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Biomedical subjects

M A Jordan

Publications and source records attributed to M A Jordan.

At least 19 recordsLinked to original sources
PubMed2000

Linkage analysis of systemic lupus erythematosus induced in diabetes-prone nonobese diabetic mice by Mycobacterium bovis.

Systemic lupus erythematosus induced by Mycobacterium bovis in diabetes-prone nonobese diabetic mice was mapped in a backcross to the BALB/c strain. The subphenotypes-hemolytic anemia, antinuclear autoantibodies, and glomerular immune complex deposition-did not cosegregate, and linkage analysis for each trait was performed independently. Hemolytic anemia mapped to two loci: Bah1 at the MHC on chromosome 17 and Bah2 on distal chromosome 16. Antinuclear autoantibodies mapped to three loci: Bana1 at the MHC on chromosome 17, Bana2 on chromosome 10, and Bana3 on distal chromosome 1. Glomerular immune complex deposition did not show significant linkage to any genomic region. Mapping of autoantibodies (Coombs' or antinuclear autoantibodies) identified two loci: Babs1 at the MHC and Babs2 on distal chromosome 1. It has previously been reported that genes conferring susceptibility to different autoimmune diseases map nonrandomly to defined regions of the genome. One possible explanation for this clustering is that some alleles at loci within these regions confer susceptibility to multiple autoimmune diseases-the "common gene" hypothesis. With the exception of the H2, this study failed to provide direct support for the common gene hypothesis, because the loci identified as conferring susceptibility to systemic lupus erythematosus did not colocalize with those previously implicated in diabetes. However, three of the four regions identified had been previously implicated in other autoimmune diseases.

Anemia, Hemolytic↗
PubMed1999

In vitro pharmacology of cryptophycin 52 (LY355703) in human tumor cell lines.

PURPOSE: Cryptophycin 52 (LY355703) is a new member of the cryptophycin family of antitumor agents that is currently undergoing clinical evaluation for cancer chemotherapy. The mechanism of action of the cryptophycin class of compounds is associated with an action on microtubules. This report details the pharmacological profile of this new clinical compound in a panel of human tumor cell lines. METHODS: Antiproliferative effects of cryptophycin 52 were measured indirectly by detection of the metabolic reduction of alamarBlue. Cytoxicity was assessed by enzymatic dye activation (calcein AM) combined with dye exclusion (ethidium homodimer) and by clonogenicity assay. Cell cycle effects were evaluated using flow cytometry and fluorescence microscopy. RESULTS: Both antiproliferative and cytotoxic effects of cryptophycin 52 were concentration- and time-dependent. IC50 values for antiproliferative activity in both solid and hematologic tumor cell lines were in the low picomolar range, and without exception, were significantly below values for the antimitotic agents paclitaxel and vinblastine. Flow cytometry and microscopic examination of tumor cells treated with cryptophycin 52 indicated that they accumulated in the mitotic phase of the cell cycle. Cryptophycin 52 was tested for its sensitivity to multidrug-resistance in several paired cell lines in which a sensitive parental line was matched with a multidrug-resistant derivative line. The resistant lines have been shown to over express Pgp and/or MRP multidrug-resistance transport factors. Compared to other antimitotic agents (paclitaxel, vinblastine, vincristine), the potency of cryptophycin 52 was shown to be minimally affected in multidrug-resistant cells compared to their sensitive parental lines. CONCLUSION: Cryptophycin 52 has potent antimitotic, antiproliferative and cytotoxic activity in in vitro human tumor cell models. It is significantly more potent and less sensitive to multidrug resistance mechanisms than other antimitotic antitumor agents currently used in cancer therapy. These characteristics may translate into therapeutic advantages for the clinical use of cryptophycin 52 in cancer chemotherapy.

Antineoplastic Agents↗
PubMed1999

Taxol suppresses dynamics of individual microtubules in living human tumor cells.

Microtubules are intrinsically dynamic polymers, and their dynamics play a crucial role in mitotic spindle assembly, the mitotic checkpoint, and chromosome movement. We hypothesized that, in living cells, suppression of microtubule dynamics is responsible for the ability of taxol to inhibit mitotic progression and cell proliferation. Using quantitative fluorescence video microscopy, we examined the effects of taxol (30-100 nM) on the dynamics of individual microtubules in two living human tumor cell lines: Caov-3 ovarian adenocarcinoma cells and A-498 kidney carcinoma cells. Taxol accumulated more in Caov-3 cells than in A-498 cells. At equivalent intracellular taxol concentrations, dynamic instability was inhibited similarly in the two cell lines. Microtubule shortening rates were inhibited in Caov-3 cells and in A-498 cells by 32 and 26%, growing rates were inhibited by 24 and 18%, and dynamicity was inhibited by 31 and 63%, respectively. All mitotic spindles were abnormal, and many interphase cells became multinucleate (Caov-3, 30%; A-498, 58%). Taxol blocked cell cycle progress at the metaphase/anaphase transition and inhibited cell proliferation. The results indicate that suppression of microtubule dynamics by taxol deleteriously affects the ability of cancer cells to properly assemble a mitotic spindle, pass the metaphase/anaphase checkpoint, and produce progeny.

Adenocarcinoma↗
PubMed1998

Suppression of microtubule dynamics by binding of cemadotin to tubulin: possible mechanism for its antitumor action.

Cemadotin (LU103793) (NSC D-669356) is a water-soluble synthetic analogue of dolastatin 15 that inhibits cell proliferation in vitro and the growth of human tumor xenografts. Cemadotin is in phase II clinical trials as a promising cancer chemotherapeutic agent. The drug blocks cells at mitosis. Its primary mode of action has been unclear but is believed to involve an action on microtubules. We have found that cemadotin binds to tubulin and strongly suppresses microtubule dynamics. Scatchard analysis of cemadotin binding to tubulin indicated that there are two affinity classes of cemadotin-binding sites with Kd values of 19.4 microM and 136 microM. Cemadotin did not inhibit the binding of vinblastine to tubulin, and, conversely, vinblastine did not inhibit the binding of cemadotin to tubulin. By quantitative video microscopy of individual microtubules, we found that cemadotin strongly suppressed dynamic instability of microtubules assembled to steady state using bovine brain tubulin devoid of microtubule-associated proteins. It reduced the rate and extent of growing and shortening, increased the rescue frequency, and increased the percentage of time the microtubules spent in an attenuated or paused state, neither growing nor shortening detectably. At the lowest effective cemadotin concentrations, dynamics were suppressed in the absence of significant microtubule depolymerization. The results suggest that cemadotin exerts its antitumor activity by suppressing spindle microtubule dynamics through a distinct molecular mechanism by binding at a novel site in tubulin.

Animals↗
PubMed1998

Antiproliferative mechanism of action of cryptophycin-52: kinetic stabilization of microtubule dynamics by high-affinity binding to microtubule ends.

Cryptophycin-52 (LY355703) is a new synthetic member of the cryptophycin family of antimitotic antitumor agents that is currently undergoing clinical evaluation. At high concentrations (>/=10 times the IC50), cryptophycin-52 blocked HeLa cell proliferation at mitosis by depolymerizing spindle microtubules and disrupting chromosome organization. However, low concentrations of cryptophycin-52 inhibited cell proliferation at mitosis (IC50 = 11 pM) without significantly altering spindle microtubule mass or organization. Cryptophycin-52 appears to be the most potent suppressor of microtubule dynamics found thus far. It suppressed the dynamic instability behavior of individual microtubules in vitro (IC50 = 20 nM), reducing the rate and extent of shortening and growing without significantly reducing polymer mass or mean microtubule length. Using [3H]cryptophycin-52, we found that the compound bound to microtubule ends in vitro with high affinity (Kd, 47 nM, maximum of approximately 19.5 cryptophycin-52 molecules per microtubule). By analyzing the effects of cryptophycin-52 on dynamics in relation to its binding to microtubules, we determined that approximately 5-6 molecules of cryptophycin-52 bound to a microtubule were sufficient to decrease dynamicity by 50%. Cryptophycin-52 became concentrated in cells 730-fold, and the resulting intracellular cryptophycin-52 concentration was similar to that required to stabilize microtubule dynamics in vitro. The data suggest that cryptophycin-52 potently perturbs kinetic events at microtubule ends that are required for microtubule function during mitosis and that it acts by forming a reversible cryptophycin-52-tubulin stabilizing cap at microtubule ends.

Antineoplastic Agents↗
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↗