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Title: Kinetics of carboplatin-DNA binding in genomic DNA and bladder cancer cells as determined by accelerator mass spectrometry

Abstract

Cisplatin and carboplatin are platinum-based drugs that are widely used in cancer chemotherapy. The cytotoxicity of these drugs is mediated by platinum-DNA monoadducts and intra- and interstrand diadducts, which are formed following uptake of the drug into the nucleus of cells. The pharmacodynamics of carboplatin display fewer side effects than for cisplatin, albeit with less potency, which may be due to differences in rates of DNA adduct formation. We report the use of accelerator mass spectrometry (AMS), a sensitive detection method often used for radiocarbon quantitation, to measure both the kinetics of [{sup 14}C]carboplatin-DNA adduct formation with genomic DNA and drug uptake and DNA binding in T24 human bladder cancer cells. Only carboplatin-DNA monoadducts contain radiocarbon in the platinated DNA, which allowed for calculation of kinetic rates and concentrations within the system. The percent of radiocarbon bound to salmon sperm DNA in the form of monoadducts was measured by AMS over 24 h. Knowledge of both the starting concentration of the parent carboplatin and the concentration of radiocarbon in the DNA at a variety of time points allowed calculation of the rates of Pt-DNA monoadduct formation and conversion to toxic cross-links. Importantly, the rate of carboplatin-DNA monoadduct formation was approximatelymore » 100-fold slower than that reported for the more potent cisplatin analogue, which may explain the lower toxicity of carboplatin. T24 human bladder cancer cells were incubated with a subpharmacological dose of [{sup 14}C]carboplatin, and the rate of accumulation of radiocarbon in the cells and nuclear DNA was measured by AMS. The lowest concentration of radiocarbon measured was approximately 1 amol/10 {micro}g of DNA. This sensitivity may allow the method to be used for clinical applications.« less

Authors:
; ; ;
Publication Date:
Research Org.:
Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
891393
Report Number(s):
UCRL-JRNL-217933
TRN: US0605385
DOE Contract Number:
W-7405-ENG-48
Resource Type:
Journal Article
Resource Relation:
Journal Name: Chemical Research in Toxicology, vol. 19, no. 5, May 15, 2006, pp. 622
Country of Publication:
United States
Language:
English
Subject:
43 PARTICLE ACCELERATORS; 37 INORGANIC, ORGANIC, PHYSICAL AND ANALYTICAL CHEMISTRY; 59 BASIC BIOLOGICAL SCIENCES; ACCELERATORS; ADDUCTS; BLADDER; CHEMOTHERAPY; DETECTION; DNA; DNA ADDUCTS; KINETICS; MASS SPECTROSCOPY; NEOPLASMS; SALMON; SENSITIVITY; SIDE EFFECTS; SPERMATOZOA; TOXICITY

Citation Formats

Hah, S S, Stivers, K M, Vere White, R, and Henderson, P T. Kinetics of carboplatin-DNA binding in genomic DNA and bladder cancer cells as determined by accelerator mass spectrometry. United States: N. p., 2005. Web.
Hah, S S, Stivers, K M, Vere White, R, & Henderson, P T. Kinetics of carboplatin-DNA binding in genomic DNA and bladder cancer cells as determined by accelerator mass spectrometry. United States.
Hah, S S, Stivers, K M, Vere White, R, and Henderson, P T. Thu . "Kinetics of carboplatin-DNA binding in genomic DNA and bladder cancer cells as determined by accelerator mass spectrometry". United States. doi:. https://www.osti.gov/servlets/purl/891393.
@article{osti_891393,
title = {Kinetics of carboplatin-DNA binding in genomic DNA and bladder cancer cells as determined by accelerator mass spectrometry},
author = {Hah, S S and Stivers, K M and Vere White, R and Henderson, P T},
abstractNote = {Cisplatin and carboplatin are platinum-based drugs that are widely used in cancer chemotherapy. The cytotoxicity of these drugs is mediated by platinum-DNA monoadducts and intra- and interstrand diadducts, which are formed following uptake of the drug into the nucleus of cells. The pharmacodynamics of carboplatin display fewer side effects than for cisplatin, albeit with less potency, which may be due to differences in rates of DNA adduct formation. We report the use of accelerator mass spectrometry (AMS), a sensitive detection method often used for radiocarbon quantitation, to measure both the kinetics of [{sup 14}C]carboplatin-DNA adduct formation with genomic DNA and drug uptake and DNA binding in T24 human bladder cancer cells. Only carboplatin-DNA monoadducts contain radiocarbon in the platinated DNA, which allowed for calculation of kinetic rates and concentrations within the system. The percent of radiocarbon bound to salmon sperm DNA in the form of monoadducts was measured by AMS over 24 h. Knowledge of both the starting concentration of the parent carboplatin and the concentration of radiocarbon in the DNA at a variety of time points allowed calculation of the rates of Pt-DNA monoadduct formation and conversion to toxic cross-links. Importantly, the rate of carboplatin-DNA monoadduct formation was approximately 100-fold slower than that reported for the more potent cisplatin analogue, which may explain the lower toxicity of carboplatin. T24 human bladder cancer cells were incubated with a subpharmacological dose of [{sup 14}C]carboplatin, and the rate of accumulation of radiocarbon in the cells and nuclear DNA was measured by AMS. The lowest concentration of radiocarbon measured was approximately 1 amol/10 {micro}g of DNA. This sensitivity may allow the method to be used for clinical applications.},
doi = {},
journal = {Chemical Research in Toxicology, vol. 19, no. 5, May 15, 2006, pp. 622},
number = ,
volume = ,
place = {United States},
year = {Thu Dec 29 00:00:00 EST 2005},
month = {Thu Dec 29 00:00:00 EST 2005}
}
  • Accelerator mass spectrometry (AMS) is used to determine the amount of carcinogen covalently bound to mouse liver DNA (DNA adduct) following very low-level exposure to a {sup 14}C-labeled carcinogen. AMS is a highly sensitive method for counting long-lived but rare cosmogenic isotopes. While AMS is a tool of importance in the earth sciences, it has not been applied in biomedical research. The ability of AMS to assay rare isotope concentrations ({sup 10}Be, {sup 14}C, {sup 26}Al, {sup 41}Ca, and {sup 129}I) in microgram amounts suggests that extension to the biomedical sciences is a natural and potentially powerful application of themore » technology. In this study, the relationship between exposure to low levels of 2-amino-3,8-dimethyl(2-{sup 14}C)imidazo(4,5-f)quinoxaline and formation of DNA adducts is examined to establish the dynamic range of the technique and the potential sensitivity for biological measurements, as well as to evaluate the relationship between DNA adducts and low-dose carcinogen exposure. Formation of adducts is linearly dependent on dose down to an exposure of 500 ng per kg of body weight. With the present measurements, the authors demonstrate at least 1 order of magnitude improvement over the best adduct detection sensitivity reported to data and 3-5 orders of magnitude improvement over other methods used for adduct measurement. Expanded applications involving human subjects, including clinical applications, are now expected because of the great detection sensitivity and small sample size requirements of AMS.« less
  • We report here genome-wide analysis of the tumor suppressor p53 binding sites in normal human cells. 743 high-confidence ChIP-seq peaks representing putative genomic binding sites were identified in normal IMR90 fibroblasts using a reference chromatin sample. More than 40% were located within 2 kb of a transcription start site (TSS), a distribution similar to that documented for individually studied, functional p53 binding sites and, to date, not observed by previous p53 genome-wide studies. Nearly half of the high-confidence binding sites in the IMR90 cells reside in CpG islands, in marked contrast to sites reported in cancer-derived cells. The distinct genomicmore » features of the IMR90 binding sites do not reflect a distinct preference for specific sequences, since the de novo developed p53 motif based on our study is similar to those reported by genome-wide studies of cancer cells. More likely, the different chromatin landscape in normal, compared with cancer-derived cells, influences p53 binding via modulating availability of the sites. We compared the IMR90 ChIPseq peaks to the recently published IMR90 methylome1 and demonstrated that they are enriched at hypomethylated DNA. Our study represents the first genome-wide, de novo mapping of p53 binding sites in normal human cells and reveals that p53 binding sites reside in distinct genomic landscapes in normal and cancer-derived human cells.« less
  • The response rates of extensively used chemotherapeutic drugs, carboplatin (Carb) or 5-fluorouracil (5-FU) are relatively disappointing because of considerable side effects associated with their high-dose regimen. In the present study, we determined whether treatment with a cholesterol depleting agent, methyl-{beta}-cyclodextrin (MCD), enhances the weak efficacy of low doses of Carb or 5-FU in human breast cancer cells. Data demonstrate that pretreatment with MCD significantly potentiates the cytotoxic activity of Carb and 5-FU in both MCF-7 and MDA-MB-231. Furthermore, we explored the molecular basis of enhanced cytotoxicity, and our data revealed that low-dose treatment with these drugs in MCD pretreated cellsmore » exhibited significantly decreased Akt phosphorylation, NF-{kappa}B activity and down-regulation in expression of anti-apoptotic protein Bcl-2. In addition, MCD pretreated cells demonstrated an increased intracellular drug accumulation as compared to cells treated with drugs alone. Taken together, our data provide the basis for potential therapeutic application of MCD in combination with other conventional cytotoxic drugs to facilitate reduction of drug dosage that offers a better chemotherapeutic approach with low toxicity.« less
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