A quasi-quantitative dual multiplexed immunoblot method to simultaneously analyze ATM and H2AX Phosphorylation in human peripheral blood mononuclear cells

Abstract

Pharmacologic inhibition of DNA repair may increase the efficacy of many cytotoxic cancer agents. Inhibitors of DNA repair enzymes including APE1, ATM, ATR, DNA-PK and PARP have been developed and the PARP inhibitor olaparib is the first-in-class approved in Europe and the USA for the treatment of advanced BRCA-mutated ovarian cancer. Sensitive pharmacodynamic (PD) biomarkers are needed to further evaluate the efficacy of inhibitors of DNA repair enzymes in clinical trials. ATM is a protein kinase that mediates cell-cycle checkpoint activation and DNA double-strand break repair. ATM kinase activation at DNA double-strand breaks (DSBs) is associated with intermolecular autophosphorylation on serine-1981. Exquisite sensitivity and high stoichiometry as well as facile extraction suggest that ATM serine-1981 phosphorylation may be a highly dynamic PD biomarker for both ATM kinase inhibitors and radiation- and chemotherapy-induced DSBs. Here we report the pre-clinical analytical validation and fit-for-purpose biomarker method validation of a quasi-quantitative dual multiplexed immunoblot method to simultaneously analyze ATM and H2AX phosphorylation in human peripheral blood mononuclear cells (PBMCs). We explore the dynamics of these phosphorylations in PBMCs exposed to chemotherapeutic agents and DNA repair inhibitors in vitro, and show that ATM serine-1981 phosphorylation is increased in PBMCs in sarcoma patients treated with DNA damaging chemotherapy.

Keywords: ATM; DNA damage response; H2AX; PBMCs; doxorubicin.

Figure 1. Effect of singleplex and multiplex antibody combinations on ATM (A) and H2AX (B) protein detection

Whole blood was irradiated with 2 Gy and PBMCs were isolated and processed for immunoblot analysis. Primary antibodies were added separately and in combination and indicated proteins were detected at 700 and 800 nm. The blots are representative images for one of three experiments.

Figure 2. Effect of PBMC lysate dilution on protein quantitation

Cellular lysates, prepared from lllltreated (A,D) and 2 Gy irradiated PBMCs (B,E,) from subject B were serially diluted and processed by ATM (A,B) and H2AX (C,D) immunoblot analysis. Fold activation ofATM (C) and H2AX (F) were analyzed as a function ofPBMCs per gel lane.

Figure 3. Effect of storage time on protein fold activation

Blood tubes from three subjects were 2 Gy irradiated and PBMCs were isolated and stored at −70°C. Immediately after irradiation and over 46 weeks, PBMC pellets were analyzed for ATM (A) and H2AX activation (B).

Figure 4. Effect of various chemotherapeutic agents on DNA damage response in PBMCs

PBMCs were incubated with the indicated agents for 6 and 24 hr. Cells were harvested and processed for immunoblot analysis. The values represent the mean ± S.D. from duplicate measurements from three independent experiments.

Figure 5. Effect of doxorubicin in combination with kinase inhibitors on DNA damage response in PBMCs

PBMCs were incubated with kinase inhibitors in the presence or absence of doxorubicin (1.7 uM) for 6 hr. Cells were harvested and processed for immunoblot analysis. Data from two separate experiments is shown.

Figure 6. Protein expression after irradiation

PBMCs and IMR90 lung fibroblasts were irradiated and processed for immunoblot analysis.

Figure 7. DNA damage response in patient PBMCs

PBMCs were obtained from three patients treated with doxorubicin/ifosfamide. Samples were processed for immunoblot analysis for detection of ATM (A) and H2AX activation (B). (C) Image of H2AX immunoblots in PBMCs from patient#2.