Quantitative analysis of ATM phosphorylation in lymphocytes

Area of Science:

• Biochemistry
• Molecular Biology
• Cancer Research

Background:

• Anticancer therapies often target DNA and DNA damage response (DDR) pathways.
• Biomarkers for DNA damage endpoints are crucial for cancer research.
• Ataxia telangiectasia-mutated (ATM) kinase is a key regulator of cellular responses to DNA double-strand breaks (DSBs).

Purpose of the Study:

• To validate a quantitative dual-labeled immunoblot assay for measuring phosphorylated ATM (p-S1981-ATM) and total ATM (pan-ATM).
• To assess the ATM kinase autophosphorylation response in human peripheral blood mononuclear cells (PBMCs) after ionizing radiation (IR) exposure.
• To investigate potential sex-based differences in the ATM DDR pathway.

Main Methods:

• Developed and validated a quantitative dual-labeled immunoblot assay.
• Simultaneously measured p-S1981-ATM and pan-ATM in human PBMCs.
• Exposed PBMCs ex vivo to 2 Gy of IR and calculated the percentage of phosphorylated ATM (%p-ATM).
• Analyzed samples from 41 healthy volunteers.

Main Results:

• The assay demonstrated analytical characteristics and fit-for-purpose validation.
• Basal %p-ATM in PBMCs was 4%, increasing to 63% after 2 Gy IR exposure.
• PBMCs from women showed a 2.6-fold higher median p-S1981-ATM level post-IR compared to men (p < 0.01).
• Women exhibited a significantly greater %p-ATM (68%) than men (49%) after IR exposure (p < 0.01).

Conclusions:

• The validated immunoblot assay provides a reliable method for quantifying ATM activation in response to DNA damage.
• The ATM DNA damage response pathway exhibits a sex-specific difference, with women showing a heightened response.
• These findings have significant implications for understanding DDR and developing personalized cancer therapies.