S-Phase-specific Activation of PKCα Induces Senescence in Non-small Cell Lung Cancer Cells

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 G 1 phase inhibits the progression into S phase, an effect mediated by PKC␦-induced up-regulation of the cell cycle inhibitor p21 Cip1 . However, PMA treatment in asynchronously growing NSCLC cells leads to accumulation of cells in G 2 /M. Studies in post-G 1 phases revealed that PMA induced an irreversible G 2 /M cell cycle arrest in NSCLC cells and conferred morphological and biochemical features of senescence, including elevated SA-␤-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 G 1 , phase. Mechanistic analysis revealed a crucial role for the classical PKC␣ isozyme as mediator of the G 2 /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␣, 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.Activation of protein kinase C (PKC) 4 with phorbol esters and related natural compounds causes an array of effects on differentiation, mitogenesis, survival, apoptosis, and transformation. The diverse effects of phorbol esters both in normal and cancerous cells is due to the existence of numerous intracellular effectors, of which PKC isozymes have been the most widely characterized. The PKC family comprises 3 subfamilies that include 10 structurally related phospholipid-dependent serine/threonine kinases (1, 2). Members of the classical cPKC (␣, ␤I, ␤II, and ␥) and the novel nPKC (␦, ⑀, , and ) subfamilies are activated by phorbol esters and their cellular analogue, the second messenger diacylglycerol, which leads to redistribution (translocation) of the enzymes from cytosol to intracellular membrane compartments, where they phosphorylate specific substrates. The marked heterogeneity in the signaling events and cell type-specific responses triggered by phorbol esters could be explained by the distinctive pattern of expression and intracellular localization of PKC isozymes and their substrates, which ultimately results in selective pathway activation.One of the paradigms that best exemplifies the functional versatility of PKC isozymes is the regulation of the cell cycle machinery. It became evident in the last years that PKCs can impact on the cell cycle both in positive and negative manners with a strict degree of cell type and isozyme specificity. PKC isozymes have been shown to regulate the progression of cells from G 1 to S phase as well as with the transition from G 2 to M phase (3) via transcriptional, translational, and post-translational mechanisms. PKCs control the activity of cyc...

Prevention of premature senescence requires JNK regulation of Bcl-2 and reactive oxygen species

“…There have been many reports of G0/G1 arrest in senescence (Kramer et al, 2001;Qian et al, 2008). However, it was also shown that senescence was accompanied by cell cycle arrest in G2/M arrest, including SP600125-induced G2/ M arrest (MacLaren et al, 2004;Mingo-Sion et al, 2004;Wada et al, 2004;Vigneron et al, 2005;Xia et al, 2006;Oliva et al, 2008). In this study, p21 has a major role in cell cycle arrest, and G2/M cell cycle arrest is associated with rapid CDC25 phosphorylation and decrease of cyclin B1 and CDK2 in SP600125-treated cells.…”

Activation of Protein Kinase C by Phorbol 12-Myristate 13-Acetate suppresses the growth of lung cancer cells through KLF6 induction

“…19 More recently, PKC-mediated induction of p21 WAF1/CIP1 was shown to be an obligatory event for PMA-induced senescence in non-small cell lung cancer cells. 20 In this study, we increase the knowledge of the mechanisms of PKC-mediated p21 induction and cell growth arrest by PMA. We show that exposure of H358 lung cancer cells to PMA increased the levels of the candidate tumor suppressor KLF6, which is downregulated in many lung cancer cell lines, concomitantly with an increase in the CDKI p21 WAF1/CIP1 .…”

“…1,20 This diverse range of effects is due to the opposing actions of various members or isozymes of the PKC protein family. 1,20 The induction of a putative tumor suppressor (i.e., KLF6) by PMA is not entirely expected because PMA is well known to act as a tumor promoting agent in two-stage carcinogenesis models (e.g., skin). 25,26 On the other hand, PMA or certain PKC activators induce cell growth arrest mediated by the cyclin dependent kinase inhibitor p21 WAF1/CIP1 .…”

Site-specific Phosphorylation of CXCR4 Is Dynamically Regulated by Multiple Kinases and Results in Differential Modulation of CXCR4 Signaling

“…PKC␦ was targeted against the following sense strands: 5Ј-CCAU-GAGUUUAUCGCCACC-3Ј and 5Ј-CAGCACAGAGCGUG-GGAAA-3Ј. The arrestin and PKC␣ siRNAs have been previously described (31,32). The PKC siRNAs were reconstituted individually at 60 pmol/l before transfection, and 300 pmol of each were combined for a total of 600 pmol/transfection.…”