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Trends in Pharmacological Sciences May 2019Although old molecules, alkylating agents and platinum derivatives are still widely used in the treatment of various solid tumors. However, systemic toxicity and... (Review)
Review
Although old molecules, alkylating agents and platinum derivatives are still widely used in the treatment of various solid tumors. However, systemic toxicity and cellular resistance mechanisms impede their efficacy. Innovative strategies, including local administration, optimization of treatment schedule/dosage, synergistic combinations, and the encapsulation of bioactive molecules in smart, multifunctional drug delivery systems, have shown promising results in potentiating anticancer activity while circumventing such hurdles. Furthermore, questioning of the old paradigm according to which nuclear DNA is the critical target of their anticancer activity has shed light on subcellular alternative and neglected targets that obviously participate in the mediation of cytotoxicity or resistance. Thus, rethinking of the use of these pivotal antineoplastic agents appears critical to improve clinical outcomes in the management of solid tumors.
Topics: Antineoplastic Agents, Alkylating; Drug Delivery Systems; Drug Synergism; Humans; Neoplasms
PubMed: 30979523
DOI: 10.1016/j.tips.2019.03.003 -
Biomedical and Environmental Sciences :... May 2022To investigate the function of primary cilia in regulating the cellular response to temozolomide (TMZ) and ionizing radiation (IR) in glioblastoma (GBM).
OBJECTIVE
To investigate the function of primary cilia in regulating the cellular response to temozolomide (TMZ) and ionizing radiation (IR) in glioblastoma (GBM).
METHODS
GBM cells were treated with TMZ or X-ray/carbon ion. The primary cilia were examined by immunostaining with Arl13b and γ-tubulin, and the cellular resistance ability was measured by cell viability assay or survival fraction assay. Combining with cilia ablation by IFT88 depletion or chloral hydrate and induction by lithium chloride, the autophagy was measured by acridine orange staining assay. The DNA damage repair ability was estimated by the kinetic curve of γH2AX foci, and the DNA-dependent protein kinase (DNA-PK) activation was detected by immunostaining assay.
RESULTS
Primary cilia were frequently preserved in GBM, and the induction of ciliogenesis decreased cell proliferation. TMZ and IR promoted ciliogenesis in dose- and time-dependent manners, and the suppression of ciliogenesis significantly enhanced the cellular sensitivity to TMZ and IR. The inhibition of ciliogenesis elevated the lethal effects of TMZ and IR the impairment of autophagy and DNA damage repair. The interference of ciliogenesis reduced DNA-PK activation, and the knockdown of DNA-PK led to cilium formation and elongation.
CONCLUSION
Primary cilia play a vital role in regulating the cellular sensitivity to TMZ and IR in GBM cells through mediating autophagy and DNA damage repair.
Topics: Antineoplastic Agents, Alkylating; Brain Neoplasms; Cell Line, Tumor; DNA; Glioblastoma; Humans; Radiation, Ionizing; Temozolomide
PubMed: 35676813
DOI: 10.3967/bes2022.058 -
Nucleic Acids Research Jul 2019Higher-ordered structure motifs of nucleic acids, such as the G-quadruplex (G-4), mismatched and bulge structures, are significant research targets because these...
Higher-ordered structure motifs of nucleic acids, such as the G-quadruplex (G-4), mismatched and bulge structures, are significant research targets because these structures are involved in genetic control and diseases. Selective alkylation of these higher-order structures is challenging due to the chemical instability of the alkylating agent and side-reactions with the single- or double-strand DNA and RNA. We now report the reactive OFF-ON type alkylating agents, vinyl-quinazolinone (VQ) precursors with a sulfoxide, thiophenyl or thiomethyl group for the OFF-ON control of the vinyl reactivity. The stable VQ precursors conjugated with aminoacridine, which bind to the G-4 DNA, selectively reacted with a T base on the G-4 DNA in contrast to the single- and double-strand DNA. Additionally, the VQ precursor reacted with the T or U base in the AP-site, G-4 RNA and T-T mismatch structures. These VQ precursors would be a new candidate for the T or U specific alkylation in the higher-ordered structures of nucleic acids.
Topics: Alkylating Agents; Alkylation; Base Pairing; DNA; DNA, Single-Stranded; G-Quadruplexes; Molecular Structure; Nuclear Magnetic Resonance, Biomolecular; Nucleic Acid Conformation; Purines; Spectrometry, Mass, Electrospray Ionization; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Structure-Activity Relationship; Triazines; Vinyl Compounds
PubMed: 31188442
DOI: 10.1093/nar/gkz512 -
Cellular and Molecular Life Sciences :... Mar 2009The genomic integrity of all living organisms is constantly jeopardized by physical [e.g. ultraviolet (UV) light, ionizing radiation] and chemical (e.g. environmental... (Review)
Review
The genomic integrity of all living organisms is constantly jeopardized by physical [e.g. ultraviolet (UV) light, ionizing radiation] and chemical (e.g. environmental pollutants, endogenously produced reactive metabolites) agents that damage the DNA. To overcome the deleterious effects of DNA lesions, nature evolved a number of complex multi-protein repair processes with broad, partially overlapping substrate specificity. In marked contrast, cells may use very simple repair systems, referred to as direct DNA damage reversal, that rely on a single protein, remove lesions in a basically error-free manner, show high substrate specificity, and do not involve incision of the sugar-phosphate backbone or base excision. This concise review deals with two types of direct DNA damage reversal: (i) the repair of alkylating damage by alkyltransferases and dioxygenases, and (ii) the repair of UV-induced damage by spore photoproduct lyases and photolyases. (Part of a Multi-author Review).
Topics: Alkyl and Aryl Transferases; Alkylating Agents; Animals; Bacterial Proteins; DNA Damage; DNA Repair; Deoxyribodipyrimidine Photo-Lyase; Dioxygenases; Models, Molecular; Phylogeny; Ultraviolet Rays
PubMed: 19153659
DOI: 10.1007/s00018-009-8735-0 -
Chemical Research in Toxicology Feb 2016N,N-Bis-(2-chloroethyl)-phosphorodiamidic acid (phosphoramide mustard, PM) and N,N-bis-(2-chloroethyl)-amine (nornitrogen mustard, NOR) are the two biologically active...
N,N-Bis-(2-chloroethyl)-phosphorodiamidic acid (phosphoramide mustard, PM) and N,N-bis-(2-chloroethyl)-amine (nornitrogen mustard, NOR) are the two biologically active metabolites of cyclophosphamide, a DNA alkylating drug commonly used to treat lymphomas, breast cancer, certain brain cancers, and autoimmune diseases. PM and NOR are reactive bis-electrophiles capable of cross-linking cellular biomolecules to form covalent DNA-DNA and DNA-protein cross-links (DPCs). In the present work, a mass spectrometry-based proteomics approach was employed to characterize PM- and NOR-mediated DNA-protein cross-linking in human cells. Following treatment of human fibrosarcoma cells (HT1080) with cytotoxic concentrations of PM, over 130 proteins were found to be covalently trapped to DNA, including those involved in transcriptional regulation, RNA splicing/processing, chromatin organization, and protein transport. HPLC-ESI(+)-MS/MS analysis of proteolytic digests of DPC-containing DNA from NOR-treated cells revealed a concentration-dependent formation of N-[2-[cysteinyl]ethyl]-N-[2-(guan-7-yl)ethyl]amine (Cys-NOR-N7G) conjugates, confirming that it cross-links cysteine thiols of proteins to the N7 position of guanines in DNA. Cys-NOR-N7G adduct numbers were higher in NER-deficient xeroderma pigmentosum cells (XPA) as compared with repair proficient cells. Furthermore, both XPA and FANCD2 deficient cells were sensitized to PM treatment as compared to that of wild type cells, suggesting that Fanconi anemia and nucleotide excision repair pathways are involved in the removal of cyclophosphamide-induced DNA damage.
Topics: Alkylating Agents; Cell Line, Tumor; Chromatography, High Pressure Liquid; DNA; DNA Adducts; Humans; Nitrogen Mustard Compounds; Peptides; Phosphoramide Mustards; Proteins; Proteomics; Spectrometry, Mass, Electrospray Ionization
PubMed: 26692166
DOI: 10.1021/acs.chemrestox.5b00430 -
Environmental Health Perspectives Oct 1993Alkylpurines are liberated from alkylated DNA by glycosylase repair enzymes and, in most cases, excreted in urine without further metabolism. This phenomenon forms the...
Alkylpurines are liberated from alkylated DNA by glycosylase repair enzymes and, in most cases, excreted in urine without further metabolism. This phenomenon forms the basis of noninvasive methods to measure DNA alkylation in vivo. In the case of methyl adducts, such as 7-methylguanine (7-MeGua), natural backgrounds exist due to RNA turnover. However, deuterated (d3) methylating agents or precursors give rise to d3-7-MeGua and d3-3-methyladenine (3-MeAde), which can be readily quantitated using gas chromatography-mass spectrometry (GC-MS). A deuterated probe drug, such as d6-aminopyrine, can be used to measure endogenous nitrosation levels in experimental animals. In contrast, for higher alkyl homologues of alkylpurines, natural backgrounds are low or nonexistent and can be directly measured by GC-MS using stable isotope labeled internal standards. For example, increased levels of urinary 3-ethyladenine were observed in cigarette smokers. Due to recent advances in analytical methodology, notably immunoaffinity cleanup of urine, measurements of excreted DNA adducts can be used in studies in human populations exposed to low levels of alkylating carcinogens.
Topics: Adenine Nucleotides; Alkylation; Animals; DNA; Genetic Markers; Humans; Methylation
PubMed: 8143609
DOI: 10.1289/ehp.93101s3151 -
Molecules and Cells Dec 1998A variety of alkylated base adducts are repaired by 3-methyladenine DNA glycosylases, one of the base excision repair enzymes. In this study, we examined the DNA adducts...
A variety of alkylated base adducts are repaired by 3-methyladenine DNA glycosylases, one of the base excision repair enzymes. In this study, we examined the DNA adducts induced by hepsulfam and determined whether alkylated base adducts can be substrates for bacterial and mammalian 3-methyladenine DNA glycosylases by electrophoresis methods. Hepsulfam, a synthetic analogue of busulfan, is known to alkylate DNA and form interstrand cross-links. The extent of DNA interstrand cross-links induced by hepsulfam and busulfan was found to be similar but significantly lower than that induced by chlorambucil, as measured by an agarose gel assay. The major monofunctional alkylation site of hepsulfam was observed at the N7 position of guanine, and not at the N3 position of adenine. Both compounds did not exhibit any sequence selective DNA alkylation patterns. The excision of hepsulfam-induced DNA adducts has been determined by treatment with homogeneous recombinant bacterial, rat and human 3-methyladenine DNA glycosylases and successive treatments by formamidopyrimidine-DNA glycosylase. The Escherichia coli alkA protein was shown to completely excise N7 guanine adducts, whereas mammalian 3-methyladenine DNA glycosylase failed to excise them. In addition, the cytotoxicity assay showed that E. coli mutant strains defective in the alkA gene or the uvrA gene were more sensitive to killing by hepsulfam than the wild type.
Topics: Alkylation; Animals; Antineoplastic Agents; Antineoplastic Agents, Alkylating; Binding Sites; Busulfan; Cross-Linking Reagents; DNA; DNA Adducts; DNA Glycosylases; DNA Repair; DNA, Bacterial; Escherichia coli; Humans; N-Glycosyl Hydrolases; Plasmids; Rats; Sulfonic Acids
PubMed: 9895121
DOI: No ID Found -
International Journal of Molecular... Mar 2023Glioblastoma is the most common malignant brain tumor in adults. Standard treatment includes tumor resection, radio-chemotherapy and adjuvant chemotherapy with...
Glioblastoma is the most common malignant brain tumor in adults. Standard treatment includes tumor resection, radio-chemotherapy and adjuvant chemotherapy with temozolomide (TMZ). TMZ methylates DNA, whereas O6-methylguanine DNA methyltransferase (MGMT) counteracts TMZ effects by removing the intended proteasomal degradation signal. Non-functional MGMT mediates the mismatch repair (MMR) system, leading to apoptosis after futile repair attempts. This study investigated the associations between promoter methylation, MGMT and MMR protein expression, and their effect on overall survival (OS) and progression-free survival (PFS) in patients with glioblastoma. promoter methylation was assessed in 42 treatment-naïve patients with glioblastoma WHO grade IV by pyrosequencing. MGMT and MMR protein expression was analyzed using immunohistochemistry. promoter methylation was present in 52%, whereas patients <70 years of age revealed a significantly longer OS using a log-rank test and a significance threshold of ≤ 0.05. MGMT protein expression and methylation status showed no correlation. MMR protein expression was present in all patients independent of MGMT status and did not influence OS and PFS. Overall, promoter methylation implicates an improved OS in patients with glioblastoma aged <70 years. In the elderly, the extent of surgery has an impact on OS rather than the promoter methylation or protein expression.
Topics: Adult; Aged; Humans; Temozolomide; Glioblastoma; Progression-Free Survival; Antineoplastic Agents, Alkylating; Dacarbazine; Methylation; DNA Mismatch Repair; DNA Modification Methylases; Brain Neoplasms; O(6)-Methylguanine-DNA Methyltransferase; DNA Repair Enzymes; DNA Methylation; Tumor Suppressor Proteins
PubMed: 37047153
DOI: 10.3390/ijms24076184 -
Molecules (Basel, Switzerland) Oct 2019A wide range of endogenous and exogenous alkylating agents attack DNA to generate various alkylation adducts. N7-methyl-2-deoxyguanosine (Fm7dG) is the most abundant...
A wide range of endogenous and exogenous alkylating agents attack DNA to generate various alkylation adducts. N7-methyl-2-deoxyguanosine (Fm7dG) is the most abundant alkylative DNA lesion. If not repaired, Fm7dG can undergo spontaneous depurination, imidazole ring-opening, or bypass by translesion synthesis DNA polymerases. Human DNA polymerase η (polη) efficiently catalyzes across Fm7dG in vitro, but its structural basis is unknown. Herein, we report a crystal structure of polη in complex with templating Fm7dG and an incoming nonhydrolyzable dCTP analog, where a 2'-fluorine-mediated transition destabilization approach was used to prevent the spontaneous depurination of Fm7dG. The structure showed that polη readily accommodated the Fm7dG:dCTP base pair with little conformational change of protein and DNA. In the catalytic site, Fm7dG and dCTP formed three hydrogen bonds with a Watson-Crick geometry, indicating that the major keto tautomer of Fm7dG is involved in base pairing. The polη-Fm7dG:dCTP structure was essentially identical to the corresponding undamaged structure, which explained the efficient bypass of the major methylated lesion. Overall, the first structure of translesion synthesis DNA polymerase bypassing Fm7dG suggests that in the catalytic site of Y-family DNA polymerases, small N7-alkylguanine adducts may be well tolerated and form the canonical Watson-Crick base pair with dCTP through their keto tautomers.
Topics: Alkylation; Base Pairing; Catalytic Domain; DNA; DNA Damage; DNA-Directed DNA Polymerase; Deoxycytosine Nucleotides; Deoxyguanosine; Humans; Kinetics; Metals; Models, Molecular; Nucleic Acid Conformation
PubMed: 31683505
DOI: 10.3390/molecules24213928 -
International Journal of Molecular... Jan 2021Sulfur mustard (SM) is a chemical warfare agent that can damage DNA via alkylation and oxidative stress. Because of its genotoxicity, SM is cancerogenic and the...
Sulfur mustard (SM) is a chemical warfare agent that can damage DNA via alkylation and oxidative stress. Because of its genotoxicity, SM is cancerogenic and the progenitor of many chemotherapeutics. Previously, we developed an SM-resistant cell line via chronic exposure of the popular keratinocyte cell line HaCaT to increasing doses of SM over a period of 40 months. In this study, we compared the genomic landscape of the SM-resistant cell line HaCaT/SM to its sensitive parental line HaCaT in order to gain insights into genetic changes associated with continuous alkylation and oxidative stress. We established chromosome numbers by cytogenetics, analyzed DNA copy number changes by means of array Comparative Genomic Hybridization (array CGH), employed the genome-wide chromosome conformation capture technique Hi-C to detect chromosomal translocations, and derived mutational signatures by whole-genome sequencing. We observed that chronic SM exposure eliminated the initially prevailing hypotetraploid cell population in favor of a hyperdiploid one, which contrasts with previous observations that link polyploidization to increased tolerance and adaptability toward genotoxic stress. Furthermore, we observed an accumulation of chromosomal translocations, frequently flanked by DNA copy number changes, which indicates a high rate of DNA double-strand breaks and their misrepair. HaCaT/SM-specific single-nucleotide variants showed enrichment of C > A and T > A transversions and a lower rate of deaminated cytosines in the CpG dinucleotide context. Given the frequent use of HaCaT in toxicology, this study provides a valuable data source with respect to the original genotype of HaCaT and the mutational signatures associated with chronic alkylation and oxidative stress.
Topics: Alkylating Agents; Cell Line; Chromosome Aberrations; Comparative Genomic Hybridization; DNA; DNA Adducts; DNA Breaks, Double-Stranded; DNA Damage; Humans; Keratinocytes; Mustard Gas; Mutation; Oxidative Stress; Radiation, Ionizing
PubMed: 33498964
DOI: 10.3390/ijms22031146