Biomedical subjects

Kathy Kamath

Publications and source records attributed to Kathy Kamath.

6 recordsLinked to original sources

PubMed2006

FTDP-17 mutations compromise the ability of tau to regulate microtubule dynamics in cells.

The neural microtubule-associated protein Tau binds directly to microtubules and regulates their dynamic behavior. In addition to being required for normal development, maintenance, and function of the nervous system, Tau is associated with several neurodegenerative diseases, including Alzheimer disease. One group of neurodegenerative dementias known as FTDP-17 (fronto-temporal dementia with Parkinsonism linked to chromosome 17) is directly linked genetically to mutations in the tau gene, demonstrating that Tau misfunction can cause neuronal cell death and dementia. These mutations result either in amino acid substitutions in Tau or in altered Tau mRNA splicing that skews the expression ratio of wild-type 3-repeat and 4-repeat Tau isoforms. Because wild-type Tau regulates microtubule dynamics, one possible mechanism underlying Tau-mediated neurodegeneration is aberrant regulation of microtubule behavior. In this study, we microinjected normal and mutated Tau protein into cultured cells expressing fluorescent tubulin and measured the effects on the dynamic instability of individual microtubules. We found that the FTDP-17 amino acid substitutions G272V (in both 3-repeat and 4-repeat Tau contexts), DeltaK280, and P301L all exhibited markedly reduced abilities to regulate dynamic instability relative to wild-type Tau. In contrast, the FTDP-17 R406W mutation (which maps in a regulatory region outside the microtubule binding domain of Tau) did not significantly alter the ability of 3-repeat or 4-repeat Tau to regulate microtubule dynamics. Overall, these data are consistent with a loss-of-function model in which both amino acid substitutions and altered mRNA splicing in Tau lead to neurodegeneration by diminishing the ability of Tau to properly regulate microtubule dynamics.

Alternative Splicing↗

PubMed2006

2-Methoxyestradiol suppresses microtubule dynamics and arrests mitosis without depolymerizing microtubules.

2-Methoxyestradiol (2ME2), a metabolite of estradiol-17beta, is a novel antimitotic and antiangiogenic drug candidate in phase I and II clinical trials for the treatment of a broad range of tumor types. 2ME2 binds to tubulin at or near the colchicine site and inhibits the polymerization of tubulin in vitro, suggesting that it may work by interfering with normal microtubule function. However, the role of microtubule depolymerization in its antitumor mechanism of action has been controversial. To determine the mechanism by which 2ME2 induces mitotic arrest, we analyzed its effects on microtubule polymerization in vitro and its effects on dynamic instability both in vitro and in living MCF7 cells. In vitro, 2ME2 (5-100 micromol/L) inhibited assembly of purified tubulin in a concentration-dependent manner, with maximal inhibition (60%) at 200 micromol/L 2ME2. However, with microtubule-associated protein-containing microtubules, significantly higher 2ME2 concentrations were required to depolymerize microtubules, and polymer mass was reduced by only 13% at 500 micromol/L 2ME2. In vitro, dynamic instability was inhibited at lower concentrations. Specifically, 4 micromol/L 2ME2 reduced the mean growth rate by 17% and dynamicity by 27%. In living interphase MCF7 cells at the IC50 for mitotic arrest (1.2 micromol/L), 2ME2 significantly suppressed the mean microtubule growth rate, duration and length, and the overall dynamicity, consistent with its effects in vitro, and without any observable depolymerization of microtubules. Taken together, the results suggest that the major mechanism of mitotic arrest at the lowest effective concentrations of 2ME2 is suppression of microtubule dynamics rather than microtubule depolymerization per se.

2-Methoxyestradiol↗

PubMed2005

BetaIII-tubulin induces paclitaxel resistance in association with reduced effects on microtubule dynamic instability.

The development of resistance to paclitaxel in tumors is one of the most significant obstacles to successful therapy. Overexpression of the betaIII-tubulin isotype has been associated with paclitaxel resistance in a number of cancer cell lines and in tumors, but the mechanism of resistance has remained unclear. Paclitaxel inhibits cancer cell proliferation by binding to the beta-subunit of tubulin in microtubules and suppressing microtubule dynamic instability, leading to mitotic arrest and cell death. We hypothesized that betaIII-tubulin overexpression induces resistance to paclitaxel either by constitutively enhancing microtubule dynamic instability in resistant cells or by rendering the microtubules less sensitive to the suppression of dynamics by paclitaxel. Using Chinese hamster ovary cells that inducibly overexpress either betaI- or betaIII-tubulin, we analyzed microtubule dynamic instability during interphase by microinjection of rhodamine-labeled tubulin and time-lapse fluorescence microscopy. In the absence of paclitaxel, there were no differences in any aspect of dynamic instability between the two beta-tubulin-overexpressing cell types. However, in the presence of 150 nm paclitaxel, dynamic instability was suppressed to a significantly lesser extent (suppressed only 12%) in cells overexpressing betaIII-tubulin than in cells overexpressing similar levels of betaI-tubulin (suppressed 47%). The results suggest that overexpression of betaIII-tubulin induces paclitaxel resistance by reducing the ability of paclitaxel to suppress microtubule dynamics. The results also suggest that endogenous regulators of microtubule dynamics may differentially interact with individual tubulin isotypes, supporting the idea that differential expression of tubulin isotypes has functional consequences in cells.

Animals↗

PubMed2004

Synergistic suppression of microtubule dynamics by discodermolide and paclitaxel in non-small cell lung carcinoma cells.

Discodermolide is a new microtubule-targeted antimitotic drug in Phase I clinical trials that, like paclitaxel, stabilizes microtubule dynamics and enhances microtubule polymer mass in vitro and in cells. Despite their apparently similar binding sites on microtubules, discodermolide acts synergistically with paclitaxel to inhibit proliferation of A549 human lung cancer cells (L. Martello et al., Clin. Cancer Res., 6: 1978-1987, 2000). To understand their synergy, we examined the effects of the two drugs singly and in combination in A549 cells and found that, surprisingly, their antiproliferative synergy is related to their ability to synergistically inhibit microtubule dynamic instability and mitosis. The combination of discodermolide and paclitaxel at their antiproliferative IC(50)s (7 nm for discodermolide and 2 nm for paclitaxel) altered all of the parameters of dynamic instability synergistically except the time-based rescue frequency. For example, together the drugs inhibited overall microtubule dynamicity by 71%, but each drug individually inhibited dynamicity by only 24%, giving a combination index (CI) of 0.23. Discodermolide and paclitaxel also synergistically blocked cell cycle progression at G(2)-M (41, 9.6, and 16% for both drugs together, for discodermolide alone, and for paclitaxel alone, respectively; CI = 0.59), and they synergistically enhanced apoptosis (CI = 0.85). Microtubules are unique receptors for drugs. The results suggest that ligands that bind to large numbers of binding sites on an individual microtubule can interact in a poorly understood manner to synergistically suppress microtubule dynamic instability and inhibit both mitosis and cell proliferation, with important consequences for combination clinical therapy with microtubule-targeted drugs.

Alkanes↗

PubMed2003

Suppression of microtubule dynamics by epothilone B is associated with mitotic arrest.

The epothilones are a group of novel microtubule-targeted, antimitotic compounds that have a paclitaxel-like, assembly enhancing effect on tubulin in vitro as well as in cultured cells. We hypothesize that epothilones induce mitotic arrest by suppressing microtubule dynamics. To test this hypothesis, we used MCF7 cells stably transfected with GFP-alpha-tubulin to analyze microtubule dynamics at three concentrations of epothilone B, one that induced no mitotic arrest (0.2 nM, 20 h), one that induced one-third maximal mitotic arrest (IC(33,) 2 nM, 20 h), and one that induced half-maximal mitotic arrest (IC(50,) 3.5 nM, 20 h). We found that epothilone B suppressed microtubule dynamics in a concentration-dependent manner coincident with mitotic block. At 0.2 nM epothilone B, dynamics were not significantly altered. At 2 nM epothilone B (IC(33)), the mean growth and shortening rates were decreased by 38 and 27%, respectively. Dynamicity was decreased by 47%. At the IC(50), 80% of the cells had nearly complete stabilization of microtubule dynamics, and no anaphase or telophase figures were observed. Comparison of the effects of epothilone B on microtubule dynamics with those of paclitaxel indicated that both drugs alter the same microtubule dynamic parameters to a similar extent. At the IC(50) for mitotic arrest, dynamicity was reduced by 54% by paclitaxel compared with 62% for epothilone B. In 65% of the cells treated with paclitaxel, the microtubules were completely stabilized. Thus, the effects of epothilone B on microtubule dynamics are remarkably similar to those of paclitaxel, suggesting that both drugs induce mitotic block by a similar mechanism.

Anaphase↗

PubMed2003

Suppression of microtubule dynamics by discodermolide by a novel mechanism is associated with mitotic arrest and inhibition of tumor cell proliferation.

Discodermolide is a new microtubule-targeted drug in Phase I clinical trials that inhibits tumor growth and induces G(2)-M cell cycle arrest. It is effective against paclitaxel-resistant cell lines and acts synergistically in combination with paclitaxel. Suppression of microtubule dynamics by microtubule-targeted drugs has been hypothesized to be responsible for their ability to inhibit mitotic progression and cell proliferation. To determine whether discodermolide blocks mitosis by an effect on microtubule dynamics, we analyzed the effects of discodermolide on microtubule dynamics in living A549 human lung cancer cells during interphase at concentrations that block mitosis and inhibit cell proliferation. We found that discodermolide (7-166 nM) significantly suppressed microtubule dynamic instability. At the IC(50) for proliferation (7 nM discodermolide, 72 h), overall dynamicity was reduced by 23%. The principal parameters of dynamic instability suppressed by discodermolide were the microtubule shortening rate and length shortened. In addition, discodermolide markedly increased the frequency of rescued catastrophes. At the discodermolide concentration that resulted in 50% of maximal mitotic block (83 nM, 20 h), most microtubules were completely non-dynamic, no anaphases occurred, and all spindles were abnormal. The dynamicity of the remaining dynamic microtubules was reduced by 62%. The results indicate that a principal mechanism of inhibition of cell proliferation and mitotic block by discodermolide is suppression of microtubule dynamics. Importantly, the results indicate significant additional stabilizing effects of discodermolide on microtubule dynamics as compared with those of paclitaxel that may in turn reflect differences in their binding sites and their effects on tubulin conformation.

Alkanes↗