Impact of structural and metabolic variations on the toxicity and carcinogenicity of hydroxy- and alkoxy-substituted allyl- and propenylbenzenes.

The metabolic fate of a compound is determined by numerous factors including its chemical structure. Although the metabolic options for a variety of functional groups are well understood and can often provide a rationale for the comparison of toxicity based on structural analogy, at times quite minor structural variations may have major consequences for metabolic outcomes and toxicity. In this perspective, the effects of structural variations on metabolic outcomes is detailed for a group of related hydroxy- and alkoxy-substituted allyl- and propenylbenzenes. These classes of compounds are naturally occurring constituents of a variety of botanical-based food items. The classes vary from one another by the presence or absence of alkylation of their para-hydroxyl substituents and/or the position of the double bond in the alkyl side chain. We provide an overview of how these subtle structural variations alter the metabolism of these important food-borne compounds, ultimately influencing their toxicity, particularly their DNA reactivity and carcinogenic potential. The data reveal that detailed knowledge of the consequences of subtle structural variations for metabolism is essential for adequate comparison of structurally related chemicals. Taken together, it is concluded that predictions in toxicological risk assessment should not be performed on the basis of structural analogy only but should include an analogy of metabolic pathways across compounds and species.

Metabolism of polycyclic aromatic hydrocarbon derivatives to ultimate carcinogens during lipid peroxidation.

Lipid peroxidation triggered by ascorbate or reduced nicotinamide adenine dinucleotide in rat liver microsomes can initiate the epoxidation of 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene. The stereochemistry of epoxidation is indicative of a peroxide-dependent free radical process. Since the epoxides formed may be the most carcinogenic derivatives of benzo[a]pyrene yet identified, lipid peroxidation can effect the metabolic activation of proximate carcinogens to ultimate carcinogens.

Detection of endogenous malondialdehyde-deoxyguanosine adducts in human liver.

Endogenous DNA adducts may contribute to the etiology of human genetic disease and cancer. One potential source of endogenous DNA adducts is lipid peroxidation, which generates mutagenic carbonyl compounds such as malondialdehyde. A sensitive mass spectrometric method permitted detection and quantitation of the major malondialdehyde-DNA adduct, a pyrimidopurinone derived from deoxyguanosine. DNA from disease-free human liver was found to contain 5400 adducts per cell, a frequency comparable to that of adducts formed by exogenous carcinogens.

Aspirin-like molecules that covalently inactivate cyclooxygenase-2.

Many of aspirin's therapeutic effects arise from its acetylation of cyclooxygenase-2 (COX-2), whereas its antithrombotic and ulcerogenic effects result from its acetylation of COX-1. Here, aspirin-like molecules were designed that preferentially acetylate and irreversibly inactivate COX-2. The most potent of these compounds was o-(acetoxyphenyl)hept-2-ynyl sulfide (APHS). Relative to aspirin, APHS was 60 times as reactive against COX-2 and 100 times as selective for its inhibition; it also inhibited COX-2 in cultured macrophages and colon cancer cells and in the rat air pouch in vivo. Such compounds may lead to the development of aspirin-like drugs for the treatment or prevention of immunological and proliferative diseases without gastrointestinal or hematologic side effects.

The FEMA GRAS assessment of alpha,beta-unsaturated aldehydes and related substances used as flavor ingredients.

This publication is the 12th in a series of safety evaluations performed by the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA). In 1993, the Panel initiated a comprehensive program to re-evaluate the safety of more than 1700 GRAS flavoring substances under conditions of intended use. Since then, the number of flavoring substances has grown to more than 2200 chemically-defined substances. Elements that are fundamental to the safety evaluation of flavor ingredients include exposure, structural analogy, metabolism, toxicodynamics and toxicology. Scientific data relevant to the safety evaluation for the use of aliphatic, linear alpha,beta-unsaturated aldehydes and structurally related substances as flavoring ingredients are evaluated. The group of substances was reaffirmed as GRAS (GRASr) based, in part, on their self-limiting properties as flavoring substances in food; their low level of flavor use; the rapid absorption and metabolism of low in vivo concentrations by well-recognized biochemical pathways; adequate metabolic detoxication at much higher levels of exposure in humans and animals; the wide margins of safety between the conservative estimates of intake and the no-observed-adverse effect levels determined from subchronic and chronic studies. While some of the compounds described here have exhibited positive in vitro genotoxicity results, evidence of in vivo genotoxicity and carcinogenicity occurs only under conditions in which animals are repeatedly and directly exposed to high irritating concentrations of the aldehyde. These conditions are not relevant to humans who consume alpha,beta-unsaturated aldehydes as flavor ingredients at low concentrations distributed in a food or beverage matrix.

Endogenous DNA damage and mutation.

In humans, approximately 10(7) cells divide per second. Estimates suggest that spontaneous mutations arise in about a third of those cells. These mutations arise as mistakes in DNA replication and when DNA polymerases copy damaged templates. The latter result from chemical hydrolysis of nucleoside bases or by reaction of DNA with electrophiles or reactive free radicals generated during metabolism (endogenous DNA damaging agents). This article highlights recent discoveries and emerging opportunities in the study of endogenous DNA damage and mutation.

Cyclooxygenase enzymes: catalysis and inhibition.

Scientists working in the field of cyclooxygenase enzymes have witnessed several major advances in the past two years. Crystal structures of fatty acid substrate and prostaglandin product complexes have been elucidated. Elegant site-directed mutagenesis studies have pinpointed the roles of key amino acids within the active site. Together, these results have provided key insights into the overall reaction mechanism. Detailed kinetics, spectroscopic and crystallographic studies have shed new light on the complex mechanism of inhibition of these fascinating enzymes. Finally, novel substrates of cyclooxygenase-2 have been identified.

The FEMA GRAS assessment of aromatic substituted secondary alcohols, ketones, and related esters used as flavor ingredients.

This publication is the 11th in a series of safety evaluations performed by the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA). In 1993, the Panel initiated a comprehensive program to re-evaluate the safety of more than 1700 GRAS flavoring substances under conditions of intended use. The list of GRAS substances has now grown to more than 2100 substances. Elements that are fundamental to the safety evaluation of flavor ingredients include exposure, structural analogy, metabolism, pharmacokinetics and toxicology. Flavor ingredients are evaluated individually and in the context of the available scientific information on the group of structurally related substances. In this monograph, a detailed interpretation is presented on the renal carcinogenic potential of the aromatic secondary alcohol alpha-methylbenzyl alcohol, aromatic ketone benzophenone, and corresponding alcohol benzhydrol. The relevance of these effects to the flavor use of these substances is also discussed. The group of aromatic substituted secondary alcohols, ketones, and related esters was reaffirmed as GRAS (GRASr) based, in part, on their rapid absorption, metabolic detoxication, and excretion in humans and other animals; their low level of flavor use; the wide margins of safety between the conservative estimates of intake and the no-observed-adverse effect levels determined from subchronic and chronic studies and the lack of significant genotoxic and mutagenic potential.

Modulation of oxidant formation in mouse skin in vivo by tumor-promoting phorbol esters.

The pathways of oxidant generation in mouse epidermis were investigated by 32P-postlabeling analysis of diastereomeric DNA adducts derived from oxidation of (7S,8S)-dihydroxy-7,8-dihydrobenzo(a)pyrene ((+)-BP-7,8-diol). The pattern of deoxynucleoside-3'-5'-bis-phosphate adducts in epidermal scrapings from female CD-1 mice indicated that cytochrome P-450 was the major oxidant. When animals were pretreated with the tumor-promoting phorbol ester, tetradecanoyl phorbol acetate (TPA), 24 h before coadministration of TPA and (+)-BP-7,8-diol, the pattern of DNA adducts indicated that peroxyl radicals made a major contribution to (+)-BP-7,8-diol epoxidation. Peroxy radical-dependent epoxidation was maximal when the time between the 2 TPA administrations was 24-72 h. No increase in radical-derived adducts was observed when the non-tumor-promoting phorbol ester 4-O-methyl-TPA was substituted for TPA. The calcium ionophore A23187 stimulated radical generation when substituted for the first, but not the second, TPA treatment. The antiinflammatory steroid fluocinolone acetonide inhibited (-)-anti-BPDE-DNA adduct formation when coadministered with the first but not the second TPA treatment. In contrast, all-trans-retinoic acid inhibited (-)-anti-BPDE-DNA adduct formation when coadministered with the second but not the first TPA treatment. These findings demonstrate that tumor promoting phorbol esters stimulate oxygen radical generation in mouse skin and that radical generation is blocked by inhibitors of tumor promotion.

Comparison of the mutagenicities of malondialdehyde and the side products formed during its chemical synthesis.

Malondialdehyde, a product of polyunsaturated fatty acid metabolism and degradation, has been reported to be mutagenic and carcinogenic. The malondialdehyde used for testing was generated by the acidic hydrolysis of tetraalkoxypropanes. We have studied the production of compounds mutagenic to Salmonella typhimurium strain his D 3052 following the hydrolysis of tetraalkoxypropanes. The major mutagenic compound produced from tetraethoxypropane is beta-ethoxy-acrolein (90 to 100 revertants/mumol) and not malondialdehyde (3 to 5 revertants/mumol). Hydrolysis of tetramethoxypropane produces two compounds, beta-methoxy-acrolein (125 to 160 revertants/mumol) and 3,3-dimethoxypropionaldehyde (105 to 135 revertants/mumol), which are more mutagenic than is malondialdehyde. Using standard conditions for the hydrolysis of tetraethoxypropane, the yield of malondialdehyde is 25%, and the yield of beta-ethoxyacrolein is 13%. Considering the differences in specific mutagenicity, the compound which accounts for the bulk of the mutagenicity of a crude hydrolsate of tetraethoxypropane is not malondialdehyde. The presence of these incomplete hydrolysis products may lead to a substantial overestimation of the actual mutagenicity and carcinogenicity of malondialdehyde.

Molecular requirements for the mutagenicity of malondialdehyde and related acroleins.

Malondialdehyde, a product of lipid peroxidation and prostaglandin biosynthesis, is mutagenic in Salmonella. To determine the molecular requirements for its mutagenicity, we tested a series of beta-substituted acroleins in Salmonella typhimurium hisD3052 . Mutagenicity is dependent on the steric bulk of the substituent (revertants/mumol) at the beta position: beta- methoxyacrolein , 220; beta- ethoxyacrolein , 110; and beta- isobutoxyacrolein , 40. A good leaving group at the beta position substantially increases the mutagenic activity (revertants/mumol): beta-(p-nitrophenoxy)acrolein, 620; beta- benzoyloxyacrolein , 320; beta- chloroacrolein , 890; and di-gamma- oxopropenyl ether, 870. These data suggest that nucleophilic attack on the beta-carbon followed by elimination of the beta substituent is important for mutagenicity. Substitution of a methyl group at the alpha-carbon abolishes mutagenicity of these compounds. This effect can be explained by the lack of chemical reactivity of the alpha-methyl analogues toward oxygen or nitrogen nucleophiles. Propynal , which can add nucleophiles to generate a substituted acrolein, exhibits the highest mutagenicity (1370 revertants/mumol) in this series. The importance of the aldehyde functionality is suggested by the nonmutagenicity of propiolonitrile , ethyl propiolate , 4-benzoyloxy-3- buten -2-one, and 4-methoxy-3- buten -2-one. Aldehyde addition subsequent to the formation of the Michael adduct is, therefore, important for mutagenesis. An investigation of the toxicity of the present series indicates that toxicity and mutagenicity are independent events based on different chemical reactions.

Peroxyl radical- and cytochrome P-450-dependent metabolic activation of (+)-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene in mouse skin in vitro and in vivo.

The role of peroxyl radicals and cytochrome P-450 in the metabolic activation of the (+)-enantiomer of 7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene [(+)-BP-7,8-diol] was investigated in the epidermis of CD-1 mice. In skin homogenates from untreated or acetone-pretreated animals [7-14C]-(+)-BP-7,8-diol (20 microM) was metabolized primarily to 7 alpha,8 beta-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydroBP [(-)-anti-BPDE] as detected by high performance liquid chromatography of the stable tetraol hydrolysis products. The amounts of anti-BPDE-tetraols increased with the length of time of incubation (0-90 min). Only small amounts of 7 beta, 8 alpha-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydroBP [(+)-syn-BPDE]-tetraols were detected. Epoxidation was not dependent upon NADPH. The addition of butylated hydroxyanisole (BHA, a free radical scavenger) decreased the formation of both anti- and syn-BPDE-tetraols (I50 less than 1 microM). In epidermal homogenates from animals pretreated with beta-naphthoflavone (beta-NF, an inducer of cytochrome P-450c), (+)-BP-7,8-diol was metabolized almost exclusively to (+)-syn-BPDE. The amounts of syn-BPDE-tetraols also increased with time of incubation with only small amounts of anti-BPDE-tetraols being detected. Epoxidation was NADPH-dependent and was not inhibited by the addition of BHA. The addition of alpha-naphthoflavone (an inhibitor of cytochrome P-450) inhibited syn-BPDE-tetraol formation (I50 approximately 2.5 microM). The DNA adducts formed in mouse epidermis after topical application of [1,3-3H]-(+)-BP-7,8-diol (200 nmol/mouse, 50 microCi/mouse) were analyzed by high performance liquid chromatography. The hydrocarbon-modified deoxyribonucleosides were identified by comparison with standards of (+)-syn-BPDE-dGuo and (-)-anti-BPDE-dGuo. In animals that received no pretreatment, similar amounts of (-)-anti-BPDE-dGuo and (+)-syn-BPDE-dGuo were formed after 3 h of exposure to (+)-BP-7,8-diol, whereas in beta-NF-pretreated animals larger proportions of (+)-syn-BPDE-dGuo were formed. Analysis of the tetraol hydrolysis products and DNA adducts formed from (+)-BP-7,8-diol in mouse skin demonstrates that two independent pathways of metabolic activation occur in vivo: in control animals peroxyl radical-mediated pathways are important contributors to metabolic activation, whereas in beta-NF-pretreated animals cytochrome P-450 is the major oxidizing agent. These results provide the first evidence that peroxyl radicals play a role in xenobiotic metabolism in vivo.

Structural requirements for stimulation of colonic cell proliferation by oxidized fatty acids.

The primary autoxidation products of polyunsaturated fatty acids are known to stimulate DNA synthesis and induce ornithine decarboxylase activity in colonic mucosa. In the present study we have determined the structural features of the oxidized fatty acids necessary for the stimulation of these two components of mitogenesis. Compounds were instilled intrarectally in either aqueous or mineral oil vehicles and 3 h later (ornithine decarboxylase activity) or 12 h later (tritiated thymidine incorporation), the animals were killed and the colonic mucosa harvested for measurement of the two parameters of cell proliferation. Hydroperoxy and hydroxy fatty acids derived from oleate and stearate were studied. Ricinoleic acid and the alpha,beta-unsaturated ketone derived from oleic acid were also investigated. The minimal requirement for stimulation of cell proliferation is the presence of an oxidized functionally adjacent to a carbon-carbon double bond. All active compounds studied were roughly equipotent, which suggests a common mediator may be involved. These results imply that, in addition to biliary steroids, the autoxidation products of unsaturated fatty acids may play a role in the enhancement of tumorigenesis by high levels of dietary fat. Furthermore, the data suggest a possible mechanism of action for the active compounds.

Thromboxane A2 is a mediator of cyclooxygenase-2-dependent endothelial migration and angiogenesis.

Cyclooxygenase-2 (COX-2) inhibitors reduce angiogenic responses to a variety of stimuli, suggesting that products of COX-2 may mediate critical steps. Here, we show that thromboxane A2 (TXA2) is one of several eicosanoid products generated by activated human microvascular endothelial cells. Selective COX-2 antagonists inhibit TXA2 production, endothelial migration, and fibroblast growth factor-induced corneal angiogenesis. Endothelial migration and corneal angiogenesis are similarly inhibited by a TXA2 receptor antagonist, SQ29548. A TXA2 agonist, U46619, reconstitutes both migration and angiogenesis responses under COX-2-inhibited conditions. These findings identify TXA2 as a COX-2 product that functions as a critical intermediary of angiogenesis.

Roles of individual human cytochrome P-450 enzymes in the bioactivation of benzo(a)pyrene, 7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene, and other dihydrodiol derivatives of polycyclic aromatic hydrocarbons.

Human liver microsomes oxidized 7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene [B(a)P-7,8-diol] to products that yield DNA adduct formation and umu gene expression in the tester system Salmonella typhimurium TA1535/pSK1002. The umu response is correlated to levels of microsomal cytochrome P-450NF (P-450NF) and nifedipine oxidation in different human liver samples used for activation, and both the (+)- and (-)-enantiomers of B(a)P-7,8-diol gave similar results in these and other assays. The microsomal umu response was inhibited by antibodies raised against P-450NF. 7,8-Benzoflavone stimulated the B(a)P-7,8-diol-dependent umu response observed with purified P-450NF and human liver and lung microsomes. Thus, P-450NF appears to be the major enzyme involved in the activation of B(a)P-7,8-diol in human liver and possibly lung. Similar results were obtained for the activation of trans-9,10-dihydroxy-9,10-dihydrobenzo(b)fluoranthene and trans-3,4-dihydroxy-3,4-dihydro-7,12-dimethylbenz(a)anthracene, compounds that are known to form highly tumorigenic diol-epoxides. The major product of the oxidation of (+)-B(a)P-7,8-diol was the cis-syn isomer of benzo(a)pyrene-7,8,9,10-tetraol[7 beta, 8 alpha, 9 beta, 10 beta-tetrahydroxy-7,8,9,10-tetrahydrobenzo(a)pyrene]. Studies on the nature of the human liver enzymes involved in the formation of B(a)P-7,8-diol [from benzo(a)pyrene] indicate that neither P-450NF, P-450PA, P-450j, P-450DB, nor P-450MP is involved. The correlation of 7,8-diol formation with phenacetin O-deethylation in a set of liver samples and the partial inhibition of the reaction by 7,8-benzoflavone and anti-rat P-450 beta NF-B suggest that the enzyme involved may be P1-450, the human ortholog of rat P-450 beta NF-B, which catalyzes both the formation of B(a)P-7,8-diol and its subsequent oxidation in tissues of polycyclic hydrocarbon-treated rats. The differential effects of inhibitors indicate that benzo(a)pyrene 3-hydroxylation, 4,5-epoxidation, and 9,10-epoxidation are catalyzed by an enzyme(s) distinct from that which forms the 7,8-epoxide. The roles of the human P-450 enzymes differ from the rodent orthologs in the paradigm for bioactivation of polycyclic hydrocarbons; further, flavones appear to have opposing effects on diol formation and further epoxidation in both human liver and lung.

In vivo stimulation of DNA synthesis and induction of ornithine decarboxylase in rat colon by fatty acid hydroperoxides, autoxidation products of unsaturated fatty acids.

The effect of intrarectal instillation of hydroperoxy and hydroxy fatty acids on colonic DNA synthesis and ornithine decarboxylase activity in male Sprague-Dawley rats was examined. A mixture of hydroperoxy-arachidonic acid isomers was prepared by methylene blue-sensitized photooxygenation. Pure 13-hydroperoxy-9,11-octadecadienoic acid was prepared by the action of soybean lipoxygenase on linoleic acid. Sodium borohydride reduction yielded the respective hydroxy fatty acids. Twelve hr after instillation of solutions of either hydroperoxy or hydroxy fatty acids, at concentrations up to 10 mM, DNA synthesis was increased in a dose-dependent fashion up to 240% above control values. The induction of ornithine decarboxylase occurred over a similar concentration range 3 hr after instillation of oxidized linoleic acid. In this case, the hydroxy acids (49-fold increase at 10 mM), were more stimulatory than the hydroperoxy derivatives (23-fold at 10 mM). Highly purified linoleic and arachidonic acids did not stimulate either activity at concentrations up to 50 mM. These data indicate that autoxidation products of unsaturated fatty acids, likely components of high-fat diets, can evoke proliferative responses in colonic mucosa. These responses may be relevant to the promotional effect of high dietary fat on colon carcinogenesis.

Summary of the round table discussion on strategies for cancer prevention: diet, food, additives, supplements, and drugs.

A Round Table Discussion was held at the Fourth International Conference on Anticarcinogenesis and Radiation Protection. Scientists from government and academia were brought together to discuss the evidence for the preventive effect of foods, specific nutrients and drugs against cancer, and the most appropriate methods of initiating nutritional cancer prevention activities to improve the health of the public. The panel reviewed the epidemiological evidence of the role of diet and specific micronutrients for the prevention of cancer, the doses of specific micronutrients required for preventive effects and their safety, the evidence for aspirin as a chemopreventive agent, the issue of foods versus specific micronutrients in the prevention of cancer, food safety, and approaches to prevention such as food fortification or dietary supplements. The remarks of the panel members are summarized.

Endogenous 12(S)-HETE production by tumor cells and its role in metastasis.

12(S)-Hydroxyeicosatetraenoic acid [12(S)-HETE] is the 12-lipoxygenase metabolite of arachidonic acid. Previously, we have demonstrated that exogenous 12(S)-HETE can activate protein kinase C, increase cell surface expression of integrins, enhance adhesion, induce endothelial cell retraction, and increase experimental metastasis of tumor cells. Because of these prominent effects of exogenous 12(S)-HETE on tumor cell metastatic potential, it is important to determine whether there is endogenous 12(S)-HETE production by tumor cells. In the present study, mRNAs from human, rat, and mouse platelets as well as human colon carcinoma (Clone A), rat Walker carcinoma (W256), and mouse melanoma (B16a) and lung carcinoma (3LL) were reverse transcribed and amplified by polymerase chain reaction with platelet 12-lipoxygenase specific primers. Identity of the polymerase chain reaction fragments was confirmed by sequencing. 12-Lipoxygenase protein was detected by Western blotting. Tumor cell-derived 12-HETE was determined by reverse phase-high performance liquid chromatography analysis. In addition, the effect of endogenous 12(S)-HETE on tumor cells was studied by using a platelet-type 12-lipoxygenase selective inhibitor (N-benzyl-N-hydroxy-5-phenylpentanamide). Our results suggest that some tumor cells express platelet-type 12-lipoxygenase mRNA, protein and metabolize arachidonic acid to 12(S)-HETE and that endogenous 12(S)-HETE, like the exogenous 12(S)-HETE, may play an important role in tumor cell adhesion to matrix in vitro and lung colonization in vivo.

Role of platelet membrane in enhancement of tumor cell adhesion to endothelial cell extracellular matrix.

Tumor cell adhesion to subendothelial matrix in the presence of platelets and plasma has been examined in vitro using an entirely homologous system of rat Walker 256 carcinosarcoma cells, matrix laid down by rat aortic endothelial cells and rat platelets and plasma. In the presence of platelets or platelets plus plasma, tumor cell adhesion was significantly enhanced when compared to adhesion in the absence of platelets. In the presence of plasma alone (0.1%), we observed no significant increase in tumor cell adhesion. In order to determine which platelet factors contribute to the enhancement of tumor cell adhesion by platelets, we subjected washed rat platelets to mechanical lysis or thrombin stimulation followed by centrifugation. The membrane fractions and supernatant fractions containing platelet attachment proteins were compared for their abilities to support tumor cell adhesion to subendothelial matrix. Platelet membranes were also recombined with platelet supernatant fractions to determine if platelet attachment proteins or platelet membranes required the presence of the other to enhance tumor cell adhesion. Platelet supernatant fractions which contained release reaction proteins (confirmed by polyacrylamide gel electrophoresis) did not enhance tumor cell adhesion. Purified thrombospondin, fibronectin, beta-thromboglobulin, platelet derived growth factor, and serotonin had no effect on tumor cell adhesion. Platelet membrane containing fractions affected tumor cell adhesion to subendothelial matrix as follows: (a) platelets formed an adhesive bridge between tumor cells and the subendothelial matrix as demonstrated by scanning electron microscopy; (b) intact platelets and thrombin stimulated platelets were the most effective at facilitating tumor cell adhesion; (c) preparations containing partially lysed platelet ghosts were more effective in supporting tumor cell adhesion to subendothelial matrix than were preparations containing completely lysed platelet membrane fragments; (d) recombination of platelet supernatant fractions with mechanically lysed platelets did not enhance their ability to support adhesion; (e) fixed platelets, either alone or in combination with platelet supernatant fractions, failed to enhance adhesion. These data indicate that platelet enhanced tumor cell adhesion appears to be dependent on platelet membrane factors including receptor mobility, rather than intraplatelet components.

Role of tumor cytoskeleton and membrane glycoprotein IRGpIIb/IIIa in platelet adhesion to tumor cell membrane and tumor cell-induced platelet aggregation.

Components of the tumor cell cytoskeleton (i.e., microtubules, microfilaments, and intermediate filaments) have been reported to affect metastatic ability, since disruption of these components leads to a decrease in metastasis. One mechanism of metastasis which has not been previously considered is the decreased interaction of tumor cells with platelets. We present evidence that disruption of the tumor cell cytoskeleton decreases the ability of tumor cells to aggregate homologous platelets. This effect is dependent upon the disruption of microfilaments/intermediate filaments but not disruption of microtubules. In addition, tumor cell platelet interactions require the lateral mobility of specific receptors (i.e., clustering) on the tumor cell plasma membrane. A membrane glycoprotein immunologically related to the platelet glycoprotein IIb/IIIa complex was identified on Walker 256 carcinosarcoma cells using specific polyclonal and monoclonal antibodies and Northern blot analysis using complementary DNA probes for IIb and IIIa. Mobility of this receptor is dependent upon tumor cell microfilaments/intermediate filaments, but not microtubules. Furthermore, treatment of tumor cells with specific antibodies to the platelet glycoprotein IIb/IIIa complex inhibits tumor cell-platelet interaction at the macroscopic level (i.e., aggregation) and at the ultrastructural level (i.e., platelet adhesion to the tumor cell surface). These results suggest that this immunologically related glycoprotein IIb/IIIa is a receptor for platelet binding to the tumor cell surface, an event which precedes overt platelet aggregation and is dependent upon an intact tumor cell microfilament and intermediate filament network. Therefore, the decreased metastasis observed by others following disruption of the tumor cell cytoskeleton may be due, in part, to a decreased tumor cell-platelet interaction.