Evidence for ongoing DNA damage in multiple myeloma cells as revealed by constitutive phosphorylation of H2AX

Abstract

DNA double-strand breaks (DSBs) are deleterious lesions that can lead to chromosomal anomalies, genomic instability and cancer. The histone protein H2AX has an important role in the DNA damage response (DDR) and the presence of phospho-H2AX (γH2AX) nuclear foci is the hallmark of DSBs. We hypothesize that ongoing DNA damage provides a mechanism by which chromosomal abnormalities and intratumor heterogeneity are acquired in malignant plasma cells (PCs) in patients with multiple myeloma (MM). Therefore, we assessed PCs from patients with the premalignant condition, monoclonal gammopathy of undetermined significance (MGUS) and MM, as well as human MM cell lines (HMCLs) for evidence of DSBs. γH2AX foci were detected in 2/5 MGUS samples, 37/40 MM samples and 6/6 HMCLs. Notably, the DSB response protein 53BP1 colocalized with γH2AX in both MM patient samples and HMCLs. Treatment with wortmannin decreased phosphorylation of H2AX and suggests phosphoinositide (PI) 3-kinases and/or PI3-kinase-like family members underlie the presence of γH2AX foci in MM cells. Taken together, these data imply that ongoing DNA damage intensifies across the disease spectrum of MGUS to MM and may provide a mechanism whereby clonal evolution occurs in the monoclonal gammopathies.

Figure 1. Detection of γH2AX nuclear foci in MM patient samples and HMCLs

A) IF detection of γH2AX nuclear foci in freshly isolated CD138+ cells of 5 MM patients and 1 MGUS patient using an antiγH2AX-FITC Ab (green), anti-Ig (H + L) Ab (red) and DAPI (blue). B) Representative IF detection of nuclear γH2AX foci in HMCLs using an anti-γH2AX-FITC Ab (green) and DAPI (blue). C) The number of γH2AX foci/cell present in 50 individual cells for each HMCL. D) Representative IF detection of nuclear γH2AX foci in normal BM-PCs and normal B cells using an anti-γH2AX-FITC Ab (green) and DAPI (blue).

Figure 2. γH2AX foci do not correlate with phase of the cell cycle and γH2AX colocalizes with 53BP1

A) IF analysis of BrdU labeled ANBL-6 cells revealed both BrdU positive (S/G2M phase) and BrdU negative (G1) cells possessing γH2AX foci. B) No correlation is observed between the % of cells possessing γH2AX foci in primary MM patient samples and their respective PCLI. C) HMCLs and primary MM patient samples were double stained with an anti-γH2AX-FITC Ab (green) and an anti-53BP1/Alexa 594 Ab (red). The resulting images were then overlaid in order to determine colocalization. Representative results are shown using one MM patient sample as well as the HMCL ANBL-6.

Figure 3. Activation of DNA damage signaling molecules in HMCLs

A) Immunoblotting demonstrates that HMCLs show evidence of constitutively activated DNA damage signaling molecules while normal control cells lack constitutive activation. Cell lines were maintained under normal culture conditions. B) Irradiation induced activation of P-ATM in the ANBL-6 HMCL. Irradiated cells were allowed to recover for 30 minutes.

Figure 4. PI3-kinase inhibition decreases γH2AX

HMCLs were either untreated or treated for 1 hr with 20 μM or 200 μM wortmannin and analyzed via immunoblot.

Figure 5. HMCLs express high levels of p53 and possess p53 mutations

A) Immunoblotting demonstrates that HMCLs generally show evidence of high levels of expression of p53. Cell lines were maintained under normal culture conditions. B) Specific p53 mutations harbored by each HCML.