Cisplatin enhances radiation-induced apoptosis in peripheral lymphocytes of cancer patients: in vivo and in vitro findings.

BACKGROUND: Concurrent cisplatin and radiotherapy frequently cause severe acute hematologic toxicity. This study investigated whether lymphocyte apoptosis, quantified via the γ-H2AX pan-nuclear immunostaining assay, can serve as a reliable biomarker for the acute hematologic side effects of combined chemoradiation. METHODS: Blood samples were collected from cancer patients and healthy controls. Lymphocyte apoptosis was evaluated using the γ-H2AX pan-nuclear assay following in vitro and in vivo exposure to cisplatin and ionizing radiation (IR). In PBMCs, γ-H2AX results strongly correlated with conventional caspase-3 (r = 0.9736) and annexin V (r = 0.9780) apoptosis assays. Additionally, in vitro experiments assessed how phytohemagglutinin-L (PHA-L)-induced lymphocyte proliferation influences the combined effects of cisplatin and IR on apoptosis, alongside the regulatory role of the ATM protein. RESULTS: In vitro, cisplatin increased lymphocyte apoptosis asymptotically, plateauing at 10 µg/ml (11.6 ± 5.0%). Combined cisplatin and IR treatment increased apoptosis in a strictly additive manner (radiation effect: p < 0.0001), with no significant synergistic interaction effect between the two modalities (p = 0.22). The apoptotic effect level of in vivo cisplatin administration did not differ significantly from the maximum effect observed following a 1-hour in vitro exposure to high cisplatin concentrations. In vivo, the additive effect of cisplatin on IR-induced apoptosis peaked up to 3 days post-administration before returning to baseline by day 6. Consistent with these findings, cisplatin and radiation induced a significant, additive increase in apoptosis within PHA-L-stimulated lymphocytes. Inhibition of ATM has no effect on apoptosis following treatment with cisplatin and IR. CONCLUSIONS: The γ-H2AX pan-nuclear assay provides a valid, reliable method for quantifying radiation- and chemotherapy-induced lymphocyte damage. The time-dependent, additive toxicity observed indicates a critical clinical window (up to 3 days post-cisplatin) for maximum hematologic damage, allowing for optimization of treatment schedules. CLINICAL TRIAL NUMBER: Not applicable.