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Reduction in the Radiation-induced Late S Phase and G₂ Blocks in HL-60 Cell Populations by Amiloride, an Efficient Inhibitor of the Na⁺/H⁺ Transporter¹

Los Alamos National Laboratory, Life Sciences Division, Los Alamos, New Mexico 87545 [B. L. S., A. M. B., J. G. V., J. A. D., H. A. C.], and Universidad de Alcalá de Henares, Alcalá de Henares, 28871 Madrid, Spain [J. M. C.]

Recent investigations that showed that amiloride delayed or inhibited apoptosis indicated it might also attenuate cell cycle checkpoints activated by ionizing radiation. In this report, single- and dual-parameter flow cytometry were used to investigate the effects of amiloride on cell cycle progression, and the effectiveness of amiloride to attenuate the S and G₂ phase checkpoint responses induced by 2.5, 5.0, and 7.5 Gy of gamma radiation. The late S-phase delay, noted at 8 h following irradiation, and a radiation-induced G₂ block, which was maximum at 16 h after irradiation, were both significantly reduced in amiloride-treated samples. Attenuation of the radiation-induced late S phase and G₂ blocks resulted in cell division without apparent apoptosis or necrosis over a 24-h period. Results presented indicate that amiloride reduces the radiation-induced G₂ block in HL-60 cell populations almost equally well as caffeine and to a greater extent than staurosporine. Immunofluorescent detection and quantitation of cyclin B1 expression demonstrated that amiloride only significantly reduced cyclin B1 expression following 5.0 Gy, when there was a notable induction of a significant G₂ delay, followed by a relatively rapid recovery in cycling potential. The results suggest that amiloride affects the radiation-triggered signaling cascades to alter the kinase activity of proteins associated with mitotic progression, particularly the cyclin B1-p34^cdc2 complex. Alternatively, alterations in intracellular ion concentrations induced by amiloride may lead to changes in Ca²⁺-dependent signaling cascades and thereby decrease the radiation-mediated cell cycle perturbations.

¹ This work was supported by United States Department of Energy and Los Alamos National Laboratory Flow Cytometry Resource (NIH Grants p41-RR01315, R01 RR06758, and R01 RR07855).

² To whom requests for reprints should be addressed, at Life Sciences Division, Los Alamos National Laboratory, LS-4 M888, Los Alamos, NM 87545 Phone: (505) 667-2791; Fax: (505) 665-3024; E-mail: crissman@telomere.lanl.gov.

Received 7/31/97. Accepted 12/ 2/97.

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