Search PubMed⌕ Search

Biomedical subjects

M A Jordan

Publications and source records attributed to M A Jordan.

At least 55 records · Page 3Linked to original sources
PubMed1993

Mechanism of mitotic block and inhibition of cell proliferation by taxol at low concentrations.

Taxol inhibited HeLa cell proliferation by inducing a sustained mitotic block at the metaphase/anaphase boundary. Half-maximal inhibition of cell proliferation occurred at 8 nM taxol, and mitosis was half-maximally blocked at 8 nM taxol. Inhibition of mitosis was associated with formation of an incomplete metaphase plate of chromosomes and an altered arrangement of spindle microtubules that strongly resembled the abnormal organization that occurs with low concentrations of vinblastine and other antimitotic compounds. No increase in microtubule polymer mass occurred below 10 nM taxol. The mass of microtubules increased half-maximally at 80 nM taxol and attained maximal levels (5 times normal) at 330 nM taxol. At submicromolar concentrations, taxol suppressed growing and shortening at the ends of microtubules reassembled in vitro from bovine brain tubulin in a manner that resembled suppression by vinblastine. Taxol was concentrated in HeLa cells several hundredfold to levels that were similar to those which suppressed dynamic instability in vitro. The results indicate that taxol shares a common antiproliferative mechanism with vinblastine. At its lowest effective concentrations, taxol appears to block mitosis by kinetically stabilizing spindle microtubules and not by changing the mass of polymerized microtubules.

Cell Division↗
PubMed1993

Kinetic stabilization of microtubule dynamic instability in vitro by vinblastine.

The antiproliferative action of vinblastine at low concentrations appears to result from modulation of the polymerization dynamics of spindle microtubules rather than from depolarization of the microtubules [Jordan, M. A., Thrower, D., & Wilson, L. (1991) Cancer Res. 51, 2212-2222; (1992) J. Cell. Sci. 102, 401-416]. In the present study, we used differential interference contrast video microscopy to analyze the effects of vinblastine on the growing and shortening dynamics (dynamic instability) of individual bovine brain microtubules in vitro. With microtubules which were either depleted of microtubule-associated proteins (MAPs) or rich in MAPs, low concentrations of vinblastine (0.2 microM-1 microM) suppressed the growing and shortening rates and increased the percentage of time that the microtubules spent a state of attenuated activity, neither growing nor shortening detectably. Vinblastine also suppressed the duration of microtubule growing and shortening, and increased the duration of the attenuated state, during which the microtubules neither grew nor shortened detectably. Consistent with previous data obtained using radiolabeled nucleotide exchange in microtubule suspensions [Jordan, M. A., & Wilson, L. (1990) Biochemistry 29, 2730-2739], vinblastine suppressed growing and shortening dynamics at the kinetically more rapid plus ends. The results suggest that vinblastine kinetically stabilizes microtubule ends by modulating the gain and loss of the stabilizing GTP or GDP-Pi "cap", which is believed to be responsible for the transitions between the growing and shortening phases. The data support the hypothesis that (1) low concentrations of vinblastine inhibit mitosis by kinetically stabilizing the polymerization dynamics of spindle microtubules and that (2) the dynamics of spindle microtubules are critical for the proper progression of mitosis.

Animals↗
PubMed1993

Mitotic block in HeLa cells by vinblastine: ultrastructural changes in kinetochore-microtubule attachment and in centrosomes.

Previous work from this laboratory has indicated that very low concentrations of vinblastine block HeLa cells at mitosis in the presence of a full complement of microtubules and without major disruption of spindle organization. In the present study we analyzed the structural organization of mitotic spindle microtubules, chromosomes and centrosomes by electron microscopy after incubating HeLa cells for one cell cycle with 2 nM vinblastine. We found that mitotic block of HeLa cells by vinblastine was associated with alterations of the fine structure of the spindle that were subtle but profound in their apparent consequences. The cell cycle was blocked in a stage that resembled prometaphase or metaphase; chromosomes had not undergone anaphase segregation. Neither the structure of the microtubules nor the structure of the kinetochores was detectably altered by the drug. However, the number of microtubules attached to kinetochores was decreased significantly. In addition, the centrosomes were altered; the normal close association of mother and daughter centriole was lost, numerous membranous vesicles were found in the centrosomal region, and many centrioles exhibited abnormal ultrastructure and had microtubules coursing through their interiors. These findings are consistent with our previous results and indicate that inhibition of the polymerization dynamics of mitotic spindle microtubules and perhaps of centriole microtubules, rather than microtubule depolymerization, is responsible for the mitotic inhibition by vinblastine.

Cell Death↗
PubMed1992

Temperature sensitivity of vinblastine-induced tubulin polymerization in the presence of microtubule-associated proteins.

The antitumor drug vinblastine has been a useful probe for examining the interaction of tubulin with the microtubule-associated proteins (MAPs), specifically with tau and MAP 2. Although tau and MAP 2 can stimulate microtubule assembly in vitro, their specific interactions with tubulin are known to differ. For example, in the presence of vinblastine, both tau and MAP 2 cause tubulin to form spirals, but tau causes formation of clustered spirals of high turbidity, while MAP 2 causes formation of loose spirals of low turbidity [Ludueña et al., J. Biol. Chem. 259, 12890-12898 (1984)]. Although cold temperatures can inhibit microtubule assembly, cold has no effect on vinblastine-induced tubulin spiral formation. Consequently, we used the vinblastine-tubulin system to examine the interactions of tau and MAP 2 with tubulin at low temperatures. We found that tau-tubulin-vinblastine complexes form about as well at 0 degree C as at 37 degrees C. In contrast, MAP 2-tubulin-vinblastine complexes form much less well at 0 degrees C than at 37 degrees C. We find, however, that MAP 2, at 0 degrees C, will strongly inhibit, and even reverse, formation of the tau-tubulin-vinblastine complex. This suggests that the temperature-sensitive factor is the MAP 2-stimulated tubulin-tubulin interaction rather than the MAP 2-tubulin interaction per se; this raises the possibility that the tubulin-tubulin interactions stimulated by tau differ in their temperature sensitivity from those stimulated by MAP 2.

Animals↗
PubMed1992

Effects of vinblastine, podophyllotoxin and nocodazole on mitotic spindles. Implications for the role of microtubule dynamics in mitosis.

Inhibition of mitosis by many drugs that bind to tubulin has been attributed to depolymerization of microtubules. However, we found previously that low concentrations of vinblastine and vincristine blocked mitosis in HeLa cells with little or no depolymerization of spindle microtubules, and spindles appeared morphologically normal or nearly normal. In the present study, we characterized the effects of vinblastine, podophyllotoxin and nocodazole over broad concentration ranges on mitotic spindle organization in HeLa cells. These three drugs are known to affect the dynamics of microtubule polymerization in vitro and to depolymerize microtubules in cells. We wanted to probe further whether mitotic inhibition by these drugs is brought about by a more subtle effect on the microtubules than net microtubule depolymerization. We compared the effects of vinblastine, podophyllotoxin and nocodazole on the organization of spindle microtubules, chromosomes and centrosomes, and on the total mass of microtubules. Spindle organization was examined by immunofluorescence microscopy, and microtubule polymer mass was assayed on isolated cytoskeletons by a quantitative enzyme-linked immunoadsorbence assay for tubulin. As the drug concentration was increased, the organization of mitotic spindles changed in the same way with all three drugs. The changes were associated with mitotic arrest, but were not necessarily accompanied by net microtubule depolymerization. With podophyllotoxin, mitotic arrest was accompanied by microtubule depolymerization. In contrast, with vinblastine and nocodazole, mitotic arrest occurred in the presence of a full complement of spindle microtubules. All three drugs induced a nearly identical rearrangement of spindle microtubules, an increasingly aberrant organization of metaphase chromosomes, and fragmentation of centrosomes. The data suggest that these anti-mitotic drugs block mitosis primarily by inhibiting the dynamics of spindle microtubules rather than by simply depolymerizing the microtubules.

Cell Cycle↗
PubMed1991

Mechanism of inhibition of cell proliferation by Vinca alkaloids.

We have used a structure-activity approach to investigate whether the Vinca alkaloids inhibit cell proliferation primarily by means of their effects on mitotic spindle microtubules or by another mechanism or by a combination of mechanisms. Five Vinca alkaloids were used to investigate the relationship in HeLa cells between inhibition of cell proliferation and blockage of mitosis, alteration of spindle organization, and depolymerization of microtubules. Indirect immunofluorescence staining of microtubules and 4,6-diamidino-2-phenylindole staining of chromatin were used to characterize the effects of the drugs on the distributions of cells in stages of the cell cycle and on the organization of microtubules and chromosomes in metaphase spindles. The microtubule polymer was isolated from cells and quantified using a competitive enzyme-linked immunoadsorbent assay for tubulin. We observed a nearly perfect coincidence between the concentration of each Vinca derivative that inhibited cell proliferation and the concentration that caused 50% accumulation of cells at metaphase, despite the fact that the antiproliferative potencies of the drugs varied over a broad concentration range. Inhibition of cell proliferation and blockage of cells at metaphase at the lowest effective concentrations of all Vinca derivatives occurred with little or no microtubule depolymerization or spindle disorganization. With increasing drug concentrations, the organization of microtubules and chromosomes in arrested mitotic spindles deteriorated in a manner that was common to all five congeners. These results indicate that the antiproliferative activity of the Vinca alkaloids at their lowest effective concentrations in HeLa cells is due to inhibition of mitotic spindle function. The results suggest further that the Vinca alkaloids inhibit cell proliferation by altering the dynamics of tubulin addition and loss at the ends of mitotic spindle microtubules rather than by depolymerizing the microtubules. The specific alterations of spindle microtubule dynamics appear to differ among the five Vinca congeners, and such differences may be responsible for differences in the antitumor specificities of the drugs.

Cell Cycle↗
PubMed1991

Quantitation of cellular tubulin in microtubules and tubulin pools by a competitive ELISA.

A comprehensive method is described for isolating microtubules from cultured mammalian cells and quantitating the tubulin content of both the microtubules and total cellular tubulin pools with a competitive enzyme-linked immunosorbent assay (ELISA). The microtubule isolation procedure involves detergent lysis of cells in a microtubule stabilizing buffer, high speed centrifugation to collect the cytoskeletons, and subsequent solubilization of tubulin from microtubule-containing pellets. The competitive immunoassay involves preincubating an anti-tubulin monoclonal antibody with an unknown quantity of tubulin in cell extracts or solubilized microtubules to quantitatively reduce the antibody available to bind to a tubulin-coated microtiter plate. Binding of remaining antibody to the microtiter plate is measured spectrophotometrically using an alkaline phosphatase-conjugated secondary antibody. Quantitation is accomplished by comparison with a known quantity of bovine brain tubulin.

Antibody Specificity↗
PubMed1990

Kinetic analysis of tubulin exchange at microtubule ends at low vinblastine concentrations.

We have investigated the effects of vinblastine at micromolar concentrations and below on the dynamics of tubulin exchange at the ends of microtubule-associated-protein-rich bovine brain microtubules. The predominant behavior of these microtubules at polymer-mass steady state under the conditions examined was tubulin flux, i.e., net addition of tubulin at one end of each microtubule, operationally defined as the assembly or A end, and balanced net loss at the opposite (disassembly or D) end. No dynamic instability behavior could be detected by video-enhanced dark-field microscopy. Addition of vinblastine to the microtubules at polymer-mass steady state resulted in an initial concentration-dependent depolymerization predominantly at the A ends, until a new steady-state plateau at an elevated critical concentration was established. Microtubules ultimately attained the same stable polymer-mass plateau when vinblastine was added prior to initiation of polymerization as when the drug was added to already polymerized microtubules. Vinblastine inhibited tubulin exchange at the ends of the microtubules at polymer-mass steady state, as determined by using microtubules differentially radiolabeled at their opposite ends. Inhibition of tubulin exchange occurred at concentrations of vinblastine that had very little effect on polymer mass. Both the initial burst of incorporation that occurs in control microtubule suspensions following a pulse of labeled GTP and the relatively slower linear incorporation of label that follows the initial burst were inhibited in a concentration-dependent manner by vinblastine. Both processes were inhibited to the same extent at all vinblastine concentrations examined. If the initial burst of label incorporation represents a low degree of dynamic instability (very short excursions of growth and shortening of the microtubules at one or both ends), then vinblastine inhibits both dynamic instability and flux to similar extents. The ability of vinblastine to inhibit tubulin exchange at microtubule ends in the micromolar concentration range appeared to be mediated by the reversible binding of vinblastine to tubulin binding sites exposed at the polymer ends. Determination by dilution analysis of the effects of vinblastine on the apparent dissociation rate constants for tubulin loss at opposite microtubule ends indicated that a principal effect of vinblastine is to decrease the dissociation rate constant at A ends (i.e., it produces a kinetic cap at A ends), whereas it has no effect on the D-end dissociation rate constant.

Animals↗
PubMed1989

Binding of vinblastine to stabilized microtubules.

Addition of 2-200 microM vinblastine to microtubules at steady state in vitro causes the microtubules to depolymerize, with the formation of protofilament spirals and other aggregated forms of microtubule protein. The presence of such spirals and protein aggregates, which are difficult to separate from microtubules, has complicated attempts to measure the binding of vinblastine to microtubules. We have found that stabilizing bovine brain microtubules in vitro with dimethyl sulfoxide, taxol, or a combination of dimethyl sulfoxide and taxol prevents or greatly retards the formation of protofilamentous spirals, thus permitting us to measure the binding of vinblastine to intact microtubules. Reciprocal plots of binding data indicate the presence of 1.4-1.7 vinblastine binding sites/mol of tubulin in the microtubule, with a Ka of approximately 3-4 x 10(3) M-1. The Ka value obtained is within 1 order of magnitude of the apparent intrinsic binding constant for the binding of vinblastine to tubulin dimers. The results support the idea that depolymerization of microtubules by intermediate and high concentrations of vinblastine occurs by stoichiometric binding of vinblastine to tubulin along the microtubule surface.

Alkaloids↗
PubMed1987

The interaction of cystamine with bovine brain tubulin.

Microtubule assembly in vitro is sensitive to a variety of non-physiological sulfhydryl-oxidizing agents, but the physiological significance of this phenomenon is unknown, since no physiological sulfhydryl-oxidizing agent has been shown to affect microtubule assembly in vitro. We have accordingly investigated the interaction of tubulin with cystamine. We have found that millimolar concentrations of cystamine inhibit microtubule assembly and induce an abnormal form of tubulin polymerization. Cystamine-induced polymerization does not occur at cold temperature. Formation of the polymer requires reaction of cystamine with two sulfhydryls which become available at 37 degrees C. In addition, cystamine reacts with about three sulfhydryls at 0 degrees C without inducing polymerization. This latter set of sulfhydryls appear to include one or both of the previously defined beta s sulfhydryls whose reaction with N, N'-ethylene-bis(iodoacetamide) is markedly inhibited by GTP, maytansine and vinblastine [Roach, M. C. & Luduena, R. F. (1984) J. Biol. Chem. 259, 12063-12071]. Cystamine's specific manner of interacting with tubulin suggests that it may mimic an endogenous sulfhydryl-directed regulator of microtubule assembly.

Animals↗
PubMed1987

Phase dynamics at microtubule ends: the coexistence of microtubule length changes and treadmilling.

The length dynamics both of microtubule-associated protein (MAP)-rich and MAP-depleted bovine brain microtubules were examined at polymer mass steady state. In both preparations, the microtubules exhibited length redistributions shortly after polymer mass steady state was attained. With time, however, both populations relaxed to a state in which no further changes in length distributions could be detected. Shearing the microtubules or diluting the microtubule suspensions transiently increased the extent to which microtubule length redistributions occurred, but again the microtubules relaxed to a state in which changes in the polymer length distributions were not detected. Under steady-state conditions of constant polymer mass and stable microtubule length distribution, both MAP-rich and MAP-depleted microtubules exhibited behavior consistent with treadmilling. MAPs strongly suppressed the magnitude of length redistributions and the steady-state treadmilling rates. These data indicate that the inherent tendency of microtubules in vitro is to relax to a steady state in which net changes in the microtubule length distributions are zero. If the basis of the observed length redistributions is the spontaneous loss and regain of GTP-tubulin ("GTP caps") at microtubule ends, then in order to account for stable length distributions the microtubule ends must reside in the capped state far longer than in the uncapped state, and uncapped microtubule ends must be rapidly recapped. The data suggest that microtubules in cells may have an inherent tendency to remain in the polymerized state, and that microtubule disassembly must be induced actively.

Animals↗
PubMed1986

Identification of a distinct class of vinblastine binding sites on microtubules.

Vinblastine, at concentrations above approximately 1 to 2 microM, causes depolymerization of steady-state bovine brain microtubules in vitro by a fraying of microtubule ends into protofilament-like spirals. Microtubule depolymerization is associated with the binding of vinblastine in approximately molar stoichiometry to tubulin in microtubules with apparent low affinity, as determined by binding experiments with radiolabeled vinblastine and by the ability of vinblastine to inhibit DEAE-dextran decoration of microtubule surfaces. Our data suggest that depolymerization occurs by a propagated mechanism, initially involving binding of vinblastine to a limited number of available sites on microtubule surfaces. This appears to cause loosening of protofilament associations which results in the exposure of new vinblastine-binding sites. Additional vinblastine binding in turn results in further loosening of protofilament associations. Such loosening, when it occurs at microtubule ends, results in protofilament-like splaying and end-wise depolymerization. Microtubule depolymerization appears mechanistically distinct from inhibition of microtubule polymerization by the drug, which is associated with the binding of vinblastine to small numbers of high-affinity binding sites on tubulin at one or both microtubule ends.

Animals↗
PubMed1986

Effects of gas tension on epidermal keratinocyte DNA synthesis and prostaglandin production.

The gas phase partial pressure of O2 (PO2) overlying mouse keratinocyte cultures controls the rate of DNA synthesis of these cells by an undefined mechanism. In these studies, both PO2 and PCO2 tensions overlying primary cell cultures were varied within the physiologic range. The prostaglandin (PG) production of cells grown under several varying gas tensions was then determined using radioimmune assay. The cultures were grown under a PO2 of either 7.5% (physiologic for in vivo epidermis) or 21% (atmospheric; culture conditions and wound healing) for 5 days. The PCO2 was either 5 or 10%, 2 CO2 tensions routinely used in tissue culture studies. DNA synthesis was quantitated using [3H]dThd uptake into DNA and autoradiography. The PGE2 and PGF2 alpha syntheses by the cultures over specific time periods were determined. Changing the PO2 from 21 to 7.5% decreased the rate of DNA synthesis, while PG production remained constant. When the PCO2 was varied from 5 to 10%, keratinocyte DNA synthesis remained unchanged but PG production was markedly stimulated. The PCO2 effect on PG production was greatest at the highest oxygen tension. The data indicated that under this set of variables, PG production by keratinocytes is not directly related to the rate of DNA synthesis of the cells. Apparently the amount of oxygen in the gas phase can have a permissive effect on epidermal keratinocyte PG production.

Carbon Dioxide↗
PubMed1986

Mechanism of assembly of sea urchin egg tubulin.

Tubulin purified from eggs of the sea urchin Strongylocentrotus purpuratus assembles efficiently in vitro to form microtubules at physiological (18 degrees C) and nonphysiological (37 degrees C) temperatures. MAPs, ring oligomers, and high concentrations of nonphysiological solvents are not required for the assembly reaction. At concentrations above 1.2 mg/ml at 18 degrees C and 0.5 mg/ml at 37 degrees C a concentration-dependent overshoot in turbidity and in light scattering at small angles was observed: turbidity and scattering increased rapidly to a peak, then decreased asymptotically toward a steady-state value. Electron microscopic analysis demonstrated that tubulin sheets were prevalent during the initial stages of overshoot assembly, whereas complete microtubules were present at steady state. Qualitative observations of solution birefringence suggested that the polymer became progressively more aligned during assembly. The overshoot cannot be explained by proteolysis or denaturation of tubulin, by depletion of GTP, by a decrease in assembled mass, or by redistribution of polymer lengths. Taken together, the results suggest that changes in the form and/or in the organization of the assembling polymer are responsible for the overshoots in turbidity and in light scattering at small angles. Our results are consistent with models of microtubule assembly that postulate nucleation by tubulin sheets and subsequent folding of the sheets to form mature microtubules.

Animals↗
PubMed1985

Mechanism of microtubule assembly. Changes in polymer structure and organization during assembly of sea urchin egg tubulin.

Assembly of tubulin, purified from eggs of the sea urchin Stronglyocentrotus purpuratus, was examined at physiological (18 degrees C) and nonphysiological (37 degrees C) temperatures. Critical concentrations for assembly were 0.71 mg/ml at 18 degrees C and 0.21 mg/ml at 37 degrees C. At tubulin concentrations above 1.2 mg/ml at 18 degrees C and 0.5 mg/ml at 37 degrees C, a concentration-dependent "overshoot" in turbidity and in small-angle light scattering was observed; turbidity and scattering increased rapidly to a peak, then decreased asymptotically toward a steady-state value. Quantitative sedimentation analysis revealed that the mass of assembled polymer reached and maintained a constant level during overshoot of turbidity. Changes in the wavelength dependence of turbidity were consistent with the initial formation of sheets of tubulin, followed by conversion of the sheets to microtubules, both at 18 and 37 degrees C. Examination by negative-stain electron microscopy showed that sheetlike structures predominated during the early stages of overshoot assembly, while complete microtubules were present at steady state. Furthermore, measurements of average polymer length revealed that the overshoots in turbidity and in light scattering are unlikely to be caused by polymer length redistribution. Qualitative observations of solution birefringence suggested that the polymer became progressively more aligned during assembly. These results suggest that the turbidity/light-scattering overshoots reflect changes in the form or in the organization of the assembling polymer, or both.

Animals↗
PubMed1985

Interaction of reduced glutathione with bovine brain tubulin.

Incubation of phosphocellulose-purified tubulin with GSH at 30 degrees C results in an inhibition of colchicine binding activity. GSSG has a protective effect against the GSH-induced loss of colchicine-binding. Incubation of tubulin with GSH at 30 degrees C results in the formation of abnormal tubulin polymers which are insensitive to cold. Such aggregation is insensitive to antimicrotubular drugs. Aggregation is inhibited by GSSG but not by DTT or mercaptoethanol. GSH-induced aggregation is very sensitive to the ionic strength of the assembly medium; both the aggregation and colchicine binding inhibition induced by GSH are inhibited at higher ionic strength. These results indicate a very complex interaction of GSH with tubulin.

Animals↗