Induction of superoxide by 12-O-tetradecanoylphorbol-13-acetate and thapsigargin, a non-phorbol ester-type tumor promoter, in peritoneal macrophages elicited from SENCAR and B6C3F1 mice: The significance of protein kinase C vs. arachidonic acid-mediated signal transduction pathways
Abstract
Local production of reactive oxygen intermediates (ROIs) such as superoxide anion by tumor promoter-stimulated inflammatory macrophages (MPs) may contribute significantly to tumor development in classical models of two-stage chemically-induced carcinogenesis in murine skin. It has been suggested that genetic damage in initiated epidermal cells may be potentiated by ROIs generated by tumor promoter-stimulated inflammatory cells. Therefore, an understanding of the signal transduction mechanisms accounting for any observed strain-dependent differences in the stimulation of MPs to produce ROIs by 12-O-tetradecanoylphorbol-13-acetate (TPA) (or other non-TPA-type tumor promoters) may lead to better understanding of the relationship between inflammation and the process of tumor promotion. In the studies reported herein, peritoneal MPs elicited from phorbol ester-sensitive SENCAR mice, demonstrated a dose-dependent release of superoxide anion (4-6 nmol/10$\sp6$ cells) when stimulated by TPA in vitro; MP superoxide production was significantly inhibited (50-70%) by preincubation with 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) (40 uM), a relatively specific protein kinase C (PKC) inhibitor. Alternatively, TPA-stimulated MPs derived from relatively-resistant B6C3F1 mice generated negligible superoxide under the same conditions. A similar strain-dependent induction of superoxide was observed when MPs were stimulated with thapsigargin (TG), a tumor promoter which has been shown to act independently of PKC. TG-stimulated SENCAR MPs released significant superoxide (2-3 nmol/10$\sp6$ cells) which was not inhibited by H-7; MPs from B6C3F1 mice demonstrated negligible stimulation by TG. Preincubation of SENCAR MPs with dibromoacetophenone (DBAP) (100 uM), an inhibitor of phospholipase A$\sb2$ (PLA$\sb2$), completely suppressed the superoxide induced by TPA and TG stimulation. Like TPA, 1-oleoyl-2-acetylglycerol (OAG) (50 uM), a diacylglycerol analogue and PKC activator, also induced significant superoxide from SENCAR MPs. In parallel with the superoxide findings, TPA and TG stimulated significantly greater ($\sp3$H) -arachidonic acid (AA) release from prelabeled SENCAR MPs (32% and 48% increase, respectively, over unstimulated controls) relative to MPs elicited from B6C3F1 mice. Two-dimensional (2-D) gel electrophoretic analysis indicated that TPA-induced phosphorylation of a 47 kDa protein (a presumed substrate for PKC previously linked to NADPH oxidase activation in guinea pig, and human polymorphonuclear leukocytes (PMNs)) occurred in MPs elicited from both SENCAR and B6C3F1 mice. Therefore, AA production may be a common biochemical pathway by which phorbol and non-phorbol ester-type tumor promoters activate MPs in SENCAR mice; such a response may be "permissive" for additive (or synergistic) interactions with PKC-driven signal pathways.
Degree
Ph.D.
Advisors
Marcus, Purdue University.
Subject Area
Pharmacology|Immunology
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