) Control group, treated with distilled water from days 1 to 5; ii) the MMS group, treated with MMS at a dose of 40 mg·kg(-1) from days 1‑5; or iii) the VE+MMS group, treated with MMS at a dose of 40 mg·kg(-1) from days 1‑5, followed by VE at a dose of 150 mg·kg(-1) from day 6 for 6 weeks. Sperm count, motility and morphology were examined following treatment with VE. The serum testosterone level and antioxidant enzyme activity were measured, and the localization of Vasa, promyelocytic leukemia zinc finger protein (Plzf) and synaptonemal complex protein 3 (Scp3) were also examined. MMS treatment decreased sperm count and motility, and the levels of immunoreactive serum testosterone and endogenous antioxidants. In addition, MMS increased the percentage of abnormal sperm and the levels of free radicals. After MMS and VE treatment, sperm count and motility were significantly higher in rats from the VE+MMS group than in the MMS group. In addition, the serum testosterone concentration, as well as the levels of Vasa and free radicals and the percentage of abnormal sperm, decreased. The results indicated that VE has protective effects against MMS-induced teratozoospermia in adult rats.

Effect of mutagens on regeneration and growth of in vitro grown epicotyl segments of rough lemon seedlings (Citrus jambhiri Lush.)

The present study on the effect of mutagens on regeneration and growth of in vitro grown epicotyls segments of rough lemon seedlings (Citrus jambhiri Lush.) was carried out during the years 2009- 2010 and 2010- 2011 in the Tissue Culture Laboratory, Department of Fruit Science, Punjab Agricultural University, Ludhiana. Developmental characteristics of the in vitro grown epicotyls segments on regeneration media were treated employing gamma radiation in Gray(Gy) at 0, 5,10,15, 20,25,30,35,40 and 45 Gy; the alkylating agent ethyl methane sulfonate (EMS) and methyl methane sulfonate (MMS) each at 0, 0.1, 0.2, 0.3, 0.4,0.5 and 0.6% (v/v) were evaluated. Epicotyl segments from one month old in vitro grown seedling were cultured in regeneration medium (MS+BAP 1.0 mglit-1) under controlled laboratory conditions (25±2? C, 16 hr photoperiod, 2000 lux light). LD50, the dose required to kill half of the tested population corresponded to 35Gy for gamma radiation, 0.3% each for EMS and MMS treatments. Number of days taken for regeneration increased with increasing dose of gamma irradiation, EMS and MMS. Percent regeneration, number of buds, number of shoots, shoot length, number of leaves, internodal length, primary root length and number of secondary roots decreased with increasing dose of gamma radiation , EMS and MMS. The study would be beneficial to induce desirable variations in plant growth characteristics of rough lemon by the use of mutagens treatment.

RNA sequencing provides insights into the toxicogenomic response of ZF4 cells to methyl methanesulfonate

Whole genome transcriptomic studies are powerful for characterizing the molecular mechanisms underlying the physiological effects of chemicals, and are informative for environmental health risk assessment. Alkylating agents are an abundant class of chemicals that can damage DNA in the environment, and are used for anticancer treatments. Currently, little is known regarding the molecular mechanisms of toxic alkylating agents in zebrafish cell lines. In this study, RNA-sequencing was used to investigate the transcriptomic responses of zebrafish ZF4 cells following exposure to the model genotoxicant methyl methanesulfonate (MMS). The half-maximal inhibitory concentration (IC50 ) of MMS was 639.16 ± 61.8 µm, and apoptosis was induced within 24 h of exposure. RNA sequencing identified 3601 differentially expressed genes (DEGs) that were upregulated and 3037 that were downregulated. Gene ontology enrichment analysis revealed that most DEGs belonged to synthesis and metabolism categories. RNA-associated processes were the most upregulated, while cell cycle and adhesion were the most repressed processes, and neuron-related processes were the most downregulated developmental process. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis identified DNA damage repair, cell cycle, apoptosis and spliceosome as overrepresented terms. Six types of alternative splicing were detected. In total, 1156 alternative splicing DEGs were specifically expressed following MMS treatment, many of which belonged to metabolism and catabolic process categories. Cluster analysis of orthologs was able to extrapolate toxicotranscriptomic data between zebrafish and yeast. These results provide insight into the genome-wide response of ZF4 cells following exposure to MMS, and this knowledge will inform future toxicogenomic data analysis and environmental health risk assessment.

AP endonuclease knockdown enhances methyl methanesulfonate hypersensitivity of DNA polymerase β knockout mouse embryonic fibroblasts

Apurinic/apyrimidinic (AP) endonuclease (Apex) is required for base excision repair (BER), which is the major mechanism of repair for small DNA lesions such as alkylated bases. Apex incises the DNA strand at an AP site to leave 3′-OH and 5′-deoxyribose phosphate (5′-dRp) termini. DNA polymerase β (PolB) plays a dominant role in single nucleotide (Sn-) BER by incorporating a nucleotide and removing 5′-dRp. Methyl methanesulfonate (MMS)-induced damage is repaired by Sn-BER, and thus mouse embryonic fibroblasts (MEFs) deficient in PolB show significantly increased sensitivity to MMS. However, the survival curve for PolB-knockout MEFs (PolBKOs) has a shoulder, and increased sensitivity is only apparent at relatively high MMS concentrations. In this study, we prepared Apex-knockdown/PolB-knockout MEFs (AKDBKOs) to examine whether BER is related to the apparent resistance of PolBKOs at low MMS concentrations. The viability of PolBKOs immediately after MMS treatment was significantly lower than that of wild-type MEFs, but there was essentially no effect of Apex-knockdown on cell viability in the presence or absence of PolB. In contrast, relative counts of MEFs after repair were decreased by Apex knockdown. Parental PolBKOs showed especially high sensitivity at >1.5 mM MMS, suggesting that PolBKOs have another repair mechanism in addition to PolB-dependent Sn-BER, and that the back-up mechanism is unable to repair damage induced by high MMS concentrations. Interestingly, AKDBKOs were hypersensitive to MMS in a relative cell growth assay, suggesting that MMS-induced damage in PolB-knockout MEFs is repaired by Apex-dependent repair mechanisms, presumably including long-patch BER.

Both the charged linker region and ATPase domain of Hsp90 are essential for Rad51-dependent DNA repair.

The inhibition of Hsp90 in cancerous cells has been correlated with the reduction in double-strand break (DSB repair) activity. However, the precise effect of Hsp90 on the DSB repair pathway in normal cells has remained enigmatic. Our results show that the Hsp82 chaperone, the ortholog of mammalian Hsp90, is indispensable for homologous-recombination (HR)-mediated DNA repair in the budding yeast Saccharomyces cerevisiae. A considerable reduction in cell viability is observed in an Hsp82-inactivated mutant upon methyl methanesulfonate (MMS) treatment as well as upon UV treatment. The loss of Hsp82 function results in a dramatic decrease in gene-targeting efficiency and a marked decrease in the endogenous levels of the key recombination proteins Rad51 and Rad52 without any notable change in the levels of RAD51 or RAD52 transcripts. Our results establish Rad51 as a client of Hsp82, since they interact physically in vivo, and also show that when Hsp82 is inhibited by 17-AAG, Rad51 undergoes proteasomal degradation. By analyzing a number of point mutants with mutations in different domains of Hsp82, we observe a strong association between the sensitivity of an ATPase mutant of Hsp82 to DNA damage and the decreases in the amounts of Rad51 and Rad52 proteins. The most significant observations include the dramatic abrogation of HR activity and the marked decrease in Rad51 focus formation in the charged linker deletion mutant of Hsp82 upon MMS treatment. The charged linker region of Hsp82 is evolutionarily conserved in all eukaryotes, but until now, no biological significance has been assigned to it. Our findings elucidate the importance of this region in DNA repair for the first time.

Mass spectrometry-based quantification of the cellular response to methyl methanesulfonate treatment in human cells

Faithful transmission of genetic material is essential for cell viability and organism health. The occurrence of DNA damage, due to either spontaneous events or environmental agents, threatens the integrity of the genome. The consequences of these insults, if allowed to perpetuate and accumulate over time, are mutations that can lead to the development of diseases such as cancer. Alkylation is a relevant DNA lesion produced endogenously as well as by exogenous agents including certain chemotherapeutics. We sought to better understand the cellular response to this form of DNA damage using mass spectrometry-based proteomics. For this purpose, we performed sub-cellular fractionation to monitor the effect of methyl methanesulfonate (MMS) treatment on protein localization to chromatin. The levels of over 500 proteins were increased in the chromatin-enriched nuclear lysate including histone chaperones. Levels of ubiquitin and subunits of the proteasome were also increased within this fraction, suggesting that ubiquitin-mediated degradation by the proteasome has an important role in the chromatin response to MMS treatment. Finally, the levels of some proteins were decreased within the chromatin-enriched lysate including components of the nuclear pore complex. Our spatial proteomics data demonstrate that many proteins that influence chromatin organization are regulated in response to MMS treatment, presumably to open the DNA to allow access by other DNA damage response proteins. To gain further insight into the cellular response to MMS-induced DNA damage, we also performed phosphorylation enrichment on total cell lysates to identify proteins regulated via post-translational modification. Phosphoproteomic analysis demonstrated that many nuclear phosphorylation events were decreased in response to MMS treatment. This reflected changes in protein kinase and/or phosphatase activity in response to DNA damage rather than changes in total protein abundance. Using these two mass spectrometry-based approaches, we have identified a novel set of MMS-responsive proteins that will expand our understanding of DNA damage signaling.

DNA Damage and Oxidative Stress in Patients with Chronic Obstructive Pulmonary Disease

Background: We aimed to assess the level of DNA damage and susceptibility to exogenous mutagens in peripheral blood cells of Chronic Obstructive Pulmonary Disease (COPD) patients and healthy individuals by comet assay. Oxidative stress was also evaluated by means of thiobarbituric acid reactive species (TBARS) in blood plasma. Methods: Case-control study enrolling 51 COPD patients and 51 controls. Peripheral blood was used to perform the alkaline (pH>13) and neutral (pH=8.5) comet assay. For the assessment of susceptibility to exogenous DNA damage, the cells were treated with methylmethane sulfonate (MMS) for 1-hour or 3-hour at 37° C. The percentage of residual DNA damage after 3-h MMS treatment was calculated using the value of 1-h MMS treatment for each subject as 100%. Lipid peroxidation was evaluated by measuring TBARS in blood plasma. Results: DNA damage in patients was significantly higher than in controls as measured by the neutral and alkaline comet assay. Residual DNA damage detected after MMS treatment increased in patients, in contrast to controls, indicating higher susceptibility to alkylation damage and/or repair inhibition. High susceptibility to exogenous DNA damage in COPD patients correlates with high amount of TBARS and low forced vital capacity and expiratory volume. Conclusion: The positive correlation between increased susceptibility to exogenous DNA damage and TBARS levels in COPD patients suggests the possible involvement of oxidative stress in damage induction and/or repair inhibition.

Abstract 1989: ASPP2 enhances DRAM induced autophagy to promote apoptosis in HepG2 cells.

Abstract Background and Aims: ASPP2 (Apoptosis Stimulating Protein 2 of p53) binds to p53 and enhances p53 pro-apoptotic function. Autophagy may play a role in this process although the mechanisms remain to be clarified. Our aims were to explore the mechanisms by which the ASPP2-p53 pathway could promote apoptosis and autophagy in hepatocellular carcinoma cells in order to identify novel therapeutic targets in this disease. Methods: HepG2 cells were transfected with GFP-LC3 plasmid and then damaged with methyl methanesulfonate (MMS). Apoptosis was detected by cleavage of CK18 and PARP by immunocytochemical staining and Western blot. Real-time PCR and immunoblot were used to analyze changes in DRAM and LC3I/II expression. DRAM siRNA was used to inhibit DRAM function. Results: After MMS exposure for 24 hours, we identified more M30 positive cells and p85 formation in HepG2 cells compared to control cells. When HepG2 cells were transfected with a GFP-LC3 expression plasmid and then treated by MMS, we found that MMS treatment induced LC3 staining punctate structures, a marker for autophagy. Moreover, enforced expression of ASPP2 using adenovirus infection led to more autophagy and apoptosis in HepG2 after MMS treatment, under conditions where ASPP2 expressing adenovirus infection alone could not induce apoptosis and autophagy. Using real time PCR, we identified that MMS treatment could induce DRAM expression and that ASPP2 overexpression prior to MMS could further enhance DRAM expression compared to MMS alone. Immunoblot assay demonstrated that ASPP2 expression increased MMS-induced DRAM expression, LC3 II and p85 formation. Importantly, a DRAM specific siRNA blocked autophagy and apoptosis in HepG2 cells under these conditions when compared to a control siRNA. Conclusions: Our results suggest that ASPP2 can enhance MMS induced apoptosis via p53-dependent DRAM-mediated autophagy. Since DRAM is an important component of p53-mediated autophagy, our results demonstrate a new role for ASPP2 in this pathway. These findings open new avenues for investigation into the mechanisms of autophagy-based therapy in hepatocellular cancer (Supported by National Natural Science Foundation of China (30870853,30770742 and 30910103915), Beijing Municipal Natural Science Foundation (7092045 and 7101005) and Basic-Clinical Collaborative Research Fund, CCMU (12JL-L05)). Citation Format: Kai Liu, Tao Wen, Ming Ji Yin, Xin Lu Qiao, Ying Shi, Li Li, Jie Dao Liu, Chao Zhang, Ning Li, Charles Lopez, Xi De Chen. ASPP2 enhances DRAM induced autophagy to promote apoptosis in HepG2 cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1989. doi:10.1158/1538-7445.AM2013-1989

Abstract LB-151: REV1 has critical roles in base excision repair intermediate-mediated transversion mutations but not for O6-methylguanine-initiated mutations in vertebrate cells.

Abstract Isogenic chicken DT40 cells deficient in various DNA repair pathways are a great tool for understanding the DNA damage response and mutagenesis caused by genotoxic agents by a comparison of mutants and parental DT40 cells. We recently found that ~55% of PIGO mutations are located at A:T sites with a high frequency of A to T transversion mutations in DT40 cells exposed to methyl methanesulfonate (MMS). These results suggest that A to T transversion mutations found in MMS exposure-derived PIGO mutant cells are due to base excision repair intermediates, such as abasic sites and single strand breaks originated from N3-methyladenine. It has been reported that DNA repair protein REV1 acts as not only as a deoxycytidine transferase across abasic sites but also as a scaffold protein to coordinate translesion DNA synthesis (TLS) polymerase switching. To address effects of REV1 on MMS-induced PIGO mutations in DT40 cells, we exposed REV1 knockout DT40 cells to low-dose MMS. REV1 knockout DT40 cells showed hyper-sensitivity in cell survival after MMS treatment with no increase in PIG-O mutations. These results indicate that PIG-O mutations in DT40 cells exposed to MMS are caused through REV1-dependent, but REV1 deoxycytidyl transferase-independent, TLS. In contrast to MMS, REV1 and parental DT40 cells showed similar toxicity and mutagenicity caused by a combination of low-dose N-methyl-N-nitrosourea and O6-benzylguanine. These results suggest that base excision repair intermediates appear to initiate REV1-dependent TLS, whereas, O6-methylguanine could be by-passed by replicative polymerases, such as polymerase delta complexes, with an associated increase in G to A transition mutations. Citation Format: Jun Nakamura, Xu Tian, James Swenberg. REV1 has critical roles in base excision repair intermediate-mediated transversion mutations but not for O6-methylguanine-initiated mutations in vertebrate cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-151. doi:10.1158/1538-7445.AM2013-LB-151

The Preference for Error-Free or Error-Prone Postreplication Repair in Saccharomyces cerevisiae Exposed to Low-Dose Methyl Methanesulfonate Is Cell Cycle Dependent

Cells employ error-free or error-prone postreplication repair (PRR) processes to tolerate DNA damage. Here, we present a genome-wide screen for sensitivity to 0.001% methyl methanesulfonate (MMS). This relatively low dose is of particular interest because wild-type cells exhibit no discernible phenotypes in response to treatment, yet PRR mutants are unique among repair mutants in their exquisite sensitivity to 0.001% MMS; thus, low-dose MMS treatment provides a distinctive opportunity to study postreplication repair processes. We show that upon exposure to low-dose MMS, a PRR-defective rad18Δ mutant stalls into a lengthy G2 arrest associated with the accumulation of single-stranded DNA (ssDNA) gaps. Consistent with previous results following UV-induced damage, reactivation of Rad18, even after prolonged G2 arrest, restores viability and genome integrity. We further show that PRR pathway preference in 0.001% MMS depends on timing and context; cells preferentially employ the error-free pathway in S phase and do not require MEC1-dependent checkpoint activation for survival. However, when PRR is restricted to the G2 phase, cells utilize REV3-dependent translesion synthesis, which requires a MEC1-dependent delay and results in significant hypermutability.

Alkyladenine DNA glycosylase (AAG) localizes to mitochondria and interacts with mitochondrial single-stranded binding protein (mtSSB)

Due to a harsh environment mitochondrial genomes accumulate high levels of DNA damage, in particular oxidation, hydrolytic deamination, and alkylation adducts. While repair of alkylated bases in nuclear DNA has been explored in detail, much less is known about the repair of DNA alkylation damage in mitochondria. Alkyladenine DNA glycosylase (AAG) recognizes and removes numerous alkylated bases, but to date AAG has only been detected in the nucleus, even though mammalian mitochondria are known to repair DNA lesions that are specific substrates of AAG. Here we use immunofluorescence to show that AAG localizes to mitochondria, and we find that native AAG is present in purified human mitochondrial extracts, as well as that exposure to alkylating agent promotes AAG accumulation in the mitochondria. We identify mitochondrial single-stranded binding protein (mtSSB) as a novel interacting partner of AAG; interaction between mtSSB and AAG is direct and increases upon methyl methanesulfonate (MMS) treatment. The consequence of this interaction is specific inhibition of AAG glycosylase activity in the context of a single-stranded DNA (ssDNA), but not a double-stranded DNA (dsDNA) substrate. By inhibiting AAG-initiated processing of damaged bases, mtSSB potentially prevents formation of DNA breaks in ssDNA, ensuring that base removal primarily occurs in dsDNA. In summary, our findings suggest the existence of AAG-initiated BER in mitochondria and further support a role for mtSSB in DNA repair.

QPCA: a scalable assay to measure the perturbation of protein–protein interactions in living cells

One of the most important challenges in systems biology is to understand how cells respond to genetic and environmental perturbations. Here we show that the yeast DHFR-PCA, coupled with high-resolution growth profiling (DHFR-qPCA), is a straightforward assay to study the modulation of protein-protein interactions (PPIs) in vivo as a response to genetic, metabolic and drug perturbations. Using the canonical Protein Kinase A (PKA) pathway as a test system, we show that changes in PKA activity can be measured in living cells as a modulation of the interaction between its regulatory (Bcy1) and catalytic (Tpk1 and Tpk2) subunits in response to changes in carbon metabolism, caffeine and methyl methanesulfonate (MMS) treatments and to modifications in the dosage of its enzymatic regulators, the phosphodiesterases. Our results show that the DHFR-qPCA is easily implementable and amenable to high-throughput. The DHFR-qPCA will pave the way to the study of the effects of drug, genetic and environmental perturbations on in vivo PPI networks, thus allowing the exploration of new spaces of the eukaryotic interactome.

Hyperactivation of PARP Triggers Nonhomologous End-Joining in Repair-Deficient Mouse Fibroblasts

Regulation of poly(ADP-ribose) (PAR) synthesis and turnover is critical to determining cell fate after genotoxic stress. Hyperactivation of PAR synthesis by poly(ADP-ribose) polymerase-1 (PARP-1) occurs when cells deficient in DNA repair are exposed to genotoxic agents; however, the function of this hyperactivation has not been adequately explained. Here, we examine PAR synthesis in mouse fibroblasts deficient in the base excision repair enzyme DNA polymerase β (pol β). The extent and duration of PARP-1 activation was measured after exposure to either the DNA alkylating agent, methyl methanesulfonate (MMS), or to low energy laser-induced DNA damage. There was strong DNA damage-induced hyperactivation of PARP-1 in pol β nullcells, but not in wild-type cells. In the case of MMS treatment, PAR synthesis did not lead to cell death in the pol β null cells, but instead resulted in increased PARylation of the nonhomologous end-joining (NHEJ) protein Ku70 and increased association of Ku70 with PARP-1. Inhibition of the NHEJ factor DNA-PK, under conditions of MMS-induced PARP-1 hyperactivation, enhanced necrotic cell death. These data suggest that PARP-1 hyperactivation is a protective mechanism triggering the classical-NHEJ DNA repair pathway when the primary alkylated base damage repair pathway is compromised.

Methyl methanesulfonate induces apoptosis in p53-deficient H1299 and Hep3B cells through a caspase 2- and mitochondria-associated pathway

Methyl methanesulfonate (MMS) has been shown to induce apoptosis in various cell types through p53-dependent pathways. Nevertheless, pharmacological and genetic blockade of p53 functions results in similar or delayed sensitivity to MMS treatment, suggesting the presence of p53-independent apoptotic mechanisms. To understand the p53-independent mechanisms that are engaged during MMS-induced apoptosis, we established MMS-induced apoptotic cell models using p53-deficient H1299 and Hep3B cells. Our results demonstrated that MMS at concentrations of 50, 100, 200, 400 and 800μM induced the formation of gammaH2AX foci, and that at higher concentrations, 400 and 800μM, MMS treatment led to apoptosis in the two cell lines. This apoptotic cell death was concurrent with the loss of mitochondrial membrane potential, nuclear-cytosolic translocation of active caspase 2, release of cytochrome c from mitochondria, and the cleavage of caspase 9, caspase 3 and PARP. However, MMS-induced DNA damage failed to stabilize the p53 family members TAp73 and DNp73. These results demonstrated a p53- and p73-independent mechanism for MMS-induced apoptosis that involves the nuclear-cytosolic translocation of active caspase 2 as well as the mitochondria-mediated pathway.

Brahmarasayana protects against Ethyl methanesulfonate or Methyl methanesulfonate induced chromosomal aberrations in mouse bone marrow cells

BackgroundAyurveda, the traditional Indian system of medicine has given great emphasis to the promotion of health. Rasayana is one of the eight branches of Ayurveda which refers to rejuvenant therapy. It has been reported that rasayanas have immuno-modulatory, antioxidant and antitumor functions, however, the genotoxic potential and modulation of DNA repair of many rasayanas have not been evaluated.MethodsThe present study assessed the role of Brahmarasayana (BR) on Ethyl methanesulfonate (EMS)-and Methyl methanesulfonate (MMS)-induced genotoxicity and DNA repair in in vivo mouse test system. The mice were orally fed with BR (5 g or 8 mg / day) for two months and 24 h later EMS or MMS was given intraperitoneally. The genotoxicity was analyzed by chromosomal aberrations, sperm count, and sperm abnormalities.ResultsThe results have revealed that BR did not induce significant chromosomal aberrations when compared to that of the control animals (p >0.05). On the other hand, the frequencies of chromosomal aberrations induced by EMS (240 mg / kg body weight) or MMS (125 mg / kg body weight) were significantly higher (p<0.05) to that of the control group. The treatment of BR for 60 days and single dose of EMS or MMS on day 61, resulted in significant (p <0.05) reduction in the frequency of chromosomal aberrations in comparison to EMS or MMS treatment alone, indicating a protective effect of BR. Constitutive base excision repair capacity was also increased in BR treated animals.ConclusionThe effect of BR, as it relates to antioxidant activity was not evident in liver tissue however rasayana treatment was observed to increase constitutive DNA base excision repair and reduce clastogenicity. Whilst, the molecular mechanisms of such repair need further exploration, this is the first report to demonstrate these effects and provides further evidence for the role of brahmarasayana in the possible improvement of quality of life.

Pph3 Dephosphorylation of Rad53 Is Required for Cell Recovery from MMS-Induced DNA Damage in Candida albicans

The pathogenic fungus Candida albicans switches from yeast growth to filamentous growth in response to genotoxic stresses, in which phosphoregulation of the checkpoint kinase Rad53 plays a crucial role. Here we report that the Pph3/Psy2 phosphatase complex, known to be involved in Rad53 dephosphorylation, is required for cellular responses to the DNA-damaging agent methyl methanesulfonate (MMS) but not the DNA replication inhibitor hydroxyurea (HU) in C. albicans. Deletion of either PPH3 or PSY2 resulted in enhanced filamentous growth during MMS treatment and continuous filamentous growth even after MMS removal. Moreover, during this growth, Rad53 remained hyperphosphorylated, MBF-regulated genes were downregulated, and hypha-specific genes were upregulated. We have also identified S461 and S545 on Rad53 as potential dephosphorylation sites of Pph3/Psy2 that are specifically involved in cellular responses to MMS. Therefore, our studies have identified a novel molecular mechanism mediating DNA damage response to MMS in C. albicans.

A New Role for Translation Initiation Factor 2 in Maintaining Genome Integrity

Escherichia coli translation initiation factor 2 (IF2) performs the unexpected function of promoting transition from recombination to replication during bacteriophage Mu transposition in vitro, leading to initiation by replication restart proteins. This function has suggested a role of IF2 in engaging cellular restart mechanisms and regulating the maintenance of genome integrity. To examine the potential effect of IF2 on restart mechanisms, we characterized its influence on cellular recovery following DNA damage by methyl methanesulfonate (MMS) and UV damage. Mutations that prevent expression of full-length IF2-1 or truncated IF2-2 and IF2-3 isoforms affected cellular growth or recovery following DNA damage differently, influencing different restart mechanisms. A deletion mutant (del1) expressing only IF2-2/3 was severely sensitive to growth in the presence of DNA-damaging agent MMS. Proficient as wild type in repairing DNA lesions and promoting replication restart upon removal of MMS, this mutant was nevertheless unable to sustain cell growth in the presence of MMS; however, growth in MMS could be partly restored by disruption of sulA, which encodes a cell division inhibitor induced during replication fork arrest. Moreover, such characteristics of del1 MMS sensitivity were shared by restart mutant priA300, which encodes a helicase-deficient restart protein. Epistasis analysis indicated that del1 in combination with priA300 had no further effects on cellular recovery from MMS and UV treatment; however, the del2/3 mutation, which allows expression of only IF2-1, synergistically increased UV sensitivity in combination with priA300. The results indicate that full-length IF2, in a function distinct from truncated forms, influences the engagement or activity of restart functions dependent on PriA helicase, allowing cellular growth when a DNA–damaging agent is present.

Abstract 4689: Stabilization of PARP-DNA complexes plays a critical role for the cytotoxic effects of the PARP inhibitors, veliparib and oliparib

Abstract Poly(ADPribose) (PAR) polymerase (PARP) inhibitors are in clinical trials with alkylating agents such as temozolomide and topoisomerase I (Top1) inhibitors such as camptothecin derivatives (topotecan and irinotecan). To elucidate the mechanism of action of PARP inhibitors and how they synergize with alkylating agents and Top1 inhibitors, we compared genetic PARP knockout versus chemical inhibition with the PARP inhibitor. DNA alkylation damage was assayed by treating wild type (WT) and PARP knockout chicken DT40 cells with methyl methane sulfonate (MMS) in the presence of oliparib or veliparib. Notably, PARP inhibitors produced a much greater cytotoxicity than knocking out the PARP gene in cells treated with low MMS concentrations that are not cytotoxic in WT cells. Moreover, knocking out PARP eliminated the cytotoxicity of the PARP inhibitors even in the absence of MMS treatment. These results suggested that PARP inhibitors not only act by abrogating the catalytic activity of PARP and blocking PAR polymer formation but also by poisoning PARP. To further investigate this possibility, we measured PARP binding to DNA. Accordingly, we found that both oliparib and veliparib trap PARP on cellular DNA. By contrast, in the case of Top1 inhibitors, the potentiation by PARP inhibitors was no greater than knocking out PARP. Together, these results demonstrate that oliparib and veliparib act primarily as catalytic PARP inhibitors for Top1-linked DNA damage, whereas, in the case of base damage, oliparib and veliparib also act by preventing the removal of PARP complexes from DNA, probably as a result of defective PARP auto-PARylation, thereby converting base damage and DNA nicks into tight PARP-DNA complexes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4689. doi:1538-7445.AM2012-4689