Cell Biology International Reports, Vol. 16, No. 9, 1992
THE EFFECTS OF STAUROSPORINE AND OKADAIC ACID ON BABY H A M S T E R KIDNEY FIBROBLAST C E L L ADHESION. LAKHDAR GASMI and ADAM CURTIS Department of Cell Biology, Glasgow University, Glasgow G12 8QQ. ABSTRACT The effects of staurosporine, a potent protein kinase C inhibitor, and okadaic acid, a non-TPA tumour promoter, on the adhesion o f BHK fibroblast were investigated. Staurosporine at 2.5 and 5 BM was found to stimulate a gradual increase in BHK cell adhesion as well as spreading in 3% serumcontaining medium. An increase of approximately 27% over the control value was found at 5 BM concentration in 20 minutes. No such effect was seen in serum-free conditions. Staurosporine at 5BM, enhanced BHK cell-cell adhesion in 3% serum and in serum-free conditions. Okadaic acid, a phosphatase inhibitor, at concentrations between 0.25 and 1 ~tg/ml, was found to inhibit BHK cell-substratum adhesion and spreading. The inhibitory effect was time and concentration dependent. These findings suggest that protein kinase C might be involved in the mechanism(s) controlling BHK cell attachment.
INTRODUCTION Protein kinase C plays a crucial role in signal transduction for a variety of biologically active substances which activate cellular functions and proliferation (Nishizuka, 1984). Like many cellular functions, it is becoming certain that cell adhesion, is regulated by the action of protein kinases (Danilov and Juliano 1989). Work on white blood cells and platelets has shown that their adhesion is conlzolled by series of activation events, in particular those involving the products of fatty acid metabolism and the protein kinase C pathway (Flower, 1975; Gordon, 1980; Nishizuka, 1984; Webster et al., 1986). In addition to work on myeloid cells, the adhesion of the cells of sponge Microciona prolifera cells were shown to be activated using agents which mobilize calcium and activate protein kinase C (Weissmann et al., 1986). Nagao et el., (1989) presented data showing a correlation between an increase in protein kinase C activity and formation of cell-cell contacts in a human carcinoma cell line. It was also demonstrated that Chinese hamster ovary cells treated with phorbol-12-myristate-13-acetate produced a remarkable increase in their ability to adhere to fibronectin (Danilov and Juliano 1989), probably due to the activation of protein kinase C and the subsequent phosphorylation of target proteins. In order to examine the role of protein kinase C in cell adhesion, we have examined the effects of staurosporine, a potent protein kinase C inhibitor (Tamaoki et aL, 1986),
© 1992 Academic Press Ltd
Cell Biology International Reports, VoL 16, No. 9, 1992
and okadaic acid, a non-TPA tumour promoter (Suganima, et al., 1988) and a phosphatase inhibitor (Haystead et al., 1989), on the adhesion and spreading of BHK fibroblasts. In the present report, we show that staurosporine stimulated cell adhesion and spreading while okadaic acid suppressed both adhesion and spreading. MATERIALS AND M E T H O D S
CELLS Baby Hamster Kidney cells (BHK21 C13) were supplied frozen from departmental stocks and recultured in BHK21 culture medium (Glasgow modified-MEM supplemented with 0.22% bicarbonate, 10% calf serum, 10% tryptose broth, 2.85 mM glutamine and antibiotics) in 75 cm 2 polystyrene tissue culture flasks (Bibby, Stone, Staffordshire ST15 OSA) in 5% CO2 atmosphere. BHK cells were trypsinised to provide cell suspensions using trypsin-versene at a trypsin activity of 250 BAEE unit per ml in 0.5 mM EDTA in Ca2÷-Mg2+-free Hanks saline for 5 minutes at 37oc. The tryptic activity was stopped by addition of either 5 ml 3% serum Ham's F-10 medium (in those experiments in which cells were tested for their adhesion in the presence of serum) or 5ml serum-free Ham's F-10 medium containing 25 gg/ml leupeptin (in experiments designed to test cell adhesion in serumfree conditions). Then, the cells were aspirated, spun at 1500 rpm. and the pellet resuspended in the appropriate medium at the required cell concentration. 3% serum Ham's F-10 medium, is Hepes buffered nutrient mixture Ham's F-10 supplemented with 3% foetal calf serum (Flow Laboratories, Rickmansworth, WD3 1PQ), 2.85 mM glutamine, antibiotics and 250 ng/ml insulin, 250 ng/ml transferrin and 0.25 ng/ml selenite. The cell viability was determined by staining the cell populations with trypan blue (Mishell and Shiigi, 1980). CULTURE DISHES 33 mm diameter polystyrene tissue culture (PTC) dishes were obtained from Falcon (Becton Dickinson U.K. Ltd) and Nunclon (PB. 280, Roskilde, Denmark). REAGENTS. Concentrate (xl0) nutrient mixture Ham's F-10 (Gibco Ltd, Paisley Scotland); Leupeptin, staurosporine, okadaic acid (Sigma Chemical Company Ltd, Poole England). Stock solutions of the drugs were made up freshly in dimethyl sulfoxide and corresponding amounts were added to controls. MEASUREMENT OF CELL ADHESION CeU-substratum adhesion assay In order to measure cell-substratum adhesion, 0.2x10 6 BHK cells were added to each
Cell Biology lnternational Reports, Vol. 16, No. 9, 1992
33 mm diameter polystyrene tissue culture dish or glass coverslip, and incubated for 20 mins (unless otherwise indicated) at 37°C. The cells that had not adhered during this period were washed off gently with Hepes saline. The cells adherent to culture dishes or glass coverslips were fixed in formal saline, stained in 0.1% Kenacid blue and washed with water. The number of cells adherent in each of 30 standard counting areas from three replicates (unless otherwise stated) was then counted using a microscope to detect the cells and an image analyzing system based on a video camera, a Matrox 512 frame grabber (Matrox Electronic Systems Ltd. Quebec, Canada) and Semper 6P software (Synoptics, Cambridge) running on a Compaq 386 computer to count the cells. The results are expressed in terms of the number of cells adhering per cm 2. Cell-cell adhesion assay Aggregation studies were made using a Couette viscometer as described previously by Curtis, (1969,1973). Briefly, 1 ml of 1 x 106 BHK cells were added to the rotating cup of the Couette viscometer at 37oc with a constant shear of 16 per second. Samples were removed at regular intervals and the total number of cells singly or in aggregate per volume from duplicate samples were counted on a haemocytometer using phase contrast microscopy. The data were plotted as incubation time versus logarithm of the number of ceils of all classes counted at time 't' (No, t), divided by the number of cells at the starting time (No,0) (Curtis, 1969). The slope of these curves is proportional to collision efficiency, ie adhesion. SPREADING ASSAY The spreading assay was performed as described by Curtis and McMurray (1986). The area of attached cells was measured with the image analysing system described above. The spread area of between 100 to 200 cells were measured for each experimental condition from at least two replicates. The mean spreading area per cell is expressed in lxm2.
RESULTS THE EFFECT OF STAUROSPORINE ON BHK CELL ADHESION In this assay, BHK cells were first preincubated in Universal bottles for 10 mins at 37oC with two different concentrations of staurosporine (2.5 and 5~tM). Then, the cells were plated into polystyrene dishes containing glass coverslips and adherent cells were counted after 20 mins incubation at 37°C. Staurosporine enhanced BHK cell adhesion and cell spreading (Fig.l&2) in a 3% serum-containing medium. The adhesion and spreading were increased by
Cell Biology International Reports, Vol. 16, No. 9, 1992
a p p r o x i m a t e l y 2 7 % a n d 4 8 % r e s p e c t i v e l y o v e r the c o n t r o l v a l u e s w h e n c e l l s w e r e t r e a t e d w i t h 5 NM s t a u r o s p o r i n e .
30000 150 ¢.q
:~ 10000 r..)
1. The effect of staurosporine on F i g u r e 2. The effect of staurosporine on BHI< BHK cell adhesion in 3% serum-containing cell spreading in 3% serum-containing medium. medium. The spreading area was measured after 20 min., The adhesion was measured after 20 mins incubation at 37oc. incubation at 37°C. Mean spreading area of ca. 200 cells for eact Means of 30 standard observations from three sample. replicate experiment. Error bars represent one standard deviation. Both means differ significantly from the control Both means differ significandy from the control 7