Effect of hydroxyurea and normal plasma on DNA synthesis in lymphocytes from Fanconi anemia patients

Frias, Sara; Gómez, Laura; Molina, Bertha; Rojas, Emilio; Ostrosky‐Wegman, Patricia; Carnevale, Alessandra

doi:10.1016/0027-5107(96)00091-7

Cited by 11 publications

(7 citation statements)

References 24 publications

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“…In their studies the complementation group of the patients was not specified. 64 In our studies, treatment of normal, FAC-, or mock-corrected FA(C) lymphoblasts with HU resulted in equivalent degrees of cell cycle arrest at the G1/S boundary. Therefore, we conclude that the S-phase checkpoint that responds to incomplete replication of DNA is intact in FA cells and find no evidence of hypersensitivity of FA(C) lymphoblasts to HU.…”

Section: Cell Cycle Control In Fa(c)mentioning

confidence: 48%

DNA Cross-Linker–Induced G2/M Arrest in Group C Fanconi Anemia Lymphoblasts Reflects Normal Checkpoint Function

Heinrich

Hoatlin

Zigler

et al. 1998

Blood

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Cells from individuals with Fanconi anemia (FA) arrest excessively in the G2/M cell cycle compartment after exposure to low doses of DNA cross-linking agents. The relationship of this abnormality to the fundamental genetic defect in such cells is unknown, but many investigators have speculated that the various FA genes directly regulate cell cycle checkpoints. We tested the hypothesis that the protein encoded by the FA group C complementing gene (FAC) functions to control a cell cycle checkpoint and that cells from group C patients (FA[C]) have abnormalities of cell cycle regulation directly related to the genetic mutation. We found that retroviral transduction of FA(C) lymphoblasts with wild-type FAC cDNA resulted in normalization of the cell cycle response to low-dose mitomycin C (MMC). However, when DNA damage was quantified in terms of cytogenetic damage or cellular cytotoxicity, we found similar degrees of G2/M arrest in response to equitoxic amounts of MMC in FA(C) cells as well as in normal lymphoblasts. Similar results were obtained using isogenic pairs of uncorrected, FAC- or mock-corrected (neo only) FA(C) cell lines. To test the function of other checkpoints we examined the effects of hydroxyurea (HU) and ionizing radiation on cell cycle kinetics of FA(C) and normal lymphoblasts as well as with isogenic pairs of uncorrected, FAC-corrected, or mock-corrected FA(C) cell lines. In all cases the cell cycle response of FA(C) and normal lymphoblasts to these two agents were identical. Based on these studies we conclude that the aberrant G2/M arrest that typifies the response of FA(C) cells to low doses of cross-linking agents does not represent an abnormal cell cycle response but instead represents a normal cellular response to the excessive DNA damage that results in FA(C) cells following exposure to low doses of cross-linking agents.

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Section: Cell Cycle Control In Fa(c)mentioning

confidence: 48%

DNA Cross-Linker–Induced G2/M Arrest in Group C Fanconi Anemia Lymphoblasts Reflects Normal Checkpoint Function

Heinrich

Hoatlin

Zigler

et al. 1998

Blood

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“…This function is dependent of the p53 status of the cell, as no increases in the frequency of chromosomal damage in mouse embryo fibroblasts exposed to bleomycin were observed in a p53 −/− background (Allio et al 2000). On the other hand, HU prevents DNA replication by selectively inhibiting ribonucleotide reductase with S-phase cell-cycle specificity and some potential to arrest cells at the G1/S boundary (Frias et al 1996). It has been shown that HU, in combination with Ara-C neither causes DNA breaks nor affected cell survival during the first 10 h (Filatov et al 1998).…”

Section: Discussionmentioning

confidence: 91%

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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“…This function of araC is dependent on the p53 status of the cell, since no increases in the frequency of chromatid aberration in mouse embryoˆbroblasts exposed to BLM were observed by addition of araC in a p53 null (p53 -/-) background, although it increased in the wild type cells to the same level as the null mutant cells (13). On the other hand, HU prevents DNA replication by selectively inhibiting ribonucleotide reductase with S-phase cell-cycle speciˆcity, and exhibits some potential to arrest cells at the G1/S boundary (14). HU inhibits DNA repair by blocking DNA synthesis and aŠecting the replication checkpoint (15), which enhances cell cytotoxicity.…”

Section: Discussionmentioning

confidence: 99%

Does p53 Status Affect the Sensitivity of the Comet Assay?

Nakamura

Yamamoto

Honda

et al. 2010

Genes and Environment

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It has been shown that cells having mutant p53 are more sensitive to genotoxic eŠects than cells having wild-type p53. To determine whether p53 status aŠects the sensitivity of the comet assay, we compared the results of the comet assay in WTK1 cells having mutant p53 and TK6 cells having wild-type p53 in the presence and absence of DNA repair inhibitors. Cells were exposed to methyl nitrosourea (MNU), ethyl nitrosourea (ENU), methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), and bleomycin (BLM) for 2 h in the presence or absence of araC and hydroxyurea (araC/HU) or irradiated to UVC. UVC irradiated cells were cultured for 2 h with or without araC/HU. Immediately after the exposure to chemical mutagens or 2 h after UVC irradiation, slides for the comet assay were prepared. In the absence of araC/HU, the lowest genotoxic doses (LGDs) were higher in TK6 than in WTK1 cells, showing that wild-type p53 reduces comet assay sensitivity. On the other hand, in the presence of araC/HU, the LGDs were almost the same in WTK1 and TK6 cells. In the presence of araC/HU, positive responses to UVC irradiation were observed earlier in TK6 than in WTK1 cells. Therefore, SSB formation followed by SSB rejoining during the nucleotide excision repair process is considered to occur earlier in TK6 than in WTK1 cells, which results in a lower comet assay sensitivity in cells having wild-type p53. Comet assay response to mutagens inducing DNA damages that are repaired by the excision repair system tended to be aŠected by p53 status.

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Effect of hydroxyurea and normal plasma on DNA synthesis in lymphocytes from Fanconi anemia patients

Cited by 11 publications

References 24 publications

DNA Cross-Linker–Induced G2/M Arrest in Group C Fanconi Anemia Lymphoblasts Reflects Normal Checkpoint Function

DNA Cross-Linker–Induced G2/M Arrest in Group C Fanconi Anemia Lymphoblasts Reflects Normal Checkpoint Function

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

Does p53 Status Affect the Sensitivity of the Comet Assay?

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