Application of a sensitive immunoassay to the study of DNA adducts formed in peripheral blood mononuclear cells of patients undergoing high-dose melphalan therapy

Tilby, M.J.; Newell, David R.; Selby, P.; Viner, C.; Dean, Christopher

doi:10.1016/s0959-8049(05)80346-6

Cited by 20 publications

(19 citation statements)

References 12 publications

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“…Monoadducts and ICL were measured following cell treatment with 10 Ag/mL melphalan for 1 hour. This concentration is of biological relevance because it gives rise to adduct levels similar to those detected in multiple myeloma patients following therapeutic treatment (23,28). In all cases, peak monoadduct levels were observed 2 hours following the end of treatment, and in XP-A cells, they reached very similar levels in all genes examined (Fig.…”

Section: Resultssupporting

confidence: 53%

Association between Transcriptional Activity, Local Chromatin Structure, and the Efficiencies of Both Subpathways of Nucleotide Excision Repair of Melphalan Adducts

Episkopou

Kyrtopoulos

Sfikakis³

et al. 2009

Cancer Research

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The repair of melphalan-induced N-alkylpurine monoadducts and interstrand cross-links was examined in different repair backgrounds, focusing on four genes (b-actin, p53, N-ras, and d-globin) with dissimilar transcription activities. Adducts were found to be substrates for both global genome repair (GGR) and transcription-coupled repair (TCR), with TCR being less efficient than GGR. In nucleotide excision repair-deficient cells, adducts accumulated to similar levels in all four genes. The repair efficiency in different gene loci varied in a qualitatively and quantitatively similar way in both GGRdeficient and TCR-deficient backgrounds and correlated with transcriptional activity and local chromatin condensation. No strand-specific repair was found in GGR + /TCR + cells, implying that GGR dominated. Adducts were lost over two sharply demarcated phases: a rapid phase resulting in the removal within 1 hour of up to f80% of the adducts, and a subsequent phase with t 1/2 f36 to 48 hours. Following pretreatment of cells with A-amanitin, the rate of transcription, the state of chromatin condensation, and the repair efficiencies (both TCR and GGR) of the transcribed b-actin, p53, and N-ras genes became similar to those of the nontranscribed d-globin gene. In conclusion, a continuous, parallel variation of the state of transcription and local chromatin condensation, on one hand, and the rates of both GGR and TCR, on the other hand, have been shown.

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Section: Resultssupporting

confidence: 53%

Association between Transcriptional Activity, Local Chromatin Structure, and the Efficiencies of Both Subpathways of Nucleotide Excision Repair of Melphalan Adducts

Episkopou

Kyrtopoulos

Sfikakis³

et al. 2009

Cancer Research

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“…Monohydroxymelphalan was prepared as described previously (Tilby et al, 1998). Solutions of melphalan (Sigma-Aldrich, St. Louis, MO) and monohydroxymelphalan were prepared in acidified ethanol (Tilby et al, 1993) immediately before use and then diluted into culture medium to give final ethanol concentrations of 1% (v/v) for controls and for all exposures to alkylating agents. Cells were incubated with drug for 1 h at 37°C, and then drug was removed from the cells by centrifugation (190g for 5 min at 25°C) and washing with PBS.…”

Section: Methodsmentioning

confidence: 99%

“…DNA extraction and competitive ELISA methods using monoclonal antibody MP5/73 were performed as described elsewhere (Tilby et al, 1987(Tilby et al, , 1991(Tilby et al, , 1993. This technique shows equal sensitivity for DNA adducts formed by melphalan and monohydroxymelphalan (Tilby et al, 1998); nevertheless, independent standards of DNA alkylated with the appropriate radioactively labeled alkylating agent were included in every assay.…”

Section: Methodsmentioning

confidence: 99%

“…We have also described a sensitive immunoassay for DNA adducts induced by melphalan (Tilby et al, 1987). This was shown to be applicable to clinical specimens (Tilby et al, 1993) and, with equal sensitivity, to adducts formed by monohydroxymelphalan (Tilby et al, 1998). These tools permit a detailed comparison of the cellular effects of chemically equivalent mono-and bifunctional adducts formed by a drug that is representative of, and relevant to, a large number of chemically and mechanistically related drugs, including agents currently being developed for targeted therapies (Melton et al, 1996).…”

mentioning

confidence: 99%

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p53 Elevation in Relation to Levels and Cytotoxicity of Mono- and Bifunctional Melphalan-DNA Adducts

Gould

Nixon²,

Tilby³

2004

Mol Pharmacol

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We tested the hypothesis that bifunctional DNA adducts formed by a nitrogen mustard-based anticancer drug were more efficient than monofunctional adducts at causing elevation of p53, consistent with the difference in cytotoxicity. Human leukemia cell line ML-1 was exposed for 1 h to melphalan or its monofunctional derivative monohydroxymelphalan. Levels of DNA adducts, measured by specific immunoassay, were linearly related to the concentration of alkylating agent. Monohydroxymelphalan formed twice as many adducts as did equal concentrations of melphalan. After the removal of the alkylating agent, adduct levels were maintained or increased slightly up to 8 h and then decreased by 27 to 44% by 24 h. Alkaline elution analyses confirmed the absence of detectable DNA interstrand cross-links in cells exposed to monohydroxymelphalan. DNA single-strand breaks were detected after monohydroxymelphalan but not after melphalan. Levels of p53 were quantified by sensitive fluorogenic enzyme-linked immunosorbent assay at intervals up to 24 h after exposure of cells to various concentrations of melphalan and monohydroxymelphalan. The level of initially formed DNA adducts needed to cause elevation of p53 from a baseline level of 0.5 ng/mg total protein to 2 ng/mg was 5-to 8-fold higher for monohydroxymelphalan than melphalan. The concentrations of melphalan and monohydroxymelphalan (ϮS.D.) causing 50% growth inhibition were 1.2 Ϯ 0.4 and 28.1 Ϯ 1.6 g/ml, respectively, a 23-fold difference. The adduct levels induced by these exposures were 9.3 and 420 nmol/g DNA for melphalan and monohydroxymelphalan, respectively, a 45-fold difference, which is considerably greater than the difference in efficacy at elevating p53.

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“…Evidently, when the total administered amount of melphalan (based on a clinically relevant dose) is equal, neither duration of melphalan exposure nor melphalan concentration affect the tumor and liver uptake of melphalan within the conditions of our experiment. Based on these results, no differences in antitumor effect would be expected after 5-or 20-min arterial infusion either; melphalan exerts its cytotoxic effect by formation of DNA-adducts (Kohn, 1981), and a clear correlation has been observed between melphalan concentration, level of melphalan-derived DNA-adducts, and (Tilby et al, 1993) cytotoxicity (Hansson et al, 1987;Tilby et al, 1993;Frank et al, 1996).…”

Section: Discussionmentioning

confidence: 99%

Melphalan Antitumor Efficacy and Hepatotoxicity: The Effect of Variable Infusion Duration in the Hepatic Artery

Rothbarth

Woutersen²,

Sparidans³

et al. 2003

J Pharmacol Exp Ther

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The optimum conditions (duration and concentration) of a fixed dose, intra-arterial melphalan infusion in relation to its antitumor effect and toxicity in the liver were investigated in a rat colon tumor model (CC531) of liver metastases. We studied the difference in tumor and liver uptake, as well as antitumor effect and hepatotoxicity after 5-and 20-min hepatic arterial infusion (HAI) of a fixed melphalan dose. Melphalan content in perfusate, liver, and tumor tissue was analyzed by high-performance liquid chromatography. The antitumor effect and hepatotoxicity in rats treated either systemically or with 5-and 20-min HAI, with a fixed dose melphalan (4.4 mol), were assessed 2 weeks after treatment. No difference in melphalan content of tumor/ liver tissue or antitumor effect was observed between rats treated with 5-and 20-min HAI. Hepatotoxicity was strongly affected by perfusion duration/concentration, however. Rats treated with 5-min HAI weighed significantly less, and liver toxicity parameters were significantly increased compared with those of all other groups; eight of nine rats showed severe cholangiofibrosis. Body weights and liver toxicity parameters of the rats treated with 20-min HAI were not statistically different from the control group. In conclusion, duration of HAI with 4.4 mol of fixed dose melphalan did not affect tumor uptake and antitumor effect, but the resulting increase in melphalan concentration had major impact on hepatobiliary toxicity. Therefore, in a clinical setting, caution should be taken when infusion duration and concentration of melphalan are changed.Chemotherapeutic treatment is the therapy of choice when curative resection of liver metastases is not possible, for instance because too many metastases are present or the localization or size of these metastases precludes total resection. In the treatment of liver tumors, hepatic artery infusion (HAI) of cytostatics is favored because liver tumors are mainly vascularized by the hepatic artery (Breedis and

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Application of a sensitive immunoassay to the study of DNA adducts formed in peripheral blood mononuclear cells of patients undergoing high-dose melphalan therapy

Cited by 20 publications

References 12 publications

Association between Transcriptional Activity, Local Chromatin Structure, and the Efficiencies of Both Subpathways of Nucleotide Excision Repair of Melphalan Adducts

Association between Transcriptional Activity, Local Chromatin Structure, and the Efficiencies of Both Subpathways of Nucleotide Excision Repair of Melphalan Adducts

p53 Elevation in Relation to Levels and Cytotoxicity of Mono- and Bifunctional Melphalan-DNA Adducts

Melphalan Antitumor Efficacy and Hepatotoxicity: The Effect of Variable Infusion Duration in the Hepatic Artery

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