Phorbol Ester Receptors and the in Vitro Effects of Tumor Promoters

Blumberg, Peter M.; Delclos, K. Barry; Dunn, Joseph A.; Jaken, Susan; Leach, Karen L.; Yeh, Erika

doi:10.1111/j.1749-6632.1983.tb47836.x

Cited by 31 publications

(15 citation statements)

References 34 publications

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“…Extensive evidence suggests the existence of multiple phorbol ester receptor subclasses in skin and certain cultured cells (50)(51)(52)(53). Although receptor subclasses could represent distinct gene products, they alternatively could reflect association of a single gene product with distinct lipid domains.…”

Section: Methodsmentioning

confidence: 99%

Specific labeling of mouse brain membrane phospholipids with [20-3H]phorbol 12-p-azidobenzoate 13-benzoate, a photolabile phorbol ester.

Delclos¹,

Yeh²,

Blumberg³

1983

Proc. Natl. Acad. Sci. U.S.A.

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As part of our effort to characterize receptors for the phorbol ester tumor promoters, a phorbol ester photoaffinity probe, [20-3H]phorbol 12-p-azidobenzoate 13-benzoate (PaBzBz), was synthesized. In the dark, PaBzBz bound reversibly to brain particulate fractions with a dissociation constant (Kd) of 0.81 ± 0.09 x 10-9 M. Specific binding of PaBzBz, at a concentration equal to its Kd, represented 85% of the total bound. At saturation, 24 ± 5 pmol of PaBzBz were bound per mg of brain protein, a level similar to that observed with [20-3H]phorbol 12,13-dibutyrate. Under the conditions used (concentrations greater than the Kd for PaBzBz), irradiation caused 45% of the PaBzBz binding to become irreversible. Most of the binding (=60%), including most of the specific irreversible binding, was to phospholipid rather than to protein. Based on susceptibility to enzymatic digestion and on chromatographic mobility, the specifically labeled phospholipids were identified as phosphatidylserine, phosphatidylethanolamine, and phosphatidylethanolamine plasmalogen. Although the PaBzBz specifically labeled lipid, labeling was blocked by pretreatment of membranes at 100IC for 5 min or by papain digestion. Therefore, it seems likely that the identified lipids are specifically associated with a protein receptor and are preferentially labeled either because of the location or reactivity of the nitrene generated on the photoaffinity probe.The phorbol esters are potent tumor promoters, which have also been shown to induce dramatic cellular and biochemical changes in vivo and in vitro (1-4). This laboratory has demonstrated specific, saturable binding of the phorbol esters to tissue particulate preparations and intact cells from a variety of organisms (5-10). The structure-activity requirements for binding are consistent with the receptors mediating the biological responses to the phorbol esters. The highest level of binding activity, 28 pmol/mg of protein, is found in brain (7). This level, which is considerably higher than that typical for hormone or neurotransmitter receptors, led us to suggest that the phorbol esters may be binding to a modulatory site on an enzyme or transport protein (7).We previously have demonstrated that phorbol ester binding in brain was sensitive to heat, protease, and phospholipase A2 treatments (7). In addition, we have shown that low concentrations of ascorbate inactivated binding with a time course and dose-response curve paralleling that for induction of lipid peroxidation (11). These results suggested either that the phorbol ester receptor is dependent on its specific lipid environment or else that lipid plays a role in the binding site itself.In an effort to obtain fiurther evidence on the nature of the phorbol ester binding site, we prepared a radioactive phorbol ester photoaffinity derivative, phorbol 12-p-azidobenzoate 13-benzoate (PaBzBz). This derivative contains an aryl azide, which is one of the widely used classes of photoaffinity labels (12, 13). We describe here specific, covalent bind...

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Section: Methodsmentioning

confidence: 99%

Specific labeling of mouse brain membrane phospholipids with [20-3H]phorbol 12-p-azidobenzoate 13-benzoate, a photolabile phorbol ester.

Delclos¹,

Yeh²,

Blumberg³

1983

Proc. Natl. Acad. Sci. U.S.A.

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“…Following the identification of phorbol esters as the active ingredient, models of skin carcinogenesis quickly established that phorbol esters are potent tumor promoters [8]. Painting sub-threshold amounts of a carcinogen such as DMBA on mouse skin had no effect, but if this was followed by closely spaced (twice a week) and repetitive applications of phorbol esters, papilomas developed (reviewed in [3]).…”

Section: Phorbol Estersmentioning

confidence: 99%

Reversing the Paradigm: Protein Kinase C as a Tumor Suppressor

Newton

Brognard

2017

Trends in Pharmacological Sciences

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The discovery in the 1980s that protein kinase C (PKC) is a receptor for the tumor-promoting phorbol esters fueled the dogma that PKC is an oncoprotein. Yet 30+ years of clinical trials for cancer using PKC inhibitors not only failed, but in some instances worsened patient outcome. The recent analysis of cancer-associated mutations, from diverse cancers and throughout the PKC family, revealed that PKC isozymes are generally inactivated in cancer, supporting a tumor suppressive function. In keeping with a bona fide tumor suppressive role, germ line causal loss of function (LOF) mutations in one isozyme have recently been identified in lymphoproliferative disorders. Thus, strategies in cancer treatment should focus on restoring, rather than inhibiting, PKC.

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“…Phorbol myristate acetate (PMA), a cocarcinogenic ingredient of croton oil, possesses diverse bioactions. In addition to promoting tumors in vivo (reviewed in Blumberg et al, 1983), PMA acts in vitro to stimulate cellular differentiation (Rovera et al, 1979), lymphocyte mitogenesis (Alb et al, 1979), and platelet aggregation (Zucker et al, 1974). Polymorphonuclear neutrophils (PMN) also respond to the phorbol diester; these cells have been useful in investigating how PMA acts.…”

mentioning

confidence: 99%

Diacylglycerols and mezerein activate neutrophils by a phorbol myristate acetate‐like mechanism

et al. 1985

Journal Cellular Physiology

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rac-1-O-Myristoyl-2-O-acetylglycerol, rac-1-O-palmitoyl-2-O-acetylglycerol, and rac-1-O-oleoyl-2-O-acetylglycerol acted like phorbol myristate acetate and mezerein in stimulating human neutrophil aggregation. Responses to these agents were equally influenced by cytochalasin B, extracellular calcium and magnesium, arachidonate antimetabolites, and procedures that rendered the cells desensitized to other agonists. The compounds also inhibited the binding of [3H]-phorbol myristate acetate to its receptor on neutrophils. Thus, these agents are biologically homologous. They act by binding to a common receptor. This receptor may function physiologically as a transducer for endogenous glycerides that form in cells challenged by other stimuli.

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Phorbol Ester Receptors and the in Vitro Effects of Tumor Promoters

Cited by 31 publications

References 34 publications

Specific labeling of mouse brain membrane phospholipids with [20-3H]phorbol 12-p-azidobenzoate 13-benzoate, a photolabile phorbol ester.

Specific labeling of mouse brain membrane phospholipids with [20-3H]phorbol 12-p-azidobenzoate 13-benzoate, a photolabile phorbol ester.

Reversing the Paradigm: Protein Kinase C as a Tumor Suppressor

Diacylglycerols and mezerein activate neutrophils by a phorbol myristate acetate‐like mechanism

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