A fluorimetric assay for the spontaneous release of an N7-alkylguanine residue from duplex DNA

Shipova, Ekaterina V.; Gates, Kent S.

doi:10.1016/j.bmcl.2005.02.058

Cited by 19 publications

(21 citation statements)

References 39 publications

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“…As shown in Figure 2C, about a three-fold increase in the FITC fluorescence signal was observed in MiaPaCa cells exposed to 100 ng/mL leinamycin for 2 h compared to the untreated cells. Overall, these data are consistent with the recent in vitro studies showing that the leinamycin-guanine adduct in double-stranded DNA undergoes unusually rapid depurination to generate AP sites in duplex DNA (19–21). …”

Section: Resultssupporting

confidence: 91%

“…In a previous study, the kinetics of abasic site formation in methyl methanesulfonate (MMS)-treated HeLa cells were investigated using the ARP, revealing that the amount of AP sites in MMS-treated cells increases up to 3 h, and then disappears with a half-life of about 20 h, which is quite different from what we observed for leinamycin (63). The relatively rapid formation and disappearance of leinamycin-derived AP sites may be due to several factors, including rapid DNA alkylation by activated leinamycin (14,17,18), rapid spontaneous depurination of the leinamycin-guanine adduct (19–21) and, finally, rapid removal of the AP site by enzymatic repair processes (64, 65). …”

Section: Discussionmentioning

confidence: 99%

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Characterization of DNA Damage Induced by a Natural Product Antitumor Antibiotic Leinamycin in Human Cancer Cells

Viswesh

Gates

Sun

2009

Chem. Res. Toxicol.

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Leinamycin is a structurally novel Streptomyces-derived natural product that displays very potent activity against various human cancer cell lines (IC 50 values in the low nanomolar range). Previous in vitro biochemical studies have revealed that leinamycin alkylates DNA, generates apurinic (AP) sites and reactive oxygen species (ROS), and causes DNA strand breaks. However, it is not clear whether these events occur inside cells. In the present study, we have determined the endogenous amount of AP sites and DNA strand breaks in genomic DNA and the amount of oxidative stress in a human pancreatic carcinoma cell line, MiaPaCa, treated with leinamycin by utilizing the aldehydereactive probe (ARP) assay, the comet assay, and fluorescent probes, respectively. We demonstrated that AP sites are formed rapidly following exposure to leinamycin, and the number of AP sites was increased up to seven-fold in a dose dependent manner. However, only 25-50% of these sites remain 2 h after media containing drug molecules was aspirated and replaced with fresh media. We also observed leinamycin induced ROS generation and a concomitant increase in apoptosis of MiaPaCa cells. Because both AP sites and ROS have the potential to generate strand breaks in cellular DNA, the comet assay was utilized to detect damage to nuclear DNA in leinamycin-treated MiaPaCa cell cultures. Both alkaline and neutral electrophoretic analysis revealed that leinamycin produces both single-and double-stranded DNA damage in drug-treated cells in a dose-dependent manner. Taken together, the results suggest that rapid conversion of leinamycin-guanine (N7) adducts into AP sites to produce DNA strand breaks, in synergy with leinamycin-derived ROS, account for the exceedingly potent biological activity of this natural product.

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Section: Resultssupporting

confidence: 91%

Section: Discussionmentioning

confidence: 99%

Characterization of DNA Damage Induced by a Natural Product Antitumor Antibiotic Leinamycin in Human Cancer Cells

Viswesh

Gates

Sun

2009

Chem. Res. Toxicol.

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“…Both adducts have rapid rates of spontaneous depurination as alkylation of purines destabilizes the N -glycosidic bond and renders these bases more susceptible to hydrolysis with half-lives of approximately 30 h and 70 h, respectively, for N3-MeA and N7-MeG at 39°C [72]. In fact, spontaneous depurination is the major fate of N7-alkyl adducts [48, 73, 74]. The resulting abasic/apurinic sites can be processed by a number of other repair pathways, including HR, NER, or translesion DNA synthesis (TLS), a DNA tolerance pathway [75-77].…”

Section: Alkylating Agentsmentioning

confidence: 99%

Contributions of DNA repair and damage response pathways to the non-linear genotoxic responses of alkylating agents

Klapacz

Pottenger

Engelward

et al. 2016

Mutation Research/Reviews in Mutation Research

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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 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.

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“…6,10 The resulting 7-alkylguanine residues undergo rapid depurination to generate a burst of cytotoxic abasic sites in duplex DNA. [11][12][13] The reaction with thiols may be central to the potent biological activity of leinamycin because cells contain millimolar concentrations of the thiol-containing tripeptide glutathione 14 that can trigger the release of cell killing reactive intermediates from this natural product.In light of the central role played by leinamycin's sulfoxide oxygen in the generation of cytotoxic reactive intermediates (Scheme 1), it is not surprising that S-deoxyleinamycin (5) is markedly less cytotoxic than leinamycin. For example, 5 displays an IC 50 value of 4 μM against HeLa S3 cells, whereas that for leinamycin is 27 nM.…”

mentioning

confidence: 99%

mentioning

confidence: 99%

Possible chemical mechanisms underlying the antitumor activity of S-deoxyleinamycin

Sivaramakrishnan

Gates

2008

Bioorganic & Medicinal Chemistry Letters

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Though less potent than the parent natural product leinamycin, S-deoxyleinamycin displays activity against human cancer cell lines that is comparable to many clinically-used agents. The results reported here suggest that the 1,2-dithiolan-3-one heterocycle found in S-deoxyleinamycin reacts with thiols to generate a persulfide intermediate (RSS − ) that could deliver biologically active polysulfides, hydrogen sulfide, and reactive oxygen species (O 2 • − , H 2 O 2 , and HO•) to the interior of cells.Leinamycin (1) is a structurally interesting natural product that displays impressive, nanomolar IC 50 -values against human cancer cell lines. 1-5 Reaction of thiols with leinamycin leads to ejection of a persulfide intermediate (2) that generates cell-killing reactive oxygen species (Scheme 1). [6][7][8][9] In addition, the 1,2-oxathiolan-5-one derivative (3) formed in this reaction undergoes further rearrangement to an episulfonium ion (4) that efficiently alkylates guanine residues in duplex DNA. 6,10 The resulting 7-alkylguanine residues undergo rapid depurination to generate a burst of cytotoxic abasic sites in duplex DNA. [11][12][13] The reaction with thiols may be central to the potent biological activity of leinamycin because cells contain millimolar concentrations of the thiol-containing tripeptide glutathione 14 that can trigger the release of cell killing reactive intermediates from this natural product.In light of the central role played by leinamycin's sulfoxide oxygen in the generation of cytotoxic reactive intermediates (Scheme 1), it is not surprising that S-deoxyleinamycin (5) is markedly less cytotoxic than leinamycin. For example, 5 displays an IC 50 value of 4 μM against HeLa S3 cells, whereas that for leinamycin is 27 nM. 3 Despite its diminished cytotoxicity relative to leinamycin, it is important to note that the activity of 5 is comparable to some clinically-used anticancer drugs. 15 Accordingly, studies of 5 have the potential to reveal new chemical processes by which organic molecules can elicit anticancer activity.Previous work has shown that 5 does not cause thiol-triggered DNA alkylation; 3 however, our previous experience with related sulfur heterocycles [16][17][18][19] led us to consider the possibility that reactions of thiols with the 1,2-dithiolan-3-one heterocycle in 5 might lead to the generation of superoxide radical (O 2 • − ). Under physiological conditions, superoxide radical decomposes to hydrogen peroxide and, ultimately, hydroxyl radical as shown in the (unbalanced) Eqns 4 -5, where M represents a transition metal such as iron or copper. 20 Intracellular production of *Corresponding author. Tel.: +1-573-882-6763; fax: +1-573-882-2754, e-mail gatesk@missouri.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in ...

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A fluorimetric assay for the spontaneous release of an N7-alkylguanine residue from duplex DNA

Cited by 19 publications

References 39 publications

Characterization of DNA Damage Induced by a Natural Product Antitumor Antibiotic Leinamycin in Human Cancer Cells

Characterization of DNA Damage Induced by a Natural Product Antitumor Antibiotic Leinamycin in Human Cancer Cells

Contributions of DNA repair and damage response pathways to the non-linear genotoxic responses of alkylating agents

Possible chemical mechanisms underlying the antitumor activity of S-deoxyleinamycin

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