Inhibition of Nucleotide Excision Repair by Fludarabine in Normal Lymphocytes <i>in vitro</i>, Measured by the Alkaline Single Cell Gel Electrophoresis (Comet) Assay

Yamauchi, Takahiro; Kawai, Yasukazu; Ueda, Takanori

doi:10.1111/j.1349-7006.2002.tb01292.x

Cited by 15 publications

(16 citation statements)

References 27 publications

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“…It is known that alkylated bases initiate several excision repair mechanisms, which involve incision of the DNA strand, excision of the damaged nucleotide, gap filling by DNA resynthesis, and rejoining by ligation (Yamauchi et al 2002). Thus, another way to detect the overall effects on DNA damage induced by alkylating agents is by using DNA repair inhibitors interfering the repair mechanism that eliminated DNA alkylations.…”

Section: Discussionmentioning

confidence: 98%

Detection of excision repaired DNA damage in the comet assay by using Ara-C and hydroxyurea in three different cell types

2007

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Because of its characteristics, the comet assay has been used to evaluate the ability of virtually any type of eukaryotic cell to repair different kinds of DNA damage, including double and single strand breaks and base damage. The ability to detect excision repair sites using the alkaline version can be enhanced by the inclusion of repair inhibitors, DNA synthesis inhibitors, or chain terminators. In this sense, we evaluated the ability of hydroxyurea (HU) and cytosine arabinoside (Ara-C), for detecting lesions produced by the alkylating agents ethyl methanesulfonate (EMS) and methyl methanesulfonate (MMS) in three different cell systems. Two hundred cells for experimental point were analyzed in the alkaline version of the comet assay, and the results are evidences of the utility of the assay to detect alkylation of bases in the cells lines MRC-5 and TK-6, as the treatment with HU +Ara-C significantly increases both the basal and induced frequency of DNA damage. The use of whole blood, although it detected the effects of MMS, with and without repair inhibitors, failed to detect the effect of the selected dose of EMS and does not permit detection increases in the background level.

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Section: Discussionmentioning

confidence: 98%

Detection of excision repaired DNA damage in the comet assay by using Ara-C and hydroxyurea in three different cell types

2007

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“…Previous studies have shown that fludarabine is capable of inhibiting DNA repair induced by carboplatin 33 or UV radiation 34 in quiescent human lymphocytes that were stimulated by 4-hydroperoxycyclophosphamide in CLL lymphocytes, 23 and cisplatin-generated damage in K562 cells. 26 As an extension of those studies, the present investigations reveal a greater than additive cytotoxic effect of fludarabine in combination with oxaliplatin in both normal lymphocytes and CLL cells.…”

Section: Discussionmentioning

confidence: 99%

Fludarabine increases oxaliplatin cytotoxicity in normal and chronic lymphocytic leukemia lymphocytes by suppressing interstrand DNA crosslink removal

Moufarij

Sampath

Keating

et al. 2006

Blood

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IntroductionChronic lymphocytic leukemia (CLL) is the most frequent type of leukemia in Western countries and it accounts for approximately 25% of all leukemias. 1 Although at the present there is no curative treatment, combinations of cytotoxic agents and of immunotherapies that generate high complete remission rates hold promise for altering the natural history of this disease. [2][3][4] Alkylating agents and nucleoside analogues are 2 classes of anticancer agents important in the treatment of chronic lymphocytic leukemia. Indolent B-cell malignancies such as CLL exhibit a high DNA damage repair capacity. 5 This biologic property provides a rationale for therapeutic strategies that combine DNA-damaging cytotoxic agents, such as oxaliplatin, with an inhibitor of DNA repair such as fludarabine.Fludarabine (9-beta-D-arabinofuranosyl-2-fluoroadenine 5Ј-phosphate) is the most effective purine nucleoside analogue for the treatment of indolent lymphoproliferative disorders, including CLL, low-grade lymphoma, and prolymphocytic leukemia. 6,7 On infusion into the bloodstream, fludarabine is dephosphorylated to the respective nucleoside (F-ara-A) which is a substrate for transporters. 8 On entering the cell, fludarabine is anabolized to its triphosphate form, its major intracellular metabolite. 9 The triphosphate can then become incorporated into DNA or RNA, actions which block further synthesis. 10 Its inhibition of ribonucleotide reductase, an action that depletes the deoxynucleotide pools required for DNA repair and replication, may favor its increased incorporation into newly synthesized DNA. 11 Further, its inhibitory actions against both DNA ligase 12 and DNA primase 13 are likely to antagonize the consequences of its incorporation. The finding that it is resistant to excision 14 is likely to contribute to its effective inhibition of DNA synthesis. Finally, in vitro studies have shown that the loss of cell viability correlates directly with the level of incorporation of the analog into cellular DNA.Oxaliplatin (DACH oxalate-platinum(II); Eloxatin) is a thirdgeneration platinum compound with a different spectrum of activity and low cross-resistance with cisplatin. 15 It is currently indicated for use with fluorouracil and leucovorin for the treatment of advanced cancer of the colon or rectum. 16 Oxaliplatin has shown in vitro and in vivo supra-additive effects in combination with several other antitumor agents, including 5-fluorouracil, topoisomerase I inhibitors, thymidylate synthase inhibitors, paclitaxel, cisplatin, and carboplatin. 15,17 The mechanism of action of oxaliplatin is mediated by the formation of DNA adducts. The main types of DNA lesions induced by oxaliplatin are intrastrand crosslinks covalently binding the platinum compound to guanine residues. 18 The other type of lesions include DNA interstrand crosslinks (ICLs) and DNA protein crosslinks. 19 Oxaliplatin-DNA adducts are suggested to exert their cytotoxicity by directly inhibiting DNA and RNA synthesis and inducing apoptosis.The nonoverlapping s...

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“…The Comet assay was used for this purpose according to the method previously described, with a slight modification. 16) Briefly, lymphocytes after treatments were mixed with agarose and placed onto a microscope slide. After solidification of the agarose, the slide was left in a lysis solution (2.5 M NaCl, 10 mM Tris, 100 mM ethylenediaminetetraacetic acid, 10% dimethylsulfoxide, and 1% Triton X-100, pH 10) for 1 h at 4°C.…”

Section: Methodsmentioning

confidence: 99%

“…15,16) In brief, normal lymphocytes (2×10 6 cells) having previously been treated and washed in fresh media were incubated with tritiated thymidine (3 µCi/ml) for 0, 1, 2, 4, and 6 h. The lymphocytes were then collected, centrifuged, and resuspended in 500 µl of 0.4 N perchloric acid. The sample was mixed, centrifuged, and resuspended again in the acid.…”

Section: )mentioning

confidence: 99%

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l‐β‐D‐Arabinofuranosylcytosine Is Cytotoxic in Quiescent Normal Lymphocytes Undergoing DNA Excision Repair

Yamauchi

Kawai

Ueda

2002

Japanese Journal of Cancer Research

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We have sought to clarify the potential activity of the S-phase-specific antileukemic agent 1-β β β β-Darabinofuranosylcytosine (ara-C), an inhibitor of DNA synthesis, in quiescent cells that are substantially non-sensitive to nucleoside analogues. It was hypothesized that the combination of ara-C with DNA damaging agents that initiate DNA repair will expand ara-C cytotoxicity to non-cycling cells. The repair kinetics, which included incision of damaged DNA, gap-filling by DNA synthesis and rejoining by ligation, were evaluated using the single cell gel electrophoresis (Comet) assay and the thymidine incorporation assay. When normal lymphocytes were treated with ultraviolet C or with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), the processes of DNA excision repair were promptly initiated and rapidly completed. When the cells were incubated with ara-C prior to irradiation or BCNU treatment, the steps of DNA synthesis and rejoining in the repair processes were both inhibited. The ara-C-mediated inhibition of the repair processes was concentration-dependent, with the effect peaking at 10 µ µ µ µM. The combination of ara-C with these DNA repair initiators exerted subsequent cytotoxicity, which was proportional to the extent of the repair inhibition in the presence of ara-C. In conclusion, ara-C was cytotoxic in quiescent cells undergoing DNA repair. This might be attributed to unrepaired DNA damage that remained in the cells, thereby inducing lethal cytotoxicity. Alternatively, ara-C might exert its own cytotoxicity by inhibiting DNA synthesis in the repair processes. Such a strategy may be effective against a dormant subpopulation in acute leukemia that survives chemotherapy.

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Inhibition of Nucleotide Excision Repair by Fludarabine in Normal Lymphocytes in vitro, Measured by the Alkaline Single Cell Gel Electrophoresis (Comet) Assay

Cited by 15 publications

References 27 publications

Detection of excision repaired DNA damage in the comet assay by using Ara-C and hydroxyurea in three different cell types

Detection of excision repaired DNA damage in the comet assay by using Ara-C and hydroxyurea in three different cell types

Fludarabine increases oxaliplatin cytotoxicity in normal and chronic lymphocytic leukemia lymphocytes by suppressing interstrand DNA crosslink removal

l‐β‐D‐Arabinofuranosylcytosine Is Cytotoxic in Quiescent Normal Lymphocytes Undergoing DNA Excision Repair

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