1. by Chryso Kanthou, and Gillian M. Tozer
The tumor vascular targeting agent combretastatin A–4-phosphate induces reorganization of the actin cytoskeleton and early membrane blebbing in human endothelial cells
by Chryso Kanthou, and Gillian M. Tozer
Blood
Volume 99(6):
March 15, 2002
©2002 by American Society of Hematology

2. CA-4-P disrupts the endothelial microtubule cytoskeleton and alters endothelial cell morphology.(A,B) Immunostaining of endothelial cells with an anti–β-tubulin antibody before (A) and after (B) exposure to CA-4-P (1 μM, 30 minutes).
CA-4-P disrupts the endothelial microtubule cytoskeleton and alters endothelial cell morphology.(A,B) Immunostaining of endothelial cells with an anti–β-tubulin antibody before (A) and after (B) exposure to CA-4-P (1 μM, 30 minutes). Bar = 60 μm. (C,D) Western blotting analysis of soluble (S) and polymeric (P) tubulin fractions. Cells were exposed to either CA-4-P (30 minutes) or Taxol (30 minutes; C) or t-CA-4-P (30 minutes) or indicated amounts of CA-4-P (30 minutes; D). Equal aliquots of protein (30 μL) were run into the gel. (E,F) Phase contrast images of endothelial cells before (E) or after (F) exposure to CA-4-P (1 μM, 30 minutes). Arrowheads indicate blebbing cells. Bar = 90 μm.
Chryso Kanthou, and Gillian M. Tozer Blood 2002;99:
©2002 by American Society of Hematology

3. CA-4-P stimulates actin stress fiber formation via Rho/Rho-kinase
CA-4-P stimulates actin stress fiber formation via Rho/Rho-kinase.Sparsely plated endothelial cells were pretreated with either vehicle control (30 minutes; A,B) or C3 exoenzyme (24 hours, 10 μg/mL; C,D) or Y (30 minutes, 10 μM; E,F) or HA1077 (30 min...
CA-4-P stimulates actin stress fiber formation via Rho/Rho-kinase.Sparsely plated endothelial cells were pretreated with either vehicle control (30 minutes; A,B) or C3 exoenzyme (24 hours, 10 μg/mL; C,D) or Y (30 minutes, 10 μM; E,F) or HA1077 (30 minutes, 10 μM; G,H) and then were exposed to 1 μM CA-4-P for 30 minutes (B,D,F,H). Actin was stained with Texas Red–conjugated phalloidin as described in “Materials and methods,” and cells were examined by fluorescence microscopy. Bar = 30 μm.
Chryso Kanthou, and Gillian M. Tozer Blood 2002;99:
©2002 by American Society of Hematology

4. CA-4-P stimulates early membrane blebbing in confluent endothelial cells by Rho/Rho-kinase.Confluent endothelial cultures were pretreated with either vehicle control (A-D) or C3 exoenzyme (24 hours, 10 μg/mL; E,F) or Y (30 minutes, 10 μM; G,H) and the...
CA-4-P stimulates early membrane blebbing in confluent endothelial cells by Rho/Rho-kinase.Confluent endothelial cultures were pretreated with either vehicle control (A-D) or C3 exoenzyme (24 hours, 10 μg/mL; E,F) or Y (30 minutes, 10 μM; G,H) and then were exposed to 1 μM CA-4-P for either 15 minutes (B) or 45 minutes (C,D,F,H). Actin was stained with Texas Red–conjugated phalloidin as described in “Materials and methods,” and cells were examined by fluorescence microscopy. Bars for panels A-C and panels E-H = 40 μm. Panel D represents a higher magnification of a characteristic blebbing cell. Bar for panel D = 15 μm.
Chryso Kanthou, and Gillian M. Tozer Blood 2002;99:
©2002 by American Society of Hematology

5. Regulation of CA-4-P–induced membrane blebbing
Regulation of CA-4-P–induced membrane blebbing.Confluent endothelial cultures grown on Permanox slides were pretreated with either C3 exoenzyme (24 hours, 10 μg/mL) or for 30 minutes with all other specified inhibitors/activators used either singly or in co...
Regulation of CA-4-P–induced membrane blebbing.Confluent endothelial cultures grown on Permanox slides were pretreated with either C3 exoenzyme (24 hours, 10 μg/mL) or for 30 minutes with all other specified inhibitors/activators used either singly or in combination at the following concentrations: Y-27632, 10 μM; HA1077, 10 μM; cytochalasin D, 10 nM; SB203580, 10 μM; PD98059, 25 μM; PDBu, 200 nM; ML-7, 10 μM; ML-9, 10 μM; and BDM, 5 mM. Cells were then exposed to CA-4-P (60 minutes, 1 μM), stained with Texas Red–conjugated phalloidin, and mounted in Vectashield with DAPI. Blebbing was quantified by counting phalloidin-stained blebbing cells in 9 random microscope fields per treatment and expressing these as a proportion of total numbers of DAPI-stained nuclei in identical fields. Values are means ± SD from 4 independent experiments using HUVEC cultures at passages 2 to 3. *P < .05 compared with vehicle + CA-4-P.
Chryso Kanthou, and Gillian M. Tozer Blood 2002;99:
©2002 by American Society of Hematology

6. CA-4-P induces an increase in endothelial monolayer permeability to dextrans.(A) Confluent endothelial monolayers grown on transwell filters were treated with vehicle control (○), CA-4-P (15 minutes, 1 μM) (●), C3 exoenzyme (24 hours, 10 μg/mL) followed by ...
CA-4-P induces an increase in endothelial monolayer permeability to dextrans.(A) Confluent endothelial monolayers grown on transwell filters were treated with vehicle control (○), CA-4-P (15 minutes, 1 μM) (●), C3 exoenzyme (24 hours, 10 μg/mL) followed by CA-4-P (15 minutes, 1 μM) (▴), and Y (30 minutes, 10 μM) followed by CA-4-P (▪). Data for inhibitors alone are not plotted and were not significantly different from inhibitors plus CA-4-P. Medium was replaced with FITC-dextran, and its passage through the filters was monitored at time intervals for 1 hour. In inset, passage of dextran through parallel cultures exposed to EGTA (5 mM, 15 minutes) (▵) is compared with vehicle (○) and CA-4-P (●). (B) Values taken at 60-minute time point in panel A are expressed as the percentage of fluorescent dextran passing through monolayers exposed to EGTA (see inset, 60-minute time point). Preincubation with HA1077 was for 30 minutes at 10 μM. Inhibitors were followed by either 15 minutes of vehicle control or 15 minutes of 1 μM CA-4-P. Mean values ± SEM were obtained from 3 separate filters from one of 3 representative experiments. *P < .05 for CA-4-P compared to vehicle or for inhibitors + CA-4-P compared to CA-4-P vehicle.
Chryso Kanthou, and Gillian M. Tozer Blood 2002;99:
©2002 by American Society of Hematology

7. SAPK2/p38 is activated by CA-4-P and regulates membrane blebbing
SAPK2/p38 is activated by CA-4-P and regulates membrane blebbing.(A) Sparse or confluent endothelial cultures were treated with 1 μM CA-4-P, cell lysates were harvested, and equal amounts of protein were analyzed by Western blotting for phospho–SAPK-2/p38 (...
SAPK2/p38 is activated by CA-4-P and regulates membrane blebbing.(A) Sparse or confluent endothelial cultures were treated with 1 μM CA-4-P, cell lysates were harvested, and equal amounts of protein were analyzed by Western blotting for phospho–SAPK-2/p38 (P-p38). (B,C) Confluent endothelial cells were preincubated with vehicle control (B) or SB (30 minutes, 10 μM; C) and then were exposed to CA-4-P (60 minutes, 1 μM). Cells were stained for actin with Texas Red–conjugated phalloidin as described in “Materials and methods” section and were examined by fluorescence microscopy. Bar = 35 μm.
Chryso Kanthou, and Gillian M. Tozer Blood 2002;99:
©2002 by American Society of Hematology

8. Regulation of membrane blebbing by ERK-1/2
Regulation of membrane blebbing by ERK-1/2.(A) Cell lysates were harvested 18 hours after the last medium change from 2 independent endothelial cultures grown either sparsely or confluent, and equal amounts of protein (40 μg) were analyzed for phosphorylate...
Regulation of membrane blebbing by ERK-1/2.(A) Cell lysates were harvested 18 hours after the last medium change from 2 independent endothelial cultures grown either sparsely or confluent, and equal amounts of protein (40 μg) were analyzed for phosphorylated ERK-1/2 (P-ERK) and total ERK-1/2 (T-ERK). (B) Confluent endothelial cells were preincubated with either vehicle control or PD98059 (30 minutes, 25 μM) and then exposed to PDBu (200 nM) for the indicated times. Cell lysates were analyzed for P-ERK activity as in panel A. (C-H) Confluent endothelial cells grown on Permanox slides were preincubated with either PDBu (15 minutes, 200 nM; C,D) or PD98059 (30 minutes, 25 μM), followed by PDBu (15 minutes, 200 nM; E,F) or Y (30 minutes, 10 μM), followed by PDBu (15 minutes, 200 nM; G,H). Cells in panels D, F, and H were then treated with CA-4-P (30 minutes, 1 μM) and stained for actin. Bar = 40 μm.
Chryso Kanthou, and Gillian M. Tozer Blood 2002;99:
©2002 by American Society of Hematology

9. CA-4-P activates MLC phosphorylation via Rho-kinase
CA-4-P activates MLC phosphorylation via Rho-kinase.(A,B) Confluent endothelial cells were exposed to 1 μM CA-4-P (A) or 30 minutes CA-4-P (B).
CA-4-P activates MLC phosphorylation via Rho-kinase.(A,B) Confluent endothelial cells were exposed to 1 μM CA-4-P (A) or 30 minutes CA-4-P (B). Cells in panels C and D were preincubated with vehicle, C3 exoenzyme (24 hours, 10 μg/mL), Y (30 minutes, 10 μM), or ML-7 (30 minutes, 10 μM) before exposure to CA-4-P (30 minutes, 1 μM). Equal amounts of cell proteins (30 μg) were analyzed by Western blotting for di-phosphorylated MLC (P-MLC).
Chryso Kanthou, and Gillian M. Tozer Blood 2002;99:
©2002 by American Society of Hematology

10. Inhibition of myosin ATPase blocks contractility and up-regulates blebbing.Confluent endothelial cells were preincubated with either vehicle control (DMSO; B) or BDM (5 mM, 30 minutes; A,C-H).
Inhibition of myosin ATPase blocks contractility and up-regulates blebbing.Confluent endothelial cells were preincubated with either vehicle control (DMSO; B) or BDM (5 mM, 30 minutes; A,C-H). In panels E and F, cells were preincubated with SB (10 μM, 30 minutes) together with BDM and in panels G and H with cytochalasin D (10 nM, 30 minutes) together with BDM. Then cells were exposed to CA-4-P (1 μM, 60 minutes; B,C,D,F,H) and fixed and stained with Texas Red phalloidin. Panel D represents a higher magnification of cells in panel C. Bar for panels A-C and panels E-F = 42 μm; bar for panel D = 25 μm.
Chryso Kanthou, and Gillian M. Tozer Blood 2002;99:
©2002 by American Society of Hematology

11. CA-4-P induces the assembly of focal adhesions
CA-4-P induces the assembly of focal adhesions.Sparse endothelial cells grown on Permanox slides were exposed to CA-4-P (60 minutes, 1 μM).
CA-4-P induces the assembly of focal adhesions.Sparse endothelial cells grown on Permanox slides were exposed to CA-4-P (60 minutes, 1 μM). Cells in panels E and F were pretreated with PD98059 (30 minutes, 25 μM) to induce blebbing. Cells were fixed and double stained with Texas Red phalloidin and antivinculin antibody. Panels A,C,E have vinculin staining; panels B,D,F, actin staining. Bar = 20 μm.
Chryso Kanthou, and Gillian M. Tozer Blood 2002;99:
©2002 by American Society of Hematology