The novel Akt inhibitor Palomid 529 (P529) enhances the effect of radiotherapy in prostate cancer.

Radiotherapy (RT) is a common treatment for localised prostate cancer, but can cause important side effects. The therapeutic efficacy of RT can be enhanced by pharmacological compounds that target specific pathways involved in cell survival. This would elicit a similar therapeutic response using lower doses of RT and, in turn, reducing side effects. This study describes the antitumour activity of the novel Akt inhibitor 8-(1-Hydroxy-ethyl)-2-methoxy-3-(4-methoxy-benzyloxy)-benzo[c]chromen-6-one (Palomid 529 or P529) as well as its ability to decrease radiation-activated phospho-Akt (p-Akt) signalling in a prostate cancer model. P529 showed a potent antiproliferative activity in the NCI-60 cell lines panel, with growth inhibitory 50 (GI50) <35 microM. In addition, P529 significantly enhanced the antiproliferative effect of radiation in prostate cancer cells (PC-3). Analysis of signalling pathways targeted by P529 exhibited a decrease in p-Akt, VEGF, MMP-2, MMP-9, and Id-1 levels after radiation treatment. Moreover, the Bcl-2/Bax ratio was also reduced. Treatment of PC-3 tumour-bearing mice with 20 mg kg(-1) P529 or 6 Gy radiation dose decreased tumour size by 42.9 and 53%, respectively. Combination of both treatments resulted in 77.4% tumour shrinkage. Decreased tumour growth was due to reduced proliferation and increased apoptosis (as assessed by PCNA and caspase-3 immunostaining). Our results show the antitumour efficacy of P529 alone, and as a radiosensitiser, and suggest that this compound could be used in the future to treat human prostate cancer.

Serotonin in the regulation of brain microcirculation.

Manipulation of brainstem serotonin (5-HT) raphe neurons induces significant alterations in local cerebral metabolism and perfusion. The vascular consequences of intracerebrally released 5-HT point to a major vasoconstrictor role, resulting in cerebral blood flow (CBF) decreases in several brain regions such as the neocortex. However, vasodilatations, as well as changes in blood-brain barrier (BBB) permeability, which are blocked by 5-HT receptor antagonists also can be observed. A lack of relationship between the changes in flow and metabolism indicates uncoupling between the two variables and is suggestive of a direct neurogenic control by brain intrinsic 5-HT neurons on the microvascular bed. In line with these functional data are the close associations that exist between 5-HT neurons and the microarterioles, capillaries and perivascular astrocytes of various regions but more intimately and/or more frequently so in those where CBF is altered significantly following manipulation of 5-HT neurons. The ability of the microvascular bed to respond directly to intracerebrally released 5-HT is underscored by the expression of distinct 5-HT receptors in the various cellular compartments of the microvascular bed. Thus, it appears that while some 5-HT-mediated microvascular functions involve directly the blood vessel wall, others would be relayed through the perivascular astrocyte. The strategic localization of perivascular astrocytes and the different 5-HT receptors that they harbor strongly emphasize their putative pivotal role in transmitting information between 5-HT neurons and microvessels. It is concluded that the cerebral circulation has full capacity to adequately and locally adapt brain perfusion to changes in central 5-HT neurotransmission either directly or indirectly via the neuronal-astrocytic-vascular tripartite functional unit. Dysfunctions in these neurovascular interactions might result in perfusion deficits and might be involved in specific pathological conditions.

Indanocine, a microtubule-binding indanone and a selective inducer of apoptosis in multidrug-resistant cancer cells.

BACKGROUND: Certain antimitotic drugs have antitumor activities that apparently result from interactions with nontubulin components involved in cell growth and/or apoptotic cell death. Indanocine is a synthetic indanone that has been identified by the National Cancer Institute's Developmental Therapeutics Program as having antiproliferative activity. In this study, we characterized the activity of this new antimitotic drug toward malignant cells. METHODS: We tested antiproliferative activity with an MTT [i.e., 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay, mitochondrial damage and cell cycle perturbations with flow cytometry, caspase-3 activation with fluorometry, alterations of the cytoskeletal components with immunofluorescence, and antimicrotubule activity with a tubulin polymerization assay. RESULTS/CONCLUSIONS: Indanocine is a cytostatic and cytotoxic indanone that blocks tubulin polymerization but, unlike other antimitotic agents, induces apoptotic cell death in stationary-phase multidrug-resistant cancer cells at concentrations that do not impair the viability of normal nonproliferating cells. Of the seven multidrug-resistant cell lines tested, three (i.e., MCF-7/ADR, MES-SA/DX5, and HL-60/ADR) were more sensitive to growth inhibition by indanocine than were their corresponding parental cells. Confluent multidrug-resistant cells (MCF-7/ADR), but not drug-sensitive cancer cells (MCF-7) or normal peripheral blood lymphocytes, underwent apoptotic cell death 8-24 hours after exposure to indanocine, as measured by sequential changes in mitochondrial membrane potential, caspase activity, and DNA fragmentation. Indanocine interacts with tubulin at the colchicine-binding site, potently inhibits tubulin polymerization in vitro, and disrupts the mitotic apparatus in dividing cells. IMPLICATIONS: The sensitivity of stationary multidrug-resistant cancer cells to indanocine suggests that indanocine and related indanones be considered as lead compounds for the development of chemotherapeutic strategies for drug-resistant malignancies.

Transforming growth factor beta as a potent promoter in two-stage BALB/c 3T3 cell transformation.

We have tested transforming growth factor beta (TGF beta) in the two-stage BALB/c 3T3 cell transformation assay for possible tumor-promoting activity, since it has several effects similar to those of tumor-promoting phorbol esters. After initiation of BALB/c 3T3 cells with 3-methylchol-anthrene, treatment with TGF beta at 1 ng/ml alone or in combination with epidermal growth factor (EGF) for 4 weeks enhanced the number of transformed foci by 5- to 6-fold in comparison with uninitiated cells. Initiation treatment alone induced no or very few transformed foci in several assays. Treatment with phorbol-12,13-didecanoate (PDD) at 100 ng/ml for 4 weeks enhanced the number of transformed foci in initiated BALB/c 3T3 cells by 4- to 5-fold in comparison with uninitiated cells. Thus, TGF beta at 1 ng/ml is as potent as PDD at 100 ng/ml for tumor-promoting activity in the two-stage BALB/c 3T3 cell transformation assay. The enhancing effect of TGF beta was dose-related in the dose range tested (0.03-1 ng/ml) and was not reversible. Some of the foci induced by combined MCA-TGF beta-EGF treatment were cloned, and eight out of nine clones tested produced tumors in nude mice. TGF beta (1 ng/ml) plus EGF (2 ng/ml) increased the saturation density to a similar extent as PDD (100 ng/ml) but did not affect the growth of BALB/c 3T3 cells. We observed no change in junctional intercellular communication, as measured by the dye transfer method, when cells were treated with TGF beta during the two-stage BALB/c 3T3 cell transformation assay. Nevertheless, there was selective communication between transformed and surrounding nontransformed cells; MCA-TGF beta transformed cells intercommunicated among themselves but not with surrounding nontransformed cells. Our results indicate that TGF beta has potent tumor-promoting activity in vitro, but that this activity is not mediated by a complete blockage of intercellular communication, as is suggested for phorbol ester tumor promoters.

Tubulin-dependent hydrolysis of guanosine triphosphate as a screening test to identify new antitubulin compounds with potential as antimitotic agents: application to carbamates of aromatic amines.

Tubulin-dependent GTP hydrolysis was evaluated for its potential as a relatively simple screening assay for new antimitotic drugs. Carbamates of aromatic amines were chosen as the test system because of the relatively diverse structures of compounds in this class already known to have antimitotic properties and because of the large number of such compounds in the NSC collection of the National Cancer Institute. Of 162 compounds evaluated, significant alterations in the GTPase reaction were observed with 26 agents. Sixteen of these had substantial inhibitory effects on tubulin polymerization (true positives), while ten did not (false positives). There were no false negatives (i.e., no agent inactive in the GTPase assay inhibited tubulin polymerization). The true positives were examined for effects on cell growth and mitosis, and four compounds had 50% inhibitory concentration values of 2 microM or less with L1210 murine leukemia cells. All four caused the accumulation of cells in metaphase arrest. We conclude that tubulin-dependent GTP hydrolysis can be used effectively to select new antitubulin compounds with potential as antimitotic agents from a large group of compounds of unknown activity.

Identification of novel antimitotic agents acting at the tubulin level by computer-assisted evaluation of differential cytotoxicity data.

Data generated in the new National Cancer Institute drug evaluation program, which are based on inhibition of cell growth in 60 human tumor cell lines, were probed with nine known antimitotic agents using the COMPARE algorithm. Cytotoxicity data were available on approximately 7000 compounds at the time of the analysis, and, based on the criteria used, 82 compounds were selected as positive by the computer search. Nine were the probe compounds themselves, and 41 were analogues of known antimitotic agents. Among the remaining 32 compounds there were 19 distinct chemical species. Agents in ten of these groups (containing 20 compounds) were effective inhibitors of in vitro tubulin polymerization and caused the mitotic arrest of cells grown in culture. Two compounds were related natural products binding in the Vinca domain of tubulin, and the others were synthetic agents which interfered with colchicine binding. The remaining 12 agents (one natural product, the remainder synthetic) fell into several groups: two compounds were weak inhibitors of tubulin polymerization, inhibited colchicine binding, and caused mitotic arrest; one compound weakly inhibited tubulin polymerization but did not cause an increase in the number of cells arrested in mitosis; two compounds caused mitotic arrest at micromolar concentrations, but thus far no in vitro interaction with tubulin has been observed; the remainder neither inhibited tubulin polymerization nor caused a rise in the number of cultured cells arrested in mitosis. Tubulin-dependent GTP hydrolysis was stimulated or inhibited by all agents which inhibited tubulin polymerization with the exception of one compound. The analysis of differential cytotoxicity data thus appears to have great promise for the identification of new antimitotic agents with antineoplastic potential.

Inhibition of angiogenesis and breast cancer in mice by the microtubule inhibitors 2-methoxyestradiol and taxol.

2-Methoxyestradiol (2-ME), an endogenous estrogen metabolite which disrupts microtubule function, has been shown to inhibit proliferating cells in vitro and suppress certain murine tumors in vivo. In vitro screening has determined that breast cancer cell lines are most sensitive to inhibition by 2-ME. Additionally, 2-ME has been shown to inhibit angiogenesis in vitro. We tested whether 2-ME suppresses cytokine-induced angiogenesis in vivo and inhibits growth of a human breast carcinoma in severe combined immunodeficient mice. A model of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF)-induced corneal neovascularization in C57BL/6 mice was used to evaluate the antiangiogenic effects of 2-ME and other microtubule inhibitors such as Taxol, vincristine, and colchicine. 2-ME (150 mg/kg p.o., n = 20) inhibited bFGF and VEGF-induced neovascularization by 39% and 54%, respectively. Taxol (6 mg/kg i.p., n = 17) inhibited bFGF and VEGF-induced neovascularization by 45% and 37%, respectively. Vincristine (0.2 mg/kg i.p., n = 8) and colchicine (0.25 mg/kg i.p., n = 8) had no effect. Treatment with 2-ME (75 mg/kg p.o., n = 9) for 1 month suppressed the growth of a human breast carcinoma in mice by 60% without toxicity. Recognition of the antiangiogenic and antitumor properties of 2-ME and Taxol may be crucial in planning clinical applications to angiogenesis-dependent diseases.

Derivatives of 5,6-diphenylpyridazin-3-one: synthetic antimitotic agents which interact with plant and mammalian tubulin at a new drug-binding site.

A series of derivatives of 5,6-diphenylpyridazin-3-one (DPP) was examined for interactions with calf brain tubulin following the demonstration that many members of the class caused significant mitotic effects in intact animals, while others had activity against murine P388 leukemia. In L1210 cells several DPP derivatives caused a rise in the mitotic index which correlated well with the cytotoxicity of the drugs. Active DPP derivatives markedly stimulated tubulin-dependent guanosine triphosphate hydrolysis and inhibited tubulin polymerization or induced tubulin oligomer formation, depending on specific reaction conditions. These new agents, however, did not interfere with the binding to tubulin of radiolabeled colchicine, vinblastine, maytansine, or guanosine triphosphate. They thus appear to bind at a previously undescribed site on the tubulin molecule. Some DPP derivatives have significant herbicidal activity, causing mitotic disruption and a rise in the mitotic index in seedling root tissues. Although the DPP derivatives most toxic to plant tissues differ from those most active in inhibiting calf brain tubulin polymerization, virtually all active compounds bear a nitrile substituent at position 4 of the pyridazinone ring. Most active derivatives also bear substituents of varying structure at position 2 of this ring, but no clear structure-function pattern is apparent at this position. The phenyl rings in the most active herbicidal DPP derivatives either are unsubstituted or bear fluorine atoms. Derivatives with chlorine substituents have no detectable herbicidal activity. In contrast, interactions with calf brain tubulin are substantially enhanced if the phenyl rings bear chlorine substituents.

Characterization of the interaction of cryptophycin 1 with tubulin: binding in the Vinca domain, competitive inhibition of dolastatin 10 binding, and an unusual aggregation reaction.

The antimitotic depsipeptide cryptophycin 1 (CP1) was compared to the antimitotic peptide dolastatin 10 (D10) as an antiproliferative agent and in its interactions with purified tubulin. The potent activity of CP1 as an inhibitor of cell growth was confirmed. The agent had an IC50 of 20 pM against L1210 murine leukemia cells versus 0.5 nM for D10. Both drugs were comparable as inhibitors of the glutamate-induced assembly of purified tubulin, with D10 being slightly more potent. CP1, like D10, was a noncompetitive inhibitor of the binding of [3H]vinblastine to tubulin (apparent Ki, 3.9 microM); and the depsipeptide was a competitive inhibitor of the binding of [3H]D10 to tubulin (apparent Ki, 2.1 microM). CP1 was less potent than D10 as an inhibitor of nucleotide exchange on tubulin, but the two drugs were equivalent in stabilizing the colchicine binding activity of tubulin. CP1, like D10, caused the formation of extensive structured aggregates of tubulin when present in stoichiometric amounts relative to the protein. Whereas at lower concentrations the drugs were equivalent in causing formation of small oligomers detected by gel permeation high-performance liquid chromatography, there were notable differences in the aggregation reactions induced by the two drugs. The electron micrographic appearance of the D10-induced aggregate differed substantially from that of the CP1-induced aggregate. With D10, but not CP1, aggregate morphology was greatly altered in the presence of microtubule-associated proteins. Finally, although CP1 caused the formation of massive aggregates, as did D10, there was little turbidity change with the depsipeptide as opposed to the peptide.

Regulation of ornithine decarboxylase induction by deguelin, a natural product cancer chemopreventive agent.

Deguelin, a plant-derived rotenoid, mediates potent chemopreventive responses through transcriptional regulation of phorbol ester-induced ornithine decarboxylase (ODC) activity. To explore the mechanism of this effect, the activity of this compound was evaluated with a number of model systems. Using cultured mouse epidermal 308 cells, the steady-state levels of both 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ODC mRNA and c-fos were decreased by treatment with deguelin. ODC activity was also inhibited by bullatacin and various antimitotic agents (podophyllotoxin, vinblastine, and colchicine), but only deguelin and bullatacin were active as inhibitors of ODC levels in a TPA-independent c-Myc-mediated induction system using cultured BALB/c c-MycER cells. These results suggest that antimicrotubule effects, as mediated by rotenone, for example, are not responsible for inhibitory activity facilitated by deguelin. This was confirmed by use of an in vitro model of tubulin polymerization in which deguelin and a variety of other rotenoids were investigated and found to be inactive. As anticipated, however, NADH dehydrogenase was inhibited by these rotenoids. Moreover, inhibition of this enzyme correlated with a rapid depletion of ATP levels and potential to inhibit either TPA- or c-Myc-induced ODC activity. It therefore seems that deguelin-mediated interference with transient requirements for elevated energy can inhibit the induction of ODC activity and thereby yield a cancer chemopreventive response.

Neuronal networks and mediators of cortical neurovascular coupling responses in normal and altered brain states.

Brain imaging techniques that use vascular signals to map changes in neuronal activity, such as blood oxygenation level-dependent functional magnetic resonance imaging, rely on the spatial and temporal coupling between changes in neurophysiology and haemodynamics, known as 'neurovascular coupling (NVC)'. Accordingly, NVC responses, mapped by changes in brain haemodynamics, have been validated for different stimuli under physiological conditions. In the cerebral cortex, the networks of excitatory pyramidal cells and inhibitory interneurons generating the changes in neural activity and the key mediators that signal to the vascular unit have been identified for some incoming afferent pathways. The neural circuits recruited by whisker glutamatergic-, basal forebrain cholinergic- or locus coeruleus noradrenergic pathway stimulation were found to be highly specific and discriminative, particularly when comparing the two modulatory systems to the sensory response. However, it is largely unknown whether or not NVC is still reliable when brain states are altered or in disease conditions. This lack of knowledge is surprising since brain imaging is broadly used in humans and, ultimately, in conditions that deviate from baseline brain function. Using the whisker-to-barrel pathway as a model of NVC, we can interrogate the reliability of NVC under enhanced cholinergic or noradrenergic modulation of cortical circuits that alters brain states.This article is part of the themed issue 'Interpreting BOLD: a dialogue between cognitive and cellular neuroscience'.

Activities of guanosine triphosphate analogues in reactions catalyzed by elongation factor Tu and initiation factor 2 of Escherichia coli.

In earlier studies two natural analogues of GTP, guanosine 3'-diphosphate 5'-triphosphate (pppGpp) and dGTP, were found to substitute for GTP in reactions catalyzed by initiation factor 2 (IF-2) and elongation factor Tu (EF-Tu), while only dGTP could replace GTP with elongation factor G. These observations with IF-2 and EF-Tu have been extended to two analogues of GTP modified at the 3' ribose hydroxyl position, 3'-deoxyguanosine 5'-triphosphate (3'dGTP) and 3'-deoxy-3'-aminoguanosine 5'-triphosphate (3'dNH2GTP). These compounds were found to be similar to GTP, dGTP, and pppGpp in IF-2-dependent formation of N-formylmethionyl-puromycin and EF-Tu-dependent formation of N-acetyl-Phe-Phe-tRNA. The apparent Km values for the five guanosine nucleotides were 2 - 10(-6)-4 - 10(-6)M in the former reaction and 2-10(-7)--6-10(-7) M in the latter. These reactions did not have an absolute requirement for either an intact pentose ring or for the guanine base in the nucleotide. Although substantially less active than the guanine nucleotides, ITP and the dialcohol derived from GTP by periodate oxidation and borohydride reduction (ox-redGTP) were partially active in both the IF-2 and EF-Tu-dependent reactions, with apparent Km values about 40-100 times those of GTP.

Di-and triphosphate derivatives of acyclo- and arabinosylguanine. Effects on the polymerization of purified tubulin.

Glutamate- and nucleotide-dependent polymerization of purified calf brain tubulin was used as a model system to study interactions of ribose-modified GDP and GTP analogs with tubulin. Earlier studies (Hamel, E., and Lin, C.M.(1981) Proc. Natl. Acad. Sci. U.S.A. 78,3368-3372) were extended to three additional sets of analogs: the di- and triphosphate derivatives of 9-beta-D-arabinofuranosylguanine (araGDP and araGTP) and acycloguanosine (9-(2-hydroxyethoxymethyl)guanine) (acycloGDP and acycloGTP), as well as the periodate-oxidized and borohydride-reduced derivatives of GDP and GTP (ox-redGDP and ox-redGTP). Disruption of the ribose ring in ox-redGTP resulted in major loss of activity relative to GTP in supporting tubulin polymerization, although the analog's deficiency may result from an inability to displace GDP from the exchangeable site rather than a direct effect on the polymerization reaction itself. The poor activity of ox-redGTP could be largely reversed if nucleoside diphosphate kinase was added to the reaction mixture. Removal of the 2' and 3' carbons entirely, in the form of acycloGTP, resulted in only minimal loss of activity relative to GTP. AraGTP, on the other hand, was more active than GTP in supporting tubulin polymerization. All three GDP analogs were much less effective than GDP in inhibiting tubulin polymerization, although araGDP was significantly more inhibitory than acycloGDP or ox-redGDP. Relative inhibitory activity of these and additional GDP analogs was the same whether GTP or a GTP analog was used to support tubulin polymerization.

Mechanism of action of the antimitotic drug 2,4-dichlorobenzyl thiocyanate: alkylation of sulfhydryl group(s) of beta-tubulin.

The compound, 2,4-dichlorobenzyl thiocyanate (DCBT) was previously shown to cause mitotic arrest, disruption of intracellular microtubules, and inhibition of tubulin polymerization, with resistance to the drug conferred by a mutation in a beta-tubulin gene (Abraham, I., Dion, R.L., Duanmu, C., Gottesman, M.M. and Hamel, E. (1986) Proc. Natl. Acad. Sci. USA 83, 6839-6843). We have now examined its mechanism of action in further detail and conclude that DCBT acts as a sulfhydryl alkylating reagent. A mixed disulfide forms between the 2,4-dichlorobenzyl mercaptan moiety of DCBT and protein sulfhydryl groups with release of cyanate anion to the medium. Gel filtration and dialysis of complexes of tubulin formed with either [nitrile-14C]DCBT, [35S]DCBT or [benzyl-3H]DCBT demonstrated persistent association of 35S and 3H with denatured tubulin, but no binding of 14C to the protein even under native conditions. With equimolar tubulin and DCBT, beta-tubulin is the predominant alkylated species. At high drug concentrations, superstoichiometric amounts of DCBT react with tubulin, and both subunits are alkylated almost equally. When extracts of drug-treated L1210 murine leukemia cells were examined by polyacrylamide gel electroporesis, we found that multiple proteins were alkylated by DCBT, but the most prominent radiolabeled band was that corresponding to beta-tubulin. Dithiothreitol partially reverses inhibition of tubulin polymerization by DCBT and removes almost all the 2,4-dichlorobenzyl mercaptan moiety covalently bound to tubulin. Mitotic arrest occurs with DCBT because tubulin is the cellular protein most sensitive to the agent, probably because of its high cysteine content (20/mol).

Inhibition of tubulin polymerization with ribose-modified analogs of GDP and GTP. Reduced inhibition with microtubule-associated proteins and magnesium.

Inhibitory effects of ribose-modified GDP and GTP analogs on tubulin polymerization were examined to explore nucleotide structural requirements at the exchangeable GTP binding site. With microtubule-associated proteins and Mg2+, GTP-supported polymerization was only modestly inhibited by GDP, and still weaker inhibitory activity was found with two analogs, dGDP and 9-beta-D-arabinofuranosylguanine-5'-diphosphate (araGDP). Omission of Mg2+ significantly enhanced the inhibitory effects of GDP, dGDP and araGDP and resulted in weak inhibition of the reaction by several other GDP analogs. The relative inhibitory activity of the GDP analogs had no discernible relationship to the relative activity of cognate GTP analogs in supporting microtubule-associated protein-dependent polymerization. One GTP analog, 2',3'-dideoxyguanosine 5'-triphosphate (ddGTP), supports polymerization both with and without microtubule-associated proteins. The inhibitory activity of GDP and GDP analogs in ddGTP-supported polymerization was much greater in the absence of microtubule-associated proteins than in their presence; and both reactions were more readily inhibited than was microtubule-associated protein-dependent, GTP-supported polymerization. Microtubule-associated protein-independent, ddGTP-supported polymerization was also potently inhibited by GTP and a number of GTP analogs. GTP was in fact twice as inhibitory as GDP. The relative inhibitory activity of the GTP analogs was comparable to the relative inhibitory activity of the cognate GDP analogs and very different from their relative activity in supporting polymerization.

Stabilization of the colchicine-binding activity of tubulin by organic acids.

A number of carboxylic acids and organic phosphates were found to be highly effective in stabilizing the colchicine-binding activity of calf brain tubulin. The most active were glutamate, glutarate, delta-aminovalerate, glucose 1-phosphate, glucose 6-phosphate, fructose 1,6-(bis)phosphate, creatine phosphate and 6-phosphogluconate Maximum effects occurred at high concentrations. Combinations of agents were also examined, and the most effective mixture for stabilizing tubulin found thus far was the combination of 1.0 M glutamate, 100 mM glucose 1-phosphate, 1 mM GTP and 0.5 mg/ml of albumin. No loss of activity occurred over 48 h at 37 degrees C with tubulin was present at a concentration of 100 microgram/ml.

Modulation of cellular phorbol ester binding and/or protein kinase C activity by human placental fractions.

We describe two factors in human placenta that modulate the interaction of phorbol ester tumor promoters with cell membranes or with protein kinase C. One, phorbol ester binding inhibitory factor, can inhibit binding of [3H]phorbol-12,13-dibutyrate to cultured cells or to a membrane fraction but does not inhibit its binding to a homogeneous C kinase preparation (phorbol ester binding sites). The other, C kinase activating factor, stimulates C kinase activity in a calcium-dependent manner. We separated these two biochemical activities from a crude human placental fraction by gel filtration.

Differential effects of magnesium on tubulin-nucleotide interactions.

Magnesium-depleted 2-(N-morpholino)ethanesulfonate (Mes), glutamate, tubulin and microtubule-associated proteins were prepared and used to study the effects of exogenously added MgCl2 on tubulin-nucleotide interactions in 0.1 M Mes with microtubule-associated proteins and in 1.0 M glutamate. Endogenous levels of Mg2+ in the systems studied were approximately stoichiometric with the tubulin concentrations and largely derived from the tubulin. We examined the effects of added Mg2+ on tubulin polymerization, GDP inhibition of polymerization, binding of GDP and GTP to tubulin, and GTP hydrolysis. Exogenously added Mg2+ had markedly different effects on these reactions. The order of their sensitivity for a requirement for added Mg2+ was as follows: GTP binding greater than GTP hydrolysis greater than polymerization greater than GDP binding. Inhibition of polymerization by GDP varied inversely with the Mg2+ concentration and was greatest in the absence of the cation. These results indicate that GDP and GDP-Mg2+ interact with similar affinity at the exchangeable site, while GTP-Mg2+ has a higher affinity for tubulin than does free GTP. Nevertheless, under appropriate conditions, free GTP can interact sufficiently well with tubulin to permit both nucleation and elongation reactions.

Tubulin polymerization with ATP is mediated through the exchangeable GTP site.

Glycerol-induced tubulin polymerization supported by non-guanine nucleotides was examined. The electrophoretically homogeneous tubulin was devoid of nucleoside diphosphate kinase activity and 95% saturated with exchangeable GDP and nonexchangeable GTP. All purine ribonucleoside 5'-triphosphates were active but no polymerization occurred with CTP or UTP. All polymerization reactions, as a function of nucleotide concentration, were similar: above a minimum (threshold) concentration, as the amount of nucleotide increased the reaction became progressively more rapid and extensive with a progressively shorter nucleation period. Threshold concentrations of ATP, XTP, ITP and GTP were 0.6 mM, 0.3 mM, 30 microM and 7 microM, respectively. Most ribose- and polyphosphate-modified ATP analogs also supported polymerization at high concentrations, but the activity of these analogs relative to ATP was very similar to the activity of cognate GTP analogs relative to GTP. Polymerization with ATP was associated with an ATPase reaction. ATP hydrolysis was potently inhibited by GDP and GTP and altered by antimitotic drugs in parallel with the effects of these agents on GTP hydrolysis. Substantial amounts of [8-14C]GDP bound in the exchangeable site of tubulin were displaced during polymerization with GTP or ATP, but much higher concentrations of ATP were required for equivalent displacement of the tubulin-bound GDP. Polymerization with GTP or ATP was inhibited in a qualitatively similar manner by GDP, with increasing concentrations of GDP causing a progressive prolongation of the nucleation period and reduction in reaction rate and extent. However, complete inhibition of polymerization required that GDP:GTP much greater than 1, but that GDP:ATP much less than 1. Inhibition appeared to be primarily competitive, since with higher triphosphate concentrations higher GDP concentrations were required for comparable inhibition. We conclude that ATP effects on tubulin polymerization are mediated through a feeble interaction at the exchangeable GTP site.

A simple method for the rapid determination of the stereospecificity of NAD-dependent dehydrogenases applied to mammalian IMP dehydrogenase and bacterial NADH peroxidase.

The stereospecificity of IMP dehydrogenase (IMP:NAD+ oxidoreductase, EC 1.1.1.205) from two different sources was determined. The enzyme preparations were obtained from murine lymphoblasts and from Escherichia coli. Both enzymes transferred the 2-3H of IMP to the pro-S position of carbon atom C-4 of the nicotinamide ring in NAD. Thus, B-sided stereospecificity is common to the enzyme from two very different species. In addition, the studies described here demonstrate that alcohol dehydrogenase and NADH peroxidase, used as auxiliary enzymes, in combination with a microdistillation procedure, should permit rapid determination of the stereospecificity of any NAD-dependent dehydrogenase for which the appropriate tritiated substrate is available.