Covalent binding of the benzamide RH-4032 to tubulin in suspension-cultured tobacco cells and its application in a cell-based competitive-binding assay

The benzamide, RH-4032, was found to be a potent antimicrotubule agent in tobacco (Nicotiana tabacum) cells. It strongly inhibited root growth and produced swollen club-shaped roots, an accumulation of cells in arrested metaphase, and loss of microtubules. RH-4032 inhibited the in vitro assembly of bovine tubulin into microtubules, with inhibition requiring a relatively long incubation period. Treatment of tobacco suspension-cultured cells or isolated bovine tubulin with [(14)C]RH-4032, and analysis of radiolabeled protein revealed a highly specific covalent attachment to beta-tubulin. Binding of [(3)H]RH-4032 in tobacco suspension-cultured cells was shown to be saturable and to be influenced by pre-incubation of the cells with various antimicrotubule agents: Binding of [(3)H]RH-4032 was inhibited by the benzamides, pronamide and zarilamide, the N-phenylcarbamate, chlorpropham, and the microtubule-stabilizing drug, paclitaxel, whereas trifluralin and amiprophosmethyl were not inhibitory. A common characteristic of agents that cause microtubule disassembly was a slight enhancement of [(3)H]RH-4032 binding at low concentrations, which did not occur with the microtubule-stabilizing agent paclitaxel. For structural analogs of RH-4032 and various N-phenylcarbamates, it was shown that the ability to inhibit binding of [(3)H]RH-4032 was correlated with the ability to inhibit tobacco root elongation. The results suggest a common binding site on beta-tubulin for RH-4032, pronamide, zarilamide, and chlorpropham, which is distinct from the binding site(s) for trifluralin and amiprophosmethyl. RH-4032 provides a unique approach to studying effects of antimicrotubule agents on plant cells by allowing competitive tubulin binding assays to be conducted in whole cells.     

Role of microtubules in surfactant secretion

In the isolated perfused rat lung and cultured type II cells, surfactant secretion and cellular adenosine 3',5'-cyclic monophosphate (cAMP) content was stimulated by beta-adrenergic agonists. Isoproterenol-induced surfactant secretion was inhibited by the antimicrotubule agents colchicine and vinblastine. Incorporation of [3H]glycerol into disaturated phosphatidylcholine was augmented by beta-adrenergic agents but was not significantly different from the enhanced incorporation rate when colchicine was present. This suggests that the augmented incorporation of [3H]glycerol into disaturated phosphatidylcholine was a secondary response to storage depletion rather than direct cAMP stimulation. beta-Adrenergic agents shifted the equilibrium in the isolated perfused rat lung and cultured type II cells to favor microtubules. The stimulatory effect of 1.0 microM isoproterenol on tubulin polymerization was observed as early as 1 min and was augmented 2.8-fold at a half-maximal stimulation of 4 nM in cultured type II cells. Cytochalasin B, an antimicrofilament agent, potentiated the isoproterenol-induced secretion. These results suggest that an intact microtubule-microfilament system may be obligatory for enhanced surfactant secretion and that beta-adrenergic agents not only induce surfactant release but also tubulin polymerization.     

Role of membrane colchicine binding proteins in the transmission of prolactin message to casein genes in the rabbit mammary gland

Previous work demonstrated that tubulin binding drugs specifically inhibit the capacity of prolactin to initiate casein and DNA synthesis in the mammary cell. It was concluded that microtubules or other tubulin containing cellular structures were involved in the transmission of the prolactin message to genes. In the present work, it is shown that griseofulvin, an antimitotic drug which alters microtubule structure and function, does not prevent prolactin actions. Autoradiographic studies showed that [3H]colchicine binds preferentially to plasma and Golgi membranes in the mammary cell. Short term cultures of mammary explants with [3H]colchicine demonstrated that the labelled drug binds to membranous cellular structures which were isolated from explants at the end of the culture. Fractions containing plasma and Golgi membranes contained the highest amount of radioactivity. Solubilisation of the membranes by Triton X-100 dissociated the [3H]colchicine from the prolactin receptors as judged by a chromatography of the soluble fraction on a Sepharose 6 B column. On the column, the labelled colchicine remains associated with a molecular entity which may be free tubulin. In all cases, the binding of [3H]colchicine was greatly attenuated by an excess of unlabelled colchicine but was only slightly affected by the competition with lumicolchicine. These results suggest that mammary membranes contain tubulin and that binding of drugs to this molecule inhibits the generation of the prolactin second messengers eliciting the hormonal actions in the mammary cell. This also suggests that microtubules are probably not involved in the mechanism of prolactin action.     

DNA synthesis and microtubule assembly-related events in fertilized Paracentrotus lividus eggs: reversible inhibition by 10 mM procaine

This report describes the effects of 10 mM procaine on microtubule assembly and on DNA synthesis, as followed by [3H]colchicine binding assays and [3H]thymidine incorporation respectively, in fertilized Paracentrotus lividus eggs. In the absence of microtubule assembly inhibitors, about 25% of the total egg tubulin is submitted to two cycles of polymerization prior to the first cell division, this polymerization process precedes DNA synthesis. If the zygotes are treated with 10 mM procaine in the course of the cell cycle, tubulin polymerization is inhibited or microtubules are disassembled. DNA synthesis is inhibited when procaine treatment is performed 10 min, before the initiation of the S-period. However, when the drug is applied in the course of this synthetic period, the process is normally accomplished, but the next S-period becomes inhibited. Moreover, procaine treatment increases the cytoplasmic pH of the fertilized eggs by about 0.6 to 0.8 pH units. This pH increase precedes microtubule disassembly and inhibition of DNA synthesis. Washing out the drug induces a decrease of the intracellular pH which returns to about the same value as that of the fertilized egg controls. This pH change is then followed by the reinitiation of microtubule assembly, DNA synthesis and cell division. Our results show that the inhibition of both tubulin polymerization and DNA synthesis in fertilized eggs treated with 10 mM procaine, appears to be related to the drug-induced increase in cytoplasmic pH.     

Quantitative biochemical analysis of microtubule content in normal and transformed 3T3 cells

Microtubules in normal and transformed BALB 3T3 cells were preserved in a stabilizing medium and measured by a [3H]colchicine-binding tubulin assay, and compared to total cellular tubulin measured under nonstabilizing conditions. Essentially no change in tubulin or microtubule content was seen with changes in cell density or with changes in cellular morphology at various stages of growth of normal or transformed cells or induced by dibutyryl cAMP treatment of transformed cells. Of five cell lines transformed by a variety of agents, four had a significantly higher total tubulin content than untransformed 3T3 cells and all of them had an increased microtubule content. None of the transformed lines had a lower fraction of tubulin recoverable as sedimentable microtubules compared to untransformed cells, and in three of them this fraction was significantly higher. These results establish that microtubules are present in transformed cells to at least the extent (if not greater) than in normal cells but that there are variations in the total amount of tubulin and microtubules as well as the fraction of the total tubulin present as microtubules which are not strictly correlated with transformation or cell morphology.     

Microtubule-disrupting agents reverse the inhibitory effect of interferon on mitogenesis in 3T3 cells

Interferon can inhibit the stimulation of DNA synthesis in quiescent 3T3 cells exposed to combinations of purified growth factors, but the extent of inhibition varies with the number and combination of mitogens used. As the number of growth factors used to stimulate the cells is increased from two to three, the inhibitory effect of IFN is reduced, and if the third mitogen is a microtubule-disrupting agent such as colchicine or nocodazole, it is abolished altogether. The antagonistic effect of microtubule-disrupting agents on interferon-induced inhibition of DNA synthesis suggests that an intact tubulin network is required for this action of interferon. Interferon and tubulin disrupting agents also show similar kinetics in establishing an effect on DNA synthesis which could imply that they have opposite effects on tubulin assembly.     

Cytoplasmic microtubule assembly-disassembly from endogenous tubulin in a Brij-lysed cell model

We studied the characteristics of cytoplasmic microtubule reassembly from endogenous tubulin pools in situ using a Brij 58-lysed 3T3 cell system. Cells that were pretreated in vivo with colcemid retain endogenous tubulin in the depolymerized state after lysis. When lysed cells were removed from colcemid block and incubated in GTP-PIPES reassembly buffer at pH 6.9, microtubules repolymerized randomly throughout the cytoplasm, appeared to be free-ended and were generally not associated with the centrosomes. However, tubulin could be induced to polymerize in an organized manner from the centrosomes by increasing the pH to 7.6 in the presence of ATP and cAMP. Microtubules polymerized in ATP had significantly longer lengths than those assembled in GTP or UTP. When cells not treated with colcemid were lysed, the integrity of the cytoplasmic microtubule complex (CMTC) was maintained during subsequent incubation in reassembly buffer. However, in contrast to unlysed, living cells, microtubules of lysed cells were stable to colchicine. A significant fraction of the CMTC was stable to cold- induced disassembly whereas microtubules reassembled after lysis were extremely cold-sensitive. When cells not treated with colcemid were lysed and incubated in millimolar Ca++, microtubules depolymerized from their distal ends and a much reduced CMTC was observed. Ca++ reversal with EGTA rapidly resulted in a reformation of the CMTC apparently by elongation of Ca++ resistant microtubules.     

Visualization of assembled and disassembled microtubule protein by double label fluorescence microscopy

Indirect immunofluorescence with rhodamine labelled antibodies and fluoresceinated colchicine (FC) are used to simultaneously localize microtubules and soluble tubulin in cultured ovarian granulosa cells. FC labelled tubulin is most concentrated in regions of the cell occupied by antitubulin stained microtubule bundles. Pretreatment of granulosa cells with colchicine results in a central accumulation of FC and antibody labelled tubulin that coincides with the disposition of 10-nm filament cables. In contrast, the microtubule disrupting agent nocodazole produces a diffuse tubulin distribution as detected with both FC and antibody probes. Taxol treatment, which enhances microtubule assembly, results in a striking concentration of microtubule bundles associated with the nucleus that avidly bind FC. These results suggest that disassembled tubulin is preferentially associated with cytoplasmic microtubules and possibly other formed elements of the cytoskeleton.     

Cystamine augments the stimulation of DNA synthesis by peptide growth factors and microtubule-disrupting agents in cultures of 3T3 mouse fibroblasts

Cystamine together with colchicine markedly enhanced the uptake of [3H]-thymidine into DNA of quiescent cultures of insulin-stimulated Swiss 3T3 mouse fibroblasts. Flow cytofluorometric analyses showed an increased rate of transition of cells from G0/G1----S + G2 in response to combinations of insulin, colchicine, and cystamine. Cystamine, the most effective of several thiol compounds, gave maximal augmentation at 200 microM and was toxic at 300-500 microM. Amplification of DNA synthesis by cystamine was also obtained with epidermal growth factor, vasopressin, and 0.5% fetal bovine serum. Combinations of cystamine and other microtubule-disrupting agents such as nocodazole, maytansine, and podophyllotoxin enhanced DNA synthesis in insulin-stimulated cells. In experiments involving sequential addition of agents, significant enhancement of DNA synthesis was observed when the addition of colchicine to cystamine-treated cells was delayed or conversely when the addition of cystamine to colchicine-treated cultures was delayed. This reciprocal interaction between cystamine and colchicine suggests that a prereplicative intermediate accumulates in response to the action of these dissimilar compounds. We consider the possibility that cystamine may act by forming mixed disulfides with thiol groups of unknown protein(s) that regulate DNA replication.     

Nordihydroguaiaretic acid,of a new family of microtubule-stabilizing agents,shows effects differentiated from paclitaxel

Nordihydroguaiaretic acid (NDGA) protected microtubules in NRK cells from depolymerization caused by structurally and functionally diverse drugs such as nocodazole, colchicine, vinblastine, and ilimaquinone. Hitherto reported drugs, although structurally unrelated to paclitaxel, stabilize microtubules in a way similar to that of paclitaxel and compete for paclitaxel binding to tubulin. However, NDGA had activity toward microtubules different from the effects of paclitaxel. In NRK cells, paclitaxel caused microtubule bundle formation in the presence and absence of microtubule-depolymerizing drugs. However, microtubule bundle did not form, and microtubules radiated from the microtubule-organizing center, in cells treated with NDGA. Acceleration of tubulin polymerization in vitro by paclitaxel was strong but that by NDGA was weak. Microtubules polymerized in vitro in the presence of paclitaxel, but not those polymerized in the presence of NDGA, resisted the effects of cold. NDGA seemed to bind to tubulin, but did not compete for [3H]paclitaxel binding to tubulin. These observations indicate that NDGA belongs to a novel family of microtubule-stabilizing drugs.     

Giardia lamblia: evaluation of the in vitro effects of nocodazole and colchicine on trophozoites

Giardia lamblia is the most commonly detected parasite in the intestinal tract of humans and other mammals causing giardiasis. Giardia presents several cytoskeletal structures with microtubules as major components such as the ventral adhesive disk, eight flagella axonemes, the median body and funis. Many drugs have already been tested as antigiardial agents, such as albendazole and mebendazole, which act by specifically inhibiting tubulin polymerization and hence microtubule assembly. In the present work, we used the microtubule inhibitors nocodazole and colchicine in order to investigate their direct and indirect effects on Giardia ultrastructure and attachment to the glass surface, respectively. Axenically grown G. lamblia trophozoites were treated with nocodazole or colchicine for different time intervals and analyzed by light and electron microscopy. It was observed that trophozoites became completely misshapen, detached from the glass surface and failed to complete cell division. The main alterations observed included disc fragmentation, presence of large vacuoles, and appearance of electrondense deposits made of tubulin. The cytokinesis was blocked, but not the karyokinesis, and membrane blebs were observed. These findings show that Giardia behavior and cytoskeleton are clearly affected by the commonly used microtubule targetting agents colchicine and nozodazole.     

Ionophore-induced disassembly of blood platelet microtubules: effect of cyclic AMP and indomethacin

Cytoplasmic calcium levels are believed to be important in blood platelet activation. Upon activation, the discrete marginal microtubule band, which maintains the discoid shape of non-activated platelets, becomes disrupted. Present studies demonstrate that the extent of assembly of the marginal microtubule band is related to cytoplasmic calcium levels. The divalent cationophore, A23187, causes platelet aggregation, secretion, and contraction by promoting calcium transport from intraplatelet storage sites into the cytoplasm. A23187 caused disassembly of platelet microtubules. Quantitation of electron micrographs revealed that numbers of microtubules were reduced by approximately 80% after A23187 treatment. Secondly, assembled microtubules in homogenates of platelets, in which microtubules were stabilized prior to homogenization, were decreased in favor of free tubulin in A23187-treated platelets. Thirdly, A23187 increased 14C-colchicine binding by intact platelets; this also indicated a shift in the microtubule subunit equilibrium to favor free, colchicine-binding tubulin subunits. In control experiments, A23187 did not affect the stability of platelet tubulin, the colchicine binding reaction, or the total tubulin content of platelets. Stimulation of colchicine binding depended on A23187 concentration (0.05-0.5 microM) and did not require extracellular calcium. A23187-stimulation of colchicine binding was blocked by dibutyryl cyclic AMP (0.80 mM) and/or 3-isobutyl-1-methylxanthine (50 microM) and by indomethacin (10 microM). Cyclic AMP or indomethacin also interferes with A23187-induced platelet activation, but indomethacin is not likely to completely inhibit the perturbation of intraplatelet calcium gradients by A23187. It is suggested that A23187-induced microtubule disassembly may be an indirect effect of calcium on microtubules.     

Nanomolar concentrations of nocodazole alter microtubule dynamic instability in vivo and in vitro.

Previous studies demonstrated that nanomolar concentrations of nocodazole can block cells in mitosis without net microtubule disassembly and resulted in the hypothesis that this block was due to a nocodazole-induced stabilization of microtubules. We tested this hypothesis by examining the effects of nanomolar concentrations of nocodazole on microtubule dynamic instability in interphase cells and in vitro with purified brain tubulin. Newt lung epithelial cell microtubules were visualized by video-enhanced differential interference contrast microscopy and cells were perfused with solutions of nocodazole ranging in concentration from 4 to 400 nM. Microtubules showed a loss of the two-state behavior typical of dynamic instability as evidenced by the addition of a third state where they exhibited little net change in length (a paused state). Nocodazole perfusion also resulted in slower elongation and shortening velocities, increased catastrophe, and an overall decrease in microtubule turnover. Experiments performed on BSC-1 cells that were microinjected with rhodamine-labeled tubulin, incubated in nocodazole for 1 h, and visualized by using low-light-level fluorescence microscopy showed similar results except that nocodazole-treated BSC-1 cells showed a decrease in catastrophe. To gain insight into possible mechanisms responsible for changes in dynamic instability, we examined the effects of 4 nM to 12 microM nocodazole on the assembly of purified tubulin from axoneme seeds. At both microtubule plus and minus ends, perfusion with nocodazole resulted in a dose-dependent decrease in elongation and shortening velocities, increase in pause duration and catastrophe frequency, and decrease in rescue frequency. These effects, which result in an overall decrease in microtubule turnover after nocodazole treatment, suggest that the mitotic block observed is due to a reduction in microtubule dynamic turnover. In addition, the in vitro results are similar to the effects of increasing concentrations of GDP-tubulin (TuD) subunits on microtubule assembly. Given that nocodazole increases tubulin GTPase activity, we propose that nocodazole acts by generating TuD subunits that then alter dynamic instability.     

Microtubule-associated proteins-dependent colchicine stability of acetylated cold-labile brain microtubules from the Atlantic cod, Gadus morhua

Assembly of brain microtubule proteins isolated from the Atlantic cod, Gadus morhua, was found to be much less sensitive to colchicine than assembly of bovine brain microtubules, which was completely inhibited by low colchicine concentrations (10 microM). The degree of disassembly by colchicine was also less for cod microtubules. The lack of colchicine effect was not caused by a lower affinity of colchicine to cod tubulin, as colchicine bound to cod tubulin with a dissociation constant, Kd, and a binding ratio close to that of bovine tubulin. Cod brain tubulin was highly acetylated and mainly detyrosinated, as opposed to bovine tubulin. When cod tubulin, purified by means of phosphocellulose chromatography, was assembled by addition of DMSO in the absence of microtubule-associated proteins (MAPs), the microtubules became sensitive to low concentrations of colchicine. They were, however, slightly more stable to disassembly, indicating that posttranslational modifications induce a somewhat increased stability to colchicine. The stability was mainly MAPs dependent, as it increased markedly in the presence of MAPs. The stability was not caused by an extremely large amount of cod MAPs, since there were slightly less MAPs in cod than in bovine microtubules. When "hybrid" microtubules were assembled from cod tubulin and bovine MAPs, these microtubules became less sensitive to colchicine. This was not a general effect of MAPs, since bovine MAPs did not induce a colchicine stability of microtubules assembled from bovine tubulin. We can therefore conclude that MAPs can induce colchicine stability of colchicine labile acetylated tubulin.     

Prior exposure of macrophages to a low temperature enhances their cyclic AMP responses due to microtubule disruption

Prior exposure of intact macrophages to a low temperature (4 degrees C) resulted in tremendous increases in their cAMP-generating responses to prostaglandin (PG) E1, epinephrine, adenosine, forskolin, and cholera toxin. The extent of the enhancement was dependent on the site of stimulation by these agents. The effect of cold exposure was (a) completely reversed by reexposure of the cold-treated cells to 37 degrees C; (b) mimicked by antimicrotubule agents, colchicine and vinblastine; (c) not further increased by colchicine or vinblastine treatment; and (d) efficiently antagonized by D2O and taxol, microtubule stabilizers. These results demonstrated that enhancement of cAMP generation by cold exposure was mediated through microtubule disruption. The effects of cold exposure and microtubule-disrupting agents on hormone-induced refractoriness was also studied. Macrophages stimulated at 37 degrees C by PGE1 became refractory to the subsequent stimulation by PGE1, regardless of whether the cells had been, or were subsequently, exposed to 4 or 37 degrees C. In contrast, cells stimulated by PGE1 in the presence of colchicine, or cells pretreated with PGE1 at 4 degrees C, responded to PGE1 rechallenge normally. The results suggested that disassembly of microtubules provides a condition unfavorable for development of receptor refractoriness, but never favors recovery therefrom.     

Role of the tubulin-microtubule system in lymphocyte activation

The role of the tubulin-microtubule system was examined in human peripheral blood leukocytes after activation with phytohemagglutinin (PHA). Soluble tubulin and microtubules were measured with a [(3)H]colchicine-binding assay. It was found that the tubulin content of PHA-activated lymphocytes was consistently increased relative to total protein content after 36 h of culture. There was no increase in the proportion of total tubulin synthesis which was present as microtubules at 36 h. Nevertheless, as a result of increased tubulin synthesis, there was a two-to three-fold increase in total microtubular mass. Colchicine, which disrupts microtubles, was used to assess the role of microtubule assembly in the sequence of events which follow lymphocyte activation, namely lymphokine release, protein synthesis, RNA synthesis, and DNA synthesis. Colchicine consistently inhibited DNA synthesis but did not inhibit release of the lymphokine, osteoclast activating factor (OAF). Protein and RNA syntheses were inhibited much less than DNA synthesis. The fact that some effects of PHA on lymphocytes appear to require intact microtubules and at least one does not suggest that the microtubule dependent step in PHA-stimulated lymphocyte activation occurs at a stage after propagation of the signal from the membrane to the cell interior.     

Distinct effects of calcium- and cyclic AMP-enhancing factors on cytoskeletal synthesis and assembly in mouse osteoblastic cells.

Previous studies have indicated that the effects of parathyroid hormone (PTH) on osteoblastic function involve alteration of cytoskeletal assembly. We have reported that after a transitory cell retraction, PTH induces respreading with stimulation of actin, vimentin and tubulins synthesis in mouse bone cells and that this effect is not mediated by cAMP. In order to further elucidate the role of intracellular cAMP and calcium on PTH action on bone cell shape and cytoskeleton we have compared the effects of calcium- and cAMP-enhancing factors on actin, tubulin and vimentin synthesis in relation with mouse bone cell morphology, DNA synthesis and alkaline phosphatase activity as a marker of differentiation. Confluent mouse osteoblastic cells were treated with 0.1 mM isobutylmethylxanthine (IBMX) for 24 h. This treatment caused an increase in the levels of cytoskeletal subunits associated with an elevation of cAMP. Under these conditions, PTH (20 nM) and forskolin (0.1 microM) produced persistent cytoplasmic retraction. PTH and forskolin treatment in presence of IBMX (24 h) induced inhibitory effects on actin and tubulin synthesis evaluated by [35S]methionine incorporation into cytoskeletal proteins identified on two-dimensional gel electrophoresis. Under these culture conditions PTH and forskolin also caused disassembly of microfilament and microtubules as shown by the marked reduction in Triton X soluble-actin and alpha- and beta-tubulins. In contrast, incubation of mouse bone cells with 1 microM calcium ionophore A23187 (24 h) resulted in increased monomeric and polymeric forms of actin and tubulin while not affecting intracellular cAMP. Alkaline phosphatase activity was increased in all conditions while DNA synthesis evaluated by [3H]thymidine incorporation into DNA was stimulated by PTH combined with forskolin and inhibited by the calcium ionophore. These data indicate that persistent elevation of cAMP levels induced by PTH and forskolin with IBMX cause cell retraction with actin and tubulin disassembly whereas rising cell calcium induces cytoskeletal protein assembly and synthesis in mouse osteoblasts. The results point to a distinct involvement of calcium and cAMP in both cytoskeletal assembly and DNA synthesis in mouse bone cells.     

Reassembly of microtubules in protoplasts ofSaccharomyces cerevisiae after nocodazole-induced depolymerization

Summary By following microtubule neoformation after their complete destruction by nocodazole, we analyzed the pattern of microtubule nucleation in protoplasts ofSaccharomyces cerevisiae. Using immunofluorescence, the drug was shown to induce rapid and complete disassembly of both cytoplasmic and spindle microtubules and to selectively block protoplast nuclear division at a defined stage of the cell cycle. Treated protoplasts placed in a drug-free environment recovered a more abundant microtubular system. The majority of microtubules re-formed at SPBs whereas a minority of free-ended microtubules nucleated in the cytoplasm of the protoplasts without any detectable association with recognizable nucleation sites. Random nucleation of free microtubules might be induced by high amounts of unpolymerized tubulin likely to be present in the protoplasts at the moment of drug release.Abbreviations MT microtubule - NOCO nocodazole - SPBs spindle pole bodies - PMSF phenylmethylsulfonyl fluoride - BSA bovine serum albumine - sMT spindle microtubule - cMT cytoplasmic microtubule - MTOC microtubule organizing center     

Cyclic AMP does not mediate inhibition of DNA synthesis by interferon in mouse Swiss 3T3 cells

The purpose of this study was to determine whether cyclic AMP (cAMP) plays any direct or indirect role in the antiproliferative effect of mouse L-cell interferon in Swiss 3T3 cells. Firstly, we found that interferon did not affect intracellular levels of cAMP in these cells in the absence or the presence of cAMP-elevating agents. Secondly, we examined the effect of interferon on the stimulation of DNA synthesis of quiescent 3T3 cells by a range of cyclic AMP-elevating agents, including cholera toxin, cAMP derivatives, and prostaglandin E, added in the presence of insulin or vasopressin. Interferon inhibited cyclic AMP-stimulated DNA synthesis as measured by incorporation of radioactive thymidine into acid-insoluble material and autoradiographic analysis of the fraction of labelled cells. Dose-response curves and kinetics of inhibition were identical to those obtained in cultures stimulated by combinations of growth factors that do not increase the intracellular level of cAMP. The inhibition by interferon of cAMP-stimulated DNA synthesis was also observed in secondary cultures of mouse embryo fibroblasts, where cAMP-elevating agents provide a mitogenic signal in the absence of other added growth factors. These results show that the inhibitory effect of interferon on DNA synthesis in Swiss 3T3 cells is not mediated by cyclic AMP.