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For the improvement of chemotherapy with platinum (Pt)-containing drugs a sensitive assay to detect the induced Pt-DNA adducts is needed. Therefore, the 32P-postlabelling assay, described by Blommaert and Saris (Nucleic Acids Res., 1995, 23, 1300-1306), to detect the major adducts Pt-GG and Pt-AG has substantially been improved and compared with EL...
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... incubated with various concentrations of cisplatin, was analyzed by postlabelling and another part by AAS. As shown in Figure 6, highly significant correlations were observed between the total Pt-DNA content (AAS data) and the post- labelling results of the Pt-AG and Pt-GG adducts with R-values of 0.99 (P 0.012) and 1.0 (P 0.003), respectively. The total adduct level measured by postlabelling, i.e. ...
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... tried to dansylate the hydroxyl groups amounts of the dinucleotides as measured by weight (input). The linearity of the method could be confirmed by analysis of in the deoxyribose moieties under non-aqueous conditions (pyridine) analogous to the binding of p-toluenesulphonyl DNA samples isolated from human IGROV-1 ovarian cancer cells treated with various concentrations of cisplatin ( Figure 6). chloride to carbohydrates. ...
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... very good and highly significant correlation was found between the levels of the two adducts in the cellular the quantification of the 32 P-labelled products two different methods have been elaborated (see Scheme I for the outline DNA samples as determined by postlabelling (with HPLC separation) and the total platination levels of the original DNA of the whole postlabelling procedure). As the first step in this assay, (isolated) platinated DNA was digested to unmodified samples measured with AAS (see Figure 6). The total amount of the two adducts measured by the postlabelling assay, as deoxynucleosides and platinated adducts. ...
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... total amount of the two adducts measured by the postlabelling assay, as deoxynucleosides and platinated adducts. For the dephos- phorylation by the enzyme alkaline phosphatase a pH of ~9 shown in Figure 6 (dashed line), was found to be ~85% of the overall platination level of the DNA samples, which is in is required, which is obtained by addition of only 1/100 volume of 1 M Tris/HCl (pH 9.0) to the digestion mixture instead of agreement with previous findings (2,26). The same holds, with a few exceptions, for the observed ratio of 3-4 between the the 1/10 volume as reported before (1). ...
Citations
... The cytotoxic efficacy for each of the different types of DNA adduct and the relationship between adduct levels in tumour cells and blood is not well understood. Intrastrand-crosslink concentrations in tumour biopsies taken from patients treated with cisplatin (70 mg m −2 ) and measured using 32 P post-labelling are reported [17] to be approximately 93, 1,2-GG and 30, 1,2-AG, cisplatin adducts per 10 7 nucleotides. This reflects adduct levels in white blood cells of approximately 14-22 and 5-8 adducts per 10 7 nucleotides, for 1,2-GG and 1,2-AG, respectively. ...
... Welters et al. [157] and Pluim et al. [158] improved the method substantially by the inclusion of an internal dinucleotide standard, improved deplatination procedures and used a more efficient chromatography. The method was recently applied to normal tissue (white blood cells and buccal cells) and tumor tissue of patients undergoing platinum-based chemotherapy [134]. ...
... Method involves digestion to nucleotides of DNA containing cisplatin-or carboplatin-induced adducts, followed by chromatographic separation of adducted nucleotides, deplatination, enzymatic labeling with [γ-32 P]-ATP and thin-layer chromatography with quantification by autoradiography LOD = 0.3 fmol per µg of DNA, approximately one adduct per 10 7 nt from 10 µg of DNA [156] Added an internal standard and improved adduct isolation and recovery. Demonstrated method on cultured cells and patient tissues 1.6 adducts per 10 7 nt using 10 µg of DNA [157] Introduced online HPLC fractionation of adducts and optimized further the method reported in. Method was applied to normal and tumor tissues LOQ for platinum-GG and platinum-AG adducts of 0.087 and 0.53 fmol per µg of DNA, respectively [134,147,158] ...
The personalized medicine revolution is occurring for cancer chemotherapy. Biomarkers are increasingly capable of distinguishing genotypic or phenotypic traits of individual tumors, and are being linked to the selection of treatment protocols. This review covers the molecular basis for biomarkers of response to targeted and cytotoxic lung and bladder cancer treatment with an emphasis on platinum-based chemotherapy. Platinum derivatives are a class of drugs commonly employed against solid tumors that kill cells by covalent attachment to DNA. Platinum-DNA adduct levels in patient tissues have been correlated to response and survival. The sensitivity and precision of adduct detection has increased to the point of enabling subtherapeutic dosing for diagnostics applications, termed diagnostic microdosing, prior to the initiation of full-dose therapy. The clinical status of this unique phenotypic marker for lung and bladder cancer applications is detailed along with discussion of future applications.
... This figure is lower than reported values of 1.9-3.0 Pt-DNA adduct per 10 6 nucleotides determined by the post-labelling assay [79] in white blood cells of patients after cisplatin chemotherapy. It is clear from Figs. 1 and 2 that the method employed here allows for the measurement of in vivo Pt-DNA adducts formed due to the administration of a variety of Pt-drugs at clinical concentrations. ...
This paper describes methodologies, based on sector field inductively-coupled plasma mass spectrometry (SF-ICP-MS), and their application in the holistic study of the fate of Pt in human cell populations following treatment with cis- or oxaliplatin and combination treatments. Pt–DNA adduct formation data at several time points has been determined in the leukocytes from patients undergoing Pt-based chemotherapy demonstrating significant inter-patient variability and excellent reproducibility of the assay. The sensitivity of the technique enabled quantitation of as little as 0.2 Pt adducts per 106 nucleotides using 10 μg of patient DNA. Further, the first ever reported in vivo sub-cellular Pt fractionation data on a patient sample is presented indicating the feasibility of applying the methods presented here in a clinical environment. For in vitro studies, three cell models were used: A549 human lung adenocarcinoma epithelial cells were exposed to 50 μM cisplatin for 1 h; HCA7 human colorectal cancer cells were treated with either FOLFOX (200 μM 5-fluorouracil, 200 μM folinic acid and 50 μM oxaliplatin) or 50 μM oxaliplatin; and HT29 human colorectal cancer cells were treated with 50 μM oxaliplatin in combination with 20 μM methaneseleninic acid, CH3SeO2H (MSA). The cells were harvested and either the DNA extracted and/or a commercially available kit used to fractionate the treated cells into four sub-cellular compartments. Each of the sub-cellular fractions and extracted DNA were digested separately, evaporated to dryness and reconstituted in 2% nitric acid for analysis by SF-ICP-MS. The sub-cellular Pt distribution for cisplatin treated A549 cells was shown to be as follows: ∼70% localized in the cytosol, ∼17% in the membrane and membrane localized fraction, ∼9% in the nuclear fraction and ∼4% in the cytoskeletal fraction. Both FOLFOX and oxaliplatin treated HCA7 cells showed comparable sub-cellular Pt distributions, and Pt–DNA adduct formation was similar for the oxaliplatin and FOLFOX treatments with adduct yields of 5.6 and 5.5 adducts per 106 nucleotides respectively. It was found that the combination of oxaliplatin with 20 μM MSA did not change the distribution of Pt or significantly alter its accumulation in the cytosol of the HT29 cells. Mass balance experiments showed a >99% recovery of the total Pt in the sub-cellular fractions. These experiments are the first to provide such a detailed quantitation of Pt-drug partitioning and they show that the Pt broadly follows the total protein content of the individual compartments with the majority being scavenged in the cytosol compartment.Graphical abstractView high quality image (197K)Research highlights▶ Partitioning of Pt-based drugs in cells of patients undergoing Pt-based chemotherapy. ▶ New data on Pt -DNA adduct formation and persistence over extended treatment cycles. ▶ Variability in patient response and evidence of adduct repair. ▶ The effect of combination therapy, such as the FOLFOX regimen. ▶ Effect of Se supplementation, and cell partitioning of drug.
... Currently, one of the most sensitive techniques for DNA adduct detection is the 32 P postlabeling assay, which has measurement sensitivity of one adduct in 10 7 -10 8 nucleotides for detecting Pt-DNA crosslinks, 17,18 about tenfold more sensitive than ELISA-based quantitative assays. [19][20][21] However, neither the postlabeling nor ELISA-based assays are useful for quantitating carboplatin-DNA monoadducts. ...
Formation and repair of platinum (Pt)-induced DNA adducts is a critical step in Pt drug-mediated cytotoxicity. Measurement of Pt-DNA adduct kinetics in tumors may be useful for better understanding chemoresistance and therapeutic response. However, this concept has yet to be rigorously tested because of technical challenges in measuring the adducts at low concentrations and consistent access to sufficient tumor biopsy material. Ultrasensitive accelerator mass spectrometry was used to detect [(14)C]carboplatin-DNA monoadducts at the attomole level, which are the precursors to Pt-DNA crosslink formation, in six cancer cell lines as a proof-of-concept. The most resistant cells had the lowest monoadduct levels at all time points over 24 hr. [(14)C]Carboplatin "microdoses" (1/100th the pharmacologically effective concentration) had nearly identical adduct formation and repair kinetics compared to therapeutically relevant doses, suggesting that the microdosing approach can potentially be used to determine the pharmacological effects of therapeutic treatment. Some of the possible chemoresistance mechanisms were also studied, such as drug uptake/efflux, intracellular inactivation and DNA repair in selected cell lines. Intracellular inactivation and efficient DNA repair each contributed significantly to the suppression of DNA monoadduct formation in the most resistant cell line compared to the most sensitive cell line studied (p < 0.001). Nucleotide excision repair (NER)-deficient and -proficient cells showed substantial differences in carboplatin monoadduct concentrations over 24 hr that likely contributed to chemoresistance. The data support the utility of carboplatin microdosing as a translatable approach for defining carboplatin-DNA monoadduct formation and repair, possibly by NER, which may be useful for characterizing chemoresistance in vivo.
... 13 The Comet assay, however, is considered qualitative as it does not directly provide information on the number of Pt adducts. In comparison, the post labelling assay (PLA) 14 provides a very sensitive measure of Pt adduct formation and can be used with relatively small DNA samples; however, it is labour intensive and requires the use of radioactive reagents. Structural characterisation using NMR 15 and crystallography 16 have also been employed for studying the conformation of oxaliplatin-DNA adducts, however such techniques require relatively large samples. ...
This paper describes a set of fast and selective high performance liquid chromatography (HPLC) methods coupled to electro-spray ionisation linear ion trap mass spectrometry (ESI-MS), sector-field inductively coupled plasma mass spectrometry (SF-ICP-MS) and UV detection for in vitro studies of the bifunctional adducts of oxaliplatin with mono-nucleotides, di-nucleotides and cellular DNA. The stationary phases and the optimised conditions used for each separation are discussed. Interaction of oxaliplatin with A and G mono-nucleotides resulted in the formation of five bifunctional platinum diaminocyclohexane (DACHPt) adducts. These were two isomers of the A-DACHPt-A and A-DACHPt-G adducts, and one G-DACHPt-G adduct, as confirmed by MS/MS spectra obtained by collision induced dissociation. These adducts were also characterised by UV absorption data and SF-ICP-MS elemental (195)Pt and (31)P signals. Further, interaction of oxaliplatin with AG and GG di-nucleotides resulted in the formation of three adducts: DACHPt-GG and two isomers of the DACHPt-AG adduct, as confirmed by ESI-MS and the complementary data obtained by UV and SF-ICP-MS. Finally, a very sensitive LC-ICP-MS method for the quantification of oxaliplatin GG intra-strand adducts (DACHPt-GG) was developed and used for monitoring the in vitro formation and repair of these adducts in human colorectal cancer cells. The method detection limit was 0.14 ppb Pt which was equivalent to 0.22 Pt adduct per 10(6) nucleotides based on a 10 μg DNA sample. This detection limit makes this method suitable for in vivo assessment of DACHPt-GG adducts in patients undergoing oxaliplatin chemotherapy.
... However, the overall recovery is only 30%, and reproducible and accurate quantitative analysis is impossible due to the absence of an internal standard. Further studies by Welters et al. [42] involved the addition of an internal standard [thymidylyl (3 0 -5 0 ) thymidine, TdT] for the labeling reaction, a more efficient isolation of the Pt adducts on the cation-exchanger and a simplification of the deplatination step. This improved method was found to be quite suitable to determine adduct levels in in vitro cisplatin-treated DNA, DNA isolated from cisplatin-treated cultured cells and DNA from human white blood cells and (tumor) tissues of cisplatin-treated patients. ...
... To allow more sensitive, selective and routine analyses of intrastrand Pt-GG and Pt-AG adducts, Pluim et al. [43] introduced some modifications in the method of Welters et al. [42], including: ...
Platinum-containing anti-cancer drugs (e.g., cisplatin) exert their biological effects by forming DNA adducts, so highly sensitive, specific quantitative methods are needed to correlate the molecular dose of these adducts with the effects of treatment. DNA adducts may also give information with regard to understanding drug resistance. In this work, we briefly review and compare existing quantitative strategies for the determination of the most important platinum-DNA adducts (e.g., immunochemical assays, 32P-post-labeling and mass spectrometry-based methods). We examine the advantages and the disadvantages of the different strategies. In addition, we report important information on the analytical figures of merit of the different methodologies and the problems associated with sample processing.
... For DNA repair studies, monitoring the formation and removal of physiologically and environmentally relevant low levels of DNA damage is feasible 35 . The 32 P-postlabeling method has also been adapted to the detection of DNA intrastrand crosslinks formed by bifunctional agents such as cisplatin 36,37 ; here the method involves enzymatic digestion of the DNA with nuclease P 1 , DNase I and alkaline phosphatase to yield normal nucleosides and platinated dinucleotides; following separation and deplatination of the adducts (PNK is inactivated by platinum), the resulting dinucleotides are 32 P-labeled, resolved on TLC and HPLC and quantified. ...
32P-postlabeling analysis is an ultrasensitive method for the detection and quantitation of carcinogen-DNA adducts. It consists of four principal steps: (i) enzymatic digestion of DNA to nucleoside 3'-monophosphates; (ii) enrichment of the adduct fraction of the DNA digest; (iii) 5'-labeling of the adducts by transfer of 32P-orthophosphate from [gamma-32P]ATP mediated by polynucleotide kinase (PNK); (iv) chromatographic or electrophoretic separation of the labeled adducts or modified nucleotides and quantitation by measurement of their radioactive decay. The assay requires only microgram quantities of DNA and is capable of detecting adducts at frequencies as low as 1 in 10(10) nt, making it applicable to the detection of events resulting from environmental exposures, or experiments using physiological concentrations of agents. It has a wide range of applications in human, animal and in vitro studies, and can be used for a wide variety of classes of compound and for the detection of adducts formed by complex mixtures. This protocol can be completed in 3 d.
... Platinum-DNA adduct measurement. Pt-DNA adduct levels were analyzed using 32 P-postlabeling as described previously (25)(26)(27). In short, DNA was digested to unmodified mononucleosides and Pt-containing (di)nucleotides. ...
Gemcitabine (dFdC) can increase the sensitivity of both cisplatin (CDDP)-sensitive and -resistant cell lines. It has been postulated that both formation and repair of platinum-(Pt)-DNA adducts are related to these effects. Therefore, we investigated the effects of dFdC on the formation and repair of Pt-DNA adducts in the human ovarian cancer cell line, A2780, and its CDDP- or dFdC-resistant variants, ADDP and AG6000, which have a different expression of various repair enzymes. Cells were exposed for 1 h to CDDP alone or combined with dFdC in IC50 concentrations, followed by a 1-h exposure to thiourea and, subsequently, by a drug-free period of 1, 3 or 23 h (i.e. 2, 4 or 24 h after CDPP +/- dFdC removal). Pt-DNA adducts were quantified with 32P-post-labeling. The gene expression of the repair enzymes, XPA and XRCC1, was the same in all 3 cell lines but ERCC1, ERCC3 and XPC were 2-6 times higher in AG6000 compared to A2780 cells. In contrast, both ERCC1 and ERCC3 were 10- and 1.5-fold lower in ADDP cells compared to A2780. The mismatch enzyme, MLH1, was lower in ADDP cells. At equally toxic CDDP concentrations, all cell lines formed comparable peak levels of total Pt-DNA adducts (36-48 fmol/microg DNA). However, the time at which peak levels were reached showed large variation. The repair of the adducts was very efficient in the resistant cell lines whereas, in A2780 cells, plateau levels were retained until 24 h after CDDP exposure. In A2780 cells, dFdC shifted the adduct peaks from 4 h to directly after CDDP exposure and increased peak levels by >3.9-fold. dFdC also enhanced the repair of adducts by >1.7-fold and increased the Pt-GG:Pt-AG ratio compared to CDDP alone by >1.4-fold. Overall, dFdC decreased the area under the Pt-DNA adduct-time curve (AUA0-25 h) in A2780 cells by 2.7-fold. In ADDP cells, dFdC shifted the adduct peaks from 2 to 4 h and increased them by >2.2-fold. dFdC also increased the Pt-GG:Pt-AG ratio during the repair process by 1.4-fold. Overall, dFdC increased the AUA0-25 h in ADDP cells by 1.7-fold. In AG6000 cells, dFdC increased the Pt-GG:Pt-AG ratio by 1.6-fold directly after exposure but did not clearly affect the AUA0-25 h. In conclusion, dFdC can affect both Pt-DNA adduct formation and repair, depending on the initial sensitivity of the cells.
... Secondly, GA adducts were never specifically targeted. Workers quantifying digested DNA fragments by HPLC or FPLC usually verified the identity of the PtÀAG fraction either by NMR spectroscopy, [9] digestion and subsequent deplatination, [10] co-elution with an authentic dinucleotide (p)ApG cross-linked with the platinum complex, [40,42] or by using monoclonal antibodies raised against cis-[Pt(NH 3 ) 2 (pApG)]. [13,39,43] Unfortunately, checks with authentic samples of cis-[Pt(NH 3 ) 2 (pGpA)] or with antibodies raised against this species were not carried out. ...
The sequence selectivity of the antitumor drug cisplatin (cis-[PtCl(2)(NH(3))(2)] (1)) between the 5'-AG-3' and 5'-GA-3' sites of DNA has been a matter of discussion for more than twenty years. In this work, we compared the reactivity of GA and AG sequences of DNA towards the aquated forms of cisplatin (cis-[PtCl(NH(3))(2)(H(2)O)](+) (2), cis-[Pt(NH(3))(2)(H(2)O)(2)](2+) (3), and cis-[Pt(OH)(NH(3))(2)(H(2)O)](+) (4)) using two sets of experiments. In the first, we investigated a DNA hairpin, whose duplex stem contained a TGAT sequence as the single reactive site, and determined the individual rate constants of platination with 2 and 3 for G and A in acidic solution. The rate constants at 20 degrees C in 0.1M NaClO(4) at pH 4.5+/-0.1 were 0.09(4) M(-1)s(-1) (G) and 0.11(3) M(-1)s(-1) (A) for 2, and 9.6(1) M(-1)s(-1) (G) and 1.7(1) M(-1)s(-1) (A) for 3. These values are similar to those obtained previously for an analogous hairpin that contained a TAGT sequence. The monoadducts formed with 2 by both GA purines are extremely long-lived, partly as a result of the slow hydrolysis of the chloro monoadduct at A, and partly because of the very low chelation rate (1.4 x 10(-5)s(-1) at 20 degrees C) of the aqua monoadduct on the guanine. In the second set of experiments, we incubated pure or enriched samples of 1, 2, 3, or 4 for 18-64 h at 25 degrees C with a 19 base pair (bp) DNA duplex, whose radiolabeled top strand contained one GA and one AG sequence as the only reactive sites. Quantification of the number of GA and AG cross-links afforded a ratio of about two in favor of AG, irrespective of the nature of the leaving ligands. These results disagree with a previous NMR spectroscopy study, and indicate that GA sequences of DNA are substantially more susceptible to attack by cisplatin than previously thought.
... Pt-DNA adduct levels in WBC were analyzed by 32 P-postlabeling as described previously [45, 46]. DNA was isolated and digested, after which 1/100 volume (V) 1 M Tris-HCl was added for complete digestion of the major intrastrand crosslinks to Pt-GG and Pt-AG dinucleotides. ...
To determine possible schedule dependent pharmacokinetic and pharmacodynamic interactions between gemcitabine (2',2'-difluorodeoxycytidine, dFdC) and cisplatin (cis-diammine-dichloroplatinum, CDDP) in patients with advanced stage solid tumors in a phase I trial.
A total of 33 patients with advanced stage solid tumors were treated with gemcitabine (30-min infusion, 800 mg/m2) and cisplatin (one-hour infusion, 50 mg/m2). Sixteen patients had a four-hour interval between gemcitabine (days 1, 8, 15) and cisplatin (days 1 and 8), followed by the reverse schedule and seventeen patients had a 24-hour interval between gemcitabine (days 1, 8, 15) and cisplatin (days 2 and 9), followed by the reverse schedule. Gemcitabine and cisplatin pharmacokinetics were measured in plasma and white blood cells (WBC), isolated from blood samples taken at several time points after the start of treatment.
A four-hour time interval between both agents did not reveal major differences in plasma pharmacokinetics of gemcitabine, dFdU (deaminated gemcitabine) and platinum (Pt), and of gemcitabine-triphosphate (dFdCTP) accumulation and Pt-DNA adduct formation in WBC between the two different sequences of gemcitabine and cisplatin. In the patients treated with the 24-hour interval, cisplatin before gemcitabine did not significantly change peak gemcitabine levels and the AUC of plasma dFdU, but tended to increase dFdCTP AUC in WBC 1.5-fold (P < 0.06). Gemcitabine before cisplatin decreased the plasma AUC of Pt 2.1-fold (P = 0.03). No significant differences in Pt-DNA adduct levels in WBC were found, although gemcitabine before cisplatin tended to increase the 24-hour retention of Pt-DNA adducts. Creatinine clearance on day 28 was related to the peak plasma levels of total Pt (linear regression coefficient (r) = 0.47, P = 0.02, n = 26). Furthermore, the increase in the Pt-GG to Pt-AG ratio 24 hours after cisplatin treatment was related to the overall response of patients (r = 0.89, P < 0.01, n = 8).
Of all schedules the treatment of patients with cisplatin 24 hours before gemcitabine led to the highest dFdCTP accumulation in WBC and total Pt levels in plasma. These characteristics formed the basis for further investigation of this schedule in a phase II clinical study.
