P53-independent caspase-mediated apoptosis in human leukaemic cells is induced by a DNA minor groove binder with antineoplastic activity.
Treatment of HL60 and Jurkat leukaemic cell lines, both not expressing p53, with the new non-covalent DNA minor groove binder alpha-bromoacryloyl-distamycin (PNU 151807), induces apoptosis as shown either morphologically or by DNA laddering formation. We evaluated the p53-independent mechanisms of activation of apoptosis in these cell systems, by determining the levels of different caspases at different times after treatment with PNU 151807. Through Western blotting analysis we could measure the cleavage of the 110 Kd form of PARP in both HL60 and Jurkat cells and correspondingly the activation of CPP32-caspase 3. The levels of caspase-1 did not change at any time after drug treatment. By using the tetrapeptidic sequence recognized by caspase-3 (DEVD-AMC) or by caspase-1 (YVAD-AMC) linked to fluorogenic substrate, we also demonstrated that only the DEVD sequence was recognized and cleaved after drug treatment, while no significant changes were found for YVAD peptides. PNU 151807-induced DNA fragmentation and DEVD-cleavage were both inhibited by concomitant treatment with the specific inhibitor DEVD-CHO, but not by YVAD-CHO, clearly demonstrating the direct activation of caspase-3-like caspases in the induction of programmed cell death in these cell lines after minor groove binder exposure.
Marchini
S
S
Molecular Pharmacology Unit, Department of Oncology, Istituto di Ricerche, Farmacologiche Mario Negri, Via Eritrea 62, 20157 Milan, Italy.
Ciro'
M
M
Broggini
M
M