DIATOM MINERALIZATION OF SILICIC ACID. VI. THE EFFECTS OF MICROTUBULE INHIBITORS ON SILICIC ACID METABOLISM IN NAVICULA SAPROPHILA1

The role of microtubules in silicon metabolism leading to valve formation was investigated in the pennate diatom Navicula saprophila Lange-Bertalot & Bonik. By using synchronized cells blocked after mitosis and cytokinesis but prior to cell wall formation, effects due to inhibition of mitosis were eliminated. Cells were treated with three anti-microtubule drugs to assess the role of microtubules. Chemical analogs to two of the drugs provided controls for inhibition not related to microtubule disruption. Although all three anti-microtubule drugs reduced cell separation at high concentrations (1 × 10^?3 M), podophyllotoxin was the only drug which reduced cell separation at concentrations lower than 1 × 10^?5 M. None of the drugs at any concentration tested affected cell viability. There was no differential inhibitory effect between the active and inactive drugs on silicic acid transport, total uptake, incorporation, or pool formation. There was no qualitative difference between silica incorporated in treated and untreated cells. A colchicine binding component was isolated from N. saprophila. The characteristics of colchicine binding suggest this component may be tubulin. Microtubules do not appear to be involved in any of the steps of silicon metabolism leading to valve formation and yet they have profound influence on the symmetry and pattern of the mineralized product, the siliceous valve.     

VALVE MORPHOGENESIS IN THE PENNATE DIATOM NAVICULA CUSPIDATA1

The deposition of siliceous valves during asexual reproduction of the pennate diatom, Navicula cuspidata Kütz., is described with emphasis on the cytoplasmic components involved. The events accompanying valve secretion are similar to those already known from other pennate species. After mitosis, the microtubule centre (MC) moves to the center of the cleavage furrow where silica deposition is initiated inside a tubular silicalemma, and it remains associated with the prospective central nodule during valve growth. Microtubules (MTs), emanating from the MC, run parallel to the prospective raphe and together with the raphe fibres, appear to be involved with raphe development. Multiple raphe fibres occupy the maturing raphe fissure, in contrast to the single fibre of Pinnularia viridis, P. maior and Hantzschia amphioxys. The fibers exhibit a periodic substructure and are often opposed to the silicalemma where they may inhibit silica deposition and control the shaping of the raphe fissure. In contrast with the above species, in N. cuspidata MTs are clustered strictly opposite the raphe and lose their association with the MC which degenerates before the valves are mature. The primary role of MTs may be the stabilization of the cytoplasmic region where initial silicification occurs. Mitochondria and endoplasmic reticulum are not involved in molding valve growth in this species. Evidence for vesicle involvement in silica transport and deposition was limited. The possible contributions provided by comparative studies on the ultrastructure of valve morphogenesis towards elucidating the control of valve formation and the taxonomy of diatoms are discussed briefly.     

The effect of drugs on diatom valve morphogenesis

Summary The effects of various drugs on cell wall (valve) morphogenesis was investigated in three species of diatoms (Pinnularia spp., Surirella robusta, andHantzschia amphioxys) using light microscopy (LM) and scanning electron microscopy (SEM). Treatment ofSurirella with the microtubule (MT) disrupting agent colchicine during early valve formation results in a characteristic malformation of the valve, whereby part of the normally circumferential raphe canal forms as an abnormal protruding lip on the valve surface, located up to 20 m from the edge of the valve. The position of this malformed lip coincides with the location of a microtubule center (MC) at the time of colchicine addition, suggesting that the MC may play a direct role in positioning the tip of the raphe canal during valve formation. The migration of this MC to the tip of the cell during early valve morphogenesis is reversibly inhibited by the metabolic inhibitor 2-4-dinitrophenol (DNP). The effect of colchicine onPinnularia valve formation is less severe, causing occasional malformation of the raphe, but little if any lateral displacement. InHantzschia, colchicine has no effect on the positioning of the raphe, but prolonged exposure causes fusion of the raphe canal with the valve face. Cochicine treatment also results in the absence of the normal curvature at the central interruption in the raphe, as well as abnormal pore formation in this central area. Addition of cytochalasin D during early valve formation inHantzschia causes the raphe canal to form in the center of the valve face, suggesting that the normal translocation of the raphe canal to the valve edge is actindependent. Comparison of valves from control and cytochalasintreatmentHantzschia suggest that the pore spacing within the valve is determined by the position relative to the raphe, and does not depend on whether to pores form on the side (mantle) or the face of the mature valve.Abbreviations DM diatom medium - DNP dinitrophenol - MT microtubule - MC microtubule center - PSS primary silicification site - SDV silica deposition vesicle     

VARIATION IN PATTERNS OF VALVE MORPHOGENESIS BETWEEN REPRESENTATIVES OF SIX BIRAPHID DIATOM GENERA (BACILLARIOPHYCEAE)

Scanning electron microscopic studies of silica valve formation in naviculoid diatoms representing six different genera revealed that the precise sequence of depositional events varied among genera. Valve deposition begins with the formation of the raphe sternum, from which virgae (lateral outgrowths) extend. Areolae (pores) are formed between the virgae by the fusion of cross-extensions (vimines). In most of the species studied ( Craticula ambigua (Kützing) D. G. Mann, Frustulia vulgaris (Thwaites) De Toni, Craspedostauros australis E. J. Cox, and Gomphonema truncatum Ehrenberg), areola (pore) formation began near the raphe sternum before completion of the valve margin, but in Pinnularia gibba Ehrenberg the valve margin fused before the areolae were formed. Silica deposition in all these taxa was mainly distal to proximal (with respect to the cytoplasm), but in Haslea sp. it was mainly proximal to distal. Haslea also differed in that areolae were defined as the valve margin was completed. These data have also contributed to the interpretation of taxonomically important features, such as raphe endings. In P. gibba the internal central raphe fissures were laterally deflected but subsequently obscured by additional silicification of the valve, whereas in G. truncatum they were initially straight, becoming laterally deflected as valves mature. External raphe fissures in Frustulia became Y-shaped only just before maturity; in immature valves they were dotlike, as in Amphipleura Kützing. The comparison of developmental pathways in diatoms is a useful adjunct to morphological and other approaches in diatom systematics and warrants renewed attention.     

STRUCTURE,LIFE HISTORY AND SYSTEMATICS OF RHOICOSPHENIA (BACILLARIOPHYTA). I. THE VEGETATIVE CELL OF RH. CURVATA1

Rhoicosphenia Grun. has been placed by some authors in the monoraphid group with Achnanthes Bory and Cocconeis Ehrenb., and by others near Gomphonema Ehrenb. In order to clarify the systematic position of the genus, the morphology and anatomy of the vegetative cells of Rh. curvata (Kütz.) Grun. were investigated using light and electron microscopy. The structure and formation of the two types of valve are described, and the heterovalvy shown to be of a different type from that of the monoraphids; on the basis of raphe, valve and girdle structure a close relationship between these and Rhoicosphenia is unlikely. Rhoicosphenia shows many resemblances to Gomphonema but the types of pore occlusion present, coupled with apparently slight differences in the mucilage-secreting structures and the girdle, suggest that classification in the same family is unwise. The cryptic asymmetry of the valves, and in particular of the raphe system, is noted and explained with reference to their formation; with respect to this asymmetry two configurations of the valves can occur (named cis and trans types) and the distribution of these in raphid genera is discussed briefly. In view of the lack of evidence in raphid diatoms supporting a classification of bands into copulae and pleurae, it is recommended that this practice be suspended.     

POLYMORPHISM OF THE DIATOM PINNULARIA BREBISSONII IN CULTURE AND A FIELD COLLECTION1,2

Two clones of Pinnularia brebissonii (Kütz.) Rabh. var. brebissonii were established and maintained in logarithmic phase of growth. Initial length of the cells was 37 μm. As cell division occurred, the mean length of cells in each population decreased as predicted by the MacDonald-Pfitzer hypothesis; however, the decrease in mean length was not uniform throughout the growth period. This nonuniformity is probably caused by nonrandom division of cells in the population or by a changing increment of size reduction due to division. The initial increment of size reduction was calculated as 0.7 μm/division. The smallest, cells observed were 8 μm long. As cells decrease in length, cell volume decreases and the proportion of cells with aberrant valve structure increases. More than 90% of the valves were abnormal in a population with mean length of 14 μm. The abnormalities of structure involved the raphe, the central area and the striae.     

Microtubular protein reaction with nucleotides.

The dissociation constants for GTP and GDP with tubulin were determined to be equal to 1.1 ± 0.4 × 10^?7 M and 1.5 ± .6 × 10^?7 (4°), respectively. A lower limit for the dissociation constant for ATP was established as equal to 6 × 10^?4 M. The equivalent binding of GTP and GDP is not readily consistent with a mechanism in which the role of GTP in microtubule assembly is to bind to the protein to induce a conformation which is able to polymerize. An ATP-induced polymerization of tubulin apparently involves a transphosphorylation reaction in which GTP is formed and mediates the assembly. For this reaction to occur with desalted tubulin trace amounts of GDP are required; in the reaction of 0.1 mM ATP with 22.0 μM tubulin, 0.1 μM GDP induces about 80% as much tubule formation as is seen with 0.1 mM GTP alone.     

Cytomorphogenetic- and anti-microtubule action of the antibiotic gougerotin inMicrasterias denticulata Bréb.

Summary Differentiating cells ofMicrasterias denticulata have been treated with aqueous solutions of the antibiotic gougerotin. Strong and characteristic cytomorphogenetic aberrations, resembling those of the anuclear type of development could be observed. It has been speculated that the aberrant growth of the growing half cell is the result of inhibition of protein synthesis by gougerotin.In addition to the morphogenetic influence, nuclear migration has been strongly inhibited by the drug. Therefore, it might be suggested that gougerotin is an active anti-microtubule agent.     

STRUCTURE,LIFE HISTORY AND SYSTEMATICS OF RHOICOSPHENIA (BACILLARIOPHYTA). V. INITIAL CELL AND SIZE REDUCTION IN RH. CURVATA AND A DESCRIPTION OF THE RHOICOSPHENIACEAE FAM. NOV.1

The initial epivalve of Rhoicosphenia curvata (Kütz.) Grun. differs from vegetative valves in having a strongly arched section, a wide hyaline marginal strip, no pseudosepta, an unthickened margin, and a terminal raphe fissure at the head pole. The initial epivalve is of the D type, with short raphe fissures. The epicingulum consists of three bands as usual, but they are narrower and more delicate than those of vegetative cells. The initial hypovalve and hypocingulum are similar in every way to those of vegetative cells, except for the rounded section of the hypovalve. During size reduction the almost isopolar outline of the initial valves and their immediate descendants gives way to an increasingly strong heteropolarity, and this is accompanied by changes in the relative lengths of the raphe slits and the shape of the central area. Different populations have different gametangium and initial cell sizes, suggesting the presence of races within the species. The structure of the initial cell indicates that Rhoicosphenia is less closely related to the monoraphid genera than to the gomphocymbelloid genera, confirming conclusions reached from studies of the vegetative cell and auxospore formation. Rhoicosphenia should therefore be separated into a new family, the Rhoicospheniaceae, which is described.     

Interaction of vinblastine with steady-state microtubules in vitro

At low concentrations, vinblastine binds rapidly and reversibly to a very limited number of high affinity sites on steady-state bovine brain microtubules (mean K_d, 1.9 × 10^?6m; 16.8 ± 4.3 vinblastine binding sites per microtubule) which appear to be located at one or both ends of the microtubules. At high concentrations, vinblastine binds to a high binding capacity class of sites of undetermined affinity, located on helical strands of protofilaments which form at the ends of depolymerizing microtubules, and/or along the surface of the microtubules. Substoichiometric inhibition of microtubule assembly, which occurs at low vinblastine concentrations, appears to be due to the binding of vinblastine to the high affinity class of sites. Fifty per cent inhibition of tubulin addition to the net assembly ends of steady-state microtubules occurred at 1.38 × 10^?7m-drug, and at this concentration, 1.16 ± 0.27 molecules of vinblastine were bound to the high affinity class of sites. Vinblastine appeared to bind directly to the microtubule ends, and our results indicate that vinblastine inhibits the assembly of steady-state bovine brain microtubules by binding rapidly and with high affinity to one or two molecules of tubulin at the net assembly ends. Splaying and peeling of protofilaments at microtubule ends and the active depolymerization of microtubules occurred only at vinblastine concentrations greater than 1 × 10^?6 to 2 × 10^?6m. This action of vinblastine is associated with and may be due to the binding of vinblastine to the high capacity class of sites. Both actions of vinblastine may be due to the binding of vinblastine to the same binding sites on the tubulin molecule, with the sites exhibiting either a high or low affinity depending upon the location in the microtubule.     

VALVE MORPHOGENESIS AND THE MICROTUBULE CENTER IN THREE SPECIES OF THE DIATOM NITZSCHIA1

The relationship of cell organelles to valve morphogenesis was investigated in three species of Nitzschia. One, N. sigmoidea (Nitzsch) W. Sm., showed consistent ability to generate both nitzschioid and hantzschioid symmetry in daughter cells following cytokinesis; the other two maintained nitzschioid symmetry stably. From previous work with Hantzschia, a certain sequence of events could be anticipated in the cytoplasm. In two significant areas–the behavior of the Microtubule Center (MC) and its microtubule (MT) system, and the central origin of the silicalemma–not only were the results unexpected, but the three species showed fundamental differences among themselves. In N. sigmoidea, the silicalemma (and the future raphe region) arises centrally on the cleavage furrow, and after some lateral expansion, the silicalemmas and their associated organelles move in opposite directions in daughter cells, so that the raphe and the raphe canals end up along the girdle side of the cell as expected. However, the MCs never become associated with their silicalemma, remaining throughout near the girdle bands. In N. sigma (Kütz) W. Sm., the silicalemmas arise centrally and after lateral growth, move in opposite directions to generate nitzschioid symmetry. In this case, the MCs move to the vicinity of but never close to the silicalemmas, and follow them distantly during their lateral movement. In N. tryblionella Hantzsch, the new silicalemmas arise opposite one another, on one side of the daughter cells; each MC soon moves very close to its silicalemma, and remains thus through most of valve morphogenesis. Later, only one silicalemma/MC complex moves laterally, establishing the nitzschioid symmetry in both daughter cells. In all three species, as in Hantzschia, linear arrays of mitochondria aligned along MTs occupy the forming raphe canal, and microfilaments line the outer edge of the expanding silicalemma. The fibulae (the wall struts arching across the raphe canal) in Hantzschia always grow from the valve surface to the girdle surface of the forming valves. In these three Nitzschiae, this invariably happens in only one daughter cell of any pair; in the other, all the fibulae grow from the girdle surface to the valve surface. An explanation of these variations is proposed: that the morphogenetic machinery of Nitzschia and Hantzschia have a common origin, with present Nitzschiae having undergone considerable diversification at the intracellular level, causing the unstable cell symmetry exhibited by several modern species. Perhaps a taxonomic distinction between Hantzschia and Nitzschia lies in whether the morphogenetic machinery associated with valve morphogenesis moves laterally in the same or in opposite directions.     

The incidence of unstable chromosome aberrations in peripheral blood lymphocytes from unirradiated and occupationally exposed people

Peripheral blood lymphocytes from unirradiated control subjects and workers exposed within permitted limits to γ-radiation, have been examined for the incidence of dicentric and acentric chromosome aberrations. The results are compared with a review of data published elsewhere. Background levels show inter-laboratory variation and possible reasons for this are discussed. By combining the present data with those from the literature the spontaneous incidence of dicentric aberrations is approx. 0.55 × 10^?3 and for acentrics is 3.7 × 10^?3. In occupationally exposed subjects a significantly higher incidence of aberrations was found. When allowance was made for the turnover of lymphocytes for the period over which each man had worked with radiation a linear dose-effect relationship was apparent. The incidence of dicentrics was 2.22 ± 0.94 × 10^?4 rad^?1 and for all unstable aberrations 8.24 ± 2.8 × 10^?4 rad^?1. These are in reasonable agreement with dose-response data obtained in vitro.     

Rabbit brain purine nucleoside phosphorylase. Physical and chemical properties. Inhibition studies with aminopterin, folic acid and structurally related compounds.

Rabbit brain purine nucleoside phosphorylase used in this study was purified 6000-fold to apparent homogeneity and a specific activity or 50 μmol min^?1 mg ^?1 protein. A molecular weight of 70.000 daltons was determined for the native enzyme by gel filtration on Sephadex. Electrophoresis on polyacrylamide gel, in presence of sodium dodecyl sulfate, gave a subunit molecular weight of 34,500 daltons, suggesting that the enzyme is dimeric with, probably, identical subunits. The relationship of the structure of certain biologically active substances to their inhibitory action on the enzyme was examined. Folic acid and the compound d,l-6-methyl 5,6,7,8-tetrahydropterine, with similar substituents on their primary ring structure, were competitive inhibitors of the enzyme. The inhibition constants calculated were 3.37 × 10^?5M for folic acid and 3.80 × 10^?5m for d,l-6-methyl 5,6,7,8-tetrahydropterine. Aminopterin and the purine analog 8-aza-2,6-diaminopurine, with similar substituents on their primary ring structure, were noncompetitive inhibitors of the enzyme. Their respective inhibition constants were 1.50 × 10^?4 and 1.95 × 10^?4m. Erythro-9-(2-hydroxy-3-nonyl) adenine, an adenosine deaminase inhibitor, was also examined for inhibitory potency with mammalian purine nucleoside phosphorylase, and was observed to be a competitive inhibitor of this enzyme, with an inhibition constant of 1.90 × 10^?4m. The Michaelis constant for the substrate guanosine was near 6.0 × 10^?5m. Physical probe of the nature of the functional groups which participate in enzymic catalysis implicated both histidine and cysteine as the essential catalytic species. Photooxidation studies suggested a pH-dependent sensitivity of an essential catalytic group, and its probable location at the active site.     

Expression Levels of a Kinesin-13 Microtubule Depolymerase Modulates the Effectiveness of Anti-Microtubule Agents

Background

Chemotheraputic drugs often target the microtubule cytoskeleton as a means to disrupt cancer cell mitosis and proliferation. Anti-microtubule drugs inhibit microtubule dynamics, thereby triggering apoptosis when dividing cells activate the mitotic checkpoint. Microtubule dynamics are regulated by microtubule-associated proteins (MAPs); however, we lack a comprehensive understanding about how anti-microtubule agents functionally interact with MAPs. In this report, we test the hypothesis that the cellular levels of microtubule depolymerases, in this case kinesin-13 s, modulate the effectiveness of the microtubule disrupting drug colchicine.

Methodology/Principal Findings

We used a combination of RNA interference (RNAi), high-throughput microscopy, and time-lapse video microscopy in Drosophila S2 cells to identify a specific MAP, kinesin-like protein 10A (KLP10A), that contributes to the efficacy of the anti-microtubule drug colchicine. KLP10A is an essential microtubule depolymerase throughout the cell cycle. We find that depletion of KLP10A in S2 cells confers resistance to colchicine-induced microtubule depolymerization to a much greater extent than depletion of several other destabilizing MAPs. Using image-based assays, we determined that control cells retained 58% (±2%SEM) of microtubule polymer when after treatment with 2 µM colchicine for 1 hour, while cells depleted of KLP10A by RNAi retained 74% (±1%SEM). Likewise, overexpression of KLP10A-GFP results in increased susceptibility to microtubule depolymerization by colchicine.

Conclusions/Significance

Our results demonstrate that the efficacy of microtubule destabilization by a pharmacological agent is dependent upon the cellular expression of a microtubule depolymerase. These findings suggest that expression levels of Kif2A, the human kinesin-13 family member, may be an attractive biomarker to assess the effectiveness of anti-microtubule chemotherapies. Knowledge of how MAP expression levels affect the action of anti-microtubule drugs may prove useful for evaluating possible modes of cancer treatment.     

HERITABILITY OF MANTLE AREOLAR CHARACTERS IN AULACOSEIRA SUBARCTICA (BACILLARIOPHYTA)1

Are size and arrangement of valve mantle areolae in Aulacoseira Thwaites adapted to light intensity? To test one criterion demonstrating an adaptation, heritability experiments were run on isolates of Aulacoseira subarctica (Müller) Haworth. Several clones of A. subarctica were isolated from Yellowstone Lake (Wyoming, USA), Lewis Lake (Wyoming), and East Rosebud Lake (Montana, USA). Two to four clones from each lake were grown in batch cultures under three irradiance levels: 2, 11.4, and 115 μmol photons·m^?2·s^?1. Five randomly chosen valves for each of two replicates of each clone were examined using a scanning electron microscope for a total of 300 valves. Size measurements were taken for each valve examined, and images of mantle areolae were captured on film at a magnification of 20,000×. Each image was digitized, and quantitative morphometric areolar characters were measured. A quantitative genetic analysis was performed within each light environment for the mean area of the external opening of mantle areolae, the mean distance between areolae within pervalvar striae, and the mean distance between pervalvar striae. Resulting estimates of heritability from among‐lake and within‐lake analyses indicate that all three mantle areolar characters could presently respond to selection and thus have potentially done so in the past.     

Studies on the biochemistry and fine structure of silicia shell formation in diatoms VII. Sequential cell wall development in the pennateNavicula pelliculosa

Summary The development of the wall of synchronized culture ofN. pelliculosa is described. The first step, modification of the 3-2 configuration of the girdle bands of the wall during interphase, occurs immediately before mitotic division by the addition of a third girdl band to the hypotheca. Following cytokenesis, the new valve is initiated when a primary central band is formed within a silica deposition vesicle. This band extends the length of the cell and contains a central nodule. Secondary arms extend from the central nodule, join with extensions of the primary central band, and constitute the raphe rib. Mounds or knolls are formed on the central nodule and disappear as the valve matures. Transapical ribs appear on both the primary central band and secondary arms, and cross extensions join to form the sieve plate areas. The wall appears to be released by a joining of the inner silicalemma and the plasmalemma. An organic coat covers the newly released wall. Two girdle bands are formed and released sequentially.     

Enzymatic synthesis of [U-14C] ribose-labeled inosine.

A very potent anticholinesterase compound, 7-(diethoxyphosphinyloxy)-N-methylquinolinium fluorosulfate, has been used to determine the normality of acetylcholinesterase solutions. The inhibitor reacts rapidly and completely with acetylcholinesterase. The bimolecular rate constant is 2.5 × 10⁸m^?1 min^?1 and the equilibrium constant is about 10⁶. The reaction produces an inactive diethylphosphoryl enzyme in which the active serine is phosphorylated. The reaction produces the highly fluorescent 1-methyl-7-hydroxyquinolinium dipolar ion as a leaving group. The inhibited enzyme is quite stable and hydrolyzes to produce active enzyme only at the rate of 0.04%/min. The inhibitor was used in two ways for measuring the normality of acetylcholinesterase solutions: (1) The very fast reaction of the inhibitor with cholinesterase makes it convenient to determine the normality of enzyme solutions by measuring the decrease in enzyme activity caused by the addition of an accurately known quantity of the inhibitor. (2) The highly fluorescent nature of the leaving group makes it possible to measure the low concentration that is produced by the reaction of excess inhibitor with the enzyme. The two methods yielded activities per site of 6.9 × 10⁵ min^?1 and 7.3 × 10⁵ min^?1 using enzyme normalities of 1–2 × 10^?8m and 1–5 × 10^?m, respectively, using a commercial 11 S enzyme preparation from electric eel and acetylthiocholine as the enzyme substrate.     

The relationship between frameshift mutagenicity and DNA-binding affinity in a series of acridine-substituted derivatives of the experimental antitumour drug 4′-(9-acridinylamino)methanesulphonanilide (AMSA)

The mutagenicity and DNA-binding affinity of members of a series of acridine-substituted derivatives of 4′-(9-acridinylamino)methanesulphonanilide (AMSA) have been compared. The series includes compounds ranging from highly active to inactive in the L1210 murine leukaemia. Binding to DNA was measured by an ethidium displacement technique, with a correction being made for acridine-induced quenching of ethidium. Mutagenicity was assessed by measuring the reversion frequencies of the frameshift tester strain Salmonella typhimurium TA1537 in liquid culture. The results indicate that maximum mutagenicity is found in a “window” of DNA-binding affinities between 10⁶ and 5 × 10⁶ M^?1 (determined at 0.01 ionic strength). Compounds with binding affinities below 10⁶ M^?1 generally lacked both antitumour and mutagenic activity, whereas those with affinities above 5 × 10⁶ M^?1 were active against L1210 leukaemia but virtually inactive in inducing frameshift mutations.     

Vascular disrupting agent drug classes differ in effects on the cytoskeleton

Vascular disrupting agents (VDAs), anti-cancer drugs that target established tumor blood vessels, fall into two main classes: microtubule targeting drugs, exemplified by combretastatin A4 (CA4), and flavonoids, exemplified by 5,6-dimethylxanthenone-4-acetic acid (DMXAA). Both classes increase permeability of tumor vasculature in mouse models, and DMXAA in particular can cause massive tumor necrosis. The molecular target of CA4 is clearly microtubules. The molecular target(s) of DMXAA remains unclear. It is thought to promote inflammatory signaling in leukocytes, and has been assumed to not target microtubules, though it is not clear from the literature how carefully this assumption has been tested. An earlier flavone analog, flavone acetic acid, was reported to promote mitotic arrest suggesting flavones might possess anti-microtubule activity, and endothelial cells are sensitive to even mild disruption of microtubules. We carefully investigated whether DMXAA directly affects the microtubule or actin cytoskeletons of endothelial cells by comparing effects of CA4 and DMXAA on human umbilical vein endothelial cells (HUVEC) using time-lapse imaging and assays for cytoskeleton integrity. CA4 caused retraction of the cell margin, mitotic arrest and microtubule depolymerization, while DMXAA, up to 500 μM, showed none of these effects. DMXAA also had no effect on pure tubulin nucleation and polymerization, unlike CA4. We conclude that DMXAA exhibits no direct anti-microtubule action and thus cleanly differs from CA4 in its mechanism of action at the molecular level.