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Abstract
Protein kinase C (PKC) has been widely implicated in positive and negative control of cell proliferation. We have recently shown that treatment of non-small cell lung cancer (NSCLC) cells with phorbol 12-myristate 13-acetate (PMA) during G1 phase inhibits the progression into S phase, an effect mediated by PKC delta-induced up-regulation of the cell cycle inhibitor p21 Cip1. However, PMA treatment in asynchronously growing NSCLC cells leads to accumulation of cells in G2/M. Studies in post-G1 phases revealed that PMA induced an irreversible G2/M cell cycle arrest in NSCLC cells and conferred morphological and biochemical features of senescence, including elevated SA-beta-Gal activity and reduced telomerase activity. Remarkably, this effect was phase-specific, as it occurred only when PKC was activated in S, but not in G1, phase. Mechanistic analysis revealed a crucial role for the classical PKC alpha isozyme as mediator of the G2/M arrest and senescence, as well as for inducing p21(Cip1) an obligatory event for conferring the senescence phenotype. In addition to the unappreciated role of PKC isozymes, and specifically PKC alpha, in senescence, our data introduce the paradigm that discrete PKCs trigger distinctive responses when activated in different phases of the cell cycle via a common mechanism that involves p21 Cip1 up-regulation.
Highlights
Protein kinase C (PKC) has been widely implicated in positive and negative control of cell proliferation
We have recently shown that treatment of non-small cell lung cancer (NSCLC) cells with phorbol 12-myristate 13-acetate (PMA) during G1 phase inhibits the progression into S phase, an effect mediated by PKC␦-induced up-regulation of the cell cycle inhibitor p21Cip1
PMA Causes Irreversible G2/M Arrest in NSCLC Cells—PKC activation with phorbol esters causes a profound inhibition of cell proliferation in lung cancer cells, and our previous work has identified a PKC␦-dependent inhibitory mechanism that limits cell progression from G1 into S phase [24]
Summary
Materials—Cell culture media was purchased from Invitrogen (Carlsbad, CA). PMA was obtained from LC Laboratories (Woburn, MA). After removal of the AdV by extensive washing, cells were incubated in complete medium for 20 h. After blocking for 30 min with 3% fetal bovine serum in PBS, a mouse anti-p21Cip monoclonal antibody (1:250) was added (1 h), followed by washing with 0.1% Tween-20 in PBS, and incubation with a CY3-conjugated anti-mouse antibody (1:1000; Jackson Immunoresearch Laboratories, Inc.) was added. Cells were stimulated with either PMA or vehicle, and 3 days later fixed with 2% formamide/0.2% glutaraldehyde in PBS (10 min, room temperature) and incubated overnight at 37 °C with a solution containing 1 mg/ml 5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-Gal), 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl, and 2 mM MgCl2 in 40 mM citric acid/sodium phosphate buffer, pH 6.0. A p value of Ͻ0.05 was considered statistically significant
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