Activation of protein kinase C by non-phorbol tumor promoter, mezerein

doi:10.1016/0006-291X(84)90231-6

Biochemical and Biophysical Research Communications

Volume 121, Issue 2, 15 June 1984, Pages 649-656

https://doi.org/10.1016/0006-291X(84)90231-6 Get rights and content

Abstract

Mezerein, classified as a second-stage tumor promoter, has no diacylglycerol-like structure in its molecule, but can activate protein kinase C both $in vitro and in vivo$ . This non-phorbol diterpene competitively inhibits the specific binding of a radioactive tumor-promoting phorbol ester to the enzyme. It is suggestive that tumor-promoting phorbol esters and mezerein cause analogous changes in the membrane to activate protein kinase C, and utilize this protein kinase as a common receptive protein for tumor promotion.

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Structural insights into C1-ligand interactions: Filling the gaps by in silico methods
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Citation Excerpt :
Mezerein and its analogs, such as thymeleatoxin (ThX) and daphnetoxin, have a daphnane-type skeleton that resembles the tigliane skeleton of the phorbol esters (Fig. 7). Members of this class compete with phorbol esters and activate PKCs in phospholipid-dependent manner both in vitro and in vivo (Brooks et al., 1989; Kazanietz et al., 1993; Miyake et al., 1984; Roivainen and Messing, 1993; Ryves et al., 1991; Saraiva et al., 2001; Slaga et al., 1980). Mezerein and ThX are classified as second-stage tumor promoters, whose effect is fully manifested only after treatment by more potent tumorigenic agents such as TPA (Fürstenberger et al., 1981; Slaga et al., 1980).
Protein Kinase C isoenzymes (PKCs) are the key mediators of the phosphoinositide signaling pathway, which involves regulated hydrolysis of phosphatidylinositol (4,5)-bisphosphate to diacylglycerol (DAG) and inositol-1,4,5-trisphosphate. Dysregulation of PKCs is implicated in many human diseases making this class of enzymes an important therapeutic target. Specifically, the DAG-sensing cysteine-rich conserved homology-1 (C1) domains of PKCs have emerged as promising targets for pharmaceutical modulation. Despite significant progress, the rational design of the C1 modulators remains challenging due to difficulties associated with structure determination of the C1-ligand complexes. Given the dearth of experimental structural data, computationally derived models have been instrumental in providing atomistic insight into the interactions of the C1 domains with PKC agonists. In this review, we provide an overview of the in silico approaches for seven classes of C1 modulators and outline promising future directions.
Gene expression analysis of terminal differentiation of human melanoma cells highlights global reductions in cell cycle-associated genes
2009, Gene
Defects in differentiation are frequently observed in cancer cells. By appropriate treatment specific tumor cell types can be induced to terminally differentiate. Metastatic HO-1 human melanoma cells treated with IFN-β plus mezerein (MEZ) undergo irreversible growth arrest and terminal differentiation followed by apoptosis. In order to define the molecular changes associated with this process, changes in gene expression were analyzed by cDNA microarray hybridization and by semi-quantitative and quantitative RT-PCRs of representative 44 genes. The expression of 210 genes was changed more than two-fold at either 8 or 24 h post-treatment (166 up and 44 down). Major biological processes associated with the up-regulated genes were response to endogenous/exogenous stimuli (38%), cell proliferation (13%), cell death (16%) and development (30%). Approximately 34% of the down-regulated genes were associated with cell cycle, 9% in DNA replication and 11% in chromosome organization, respectively. Suppression of cell cycle associated genes appeared to directly correlate with growth arrest observed in the terminal differentiation process. Expression of Calpain 3 (CAPN3) variant 6 was suppressed by the combined treatment and maintained high in various melanoma cell lines. However, over-expression of the CAPN3 did not significantly affect growth kinetics and cell viability, suggesting that up-regulation of CAPN3 alone may not be a causative, but an associated change with melanoma development. This analysis provides further insights into the spectrum of up-regulated and the first detailed investigation of down-regulated gene changes associated with and potentially causative of induction of loss of proliferative capacity and terminal differentiation in human melanoma cells.
Reversible suppression of in vitro biomineralization by activation of protein kinase A
2000, Journal of Biological Chemistry
Parathyroid hormone (PTH-(1–34)) potently suppresses apatite deposition in osteoblastic cultures. These inhibitory effects are mediated through signaling events following PTH receptor binding. Using both selective inhibitors and activators of protein kinase A (PKA), this study shows that a transient activation of PKA is sufficient to account for PTH's inhibition of apatite deposition. This inhibition is not a result of reduced cell proliferation, reduced alkaline phosphatase activity, increased collagenase production, or lowering medium pH. Rather, data suggest a functional relationship between matrix assembly and apatite depositionin vitro. Bone sialoprotein (BSP) and apatite co-localize in the extracellular matrix of mineralizing cultures, with matrix deposition of BSP temporally preceding that of apatite. Transient activation of PKA by either PTH-(1–34) or short term cAMP analog treatment blocks the deposition of BSP in the extracellular matrix without a significant reduction in the total amount of BSP synthesized and secreted. This effect is reversible after allowing the cultures to recover in the absence of PKA activators for several days. Thus, a transient activation of PKA may suppress mineral deposition in vitro as a consequence of altering the assembly of an extracellular matrix permissive for apatite formation.
Inverse regulation of oestrogen receptor and epidermal growth factor receptor genbe expression in MCF-7 breast cancer cells treated with phorbol ester
1996, Journal of Steroid Biochemistry and Molecular Biology
In human breast cancer cell lines, an inverse relationship exists between the basal levels of oestrogen receptor (ER) and epidermal growth factor receptor (EGF-R) gene expression. In addition, the tumour-promoting phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) inhibits ER and stimulates EGF-R expression in MCF-7 breast cancer cells. This study aimed to define further the potential mechanisms involved in the modulation of ER and EGF-R gene expression by TPA. ER mRNA levels were reduced after 3 h and declined to 30% of control between 12 and 72 h after exposure to 10 nM TPA. This decrease in mRNA levels was preceded by an apparent fall in ER transcription rate. There was no effect on the stability of ER mRNA following pretreatment for 3–24 h with TPA, supporting the conclusion that the fall in ER mRNA levels was predominantly due to a decrease in ER transcription rate. Levels of EGF-R mRNA increased 10-fold by 12 h due predominantly to an increased transcription rate. The TPA-induced decrease in ER mRNA was unaffected by the simultaneous administration of the protein synthesis inhibitor cycloheximide, whereas the increase in EGF-R mRNA was inhibited by co-incubation with cycloheximide. These data indicate a requirement for continuing protein synthesis for the TPA effect on EGF-R but not on ER mRNA levels. Because the modulation of ER and EGF-R gene expression by TPA is likely to involve the protein kinase C (PKC) signal transduction pathway, the effects of other known activators of PKC were investigated. The non-phorboid tumour promoter mezerein modulated ER (an 80% decrease) and EGF-R (a 20-fold increase) mRNA levels in a similar manner to TPA. In contrast, neither 1,2-dioctanoyl-sn-glycerol (DiC₈) nor 1-oleoyl-2-acetyl-sn-glycerol (OAG), both permeant analogues of the endogenous physiological activators of PKC, affected ER and EGF-R mRNA levels. These latter results were not due to a lack of efficacy because a single administration of DiC₈ was as effective as TPA in inducing c-fos mRNA at 30 min. However DiC₈ was less active in the later induction of c-myc mRNA. These data demonstrate reciprocal regulation of ER and EGF-R gene expression by TPA, involving effects on transcriptional events, which appear to be mediated by sustained activation of PKC.
12-O-tetradecanoyl-phorbol-13-acetate (TPA) counteracts the cAMP up-regulation of the expression of the stimulatory guanine nucleotide binding protein (Gsα) and Gsα messenger RNA in cultured pig thyroid cells
1994, Molecular and Cellular Endocrinology
In this study, we examined whether the protein kinase C (PKC) pathway could interfere with the regulation of Gs protein in porcine thyroid cells. The two days culture of cells with 12-O-tetradecanoylphorbol 13-acetate (TPA) (0.1 μM) alone neither affected adenylyl cyclase activity, nor the level of Gsα protein in membranes when compared with control cells. The co-addition of TPA with thyrotropin (TSH) (1 mU/ml) or forskolin (fk) (10 μM) in the culture medium, abolished the stimulatory effects of either agonists on the activation of adenylyl cyclase by fk or [AlF₄]⁻ and on the increase of Gsα protein. By contrast, TPA had effects neither on the Gi-dependent inhibition of adenylyl cyclase nor on Giα proteins levels. The level of Gsα mRNA measured by Northern blot analysis was increased (200%) in TSH- or fk-treated cells and this increase was counteracted by TPA. The effects of TPA observed after 6–9 h of contact with cells were mimicked by mezerein, a non-phorbol protein kinase C activator and blocked by bisindolylmaleimide a specific protein kinase C inhibitor (GF 109203X). These results suggest that the activation of the PKC pathway prevents the cAMP-dependent up-regulation of Gsα protein and Gsα mRNA expression.
Different mechanisms regulate the monoclonal antibody-induced modulation of cd2, cd3, and cd5 in human lymphocytes
1993, Cellular Immunology
The CD2, CD3, and CD5 antigens are down-modulated from the cell surface of peripheral blood mononuclear cells after a 24-hr incubation with specific monoclonal antibodies (mAb). Here we show that active (phorbol myristate acetate, phorbol dibutyrate acetate, and mezerein) but not inactive (4β-phorbol) tumor-promoting agents inhibit the mAb-induced modulation of CD2 and CD5, but not CD3, without concomitant changes in the surface distribution of these antigens (such as capping). This inhibitory effect is not protein synthesis dependent and is reversed by protein kinase C inhibitors (staurosporine and H-7). The use of cytoskeleton-disrupting agents shows the existence of different cytoskeletal interactions driving the mAb-induced modulation of CD2 and CD5 with respect to CD3. Treatment with cytochalasin D (an agent that inhibits microfilament polymerization) but not colchicine (an agent that inhibits microtubule polymerization) reproduced the effect of TPA on the mAb-induced modulation of CD2, CD3, and CD5. Our results indicate that the mAb-induced modulation of CD2 and CD5 is dependent on microfilament (namely actin) polymerization and PKC activation, while the modulation of CD3 is not.

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^☆: This investigation was supported in part by research grants from the Scientific Research Fund of the Ministry of Education, Science and Culture, Japan 1982–1984, the Intractable Diseases Division, Public Health Bureau, the Ministry of Health and Welfare, Japan 1982–1984, a Grant-in-Aid of New Drug Development from the Ministry of Health and Welfare, Japan 1983, the Science and Technology Agency 1983, the Yamanouchi Foundation for Research on Metabolic Disorders 1982–1983, and Mitsuhisa Memorial Cancer Research Fund 1983. The data are taken in part from the dissertation that will be submitted by R. Miyake to Kobe University School of Medicine in partial fulfilment of the requirement for the degree of Doctor of Medical Science.

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Activation of protein kinase C by non-phorbol tumor promoter, mezerein☆

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