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Rho-Kinase Inhibitor Retards Migration and in Vivo Dissemination of Human Prostate Cancer Cells
Biochemical and Biophysical Research Communications 269, 652– 659 (2000) doi:10.1006/bbrc.2000.2343, available online at http://www.idealibrary.com on
Rho-Kinase Inhibitor Retards Migration and in Vivo Dissemination of Human Prostate Cancer Cells Avril V. Somlyo,* ,1 Dawn Bradshaw,† Susan Ramos,* Cheryl Murphy,† Charles E. Myers,† and Andrew P. Somlyo* *Department of Molecular Physiology and Biological Physics and †Cancer Center, University of Virginia Health System, Charlottesville, Virginia 22906
Received February 2, 2000
The Rho-kinase inhibitor, Y-27632, inhibited in vitro chemotactic migration to bone marrow fibroblast conditioned media and metastatic growth in immunecompromised mice of highly invasive human prostatic cancer (PC3) cells. Y-27632 also reduced myosin light chain phosphorylation and markedly altered the morphology of cells that developed numerous processes containing microtubules. A strikingly different, rounded phenotype was induced by an inhibitor of myosin light chain kinase, ML9. The M 110 –130 subunit of the myosin phosphatase that is regulated by Rhokinase was present in PC3 cells that contained significantly more RhoA than the less invasive, LNCaP cells. Y-27632 also inhibited angiogenesis as measured by endothelial cell tube formation on Matrigel. We conclude that invasiveness of human prostate cancer is facilitated by the Rho/Rho-kinase pathway, and exploration of selective Rho-kinase inhibitors for limiting invasive progress of prostate cancer is warranted. © 2000 Academic Press
Key Words: Rho-kinase; prostate cancer; metastasis; cell migration; Y-27632; angiogenesis; myosin phosphatase.
of the Rho/Rho-kinase pathway inhibits SMPP-1M and increases actomyosin-based motility (10; rev. in 11, 12), whereas the highly selective Rho-kinase inhibitor, Y-27632 (13), inhibits it. We chose to evaluate the effect of Y-27632 in human prostate cancer cell lines. In a significant number of prostate cancer patients in whom the cancer grows and spreads slowly, watchful observation is a reasonable alternative to surgery or radiation therapy. An orally active drug of low toxicity that further slows the spread of prostate cancer may well make conservative treatment more effective by preventing the development of metastatic disease, commonly to bone. In this paper, we present evidence that Y-27632 may well be such a compound: it blocks tumor cell motility and chemotaxis induced by bone fibroblast conditioned media, as well as intravascularly disseminated growth of PC3 cells in vivo, angiogenesis by endothelial cell tube formation on Matrigel and produces a phenotype that is strikingly different from that induced by the MLCK inhibitor, ML9. Preliminary results of some of these findings have been presented to the American Society of Cell Biology (14). MATERIALS AND METHODS
Abnormal proliferation and migration of cancer cells are both strongly dependent on processes requiring myosin II activity. Non-muscle, like smooth muscle, myosin II is activated by phosphorylation by myosin light chain kinase (MLCK; rev. in 1, 2) and inactivated by dephosphorylation of its regulatory light chain (MLC 20 ; rev. in 1). MLC 20 phosphorylation can also be increased by inhibiting (3, 4) the protein phosphatase (SMPP-1M) that normally dephosphorylates myosin II (5– 8; rev. in 9). Activation 1 To whom correspondence should be addressed at University of Virginia Health System, Department of Molecular Physiology and Biological Physics, P. O. Box 10011, Charlottesville, Virginia 229060011. Fax: (804) 982-1616. E-mail: [email protected].
0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
Cell culture. PC3 and LNCaP human prostate cancer cells (American Type Culture Collection) were cultured in RPMI medium 1640 supplemented with 10% fetal bovine serum (FBS), plus 100 g/ml streptomycin and 100 units/ml penicillin. Cell proliferation assay. Cell proliferation in the presence or absence of 25 M Y-27632 was assayed using the nonradioactive cell proliferation assay (Promega Corp., Madison, WI), 5,000 cells/well, which is based on the ability of viable cells to reduce tetrazolium salt to a colored formazin product. Cell migration and cell movement. Cell migration was assayed in Boyden chambers [8.0 m pore size polyethylene terephthalate (PET) membrane, FALCON cell culture insert (Becton-Dickinson)]. 200 l of 4.0 ⫻ 10 4 PC3 cells/ml ⫾ 25 M Y-27632 in serum-free media (SFM) were added to the upper chamber. Transwells were incubated overnight at 37°C with 500 l serum-free RPMI in the lower chamber. The top chambers containing the cells were then
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transferred to new lower chambers with or without 500 l BMFB-CM or 10 nM epidermal growth factor (EGF) in serum-free RPMI and incubated for 1 h. Cells on the inside of the transwell inserts were removed with a cotton swab, fixed, and stained with crystal violet. Five regions were randomly chosen from each insert and the number of cells counted. In controls performed to test for the possibility that treatment affected adherence to the PET membrane, adherence of control and Y-27632-treated (25 M) cells (63 ⫾ 10.6 S.D. and 66 ⫾ 5.5. S.D. cells/field, respectively) was not different when the experiment was repeated using 3.0 m pore size PET membranes, which preclude transmigration. BMFB-CM was obtained by growing CRL7298 cells to confluence with FBS, washing 3 times in SFM, incubating the cells in 5 ml of SFM for 18 –24 h, centrifuging at 1,500 rpm and used full strength in the transwell. For time-lapse images, cells were plated at 2 ⫻ 10 4 cells/ml on gridded coverslips and incubated overnight. Randomly selected areas were photographed every hour. MLC 20 phosphorylation. PC3 cells were plated at 3 ⫻ 10 6 or 6 ⫻ 10 5 cells/mL in SFM, and incubated overnight to mimic the migration assay conditions; subsequently 25 M Y-27632 was added to half the dishes for 18 h. In some cases the MLCK inhibitor ML9 was added to control and Y-27632-treated cells for 2 h prior to stimulation with 10 mM EGF or BMFB-CM for 2 min, followed by ice-cold 10% TCA. Cells were scraped and cell pellets washed three times in absolute acetone with 10 mM dithiothreitol (DTT), and spun at 11,000 rpm for 10 min. The air-dried pellet was resuspended in urea buffer containing 6.7 M urea, 20 mM Tris base, 22 mM glycine, and 10 mM DTT and unphosphorylated (U) and singly and doubly (P1 and P2) phosphorylated forms of MLC 20 separated on onedimensional isoelectric focusing acrylamide gels (ampholytes pH 4.5–5.4) using a five-well comb and run on a Hoefer minigel system. Gels were transferred to nitrocellulose and Western blot analysis was carried out using the mini-Protein II multi-screen apparatus (2.5 mm diameter gel lanes in triplicate). Lane 1, secondary antibody only; lane 2, primary polyclonal antibodies specific for phosphorylated Ser 19 of MLC 20 (15), a gift of Dr. F. Matsumura; lane 3, 1:2,000 dilution of a polyclonal antibody to MLC 20, a gift of Dr. K. Kamm, reactive with both P1, P2, and U forms of MLC 20. A donkey antirabbit HRP secondary antibody was used at 1:5000 dilution and the reactive bands detected by ECL and quantitated on a Bio-Rad GS670 densitometer. Control lanes included thiophosphorylated and nonphosphorylated turkey gizzard myosin. Percent phosphorylation ⫽ P1 ⫹ P2/(U ⫹ P1 ⫹ P2) ⫻ 100. Protein concentrations were determined by the Bradford assay. Immunolabeling. Cells were fixed with 3% paraformaldehyde in PBS or 0.5% glutaraldehyde in PIPES saline, permeabilized with 0.5% Triton X-100, rinsed and blocked for 30 min in 20% goat serum. The following antibodies were used: ␣-tubulin mouse monoclonal (Sigma) at 1:500 dilution, monoclonal anti-human vinculin (Sigma) at 1:750 dilution, FITC or TRITC goat anti-mouse secondary antibodies at 1:200 dilution. Human MAP4 (microtubule-associated protein) mouse monoclonal antibody at 1:500 dilution. Actin was stained with rhodamine- or fluorescein-labeled phalloidin at a 1:1000 dilution. Cells were washed and mounted in 1 mg/ml 1,4 diazabicylclo[2.2.2]octane. Western blotting for detection of RhoA, Rho-kinase, tubulin isoforms, myosin phosphatase, MAP4. Cells were scraped in lysis buffer (50 mM Tris, pH 7.5, 150 NaCl, 2 mM EDTA, 1% Triton X-100, protease inhibitors AEBSF, 1 mM, leupeptin, 20 g/ml, and aprotinin, 20 g/ml) and protein concentrations assayed. Proteins in Laemmli sample buffer were separated by SDS-PAGE, and transferred to nitrocellulose or polyvinylidene difluoride membranes, as previously described (10). The following antibodies were used: AA-2, which recognizes all  isoforms of tubulin, and -II, -III, and -IV, at 1:1000 dilution (gift of Dr. T. Frankfurter); ␣-tubulin (Sigma) at 1:10,000; polyclonal regulatory subunit of smooth muscle myosin
phosphatase, which recognizes the muscle and nonmuscle isoform at 1:2000, gift of Dr. D. Hartshorne, monoclonal anti-RhoA generated to amino acids 120 –150 at 1:5000 dilution, Rho-kinase at 1:1000 dilution, MAP4 at 1:1000 dilution. Secondary anti-mouse antibody 1:32,000 or a goat anti-rabbit at 1:5000. Quantitation as above. Quantitation of RhoA content of PC3 and LNCaP cells by Western blots utilized known concentrations of recombinant prenylated G14V RhoA. The linear range of protein loads and antibody labeling was used. Intracardiac injection of PC3 cells and treatment of injected mice with Y-27632. Alzet osmotic pumps (100TD) were implanted interperitoneally in 30 Rag2-pfp, mice anesthetized with methoxyfluorane and exchanged weekly over a 4-week period. Y-27632, delivering at 80 mg/kg/day in 15 pumps, and 15 pumps contained H 2O. After 2-day recovery, 50 l containing 5 ⫻ 10 5 PC3 cells were injected into the heart with an external puncture in lightly anesthetized animals in accordance with protocols approved by the University of Virginia. Animals, checked daily, that had lost over 25% of their body weight and/or had difficulty in moving, paraplegia or respiratory distress were killed by an overdose of anesthesia. All dead animals were autopsied. The extent of tumor load was scored (Table I). Tissues were fixed in 2% formaldehyde for histological studies. Survival distributions were estimated using the product-limit method of Kaplan and Meier, and the log rank test was used to compare the distributions. Angiogenesis assay. Bovine endothelial cells were cultured in Waymouth’s media plus endothelial growth supplement F12K and heparin. 10 5 cell/ml SFM ⫾ 10 or 25 M Y-27632 was added to 250 l of Matrigel/well in 24 well plates. After 11 h incubation, tube formation was assayed. The number of tubes per field in 4 wells with 5 fields/well, 200 ⫻ magnification, as well as the total number of cells/field, were counted.
RESULTS Morphological Changes Induced by the Rho-Kinase Inhibitor By 1 h, 25 M Y-27632 caused a dramatic change in PC3 cell shape, with frequently branching multiple long cell processes often three to six times, with some up to 10 times, the body length and staining very brightly for longitudinally oriented tubulin (Figs. 1b and 1d), while in untreated cells the microtubules formed a dense, mesh-like network excluding the nuclei, with occasional long processes 24 h after plating (Fig. 1a), but these were generally single extensions (not shown). Processes with microtubules also developed in Y-27632 treated LNCaP cells but were shorter. MAP4 immunolabeling was intense in the cytoplasm, colocalizing with tubulin. In Y-27632-treated cells, MAP4 staining, like tubulin staining, was particularly bright in the long cell processes. Unlike control Swiss 3T3 cells, neither cell line contained prominent fluorescent phalloidin-labeled stress fibers in the presence or absence of Y-27632, nor focal adhesions visualized with vinculin antibody. Following treatment with Y-27632, diffuse pale actin staining remained distributed throughout the cytosol; its distribution at the cell periphery was variable, with frequent discrete bright patches present at multiple points on the cell periphery and at the base of the long processes and within fine filopodia (Fig. 1e).
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FIG. 2. Time-lapse images of PC3 cells treated with 25 M Y-27632 in the presence of 10% FCS. The cell body of cell 1 has moved ⬃150 m over the 5-h period (grid size 170 M), but the tail of the cell has remained stationary and connected to the cell body by a long, thin strand of cytoplasm at 5 h. Cells 3 and 4 are undergoing the same process.
Morphology of PC3 Cells Treated with the MLCK Inhibitor, ML9 PC3 cells treated for 15 h with 50 M ML9 or with 0.1% ETOH diluent were not different in appearance from untreated cells (Fig. 1), except for a slight increase in the number of rounded cells. Treatment with 100 M ML9 for 2 h resulted in a marked rounding up and detachment of cells. Analysis of PC3 Cell Movement, Cell Migration, and Proliferation The generation of Y-27632-induced cell processes was followed by periodically photographing randomly selected cells on microgrid coverslips (Fig. 2). Two manners of cell migration were observed in treated cells. In one case, cells developed lamellopodia on the leading edge and moved forward, leaving a long tail of cytoplasm (Figs. 1b and 1d, and Fig. 2). These cells appeared unable to detach their tails as they moved forward (see cells 1, 3, and 4 in Fig. 2). In the other case, treated cells extended a narrow process on which the cell body moved forward, leaving a tail as the cell body advanced. Once the processes had formed, these cells could be stationary for up to 20 h (the longest time period monitored). In some instances, the cell body would move back and forth along the stable processes. In some images the long tails eventually sprang free and appeared as a refractile blob at the cell periphery, FIG. 1. Human prostatic cancer (PC3) cells grown in the presence of 10% FCS for 17 h without (a) or with (b) 25 M of the Rho-kinase inhibitor Y-27632 or for 15 h with the myosin light chain kinase inhibitor, ML9 (50 M) (c), immunolabeled with an ␣-tubulin
antibody. Higher magnification of (b) stained for tubulin (d) and actin (e). Note that Y-27632, but not ML9, induced multiple long, frequently branching, cell processes in the majority of the cells.
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FIG. 3. Y-27632-induced inhibition by 75% in 1 h of EGF- or bone marrow fibroblast-conditioned media (BMFB-CM)-stimulated migration of PC3 cells determined in trans-well assays. 10 nM EGF alone increased migration, which went to completion by 1 h.
or fragments of the tail were left behind. Untreated moving cells generally had a triangular or ribbon shape, with lamellipodia on the leading edge. We note that these observations, unlike the migration assays performed with Boyden chambers (see below), did not involve directed chemotaxis. Y-27632 (25 M) inhibited by 75% ( p ⬍ 0.0001) both the BMFB-CM and the EGF-stimulated migration of the PC3 cells determined by a transwell assay (Fig. 3). Nonradioactive proliferation assays of LNCaP and PC3 cells treated with 1 to 25 M Y-27632 over 3 days with the PC3 cells followed for up to 5 days showed no significant difference in growth compared to controls (n ⫽ 8 for PC3 cells, n ⫽ 8 for LNCaP cells). Therefore, the Rho-kinase inhibitor is neither cytotoxic, nor does it impede cell division.
mated by comparison with known amounts of recombinant prenylated RhoA, was 22.5 ng/100 g cell protein in PC3 cells and 15.0 ng/100 g LNCaP cell protein. The concentration of RhoA estimated by comparison of the optical density (OD) of RhoA bands within the linear range at the same protein loads was significantly lower in LNCaP cells than in the highly invasive PC3 cells (OD PC3/OD LNCaP ⫽ 1.76 ⫾ 0.2 S.E.M., p ⬍ 0.003, n ⫽ 17 pairs). Overnight treatment with 25 M Y-27632 did not affect the RhoA content of PC3 cells. In both control and treated cells Rho was predominantly in the supernatant following a 100,000g spin, whereas ROK-␣ distributed in both the supernatant and the high-speed spin pellet (Fig. 5b). Homogenates of both PC3 cells and of longitudinal smooth muscle of the rabbit ileum, known to contain the regulatory, M 110 –130 subunit of the smooth muscle myosin phosphatase (SMPP-1M), showed a doublet at 130 kDa when blotted with an antibody which recognizes both the smooth muscle and nonmuscle isoforms of this subunit (Fig. 6c). In Vivo Inhibition of Disseminated Growth of PC3 Cells by Y-27632 The number and size of tumors following intracardiac injection into 30 mice was markedly reduced during and, for a period after, treatment of the 15 mice that received Y-27632 (Table I). Survival of treated animals was also significantly ( p ⫽ 0.01) prolonged (Fig. 6). Median survival is estimated to be 37 days for the control group and 49 days for the treated group. Treatment is estimated to increase median survival by 32%. Assuming constant hazards, treatment is estimated to reduce the risk of death at any fixed timepoint by 67%.
Y-27632 Reduces MLC 20 Phosphorylation Myosin light chain phosphorylation was measured under serum-free conditions, in keeping with the conditions used for migration assays. Following culture with or without 25 M Y-27632 for 24 h, the level of MLC 20 phosphorylation in PC3 cells subsequently stimulated with 10 nM EGF for 2 min was 65% ⫾ 4.1 S.D. in untreated and 37% ⫾ 5.5 S.D. in Y-27632treated cells ( p ⬍ 0.01). A typical Western blot of MLC 20 phosphorylation is shown in Fig. 4. Tubulin Isoforms, RhoA, ROK-␣, and SMPP-1M Content in PC3 Cells: Higher Content of RhoA in the More Invasive PC3 Than LNCaP Cells Neither the total amount nor the stoichiometry of different (-II, -III, and -IV) isoforms of tubulin changed during overnight treatment with 25 M Y-27632 (n ⫽ 3; Fig. 5a). The total Rho content, esti-
FIG. 4. Typical Western blot illustrating the decreased MLC 20 phosphorylation in EGF-stimulated PC3 cells treated with 25 M Y-27632 for 18 h. The singly and doubly phosphorylated (P1 and P2) and unphosphorylated (U) forms of MLC 20 immunolabeled with an antibody specific for phosphorylated Ser 19 (lane b) or a polyclonal antibody to MLC 20, to both the P and U forms (lane c). Lane a is stained with secondary antibody only. The increased MLC 20 phosphorylation elicited by EGF was significantly ( p ⬍ 0.01) reduced in cells pretreated with Y-27632 (25 M). Unphosphorylated and phosphorylated gizzard myosins are shown as controls (the gizzard myosin runs somewhat higher than the non-muscle myosin from the PC3 cells).
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FIG. 5. Western blots for tubulin isoforms, ROK␣, RhoA, and myosin phosphatase in PC3 cells. (a) Neither the total content nor the stoichiometry of different isoforms of tubulin is changed with Y-27632 treatment (identical protein loads with five channels per sample). (b) Following fractionation, RhoA is found predominantly in the supernatant (S). Initial (800g) spin contains nuclei and large cell fragments. ROK-␣ distributes in both S and P (pellet). (c) The smooth and non-muscle phosphatase which runs as a doublet at 130 kDa is present in PC3 cells and in the (control) rabbit ileum smooth muscle. Proteolysis of SMPP-1M accounts for the additional immunoreactive bands.
migration and in vivo disseminated growth of the highly invasive PC3 prostatic carcinoma cells, alters their cell morphology, including the distribution of microtubules, reduces phosphorylation of the regulatory light chain (MLC 20) of myosin II, and inhibits endothelial cell tube formation in Matrigel assays of angiogenesis; (2) the phenotypes induced by Y-27632 and the MLCK inhibitor, ML9, are highly different; and (3) RhoA, Rho-kinase, and the regulatory subunit of myosin phosphatase (SMPP-1M) are present in PC3 cells that, furthermore, contain more RhoA than the less invasive LNCaPs. A major determinant of cell motility and migration is actin activation of the myosin II motor (rev. in 16) that, in both nonmuscle (17) and smooth muscle (see Introduction), is mediated by phosphorylation of the Ser 19 residue of MLC 20 by myosin light chain kinase (MLCK). Inhibition of SMPP-1M that inactivates (dephosphorylates) myosin can also increase MLC 20 phosphorylation and contraction/motility (3, 4, 10; rev. in 1, 9). The small GTPase, RhoA-GTP, activates Rhokinase (ROK-␣ and isoforms, 12, 18 –20) that phosphorylates the regulatory subunit and inhibits the catalytic activity of SMPP-1M (11, 21–23). Conversely, inhibition of Rho-kinase reduces MLC 20 phosphorylation and actomyosin activity (13, 24). Thus Y-27632 inhibits the contractility/motility enhanced by upregulation of the RhoA pathway, such as in malignant cells, without affecting physiological activation of Ca 2⫹calmodulin-activated MLCK.
Y-27632 Inhibits Endothelial Tube Formation Endothelial cell tube formation on Matrigel was inhibited by 10 M Y-27632 (Fig. 7). The mean number of tubes/field in 4 wells with 5 fields per well was 4.2 ⫾ 0.44 SEM in control and 0.6 ⫾ 0.24 ( p ⬍ 0.00001) with 10 M Y-27632, and 0.3 ⫾ 0.13 SEM ( p ⬍ 0.00001 compared to control) with 25 M Y-27632 when compared by Student’s t test. The total average number of cells/field in control, 10 M and 25 M Y-27632 were 177 ⫾ 10, 230 ⫾ 23, and 175 ⫾ 19 SEM, respectively. DISCUSSION The main findings of our study are that (1) the selective Rho-kinase inhibitor, Y-27632, inhibits in vitro
FIG. 6. Kaplan-Meier survival distributions of immunodeficient mice with osmotic pumps implanted in the peritoneal cavity, with 80 mg/kg/day Y-27632 or diluent delivered over a 4-week period. On Day 2, 5 ⫻ 10 5 PC3 cells were injected into the hearts of all mice. Three of the animals during the 4-week treatment period were lost due to anesthesia or surgical complications of weekly pump exchange. The median survival is estimated to be 37 days for the control group, and 49 days for the treated group. Treatment is estimated to increase median survival by 32%. Assuming constant hazards, treatment is estimated to reduce the risk of death at any fixed time point by 67%.
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Estimate of Tumor Load and Distribution at Day 45 Following Intracardiac Injection of PC3 Cells into Control and Y-27632-Treated Mice Control Days survival # of regions Lungs Chest Diaphragm Liver Mandible Legs Spine Kidneys Adrenal Heart Lymph nodes
5 ND
22 ND
26 ND
36 6 ⫹ ⫹⫹
36 3 ⫹⫹⫹⫹ ⫹⫹⫹⫹⫹
⫹⫹ ⫹⫹⫹⫹ ⫹⫹ ⫹
Treated
36 4
36 3
38 4
38 4
43 2
⫹⫹⫹⫹ ⫹⫹⫹
⫹⫹⫹
⫹⫹⫹⫹⫹
⫹⫹⫹⫹ ⫹⫹⫹
⫹⫹
⫹⫹
⫹ ⫹⫹⫹⫹
⫹⫹
⫹⫹⫹⫹⫹
⫹ ⫹⫹
⫹
⫹⫹⫹
⫹
⫹⫹⫹⫹⫹
44 8 ⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹⫹ ⫹⫹
18 ND
41 3
45 3
⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹
⫹ ⫹⫹
Note. The number of “⫹” signs reflects both the number and size of tumors.
Inhibition of Rho-kinase is, therefore, expected to inhibit migration by cancer cells. Indeed, Y-27632, a selective Rho-kinase inhibitor, inhibited transcellular invasion and reduced the extent of local peritoneal metastases by MM1 rat hepatoma (25) and migration and in vivo dissemination of prostate cancer cells (present study). Invasiveness of MM1 rat hepatoma cells is promoted by RhoA and reduced by an inhibitor of MLCK (26, 27). Guanine nucleotide exchange factors (GEFs) that activate RhoA and its upstream trimeric (G␣ 12,13) activators (28) increase metastatic potential and/or are also oncogenic (29), and some human tumors overexpress RhoA (30), findings paralleled by the higher Rho content of the more invasive (PC3) than LNCaP cells (present study). Given the frequent metastasis of prostate cancers to bone, the 75% inhibition by Y-27632 of migration of prostate cancer cells to bone marrow fibroblastconditioned medium indicates the therapeutic potential of Rho-kinase inhibitors. However, signal trans-
FIG. 7. Matrigel gel angiogenesis assay of bovine endothelial cells. 10 M Y-27632 inhibits the formation of endothelial tubes.
duction pathways regulating cell motility vary and, in contrast to the inhibition by Y-27632 of migration of prostate cancer (present study), MM1 cells (25) and vascular smooth muscle (31), Y-27632 accelerates wound healing by migrating normal fibroblasts (32). Therefore, therapeutic kinase inhibitors will have to be individualized to cellular phenotypes. Profound morphological changes, independent of stress fibers, were induced by Y-27632 in prostate cancer cells (Fig. 1) that metamorphosed, within one hour, to develop extremely long, frequently multiple, processes, accompanied by a striking reorganization of microtubules to these processes (Fig. 1). No change in total tubulin or ratio of the -II, -III, and -IV tubulin, isoforms occurred. These processes strongly resembled those induced by Y-27632 in neuroblastoma N1E115 cells (33), by cyclic AMP(cAMP) in LNCaP or PC3 cells (34), and the dendrites growing from melanoma cells during cAMP-induced differentiation (35, 36). It appears Rho-kinase can regulate tubulin organization independently of stress fibers, perhaps by increasing the phosphorylation of microtubule-associated proteins (MAPs) that modulate microtubule stability. At least one MAP, Tau, targets the catalytic phosphatase-1 subunit to microtubules (37), and phosphorylation of a PP1-targeting MAP by Rho-kinase could regulate phosphatase activity in a manner similar to the 110 – 130 kDa subunit of SMPP-1M. MAP4 is present in prostate cancer cells and colocalizes with tubulin (present study), but whether it is phosphorylated by Rho-kinase and, if so, its effect on microtubule stability remain to be determined. MLC 20 phosphorylation, although reduced, remained at significant levels (36%) in Y-27632-treated cells. This incomplete inhibition of “bulk” MLC 20 phosphorylation is unlikely to account by itself for the marked inhibition of cell migration. The lack of effect of
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Y-27632 on the leading edge of moving cells is consistent with this movement being associated with ruffling due to actin reorganization (16, 38) controlled by Rac, rather than RhoA (39). The presence of very long (⬃150 M) and occasionally broken tails of the trailing edge of treated migrating cells (Figs. 1 and 2) suggested that failure of the trailing end to detach, the “deadhesion and retraction” component of cell movement (16, 38), could contribute to the inhibition of migration by Y-27632. This may also involve inhibition of phosphorylation of Rho-kinase substrates other than MLC 20, such as the protein that localizes PP1␦ to focal adhesions (40). The dramatic inhibition of metastases (Table I) by Y-27632 may also have involved blood platelet- and angiogenesis-related mechanisms. Platelet and endothelial cell myosin II is regulated by the same phosphorylation/dephosphorylation mechanisms as smooth muscle, and platelets contain the myosin phosphatase that can be inhibited by Rho-kinase (41, 42). Upstream trimeric G-protein (G␣ 12,13) activators of the RhoA pathway (26) cause shape changes in blood platelets (43) that are inhibited by C3 or Y-27632 (41) and the Rho-kinase inhibitor inhibits platelet secretion associated with aggregation (41). Since hematogenous dissemination of cancer cells is facilitated by deposition of platelet/tumor thrombi in capillaries (44), inhibition of platelet aggregation may also have contributed to the inhibition of metastasis and improved survival by Y-27632. However, the beneficial effects of the drug occurred only during treatment and for some time thereafter, consistent with the in vitro findings that Y-27632 is not cytotoxic. Therefore, we anticipate that maximal therapeutic benefits will be derived by combining, with cytotoxic agents, selective inhibitors, such as Y-27632, of kinases that are overactive specifically in cancer cells, such as Rho-kinase, MAP-kinase and other, Ca-independent kinases (rev. in 45). Studies exploring the benefits of such combined therapies are in progress. ACKNOWLEDGMENTS We thank Dr. Gina Petroni for statistical analysis, Dr. Paul Read for recombinant RhoA protein, Mrs. Akiko Yoshimura of Yoshitomi Pharmaceutical Industries, Ltd. for generous gifts of Y-27632, and gratefully acknowledge Jama Coartney for preparation of the illustrations. This work was supported by Grants PO1 HL48807 and PO1 HL19242 from the NHLBI, and Grant P30 CA44579 from NCI.
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38. Mitchison, T. J., and Cramer, L. P. (1996) Cell 84, 371–379. 39. Hall, A. (1994) Annu. Rev. Cell Biol. 10, 31–54. 40. Villa-Moruzzi, E., Tognarini, M., Cecchini, G., and Marchisio, P. C. (1998) Cell Adhes. Commun. 5, 297–305. 41. Suzuki, Y., Yamamoto, M., Wada, H., Ito, M., Nakano, M., Sasaki, Y., Narumiya, S., Shiku, H., and Nishikawa, M. (1999) Blood 93, 3408 –3417. 42. Klages, B., Brandt, U., Simon, M. I., Schultz, G., and Offermanns, S. (1999) J. Cell Biol. 144, 745–754. 43. Essler, M., Amano, M., Kruse, H.-J., Kaibuchi, K., Weber, P. C., and Aepfelbacher, M. (1998) J. Biol. Chem. 273, 21867–21874. 44. Crissman, J. D., Hatfield, J., Schaldenbrand, M., Sloane, B. F., and Honn, K. V. (1985) Lab. Invest. 53, 470 – 478. 45. Somlyo, A. P. (1999) J. Physiol. 516, 630.
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Rho-Kinase Inhibitor Retards Migration and in Vivo Dissemination of Human Prostate Cancer Cells Avril V. Somlyo,* ,1 Dawn Bradshaw,† Susan Ramos,* Cheryl Murphy,† Charles E. Myers,† and Andrew P. Somlyo* *Department of Molecular Physiology and Biological Physics and †Cancer Center, University of Virginia Health System, Charlottesville, Virginia 22906
Received February 2, 2000
The Rho-kinase inhibitor, Y-27632, inhibited in vitro chemotactic migration to bone marrow fibroblast conditioned media and metastatic growth in immunecompromised mice of highly invasive human prostatic cancer (PC3) cells. Y-27632 also reduced myosin light chain phosphorylation and markedly altered the morphology of cells that developed numerous processes containing microtubules. A strikingly different, rounded phenotype was induced by an inhibitor of myosin light chain kinase, ML9. The M 110 –130 subunit of the myosin phosphatase that is regulated by Rhokinase was present in PC3 cells that contained significantly more RhoA than the less invasive, LNCaP cells. Y-27632 also inhibited angiogenesis as measured by endothelial cell tube formation on Matrigel. We conclude that invasiveness of human prostate cancer is facilitated by the Rho/Rho-kinase pathway, and exploration of selective Rho-kinase inhibitors for limiting invasive progress of prostate cancer is warranted. © 2000 Academic Press
Key Words: Rho-kinase; prostate cancer; metastasis; cell migration; Y-27632; angiogenesis; myosin phosphatase.
of the Rho/Rho-kinase pathway inhibits SMPP-1M and increases actomyosin-based motility (10; rev. in 11, 12), whereas the highly selective Rho-kinase inhibitor, Y-27632 (13), inhibits it. We chose to evaluate the effect of Y-27632 in human prostate cancer cell lines. In a significant number of prostate cancer patients in whom the cancer grows and spreads slowly, watchful observation is a reasonable alternative to surgery or radiation therapy. An orally active drug of low toxicity that further slows the spread of prostate cancer may well make conservative treatment more effective by preventing the development of metastatic disease, commonly to bone. In this paper, we present evidence that Y-27632 may well be such a compound: it blocks tumor cell motility and chemotaxis induced by bone fibroblast conditioned media, as well as intravascularly disseminated growth of PC3 cells in vivo, angiogenesis by endothelial cell tube formation on Matrigel and produces a phenotype that is strikingly different from that induced by the MLCK inhibitor, ML9. Preliminary results of some of these findings have been presented to the American Society of Cell Biology (14). MATERIALS AND METHODS
Abnormal proliferation and migration of cancer cells are both strongly dependent on processes requiring myosin II activity. Non-muscle, like smooth muscle, myosin II is activated by phosphorylation by myosin light chain kinase (MLCK; rev. in 1, 2) and inactivated by dephosphorylation of its regulatory light chain (MLC 20 ; rev. in 1). MLC 20 phosphorylation can also be increased by inhibiting (3, 4) the protein phosphatase (SMPP-1M) that normally dephosphorylates myosin II (5– 8; rev. in 9). Activation 1 To whom correspondence should be addressed at University of Virginia Health System, Department of Molecular Physiology and Biological Physics, P. O. Box 10011, Charlottesville, Virginia 229060011. Fax: (804) 982-1616. E-mail: [email protected].
0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
Cell culture. PC3 and LNCaP human prostate cancer cells (American Type Culture Collection) were cultured in RPMI medium 1640 supplemented with 10% fetal bovine serum (FBS), plus 100 g/ml streptomycin and 100 units/ml penicillin. Cell proliferation assay. Cell proliferation in the presence or absence of 25 M Y-27632 was assayed using the nonradioactive cell proliferation assay (Promega Corp., Madison, WI), 5,000 cells/well, which is based on the ability of viable cells to reduce tetrazolium salt to a colored formazin product. Cell migration and cell movement. Cell migration was assayed in Boyden chambers [8.0 m pore size polyethylene terephthalate (PET) membrane, FALCON cell culture insert (Becton-Dickinson)]. 200 l of 4.0 ⫻ 10 4 PC3 cells/ml ⫾ 25 M Y-27632 in serum-free media (SFM) were added to the upper chamber. Transwells were incubated overnight at 37°C with 500 l serum-free RPMI in the lower chamber. The top chambers containing the cells were then
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transferred to new lower chambers with or without 500 l BMFB-CM or 10 nM epidermal growth factor (EGF) in serum-free RPMI and incubated for 1 h. Cells on the inside of the transwell inserts were removed with a cotton swab, fixed, and stained with crystal violet. Five regions were randomly chosen from each insert and the number of cells counted. In controls performed to test for the possibility that treatment affected adherence to the PET membrane, adherence of control and Y-27632-treated (25 M) cells (63 ⫾ 10.6 S.D. and 66 ⫾ 5.5. S.D. cells/field, respectively) was not different when the experiment was repeated using 3.0 m pore size PET membranes, which preclude transmigration. BMFB-CM was obtained by growing CRL7298 cells to confluence with FBS, washing 3 times in SFM, incubating the cells in 5 ml of SFM for 18 –24 h, centrifuging at 1,500 rpm and used full strength in the transwell. For time-lapse images, cells were plated at 2 ⫻ 10 4 cells/ml on gridded coverslips and incubated overnight. Randomly selected areas were photographed every hour. MLC 20 phosphorylation. PC3 cells were plated at 3 ⫻ 10 6 or 6 ⫻ 10 5 cells/mL in SFM, and incubated overnight to mimic the migration assay conditions; subsequently 25 M Y-27632 was added to half the dishes for 18 h. In some cases the MLCK inhibitor ML9 was added to control and Y-27632-treated cells for 2 h prior to stimulation with 10 mM EGF or BMFB-CM for 2 min, followed by ice-cold 10% TCA. Cells were scraped and cell pellets washed three times in absolute acetone with 10 mM dithiothreitol (DTT), and spun at 11,000 rpm for 10 min. The air-dried pellet was resuspended in urea buffer containing 6.7 M urea, 20 mM Tris base, 22 mM glycine, and 10 mM DTT and unphosphorylated (U) and singly and doubly (P1 and P2) phosphorylated forms of MLC 20 separated on onedimensional isoelectric focusing acrylamide gels (ampholytes pH 4.5–5.4) using a five-well comb and run on a Hoefer minigel system. Gels were transferred to nitrocellulose and Western blot analysis was carried out using the mini-Protein II multi-screen apparatus (2.5 mm diameter gel lanes in triplicate). Lane 1, secondary antibody only; lane 2, primary polyclonal antibodies specific for phosphorylated Ser 19 of MLC 20 (15), a gift of Dr. F. Matsumura; lane 3, 1:2,000 dilution of a polyclonal antibody to MLC 20, a gift of Dr. K. Kamm, reactive with both P1, P2, and U forms of MLC 20. A donkey antirabbit HRP secondary antibody was used at 1:5000 dilution and the reactive bands detected by ECL and quantitated on a Bio-Rad GS670 densitometer. Control lanes included thiophosphorylated and nonphosphorylated turkey gizzard myosin. Percent phosphorylation ⫽ P1 ⫹ P2/(U ⫹ P1 ⫹ P2) ⫻ 100. Protein concentrations were determined by the Bradford assay. Immunolabeling. Cells were fixed with 3% paraformaldehyde in PBS or 0.5% glutaraldehyde in PIPES saline, permeabilized with 0.5% Triton X-100, rinsed and blocked for 30 min in 20% goat serum. The following antibodies were used: ␣-tubulin mouse monoclonal (Sigma) at 1:500 dilution, monoclonal anti-human vinculin (Sigma) at 1:750 dilution, FITC or TRITC goat anti-mouse secondary antibodies at 1:200 dilution. Human MAP4 (microtubule-associated protein) mouse monoclonal antibody at 1:500 dilution. Actin was stained with rhodamine- or fluorescein-labeled phalloidin at a 1:1000 dilution. Cells were washed and mounted in 1 mg/ml 1,4 diazabicylclo[2.2.2]octane. Western blotting for detection of RhoA, Rho-kinase, tubulin isoforms, myosin phosphatase, MAP4. Cells were scraped in lysis buffer (50 mM Tris, pH 7.5, 150 NaCl, 2 mM EDTA, 1% Triton X-100, protease inhibitors AEBSF, 1 mM, leupeptin, 20 g/ml, and aprotinin, 20 g/ml) and protein concentrations assayed. Proteins in Laemmli sample buffer were separated by SDS-PAGE, and transferred to nitrocellulose or polyvinylidene difluoride membranes, as previously described (10). The following antibodies were used: AA-2, which recognizes all  isoforms of tubulin, and -II, -III, and -IV, at 1:1000 dilution (gift of Dr. T. Frankfurter); ␣-tubulin (Sigma) at 1:10,000; polyclonal regulatory subunit of smooth muscle myosin
phosphatase, which recognizes the muscle and nonmuscle isoform at 1:2000, gift of Dr. D. Hartshorne, monoclonal anti-RhoA generated to amino acids 120 –150 at 1:5000 dilution, Rho-kinase at 1:1000 dilution, MAP4 at 1:1000 dilution. Secondary anti-mouse antibody 1:32,000 or a goat anti-rabbit at 1:5000. Quantitation as above. Quantitation of RhoA content of PC3 and LNCaP cells by Western blots utilized known concentrations of recombinant prenylated G14V RhoA. The linear range of protein loads and antibody labeling was used. Intracardiac injection of PC3 cells and treatment of injected mice with Y-27632. Alzet osmotic pumps (100TD) were implanted interperitoneally in 30 Rag2-pfp, mice anesthetized with methoxyfluorane and exchanged weekly over a 4-week period. Y-27632, delivering at 80 mg/kg/day in 15 pumps, and 15 pumps contained H 2O. After 2-day recovery, 50 l containing 5 ⫻ 10 5 PC3 cells were injected into the heart with an external puncture in lightly anesthetized animals in accordance with protocols approved by the University of Virginia. Animals, checked daily, that had lost over 25% of their body weight and/or had difficulty in moving, paraplegia or respiratory distress were killed by an overdose of anesthesia. All dead animals were autopsied. The extent of tumor load was scored (Table I). Tissues were fixed in 2% formaldehyde for histological studies. Survival distributions were estimated using the product-limit method of Kaplan and Meier, and the log rank test was used to compare the distributions. Angiogenesis assay. Bovine endothelial cells were cultured in Waymouth’s media plus endothelial growth supplement F12K and heparin. 10 5 cell/ml SFM ⫾ 10 or 25 M Y-27632 was added to 250 l of Matrigel/well in 24 well plates. After 11 h incubation, tube formation was assayed. The number of tubes per field in 4 wells with 5 fields/well, 200 ⫻ magnification, as well as the total number of cells/field, were counted.
RESULTS Morphological Changes Induced by the Rho-Kinase Inhibitor By 1 h, 25 M Y-27632 caused a dramatic change in PC3 cell shape, with frequently branching multiple long cell processes often three to six times, with some up to 10 times, the body length and staining very brightly for longitudinally oriented tubulin (Figs. 1b and 1d), while in untreated cells the microtubules formed a dense, mesh-like network excluding the nuclei, with occasional long processes 24 h after plating (Fig. 1a), but these were generally single extensions (not shown). Processes with microtubules also developed in Y-27632 treated LNCaP cells but were shorter. MAP4 immunolabeling was intense in the cytoplasm, colocalizing with tubulin. In Y-27632-treated cells, MAP4 staining, like tubulin staining, was particularly bright in the long cell processes. Unlike control Swiss 3T3 cells, neither cell line contained prominent fluorescent phalloidin-labeled stress fibers in the presence or absence of Y-27632, nor focal adhesions visualized with vinculin antibody. Following treatment with Y-27632, diffuse pale actin staining remained distributed throughout the cytosol; its distribution at the cell periphery was variable, with frequent discrete bright patches present at multiple points on the cell periphery and at the base of the long processes and within fine filopodia (Fig. 1e).
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FIG. 2. Time-lapse images of PC3 cells treated with 25 M Y-27632 in the presence of 10% FCS. The cell body of cell 1 has moved ⬃150 m over the 5-h period (grid size 170 M), but the tail of the cell has remained stationary and connected to the cell body by a long, thin strand of cytoplasm at 5 h. Cells 3 and 4 are undergoing the same process.
Morphology of PC3 Cells Treated with the MLCK Inhibitor, ML9 PC3 cells treated for 15 h with 50 M ML9 or with 0.1% ETOH diluent were not different in appearance from untreated cells (Fig. 1), except for a slight increase in the number of rounded cells. Treatment with 100 M ML9 for 2 h resulted in a marked rounding up and detachment of cells. Analysis of PC3 Cell Movement, Cell Migration, and Proliferation The generation of Y-27632-induced cell processes was followed by periodically photographing randomly selected cells on microgrid coverslips (Fig. 2). Two manners of cell migration were observed in treated cells. In one case, cells developed lamellopodia on the leading edge and moved forward, leaving a long tail of cytoplasm (Figs. 1b and 1d, and Fig. 2). These cells appeared unable to detach their tails as they moved forward (see cells 1, 3, and 4 in Fig. 2). In the other case, treated cells extended a narrow process on which the cell body moved forward, leaving a tail as the cell body advanced. Once the processes had formed, these cells could be stationary for up to 20 h (the longest time period monitored). In some instances, the cell body would move back and forth along the stable processes. In some images the long tails eventually sprang free and appeared as a refractile blob at the cell periphery, FIG. 1. Human prostatic cancer (PC3) cells grown in the presence of 10% FCS for 17 h without (a) or with (b) 25 M of the Rho-kinase inhibitor Y-27632 or for 15 h with the myosin light chain kinase inhibitor, ML9 (50 M) (c), immunolabeled with an ␣-tubulin
antibody. Higher magnification of (b) stained for tubulin (d) and actin (e). Note that Y-27632, but not ML9, induced multiple long, frequently branching, cell processes in the majority of the cells.
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FIG. 3. Y-27632-induced inhibition by 75% in 1 h of EGF- or bone marrow fibroblast-conditioned media (BMFB-CM)-stimulated migration of PC3 cells determined in trans-well assays. 10 nM EGF alone increased migration, which went to completion by 1 h.
or fragments of the tail were left behind. Untreated moving cells generally had a triangular or ribbon shape, with lamellipodia on the leading edge. We note that these observations, unlike the migration assays performed with Boyden chambers (see below), did not involve directed chemotaxis. Y-27632 (25 M) inhibited by 75% ( p ⬍ 0.0001) both the BMFB-CM and the EGF-stimulated migration of the PC3 cells determined by a transwell assay (Fig. 3). Nonradioactive proliferation assays of LNCaP and PC3 cells treated with 1 to 25 M Y-27632 over 3 days with the PC3 cells followed for up to 5 days showed no significant difference in growth compared to controls (n ⫽ 8 for PC3 cells, n ⫽ 8 for LNCaP cells). Therefore, the Rho-kinase inhibitor is neither cytotoxic, nor does it impede cell division.
mated by comparison with known amounts of recombinant prenylated RhoA, was 22.5 ng/100 g cell protein in PC3 cells and 15.0 ng/100 g LNCaP cell protein. The concentration of RhoA estimated by comparison of the optical density (OD) of RhoA bands within the linear range at the same protein loads was significantly lower in LNCaP cells than in the highly invasive PC3 cells (OD PC3/OD LNCaP ⫽ 1.76 ⫾ 0.2 S.E.M., p ⬍ 0.003, n ⫽ 17 pairs). Overnight treatment with 25 M Y-27632 did not affect the RhoA content of PC3 cells. In both control and treated cells Rho was predominantly in the supernatant following a 100,000g spin, whereas ROK-␣ distributed in both the supernatant and the high-speed spin pellet (Fig. 5b). Homogenates of both PC3 cells and of longitudinal smooth muscle of the rabbit ileum, known to contain the regulatory, M 110 –130 subunit of the smooth muscle myosin phosphatase (SMPP-1M), showed a doublet at 130 kDa when blotted with an antibody which recognizes both the smooth muscle and nonmuscle isoforms of this subunit (Fig. 6c). In Vivo Inhibition of Disseminated Growth of PC3 Cells by Y-27632 The number and size of tumors following intracardiac injection into 30 mice was markedly reduced during and, for a period after, treatment of the 15 mice that received Y-27632 (Table I). Survival of treated animals was also significantly ( p ⫽ 0.01) prolonged (Fig. 6). Median survival is estimated to be 37 days for the control group and 49 days for the treated group. Treatment is estimated to increase median survival by 32%. Assuming constant hazards, treatment is estimated to reduce the risk of death at any fixed timepoint by 67%.
Y-27632 Reduces MLC 20 Phosphorylation Myosin light chain phosphorylation was measured under serum-free conditions, in keeping with the conditions used for migration assays. Following culture with or without 25 M Y-27632 for 24 h, the level of MLC 20 phosphorylation in PC3 cells subsequently stimulated with 10 nM EGF for 2 min was 65% ⫾ 4.1 S.D. in untreated and 37% ⫾ 5.5 S.D. in Y-27632treated cells ( p ⬍ 0.01). A typical Western blot of MLC 20 phosphorylation is shown in Fig. 4. Tubulin Isoforms, RhoA, ROK-␣, and SMPP-1M Content in PC3 Cells: Higher Content of RhoA in the More Invasive PC3 Than LNCaP Cells Neither the total amount nor the stoichiometry of different (-II, -III, and -IV) isoforms of tubulin changed during overnight treatment with 25 M Y-27632 (n ⫽ 3; Fig. 5a). The total Rho content, esti-
FIG. 4. Typical Western blot illustrating the decreased MLC 20 phosphorylation in EGF-stimulated PC3 cells treated with 25 M Y-27632 for 18 h. The singly and doubly phosphorylated (P1 and P2) and unphosphorylated (U) forms of MLC 20 immunolabeled with an antibody specific for phosphorylated Ser 19 (lane b) or a polyclonal antibody to MLC 20, to both the P and U forms (lane c). Lane a is stained with secondary antibody only. The increased MLC 20 phosphorylation elicited by EGF was significantly ( p ⬍ 0.01) reduced in cells pretreated with Y-27632 (25 M). Unphosphorylated and phosphorylated gizzard myosins are shown as controls (the gizzard myosin runs somewhat higher than the non-muscle myosin from the PC3 cells).
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FIG. 5. Western blots for tubulin isoforms, ROK␣, RhoA, and myosin phosphatase in PC3 cells. (a) Neither the total content nor the stoichiometry of different isoforms of tubulin is changed with Y-27632 treatment (identical protein loads with five channels per sample). (b) Following fractionation, RhoA is found predominantly in the supernatant (S). Initial (800g) spin contains nuclei and large cell fragments. ROK-␣ distributes in both S and P (pellet). (c) The smooth and non-muscle phosphatase which runs as a doublet at 130 kDa is present in PC3 cells and in the (control) rabbit ileum smooth muscle. Proteolysis of SMPP-1M accounts for the additional immunoreactive bands.
migration and in vivo disseminated growth of the highly invasive PC3 prostatic carcinoma cells, alters their cell morphology, including the distribution of microtubules, reduces phosphorylation of the regulatory light chain (MLC 20) of myosin II, and inhibits endothelial cell tube formation in Matrigel assays of angiogenesis; (2) the phenotypes induced by Y-27632 and the MLCK inhibitor, ML9, are highly different; and (3) RhoA, Rho-kinase, and the regulatory subunit of myosin phosphatase (SMPP-1M) are present in PC3 cells that, furthermore, contain more RhoA than the less invasive LNCaPs. A major determinant of cell motility and migration is actin activation of the myosin II motor (rev. in 16) that, in both nonmuscle (17) and smooth muscle (see Introduction), is mediated by phosphorylation of the Ser 19 residue of MLC 20 by myosin light chain kinase (MLCK). Inhibition of SMPP-1M that inactivates (dephosphorylates) myosin can also increase MLC 20 phosphorylation and contraction/motility (3, 4, 10; rev. in 1, 9). The small GTPase, RhoA-GTP, activates Rhokinase (ROK-␣ and isoforms, 12, 18 –20) that phosphorylates the regulatory subunit and inhibits the catalytic activity of SMPP-1M (11, 21–23). Conversely, inhibition of Rho-kinase reduces MLC 20 phosphorylation and actomyosin activity (13, 24). Thus Y-27632 inhibits the contractility/motility enhanced by upregulation of the RhoA pathway, such as in malignant cells, without affecting physiological activation of Ca 2⫹calmodulin-activated MLCK.
Y-27632 Inhibits Endothelial Tube Formation Endothelial cell tube formation on Matrigel was inhibited by 10 M Y-27632 (Fig. 7). The mean number of tubes/field in 4 wells with 5 fields per well was 4.2 ⫾ 0.44 SEM in control and 0.6 ⫾ 0.24 ( p ⬍ 0.00001) with 10 M Y-27632, and 0.3 ⫾ 0.13 SEM ( p ⬍ 0.00001 compared to control) with 25 M Y-27632 when compared by Student’s t test. The total average number of cells/field in control, 10 M and 25 M Y-27632 were 177 ⫾ 10, 230 ⫾ 23, and 175 ⫾ 19 SEM, respectively. DISCUSSION The main findings of our study are that (1) the selective Rho-kinase inhibitor, Y-27632, inhibits in vitro
FIG. 6. Kaplan-Meier survival distributions of immunodeficient mice with osmotic pumps implanted in the peritoneal cavity, with 80 mg/kg/day Y-27632 or diluent delivered over a 4-week period. On Day 2, 5 ⫻ 10 5 PC3 cells were injected into the hearts of all mice. Three of the animals during the 4-week treatment period were lost due to anesthesia or surgical complications of weekly pump exchange. The median survival is estimated to be 37 days for the control group, and 49 days for the treated group. Treatment is estimated to increase median survival by 32%. Assuming constant hazards, treatment is estimated to reduce the risk of death at any fixed time point by 67%.
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Estimate of Tumor Load and Distribution at Day 45 Following Intracardiac Injection of PC3 Cells into Control and Y-27632-Treated Mice Control Days survival # of regions Lungs Chest Diaphragm Liver Mandible Legs Spine Kidneys Adrenal Heart Lymph nodes
5 ND
22 ND
26 ND
36 6 ⫹ ⫹⫹
36 3 ⫹⫹⫹⫹ ⫹⫹⫹⫹⫹
⫹⫹ ⫹⫹⫹⫹ ⫹⫹ ⫹
Treated
36 4
36 3
38 4
38 4
43 2
⫹⫹⫹⫹ ⫹⫹⫹
⫹⫹⫹
⫹⫹⫹⫹⫹
⫹⫹⫹⫹ ⫹⫹⫹
⫹⫹
⫹⫹
⫹ ⫹⫹⫹⫹
⫹⫹
⫹⫹⫹⫹⫹
⫹ ⫹⫹
⫹
⫹⫹⫹
⫹
⫹⫹⫹⫹⫹
44 8 ⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹⫹ ⫹⫹
18 ND
41 3
45 3
⫹⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹
⫹ ⫹⫹
Note. The number of “⫹” signs reflects both the number and size of tumors.
Inhibition of Rho-kinase is, therefore, expected to inhibit migration by cancer cells. Indeed, Y-27632, a selective Rho-kinase inhibitor, inhibited transcellular invasion and reduced the extent of local peritoneal metastases by MM1 rat hepatoma (25) and migration and in vivo dissemination of prostate cancer cells (present study). Invasiveness of MM1 rat hepatoma cells is promoted by RhoA and reduced by an inhibitor of MLCK (26, 27). Guanine nucleotide exchange factors (GEFs) that activate RhoA and its upstream trimeric (G␣ 12,13) activators (28) increase metastatic potential and/or are also oncogenic (29), and some human tumors overexpress RhoA (30), findings paralleled by the higher Rho content of the more invasive (PC3) than LNCaP cells (present study). Given the frequent metastasis of prostate cancers to bone, the 75% inhibition by Y-27632 of migration of prostate cancer cells to bone marrow fibroblastconditioned medium indicates the therapeutic potential of Rho-kinase inhibitors. However, signal trans-
FIG. 7. Matrigel gel angiogenesis assay of bovine endothelial cells. 10 M Y-27632 inhibits the formation of endothelial tubes.
duction pathways regulating cell motility vary and, in contrast to the inhibition by Y-27632 of migration of prostate cancer (present study), MM1 cells (25) and vascular smooth muscle (31), Y-27632 accelerates wound healing by migrating normal fibroblasts (32). Therefore, therapeutic kinase inhibitors will have to be individualized to cellular phenotypes. Profound morphological changes, independent of stress fibers, were induced by Y-27632 in prostate cancer cells (Fig. 1) that metamorphosed, within one hour, to develop extremely long, frequently multiple, processes, accompanied by a striking reorganization of microtubules to these processes (Fig. 1). No change in total tubulin or ratio of the -II, -III, and -IV tubulin, isoforms occurred. These processes strongly resembled those induced by Y-27632 in neuroblastoma N1E115 cells (33), by cyclic AMP(cAMP) in LNCaP or PC3 cells (34), and the dendrites growing from melanoma cells during cAMP-induced differentiation (35, 36). It appears Rho-kinase can regulate tubulin organization independently of stress fibers, perhaps by increasing the phosphorylation of microtubule-associated proteins (MAPs) that modulate microtubule stability. At least one MAP, Tau, targets the catalytic phosphatase-1 subunit to microtubules (37), and phosphorylation of a PP1-targeting MAP by Rho-kinase could regulate phosphatase activity in a manner similar to the 110 – 130 kDa subunit of SMPP-1M. MAP4 is present in prostate cancer cells and colocalizes with tubulin (present study), but whether it is phosphorylated by Rho-kinase and, if so, its effect on microtubule stability remain to be determined. MLC 20 phosphorylation, although reduced, remained at significant levels (36%) in Y-27632-treated cells. This incomplete inhibition of “bulk” MLC 20 phosphorylation is unlikely to account by itself for the marked inhibition of cell migration. The lack of effect of
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Y-27632 on the leading edge of moving cells is consistent with this movement being associated with ruffling due to actin reorganization (16, 38) controlled by Rac, rather than RhoA (39). The presence of very long (⬃150 M) and occasionally broken tails of the trailing edge of treated migrating cells (Figs. 1 and 2) suggested that failure of the trailing end to detach, the “deadhesion and retraction” component of cell movement (16, 38), could contribute to the inhibition of migration by Y-27632. This may also involve inhibition of phosphorylation of Rho-kinase substrates other than MLC 20, such as the protein that localizes PP1␦ to focal adhesions (40). The dramatic inhibition of metastases (Table I) by Y-27632 may also have involved blood platelet- and angiogenesis-related mechanisms. Platelet and endothelial cell myosin II is regulated by the same phosphorylation/dephosphorylation mechanisms as smooth muscle, and platelets contain the myosin phosphatase that can be inhibited by Rho-kinase (41, 42). Upstream trimeric G-protein (G␣ 12,13) activators of the RhoA pathway (26) cause shape changes in blood platelets (43) that are inhibited by C3 or Y-27632 (41) and the Rho-kinase inhibitor inhibits platelet secretion associated with aggregation (41). Since hematogenous dissemination of cancer cells is facilitated by deposition of platelet/tumor thrombi in capillaries (44), inhibition of platelet aggregation may also have contributed to the inhibition of metastasis and improved survival by Y-27632. However, the beneficial effects of the drug occurred only during treatment and for some time thereafter, consistent with the in vitro findings that Y-27632 is not cytotoxic. Therefore, we anticipate that maximal therapeutic benefits will be derived by combining, with cytotoxic agents, selective inhibitors, such as Y-27632, of kinases that are overactive specifically in cancer cells, such as Rho-kinase, MAP-kinase and other, Ca-independent kinases (rev. in 45). Studies exploring the benefits of such combined therapies are in progress. ACKNOWLEDGMENTS We thank Dr. Gina Petroni for statistical analysis, Dr. Paul Read for recombinant RhoA protein, Mrs. Akiko Yoshimura of Yoshitomi Pharmaceutical Industries, Ltd. for generous gifts of Y-27632, and gratefully acknowledge Jama Coartney for preparation of the illustrations. This work was supported by Grants PO1 HL48807 and PO1 HL19242 from the NHLBI, and Grant P30 CA44579 from NCI.
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