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Proceedings of the National Academy of... Aug 2017Alkylated DNA lesions, induced by both exogenous chemical agents and endogenous metabolites, interfere with the efficiency and accuracy of DNA replication and...
Alkylated DNA lesions, induced by both exogenous chemical agents and endogenous metabolites, interfere with the efficiency and accuracy of DNA replication and transcription. However, the molecular mechanisms of DNA alkylation-induced transcriptional stalling and mutagenesis remain unknown. In this study, we systematically investigated how RNA polymerase II (pol II) recognizes and bypasses regioisomeric -, 3-, and -ethylthymidine (-, 3-, and -EtdT) lesions. We observed distinct pol II stalling profiles for the three regioisomeric EtdT lesions. Intriguingly, pol II stalling at -EtdT and 3-EtdT sites is exacerbated by TFIIS-stimulated proofreading activity. Assessment for the impact of the EtdT lesions on individual fidelity checkpoints provided further mechanistic insights, where the transcriptional lesion bypass routes for the three EtdT lesions are controlled by distinct fidelity checkpoints. The error-free transcriptional lesion bypass route is strongly favored for the minor-groove -EtdT lesion. In contrast, a dominant error-prone route stemming from GMP misincorporation was observed for the major-groove -EtdT lesion. For the 3-EtdT lesion that disrupts base pairing, multiple transcriptional lesion bypass routes were found. Importantly, the results from the present in vitro transcriptional studies are well correlated with in vivo transcriptional mutagenesis analysis. Finally, we identified a minor-groove-sensing motif from pol II (termed Pro-Gate loop). The Pro-Gate loop faces toward the minor groove of RNA:DNA hybrid and is involved in modulating the translocation of minor-groove alkylated DNA template after nucleotide incorporation opposite the lesion. Taken together, this work provides important mechanistic insights into transcriptional stalling, lesion bypass, and mutagenesis of alkylated DNA lesions.
Topics: Alkylation; DNA; DNA Repair; DNA Replication; Humans; Mutagenesis; RNA Polymerase II; Saccharomyces cerevisiae; Transcription, Genetic
PubMed: 28784758
DOI: 10.1073/pnas.1708748114 -
Analytical Chemistry Jan 2018
Review
Topics: Alkylating Agents; Amines; Animals; Chemistry Techniques, Analytical; DNA; DNA Adducts; DNA Damage; Heterocyclic Compounds; Humans; Reactive Oxygen Species
PubMed: 29084424
DOI: 10.1021/acs.analchem.7b04247 -
JAMA Network Open Jan 2023High-grade gliomas (HGGs) constitute the most common and aggressive primary brain tumor, with 5-year survival rates of 30.9% for grade 3 gliomas and 6.6% for grade 4... (Randomized Controlled Trial)
Randomized Controlled Trial
IMPORTANCE
High-grade gliomas (HGGs) constitute the most common and aggressive primary brain tumor, with 5-year survival rates of 30.9% for grade 3 gliomas and 6.6% for grade 4 gliomas. The add-on efficacy of interferon alfa is unclear for the treatment of HGG.
OBJECTIVES
To compare the therapeutic efficacy and toxic effects of the combination of temozolomide and interferon alfa and temozolomide alone in patients with newly diagnosed HGG.
DESIGN, SETTING, AND PARTICIPANTS
This multicenter, randomized, phase 3 clinical trial enrolled 199 patients with newly diagnosed HGG from May 1, 2012, to March 30, 2016, at 15 Chinese medical centers. Follow-up was completed July 31, 2021, and data were analyzed from September 13 to November 24, 2021. Eligible patients were aged 18 to 75 years with newly diagnosed and histologically confirmed HGG and had received no prior chemotherapy, radiotherapy, or immunotherapy for their HGG.
INTERVENTIONS
All patients received standard radiotherapy concurrent with temozolomide. After a 4-week break, patients in the temozolomide with interferon alfa group received standard temozolomide combined with interferon alfa every 28 days. Patients in the temozolomide group received standard temozolomide.
MAIN OUTCOMES AND MEASURES
The primary end point was 2-year overall survival (OS). Secondary end points were 2-year progression-free survival (PFS) and treatment tolerability.
RESULTS
A total of 199 patients with HGG were enrolled, with a median follow-up time of 66.0 (95% CI, 59.1-72.9) months. Seventy-nine patients (39.7%) were women and 120 (60.3%) were men, with ages ranging from 18 to 75 years and a median age of 46.9 (95% CI, 45.3-48.7) years. The median OS of patients in the temozolomide plus interferon alfa group (26.7 [95% CI, 21.6-31.7] months) was significantly longer than that in the standard group (18.8 [95% CI, 16.9-20.7] months; hazard ratio [HR], 0.64 [95% CI, 0.47-0.88]; P = .005). Temozolomide plus interferon alfa also significantly improved median OS in patients with O6-methylguanine-DNA methyltransferase (MGMT) unmethylation (24.7 [95% CI, 20.5-28.8] months) compared with temozolomide (17.4 [95% CI, 14.1-20.7] months; HR, 0.57 [95% CI, 0.37-0.87]; P = .008). Seizure and influenzalike symptoms were more common in the temozolomide plus interferon alfa group, with 2 of 100 (2.0%) and 5 of 100 (5.0%) patients with grades 1 and 2 toxic effects, respectively (P = .02). Finally, results suggested that methylation level at the IFNAR1/2 promoter was a marker of sensitivity to temozolomide plus interferon alfa.
CONCLUSIONS AND RELEVANCE
Compared with the standard regimen, temozolomide plus interferon alfa treatment could prolong the survival time of patients with HGG, especially the MGMT promoter unmethylation variant, and the toxic effects remained tolerable.
TRIAL REGISTRATION
ClinicalTrials.gov Identifier: NCT01765088.
Topics: Female; Humans; Male; Middle Aged; Antineoplastic Agents, Alkylating; Brain Neoplasms; Dacarbazine; Glioma; Interferon-alpha; Temozolomide; Adolescent; Young Adult; Adult; Aged
PubMed: 36705923
DOI: 10.1001/jamanetworkopen.2022.53285 -
Cancer Research Feb 2017In the January 1, 2017, issue of , Nagel and colleagues demonstrate the value of assays that determine the DNA repair capacity of cancers in predicting response to... (Review)
Review
In the January 1, 2017, issue of , Nagel and colleagues demonstrate the value of assays that determine the DNA repair capacity of cancers in predicting response to temozolomide. Using a fluorescence-based multiplex flow cytometric host cell reactivation assay that provides simultaneous readout of DNA repair capacity across multiple pathways, they show that the multivariate drug response models derived from cell line data were applicable to patient-derived xenograft models of glioblastoma. In this commentary, we first outline the mechanism of activity and current clinical application of temozolomide, which, until now, has been largely limited to glioblastoma. Given the challenges of clinical application of functional assays, we argue that functional readouts be approximated by genomic signatures. In this context, a combination of MGMT activity and mismatch repair (MMR) status of the tumor are important parameters that determine sensitivity to temozolomide. More reliable methods are needed to determine MGMT activity as DNA methylation, the current standard, does not accurately reflect the expression of MGMT. Also, genomics for MMR are warranted. Furthermore, based on patterns of MGMT expression across different solid tumors, we make a case for revisiting temozolomide use in a broader spectrum of cancers based on our current understanding of its molecular basis of activity. .
Topics: Antineoplastic Agents, Alkylating; DNA Methylation; DNA Mismatch Repair; DNA Modification Methylases; DNA Repair Enzymes; Dacarbazine; Humans; Neoplasms; Precision Medicine; Temozolomide; Tumor Suppressor Proteins; Xenograft Model Antitumor Assays
PubMed: 28159862
DOI: 10.1158/0008-5472.CAN-16-2983 -
The Journal of Biological Chemistry Apr 2024DNA modifications add another layer of complexity to the eukaryotic genome to regulate gene expression, playing critical roles as epigenetic marks. In eukaryotes, the... (Review)
Review
DNA modifications add another layer of complexity to the eukaryotic genome to regulate gene expression, playing critical roles as epigenetic marks. In eukaryotes, the study of DNA epigenetic modifications has been confined to 5mC and its derivatives for decades. However, rapid developing approaches have witnessed the expansion of DNA modification reservoirs during the past several years, including the identification of 6mA, 5gmC, 4mC, and 4acC in diverse organisms. However, whether these DNA modifications function as epigenetic marks requires careful consideration. In this review, we try to present a panorama of all the DNA epigenetic modifications in eukaryotes, emphasizing recent breakthroughs in the identification of novel DNA modifications. The characterization of their roles in transcriptional regulation as potential epigenetic marks is summarized. More importantly, the pathways for generating or eliminating these DNA modifications, as well as the proteins involved are comprehensively dissected. Furthermore, we briefly discuss the potential challenges and perspectives, which should be taken into account while investigating novel DNA modifications.
Topics: Epigenesis, Genetic; Humans; Eukaryota; DNA Methylation; Animals; DNA
PubMed: 38403247
DOI: 10.1016/j.jbc.2024.106791 -
Mutation Research. Reviews in Mutation... 2016From a risk assessment perspective, DNA-reactive agents are conventionally assumed to have genotoxic risks at all exposure levels, thus applying a linear extrapolation... (Review)
Review
From a risk assessment perspective, DNA-reactive agents are conventionally assumed to have genotoxic risks at all exposure levels, thus applying a linear extrapolation for low-dose responses. New approaches discussed here, including more diverse and sensitive methods for assessing DNA damage and DNA repair, strongly support the existence of measurable regions where genotoxic responses with increasing doses are insignificant relative to control. Model monofunctional alkylating agents have in vitro and in vivo datasets amenable to determination of points of departure (PoDs) for genotoxic effects. A session at the 2013 Society of Toxicology meeting provided an opportunity to survey the progress in understanding the biological basis of empirically-observed PoDs for DNA alkylating agents. Together with the literature published since, this review discusses cellular pathways activated by endogenous and exogenous alkylation DNA damage. Cells have evolved conserved processes that monitor and counteract a spontaneous steady-state level of DNA damage. The ubiquitous network of DNA repair pathways serves as the first line of defense for clearing of the DNA damage and preventing mutation. Other biological pathways discussed here that are activated by genotoxic stress include post-translational activation of cell cycle networks and transcriptional networks for apoptosis/cell death. The interactions of various DNA repair and DNA damage response pathways provide biological bases for the observed PoD behaviors seen with genotoxic compounds. Thus, after formation of DNA adducts, the activation of cellular pathways can lead to the avoidance of a mutagenic outcome. The understanding of the cellular mechanisms acting within the low-dose region will serve to better characterize risks from exposures to DNA-reactive agents at environmentally-relevant concentrations.
Topics: Alkylating Agents; Alkylation; Apoptosis; DNA Adducts; DNA Damage; DNA Repair; Dose-Response Relationship, Drug; Humans; Mutagenicity Tests
PubMed: 27036068
DOI: 10.1016/j.mrrev.2015.11.001 -
International Journal of Molecular... Mar 2024A glioblastoma (GBM) is one of the most aggressive, infiltrative, and treatment-resistant malignancies of the central nervous system (CNS). The current standard of care... (Review)
Review
A glioblastoma (GBM) is one of the most aggressive, infiltrative, and treatment-resistant malignancies of the central nervous system (CNS). The current standard of care for GBMs include maximally safe tumor resection, followed by concurrent adjuvant radiation treatment and chemotherapy with the DNA alkylating agent temozolomide (TMZ), which was approved by the FDA in 2005 based on a marginal increase (~2 months) in overall survival (OS) levels. This treatment approach, while initially successful in containing and treating GBM, almost invariably fails to prevent tumor recurrence. In addition to the limited therapeutic benefit, TMZ also causes debilitating adverse events (AEs) that significantly impact the quality of life of GBM patients. Some of the most common AEs include hematologic (e.g., thrombocytopenia, neutropenia, anemia) and non-hematologic (e.g., nausea, vomiting, constipation, dizziness) toxicities. Recurrent GBMs are often resistant to TMZ and other DNA-damaging agents. Thus, there is an urgent need to devise strategies to potentiate TMZ activity, to overcome drug resistance, and to reduce dose-dependent AEs. Here, we analyze major mechanisms of the TMZ resistance-mediated intracellular signaling activation of DNA repair pathways and the overexpression of drug transporters. We review some of the approaches investigated to counteract these mechanisms of resistance to TMZ, including the use of chemosensitizers and drug delivery strategies to enhance tumoral drug exposure.
Topics: Humans; Temozolomide; Glioblastoma; Antineoplastic Agents, Alkylating; Quality of Life; Brain Neoplasms; Neoplasm Recurrence, Local; DNA; Drug Resistance, Neoplasm; Cell Line, Tumor
PubMed: 38542190
DOI: 10.3390/ijms25063217 -
Chemistry (Weinheim An Der Bergstrasse,... Feb 2021Hairpin pyrrole-imidazole polyamides (hPIPs) and their chlorambucil (Chb) conjugates (hPIP-Chbs) can alkylate DNA in a sequence-specific manner, and have been studied as...
Hairpin pyrrole-imidazole polyamides (hPIPs) and their chlorambucil (Chb) conjugates (hPIP-Chbs) can alkylate DNA in a sequence-specific manner, and have been studied as anticancer drugs. Here, we conjugated Chb to a cyclic PIP (cPIP), which is known to have a higher binding affinity than the corresponding hPIP, and investigated the DNA alkylation properties of the resulting cPIP-Chb using the optimized capillary electrophoresis method and conventional HPLC product analysis. cPIP-Chb conjugate 3 showed higher alkylation activity at its binding sites than did hPIP-Chb conjugates 1 and 2. Subsequent HPLC analysis revealed that the alkylation site of conjugate 3, which was identified by capillary electrophoresis, was reliable and that conjugate 3 alkylates the N3 position of adenine as do hPIP-Chbs. Moreover, conjugate 3 showed higher cytotoxicity against LNCaP prostate cancer cells than did conjugate 1 and cytotoxicity comparable to that of conjugate 2. These results suggest that cPIP-Chbs could be novel DNA alkylating anticancer drugs.
Topics: Alkylation; Chlorambucil; DNA; Imidazoles; Nylons; Pyrroles
PubMed: 33145851
DOI: 10.1002/chem.202004421 -
Acta Neuropathologica Communications Nov 2020The clinical implications of plasmatic cell-free and tumor DNA (cfDNA and ctDNA) are challenging in glioblastoma. This prospective study included 52 consecutive newly...
The clinical implications of plasmatic cell-free and tumor DNA (cfDNA and ctDNA) are challenging in glioblastoma. This prospective study included 52 consecutive newly diagnosed glioblastoma (n = 49) or gliosarcoma (n = 3) patients treated with concomitant temozolomide and radiotherapy (RT-TMZ), followed by a TMZ maintenance phase. Plasma samples were collected at baseline, before RT-TMZ (pre-RT-TMZ) and at the end of adjuvant TMZ, or at the time of progression in cases of progressive disease (PD). The cfDNA concentration was measured with a fluorometric method, and ctDNA was detected using targeted droplet digital PCR. The main objectives were to analyze the associations between cfDNA and ctDNA measurements during the course of treatment with PD and survival. There was a significant decrease in median cfDNA concentration from baseline to pre-RT-TMZ-19.4 versus 9.7 ng/mL (p < 0.0001)-in the entire cohort. In patients with PD, a significant increase in cfDNA concentration from pre-RT-TMZ to time of PD was observed, from 9.7 versus 13.1 ng/mL (p = 0.037), respectively, while no difference was observed for nonprogressive patients. Neither the cfDNA concentration at baseline nor its kinetics correlated with survival. ctDNA was detected in 2 patients (3.8%) and only in gliosarcoma subtypes.Trial registration ClinicalTrial, NCT02617745. Registered 1 December 2015, https://clinicaltrials.gov/ct2/show/NCT02617745?term=glioplak&draw=2&rank=1 .
Topics: Aged; Antineoplastic Agents, Alkylating; Brain Neoplasms; Cell-Free Nucleic Acids; Chemoradiotherapy; Circulating Tumor DNA; Female; Glioblastoma; Gliosarcoma; Humans; Male; Middle Aged; Mutation; Neurosurgical Procedures; Promoter Regions, Genetic; Telomerase; Temozolomide
PubMed: 33148330
DOI: 10.1186/s40478-020-01057-7 -
Journal of Cancer Research and Clinical... Sep 2021Angiosarcoma (AS) is a rare vasoformative sarcoma, with poor overall survival and a high need for novel treatment options. Clinically, AS consists of different subtypes,...
PURPOSE
Angiosarcoma (AS) is a rare vasoformative sarcoma, with poor overall survival and a high need for novel treatment options. Clinically, AS consists of different subtypes, including AS related to previous UV exposure (UV AS) which could indicate susceptibility to DNA damage repair inhibition. We, therefore, investigated the presence of biomarkers PARP1 (poly(ADP-ribose)polymerase-1) and Schlafen-11 (SLFN11) in UV AS. Based on experiences in other sarcomas, we examined (combination) treatment of PARP inhibitor (PARPi) olaparib and temozolomide (TMZ) in UV AS cell lines.
METHODS
Previously collected UV AS (n = 47) and non-UV AS (n = 96) patient samples and two UV AS cell lines (MO-LAS and AS-M) were immunohistochemically assessed for PARP1 and SLFN11 expression. Both cell lines were treated with single agents PARPi olaparib and TMZ, and the combination treatment. Next, cell viability and treatment synergy were analyzed. In addition, effects on apoptosis and DNA damage were examined.
RESULTS
In 46/47 UV AS samples (98%), PARP1 expression was present. SLFN11 was expressed in 80% (37/46) of cases. Olaparib and TMZ combination treatment was synergistic in both cell lines, with significantly increased apoptosis compared to single agent treatment. Furthermore, a significant increase in DNA damage marker γH2AX was present in both cell lines after combination therapy.
CONCLUSION
We showed combination treatment of olaparib with TMZ was synergistic in UV AS cell lines. Expression of PARP1 and SLFN11 was present in the majority of UV AS tumor samples. Together, these results suggest combination treatment of olaparib and TMZ is a potential novel AS subtype-specific treatment option for UV AS patients.
Topics: Antineoplastic Agents, Alkylating; Apoptosis; Cell Proliferation; Drug Combinations; Drug Evaluation, Preclinical; Drug Synergism; Hemangiosarcoma; Humans; Nuclear Proteins; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerase Inhibitors; Prognosis; Temozolomide; Tumor Cells, Cultured; Ultraviolet Rays
PubMed: 34085099
DOI: 10.1007/s00432-021-03678-4