Transfer of Ku86 RNA antisense decreases the radioresistance of human fibroblasts

Marangoni, Elisabetta; Romancer, Muriel Le; Foray, Nicolas; Müller, Cathérine; Douc‐Rasy, Sétha; Vaganay, Sabine; Abdulkarim, Bassam; Barrois, Michel; Calsou, Patrick; Bernier, Jacques; Salles, Bernard; Bourhis, Jean

doi:10.1038/sj.cgt.7700111

Cited by 41 publications

(27 citation statements)

References 35 publications

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“…4 and 5). These experiments are consistent with the demonstration that the expression of Ku86 antisense RNA in human cells leads to cellular defects in DNA DSB repair and cell proliferation (50,51). Thus, our data suggest that species differences exist in the requirement for Ku86 and that Ku86 performs an essential function in human cells.…”

Section: Discussionsupporting

confidence: 91%

Ku86 is essential in human somatic cells

Nelsen

Hendrickson

2002

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

174

166

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Ku86 plays a key role in nonhomologous end joining in mammals. Functional inactivation in rodents of either Ku86 or Ku70, which form the heterodimeric DNA end-binding subunit of the DNAdependent protein kinase complex, is nevertheless compatible with viability. In contrast, no human patient has been described with mutations in either Ku86 or Ku70. This has led to the hypotheses that either these genes are performing an additional essential role(s) and͞or redundant pathways exist that mask the phenotypic expression of these genes when they are mutated in humans. To address this issue, we describe here the construction of human somatic cell lines containing a targeted disruption of the Ku86 locus. Human HCT116 colon cancer cells heterozygous for Ku86 were haploinsufficient with an increase in polyploid cells, a reduction in cell proliferation, elevated p53 levels, and a slight hypersensitivity to ionizing radiation. Functional inactivation of the second Ku86 allele resulted in cells with a drastically reduced doubling time. These cells were capable of undergoing only a limited number of cell divisions, after which they underwent apoptosis. These experiments demonstrate that the Ku86 locus is essential in human somatic tissue culture cells.

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Section: Discussionsupporting

confidence: 91%

Ku86 is essential in human somatic cells

Nelsen

Hendrickson

2002

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

174

166

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“…Previous studies indicated that Ku is the major DNA end binding activity in cell extracts (30). As shown in Fig.…”

Section: Ku Represses the Transcription Controlled By The Hiv-1 Ltr Imentioning

confidence: 77%

“…Thus, to address that issue in human cells, chronically HIV-1-infected U1 cells bearing integrated proviruses were transduced with a retroviral vector carrying a Ku80 full-length antisense RNA. Indeed, the antisense strategy can be used to study the effect of Ku depletion in human cells, as it was demonstrated that antisense sequences directed against Ku80 were capable of impairing the expression of the protein in human cell lines, notably human colon carcinoma cells HCT116 (31) and human fibroblast cells MRC5V1 (30). Antisense directed against Ku70 were also used to deplete human T cell lymphoma MT2 cells (32).…”

Section: Discussionmentioning

confidence: 99%

Ku Represses the HIV-1 Transcription

Jeanson

Mouscadet

2002

Journal of Biological Chemistry

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Ku has been implicated in nuclear processes, including DNA break repair, transcription, V(D)J recombination, and telomere maintenance. Its mode of action involves two distinct mechanisms: one in which a nonspecific binding occurs to DNA ends and a second that involves a specific binding to negative regulatory elements involved in transcription repression. Such elements were identified in mouse mammary tumor virus and human T cell leukemia virus retroviruses. The purpose of this study was to investigate a role for Ku in the regulation of human immunodeficiency virus (HIV)-1 transcription. First, HIV-1 LTR activity was studied in CHO-K1 cells and in CH0-derived xrs-6 cells, which are devoid of Ku80. LTR-driven expression of a reporter gene was significantly increased in xrs-6 cells. This enhancement was suppressed after re-expression of Ku80. Second, transcription of HIV-1 was followed in U1 human cells that were depleted in Ku by using a Ku80 antisense RNA. Ku depletion led to a increase of both HIV-1 mRNA synthesis and viral production compared with the parent cells. These results demonstrate that Ku acts as a transcriptional repressor of HIV-1 expression. Finally, a putative Ku-specific binding site was identified within the negative regulatory region of the HIV-1 long terminal repeat, which may account for this repression of transcription.

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“…Antisense and siRNA strategies Marangoni et al (2000b) used a full-length inverted cDNA to target Ku80 in human fibroblast cell lines. This resulted in a DRF at 10% IR survival of B1.7 for stable cell lines expressing the Ku80 antisense compared to control cell lines, which was likely a consequence of reduced DNA end-binding capacity and DSB repair rates observed in these cells.…”

Section: Molecular Targeting Of Dna-pkmentioning

confidence: 99%

The life and death of DNA-PK

et al. 2004

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Double-strand breaks (DSBs) arise endogenously during normal cellular processes and exogenously by genotoxic agents such as ionizing radiation (IR). DSBs are one of the most severe types of DNA damage, which if left unrepaired are lethal to the cell. Several different DNA repair pathways combat DSBs, with nonhomologous endjoining (NHEJ) being one of the most important in mammalian cells. Competent NHEJ catalyses repair of DSBs by joining together and ligating two free DNA ends of little homology (microhomology) or DNA ends of no homology. The core components of mammalian NHEJ are the catalytic subunit of DNA protein kinase (DNA-PK cs ), Ku subunits Ku70 and Ku80, Artemis, XRCC4 and DNA ligase IV. DNA-PK is a nuclear serine/threonine protein kinase that comprises a catalytic subunit (DNA-PK cs ), with the Ku subunits acting as the regulatory element. It has been proposed that DNA-PK is a molecular sensor for DNA damage that enhances the signal via phosphorylation of many downstream targets. The crucial role of DNA-PK in the repair of DSBs is highlighted by the hypersensitivity of DNA-PK À/À mice to IR and the high levels of unrepaired DSBs after genotoxic insult. Recently, DNA-PK has emerged as a suitable genetic target for molecular therapeutics such as siRNA, antisense and novel inhibitory small molecules. This review encompasses the recent literature regarding the role of DNA-PK in the protection of genomic stability and focuses on how this knowledge has aided the development of specific DNA-PK inhibitors, via both small molecule and directed molecular targeting techniques. This review promotes the inhibition of DNA-PK as a valid approach to enhance the tumor-cell-killing effects of treatments such as IR. The double-strand break (DSB) is generally regarded as the most lethal of all DNA lesions, which if unrepaired severely threatens not only the integrity of the genome but also survival of the organism (Hoeijmakers, 2001;van Gent et al., 2001;Vilenchik and Knudson, 2003). DSBs can arise endogenously by cellular processes such as cleavage during immunoglobulin gene rearrangement (V(D)J recombination) and meiotic recombination. DSBs are also produced by exposure to ionizing radiation (IR), radiomimetic drugs, such as bleomycin, and the collapse of replication forks when the replication machinery encounters singlestranded breaks (SSBs) (Haber, 2000;Karran, 2000;Norbury and Hickson, 2001;Bassing et al., 2002). Unrepaired DSBs can activate cell cycle checkpoint arrests and signal for cell death (Jackson, 2001;Norbury and Hickson, 2001). Possibly even more detrimental to the cell are the unrepaired or misrepaired DSBs that lead to genomic rearrangements which ultimately destabilize the genome, a phenotype observed in a large number of malignancies (Lengauer et al., 1998;Hoeijmakers, 2001;Elliott and Jasin, 2002;Thompson and Schild, 2002;Shiloh, 2003;Vilenchik and Knudson, 2003). To complicate matters further, the location of the DSBs and the structure of the damaged DNA ends can vary depending on the damaging agent....

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Transfer of Ku86 RNA antisense decreases the radioresistance of human fibroblasts

Cited by 41 publications

References 35 publications

Ku86 is essential in human somatic cells

Ku86 is essential in human somatic cells

Ku Represses the HIV-1 Transcription

The life and death of DNA-PK

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