Impaired NHEJ function in multiple myeloma

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 beta-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 gamma-H2AX response in OPM2 but only a modest increase in each radiation sensitive cell line perhaps related to the high level of gamma-H2AX in freshly plated cells. Examination of gamma-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 24h 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.

Fig. 1. Effect of NU7026 on cell survival after irradiation

Cells were pre-treated with 0µM (●), 2.5µM (♦) or 10µM (■) of NU7026 and then irradiated with 0, 2, 4, 6, 8 Gy. A) RPMI 8226, B) U266, C) OPM2. Only OPM2 cells showed the predicted decrease in survival after treatment with the DNA-PK inhibitor. Each point on the survival curve represents the surviving fraction ± SE from three separate experiments.

Fig. 2

Fig. 2A. Plasmid based in-vitro rejoining assay to detect repair. Here LM-PCR is performed with cellular extracts from specific cells. W = water control, L = DNA ligase, MJ = MO59J, MK = MO59K, Co = Uncut vector, U2 = U266, O = OPM2, RP = RPMI 8226. DNA-PK deficient MO59J gives no product though DNA-PK proficient MO59K does. Only U226 and OPM2 gives product and not RPMI 8226, indicating deficit in RPMI 8226 rejoining. Wortmannin eliminates all evidence of rejoining confirming NHEJ pathway is being used. Representative reaction of 3 individual experiments is shown. Fig. 2B. Plasmid in-vitro rejoining assay to determine repair fidelity. i) Schematic representation of the experimental design for plasmid in-vitro rejoining assay. ii) Percent of white colonies obtained from different cell lines. Here 1 = MO59J, 2 = MO59K, 3 = RPMI 8226, 4 = U266, 5 = OPM2, 6 = T4 ligase. Thus DNA-PK deficient MO59J, RPMI 8226 and OPM-2 exhibit significantly reduced repair fidelity compared to MO59K, U266 and T4 ligase groups.

Fig. 3. Protein levels of γ-H2AX

Representative western blot of γ-H2AX levels both before and after 4 Gy irradiation. A) RPMI 8226, B) U266, C) OPM2. Lane 1: No irradiation, Lane 2: 0 h, 4 Gy Lane 3: 2 h, 4 Gy, Lane 4: 24 h, 4 Gy. Histogram beneath each lane shows fold changes in γ-H2AX expression, determined by densitometry, for each data point compared to actin controls run with each experiment.

Fig. 4. Effect of radiation on γ-H2AX foci formation in RPMI 8226 cells

A) Cells were irradiated with either 0 or 4 Gy and fixed after different time points for immunocytochemical analysis of γ-H2AX foci. A. Foci detected either 1 h or 24 h after either no treatment (left) or exposure to 4 Gy (right). B) Numerical analysis of foci numbers after either no treatment (white) or exposure to 4 Gy (gray). Data illustrates average of three separate experiments.