DNA damage and repair in human lymphocytes exposed to three anticancer platinum drugs

Błasiak, Janusz; Kowalik, Joanna; Małecka‐Panas, Ewa; Drzewoski, Józef; Wojewódzka, Maria

doi:10.1002/(sici)1520-6866(2000)20:3<119::aid-tcm3>3.0.co;2-z

Cited by 33 publications

(27 citation statements)

References 30 publications

(38 reference statements)

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“…Comet assays were performed as described by Blasiak et al (33). Phosphorylated ATM and H2AX were detected by immunofluorescence using specific antibodies (Upstate and Novus Biologicals, respectively) and goat antirabbit or goat antimouse IgG Alexa Fluor 488 (Molecular Probes, Invitrogen).…”

Section: Methodsmentioning

confidence: 99%

The Epstein–Barr virus nuclear antigen-1 promotes genomic instability via induction of reactive oxygen species

Gruhne

Sompallae

Marescotti

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

213

189

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The Epstein-Barr virus (EBV) nuclear antigen (EBNA)-1 is the only viral protein expressed in all EBV-carrying malignancies, but its contribution to oncogenesis has remained enigmatic. We show that EBNA-1 induces chromosomal aberrations, DNA double-strand breaks, and engagement of the DNA damage response (DDR). These signs of genomic instability are associated with the production of reactive oxygen species (ROS) and are reversed by antioxidants. The catalytic subunit of the leukocyte NADPH oxidase, NOX2/gp91 phox , is transcriptionally activated in EBNA-1-expressing cells, whereas inactivation of the enzyme by chemical inhibitors or RNAi halts ROS production and DDR. These findings highlight a novel function of EBNA-1 and a possible mechanism by which expression of this viral protein could contribute to malignant transformation and tumor progression.is a human gamma-herpesvirus that establishes latent infections in B lymphocytes, where only a subset of viral genes is expressed and virus replication is suppressed (1). The proteins encoded by the latency genes, including 6 EBV-encoded nuclear antigens (EBNA-1, -2, -3A, -3B, -3C, and -5) and 3 latent membrane proteins (LMP1, -2A, and -2B), induce growth transformation by capturing multiple signaling pathways that control B cell proliferation and apoptosis. It is generally assumed that the continuous expression of viral genes underlies the association of EBV with a variety of human malignancies, including Burkitt's lymphoma (BL), Hodgkin's disease (HD), nasopharyngeal carcinoma (NPC), and posttransplant lymphoproliferative disease (PTLD) (2). Some EBVpositive tumors do not express all of the latency proteins, leading to restricted forms of latency in which EBNA-1 is detected either alone (latency I, found in BL) or together with the LMPs (latency II, found in HD and NPC). Thus, EBNA-1 is the only viral protein regularly expressed in all EBV-carrying malignancies.EBNA-1 binds to the viral origin of replication (oriP) and is required for the correct partitioning of the viral episomes in proliferating cells (3). It may confer a growth advantage to BL cells (4) and protect them from apoptosis (5) but does not act as an autonomous oncogene (6) and seems to be dispensable for B cell immortalization in vitro (7). Hence, the mechanism by which EBNA-1 may contribute to malignant transformation is not understood.Genomic instability is common in malignant cells and was observed in EBV-carrying tumors (8-10). EBNA-3C (11) and LMP-1 (12) may promote this phenotype through inhibition of DNA repair or inactivation of cell cycle checkpoints, which allow the propagation of DNA damage. However, these viral proteins are not expressed in EBV-carrying BLs, and only half of HDs and NPCs express detectable levels of LMP1, suggesting a limited role in EBV oncogenesis. A possible involvement of EBNA-1 in the induction of genomic instability is suggested by a significant increase of transient chromosomal aberrations, such as dicentric chromosomes, chromosome fragments, and gaps, in EBVpositiv...

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Section: Methodsmentioning

confidence: 99%

The Epstein–Barr virus nuclear antigen-1 promotes genomic instability via induction of reactive oxygen species

Gruhne

Sompallae

Marescotti

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

213

189

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“…For the patients with melanoma, even considering the high heterogeneity identified in the basal patient's cells, it did not increase significantly the frequency of Comets after in-vitro cisplatin treatment. These data could not be compared with other published papers that evaluated the cisplatin action in vitro only in control groups and not in patients with melanoma before starting the treatment [22,28]. The patient's resistance to cisplatin treatment represents the main obstacle for a successful chemotherapy.…”

Section: Discussionmentioning

confidence: 91%

“…In this study, the 5-h repair time after cisplatin treatment apparently was not sufficient to identify whether an efficient repair would have been achieved. Even considering that similar results were published earlier [28], the longer in-vitro leukocyte cell culture maintenance was not preferred because it could interfere in the final Comet evaluation showing damage other than DNA related to cisplatin. In fact, patients who had metastatic colorectal cancer and were undergoing oxaliplatin chemotherapy presented different responses to the efficiency of DNA repair by the Comet assay [35].…”

Section: Discussionmentioning

confidence: 99%

DNA damage and repair in leukocytes of melanoma patients exposed in vitro to cisplatin

Shimabukuro¹,

Neto²,

Sanches³

et al. 2011

Melanoma Research

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Resistance to chemotherapeutic drugs can be an obstacle to a successful treatment of cancer patients in part associated with individual response and differences in the DNA repair system. The Comet assay is an informative test to investigate DNA damage and repair in cells in response to a variety of DNA-damaging agents, including chemotherapeutic drugs. The aim of this study was to assess leukocytes damage after in-vitro cisplatin treatment and DNA repair action using the Comet assay in 20 patients with melanoma and 20 cancer-free individuals. Leukocytes' DNA damage before and after cisplatin treatment, in three different concentrations, was analyzed. The DNA repair capability was investigated after 1-5 h of in-vitro cells growing without cisplatin. The Comet score of the patients' basal DNA damage was higher than that observed in controls, but the difference was not statistically significant (P=0.85). Although both groups had similar Comet scores to all cisplatin concentrations tested and the DNA repair times, the basal DNA damage (P<0.001) and cisplatin damages (P<0.005) were statistically lower than the different repair times investigated. Considering the progressive increase in the Comet score due to repair time, the negative results here observed could be associated with the reduced cell culture incubation that should be better evaluated. Considering the mutagenic action of cisplatin on tumor cells and the importance of individual DNA repair mechanisms in the chemotherapeutic melanoma treatment, the peripheral leukocytes could be particularly useful as a tool for DNA repair response identified by the Comet assay.

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“…After cisplatin treatment, cells were incubated with 10µM H 2 O 2 , which introduces a number of strand breaks into the DNA and allows for the identification of cross-linking damage by the assay (Blasiak et al, 1999;Heringova et al, 2006;Shimabukuro et al, 2011). Briefly, 10µl of cells was added to 100µl 0.5% low-melting-point agarose at 37ºC.…”

Section: Comet Assaymentioning

confidence: 99%

No Relationship between the Amount of DNA Damage and the Level of hMLH1 and RASSF1A Gene Expression in Bladder Cancer Cells Treated with Cisplatin and Gemcitabine

Camargo

Silva²,

Gobette³

et al. 2013

Asian Pacific Journal of Cancer Prevention

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Tumor response to antineoplastic drugs is not always predictable. This is also true for bladder carcinoma, a highly recurrent neoplasia. Currently, the combination of cisplatin and gemcitabine is well accepted as a standard protocol for treating bladder carcinoma. However, in some cases, this treatment protocol causes harmful side effects. Therefore, we investigated the roles of the genes TP53, RASSF1A (a tumor suppressor gene) and hMLH1 (a gene involved in the mismatch repair pathway) in cell susceptibility to cisplatin/gemcitabine treatment. Two bladder transitional carcinoma cell (TCC) lines, RT4 (wild-type TP53) and 5637 (mutated TP53), were used in this study. First, we evaluated whether the genotoxic potential of cisplatin/gemcitabine was dependent on TP53 status. Then, we evaluated whether the two antineoplastic drugs modulated RASSF1A and hMLH1 expression in the two cell lines. Increased DNA damage was observed in both cell lines after treatment with cisplatin or gemcitabine and with the two drugs simultaneously, as depicted by the comet assay. A lack of RASSF1A expression and hypermethylation of its promoter were observed before and after treatment in both cell lines. On the other hand, hMLH1 downregulation, unrelated to methylation status, was observed in RT4 cells after treatment with cisplatin or with cisplatin and gemcitabine simultaneously (wild-type TP53); in 5637 cells, hMLH1 was upregulated only after treatment with gemcitabine. In conclusion, the three treatment protocols were genotoxic, independent of TP53 status. However, cisplatin was the most effective, causing the highest level of DNA damage in both wild-type and mutated TP53 cells. Gemcitabine was the least genotoxic agent in both cell lines. Furthermore, no relationship was observed between the amount of DNA damage and the level of hMLH1 and RASSF1A expression. Therefore, other alternative pathways might be involved in cisplatin and gemcitabine genotoxicity in these two bladder cancer cell lines.

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DNA damage and repair in human lymphocytes exposed to three anticancer platinum drugs

Cited by 33 publications

References 30 publications

The Epstein–Barr virus nuclear antigen-1 promotes genomic instability via induction of reactive oxygen species

The Epstein–Barr virus nuclear antigen-1 promotes genomic instability via induction of reactive oxygen species

DNA damage and repair in leukocytes of melanoma patients exposed in vitro to cisplatin

No Relationship between the Amount of DNA Damage and the Level of hMLH1 and RASSF1A Gene Expression in Bladder Cancer Cells Treated with Cisplatin and Gemcitabine

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