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Nature Communications Dec 2019Oxidation and alkylation of nucleobases are known to disrupt their base-pairing properties within RNA. It is, however, unclear whether organisms have evolved general...
Oxidation and alkylation of nucleobases are known to disrupt their base-pairing properties within RNA. It is, however, unclear whether organisms have evolved general mechanism(s) to deal with this damage. Here we show that the mRNA-surveillance pathway of no-go decay and the associated ribosome-quality control are activated in response to nucleobase alkylation and oxidation. Our findings reveal that these processes are important for clearing chemically modified mRNA and the resulting aberrant-protein products. In the absence of Xrn1, the level of damaged mRNA significantly increases. Furthermore, deletion of LTN1 results in the accumulation of protein aggregates in the presence of oxidizing and alkylating agents. This accumulation is accompanied by Hel2-dependent regulatory ubiquitylation of ribosomal proteins. Collectively, our data highlight the burden of chemically damaged mRNA on cellular homeostasis and suggest that organisms evolved mechanisms to counter their accumulation.
Topics: 4-Nitroquinoline-1-oxide; Alkylation; DNA Adducts; DNA Damage; HEK293 Cells; Humans; Methyl Methanesulfonate; Mutation; Oxidation-Reduction; Oxidative Stress; Peptides; Polyribosomes; Protein Aggregates; Quinolones; RNA Stability; RNA, Messenger; Ribosomal Proteins; Ribosomes; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Ubiquitin; Ubiquitin-Protein Ligases; Ubiquitination
PubMed: 31819057
DOI: 10.1038/s41467-019-13579-3 -
Chemistry (Weinheim An Der Bergstrasse,... Sep 2018Mitomycin C (MC), an antitumor drug, and decarbamoylmitomycin C (DMC), a derivative of MC, alkylate DNA and form deoxyguanosine monoadducts and interstrand crosslinks...
Mitomycin C (MC), an antitumor drug, and decarbamoylmitomycin C (DMC), a derivative of MC, alkylate DNA and form deoxyguanosine monoadducts and interstrand crosslinks (ICLs). Interestingly, in mammalian culture cells, MC forms primarily deoxyguanosine adducts with a 1"-R stereochemistry at the guanine-mitosene bond (1"-α) whereas DMC forms mainly adducts with a 1"-S stereochemistry (1"-β). The molecular basis for the stereochemical configuration exhibited by DMC has been investigated using biomimetic synthesis. Here, we present the results of our studies on the monoalkylation of DNA by DMC. We show that the formation of 1"-β-deoxyguanosine adducts requires bifunctional reductive activation of DMC, and that monofunctional activation only produces 1"-α-adducts. The stereochemistry of the deoxyguanosine adducts formed is also dependent on the regioselectivity of DNA alkylation and on the overall DNA CG content. Additionally, we found that temperature plays a determinant role in the regioselectivity of duplex DNA alkylation by mitomycins: At 0 °C, both deoxyadenosine (dA) and deoxyguanosine (dG) alkylation occur whereas at 37 °C, mitomycins alkylate dG preferentially. The new reaction protocols developed in our laboratory to investigate DMC-DNA alkylation raise the possibility that oligonucleotides containing DMC 1"-β-deoxyguanosine adducts at a specific site may be synthesized by a biomimetic approach.
Topics: Alkylation; Animals; Base Sequence; Chromatography, High Pressure Liquid; DNA; DNA Adducts; DNA, Bacterial; Deoxyadenosines; Deoxyguanosine; Mice; Micrococcus luteus; Mitomycin; Mitomycins; Stereoisomerism; Temperature
PubMed: 29958326
DOI: 10.1002/chem.201802038 -
SLAS Technology Jun 2022Variation in methylcytosine is perhaps the most well-studied epigenetic mechanism of gene regulation. Methods that have been developed and implemented for assessing DNA...
Variation in methylcytosine is perhaps the most well-studied epigenetic mechanism of gene regulation. Methods that have been developed and implemented for assessing DNA methylation require sample DNA to be extracted, purified and chemically-processed through bisulfite conversion before downstream analysis. While some automated solutions exist for each of these individual process steps, a fully integrated solution for accomplishing the entire process in a high-throughput manner has yet to be demonstrated. Thus, sample processing methods still require numerous manual steps that may reduce sample throughput and precision, while increasing the risk of contamination and human error. In this work, we present an integrated, automated solution for performing the entire sample preparation process, including DNA extraction, purification, bisulfite conversion and PCR plate preparation within in an enclosed environment. The method employs silica-coated magnetic particles that eliminate the need for a centrifuge or vacuum manifold, thereby reducing the complexity and cost of the required automation platform. Toward this end, we also compare commercial DNA extraction and bisulfite conversion kits to identify a protocol suitable for automation to significantly improve genomic and bisulfite-treated DNA yields over manufacturer protocols. Overall, this research demonstrated development of an automated protocol that offers the ability to generate high-quality, bisulfite-treated DNA samples in a high-throughput and clean environment with minimal user intervention and comparable yields to manual processing.
Topics: DNA; DNA Methylation; Humans; Polymerase Chain Reaction; Specimen Handling
PubMed: 35058199
DOI: 10.1016/j.slast.2021.12.002 -
Chemical Research in Toxicology Sep 2020The cellular outcomes of chemical exposure are as much about the cellular to the chemical as it is an of the chemical. We are growing in our understanding of the... (Review)
Review
The cellular outcomes of chemical exposure are as much about the cellular to the chemical as it is an of the chemical. We are growing in our understanding of the genotoxic interaction between chemistry and biology. For example, recent data has revealed the biological basis for mutation induction curves for a methylating chemical, which has been shown to be dependent on the repair capacity of the cells. However, this is just one end point in the toxicity pathway from chemical exposure to cell death. Much remains to be known in order for us to predict how cells will respond to a certain dose. Methylating agents, a subset of alkylating agents, are of particular interest, because of the variety of adverse genetic end points that can result, not only at increasing doses, but also over time. For instance, methylating agents are mutagenic, their potency, for this end point, is determined by the cellular repair capacity of an enzyme called methylguanine DNA-methyltransferase (MGMT) and its ability to repair the induceed methyl adducts. However, methyl adducts can become clastogenic. Erroneous biological processing will convert mutagenic adducts to clastogenic events in the form of double strand breaks (DSBs). How the cell responds to DSBs is via a cascade of protein kinases, which is called the DNA damage response (DDR), which will determine if the damage is repaired effectively, via homologous recombination, or with errors, via nonhomologous end joining, or whether the cell dies via apoptosis or enters senescence. The fate of cells may be determined by the extent of damage and the resulting strength of DDR signaling. Therefore, thresholds of damage may exist that determine cell fate. Such thresholds would be dependent on each of the repair and response mechanisms that these methyl adducts stimulate. The molecular mechanism of how methyl adducts kill cells is still to be fully resolved. If we are able to quantify each of these thresholds of damage for a given cell, then we can ascertain, of the many adducts that are induced, what proportion of them are mutagenic, what proportion are clastogenic, and how many of these clastogenic events are toxic. This review examines the possibility of dose and damage thresholds for methylating agents, from the perspective of the underlying evolutionary mechanisms that may be accountable.
Topics: Alkylating Agents; Animals; Enzyme Inhibitors; Humans; Methylation; O(6)-Methylguanine-DNA Methyltransferase
PubMed: 32388971
DOI: 10.1021/acs.chemrestox.0c00052 -
Oncotarget May 2023
Topics: Humans; Temozolomide; Glioblastoma; DNA Repair; Dacarbazine; Autophagy; DNA Modification Methylases; DNA Repair Enzymes; Antineoplastic Agents, Alkylating; Brain Neoplasms; Drug Resistance, Neoplasm; GTP-Binding Proteins
PubMed: 37204260
DOI: 10.18632/oncotarget.28370 -
Annals of Palliative Medicine Jan 2021High grade gliomas (HGG) include World Health Organization (WHO) grade III anaplastic astrocytoma (AA) and WHO grade IV glioblastoma (GBM). As genomic alterations are... (Review)
Review
High grade gliomas (HGG) include World Health Organization (WHO) grade III anaplastic astrocytoma (AA) and WHO grade IV glioblastoma (GBM). As genomic alterations are prognostic, even WHO grade II, IDH-wildtype gliomas may be considered as HGG. Current management of HGG include best supportive care (BSC), surgery, radiation therapy (RT), chemotherapy, and a combination. Elderly patients (defined here as age ≥65) with GBM have significantly worse survival compared to younger patients. Similarly, patients with poor performance status [defined as Karnofsky performance status (KPS) <60 or ECOG performance status (PS) >2], regardless of age have worse outcomes. The standard of care for treatment of HGG involves surgery and chemoradiation. However, the optimal treatment in terms of efficacy, safety and maintaining quality of life (QoL), remains a matter of debate in the elderly and/or poor performing patients due to their worse prognosis. Less aggressive interventions are usually reserved for these patients despite surgery providing a survival and neurologic benefit. Improved survival has been noted in elderly patients treated with RT in comparison with those receiving best supportive care (BSC) alone, with similar survival for patients undergoing standard RT (60 Gy/30 fractions) and hypofractionated RT (25-40 Gy in 5-15 daily fractions). An alkylating agent, temozolomide (TMZ), represents a safe and effective option in select patients with promoter methylation of O6-methylguanine-DNA-methyltransferase (MGMT) gene. A recent phase III randomized trial for GBM patients (age ≥65 years, ECOG PS 0-2) demonstrated a significant improvement in progression-free survival (PFS) and overall survival (OS) with hypofractionated RT (40 Gy/15 fractions) with concurrent and adjuvant TMZ vs. RT alone, without adversely impacting either QoL or functional status. Despite chemoradiation becoming the recommended treatment in GBM patients who are elderly but fit, several questions remain unanswered. This includes the survival impact of chemoradiation in patients with severe comorbidities or with ECOG PS >2 or a combination of poor prognostic features such as male gender, poor neurocognition, biopsy only and lack of MGMT methylation. Personalized management of patients with HGG is warranted in the modern era as we attempt to balance the benefit of efficacious treatment with potential toxicity while appreciating the many nuances associated with multiple prognostic factors on anticipated survival. Here, we aim to review the palliative management options available for HGG patients with an emphasis on the role of RT.
Topics: Aged; Antineoplastic Agents, Alkylating; Brain Neoplasms; Glioma; Humans; Male; Quality of Life; Temozolomide
PubMed: 33040565
DOI: 10.21037/apm-20-1246 -
CNS Neuroscience & Therapeutics Apr 2024To elucidate the relationship between USP19 and O(6)-methylguanine-DNA methyltransferase (MGMT) after temozolomide treatment in glioblastoma (GBM) patients with...
OBJECTIVE
To elucidate the relationship between USP19 and O(6)-methylguanine-DNA methyltransferase (MGMT) after temozolomide treatment in glioblastoma (GBM) patients with chemotherapy resistance.
METHODS
Screening the deubiquitinase pannel and identifying the deubiquitinase directly interacts with and deubiquitination MGMT. Deubiquitination assay to confirm USP19 deubiquitinates MGMT. The colony formation and tumor growth study in xenograft assess USP19 affects the GBM sensitive to TMZ was performed by T98G, LN18, U251, and U87 cell lines. Immunohistochemistry staining and survival analysis were performed to explore how USP19 is correlated to MGMT in GBM clinical management.
RESULTS
USP19 removes the ubiquitination of MGMT to facilitate the DNA methylation damage repair. Depletion of USP19 results in the glioblastoma cell sensitivity to temozolomide, which can be rescued by overexpressing MGMT. USP19 is overexpressed in glioblastoma patient samples, which positively correlates with the level of MGMT protein and poor prognosis in these patients.
CONCLUSION
The regulation of MGMT ubiquitination by USP19 plays a critical role in DNA methylation damage repair and GBM patients' temozolomide chemotherapy response.
Topics: Humans; Temozolomide; DNA Repair Enzymes; DNA Modification Methylases; Antineoplastic Agents, Alkylating; Animals; Cell Line, Tumor; Drug Resistance, Neoplasm; Tumor Suppressor Proteins; DNA Methylation; Mice, Nude; Brain Neoplasms; Glioblastoma; Mice; Male; Female; Dacarbazine; DNA Repair; Endopeptidases; Xenograft Model Antitumor Assays; Ubiquitination
PubMed: 38644551
DOI: 10.1111/cns.14711 -
-alkylguanine-DNA Alkyltransferases in Microbes Living on the Edge: From Stability to Applicability.International Journal of Molecular... Apr 2020The genome of living cells is continuously exposed to endogenous and exogenous attacks, and this is particularly amplified at high temperatures. Alkylating agents cause... (Review)
Review
The genome of living cells is continuously exposed to endogenous and exogenous attacks, and this is particularly amplified at high temperatures. Alkylating agents cause DNA damage, leading to mutations and cell death; for this reason, they also play a central role in chemotherapy treatments. A class of enzymes known as AGTs (alkylguanine-DNA-alkyltransferases) protects the DNA from mutations caused by alkylating agents, in particular in the recognition and repair of alkylated guanines in -position. The peculiar irreversible self-alkylation reaction of these enzymes triggered numerous studies, especially on the human homologue, in order to identify effective inhibitors in the fight against cancer. In modern biotechnology, engineered variants of AGTs are developed to be used as for the attachment of chemical ligands. In the last decade, research on AGTs from (hyper)thermophilic sources proved useful as a model system to clarify numerous phenomena, also common for mesophilic enzymes. This review traces recent progress in this class of , emphasizing their usefulness in basic research and their consequent advantages for in vivo and in vitro biotechnological applications.
Topics: Alkylation; Biotechnology; DNA Damage; DNA Repair; DNA Replication; Molecular Targeted Therapy; Neoplasms; O(6)-Methylguanine-DNA Methyltransferase; Structure-Activity Relationship; Thermodynamics; Thermoproteus
PubMed: 32326075
DOI: 10.3390/ijms21082878 -
JAMA Oncology Jul 2023O6-methylguanine-DNA methyltransferase (MGMT [OMIM 156569]) promoter methylation (mMGMT) is predictive of response to alkylating chemotherapy for glioblastomas and is...
IMPORTANCE
O6-methylguanine-DNA methyltransferase (MGMT [OMIM 156569]) promoter methylation (mMGMT) is predictive of response to alkylating chemotherapy for glioblastomas and is routinely used to guide treatment decisions. However, the utility of MGMT promoter status for low-grade and anaplastic gliomas remains unclear due to molecular heterogeneity and the lack of sufficiently large data sets.
OBJECTIVE
To evaluate the association of mMGMT for low-grade and anaplastic gliomas with chemotherapy response.
DESIGN, SETTING, AND PARTICIPANTS
This cohort study aggregated grade II and III primary glioma data from 3 prospective cohort studies with patient data collected from August 13, 1995, to August 3, 2022, comprising 411 patients: MSK-IMPACT, EORTC (European Organization of Research and Treatment of Cancer) 26951, and Columbia University. Statistical analysis was performed from April 2022 to January 2023.
EXPOSURE
MGMT promoter methylation status.
MAIN OUTCOMES AND MEASURES
Multivariable Cox proportional hazards regression modeling was used to assess the association of mMGMT status with progression-free survival (PFS) and overall survival (OS) after adjusting for age, sex, molecular class, grade, chemotherapy, and radiotherapy. Subgroups were stratified by treatment status and World Health Organization 2016 molecular classification.
RESULTS
A total of 411 patients (mean [SD] age, 44.1 [14.5] years; 283 men [58%]) met the inclusion criteria, 288 of whom received alkylating chemotherapy. MGMT promoter methylation was observed in 42% of isocitrate dehydrogenase (IDH)-wild-type gliomas (56 of 135), 53% of IDH-mutant and non-codeleted gliomas (79 of 149), and 74% of IDH-mutant and 1p/19q-codeleted gliomas (94 of 127). Among patients who received chemotherapy, mMGMT was associated with improved PFS (median, 68 months [95% CI, 54-132 months] vs 30 months [95% CI, 15-54 months]; log-rank P < .001; adjusted hazard ratio [aHR] for unmethylated MGMT, 1.95 [95% CI, 1.39-2.75]; P < .001) and OS (median, 137 months [95% CI, 104 months to not reached] vs 61 months [95% CI, 47-97 months]; log-rank P < .001; aHR, 1.65 [95% CI, 1.11-2.46]; P = .01). After adjusting for clinical factors, MGMT promoter status was associated with chemotherapy response in IDH-wild-type gliomas (aHR for PFS, 2.15 [95% CI, 1.26-3.66]; P = .005; aHR for OS, 1.69 [95% CI, 0.98-2.91]; P = .06) and IDH-mutant and codeleted gliomas (aHR for PFS, 2.99 [95% CI, 1.44-6.21]; P = .003; aHR for OS, 4.21 [95% CI, 1.25-14.2]; P = .02), but not IDH-mutant and non-codeleted gliomas (aHR for PFS, 1.19 [95% CI, 0.67-2.12]; P = .56; aHR for OS, 1.07 [95% CI, 0.54-2.12]; P = .85). Among patients who did not receive chemotherapy, mMGMT status was not associated with PFS or OS.
CONCLUSIONS AND RELEVANCE
This study suggests that mMGMT is associated with response to alkylating chemotherapy for low-grade and anaplastic gliomas and may be considered as a stratification factor in future clinical trials of patients with IDH-wild-type and IDH-mutant and codeleted tumors.
Topics: Male; Humans; Adult; Prognosis; Cohort Studies; Brain Neoplasms; Methylation; Prospective Studies; Glioma; Antineoplastic Agents, Alkylating; DNA Modification Methylases; Tumor Suppressor Proteins; DNA Repair Enzymes
PubMed: 37200021
DOI: 10.1001/jamaoncol.2023.0990 -
International Journal of Molecular... May 2023Chemoresistance blunts the efficacy of temozolomide (TMZ) in the treatment of glioblastoma (GBM). Elevated levels of O6-methylguanine-DNA methyltransferase (MGMT) and...
Chemoresistance blunts the efficacy of temozolomide (TMZ) in the treatment of glioblastoma (GBM). Elevated levels of O6-methylguanine-DNA methyltransferase (MGMT) and activation of signal transducer and of transcription 3 (STAT3) have been reported to correlate with GBM resistance to alkylator chemotherapy. Resveratrol (Res) inhibits tumor growth and improves drug chemosensitivity by targeting STAT3 signaling. Whether the combined therapy of TMZ and Res could enhance chemosensitivity against GBM cells and the underlying molecular mechanism remains to be determined. In this study, Res was found to effectively improve chemosensitivities of different GBM cells to TMZ, which was evaluated by CCK-8, flow cytometry, and cell migration assay. The combined use of Res and TMZ downregulated STAT3 activity and STAT3-regulated gene products, thus inhibited cell proliferation and migration, as well as induced apoptosis, accompanied by increased levels of its negative regulators: PIAS3, SHP1, SHP2, and SOCS3. More importantly, a combination therapy of Res and TMZ reversed TMZ resistance of LN428 cells, which could be related to decreased MGMT and STAT3 levels. Furthermore, the JAK2-specific inhibitor AG490 was used to demonstrate that a reduced MGMT level was mediated by STAT3 inactivation. Taken together, Res inhibited STAT3 signaling through modulation of PIAS3, SHP1, SHP2, and SOCS3, thereby attenuating tumor growth and increasing sensitivity to TMZ. Therefore, Res is an ideal candidate to be used in TMZ combined chemotherapy for GBM.
Topics: Humans; Antineoplastic Agents, Alkylating; Brain Neoplasms; Cell Line, Tumor; DNA Modification Methylases; DNA Repair Enzymes; Drug Resistance, Neoplasm; Glioblastoma; Molecular Chaperones; Protein Inhibitors of Activated STAT; Resveratrol; STAT3 Transcription Factor; Suppressor of Cytokine Signaling Proteins; Temozolomide
PubMed: 37298405
DOI: 10.3390/ijms24119453