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- Elmroth, Kerstin, 1970, et al.
(författare)
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Cleavage of cellular DNA by calicheamicin γ1
- 2003
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Ingår i: DNA Repair. - 1568-7864 .- 1568-7856. ; 2:4, s. 363-374
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Tidskriftsartikel (refereegranskat)abstract
- It is assumed that the efficient antitumor activity of calicheamicin γ1 is mediated by its ability to introduce DNA double-strand breaks in cellular DNA. To test this assumption we have compared calicheamicin γ1-mediated cleavage of cellular DNA and purified plasmid DNA. Cleavage of purified plasmid DNA was not inhibited by excess tRNA or protein indicating that calicheamicin γ1 specifically targets DNA. Cleavage of plasmid DNA was not affected by incubation temperature. In contrast, cleavage of cellular DNA was 45-fold less efficient at 0°C as compared to 37° due to poor cell permeability at low temperatures. The ratio of DNA double-strand breaks (DSB) to single-stranded breaks (SSB) in cellular DNA was 1:3, close to the 1:2 ratio observed when calicheamicin γ1 cleaved purified plasmid DNA. DNA strand breaks introduced by calicheamicin γ1 were evenly distributed in the cell population as measured by the comet assay. Calicheamicin γ1-induced DSBs were repaired slowly but completely and resulted in high levels of H2AX phosphorylation and efficient cell cycle arrest. In addition, the DSB-repair deficient cell line Mo59J was hyper sensitive to calicheamicin γ. The data indicate that DSBs is the crucial damage after calicheamicin γ1 and that calicheamicin γ1-induced DSBs are recognized normally. The high DSB:SSB ratio, specificity for DNA and the even damage distribution makes calicheamicin γ1 a superior drug for studies of the DSB-response and emphasizes its usefulness in treatment of malignant disease.
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| 2. |
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| 3. |
- Zander, Linda, 1976, et al.
(författare)
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Immortalized mouse cell lines that lack a functional Rev3 gene are hypersensitive to UV irradiation and cisplatin treatment.
- 2004
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Ingår i: DNA repair. - : Elsevier BV. - 1568-7864. ; 3:7, s. 743-52
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Tidskriftsartikel (refereegranskat)abstract
- The catalytic subunit of polymerase zeta is encoded from the Rev3 gene. The enzyme is conserved through eukaryotic evolution and its main function appears to be translesion synthesis (TLS) over damaged bases that stall DNA replication. In non-vertebrate cells, inactivation of polymerase zeta results in a moderate hypersensitivity to DNA damage but no proliferative defect in the absence of exogenous damage. Mouse embryos that lack Rev3 however have a severe growth defect and are aborted at midgestation. This has suggested that polymerase zeta may be involved in vital processes in mammalian cells. Here we describe the establishment of immortalized mouse fibroblast cell lines that lack a functional Rev3 gene. These were established from homozygously Rev3-targeted mouse embryos that were also heterozygously targeted at the p53 locus, but the cell lines lost the wild type p53 allele during transformation. Cell lines in which the Rev3 gene is targeted on both alleles grow more slowly than control lines and the deficiency is also associated with an increased frequency of cells at the G2/M phase of the cell cycle and augmented apoptosis. Targeted cells are hypersensitive to UV irradiation and cisplatin treatment and arrest at the S or G2/M phase of the cell cycle if exposed to these treatments. Thus, although vital for murine embryonic development, polymerase zeta activity is not essential for continuous proliferation of transformed mammalian cells that lack p53. It does, however, appear to play an important role in allowing mammalian cells to tolerate DNA damage.
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