Cytochalasin inhibition of isolated rat gastric parietal cell function

Submicrogram concentrations (0.04-0.29 microM) of the microfilament disrupting agents cytochalasins D, E, and B (CD, CE, CB) were shown to inhibit secretagogue-stimulated 14C-aminopyrine accumulation (AP) in isolated rat gastric mucosal parietal cells. The microtubule disrupting agent colchicine had little influence on AP accumulation. Histamine- and dibutyryl cyclic AMP (DbcAMP)-stimulated AP accumulation was inhibited with an order of potency CD greater than CE approximately equal to CB. CB inhibition of these secretagogue actions was, however, only approximately 65-70% of the maximal stimulated response, whereas CD and CE caused 100% inhibition. On the other hand, carbamylcholine-stimulated AP accumulation was inhibited 100% by all cytochalasins tested with an order of potency CD approximately equal to CE greater than CB. These data are discussed in relation to acid secretagogue-induced morphological changes involving actin filament organization in parietal cells.     

The cytoskeleton and cell volume regulation

Although the precise mechanisms have yet to be elucidated, early events in osmotic signal transduction may involve the clustering of cell surface receptors, initiating downstream signaling events such as assembly of focal adhesion complexes, and activation of, e.g. Rho family GTPases, phospholipases, lipid kinases, and tyrosine- and serine/threonine protein kinases. In the present paper, we briefly review recent evidence regarding the possible relation between such signaling events, the F-actin cytoskeleton, and volume-regulatory membrane transporters, focusing primarily on our own work in Ehrlich ascites tumer cells (EATC). In EATC, cell shrinkage is associated with an increase, and cell swelling with a decrease in F-actin content, respectively. The role of the F-actin cytoskeleton in cell volume regulation in various cell types has largely been investigated using cytochalasins to disrupt F-actin and highly varying effects have been reported. Findings in EATC show that the effect of cytochalasin treatment cannot always be assumed to be F-actin depolymerization, and that, moreover, there is no well-defined correlation between effects of cytochalasins on F-actin content and their effects on F-actin organization and cell morphology. At a concentration verified to depolymerize F-actin, cytochalasin B (CB), but not cytochalasin D (CD), inhibited the regulatory volume decrease (RVD) and regulatory volume increase (RVI) processes in EATC. This suggests that the effect of CB is related to an effect other than F-actin depolymerization, possibly its F-actin severing activity.     

Role of the F-actin cytoskeleton in the RVD and RVI processes in Ehrlich ascites tumor cells.

The role of the F-actin cytoskeleton in cell volume regulation was studied in Ehrlich ascites tumor cells, using a quantitative rhodamine-phalloidin assay, confocal laser scanning microscopy, and electronic cell sizing. A hypotonic challenge (160 mOsm) was associated with a decrease in cellular F-actin content at 1 and 3 min and a hypertonic challenge (600 mOsm) with an increase in cellular F-actin content at 1, 3, and 5 min, respectively, compared to isotonic (310 mOsm) control cells. Confocal visualization of F-actin in fixed, intact Ehrlich cells demonstrated that osmotic challenges mainly affect the F-actin in the cortical region of the cells, with no visible changes in F-actin in other cell regions. The possible role of the F-actin cytoskeleton in RVD was studied using 0. 5 microM cytochalasin B (CB), cytochalasin D (CD), or chaetoglobosin C (ChtC), a cytochalasin analog with little or no affinity for F-actin. Recovery of cell volume after hypotonic swelling was slower in cells pretreated for 3 min with 0.5 microM CB, but not in CD- and ChtC-treated cells, compared to osmotically swollen control cells. Moreover, the maximal cell volume after swelling was decreased in CB-treated, but not in CD- or Chtc-treated cells. Following a hypertonic challenge imposed using the RVD/RVI protocol, recovery from cell shrinkage was slower in CB-treated, but not in CD- or Chtc-treated cells, whereas the minimal cell volume after shrinkage was unaltered by either of these treatments. It is concluded that osmotic cell swelling and shrinkage elicit a decrease and an increase in the F-actin content in Ehrlich cells, respectively. The RVD and RVI processes are inhibited by 0.5 microM CB, but not by 0.5 microM CD, which is more specific for actin.     

Relation between cytoskeleton,hypo-osmotic treatment and volume regulation in Ehrlich ascites tumor cells

Summary Pretreatment with cytochalasin B, which is known to disrupt microfilaments, significantly inhibits regulatory volume decrease (RVD) in Ehrlich ascites tumor cells, suggesting that an intact microfilament network is a prerequisite for a normal RVD response. Colchicine, which is known to disrupt microtubules, has no significant effect on RVD. Ehrlich cells have a cortical three-dimensional, orthogonal F-actin filament network which makes the cells look completely black in light microscopy following immunogold/silver staining using anti-actin antibodies. After addition of cytochalasin B, the stained cells get lighter with black dots localized to the plasma membrane and appearance of multiple knobby protrusions at cell periphery. Also, a significant decrease in the staining of the cells is seen after 15 min of RVD in hypotonic medium. This microfilament reorganization appears during RVD in the presence of external Ca²⁺ or Ca²⁺-ionophore A23187. It is, however, abolished in the absence of extracellular calcium, with or without prior depletion of intracellular Ca²⁺ stores. An effect of increased calcium influx might therefore be considered. The microfilament reorganization during RVD is abolished by the calmodulin antagonists pimozide and trifluoperazine, suggesting the involvement of calmodulin in the process. The microfilament reorganization is also prevented by addition of quinine. This quinine inhibition is overcome by addition of the K⁺ ionophore valinomycin.     

Specific interaction of cytochalasins with muscle and platelet actin filaments in vitro

The cytochalasins (CE, CD, CB and H₂CB) inhibit numerous cellular processes which require the interaction of actin with other structural and contractile proteins. In this report we describe the effects of the cytochalasins on the viscosity and morphology of muscle and platelet actin. The cytochalasins decreased the viscosity of F-actin solutions. The effect of H₂CB, CB and CD on F-actin viscosity was maximal at concentrations of 20–50μM and did not increase with time. In contrast, CE caused a progressive decrease in the viscosity of F-actin solutions which was dependent upon the concentration of CE and the duration of incubation of the CE-actin mixture. After two hours of incubation of drug-actin mixtures, the relative effectiveness of the cytochalasins in reducing the viscosity of F-actin was CE > CD > CB = H₂CB. The effects of CD and CE were paralleled by morphologic changes in negatively stained actin filaments. The effects of the cytochalasins on the viscosity and morphology of muscle and platelet actin were the same whether the drugs were added before or after the polymerization of the protein. These studies show that the interaction of the cytochalasins with actin is highly specific. Because the relative potencies of these drugs for affecting motile processes and the relative affinities of the drugs for binding sites within a variety of cells are CE > CD > CB = H₂CB, the effects of cytochalasins on actin described here may contribute to some of the biological effects of the drugs on motile processes.     

Role of cytoskeleton in cell shaping of developing mesophyll of wheat (Triticum aestivum L.).

The effects of oryzalin and cytochalasin B (CB) on microtubule and actin microfilament arrays and on cell shaping were investigated in developing wheat mesophyll. Excised immature leaf sections capable of differentiating were incubated with the drugs. The behavior of the cytoskeleton was monitored by fluorescence microscopy after labeling with fluorescent dyes. Brief incubation with oryzalin (40 min, 10 microM) caused disassembly of microtubules. Recovery of microtubule arrays was comparatively slow after removal of the drug. Cells failed to establish transverse cortical bands of microtubules and transverse hoops of wall reinforcement. They expanded isodiametrically rather than longitudinally without forming lobes typical of wheat mesophyll cells. Brief treatment with CB (60 min, 20 micrograms ml-1) appeared to disrupt the microfilament arrays. Filaments recovered rapidly after removal of CB, and cells were able to shape in an apparently normal fashion. Continuous incubation at comparatively low concentration of CB (4 micrograms ml-1) appeared to cause selective loss of the fine transverse cortical microfilament arrays. Cortical transverse microtubule arrays persisted, but failed to form distinct bands in the majority of the cells. Cells were able to elongate in an almost normal fashion, but no lobes were formed.     

Actin cytoskeleton disassembly affects conductive properties of stretch-activated cation channels in leukaemia cells

Mechanosensitive channels in various eucaryotic cells are thought to be functionally and structurally coupled to the cortical cytoskeleton. However, the results of electrophysiological studies are rather controversial and the functional impact of cytoskeleton assembly-disassembly on stretch-activated channel properties remains unclear. Here, the possible involvement of cytoskeletal elements in the regulation of stretch-activated Ca2+-permeable channels was studied in human leukaemia K562 cells with the use of agents that selectively modify the actin or tubulin system. F-actin disassembly resulted in a considerable reduction of the amplitude of stretch-activated currents without significant change in channel open probability. The effects of treatments with cytochalasins or latrunculin were principally similar, developed gradually and consisted a strong decrease of single channel conductance. Microtubule disruption did not affect stretch-activated channels. The data presented here are in principal agreement with the general conclusion that mechanosensitive channel functions are largely dependent on the integrity of the cortical actin cytoskeleton. Specifically, changes in conductive properties of the pore may provide an essential mechanism of channel regulation underlying functional modulation of membrane currents. Our results allow one to speculate that microfilament organization may be an important determinant in modulating biophysical characteristics of stretch-activated cation channels in cells of blood origin.     

Actin cytoskeleton disassembly affects conductive properties of stretch-activated cation channels in leukaemia cells

Mechanosensitive channels in various eucaryotic cells are thought to be functionally and structurally coupled to the cortical cytoskeleton. However, the results of electrophysiological studies are rather controversial and the functional impact of cytoskeleton assembly-disassembly on stretch-activated channel properties remains unclear. Here, the possible involvement of cytoskeletal elements in the regulation of stretch-activated Ca²⁺-permeable channels was studied in human leukaemia K562 cells with the use of agents that selectively modify the actin or tubulin system. F-actin disassembly resulted in a considerable reduction of the amplitude of stretch-activated currents without significant change in channel open probability. The effects of treatments with cytochalasins or latrunculin were principally similar, developed gradually and consisted a strong decrease of single channel conductance. Microtubule disruption did not affect stretch-activated channels. The data presented here are in principal agreement with the general conclusion that mechanosensitive channel functions are largely dependent on the integrity of the cortical actin cytoskeleton. Specifically, changes in conductive properties of the pore may provide an essential mechanism of channel regulation underlying functional modulation of membrane currents. Our results allow one to speculate that microfilament organization may be an important determinant in modulating biophysical characteristics of stretch-activated cation channels in cells of blood origin.     

Primary granule exocytosis in human neutrophils is regulated by Rac-dependent actin remodeling

The actin cytoskeleton regulates exocytosis in all secretory cells. In neutrophils, Rac2 GTPase has been shown to control primary (azurophilic) granule exocytosis. In this report, we propose that Rac2 is required for actin cytoskeletal remodeling to promote primary granule exocytosis. Treatment of neutrophils with low doses (< or = 10 microM) of the actin-depolymerizing drugs latrunculin B (Lat B) or cytochalasin B (CB) enhanced both formyl peptide receptor- and Ca(2+) ionophore-stimulated exocytosis. Higher concentrations of CB or Lat B, or stabilization of F-actin with jasplakinolide (JP), inhibited primary granule exocytosis measured as myeloperoxidase release but did not affect secondary granule exocytosis determined by lactoferrin release. These results suggest an obligatory role for F-actin disassembly before primary granule exocytosis. However, lysates from secretagogue-stimulated neutrophils showed enhanced actin polymerization activity in vitro. Microscopic analysis showed that resting neutrophils contain significant cortical F-actin, which was redistributed to sites of primary granule translocation when stimulated. Exocytosis and actin remodeling was highly polarized when cells were primed with CB; however, polarization was reduced by Lat B preincubation, and both polarization and exocytosis were blocked when F-actin was stabilized with JP. Treatment of cells with the small molecule Rac inhibitor NSC23766 also inhibited actin remodeling and primary granule exocytosis induced by Lat B/fMLF or CB/fMLF, but not by Ca(2+) ionophore. Therefore, we propose a role for F-actin depolymerization at the cell cortex coupled with Rac-dependent F-actin polymerization in the cell cytoplasm to promote primary granule exocytosis.     

Effect of cytochalasins B and D on the developmental competence of somatic cell nuclear transfer embryos in miniature pigs

In many animals, cytochalasins have generally been used as cytoskeletal inhibitors for the diploid complement retention of somatic cell nuclear transfer (SCNT) embryos. However, limited information is available on the effects of cytochalasins on the in vitro development of SCNT embryos. Hence, we compared the effects of cytochalasin B (CB) and cytochalasin D (CD) on pseudo-polar body (pPB) extrusion, cortical actin filament (F-actin) distribution in porcine parthenogenetic oocytes and in vitro development of SCNT embryos that were reconstructed using foetal fibroblasts in the G0/G1 phase derived from miniature pigs. CB (7.5 microg/ml) and CD (2.5 microg/ml) treatments effectively inhibited pPB extrusion in SCNT embryos. CB (2.5 microg/ml) treatment could not inhibit pPB extrusion and insufficiently destabilized F-actin immediately following artificial activation. In parthenogenetic oocytes treated with 2.5 microg/ml CD, normal reorganization and uniform distribution of cortical F-actin at the cytoplasmic membrane were observed at 8 h after artificial activation; this finding was similar to that of control oocytes. In contrast, parthenogenetic oocytes treated with 7.5 microg/ml CB showed non-uniform distribution of F-actin at 8 h after artificial activation. On day 5 after in vitro cultivation, the blastocyst formation rate of SCNT embryos treated with 2.5 microg/ml CD was significantly higher than that of SCNT embryos treated with 2.5 and 7.5 microg/ml CB (p < 0.05). Hence, the present findings suggest that CD is more effective than CB as the cytoskeletal inhibitor for the production of SCNT embryos in miniature pigs.     

Cytochalasins induce actin polymerization in human leukocytes.

We studied the effect of cytochalasins (B, D, and E) on the F-actin content in human neutrophils and lymphocytes using NBD-phallacidin labeling followed by flow cytometry. All three cytochalasins induced a concentration- and time-dependent increase in the F-actin content in both cell types. The order of potency was cytochalasin D greater than E greater than B. The increase in F-actin content was accompanied by a decrease in the G-actin content as measured by DNase I inhibition assay. These observations suggest that in intact cells cytochalasins may function differently compared to purified and semipurified systems, and their effects may be modified through other actin-binding or sequestering proteins. 2-deoxyglucose (20 mM) caused a decrease in the basal F-actin content and significantly reduced the change induced by the cytochalasins. These results suggest that the state of actin in intact cells is regulated by cytosolic ATP levels, primarily by the integrity of the glycolytic pathway. Based on these observations, we conclude that the mechanism of action of cytochalasins in intact cells is more complex than current models suggest.     

Interaction between rabbit muscle actin and several chaetoglobosins or cytochalasins.

The mold metabolites chaetoglobosins Ch-A, B, C, E, F, and J exert, as do the cytochalasins CB, CD, CE, and CG, enhancing effects of various strength on the polymerization of rabbit muscle G-actin. The polymers formed differ widely in their viscosity, Ch-B and Ch-J leading to the least viscous actins. Equal states of viscosity are arrived at by interaction of F-actin with the respective drugs. There is no correlation between the ATP hydrolyzing activity of F-actin elicited by the various cytochalasins and their influence on the viscosity.     

Distribution of actin and tubulin in cells and in glycerinated cell models after treatment with cytochalasin B (CB)

The organization of microfilaments and microtubules in cultured cells before and after the addition of cytochalasin B (CB) was studied both by electron microscopy and immunofluorescence microscopy using antibodies specific for actin, tubulin and tropomyosin. CB induces a rapid disorganization of normal microfilament bundles. Star-like patches of actin and tropomyosin are visualized in immunofluorescence microscopy and dense aggregates of condensed microfilaments are seen in electron microscopy. The integrity of the microtubules is not changed by CB treatment. Addition of CB to glycerinated cells, in contrast to normal cells, does not result in the disorganization of microfilament bundles. CB-treated glycerinated models can still contract upon addition of ATP. Thus the CB-induced rearrangement of microfilament bundles occurs only in vivo and not in glycerinated cell contractility models.     

Effect of cytochalasins on cytosolic-free calcium concentration and phosphoinositide metabolism in leukocytes

Cytochalasins are routinely used to stimulate a variety of functions in eukaryotic cells even though their precise mode of action remains to be elucidated. In the present work we used the fluorescent Ca2+ indicator quin2 to study the effect of various cytochalasins, cytochalasins A, B, C, D, E (CA, CB, CC, CD, CE) and dihydrocytochalasin B (dhCB) on the intracellular Ca2+ concentration ([Ca2+]i) in various types of leukocytes, viz, neutrophils and lymphocytes. In human neutrophils, cytochalasins increase [Ca2+]i mainly by releasing Ca2+ from membrane-bound, intracellular stores. Thus, in order to readily appreciate the effect of cytochalasins on [Ca2+ )i, these cells must be loaded with low intracellular quin2 concentrations. On the other hand, in peripheral blood lymphocytes, splenocytes and thymocytes, the increase in [Ca2+]i is predominantly due to an increased Ca2+ influx from the extracellular medium. In addition, we found that in neutrophils these drugs prolong the increase in [Ca2+]i induced by chemotactic peptides, probably by increasing the cell permeability to Ca2+. Finally, in thymocytes, cytochalasins potentiate the production of inositol phosphates induced by the polyclonal mitogen concanavalin A (conA).     

Importance of cytoskeletal elements in volume regulatory responses of trout hepatocytes

The role of cytoskeletal elements in volume regulation was studied in trout hepatocytes by investigating changes in F-actin distribution during anisotonic exposure and assessing the impact of cytoskeleton disruption on volume regulatory responses. Hypotonic challenge caused a significant decrease in the ratio of cortical to cytoplasmic F-actin, whereas this ratio was unaffected in hypertonic saline. Disruption of microfilaments with cytochalasin B (CB) or cytochalasin D significantly slowed volume recovery following hypo- and hypertonic exposure in both attached and suspended cells. The decrease of net proton release and the intracellular acidification elicited by hypotonicity were unaltered by CB, whereas the increase of proton release in hypertonic saline was dramatically reduced. Because amiloride almost completely blocked the hypertonic increase of proton release and cytoskeleton disruption diminished the associated increase of intracellular pH (pH(i)), we suggest that F-actin disruption affected Na(+)/H(+) exchanger activity. In line with this, pH(i) recovery after an ammonium prepulse was significantly inhibited in CB-treated cells. The increase of cytosolic Na(+) under hypertonic conditions was not diminished but, rather, enhanced by F-actin disruption, presumably due to inhibited Na(+)-K(+)-ATPase activity and stimulated Na(+) channel activity. The elevation of cytosolic Ca(2+) in hypertonic medium was significantly reduced by CB. Altogether, our results indicate that the F-actin network is of crucial importance in the cellular responses to anisotonic conditions, possibly via interaction with the activity of ion transporters and with signalling cascades responsible for their activation. Disruption of microtubules with colchicine had no effect on any of the parameters investigated.     

The macaque sperm actin cytoskeleton reorganizes in response to osmotic stress and contributes to morphological defects and decreased motility

Sperm undergo extreme variations in temperature and osmolality during cryopreservation, resulting in cell damage that includes plasma membrane defects, changes in cell volume, decreased motility, and flagellar defects. However, the fundamental biologic mechanisms underlying these events are poorly understood. We investigated the effects of osmotic stress and cytochalasins b (CB) and d (CD), naturally occurring toxins that disrupt actin organization, on the actin cytoskeleton and motility of Rhesus macaque sperm (Macaca mulatta). Sperm were diluted in media of low, medium, or high osmolality, or medium-osmolality media containing CB or CD, were stained with phalloidin-fluorescein isothiocyanate, and were processed for microscopy. The majority of sperm incubated in medium-osmolality media exhibited postacrosomal stain, whereas the minority displayed banding patterns of F-actin stain in the head. High-osmolality media, as well as CB and CD incubation, resulted in reorganization of F-actin into bands of stain in the majority of sperm heads. Cytochalasin b treatment also resulted in curled and looped tails, a phenomenon of hyposmotic stress, and CB and CD caused significant, dose-dependent decreases in motility determined by computer-assisted sperm assessment. Rho A cell populations were determined using flow cytometry, and immunocytochemistry analysis demonstrated that Rho A localization was altered after osmotic stress. Together, our results support a mechanism in which reorganization of the actin cytoskeleton induced by osmotic stress and potentially mediated by a Rho A signaling pathway contributes to sublethal sperm flagellar and motility defects.     

Stimulation of regulatory volume decrease (RVD) by isolated bovine articular chondrocytes following F-actin disruption using latrunculin B

Articular chondrocytes are exposed to significant changes in extracellular osmolarity during normal joint activity, which can lead to changes in cell volume and metabolism of the extracellular matrix (ECM). Chondrocytes can respond to cell swelling/shrinking by volume regulatory pathways, but the signalling pathways are poorly understood although a role for the cytoskeleton is frequently implicated. Here, we have investigated the effects of disruption of the chondrocyte F-actin cytoskeleton on the recovery of cell volume by RVD. The cytoskeleton was perturbed using the relatively specific agent latrunculin B (5 microM; 30 min) and loss of F-actin integrity quantified using fluorescent phalloidin-labelling and confocal laser scanning microscopy (CLSM). Imaging of isolated chondrocytes labelled with Fura-2 to measure the fluorescence associated with cell volume changes, showed that the extent of hypo-osmotic swelling was unaffected by latrunculin B treatment. Two categories of the chondrocyte RVD response were observed: 'fast' RVD where at 3 min post-osmotic challenge there was a recovery in cell fluorescence of >or=80%, whereas other cells exhibited 'slow' RVD. Latrunculin B increased the proportion of chondrocytes demonstrating 'fast' RVD by approximately 10 fold and reduced those cells showing 'slow' RVD. An inhibitor of chondrocyte RVD (REV 5901) had no significant effect on the integrity of the cytoskeleton showing that the RVD response could be inhibited independent of the state of the F-actin cytoskeleton. These results suggest that the intact cortical F-actin cytoskeleton has a restraining effect on the RVD response of isolated bovine articular chondrocytes.     

Hydrostatic pressure induced changes in the cytoarchitecture of pheochromocytoma (PC-12) cells.

Confocal microscopy, in association with three-dimensional reconstruction, revealed that microtubules and microfilaments in differentiating PC-12 cells were disrupted in a dose-dependent manner following pressure treatment. Hydrostatic pressure caused cell rounding, microtubule and microfilament disorganization, neurite retraction and the formation of a microtubule ring adjacent to the cell surface. Volume analysis from computer-generated reconstructed cells, at atmospheric pressure, showed that the apparent volume of microtubules and microfilaments, normalized to 100 units, was 22 and 11 respectively. At 4000 and 8000 psi, the apparent microtubule volume was reduced to 16 and 12 units, respectively, and the apparent microfilament volume was reduced to 8 and 5 units, respectively. Thus, the apparent microtubule and microfilament volumes in PC-12 cells decreased as pressure increased. In the presence of taxol and phalloidin which stabilize the cytoarchitecture, cells resist the effects of hydrostatic pressure. In the presence of colchicine and cytochalasin D compounds which destabilize the cytoarchitecture, cells are more susceptible to the disrupting effects of hydrostatic pressure. The effects of hydrostatic pressure on cell morphology were reversible.     

Activation of neutrophils: measurement of actin conformational changes by flow cytometry

Actin, which comprises approximately 10% of the weight of cytoplasmic protein of neutrophils, is the principal component of the cytoplasmic microfilament lattice. It can exist in either of two physical states, G-actin, which is monomeric, or F-actin, which is polymeric or filamentous. Actin microfilaments support many forms of cell movement. Continuous remodeling of the microfilament lattice, which seems integral to sustained movement, is possible in part because of the ability of actin to change rapidly between its monomeric G-state and its filamentous F-state. Changes in the G- and F-actin equilibrium may be studied by flow analysis using a fluorescent probe which is specific for F-actin, 7-nitrobenz-2-oxa-1,3-diazole-(NBD)-phallacidin. Alterations in neutrophil F-actin have been measured in response to chemotactic agents (e.g., formyl peptides and leukotriene B4), inhibitors of cell movement (e.g., N-ethylmaleimide and cytochalasin B), agents that promote the oxidative burst (e.g., formyl peptides and phorbol esters), and priming agents [e.g., tumor necrosis factor (TNF)]. Measurements may be taken at intervals of a few seconds, allowing comparison of rapid changes in the F-actin content to other rapidly occurring changes, such as altered membrane ion permeability and activation of cellular enzymes. The use of metabolic inhibitors has allowed dissection of some of the biochemical pathways involved in actin assembly in living cells. Although clinical studies are few thus far, the technique has also been used to study basal and stimulated F-actin levels in circulating neutrophils in neonates and in family members of patients with neutrophil-actin dysfunction.     

‘Rings’ of F-actin form around the nucleus in cultured human MCF7 adenocarcinoma cells upon exposure to both taxol and taxotere

The anti-cancer taxoids, Taxol® (paclitaxel) and Taxotere® (docetaxel), are the most promising anti-mitotic agents developed for cancer treatment in the past decade. The effectiveness of this new class of compounds lies in their unique mechanism of action on the cytoskeleton. Both taxol and taxotere bind to microtubules and shift the normal equilibrium between monomeric and polymerized tubulin to favor the polymerized form by strongly promoting tubulin assembly and inhibiting microtubule depolymerization. Although very similar in structure, these two compounds have recently demonstrated different in vitro, in vivo, and clinical activities; however, no study to date has effectively compared specific cytoskeletal alterations induced by taxol and taxotere in cultured cells. Using specific staining techniques for both F-actin and α-tubulin, this study provides the first detailed immunohistochemical comparison of the effects of equimolar concentrations of taxol and taxotere on both the microfilament and microtubule networks in a cultured cell line. Using human MCF7 breast adenocarcinoma cells, new observations of taxotere/taxol alterations of the cytoskeleton include: an increased abundance of parallel microtubule ‘bundles’ in taxotere treated cells and a definitive reorganization of the microfilament network which results in novel ring-like formations of F-actin condensed exclusively in the perinuclear zone. Reorganization of the actin cytoskeleton induced by a taxoid disruption of the microtubule equilibrium is indicative of the interdependence between microtubules and microfilaments in this transformed cell line and suggests that the indirect role of the taxoids on the microfilament network may have been overlooked in their mechanism of action as chemotherapeutic agents.