We have investigated the effect of neurokinin 1 receptor (NK1R) agonists on HEK293 cells transfected with the NK1R receptor. The NK1R receptor mediates dramatic shape changes that include contractions of the membrane cortex resulting in membrane bleb formation. We have found that the cell shape changes correlate with changes in electrical impedance measured in cellular monolayers. The shape and impedance changes were prevented after preincubation with NK1R antagonists aprepitant and L-73060. Although bleb formation usually heralds apoptotic cell death, we have found that NK1R-mediated cellular blebbing does not associate with apoptosis. Preincubation with a cell-permeable derivative of C3 transferase that blocks Rho or with the Rho-associated coiled-coil kinase inhibitor Y27632 completely prevented NK1R-induced shape and impedance changes. Blebbing was also completely inhibited by ML-9, a myosin light chain kinase inhibitor. Furthermore, the phospholipase C inhibitor U73,122 did not interfere with the effect of Substance P (SP) on cellular morphology and cellular impedance but completely blocked SP-induced intracellular calcium increase, indicating that the blebbing is a process independent of intracellular calcium elevations. Blebbing is a protein kinase C-independent process, since the nonselective protein kinase C inhibitor GF109203X did not interfere with SP-induced effects. Based on these results, we provide the first evidence that NK1R receptorligand interaction can cause apoptosis-independent cellular blebbing and that this process is mediated by the Rho/Rho-associated coiled-coil kinase pathway.

otides are released during vascular injury from activated platelets and broken cells, which could stimulate human neutrophils. In this study, we characterized the P2Y receptors and investigated the functional effects of extracellular nucleotides on human neutrophils. Pharmacological characterization using selective agonists and pertussis toxin revealed that human neutrophils express only functional P2Y 2 receptors. However, P2Y 2 receptor agonists ATP or uridine triphosphate (UTP) caused intracellular Ca 2ϩ increases in isolated human neutrophils with an EC 50 of 1 M but failed to cause release of primary granules from human neutrophils. ATP and UTP were equally potent in causing elastase release from human neutrophils in the presence of exogenous soluble fibrinogen, whereas ADP and UDP were without effect. We investigated whether nucleotides depend on generated arachidonic acid metabolites to cause degranulation. However, phenidone and MK-886, inhibitors of the 5-lipoxygenase pathway, failed to block nucleotide-induced intracellular calcium mobilization and elastase release. ATP and UTP caused activation of p38 MAPK and ERK1/2 in human neutrophils. In addition, the inhibitors of the MAPK pathway, SB-203580 and U-0126, inhibited nucleotide-induced elastase release. We conclude that fibrinogen is required for nucleotide-induced primary granule release from human neutrophils through the P2Y 2 receptor without a role for arachidonic acid metabolites. Both ERK1/2 and p38 MAPK play an important role in nucleotide-induced primary granule release from human neutrophils.

Protein kinase C (PKC) isozymes, a family of serine-threonine kinases, are important regulators of cell proliferation and malignant transformation. Phorbol esters, the prototype PKC activators, cause PKC translocation to the plasma membrane in prostate cancer cells, and trigger an apoptotic response. Studies in recent years have determined that each member of the PKC family exerts different effects on apoptotic or survival pathways. PKCdelta, one of the novel PKCs, is a key player of the apoptotic response via the activation of the p38 MAPK pathway. Studies using RNAi revealed that depletion of PKCdelta totally abolishes the apoptotic effect of the phorbol ester PMA. Activation of the classical PKCalpha promotes the dephosphorylation and inactivation of the survival kinase Akt. Studies have assigned a pro-survival role to PKCepsilon, but the function of this PKC isozyme remains controversial. Recently, it has been determined that the PKC apoptotic effect in androgen-dependent prostate cancer cells is mediated by the autocrine secretion of death factors. PKCdelta stimulates the release of TNFalpha from the plasma membrane, and blockade of TNFalpha secretion or TNFalpha receptors abrogates the apoptotic response of PMA. Molecular analysis indicates the requirement of the extrinsic apoptotic cascade via the activation of death receptors and caspase-8. Dissecting the pathways downstream of PKC isozymes represents a major challenge to understanding the molecular basis of phorbol ester-induced apoptosis.

Cellular senescence is a potent anti-cancer mechanism controlled by tumor suppressor genes, particularly p53 and pRb, which is characterized by the irreversible loss of proliferation. Senescence induced by DNA damage, oncogenic stimulation, or excessive mitogenic input, serves as a barrier that counteracts cancer progression. Emerging evidence in cellular and in in vivo models revealed the involvement of additional signaling players in senescence, including PML, CK2, Bcl-2, PI3K effectors such as Rheb, Rho small GTPases, and cytokines. Recent studies have also implicated protein kinase C (PKC) isozymes as modulators of senescence phenotypes and showed that phorbol esters, widely used PKC activators, can induce senescence in a number of cancer cells. These novel findings suggest a complex array of cross-talks between senescence pathways and may have significant implications in cancer therapy.

Protein kinase C⑀ (PKC⑀), a diacyglycerol-and phorbol esterresponsive serine-threonine kinase, has been implicated in mitogenic and survival control, and it is markedly overexpressed in human tumors, including in prostate cancer. Although prostate cancer cells undergo apoptosis in response to phorbol ester stimulation via PKC␦-mediated release of death factors, the involvement of PKC⑀ in this response is not known. PKC⑀ depletion by RNAi Protein kinase C (PKC), 3 a family of serine-threonine kinases that comprises the classical (cPKCs ␣, ␤, and ␥), novel (nPKCs ␦, ⑀, , and ), and atypical (aPKCs and ) PKCs, is a key signaling component of growth factor and cytokine pathways. Despite their structural similarities, PKC isozymes have unique modes of regulation as well as differential patterns of cell and tissue expression (1, 2). Only cPKCs and nPKCs are regulated by phorbol esters and diacylglycerol, a lipid second messenger generated upon activation of G protein-coupled receptors and tyrosine kinases (1-3). Phorbol esters are capable of promoting opposite responses in different cell types, such as mitogenesis/ survival versus growth arrest/apoptosis. This paradigm of functional diversity is exemplified by the nPKCs: whereas in most cases PKC⑀ acts as a mitogenic or antiapoptotic kinase, activation of PKC␦ inhibits proliferation or triggers an apoptotic response (4 -8). PKC⑀ can signal to mitogenesis via Raf/MEK/ ERK and cyclin D1 induction (9, 10) or can even transform cells (4, 6, 11). In addition, PKC⑀ has been linked to cell survival through the activation of Akt and Bax (12,13).Tumor cells display in many cases an altered balance in PKC isozyme expression, potentially reflecting the involvement of PKCs in the etiology and progression of cancer. Most notably, many cancer cells show marked up-regulation of PKC⑀. PKC⑀ is elevated in prostate cancer, particularly in high grade tumors, and is implicated in prostate tumor progression and the transition to androgen-independence (14 -17). The functional complexity of PKC in prostate cancer cells is nonetheless highlighted by the fact that phorbol esters promote an apoptotic response in androgen-dependent prostate cancer cells (18,19). Previous work from our laboratory and others established that PKC␦ is the major mediator of the death effect of PMA in LNCaP cells (19 -21). In this context, the role of PKC⑀ remains controversial, as unlike in other cell types, PKC⑀ was found to be either dispensable for prostate cancer cell survival or even contribute to the proapoptotic effects of PKC activators (22-24). Given our limited understanding of the mechanistic insights of PKC-driven responses in prostate cancer cells, additional studies would be required to ascertain the specific contribution of PKC isozymes to this paradox.We have recently demonstrated that PKC␦-mediated prostate cancer cell death involves the activation of an apoptotic autocrine loop through the release of death factors (primarily TNF␣), and the subsequent activation of the extrinsic apoptotic cascade ...