Effect of DNA polymerase inhibitors on repair of γ ray-induced DNA damage in proliferating (intact versus permeable) human fibroblasts: evidence for differences in the modes of action of aphidicolin and 1-β-d-arabinofuranosylcytosine

Mirzayans, Razmik; Enns, Louise; Cubitt, S.; Karimian, Kashayar; Radatus, Bruno; Paterson, M. C.

doi:10.1016/0925-4439(94)90112-0

Cited by 8 publications

(3 citation statements)

References 22 publications

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“…As a major mechanism of ara-C resistance is conferred by alterations in the enzymes that influence the conversion of ara-C to ara-CTP, it is possible that aphidicolin modulates the rate of intracellular accumulation of ara-CTP. However, studies on proliferating human fibroblasts suggest that this is not the case (Mirzayans et al, 1994). Another mechanism of action for the synergy between ara-C and aphidicolin has been postulated by Kuwakado et al (1995).…”

Section: Discussionmentioning

confidence: 98%

Circumvention of ara-C resistance by aphidicolin in blast cells from patients with AML

Sargent¹,

Elgie²,

Williamson³

et al. 2001

Br J Cancer

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Summary Treatment failure in AML is often attributed to P-glycoprotein-associated multidrug resistance. However, the importance of increased DNA repair in resistant cells is becoming more apparent. In order to investigate the ability of the DNA repair inhibitor aphidicolin to modulate drug resistance, we continually exposed blasts cells, isolated from 22 patients with AML, to a variety of agents ± 15 µM aphidicolin for 48 hours. Cell survival was measured using the MTT assay. Overall, there was no significant effect of aphidicolin on sensitivity to daunorubicin, doxorubicin, etoposide or fludarabine. However, there was a marked increase in sensitivity to ara-C with a median 4.75-fold increase overall (range 0.8-80-fold; P < 0.005). The effect of aphidicolin was significantly greater in blast cells found resistant in vitro to ara-C (8.9-fold compared to 2.12-fold, P < 0.01). This observation was further validated by the correlation between ara-C LC 50 and extent of modulation effect (P < 0.05). Cells isolated from 10 cord blood samples were also tested in order to establish the haematological toxicity of combining ara-C and aphidicolin. The therapeutic index (LC 50 normal cells/tumour cells) for ara-C + aphidicolin was higher than that for ara-C alone suggesting no increased myelotoxicity for the combination. Increased cytotoxicity without increased haematotoxicity makes the combination of ara-C plus aphidicolin ideal for inclusion in future clinical trials.

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Section: Discussionmentioning

confidence: 98%

Circumvention of ara-C resistance by aphidicolin in blast cells from patients with AML

Sargent¹,

Elgie²,

Williamson³

et al. 2001

Br J Cancer

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“…Three DNA polymerase inhibitors were tested. These included aphidicolin, an inhibitor of DNA polymerase-␣, -␦, and -⑀ (Mirzayans et al, 1994); the specific DNA polymerase-␥ inhibitor N-ethylmaleimide (NEM); and the DNA polymerase-␥ and -␤ inhibitor ddNTP (in this case, ddCTP). The repair activity of mitochondrial protein was not affected by aphidicolin (data not shown).…”

Section: Dna Polymerase Activity In Brain Mitochondrial Extractsmentioning

confidence: 99%

Detection of DNA base-excision repair activity for oxidative lesions in adult rat brain mitochondria

et al. 2000

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“…The results provide evidence that this cellfree approach can be exploited as an in vitro assay for investigating DNA repair activity. Specificity of the damage response in this nucleus-based model was demonstrated by the absence of dUTP signals in nuclei incubated in buffer, by the significant inhibition of DNA synthesis in the presence of Aph and Ara-C, either alone or in combination (Crute et al, 1986;Huberman, 1981;Mirzayans et al, 1994), and especially by the absence of a DNA repair response in G1 nuclei isolated from NER-defective XPC14BR cells. Moreover, the high percentage (98%) of dUTP-positive G1 nuclei, relative to unirradiated samples, as well as the absence of BrdU incorporation in living cells before nuclei isolation indicated that DNA synthesis initiated in vitro in UVC-exposed nuclei is due to DNA repair and not to initiation of DNA replication.…”

Section: Discussionmentioning

confidence: 95%

A functional in vitro cell-free system for studying DNA repair in isolated nuclei

Guardamagna

Bassi

Savio

et al. 2020

Journal of Cell Science

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Assessment of DNA repair is an important endpoint measurement when studying the biochemical mechanisms of the DNA damage response and when investigating the efficacy of chemotherapy, which often uses DNA-damaging compounds. Numerous in vitro methods to biochemically characterize DNA repair mechanisms have been developed so far. However, such methods have some limitations, which are mainly due to the lack of chromatin organization in the DNA templates used. Here we describe a functional cell-free system to study DNA repair synthesis in vitro, using G1-phase nuclei isolated from human cells treated with different genotoxic agents. Upon incubation in the corresponding damage-activated cytosolic extracts, containing biotinylated dUTP, nuclei were able to initiate DNA repair synthesis. The use of specific DNA synthesis inhibitors markedly decreased biotinylated dUTP incorporation, indicating the specificity of the repair response. Exogenously added human recombinant PCNA protein, but not the sensors of UV-DNA damage DDB2 and DDB1, stimulated UVC-induced dUTP incorporation. In contrast, a DDB2 PCNA− mutant protein, unable to associate with PCNA, interfered with DNA repair synthesis. Given its responsiveness to different types of DNA lesions, this system offers an additional tool to study DNA repair mechanisms. This article has an associated First Person interview with the first author of the paper.

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Effect of DNA polymerase inhibitors on repair of γ ray-induced DNA damage in proliferating (intact versus permeable) human fibroblasts: evidence for differences in the modes of action of aphidicolin and 1-β-d-arabinofuranosylcytosine

Cited by 8 publications

References 22 publications

Circumvention of ara-C resistance by aphidicolin in blast cells from patients with AML

Circumvention of ara-C resistance by aphidicolin in blast cells from patients with AML

Detection of DNA base-excision repair activity for oxidative lesions in adult rat brain mitochondria

A functional in vitro cell-free system for studying DNA repair in isolated nuclei

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