Impaired NHEJ function in multiple myeloma

Clara Yang, Christopher Betti, Sheetal Singh, Amir Toor, Andrew T M Vaughan

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

Multiple myeloma (MM) is characterized by multiple chromosomal aberrations. To assess the contribution of DNA repair to this phenotype, ionizing radiation was used to induce DNA double strand breaks in three MM cell lines. Clonogenic survival assays showed U266 (SF4 = 15.3 + 6.4%) and RPMI 8226 (SF4 = 12.6.0 + 1.7%) were radiation sensitive while OPM2 was resistant (SF4 = 78.9 + 4.1%). Addition of the DNA-PK inhibitor NU7026 showed the expected suppression in radiation survival in OPM2 but increased survival in both radiation sensitive cell lines. To examine non-homologous end joining (NHEJ) repair in these lines, the ability of protein extracts to support in vitro DNA repair was measured. Among the three MM cell lines analyzed, RPMI 8226 demonstrated impaired blunt ended DNA ligation using a ligation-mediated PCR technique. In a bacterial based functional assay to rejoin a DNA break within the β-galactosidase gene, RPMI 8226 demonstrated a 4-fold reduction in rejoining fidelity compared to U266, with OPM2 showing an intermediate capacity. Ionizing radiation induced a robust γ-H2AX response in OPM2 but only a modest increase in each radiation sensitive cell line perhaps related to the high level of γ-H2AX in freshly plated cells. Examination of γ-H2AX foci in RPMI 8226 cells confirmed data from Western blots where a significant number of foci were present in freshly plated untreated cells which diminished over 24 h of culture. Based on the clonogenic survival and functional repair assays, all three cell lines exhibited corrupt NHEJ repair. We conclude that suppression of aberrant NHEJ function using the DNA-PK inhibitor NU7026 may facilitate access of DNA ends to an intact homologous recombination repair pathway, paradoxically increasing survival after irradiation. These data provide insight into the deregulation of DNA repair at the site of DNA breaks in MM that may underpin the characteristic genomic instability of this disease.

Original languageEnglish (US)
Pages (from-to)66-73
Number of pages8
JournalMutation Research - Fundamental and Molecular Mechanisms of Mutagenesis
Volume660
Issue number1-2
DOIs
StatePublished - Jan 15 2009

Fingerprint

Multiple Myeloma
Cell Line
DNA Repair
Radiation
DNA Breaks
DNA
Ionizing Radiation
Ligation
Galactosidases
Recombinational DNA Repair
Double-Stranded DNA Breaks
Genomic Instability
Chromosome Aberrations
Western Blotting
Phenotype
Polymerase Chain Reaction
Genes
sulfur tetrafluoride
Proteins
2-(morpholin-4-yl)benzo(h)chromen-4-one

Keywords

  • γ-H2AX foci
  • DNA double strand break
  • LM-PCR
  • Multiple myeloma
  • Non-homologous end joining

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Health, Toxicology and Mutagenesis

Cite this

Impaired NHEJ function in multiple myeloma. / Yang, Clara; Betti, Christopher; Singh, Sheetal; Toor, Amir; Vaughan, Andrew T M.

In: Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis, Vol. 660, No. 1-2, 15.01.2009, p. 66-73.

Research output: Contribution to journalArticle

Yang, Clara ; Betti, Christopher ; Singh, Sheetal ; Toor, Amir ; Vaughan, Andrew T M. / Impaired NHEJ function in multiple myeloma. In: Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis. 2009 ; Vol. 660, No. 1-2. pp. 66-73.
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AB - Multiple myeloma (MM) is characterized by multiple chromosomal aberrations. To assess the contribution of DNA repair to this phenotype, ionizing radiation was used to induce DNA double strand breaks in three MM cell lines. Clonogenic survival assays showed U266 (SF4 = 15.3 + 6.4%) and RPMI 8226 (SF4 = 12.6.0 + 1.7%) were radiation sensitive while OPM2 was resistant (SF4 = 78.9 + 4.1%). Addition of the DNA-PK inhibitor NU7026 showed the expected suppression in radiation survival in OPM2 but increased survival in both radiation sensitive cell lines. To examine non-homologous end joining (NHEJ) repair in these lines, the ability of protein extracts to support in vitro DNA repair was measured. Among the three MM cell lines analyzed, RPMI 8226 demonstrated impaired blunt ended DNA ligation using a ligation-mediated PCR technique. In a bacterial based functional assay to rejoin a DNA break within the β-galactosidase gene, RPMI 8226 demonstrated a 4-fold reduction in rejoining fidelity compared to U266, with OPM2 showing an intermediate capacity. Ionizing radiation induced a robust γ-H2AX response in OPM2 but only a modest increase in each radiation sensitive cell line perhaps related to the high level of γ-H2AX in freshly plated cells. Examination of γ-H2AX foci in RPMI 8226 cells confirmed data from Western blots where a significant number of foci were present in freshly plated untreated cells which diminished over 24 h of culture. Based on the clonogenic survival and functional repair assays, all three cell lines exhibited corrupt NHEJ repair. We conclude that suppression of aberrant NHEJ function using the DNA-PK inhibitor NU7026 may facilitate access of DNA ends to an intact homologous recombination repair pathway, paradoxically increasing survival after irradiation. These data provide insight into the deregulation of DNA repair at the site of DNA breaks in MM that may underpin the characteristic genomic instability of this disease.

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