Formation Of Microtubule Bundles Research Articles

Weekly administration of sagopilone (ZK-EPO), a fully synthetic epothilone, in patients with refractory solid tumours: results of a phase I trial

Background:Epothilones are a novel class of microtubule-stabilising agents, and sagopilone is a fully synthetic epothilone that has shown marked in vivo and in vitro preclinical activity.Methods:This phase I, open-label study investigated the maximum tolerated dose (MTD) and dose-limiting toxicities (DLTs) of weekly sagopilone. Twenty-three patients with malignancy resistant or refractory to standard treatment were enrolled into this study evaluating sagopilone doses from 0.6 to 7.0 mg m−2.Results:The incidence of drug-related haematological adverse events (AEs) was low, with two grade 3 events observed. Nonhaematological AEs were generally mild and reversible; increased γ-GT was the only grade 4 event and grade 3 events comprised peripheral neuropathy (n=2), diarrhoea (n=1) and fatigue (n=1). Two grade 3 events were DLTs (diarrhoea and peripheral neuropathy at 7.0 mg m−2). The MTD of weekly sagopilone was therefore established as 5.3 mg m−2. Stable disease was the best overall response (n=3). Microtubule bundle formation in peripheral blood mononuclear cells increased post-treatment, peaking after 1 h. Sagopilone disposition was similar across treatment courses and showed rapidly decreasing serum concentrations after infusion end and a long terminal disposition phase with no obvious accumulation in the serum, probably reflecting a fast uptake into tissues followed by a slow release.Conclusion:Weekly administration of sagopilone could represent an alternative to the 3-weekly administration currently evaluated in phase II trials.

Interaction of CDK5RAP2 with EB1 to Track Growing Microtubule Tips and to Regulate Microtubule Dynamics

Mutations in cdk5rap2 are linked to autosomal recessive primary microcephaly, and attention has been paid to its function at centrosomes. In this report, we demonstrate that CDK5RAP2 localizes to microtubules and concentrates at the distal tips in addition to centrosomal localization. CDK5RAP2 interacts directly with EB1, a prototypic member of microtubule plus-end tracking proteins, and contains the basic and Ser-rich motif responsible for EB1 binding. The EB1-binding motif is conserved in the CDK5RAP2 sequences of chimpanzee, bovine, and dog but not in those of rat and mouse, suggesting a function gained during the evolution of mammals. The mutation of the Ile/Leu-Pro dipeptide within the motif abolishes EB1 interaction and plus-end attachment. In agreement with the mutational analysis, suppression of EB1 expression inhibits microtubule tip-tracking of CDK5RAP2. We have also found that the CDK5RAP2-EB1 complex regulates microtubule dynamics and stability. CDK5RAP2 depletion by RNA interference impacts the dynamic behaviors of microtubules. The CDK5RAP2-EB1 complex induces microtubule bundling and acetylation when expressed in cell cultures and stimulates microtubule assembly and bundle formation in vitro. Collectively, these results show that CDK5RAP2 targets growing microtubule tips in association with EB1 to regulate microtubule dynamics.

Demonstration of microtubule-like structures formed with (−)-rhazinilam from purified tubulin outside of cells and a simple tubulin-based assay for evaluation of analog activity

(−)-Rhazinilam was spontaneously generated from a natural product during isolation. In cultured cells, it causes microtubule bundle formation, like those caused by paclitaxel. With tubulin, (−)-rhazinilam causes formation of an aberrant spiral polymer. Using glutamate and GTP, we developed an assay for spiral formation and applied it to 17 new (±)-rhazinilam analogs with either a modified side chain or a different size D ring. There was reasonable correlation between spiral formation and inhibition of human MCF-7 breast carcinoma cell growth. Only one side chain analog was as active as (±)-rhazinilam. During these studies, we observed that omitting GTP from the reaction mixture caused a major change in the morphology of the (−)-rhazinilam-induced polymer, with half the observed polymer being microtubule-like and half being spirals. This mixed polymer slowly disassembled at 0 °C, but there was no apparent difference in the lability of the microtubules versus the spirals.

Total synthesis and biological evaluation of novel C2–C6 region analogues of dictyostatin

By exploiting a Still–Gennari HWE coupling with a common C11–C26 aldehyde, a series of C2–C6 modified analogues of the microtubule-stabilising marine natural product dictyostatin were synthesised and evaluated in vitro for growth inhibition against a range of human cancer cell lines, including the (P-glycoprotein efflux-mediated) Taxol-resistant NCI/ADR cell line. Removal of the C6 methyl substituent in dictyostatin was found to be well tolerated and led to the retention of antiproliferative activity in the low nanomolar range (IC 50 = 43 nM in the NCI/ADR cell line), while partial and full saturation of the (2 Z, 4 E)-dienoate region led to a progressive reduction in biological potency. The lactone ring size was found to be critical, as C21 to C19 translactonisation to afford 20-membered isodictyostatin analogues led to a significant loss of cytotoxicity. In a series of incubatory experiments performed on the PANC-1 cell line, all three of the 22-membered macrolide analogues acted in an analogous fashion to dictyostatin, through a mechanism of microtubule stabilization, causing both an accumulation of cells at the G2/M phase and formation of characteristic dense intracellular microtubule bundles.

The Effect of Ketoconazole on the Pharmacokinetics and Pharmacodynamics of Ixabepilone: A First in Class Epothilone B Analogue in Late-Phase Clinical Development

To determine if ixabepilone is a substrate for cytochrome P450 3A4 (CYP3A4) and if its metabolism by this cytochrome is clinically important, we did a clinical drug interaction study in humans using ketoconazole as an inhibitor of CYP3A4. Human microsomes were used to determine the cytochrome P450 enzyme(s) involved in the metabolism of ixabepilone. Computational docking (CYP3A4) studies were done for epothilone B and ixabepilone. A follow-up clinical study was done in patients with cancer to determine if 400 mg/d ketoconazole (inhibitor of CYP3A4) altered the pharmacokinetics, drug-target interactions, and pharmacodynamics of ixabepilone. Molecular modeling and human microsomal studies predicted ixabepilone to be a good substrate for CYP3A4. In patients, ketoconazole coadministration resulted in a maximum ixabepilone dose administration to 25 mg/m(2) when compared with single-agent therapy of 40 mg/m(2). Coadministration of ketoconazole with ixabepilone resulted in a 79% increase in AUC(0-infinity). The relationship of microtubule bundle formation in peripheral blood mononuclear cells to plasma ixabepilone concentration was well described by the Hill equation. Microtubule bundle formation in peripheral blood mononuclear cells correlated with neutropenia. Ixabepilone is a good CYP3A4 substrate in vitro; however, in humans, it is likely to be cleared by multiple mechanisms. Furthermore, our results provide evidence that there is a direct relationship between ixabepilone pharmacokinetics, neutrophil counts, and microtubule bundle formation in PBMCs. Strong inhibitors of CYP3A4 should be used cautiously in the context of ixabepilone dosing.

Microtubule anchoring by cortical actin bundles prevents streaming of the oocyte cytoplasm

The localisation of the determinants of the body axis during Drosophila oogenesis is dependent on the microtubule (MT) cytoskeleton. Mutations in the actin binding proteins Profilin, Cappuccino (Capu) and Spire result in premature streaming of the cytoplasm and a reorganisation of the oocyte MT network. As a consequence, the localisation of axis determinants is abolished in these mutants. It is unclear how actin regulates the organisation of the MTs, or what the spatial relationship between these two cytoskeletal elements is. Here, we report a careful analysis of the oocyte cytoskeleton. We identify thick actin bundles at the oocyte cortex, in which the minus ends of the MTs are embedded. Disruption of these bundles results in cortical release of the MT minus ends, and premature onset of cytoplasmic streaming. Thus, our data indicate that the actin bundles anchor the MTs minus ends at the oocyte cortex, and thereby prevent streaming of the cytoplasm. We further show that actin bundle formation requires Profilin but not Capu and Spire. Thus, our results support a model in which Profilin acts in actin bundle nucleation, while Capu and Spire link the bundles to MTs. Finally, our data indicate how cytoplasmic streaming contributes to the reorganisation of the MT cytoskeleton. We show that the release of the MT minus ends from the cortex occurs independently of streaming, while the formation of MT bundles is streaming dependent.

Microtubule Motor Ncd Induces Sliding of Microtubules In Vivo

The mitotic spindle is a microtubule (MT)-based molecular machine that serves for equal segregation of chromosomes during cell division. The formation of the mitotic spindle requires the activity of MT motors, including members of the kinesin-14 family. Although evidence suggests that kinesins-14 act by driving the sliding of MT bundles in different areas of the spindle, such sliding activity had never been demonstrated directly. To test the hypothesis that kinesins-14 can induce MT sliding in living cells, we developed an in vivo assay, which involves overexpression of the kinesin-14 family member Drosophila Ncd in interphase mammalian fibroblasts. We found that green fluorescent protein (GFP)-Ncd colocalized with cytoplasmic MTs, whose distribution was determined by microinjection of Cy3 tubulin into GFP-transfected cells. Ncd overexpression resulted in the formation of MT bundles that exhibited dynamic "looping" behavior never observed in control cells. Photobleaching studies and fluorescence speckle microscopy analysis demonstrated that neighboring MTs in bundles could slide against each other with velocities of 0.1 microm/s, corresponding to the velocities of movement of the recombinant Ncd in in vitro motility assays. Our data, for the first time, demonstrate generation of sliding forces between adjacent MTs by Ncd, and they confirm the proposed roles of kinesins-14 in the mitotic spindle morphogenesis.

Microtubule Association of the Neuronal p35 Activator of Cdk5

Cdk5 and its neuronal activator p35 play an important role in neuronal migration and proper development of the brain cortex. We show that p35 binds directly to alpha/beta-tubulin and microtubules. Microtubule polymers but not the alpha/beta-tubulin heterodimer block p35 interaction with Cdk5 and therefore inhibit Cdk5-p35 activity. p25, a neurotoxin-induced and truncated form of p35, does not have tubulin and microtubule binding activities, and Cdk5-p25 is inert to the inhibitory effect of microtubules. p35 displays strong activity in promoting microtubule assembly and inducing formation of microtubule bundles. Furthermore, microtubules stabilized by p35 are resistant to cold-induced disassembly. In cultured cortical neurons, a significant proportion of p35 localizes to microtubules. When microtubules were isolated from rat brain extracts, p35 co-assembled with microtubules, including cold-stable microtubules. Together, these findings suggest that p35 is a microtubule-associated protein that modulates microtubule dynamics. Also, microtubules play an important role in the control of Cdk5 activation.

Peripheral blood mononuclear and tumor cell pharmacodynamics of the novel epothilone B analogue, ixabepilone

We previously demonstrated that peak microtubule bundle formation (MBF) in peripheral blood mononuclear cells (PBMCs) occurs at the end of drug infusion and correlates with drug pharmacokinetics (PK). In the current study, a new expanded evaluation of drug target effect was undertaken. Patients with advanced solid malignancies were treated with ixabepilone 40 mg/m2 administered as a 1-h i.v. infusion every 3 weeks. Blood, plasma, and tumor tissue sampling was carried out to characterize pharmacodynamics and PK. Forty-seven patients were treated with 141 cycles of ixabepilone. In both PBMCs (n=27) and tumor cells (n=9), peak MBF occurred at the end of infusion; however, at 24-72 h after drug infusion, the number of cells with MBF was significantly greater in tumor cells, relative to PBMCs. A Hill model (EC50=109.65 ng/ml; r2=0.94) was fitted, which demonstrated a relationship between percentage of PBMCs with MBF and plasma ixabepilone concentration. The percentage of PBMCs with MBF at the end of infusion also correlated with severity of neutropenia (P=0.050). Plasma ixabepilone concentration and severity of neutropenia correlate with the level of MBF in PBMCs. Therefore, this technically straightforward assay should be considered as a complement to the clinical development of novel microtubule-binding agents.

Dynactin targets Pavarotti-KLP to the central spindle during anaphase and facilitates cytokinesis inDrosophilaS2 cells

The dynactin complex cooperates with the dynein complex in various systems for mitotic completion. Here we analysed the mitotic phenotype of Drosophila S2 cells following the knockdown of the dynactin subunit p150(Glued). We found that p150(Glued)-depleted cells were delayed in metaphase and that the centrosomes were poorly connected to mitotic spindle poles. In addition, anaphase occurred with asynchronous chromosome segregation. Although cyclin B was degraded in these anaphase cells, Aurora B, MEI-S322 and BubR1 were not released from the non-segregating chromosomes. We also found that the density and organisation of the central spindle were compromised, with Aurora B and polo kinases absent from the diminished number of microtubules. Pavarotti-KLP, a component of the centralspindlin complex required for the formation of stable microtubule bundles, was not immediately targeted to the plus ends of the microtubules following anaphase onset as happened in controls. Instead, it accumulated transiently at the cell cortex during early anaphase and its targeting to the central spindle was delayed. These data suggest that the dynactin complex contributes to cytokinesis by promoting stable targeting of the centralspindlin complex to microtubule plus ends at anaphase onset. The contribution of the dynein-dynactin complex to synchronous chromosome segregation and cytokinesis is discussed.

First-in-human phase I trial of a novel epothilone, KOS-1584

2003 Background: KOS-1584 (9,10-didehydroepothilone D) was discovered as part of a screening program to develop a new generation of epothilones with higher potency and an improved pharmacologic/pharmacokinetic (PK) profile. Epothilones stabilize microtubule polymerization, inducing rapid G2/M arrest and apoptosis. Antitumor activity of KOS-1584 (Chou et al 2003) is approximately 3–12 fold more potent when compared to the structurally related Epothilone D. KOS-1584 demonstrates enhanced tumor tissue penetration and reduced exposure to selected tissues (including CNS). We report the results of the initial dose-escalation trial in which KOS-1584 was administered to pts with advanced solid malignancies. Methods: Define the MTD, toxicity profile and PK of KOS-1584 when administered via 3-hour infusion every 3 weeks. PK was determined after the 1st and 2nd infusion. Pharmacodynamics were assessed by serial sampling of PBMCs for microtubule bundle formation. Results: 27 pts (17 F; median age 60; median ECOG PS 1; median prior regimens 3, range 0–7) enrolled in 8 dose levels (between 0.8 - 11.3 mg/m2). To date, no Cycle 1 DLT has been seen. Toxicities (n=24) did not show obvious dose dependency; common toxicity (Grade 1–2) included gastrointestinal (diarrhea, constipation, nausea), fatigue, and ↑AST. Drug-related Grade 3 toxicity: constipation, fatigue and ↑AST (1 each). Drug-related neurotoxicity was not notable. PK/parent (n=25): t½ 17.7 ± 4.6h, Vz 741±330 L and CL 30.2± 16.5 L/h. At 8.5 mg/m2 Cmax 78 ± 29 ng/mL; AUCtot 631 ± 337 ng*h/mL. Cmax and AUCtot increased linearly with dose over the range tested. Vz is ∼5-fold and t½ 2-fold higher than that of Epothilone D. Dose dependent increases in microtubule bundle formation were observed (8.5 mg/m2: 40–50% at end of infusion, compared to 60–65% for ixabepilone and 50–60% for Epothilone D using the same assay at their phase 2 dose). A sigmoidal Emax model described the relationship between plasma concentration and microtubule bundle formation. Activity consisted of 4 pts with extended stable disease (6 cycles leiomyosarcoma and ovarian cancer; 5 cycles colon cancer; a 2nd patient with ovarian cancer is active at 5 cycles). Of these, 3 had document progressive disease prior to study. Conclusions: Accrual is continuing in order to define the optimal dose on the 3-week regimen. [Table: see text]

Making Microtubules and Mitotic Spindles in Cells without Functional Centrosomes

Centrosomes are considered to be the major sites of microtubule nucleation in mitotic cells (reviewed in ), yet mitotic spindles can still form after laser ablation or disruption of centrosome function . Although kinetochores have been shown to nucleate microtubules, mechanisms for acentrosomal spindle formation remain unclear. Here, we performed live-cell microscopy of GFP-tubulin to examine spindle formation in Drosophila S2 cells after RNAi depletion of either gamma-tubulin, a microtubule nucleating protein, or centrosomin, a protein that recruits gamma-tubulin to the centrosome. In these RNAi-treated cells, we show that poorly focused bipolar spindles form through the self-organization of microtubules nucleated from chromosomes (a process involving gamma-tubulin), as well as from other potential sites, and through the incorporation of microtubules from the preceding interphase network. By tracking EB1-GFP (a microtubule-plus-end binding protein) in acentrosomal spindles, we also demonstrate that the spindle itself represents a source of new microtubule formation, as suggested by observations of numerous microtubule plus ends growing from acentrosomal poles toward the metaphase plate. We propose that the bipolar spindle propagates its own architecture by stimulating microtubule growth, thereby augmenting the well-described microtubule nucleation pathways that take place at centrosomes and chromosomes.

Assembly of Tubulin by Classic Myelin Basic Protein Isoforms and Regulation by Post-Translational Modification

Myelin basic protein (MBP), a highly cationic protein that maintains the structure of the myelin sheath, associates with tubulin in vivo. The in vitro assembly of tubulin by MBP was examined here using several assays. The unmodified C1 component of 18.5 kDa bovine MBP (bC1) assembled tubulin into microtubules in a dose-dependent manner via filamentous intermediates, and was able simultaneously to promote the formation of microtubule bundles. The critical tubulin concentration in the presence of bC1 was 0.69 +/- 0.05 microM. The effects of post-translational modifications (such as deamidation and phosphorylation) were assayed by comparing the bC1-bC6 components of 18.5 kDa bovine MBP; an increasing level of modification enhanced the ability of MBP to assemble tubulin. The effects of charge reduction via deimination were examined using recombinant murine isoforms emulating the unmodified C1 and deiminated C8 isoforms of 18.5 kDa MBP; both rmC1 and rmC8 exhibited a comparable ability to assemble tubulin. The effects of alternate exon recombination of the classic MBP variants were tested using the recombinant murine 21.5, 17.22, and 14 kDa isoforms. The isoforms containing regions derived from exon II of the classic MBP gene, 21.5 and 17.22 kDa MBP, showed no substantial difference in the extent of tubulin polymerization and bundling when compared to those of 18.5 kDa MBP. The 14 kDa isoform and two terminal deletion mutants of rmC1 were able to induce microtubule polymerization, but not bundling, to the same degree as the longer proteins. Finally, bC1 was shown to disrupt and aggregate planar sheets of crystalline tubulin stabilized by paclitaxel, establishing that these structures are not suitable substrates for the formation of MBP cocrystals.

The Dual-Specificity Phosphatase CDC14B Bundles and Stabilizes Microtubules

The Cdc14 dual-specificity phosphatases regulate key events in the eukaryotic cell cycle. However, little is known about the function of mammalian CDC14B family members. Here, we demonstrate that subcellular localization of CDC14B protein is cell cycle regulated. CDC14B can bind, bundle, and stabilize microtubules in vitro independently of its catalytic activity. Basic amino acid residues within the nucleolar targeting domain are important for both retaining CDC14B in the nucleolus and preventing microtubule bundling. Overexpression of CDC14B resulted in the formation of cytoplasmic CDC14B and microtubule bundles in interphase cells. These microtubule bundles were resistant to microtubule depolymerization reagents and enriched in acetylated alpha-tubulin. Expression of cytoplasmic forms of CDC14B impaired microtubule nucleation from the microtubule organization center. CDC14B is thus a novel microtubule-bundling and -stabilizing protein, whose regulated subcellular localization may help modulate spindle and microtubule dynamics in mitosis.

Two Microtubule-Associated Proteins of the Arabidopsis MAP65 Family Function Differently on Microtubules

The organization and dynamics of microtubules are regulated by microtubule-associated proteins, or MAPs. In Arabidopsis (Arabidopsis thaliana), nine genes encode proteins of the evolutionarily conserved MAP65 family. We proposed that different MAP65s might have distinct roles in the interaction with microtubules. In this study, two AtMAP65 proteins, AtMAP65-1 and AtMAP65-6, were chosen to test this hypothesis in vitro. Although both fusion proteins were able to cosediment with microtubules in vitro, different properties on tubulin polymerization and microtubule bundling were observed. AtMAP65-1 was able to promote tubulin polymerization, enhance microtubule nucleation, and decrease the critical concentration for tubulin polymerization. It also induced the formation of large microtubule bundles by forming cross-bridges between microtubules evenly along the whole length of microtubules. In the presence of AtMAP65-1, microtubule bundles were more resistant to cold and dilution treatments. AtMAP65-6, however, demonstrated no activity in promoting tubulin polymerization and stabilizing preformed microtubules. AtMAP65-6 induced microtubules to form a mesh-like network with individual microtubules. Cross-bridge-like interactions were only found at regional sites between microtubules. The microtubule network induced by AtMAP65-6 was more resistant to high concentration of NaCl than the bundles induced by AtMAP65-1. Purified monospecific anti-AtMAP65-6 antibodies revealed that AtMAP65-6 was associated with mitochondria in Arabidopsis cells. It was concluded that these two MAP65 proteins were targeted to distinct sites, thus performing distinct functions in Arabidopsis cells.

The Synergistic Combination of the Farnesyl Transferase Inhibitor Lonafarnib and Paclitaxel Enhances Tubulin Acetylation and Requires a Functional Tubulin Deacetylase

Farnesyl transferase (FT) inhibitors (FTI) are anticancer agents developed to target oncogenic Ras proteins by inhibiting Ras farnesylation. FTIs potently synergize with paclitaxel and other microtubule-stabilizing drugs; however, the mechanistic basis underlying this synergistic interaction remains elusive. Here we show that the FTI lonafarnib affects the microtubule cytoskeleton resulting in microtubule bundle formation, increased microtubule stabilization and acetylation, and suppression of microtubule dynamics. Notably, treatment with the combination of low doses of lonafarnib with paclitaxel markedly enhanced tubulin acetylation (a marker of microtubule stability) as compared with either drug alone. This synergistic effect correlated with FT inhibition and was accompanied by a synergistic increase in mitotic arrest and cell death. Mechanistically, we show that the combination of lonafarnib and paclitaxel inhibits the in vitro deacetylating activity of the only known tubulin deacetylase, histone deacetylase 6 (HDAC6). In addition, the lonafarnib/taxane combination is synergistic only in cells lines expressing the wild-type HDAC6, but not a catalytic-mutant HDAC6, revealing that functional HDAC6 is required for the synergy of lonafarnib with taxanes. Furthermore, tubacin, a specific HDAC6 inhibitor, synergistically enhanced tubulin acetylation in combination with paclitaxel, similar to the combination of lonafarnib and paclitaxel. Taken together, these data suggest a relationship between FT inhibition, HDAC6 function, and cell death, providing insight into the putative molecular basis of the lonafarnib/taxane synergistic antiproliferative combination.

Microtubule Bundle Formation and Cell Death Induced by the Human CLASP/Orbit N-Terminal Fragment

Previously we have identified the Drosophila orbit gene whose hypomorphic mutations cause abnormal chromosome segregation (Inoue et al., 2000). The orbit encodes Orbit/Mast, a 165-kDa microtubule-associated protein (MAP) with GTP-binding motifs. Two human homologues of the Orbit/Mast, CLASP1 (hOrbit1) and CLASP2 (hOrbit2) have been identified. Using an antibody to CLASP1/hOrbit1 polypeptide, we confirmed that the polypeptide of about 150 kDa associates with microtubule purified from the porcine brain. Thus, we conjectured that CLASP1 may be a human orthologue of the Drosophila Orbit/Mast, and therefore we named it h (human) Orbit1. We constructed the plasmid for expression of a fusion protein of the putative microtubule-binding domain (1-662 out of 1289 residues) of hOrbit1 and the green fluorescent protein (GFP), and then, transfected the plasmid into Tet off cells derived from HeLa cell. Confocal laser scanning microscopic observation revealed that the GFP-fluorescence associated with short and thin filaments in the perinuclear region during the short period after plasmid transfection, and colocalized with only part of the microtubules. GFP fluorescence was later detected on the abnormally longer and thick bundles of microtubule filaments. Finally the bundles formed networks in the perinuclear region. The results suggest that the GFP-hOrbit1 N-terminal fragment (GFP-hOrbit1 NF) binds to the newly formed microtubules rather than the pre-formed ones, and that displacement of the endogenous hOrbit by the fragment might cause abnormal bundling of microtubules. Interestingly, the expression of the GFP-hOrbit1 NF results in cell death associated with nuclear fragmentation.

Phase I study using continuous intravenous (CI) KOS-862 (Epothilone D) in patients with solid tumors

2024 Background: Similar to taxanes, epothilones are a class of naturally occurring cytotoxic macrolides that stabilize microtubules and induce mitotic arrest, retaining potency in P-glycoprotein MDR cell lines. 6 schedules of KOS-862 were investigated in phase I trials. Methods: As the optimal schedule in the mouse xenograft model used prolonged exposure (6-hr IV infusion at 30 mg/kg) to reach plasma steady state Css=425 ng/mL, two CI schedules were attempted: a 24-hr CI (Schedule A) and up to 72-hr CI (Schedule B) q 2 weeks. Steady state is difficult to maintain for a drug with a rapid alpha distribution, hence a loading dose (75 mg/hr for 30 min) was incorporated, followed by maintenance dose. Results: 24 pts have been enrolled (median age 62 yrs, range 28–86 yrs; 7 F/17 M; median KPS 80; median prior regimens 3, range 0–10): 17 on Schedule A, 7 on Schedule B. Eighty total infusions have been administered. 4 dose levels have been tested on Schedule A (1, 2, 4 and 6 mg/hr rates); 2 dose levels on Schedule B (1 and 1.7 mg/hr). Drug-related toxicity includes mild-to-moderate fatigue, nausea/abdominal pain, dizziness, and neurosensory. One episode of grade 3 neurosensory occurred at 6 mg/hr x 24-hr CI; this dose group is being expanded. AUC=Css(SD) at this dose was 12300 ng/mL*h; constant dosing at 7 mg/h for 23.5 h would theoretically achieve a Css=423 ng/mL (Css=AUCSDτ n →τ n+1 /τ ). Pts dosed at 4 mg/h for 23.5h achieved Css=200 ng/mL. Comparison of PK between short infusion and CI showed similar results (mean±SD) CL=6.7±2.7 v 8.7±2.9 L/h/m2; half-life 10.3±2.9 v 10.8±2.3 hours; Vz 96±41 v136±59 L/m2; MRT 10.0±3.6 v 17.1±4.5 hours. As a marker of biologic activity, PBMCs were examined for microtubule bundle formation at the end of the CI. On Schedule A, 15 and 20% bundle formation at 4 and 6 mg/h was observed, respectively, indicating that there was significant polymerization over this period of time. Signs of antitumor activity included 1 pt with bladder ca (shrinkage of primarily nodal disease) and 1 pt with prostate ca (25% decline in PSA). Conclusions: Biologic activity at tolerable doses has been noted and accrual continues to define the recommended dose. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Kosan Biosciences, Inc. Kosan Biosciences, Inc. Kosan Biosciences, Inc. Kosan Biosciences, Inc. Kosan Biosciences, Inc.

The EF-hand Ca2+-binding protein p22 plays a role in microtubule and endoplasmic reticulum organization and dynamics with distinct Ca2+-binding requirements.

We have reported that p22, an N-myristoylated EF-hand Ca(2+)-binding protein, associates with microtubules and plays a role in membrane trafficking. Here, we show that p22 also associates with membranes of the early secretory pathway membranes, in particular endoplasmic reticulum (ER). On binding of Ca(2+), p22's ability to associate with membranes increases in an N-myristoylation-dependent manner, which is suggestive of a nonclassical Ca(2+)-myristoyl switch mechanism. To address the intracellular functions of p22, a digitonin-based "bulk microinjection" assay was developed to load cells with anti-p22, wild-type, or mutant p22 proteins. Antibodies against a p22 peptide induce microtubule depolymerization and ER fragmentation; this antibody-mediated effect is overcome by preincubation with the respective p22 peptide. In contrast, N-myristoylated p22 induces the formation of microtubule bundles, the accumulation of ER structures along the bundles as well as an increase in ER network formation. An N-myristoylated Ca(2+)-binding p22 mutant, which is unable to undergo Ca(2+)-mediated conformational changes, induces microtubule bundling and accumulation of ER structures along the bundles but does not increase ER network formation. Together, these data strongly suggest that p22 modulates the organization and dynamics of microtubule cytoskeleton in a Ca(2+)-independent manner and affects ER network assembly in a Ca(2+)-dependent manner.

An evolutionarily conserved fission yeast protein, Ned1, implicated in normal nuclear morphology and chromosome stability, interacts with Dis3, Pim1/RCC1 and an essential nucleoporin.

We identified a novel fission yeast gene, ned1(+), with pleiotropic mutations that have a high incidence of chromosome missegregation, aberrantly shaped nuclei, overdeveloped endoplasmic reticulum-like membranes, and increased sensitivity to a microtubule destabilizing agent. Ned1 protein, which was phosphorylated in a growth-related manner, interacted in a yeast two-hybrid system with Dis3 as well as with Pim1/RCC1 (nucleotide exchange factor for Ran). Ned1 also interacted with an essential nucleoporin, a probable homologue of mammalian Nup98/96. The ned1 gene displayed a variety of genetic interactions with factors involved in nuclear transport and chromosome segregation, including the crm1 (exportin), spi1 (small GTPase Ran), pim1, and dis genes. A substitution mutation that affected the two-hybrid interaction with Dis3 increased chromosome instability, suggesting the functional importance of the interaction. Overproduction of Ned1 protein induced formation of an abnormal microtubule bundle within the nucleus, apparently independently of the spindle pole body, but dependent on pim1(+) activity. The ned1(+) gene belongs to an evolutionarily conserved gene family, which includes the mouse Lpin genes, one of whose mutations is responsible for lipodystrophy.