Staphylococcal delta toxin stimulates endogenous phospholipase A2 activity and prostaglandin synthesis in fibroblasts.

Delta toxin, one of at least four toxins produced by pathogenic strains of the skin bacterium Staphylococcus aureus, is an amphipathic polypeptide possessing hemolytic and cytolytic activity. Delta toxin stimulates high levels of phospholipase A2 activity in 3T3 mouse fibroblasts with concomitant synthesis and release of prostaglandins. Alpha toxin, another hemolytic toxin produced by strains of S. aureus, did not stimulate phospholipase A2 or prostaglandin release in these cells. Analysis of the release of lactate dehydrogenase and beta-galactosidase (cytoplasmic and lysosomal marker enzymes, respectively) from delta-toxin-treated cells indicated that cytolytic concentrations of the toxin damage the cell-surface membrane more extensively than lysosomal membranes. During a 30 min exposure, delta toxin stimulated 3T3 cells to hydrolyze up to 32% of the lipids biosynthetically labeled by incorporation of [3H]arachidonic acid. A relatively high percentage of the free arachidonic acid formed in delta-toxin-treated 3T3 cells was converted to prostaglandins (up to 41.3% and 8.3% converted to chromatographically identifiable prostaglandins E2 and F2 alpha, respectively, in 30 min), with optimal conversion occurring at sublytic toxin concentrations. The degree of activation of phospholipase A2 in 3T3 cells by a range of concentrations of delta toxin correlates with cytotoxicity assessed by failure to exclude trypan blue dye. Analysis of the calcium dependency of the toxin-activated phospholipase A2 was consistent with a cell-surface, Ca2+-dependent enzyme. The phospholipase A2 exhibits a degree of specificity for substrate lipids containing polyunsaturated fatty acid residues which can serve as precursors for prostaglandin formation. Enzymatic activity was not inhibited by diisopropylfluorophosphate (5 mM), N-ethylmaleimide (5 mM) or p-bromophenacylbromide (0.1 mM). Delta toxin did not activate detectable phospholipase A2 in subcellular preparations containing plasma membrane.

Stimulation of cell surface phospholipase A2 and prostaglandin synthesis in 3T2 mouse fibroblasts by phallolysin, a toxin from Amanita phalloides.

Phallolysin, a cytotoxic glycoprotein from the poisonous mushroom Amanita phalloides, stimulates high levels of cellular phospholipase A2 in 3T3 Swiss mouse fibroblasts biosynthetically labeled by incorportion of [5,6,8,9,11,12,14,15-3H]arachiodonic acid. Up to 39.9% of labeled cell lipids were hydrolyzed in 30 min with concomitant synthesis of prostaglandins (up to 5.3 and 1.7% of incorporated label released in 30 min as chromatographically identifiable prostaglandins E2 and F2 alpha, respectively). A similar extent of hydrolysis of labeled phosphatidylcholine was observed in cells biosynthetically labeled with [1,2-14C]choline, with production of an equivalent amount of lysophosphatidylcholine. No detectable hydrolysis of sphingomyelin was observed, indicating (1) that phospholipases B, C and D and sphingomyelinases were not significantly activated, and (2) that the phospholipase A2 activated did not exhibit a high degree of specificity for arachidonic acid moieties in its substrates. An analysis of the Ca2+ requirement for activation suggests that phallolysin activates primarily cell surface, Ca2+-requiring phospholipase A2. Several lines of evidence suggest that stimulation of phospholipase A2 by phalolysin involves binding to receptor sites which are also sites for wheat germ agglutinin. The extent of cell death, as assessed by failure to exclude trypan blue dye, correlated with the percent of maximal activation of phospholipase A2 by a range of concentrations of phallolysin, suggesting that the toxin kills cells by inducing hydrolysis of membrane phospholipids.

Polycations as prostaglandin synthesis inducers. Stimulation of arachidonic acid release and prostaglandin synthesis in cultured fibroblasts by poly(L-lysine) and other synthetic polycations.

Poly(L-lysine) hydrobromide stimulates arachidonic acid release with concomitant synthesis and release of prostaglandins and lipoxygenase-mediated metabolites (hydroxyeicosatetraenoic acids) in cultures of 3T3 Swiss mouse fibroblasts biosynthetically labeled with [3H]arachidonic acid. The response is rapid, reversible with trypsin and persists for at least 50 min. An evaluation of the calcium dependence of the hydrolytic process was consistent with the rate-limiting step involving a cell-surface, calcium-dependent enzyme. The response involves stimulated hydrolysis of arachidonic acid-containing phospholipids, implying the activation of a phospholipase. Arachidonic acid release is stimulated only by poly(L-lysine) hydrobromide preparations with a molecular weight greater than 30 000, which corresponds to a polypeptide chain of more than 140 lysine hydrobromide residues. A variety of other polycations (Mr greater than 30 000), but not polyanions or neutral polymers, stimulated arachidonic acid release and prostaglandin synthesis. The results are consistent with an activation mechanism involving cross-linking of anionic sites on the cell surface. Poly(L-lysine) hydrobromide is also cytotoxic, but the cytotoxic response occurs at 10-fold higher concentrations than arachidonic acid release.

Metabolism of [3H]dopamine following intracerebroventricular injection in rats pretreated with ethanol or chloral hydrate.

The metabolism of [3H]dopamine injected into the lateral cerebroventricles, was studied in rats after treatment with either ethanol or chloral hydrate. The experimental system was designed primarily to detect the possible conversion of dopamine to the tetrahydroisoquinoline derivatives, tetrahydropapaveroline, salsolinol and O-methylsalsolinol. With or without pretreatment with ethanol or chloral hydrate, no conversion of [3H]dopamine to [3H]tetrahydroisoquinolines was detected. The limits of detection per rat brain were: tetrahydropapaveroline less than 1.6 X 10(-14) moles (0.00032% conversion), salsolinol less than 1.6 X 10(-13) moles (0.00032% conversion), and 6- and 7-O-methylsalsolinol less than 3.2 X 10(-14) moles (0.00032% conversion). Two consistent ethanol- or chloral hydrate-induced alterations in [3H]dopamine metabolism were noted: (1) small but statistically significant increases in the ratios of reduced to oxidized metabolites resulting from monoamine oxidase action; and (2) decreased relative amounts of N-methylated metabolites.

Biological activities of synthetic analogues of Alternaria alternata toxin (AAL-toxin) and fumonisin in plant and mammalian cell cultures.

In a search for an analogue of AAL-toxin with high phytotoxicity and low mammalian toxicity, aminopentols [(AP1), hexacetyl AP1 and N-acetyl AP1], and nine analogues (1-9), were tested for toxicity to duckweed (Lemna pausicostata), susceptible tomato (asc/asc) leaf discs, black nightshade leaf discs and mammalian cell lines, including dog kidney (MDCK), rat liver hepatoma (H4TG) and mouse fibroblasts (NIH3T3). These were compared with AAL-toxin and fumonisin B1 (FB1). Analogue 9 at 10 microM increased cellular leakage and chlorophyll loss from both tomato and black nightshade leaf discs. The diester 9 was the most active in the duckweed bioassay, but it was much less toxic to MDCK and H4TG cells with an IC50 of 200 microM compared to 10 microM for FB1. Analogue 9 and FB1 showed similar low toxicities (IC50 = 150 microM) to NIH3T3 cells. Among the substances tested, only analogue 9 had significant phytotoxicity and low mammalian toxicity, indicating some potential for development of safe and effective natural herbicides.

Macrocyclic trichothecenes are undetectable in kudzu (Pueraria montana) plants treated with a high-producing isolate of Myrothecium verrucaria.

Myrothecium verrucaria was found to be an effective pathogen against kudzu grown in the greenhouse and the field. M. verrucaria produced large amounts of macrocyclic trichothecenes when cultured on solid rice medium, including epiroridin E (16.8 mg/g crude extract), epiisororidin E (1 mg/g), roridin E (8.7 mg/g), roridin H (31.3 mg/g), trichoverrin A (0.6 mg/g), trichoverrin B (0.1 mg/g), verrucarin A (37.4 mg/g), and verrucarin J (2.2 mg/g). Most of these toxins were also isolated from M. verrucaria spores and mycelia grown on potato dextrose agar medium, including epiroridin E (32.3 mg/g), epiisororidin E (28.6 mg/g), roridin E (0 mg/g), roridin H (60 mg/g), trichoverrin A (1.3 mg/g), trichoverrin B (1.8 mg/g), verrucarin A (13.8 mg/g), and verrucarin J (131 mg/g). When M. verrucaria was cultured on liquid media, the numbers but not the amounts of toxins decreased. Only epiroridin E (28.3 mg/g), epiisororidin E (29.6 mg/g), verrucarin B (195 mg/g) and verrucarin J (52.6 mg/g) were measured when the fungus was cultured on cornsteep medium. On soyflour-cornmeal broth M. verrucaria produced several toxins, including epiroridin E (58.1 mg/g), epiisororidin E (5.8 mg/g), verrucarin B (29.9 mg/g) and verrucarin J (32 mg/g). In contrast, no macrocyclic trichothecenes were detected by HPLC analysis of plant tissues of kudzu, sicklepod, and soybean treated with aqueous suspensions of M. verrucaria spores formulated with a surfactant. Chloroform-methanol extracts of kudzu leaves and stems treated with M. verrucaria spores were less cytotoxic to four cultured mammalian cell lines than the corresponding extracts from control plants. Purified macrocyclic trichothecenes (verrucarin A and T-2 toxin) were very cytotoxic to the same cell lines (< or = 2 ng/ml). These results show that neither intact macrocyclic trichothecenes nor toxic metabolites could be detected in plant tissues after treatment with M. verrucaria spores. These results argue for both safety and efficacy for the use of M. verrucaria in biological control of kudzu and other noxious weeds, and support proceeding to animal feeding trials for further evaluation of safety.

Phytotoxicity and ultrastructural effects of gymnopusin from the orchid Maxillaria densa on duckweed (Lemna pausicostata) frond and root tissues.

Two phenanthrene derivatives, characterized as erianthridin (9,10-dihydro-2,7-dihydroxy-3,4-dimethoxyphenanthrene) and gymnopusin (2,7-dihydroxy-3,4,9-trimethoxyphenanthrene), were isolated from an extract of the orchid Maxillaria densa, using phytotoxicity with amaranth (Amaranthus hypochondriacus) to guide fractionation. Gymnopusin and erianthridin inhibited radicle elongation of A. hypochondriacus seedlings with IC(50) values of 330 and 58.2 microM, respectively. The phytoxicity of the two phenanthrene derivatives was also assessed on duckweed (Lemna pausicostata), and compared with mammalian toxicity estimated in vitro with four mammalian cell lines. On duckweed, both phenanthrene derivatives caused electrolyte leakage, chlorophyll loss and photobleaching. Ultrastructural examination of duckweed frond and root tissues treated with gymnopusin (100 microM) revealed membrane damage to the tonoplast after 12 h of exposure. Effects on membrane integrity followed a time course similar to that of electrolyte leakage. Both phenanthrene derivatives exhibited moderate cytotoxicity to all mammalian cells tested, which precludes their use as a bioherbicide.

Phytotoxicity and mammalian cytotoxicity of macrocyclic trichothecene mycotoxins from Myrothecium verrucaria.

Macrocyclic trichothecene toxins produced by Myrothecium verrucaria (a phytopathogen of interest in biological weed control) and the non-trichothecene toxin atranone B from Stachybotiys atra were tested for phytotoxicity in duckweed (Lemna pausicostata L.) plantlet cultures and kudzu (Pueraria lobata L.) leaf disc assays, and for mammalian cytotoxicity in four cultured cell lines. Roridin E and H, epi-isororidin E, and verrucarin A and J were phytotoxic (half-maximal effect in the concentration range 0.1-9.7 microM on duckweed and 1.5->80 microM on kudzu) and cytotoxic to mammalian cell lines (half-maximal inhibition of proliferation in the concentration range 1-35 nM). Trichoverrins A and B and atranone B were moderately phytotoxic (half-maximal effect in the concentration range 1 9-69 microM on duckweed and 13->80 microM on kudzu) and weakly cytotoxic with mammalian cell lines (half-maximal inhibition of proliferation in the concentration range 0.3->2 microM).

Occurrence of the mycotoxin chlamydosporol in Fusarium species.

The mycotoxin chlamydosporol (C11H14O5) has been independently isolated from three different Fusarium species (F. chlamydosporum, F. acuminatum and F. culmorum), suggesting that its distribution may be widespread. We have developed a sensitive, specific fluorometric assay for chlamydosporol that allows quantitation in crude culture extracts. Using this assay we have observed production of chlamydosporol on rice medium by 23 of 40 isolates (58%) of Fusarium sp. examined in amounts as high as 0.8% (wt/wt) dried culture medium.

Selection for resistance to phallolysin, a cytolytic toxin from the death-cap mushroom (Amanita phalloides).

Phallolysin-resistant variants are readily selected from unmutagenized populations of mouse fibroblasts. They exhibit partial cross-resistance to wheat germ agglutinin, a lectin with a saccharide specificity similar to phallolysin. These results are consistent with phallolysin acting by binding to cell surface glycoprotein receptors rather than by interacting directly with lipid components of the membrane.

Molecular weight variations in the diversity of phospholipase A2 forms in reptile venoms.

Reptile venoms exhibit a wide diversity of phospholipase A2 forms when examined by electrophoretic and chromatographic techniques which separate on the basis of net charge. In principle, diversity in charge among the enzyme forms could result from two types of structural modifications: (i) pretranslational modifications, such as differences in amino acid sequences; (ii) post-translational modifications, such as partial proteolysis or hydrolysis of amide functions of asparagine and glutamine residues. Some types of modifications alter both charge and molecular weight. A radiolabeling technique has been developed which allows detection of phospholipase A2 enzymes after electrophoretic separation on the basis of molecular weight in polyacrylamide gels containing sodium dodecylsulfate. Venom preparations were radiolabeled by incubation with p-dodecylphenacylbromide, which was prepared radiolabeled to high specific activity with tritium. The labeling agent was dispersed in phosphatidylcholine liposomes and incubated with venom preparations under conditions of optimal enzyme activity. The preparations were then extracted with ether and hexane to remove substrate and unbound label and subjected to polyacrylamide gel electrophoresis under standard conditions. Protein-bound radiolabel was detected by fluorography and autoradiography. Patterns of labeled species obtained by gel electrophoresis of selected venoms using separation on the basis of charge were similar to the patterns of phospholipase A2 variants obtained under the same electrophoretic conditions using enzymatic detection in phosphatidylcholine-containing gels. Examination of a variety of snake and lizard venoms using this technique revealed the presence of multiple molecular weight forms of labeled enzymes, but the extent of diversity was less than that observed with the same venoms using separation of phospholipase electrophoretic variants on the basis of charge. The results are consistent with diversity in reptile venom phospholipase A2 forms being derived from differences in both molecular weight and charge.

Phospholipase activation in the cytotoxic mechanism of thionin purified from nuts of Pyrularia pubera.

Treating NIH3T3 fibroblast cells with Pyrularia pubera thionin (100 micrograms/ml) stimulated release of labelled free fatty acids from phospholipids biosynthetically labelled by incorporation of [3H]arachidonic acid. Since Pyrularia thionin exhibited no detectable phospholipase activity, it was concluded that the release response represented activation of endogenous phospholipases in the cells. The phospholipase activated by Pyrularia thionin (100 micrograms/ml) stimulated the release of 61% of the incorporated [3H]arachidonate in the presence of 1.8 mM extracellular calcium with maximum activation at 90 min following an initial lag period of about 20 min. The release response exhibited little dependence on extracellular calcium at this thionin concentration, but at concentrations 20 micrograms/ml, extracellular calcium appeared to be inhibitory to phospholipase activation. Some characteristics of the fatty acid release response are consistent with activation of a lysosomal phospholipase being part of the cellular response to Pyrularia thionin. Activation of a lysosomal enzyme can occur independently or as a result of coordinate activation of the whole lysosome, which would expose other cellular components of degradative lysosomal enzymes. Consistent with coordinate activation of lysosomal enzymes, Pyrularia thionin also stimulates the production of small, trichloroacetic acid-soluble peptides and nucleic acid fragments from biosynthetically-labelled RNA and proteins in treated cells. It is not clear from the results obtained what role, if any, activation of lysosomal enzymes plays in the overall toxic response to Pyrularia thionin in NIH3T3 cells. However, Pyrularia pubera thionin may represent a useful tool for studying the regulation of lysosomal enzymes and their roles in cells.

Structure-activity relationships for human estrogenic activity in zearalenone mycotoxins.

Zearalenones are estrogenic Fusarium mycotoxins consisting of a resorcinol moiety fused to a 14-member macrocyclic lactone. Using an improved MCF7 human breast cell proliferation assay, we have compared the estrogenicity of 17 chromatographically-homogeneous zearalenones. Both similarities and substantial differences from published results in intact animal systems were observed. Substantial human estrogenicity was retained even in analogs lacking hydroxylation on the aromatic and macrocyclic rings.

Biological activities of fumonisins, mycotoxins from Fusarium moniliforme, in jimsonweed (Datura stramonium L.) and mammalian cell cultures.

Fumonisins A1, A2, B1, B2 and B3 are a series of mycotoxins produced by strains of Fusarium moniliforme. Fumonisins are hydroxylated long-chain alkylamines esterified with propanetricarboxylic acid moieties that represent approximately half the mol. wt of the toxins. The A-series fumonisins are N-acetylated, whereas the B series contains free amino groups. Hydrolytic removal of the propanetricarboxylic acid moieties from fumonisins B1 and B2 yields the corresponding aminopentols, AP1 and AP2, respectively. These compounds were tested for toxicity on widely differing bioassay systems, representing plant and animal systems. The plant bioassay system employed jimsonweed (Datura stramonium L.) leaves and leaf discs in which toxicity was detected as electrolyte leakage, photobleaching and quantitation of chlorophyll reduction. The animal bioassay system employed cultured mammalian cell lines in which toxicity was detected as inhibition of cell proliferation. Fumonisins B1, B2 and B3 at 50 micrograms/ml or less were effective toxins after exposure periods greater than 24 hr in all plant and animal bioassay systems examined, except 3T3 mouse fibroblasts, whereas fumonisins A1 and A2 exhibited little or no activity. However, the hydrolytic degradation products AP1 and AP2 exhibited toxicity similar to or greater than B-series fumonisins in all test systems, including substantial toxicity to 3T3 mouse fibroblasts.

Ultrastructural effects of AAL-toxin TA from the fungus Alternaria alternata on black nightshade (Solanum nigrum L.) leaf discs and correlation with biochemical measures of toxicity.

Ultrastructural effects of AAL-toxin TA from Alternaria alternata on black nightshade (Solanum, nigrum L.) leaf discs and correlation with biochemical measures of toxicity. In black nightshade (Solanum nigrum L.) leaf discs floating in solutions of AAL-toxin TA (0.01-200 microM) under continuous light at 25 degrees C, electrolyte leakage, chlorophyll loss, autolysis, and photobleaching were observed within 24 h. Electrolyte leakage, measured by the conductivity increase in the culture medium, began after 12 h with 200 microM AAL-toxin T(A), but was observed after 24 h with 0.01 to 50 microM AAL-toxin T(A), when it ranged from 25%) to 63% of total releasable electrolytes, respectively. After 48 h incubation, leakage ranged from 39% to 79% of total for 0.01 to 200 microM AAL-toxin T(A), respectively, while chlorophyll loss ranged from 5% to 32% of total, respectively. Ultrastructural examination of black night-shade leaf discs floating in 10 microM AAL-toxin TA under continuous light at 25 degrees C revealed cytological damage beginning at 30 h, consistent with the time electrolyte leakage and chlorophyll reduction were observed. After 30 h incubation chloroplast starch grains were enlarged in control leaf discs, but not in AAL-toxin T(A)-treated discs, and the thylakoids of treated tissue contained structural abnormalities. After 36-48 h incubation with 10 microM AAL-toxin T(A), all tissues were destroyed with only cell walls, starch grains, and thylakoid fragments remaining. Toxicity was light-dependent, because leaf discs incubated with AAL-toxin T(A) in darkness for up to 72 h showed little phytotoxic damage. Within 6 h of exposure to > or =0.5 microM toxin, phytosphingosine and sphinganine in black nightshade leaf discs increased markedly, and continued to increase up to 24 h exposure. Thus, phy siological and ultrastructural changes occurred in parallel with disruption of sphingolipid synthesis, consistent with the hypothesis that AAL-toxin T(A) causes phytotoxicity by interrupting sphingolipid biosynthesis, thereby damaging cellular membranes.

Phytotoxicity and cytotoxicity of the fumonisin C and P series of mycotoxins from Fusarium spp. fungi.

Fumonisin C (FC) and P (FP) are two recently identified series of sphingosine-analog mycotoxins, for which biological activities have not previously been reported. FC1, FC2 and OH-FC1 (1 microM) exhibited strong phytotoxicity comparable to the standard FB1 in duckweed (Lemna pausicotata L.) cultures, whereas FC3 and FC4 were moderately phytotoxic. Conversely, FP1 exhibited weak phytotoxicity only at higher concentrations (> or =10 microM). These mycotoxins exhibited a similar pattern of cytotoxicity with FB1-sensitive cultured mammalian cell lines, H4TG and MDCK.

Role of stress in the initial injury stages of cell killing by altered intracellular calcium.

A variety of cultured cell types are effectively killed by Ca2+-carrying ionophores (A23187, ionomycin and lasalocid) in the presence of adequate extracellular concentrations of Ca2+ and Na+, although cell killing mechanisms independent of these ions also exist. Previous studies identified 2 injury stages (termed stage I and stage II injury) at which the A23187-induced killing process in 3T3 fibroblasts is interrupted in the presence of low extracellular Ca2+ concentrations and in the absence of extracellular Na+, respectively. The present studies confirm the generality of stage II injury in Ca2+-mediated cell killing, but demonstrate a requirement for concomitant stress conditions (e.g., osmotic or oxidative stress) for expression of stage I injury.

Immunohistochemical localization of insulin-degrading enzyme along the rat intestine, in the human colon adenocarcinoma cell line (Caco-2), and in human ileum.

Insulin-degrading enzyme (IDE) has been implicated in the intracellular degradation of insulin in insulin target cells. Knowledge of the existence of this enzyme in the intestine will be beneficial to the achievement of clinical oral efficacy of insulin. A comparative study was conducted with rat intestine, human colon adenocarcinoma (Caco-2) cells, and human ileum. Confocal microscopy analysis using the anti-IDE antibody showed that IDE was localized in the mucosal cells of rat and human intestines, as well as in Caco-2 cells. Immunostaining of this enzyme was homogeneous throughout the cell excluding nucleus, indicating a typical cytosolic distribution in rat and human enterocytes and in Caco-2 cells.

Ethanol and some tetrahydroisoquinolines alter the discharge of cortical and hippocampal neurons: relationship to endogenous opioids.

The activity of single neurons in rat cortex or hippocampus (HPC) was recorded to test two hypotheses: (1) neuronal effects of ethanol are mediated by an endogenous opiate-like mechanism (for example, by release of an endogenous opioid peptide), and; (2) ethanol-induced formation of aldehyde-catecholamine condensation products (tetrahydroisoquinolines; TIQs) might contribute to some acute actions of ethanol. Ethanol and all TIQs were applied to single neurons from multibarrel micropipettes by electroosmosis or pressure. Ethanol most often inhibited neurons of the parietal cortex, while activating most HPC pyramidal neurons. Tetrahydropapaveroline (THP) most often inhibited the spontaneous and glutamate- or acetylcholine (ACh)-induced firing of neurons in both these regions, although some excitations were also seen. In contrast, salsolinol and 7-O-methyl-salsolinol predominantly excited HPC pyramidal neurons, but depressed most parietal cortical neurons. Iontophoretic or SC naloxone usually antagonized the excitatory actions of ethanol, salsolinol and methionine5-enkephalin on HPC pyramidal cells; however, ACh-induced speeding also was antagonized occasionally. Conversely, the antimuscarinic agent scopolamine antagonized the excitatory actions of salsolinol, but not those of met-enkephalin, in some HPC pyramidal cells. These results and those of previous studies show that acutely applied ethanol or salsolinol elicits a region-specific pattern of neuronal effects in brain similar to that previously described for opiates: activity is inhibited in several tested brain areas but excited in hippocampus. Furthermore, these excitatory effects are antagonized by naloxone. However, because of the occasional apparent non-specific effects of naloxone and the puzzling antagonism of the salsolinol-induced excitations by scopolamine, some doubt remains whether the opiate-like actions of these substances can be completely attributed to mediation by opiate receptors.

A proposed mechanism for cooperativity among oncogene products in cancer cells.

A widely studied model for the mechanism of oncogenic transformation involves the insertion of an oncogene or the activation of a proto-oncogene in a normal cell. Both the nature of the oncogene- and proto-oncogene-products and the mechanisms by which they act to transform the cell are poorly understood. Oncogenic transformation in tissues is believed to involve a phenomenon called cooperativity in which more than one oncogene or proto-oncogene must be activated to effect transformation. It is proposed that the various oncogene and proto-oncogene-products that lead to the transformation of a given cell type either mimic or are the enzymes and other proteins involved in mediating the mitogenic signal to which that cell type responds. Cooperativity among oncogenes follows from this because increased activity or increased levels of mediators in the mitogenic sequence could increase the efficiency of transmission of mitogenic signals from the cell surface, thereby lowering the threshold level of growth factor activity required to initiate DNA synthesis.