Protein kinase C-dependent and -independent signaling in genotoxic response to treatment of desferroxamine, a hypoxia-mimetic agent

Carlos Clavijo,^1,6 Jo-Lin Chen,^1,4 Kwang-Jin Kim,^1,2,3 Mary E. Reyland,⁵ , and David K. Ann^1,2,4

Departments of ¹Molecular Pharmacology and Toxicology and ²Medicine, the ³Will Rogers Institute Pulmonary Research Center, University of Southern California, Los Angeles; and ⁴Department of Clinical and Molecular Pharmacology, City of Hope National Medical Center, Duarte, California; ⁵Department of Craniofacial Biology/Cell and Developmental Biology, University of Colorado at Denver and Health Sciences Center, Aurora, Colorado; and ⁶Department of Biology, Universidad Nacional de Colombia, Bogota, Colombia

Abstract: Protein kinase C (PKC) plays a critical role in diseases such as cancer, stroke, and cardiac ischemia and participates in a variety of signal transduction pathways including apoptosis, cell proliferation, and tumor suppression. Here, we demonstrate that PKC is proteolytically cleaved and translocated to the nucleus in a time-dependent manner on treatment of desferroxamine (DFO), a hypoxia-mimetic agent. Specific knockdown of the endogenous PKC by RNAi (sh-PKC) or expression of the kinase-dead (Lys376Arg) mutant of PKC (PKCKD) conferred modulation on the cellular adaptive responses to DFO treatment. Notably, the time-dependent accumulation of DFO-induced phosphorylation of Ser-139-H2AX (-H2AX), a hallmark for DNA damage, was altered by sh-PKC, and sh-PKC completely abrogated the activation of caspase-3 in DFO-treated cells. Expression of Lys376Arg-mutated PKC-enhanced green fluorescent protein (EGFP) appears to abrogate DFO/hypoxia-induced activation of endogenous PKC and caspase-3, suggesting that PKCKD-EGFP serves a dominant-negative function. Additionally, DFO treatment also led to the activation of Chk1, p53, and Akt, where DFO-induced activation of p53, Chk1, and Akt occurred in both PKC-dependent and -independent manners. In summary, these findings suggest that the activation of a PKC-mediated signaling network is one of the critical contributing factors involved in fine-tuning of the DNA damage response to DFO treatment.

Key Words: DNA damage • caspase-3 • Akt

Address for reprint requests and other correspondence: D. K. Ann, City of Hope National Medical Center, KCRB Rm. 1004, 1500 E. Duarte Road, Duarte, CA 91010 (e-mail: dann{at}coh.org)