Emerging evidence suggests that activation of adenosine monophosphate-activated protein kinase (AMPK), an energy gauge and redox sensor, controls the cell cycle and protects against DNA damage. However, the molecular mechanisms by which AMPK alpha isoform regulates DNA damage remain largely unknown. The aim of this study was to determine if AMPK alpha deletion contributes to cellular hyperproliferation by reducing p21(WAF1/Cip1) (p21) expression thereby leading to accumulated DNA damage. The markers for DNA damage, cell cycle proteins, and apoptosis were monitored in cultured mouse embryonic fibroblasts (MEFs) isolated from wild type (WT, C57BL/6J), AMPK alpha 1, or AMPK alpha 2 homozygous deficient (AMPK alpha 1(-/-), AMPK alpha 2(-/-)) mice by Western blot, flow cytometry, and cellular immunofluorescence staining. Deletion of AMPK alpha 1, the predominant AMPK alpha isoform, but not AMPK alpha 2 in immortalized MEFs led to spontaneous DNA double-strand breaks (DSB) which corresponded to repair protein p53-binding protein 1 (53BP1) foci formation and subsequent apoptosis. Furthermore, AMPK alpha 1 localizes to chromatin and AMPK alpha 1 deletion down-regulates cyclin-dependent kinase inhibitor, p21, an important protein that plays a role in decreasing the incidence of spontaneous DSB via inhibition of cell proliferation. In addition, AMPK alpha 1 null cells exhibited enhanced cell proliferation. Finally, p21 overexpression partially blocked the cellular hyperproliferation of AMPK alpha 1-deleted MEFs via the inhibition of cyclin-dependent kinase 2 (CDK2). Taken together, our results suggest that AMPK alpha 1 plays a fundamental role in controlling the cell cycle thereby affecting DNA damage and cellular apoptosis. (C) 2014 Elsevier B.V. All rights reserved.