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Biomedical subjects

M A Jordan

Publications and source records attributed to M A Jordan.

At least 37 records · Page 2Linked to original sources
PubMed1998

Low potency of taxol at microtubule minus ends: implications for its antimitotic and therapeutic mechanism.

In many cells, low concentrations of Taxol potently block mitosis at the transition from metaphase to anaphase, with no change in microtubule polymer mass and no microtubule bundling. Mitotic block ultimately results in apoptotic cell death and appears to be the most potent antitumor mechanism of Taxol (M. A. Jordan et al., Cancer Res. 56: 816-825, 1996). Mitotic inhibition results, at least in part, from stabilization of growing and shortening dynamics, specifically at the plus ends of microtubules, by the binding of very few Taxol molecules to the microtubule surface (M. A. Jordan et al., Proc. Natl. Acad. Sci. USA, 90: 9552-9556, 1993; W. B. Derry et al., Biochemistry, 34: 2203-2211, 1995). A number of actions of Taxol on mitotic spindle function may be due to its effects on microtubule dynamics at the minus ends of microtubules, effects that previously have not been described. Here, we determined the effects of Taxol on minus ends of purified microtubules at steady state. In contrast to the strong stabilizing effects on plus ends, substoichiometric ratios of Taxol bound to tubulin in microtubules did not affect growing, shortening, or dynamicity at minus ends. Thus, in blocked mitotic cells, Taxol can potently suppress dynamics at plus ends of spindle microtubules, whereas its impotence at minus ends permits continued microtubule depolymerization at the spindle poles. Differential effects of Taxol at opposite microtubule ends may explain Taxol's actions on spindle structure and function and its unique potent antitumor action.

Animals↗
PubMed1998

Microtubules and actin filaments: dynamic targets for cancer chemotherapy.

Microtubules and actin filaments play important roles in mitosis, cell signaling, and motility. Thus these cytoskeletal filaments are the targets of a growing number of anti-cancer drugs. In this review we summarize the current understanding of the mechanisms of these drugs in relation to microtubule and actin filament polymerization and dynamics. In addition, we outline how, by targeting microtubules, drugs inhibit cell proliferation by blocking mitosis at the mitotic checkpoint and inducing apoptosis. The beta-tubulin isotype specificities of new anticancer drugs and the antitumor potential of agents that act on the actin cytoskeleton are also discussed.

Actin Cytoskeleton↗
PubMed1997

Mechanism of action of the unusually potent microtubule inhibitor cryptophycin 1.

Cryptophycin 1 is a remarkably potent antiproliferative compound that shows excellent antitumor activity against mammary, colon, and pancreatic adenocarcinomas in mouse xenographs. At picomolar concentrations, cryptophycin 1 blocks cells in the G2/M phase of the cell cycle by an apparent action on microtubules. The compound binds to tubulin, inhibits microtubule polymerization, and depolymerizes preformed microtubules in vitro. Its exceptionally powerful antitumor activity (many-fold greater than paclitaxel or the vinca alkaloids) raises important questions about its mechanism of action. By quantitative video microscopy, we examined the effects of cryptophycin 1 on the dynamics of individual microtubules assembled to steady state from bovine brain tubulin. At low nanomolar concentrations, in the absence of net microtubule depolymerization, cryptophycin 1 potently stabilized microtubule dynamics. It reduced the rate and extent of microtubule shortening and growing and increased the frequency of rescue. The results suggest that cryptophycin 1 exerts its antiproliferative and antimitotic activity by binding reversibly and with high affinity to the ends of microtubules, perhaps in the form of a tubulin-cryptophycin 1 complex, resulting in the most potent suppression of microtubule dynamics yet described.

Animals↗
PubMed1997

Taxol differentially modulates the dynamics of microtubules assembled from unfractionated and purified beta-tubulin isotypes.

Substoichiometric binding of taxol to tubulin in microtubules potently suppresses microtubule dynamics, which appears to be the most sensitive antiproliferative mechanism of taxol. To determine whether the beta-tubulin isotype composition of a microtubule can modulate sensitivity to taxol, we measured the effects of substoichiometric ratios of taxol bound to tubulin in microtubules on the dynamics of microtubules composed of purified alphabeta(II)-, alphabeta(III)-, or alphabeta(IV)-tubulin isotypes and compared the results with the effects of taxol on microtubules assembled from unfractionated tubulin. Substoichiometric ratios of bound taxol in microtubules assembled from purified beta-tubulin isotypes or unfractionated tubulin potently suppressed the shortening rates and the lengths shortened per shortening event. Correlation of the suppression of the shortening rate with the stoichiometry of bound taxol revealed that microtubules composed of purified alphabeta(II)-, alphabeta(III)-, and alphabeta(IV)-tubulin were, respectively, 1.6-, 7.4-, and 7.2-fold less sensitive to the effects of bound taxol than microtubules assembled from unfractionated tubulin. These results indicate that taxol differentially modulates microtubule dynamics depending upon the beta-tubulin isotype composition. The results are consistent with recent studies correlating taxol resistance in tumor cells with increased levels of beta(III0- and beta(IV)-tubulin expression and suggest that altered cellular expression of beta-tubulin isotypes can be an important mechanism by which tumor cells develop resistance to taxol.

Animals↗
PubMed1997

Inhibition of mitosis and microtubule function through direct tubulin binding by a novel antiproliferative naphthopyran LY290181.

The mechanism of action of a novel antiproliferative compound LY290181 [2-amino-4-(3-pyridyl)-4H-naphtho(1,2-b)pyran-3-carbonitrile] was characterized. LY290181 is a potent inhibitor of cell proliferation, producing 50% inhibition of vascular smooth muscle, endothelial, Chinese hamster ovary, HeLa, and human erythroleukemia cells at concentrations of 8-40 nM. Cell cycle analysis showed that LY290181 caused accumulation of smooth muscle cells at the G2/M phase and induced mitotic arrest in Chinese hamster ovary cells and HeLa cells. At low concentrations (3-30 nM), LY290181 blocked transition of cells from metaphase to anaphase and disrupted mitotic spindle organization. At high concentrations (>/=100 nM), LY290181 produced a concentration-dependent loss of cytoplasmic and spindle microtubules. LY290181 inhibited the polymerization of purified bovine brain microtubule protein into microtubules, and it depolymerized preformed microtubules. Using tubulin-1-anilino-8-naphthalene sulfonate complex fluorescence, we have shown that LY290181 directly interacted with tubulin in a unique manner. These studies show that LY290181 induces cell growth arrest in prometaphase/metaphase, and tubulin appears to be its molecular target.

Animals↗
PubMed1996

Differential effects of vinblastine on polymerization and dynamics at opposite microtubule ends.

We have characterized the effects of vinblastine on the growing and shortening dynamics at opposite ends of individual bovine brain microtubules at steady state in vitro by video microscopy. Vinblastine exerted strikingly different effects on the dynamics and polymer mass at the plus and minus ends of microtubules. At concentrations between 0.1 and 0.4 microM, the drug strongly depolymerized microtubules at minus ends, whereas it did not significantly depolymerize microtubules at plus ends. Vinblastine stabilized plus ends by suppressing the rate and extent of growth and shortening, decreasing the catastrophe frequency, and increasing the rescue frequency. In contrast, vinblastine destabilized minus ends by increasing the catastrophe frequency and decreasing the rescue frequency, whereas it had no effect on the rate or extent of growth or shortening. Thus, vinblastine moderately increased the overall dynamicity at minus ends while strongly suppressing dynamicity at plus ends. Both the kinetic destabilization of microtubules at minus ends and the stabilization at plus ends may contribute to the altered function of mitotic spindle microtubules of cells blocked in mitosis by low concentrations of vinblastine.

Animals↗
PubMed1996

Mitotic block induced in HeLa cells by low concentrations of paclitaxel (Taxol) results in abnormal mitotic exit and apoptotic cell death.

Paclitaxel at low concentrations (10 nM for 20 h) induces approximately 90% mitotic block at the metaphase/anaphase transition in HeLa cells, apparently by suppressing dynamics of spindle microtubules (M. A. Jordan et al., Proc. Natl. Acad. Sci. USA, 90: 9552-9556, 1993). It is not known, however, whether inhibition of mitosis by such low paclitaxel concentrations results in cell death. In the present work, we found that after removal of paclitaxel (10 nM-1 microM), blocked cells did not resume proliferation. Instead, cells exited mitosis abnormally within 24 h. They did not progress through anaphase or cytokinesis but entered an interphase-like state (chromatin decondensed, and an interphase-like microtubule array and nuclear membranes reformed). Many cells (> or = 55%) contained multiple nuclei. Additional DNA synthesis and polyploidy did not occur. DNA degradation into nucleosome-sized fragments characteristic of apoptosis began during drug incubation and increased after drug removal. Cells died within 48-72 h. Incubation with paclitaxel (10 nM for 20 h) resulted in high intracellular drug accumulation (8.3 microM) and little efflux after paclitaxel removal; intracellular retention of paclitaxel may contribute to its efficacy. The results support the hypothesis that the most potent chemotherapeutic mechanism of paclitaxel is kinetic stabilization of spindle microtubule dynamics.

Anaphase↗
PubMed1996

Modulation of CENP-E organization at kinetochores by spindle microtubule attachment.

CENP-E is a protein of the kinesin superfamily that appears as small paired globules at kinetochores of chromosomes in mammalian cells during prometaphase and metaphase of mitosis [Yen et al., 1992: Nature 359:536-539]. In the present study we found that a significant number of chromosomes during early prometaphase in HeLa cells (approximately 30%) were stained with a CENP-E antibody in the form of large C-shaped "collars" that partially encircled the chromosomes. The C-shaped CENP-E collars were present only transiently and were completely replaced by small paired globular forms prior to metaphase. Most chromosomes had persistent CENP-E collars in cells blocked at mitosis with a vinblastine concentration sufficient to prevent all microtubule formation. Attachment of newly formed microtubules to the kinetochores after removal of vinblastine resulted in loss of the collars and replacement with small paired globules. Similarly, a higher proportion of chromosomes isolated from vinblastine-treated cells contained CENP-E collars (73%), and the "capture" (i.e., attachment) of microtubules by the chromosomes resulted in conversion of the collars into small paired globules in vitro. Thus, the CENP-E collars form prior to microtubule attachment and disappear after attachment of the chromosomes to the spindle. The CENP-E collars may facilitate capture of microtubules by chromosomes during prometaphase.

Antibodies↗
PubMed1995

Kinetic stabilization of microtubule dynamics at steady state in vitro by substoichiometric concentrations of tubulin-colchicine complex.

We have analyzed the effects of tubulin-colchicine (TC)-complex on the dynamic instability behavior of bovine brain microtubules at steady state in vitro using video microscopy. Incorporation of low numbers of TC-complexes per microtubule strongly suppressed dynamics at the plus ends by reducing the rate and extent of growing and shortening and by increasing the time microtubules spent in an attenuated state, neither growing nor shortening detectably. In addition, TC-complex strongly suppressed the catastrophe frequency and increased the rescue frequency. At low concentrations (0.02-0.05 microM), TC-complex suppressed dynamics without reducing the polymer mass or the mean microtubule length. Such strong suppression of microtubule dynamics by low TC-complex concentrations in the absence of polymer mass changes demonstrates that microtubule dynamics are more sensitive to the actions of TC-complex than the polymer mass. Significant reduction of polymer mass occurred at relatively high TC-complex concentration (> 0.05 microM). However, the surviving microtubules were extremely stable. Thus, TC-complex stabilizes microtubules even though the microtubules can transiently depolymerize when TC-complex is added. The data also directly establish that kinetic suppression of dynamics by colchicine at low concentrations is effected by a low number of TC-complexes at the microtubule ends.

Animals↗
PubMed1995

Mitotic HeLa cells contain a CENP-E-associated minus end-directed microtubule motor.

A minus end-directed microtubule motor activity from extracts of HeLa cells blocked at prometaphase/metaphase of mitosis with vinblastine has been partially purified and characterized. The motor activity was eliminated by immunodepletion of Centromere binding protein E (CENP-E). The CENP-E-associated motor activity, which was not detectable in interphase cells, moved microtubules at mean rates of 0.46 micron/s at 37 degrees C and 0.24 micron/s at 25 degrees C. The motor activity co-purified with CENP-E through several purification procedures. Motor activity was clearly not due to dynein or to kinesin. The microtubule gliding rates of the CENP-E-associated motor were different from those of dynein and kinesin. In addition, the pattern of nucleotide substrate utilization by the CENP-E-associated motor and the sensitivity to inhibitors were different from those of dynein and kinesin. The CENP-E-associated motor had an apparent native molecular weight of 874,000 Da and estimated dimensions of 2 nm x 80 nm. This is the first demonstration of motor activity associated with CENP-E, strongly supporting the hypothesis that CENP-E may act as a minus end-directed microtubule motor during mitosis.

Cell Movement↗
PubMed1995

Substoichiometric binding of taxol suppresses microtubule dynamics.

We have measured the effects of taxol (10 nM to 1 microM) on the growing and shortening dynamics at the ends of individual bovine brain microtubules in vitro and have correlated the effects both with the stoichiometry of taxol binding to tubulin in microtubules and with the changes in the microtubule polymer mass. The results indicate that taxol suppresses microtubule dynamic instability differently depending upon the stoichiometry of taxol binding to the microtubules. At the lowest effective concentrations (< or = 100 nM), substoichiometric binding of taxol to tubulin in microtubules (between 0.001 and 0.01 mol of bound taxol/mol of tubulin in microtubules) potently and selectively suppresses the rate and extent of shortening at plus ends in association with some increase (28% to 60%) in the mass of microtubule polymer. At intermediate taxol concentrations (between 100 nM and 1 microM), the binding of additional taxol molecules to the microtubules (between 0.01 and 0.1 mol of taxol bound/mol of tubulin in microtubules) inhibits both growing and shortening events at both microtubule ends with no additional increase in microtubule polymer mass. At high taxol concentrations and high taxol binding stoichiometries (> or = 1 microM taxol and > or = 0.1 mol of taxol bound/mol of tubulin in microtubules), microtubule mass increases sharply and dynamics is almost completely suppressed. The data support the hypothesis that binding of a molecule of taxol to a tubulin subunit in microtubules induces a conformational change in that subunit that strongly reduces its ability to dissociate when the subunit becomes exposed at the microtubule end.

Animals↗
PubMed1995

Microtubule dynamics: taking aim at a moving target.

Antitumor drugs of the vinca alkaloid and taxane classes function by suppressing the dynamics of microtubules in spindles, blocking cell division at metaphase. The drugs bind to various sites on the tubulin dimer and at different positions within the microtubule, suggesting that there are many unexplored targets for the design of novel drugs of this type.

Antineoplastic Agents, Phytogenic↗
PubMed1995

Vinblastine suppresses dynamics of individual microtubules in living interphase cells.

We have characterized the effects of vinblastine on the dynamic instability behavior of individual microtubules in living BS-C-1 cells microinjected with rhodamine-labeled tubulin and have found that at low concentrations (3-64 nM), vinblastine potently suppresses dynamic instability without causing net microtubule depolymerization. Vinblastine suppressed the rates of microtubule growth and shortening, and decreased the frequency of transitions from growth or pause to shortening, also called catastrophe. In vinblastine-treated cells, both the average duration of a pause (a state of attenuated dynamics where neither growth nor shortening could be detected) and the percentage of total time spent in pause were significantly increased. Vinblastine potently decreased dynamicity, a measure of the overall dynamic activity of microtubules, reducing this parameter by 75% at 32 nM. The present work, consistent with earlier in vitro studies, demonstrates that vinblastine kinetically caps the ends of microtubules in living cells and supports the hypothesis that the potent chemotherapeutic action of vinblastine as an antitumor drug is suppression of mitotic spindle microtubule dynamics. Further, the results indicate that molecules that bind to microtubule ends can regulate microtubule dynamic behavior in living cells and suggest that endogenous regulators of microtubule dynamics that work by similar mechanisms may exist in living cells.

Animals↗
PubMed1995

The Pallister-Killian syndrome in an African individual.

We report for the first time an individual of Zulu origin with the Pallister-Killian syndrome. Apart from the commonly reported clinical signs, he also had frenula in all four quadrants of the mouth. A broad, short hallux was present. An unusually high level of mosaicism for the isochromosome 12p was found in the lymphocytes.

Abnormalities, Multiple↗
PubMed1995

Novel mechanism of resistance to paclitaxel (Taxol) in human K562 leukemia cells by combined selection with PSC 833.

A paclitaxel-resistant cell line, KPTA5, was established by co-selecting the parental erythroleukemic cell line K562 with stepwise increased concentrations of paclitaxel (Taxol) in the presence of the cyclosporin D analogue PSC 833 (2 microM), a potent modulator of the multidrug resistance phenotype. KPTA5 cells are 9-fold resistant to paclitaxel and taxotere, but do not exhibit significant resistance to Vinca alkaloids, etoposide, anthracyclines, antimetabolites, or alkylating agents. Doubling time and morphology were similar to the parental K562 cells. Reverse transcriptase-polymerase chain reaction (rt-PCR) analysis revealed no alterations in the expression of the mdr1 and MRP genes. Cellular paclitaxel accumulation was unchanged. Cell cycle analyses showed that at 20 nM there was a significantly higher proportion of K562 cells blocked in G2/M, in comparison with KPTA5 cells. In both cases, disruption of the mitotic spindles and the presence of multiple mitotic asters were comparable but occurred at lower paclitaxel concentrations in K562 cells than in KPTA5 cells. There was no difference in total tubulin content between K562 and KPTA5 cells as analyzed by immunoblotting with an anti-beta-tubulin monoclonal antibody. However, we found that KPTA5 cells presented a 2-fold increase both in 5 beta-tubulin mRNA expression and in the corresponding tubulin protein Class IV isotype content, as evaluated by rt-PCR and immunostaining. In conclusion, the KPTA5 cell line displays a novel mechanism of resistance to paclitaxel which does not involve altered cellular drug accumulation. The data presented suggest that alterations in expression of the 5 beta-tubulin gene may be involved in paclitaxel resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Removal of the projection domain of microtubule-associated protein 2 alters its interaction with tubulin.

Microtubule-associated proteins (MAPs) can promote microtubule assembly in vitro. One of these MAPs (MAP2) consists of a short promoter domain which binds to the microtubule and promotes assembly and a long projection domain which projects out from the microtubule and may interact with other cytoskeletal elements. We have previously shown that MAP2 and another MAP, tau, differ in their interactions with tubulin in that tau, but not MAP2, promotes extensive aggregation of tubulin into spiral clusters in the presence of vinblastine and that microtuubles formed with MAP2 are more resistant than those formed with tau to the antimitotic drug maytansine [Luduena, R. F., et al. (1984), J. Biol. Chem. 259, 12890-12898; Fellous, A., et al. (1985), Cancer Res. 45, 5004-5010]. Here we have used chymotryptic digestion to remove the projection domain of MAP2 and examined the interaction of the digested MAP2 (ctMAP2) with tubulin in the presence of vinblastine and maytansine. We have found that ctMAP2 behaves very much like tau, but not like undigested MAP2, in the presence of vinblastine, in that ctMAP2 causes tubulin to polymerize into large clusters of spirals. In contrast, microtubule assembly in the presence of ctMAP2 is much more resistant to maytansine inhibition than is assembly in the presence of tau or undigested MAP2. Our results suggest that the projection domain of MAP2 may play a role in the interaction of tubulin with MAP2 during microtubule assembly.

Binding Sites↗