- Email: [email protected]
Cisplatin inhibits folic acid chemotaxis and phagocytotic functions in dictyostelium discoideum
Cell Biology International 1999, Vol. 23, No. 3, 227–233 Article No. cbir.1998.0335, available online at http://www.idealibrary.com on
CISPLATIN INHIBITS FOLIC ACID CHEMOTAXIS AND PHAGOCYTOTIC FUNCTIONS IN DICTYOSTELIUM DISCOIDEUM T. B. K. REDDY* and S. CHATTERJEE School of Life Sciences, Jawaharlal Nehru University, New Delhi-110067, India Received 23 September 1998, accepted 25 November 1998
Administration of 100 and 200 g/ml of cisplatin [cis-diammine dichloro platinum (II)] for 1 h to growing Dictyostelium discoideum cells severely affects folic acid chemotaxis and phagocytotic function in this organism. Following cisplatin treatment, cells show a much lower uptake of FITC labelled bacteria and a reduced plaque forming ability when plated on Eschericia coli seeded normal agar. Folic acid chemotaxis and folate deaminase activity are greatly inhibited in cisplatin-treated Dictyostelium cells. SDS-PAGE analysis shows a greater association of actin and myosin with the cell cortex of treated cells. These results have been discussed in relation to 1999 Academic Press cisplatin’s known ability to raise the levels of cytosolic calcium. K: cisplatin; dictyostelium; chemotaxis; phagocytosis.
INTRODUCTION The cellular slime mould Dictyostelium discoideum, is a simple soil dwelling eucaryotic microbe which feeds on bacteria and yeasts, continuing to grow as long as food is available. Growing Dictyostelium cells exhibit phagocytosis, the uptake of substrate particles from the external environment into cell in plasma membrane derived vesicles. This constitutes the primary mode of nutrient uptake of this organism. Growing Dictyostelium cells sense, and then move towards the folic acid source generally released by its bacterial prey into its surroundings. Folate deaminase produced by Dictyostelium amoebae help in the establishment of a folic acid gradient and which facilitates chemotaxis towards the increasing source of folic acid (Pan et al., 1972; Bonner et al., 1979). Dictyostelium cell membranes contain different cell surface receptors which facilitate the binding of substrate particles (Vogel et al., 1980). The binding of prey particles trigger a transmembrane signal that leads to the rapid *To whom correspondence should be addressed: Dr T. B. K. Reddy, Norris Cancer Center, Room 6429 University of Southern California 1441 Eastlake Avenue Mail stop 73 Los Angeles CA 90033 U.S.A. E-mail: [email protected] 1065–6995/99/030227+07 $30.00/0
formation of pseudopodia, their circumferential attachment to the prey particle and its subsequent internalization by membrane fusion (for reviews see Besterman and Low, 1983; Stossel, 1989). cis-Diammine dichloro platinum (II) [Cisplatin] is a potential anti neoplastic agent used against a broad spectrum of cancers (Fram, 1992). Cisplatin reacts with DNA and inhibits its replication leading eventually to eventual cell death (Roberts and Thomson, 1979). Cisplatin is mutageneic (Bradley et al., 1993), induces morphological transformations in mammalian cells (Turnbull et al., 1979) and produces sister chromatid exchanges (Bradley et al., 1979). Cisplatin in a hydrolysed state, uncouples oxidative phosphorylation in mitochondria, increases oxygen consumption, inhibits ATPase activity and produces a net efflux of calcium ions (Aggarwal, 993). Cisplatin crosslinks sulfhydryl rich groups in proteins (Litterst et al., 1979) and alters membrane configuration (Sinha and Chignell, 1979). Previously we have shown the inhibitory effects of cisplatin on pinocytotic functions in growing Dictyostelium cells (Reddy and Chatterjee, 1997). In the present study, the action of cisplatin on folic acid chemotaxis and phagocytotic functions in Dictyostelium discoideum cells is reported. 1999 Academic Press
228
MATERIALS AND METHODS Organism and culture conditions The axenic strain (AX2) of Dictyostelium discoideum, originally obtained from Dr Robert Kay (Cambridge, U.K.) was used in the present study. The Dictyostelium amoebae were grown in axenic medium according to Ashworth and Watts (1970) at 22C on a rotary shaker at 120 rpm. Exponentially growing cells were harvested at a density of 5–7106 cells/ml. Cisplatin treatment Dictyostelium cells were treated with cisplatin for 1 h at a concentration of 100 and 200 g/ml. cisDiammine dichloro platinum(II), (Cisplatin, obtained from Sigma Chemical Company, U.S.A.) was dissolved, just prior to use, in Sorenson’s phosphate buffer at a concentration of 1000 g/ml and filter sterilized. Growing Dictyostelium cells were harvested by centrifuging at 2000 rpm for 2 min and were resuspended in 0.017 Sorenson’s phosphate buffer (P-buffer, pH 6.2). Appropriate amounts of cisplatin stock solution was added to give a final concentration of 100 and 200 g cisplatin/ml. Following the treatment for 1 h at 22C, cells were washed three times in ice-cold phosphate buffer. Folic acid chemotaxis Small population assay. Folic acid chemotaxis was monitored according to the method of Pan et al. (1972). Control and cisplatin treated Dictyostelium cells were harvested and suspended in phosphate buffer to give a thick slurry. Two microlitres of this cell suspension were placed as a small droplet on folic acid-agar plate (110 5 ). Folic acid-agar was prepared by adding the appropriate amount of folic acid solution to warm molten agar, just prior to plate pouring. The cells were scored positive to chemotaxis assay when they moved out of the droplet. Folate deaminase assay. Folate deaminase activity was assayed according to the method of Bernstein and Vandriel (1980). Control and cisplatin treated Dictyostelium cells were resuspended in P-buffer (pH 6.2), at a density of 1107 cells/ml. Ehrlenmeyer flasks containing the cells were placed on an orbital shaker at 120 rpm, 22C under fluorescent light. One millilitre samples were removed at 2 h intervals until 12 h. Cells were pelleted and the
Cell Biology International, Vol. 23, No. 3, 1999
supernatant collected was assayed immediately. Folate deaminase activity was assayed following the initial rate of change of absorbance of folic acid solution at 325 nm in a spectrophotometer (UV200, Hitachi). Samples were measured in 100 folic acid, 0.1 imidazole-HCl (pH 7.2) at 23C with respect to a reference containing similar solution without the enzyme. One unit of folate deaminase activity was defined as 1.0 nmol of folic acid converted to product (2-hydroxy 2-deamino folic acid) per minute. Colony blots Control and cisplatin treated Dictyostelium cells were plated as a small droplet on nutrient agar plates seeded with E. coli, the plates were then incubated at 22C. Upon appearance of plaques, 25 mm Millipore filters [Millipore (I), 0.45 m] were placed on top of the plaques for 3 min and then immediately frozen in liquid nitrogen. The filters were then stained with 0.2% ponceau ‘S’ (Sigma, U.S.A.) and post-fixed with 3% TCA. Phagocytosis assay Phagocytosis was measured by administering Fluorescein isothiocynate (FITC) labelled bacteria as substrate particles according to the method describe by Vogel (1987). Preparation of FITC labelled bacteria. FITCbacteria were prepared by suspending exponentially grown E. coli (K-802) in 50 m sodium phosphate, pH 9.2 (OD420 =20) and by the addition of FITC (isomer I, Sigma) stock solution (10 mg/ ml) to give a final FITC concentration of 0.1 mg/ ml. After 3 h incubation at 37C on a rotary shaker at 100 rpm, bacteria were collected and washed six times with P-buffer to remove unlabelled FITC from bacterial cell suspension. Phagocytosis assay. Control and cisplatin-treated Dictyostelium cells were suspended in P-buffer along with FITC-labelled bacteria in a 1:200 ratio, they were then incubated on a rotary shaker at 100 rpm. One millilitre samples were withdrawn at 10 min intervals for 1 h, phagocytosis was stopped by dilution of the sample with 4 ml of chilled P-buffer (pH 6.2). Uningested bacteria were removed by washing Dictyostelium cells three times in P-buffer. Washed cells were resuspended in sodium phosphate buffer (pH 9.2) and cell numbers were determined. The cells were lysed by the addition of Triton X-100 (0.2% final concen-
Cell Biology International, Vol. 23, No. 3, 1999
229
Fig. 1. Folic acid chemotaxis by control and cisplatin treated Dictyostelium cells. C, Control, T1, 100 g/ml dose, T2, 200 g/mldose. h, Time in hours after plating on folic acid-agar.
tration). The fluorescence intensity was measured using a fluorescence spectrophotometer (RF-540, Shimadzu) with exitation and emission wave lengths of 470 and 520 nm respectively. The number of phagocytosed bacteria was determined by comparison with a standard curve. A standard curve was obtained by lysing a known number of FITC-bacteria in 50 m sodium phosphate buffer (pH 9.2) containing 1% SDS solution by heating for 2 min at 90C and then measuring the fluorescence intensity as described above.
The supernatant was discarded and the pellet containing cytoskeletal proteins was lyophylized. Cytoskeletal proteins isolated from a fixed number of cells by the above methods were boiled for 1 min in 1 ml sample buffer. Ten microlitres of sample were loaded on to a 6–16% polyacrylamide gel and were electrophoresed at a constant voltage of 120 volts (Laemmli, 1970). The proteins were stained with 0.025% Coomassie brilliant blue R-250 in 40% methanol and 7% acetic acid for 2 h, then destained in destaining solution containing 50% methanol and 10% acetic acid.
Isolation and analysis of cytoskeletal proteins Cytoskeletal proteins were isolated as Triton X-100 insoluble fractions according to the method described by Maeda (1988). Dictyostelium cells were suspended at a density of 1107 cells/ml in 5 ml of lysis solution containing 1% Triton X-100, 10 m KCl, 10 m imidazole, 2 m sodium azide, 5 m EGTA in 8.5 m phosphate buffer (pH 6.2). Cell suspension was kept at room temperature for 10 min with occasional agitation, the samples were then centrifuged for 4 min at 8000g (4C).
RESULTS Folic acid chemotaxis In the small population assay, Dictyostelium amoebae move out of the droplet in the direction of the folic acid gradient generated by the action of folate deaminase released by Dictyostelium cells into the surroundings. As folate deaminase released by Dictyostelium cells inactivates the folic acid
230
Cell Biology International, Vol. 23, No. 3, 1999
FDA activity in units/5 × 106 cells
6.0
5.0
4.0
3.0
2.0
1.0
0
2
4
6 8 Time (h)
10
12
Fig. 2. Folate deaminase activity in control and cisplatin treated Dictyostelium cells. Each value is the mean (..) of three independent experiments. , Control; , 100 g/ml; , 200 g/ml.
near the droplet, a higher concentration of folic acid is found away from the droplet and cells therefore move in the direction of the increasing concentration of folic acid. Cisplatin-treated Dictyostelium cells showed dose dependent inhibition in folic acid chemostaxis. As shown in Fig. 1, by 3 h of plating on folic acid-agar plates a fair number of control cells moved out of the droplet. In cisplatin-treated cells, fewer cells moved shorter distances. By 6 h, control cells completely spread out of the droplet. Cells treated with 100 g/ml of cisplatin also appeared to spread by 6 h, but the cell number was considerably less than control. In cells treated with 200 g/ml of cisplatin, by 6 h only a few cells moved shorter distances compared the distance travelled by control and cells treated with 100 g/ml dose. The extracellular folate deaminase of Dictyostelium cells facilitates the establishment of a folic acid gradient. As shown in Fig. 2, cisplatin treatment inhibited the folate deaminase activity to different extents depending upon the dose. Both 100 and 200 g/ml dose showed maximum inhibition at 4 h after the treatment, as compared to control. Colony blots Dictyostelium cells are plated on an E. coli lawn where a plaque will form representing the feeding amoebae. As cells feed on bacteria at the periphery of the plaque, the colony expands in diameter. As
Fig. 3. Colony blots of control and cisplatin treated cells. (1) Control, (2) 100 g/ml dose, (3) 200 g/ml dose. H, Time in hours from the time of plating on E. coli seeded nutrient-agar.
shown in Fig. 3, plaques started to appear both in control and cisplatin treated cells by 24 h of plating. In cisplatin treated cells, the size of the plaques and the rate at which they grew was less when compared to control cells. Phagocytosis assay Cisplatin treated Dictyostelium cells showed reduced rates of FITC-labelled bacterial uptake in a dose-dependent manner. As shown in Fig. 4, there is approximately 25 and 45% inhibition of phagocytosis in cells treated with 100 and 200 g/ml cisplatin respectively. Analysis of cytoskletal proteins The SDS-PAGE analysis of cytoskeletal proteins revealed association of more actin and myosin with cell cortex. The effect of cisplatin on the increasing association of actin with cell cortex was found to be dose dependent. As shown in Fig. 5, the higher dose (200 g/ml) showed more actin with cell cortex than in the lower dose. DISCUSSION Phagocytosis is an essential biological process in eucaryotes. It serves as the sole method of food
Cell Biology International, Vol. 23, No. 3, 1999
231
Number of bacteria phagocytosed/cell
90
kDa 205
80
(1)
(2)
(3)
(4)
(5) My
70 60
116
50 40
66
30 20 10
0
10
20
30 40 Time (h)
50
60
45
Ac
Fig. 4. Uptake of FITC-labelled bacteria by control and cisplatin treated Dictyostelium cells. Each value is the mean (..) of three independent experiments. , control; , 100 g/ml; , 200 g/ml.
acquisition in some single celled protozoa and functions as an important part of immune defense mechanisms in metazoa. The phagocytotic process is a complex phenomena where several cellular events take place in a coordinated fashion in order to facilitate the uptake of substrate particles by a phagocyte (reviewed by Lewis and O’Day, 1996). Folic acid chemotaxis towards bacteria enable Dictyostelium cells to recognize prey. The present results indicate the impairment in folic acid chemotaxis in cisplatin-treated cells. The colony blots (Fig. 3) show the slow plaque clearing ability of cisplatin-treated cells, indicating their low phagocytotic ability. Protein synthesis inhibitors, like cycloheximide, anisomycin and puromycin (Gonzalez and Satre, 1991) and caffeine (Gonzalez et al., 1990), and antibodies directed against certain common carbohydrate epitopes of a major cell surface glycoprotein (Chia and Luna, 1989), inhibit phagocytosis in Dictyostelium discoideum. Our unpublished results also show a net inhibition in protein synthesis and growth of cisplatin treated Dictyostelium cells. Endocytosis is an energydependent process which is completely inhibited by 10 m azide in Dictyostelium cells (Rossomando et al., 1981). Both chemotaxis and phagocytosis represent the dynamic state of the cell and a rapid reorganization of the cytoskeletal network takes place following either chemotactic stimulation and/or binding of the prey to a phagocyte. Cislatin causes uncoupling of oxidative phosphoryl-
Fig. 5. SDS-PAGE analysis of cytoskeletal proteins. Lanes (1) and (2) low and high molecular weight markers respectively. K Da, Molecular weight in Kilo Daltons. Lane (3) Control, lane (4) 100 g/ml dose, lane (5) 200 g/ml dose. Ac, Actin. My, Myosin.
ation and calcium efflux from the mitochondria (Aggarwal, 1993). The higher levels of cytosolic calcium results in increased association of actin with the cell cortex (Maeda, 1988). The present results indicate an increased association of actin with the cell cortex following cisplatin treatment (Fig. 5), which could be due to increased cytosolic calicum. Unterweger and Schlatterer (1995) reported the inhibition of pseudopod formation and chemotaxis in Dictyostelium amoebae following the introduction of calcium ions into the cytosol. Caffeine releases calcium from intracellular stores leading to the perturbation of calcium homeostasis (Abe et al., 1988) and the inhibition endocytosis in Dictyostelium. Cisplatin effectively cross links the sulfhydryl rich groups in membrane proteins (Peyrot et al., 1983). The cell surface receptors that mediate the recognition of prey (Vogel et al., 1980; Chia and Luna, 1989) could be the likely target of cisplatin which leads to the inhibition of phagocytosis. Studies by Fumarulo et al. (1985) indicate sulfhydryl groups as being the possible cisplatin target to bring a net inhibitory effect in chemotaxis and phagocytotic functions in guinea-pig polymorphonuclear leukocytes. Once the prey is
232
internalized, the endocytic cargo is channeled from acidified endosomes to a lysosomal like compartment, where digestion of nutrients takes place. Undigested material is returned to the cell surface through near neutral postlysomal vacuoles (Padh et al., 1993). Endocytotic circuit requires the function and association of a number of proteins associated with endocytotic vesicles (Adessi et al., 1995). Cisplatin forms adducts with various cellular components and is known to cross-link several proteins. The receptor modifying and cross linking ability of cisplatin may affect the endocytic circuit resulting in the inhibition of phagocytosis in Dictyostelium cells. Thus, cisplatin appears to interact with the cytoskeleton and cell membrane leading to impairment in chemotaxis and subsequently phagocytotic functions.
ACKNOWLEDGEMENTS T.B.K.R. is thankful to the University Grants Commission, New Delhi for extending financial assistance in the form of a Research Fellowship during the tenure of this work. We are thankful to Dr R. van Driel for providing useful information for the assay of folate deaminase activity. REFERENCES A T, M Y, L T, 1988. Transient increase of the intracellular calcium concentration during chemotactic signal transduction in Dictyostelium discoideum cells. Differ 39: 90–96. A C, C A, V M, R T, K G, S M, G J, 1995. Identification of major proteins associated with Dictyostelium discoideum endocytic vesicles. J Cell Sci 108: 3331–3337. A SK, 1993. A histochemical approach to the mechanism of action of cisplatin and its analogs. J Histochem Cytochem 44: 1053–1073. A JM, W DJ, 1970. Metabolism of the slime mould Dictyostelium discoideum grown in axenic culture. Biochem J 119: 175–182. B RL, V D R, 1980. Control of folate deaminase activity of Dictyostelium discoideum by cyclic AMP. FEBS Lett 119: 249–253. B JM, L RB, 1983. Endocytosis: a review of mechanisms and plasma membrane dynamics. Biochem J 210: 1–13. B JT, H EM, S W, W BK, 1979. Evidence for a second chemotactic system in the cellular slime mould, Dictyostelium discoideum. J Bacteriol 102: 682–687. B MO, H IC, H CC, 1979. Relationship between sister chromatid exchange and mutagenicity, toxicity, and DNA damage. Nature 282: 318–320.
Cell Biology International, Vol. 23, No. 3, 1999
B LJN, Y KJ, L SJ, E JM, 1993. Mutagenicity and genotoxicity of the major DNA adduct of the antitumor drug cis-diammine dichloroplatinum (II). Biochemistry 32: 982–988. C CP, L EJ, 1989. Phagocytosis in Dictyostelium discoideum is inhibited by antibodies directed primarily against common carbohydrate epitopes of major cell-surface plasma membrane glycoprotein. Exp Cell Res 181: 11–26. F RJ, 1992. Cisplatin and platinum analogues; recent advances. Curr Opin Oncol 4: 1073–1079. F R, R S, P R, C M, G D, A M, 1985. In vitro cisplatin effects on phagocyte functions. Int J Cancer 35: 777–780. G C, G K, S M, 1990. Caffeine an inhibitor of endocytosis in Dictyostelium discoideum amoebae. J Cell Physiol 144: 408–415. G C, S M, 1991. Endocytosis in Dictyostelium discoideum amoebae: inhibition by cycloheximide and other inhibitors of protein synthesis. J Cell Sci 99: 535–543. L UK, 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685. L KL, O’D DH, 1996. Phagocytosis in Dictyostelium discoideum: Nibbling, eating and cannibalism. J Eucary Microbio 43: 65–69. L CL, L AF, G AM, 1979. The disposition and distribution of platinum following parentol administration to animals of cis-dichloride diammine platinum (II). Cancer Treat Rep 63: 1485–1492. M Y, 1988. Changes of endocytic activities during the cell cycle of Dictyostelium cells. Dev Growth Diff 30: 15–24. P H, H J, L M, S TL, 1993. A post lysosomal compartment in Dictyostelium discoideum. J Biol Chem 268: 6742–6747. P P, H EM, B JT, 1972. Folic acid as second chemotactic substance in the cellular slime moulds. Nature 237: 181–182. P U, B C, C A, B D, CM AM, S JC, 1983. Action of hydrolyzed cisplatin and some analogues on microtubule protein polymerization in vitro. Cancer Treat Rep 67: 641–646. R TBK, C S, 1997. Cisplatin inhibits pinocytosis in Dictyostelium discoideum. Cell Biol Int 21: 237–241. R JJ, T AJ, 1979. The mechanism of action and antitumor platinum compounds. Prog Nucl Acid Res Molec Biol 22: 71–133. R EF, J EG, V B, S DR, 1981. Inhibition of a nutrient-dependent pinocytosis in Dictyostelium discoideum by the amino acid analogue Hadacidin. J Cell Biol 91: 227–231. S BK, C CF, 1979. Interaction of anti tumor drugs with human erythrocyte ghost membranes and mastocytoma P815: a spin label study. Biochem Biophys Res Commun 86: 1051–1057. S TP, 1989. From signal to pseudopod. How cells control cytoplasmic assembly. J Biol Chem 264: 18,261– 18,264. T D, P NC, D JA, M BC, 1979. Cis-platinum (II) diammine dichloride causes mutation, transformation, and sister chromatid exchanges in cultured mammalian cells. Mutat Res 66: 267–275. U N, S C, 1995. Introduction of calcium buffers into the cytosol of Dictyostelium discoideum amoebae alters cell morphology and inhibits chemotaxis. Cell Calcium 17: 97–110.
Cell Biology International, Vol. 23, No. 3, 1999
V G, 1987. Endocytosis and recognition mechanisms in Dictyostelium discoideum. Methods Cell Biol 28: 129–137. V G, T L, S H, S, 1980. Mechanism of phagocytosis in Dictyostelium discoideum: Phagocytosis is
233
mediated by different recognition sites as disclosed by mutants with altered phagocytotic properties. J Cell Biol 86: 456–465.
CISPLATIN INHIBITS FOLIC ACID CHEMOTAXIS AND PHAGOCYTOTIC FUNCTIONS IN DICTYOSTELIUM DISCOIDEUM T. B. K. REDDY* and S. CHATTERJEE School of Life Sciences, Jawaharlal Nehru University, New Delhi-110067, India Received 23 September 1998, accepted 25 November 1998
Administration of 100 and 200 g/ml of cisplatin [cis-diammine dichloro platinum (II)] for 1 h to growing Dictyostelium discoideum cells severely affects folic acid chemotaxis and phagocytotic function in this organism. Following cisplatin treatment, cells show a much lower uptake of FITC labelled bacteria and a reduced plaque forming ability when plated on Eschericia coli seeded normal agar. Folic acid chemotaxis and folate deaminase activity are greatly inhibited in cisplatin-treated Dictyostelium cells. SDS-PAGE analysis shows a greater association of actin and myosin with the cell cortex of treated cells. These results have been discussed in relation to 1999 Academic Press cisplatin’s known ability to raise the levels of cytosolic calcium. K: cisplatin; dictyostelium; chemotaxis; phagocytosis.
INTRODUCTION The cellular slime mould Dictyostelium discoideum, is a simple soil dwelling eucaryotic microbe which feeds on bacteria and yeasts, continuing to grow as long as food is available. Growing Dictyostelium cells exhibit phagocytosis, the uptake of substrate particles from the external environment into cell in plasma membrane derived vesicles. This constitutes the primary mode of nutrient uptake of this organism. Growing Dictyostelium cells sense, and then move towards the folic acid source generally released by its bacterial prey into its surroundings. Folate deaminase produced by Dictyostelium amoebae help in the establishment of a folic acid gradient and which facilitates chemotaxis towards the increasing source of folic acid (Pan et al., 1972; Bonner et al., 1979). Dictyostelium cell membranes contain different cell surface receptors which facilitate the binding of substrate particles (Vogel et al., 1980). The binding of prey particles trigger a transmembrane signal that leads to the rapid *To whom correspondence should be addressed: Dr T. B. K. Reddy, Norris Cancer Center, Room 6429 University of Southern California 1441 Eastlake Avenue Mail stop 73 Los Angeles CA 90033 U.S.A. E-mail: [email protected] 1065–6995/99/030227+07 $30.00/0
formation of pseudopodia, their circumferential attachment to the prey particle and its subsequent internalization by membrane fusion (for reviews see Besterman and Low, 1983; Stossel, 1989). cis-Diammine dichloro platinum (II) [Cisplatin] is a potential anti neoplastic agent used against a broad spectrum of cancers (Fram, 1992). Cisplatin reacts with DNA and inhibits its replication leading eventually to eventual cell death (Roberts and Thomson, 1979). Cisplatin is mutageneic (Bradley et al., 1993), induces morphological transformations in mammalian cells (Turnbull et al., 1979) and produces sister chromatid exchanges (Bradley et al., 1979). Cisplatin in a hydrolysed state, uncouples oxidative phosphorylation in mitochondria, increases oxygen consumption, inhibits ATPase activity and produces a net efflux of calcium ions (Aggarwal, 993). Cisplatin crosslinks sulfhydryl rich groups in proteins (Litterst et al., 1979) and alters membrane configuration (Sinha and Chignell, 1979). Previously we have shown the inhibitory effects of cisplatin on pinocytotic functions in growing Dictyostelium cells (Reddy and Chatterjee, 1997). In the present study, the action of cisplatin on folic acid chemotaxis and phagocytotic functions in Dictyostelium discoideum cells is reported. 1999 Academic Press
228
MATERIALS AND METHODS Organism and culture conditions The axenic strain (AX2) of Dictyostelium discoideum, originally obtained from Dr Robert Kay (Cambridge, U.K.) was used in the present study. The Dictyostelium amoebae were grown in axenic medium according to Ashworth and Watts (1970) at 22C on a rotary shaker at 120 rpm. Exponentially growing cells were harvested at a density of 5–7106 cells/ml. Cisplatin treatment Dictyostelium cells were treated with cisplatin for 1 h at a concentration of 100 and 200 g/ml. cisDiammine dichloro platinum(II), (Cisplatin, obtained from Sigma Chemical Company, U.S.A.) was dissolved, just prior to use, in Sorenson’s phosphate buffer at a concentration of 1000 g/ml and filter sterilized. Growing Dictyostelium cells were harvested by centrifuging at 2000 rpm for 2 min and were resuspended in 0.017 Sorenson’s phosphate buffer (P-buffer, pH 6.2). Appropriate amounts of cisplatin stock solution was added to give a final concentration of 100 and 200 g cisplatin/ml. Following the treatment for 1 h at 22C, cells were washed three times in ice-cold phosphate buffer. Folic acid chemotaxis Small population assay. Folic acid chemotaxis was monitored according to the method of Pan et al. (1972). Control and cisplatin treated Dictyostelium cells were harvested and suspended in phosphate buffer to give a thick slurry. Two microlitres of this cell suspension were placed as a small droplet on folic acid-agar plate (110 5 ). Folic acid-agar was prepared by adding the appropriate amount of folic acid solution to warm molten agar, just prior to plate pouring. The cells were scored positive to chemotaxis assay when they moved out of the droplet. Folate deaminase assay. Folate deaminase activity was assayed according to the method of Bernstein and Vandriel (1980). Control and cisplatin treated Dictyostelium cells were resuspended in P-buffer (pH 6.2), at a density of 1107 cells/ml. Ehrlenmeyer flasks containing the cells were placed on an orbital shaker at 120 rpm, 22C under fluorescent light. One millilitre samples were removed at 2 h intervals until 12 h. Cells were pelleted and the
Cell Biology International, Vol. 23, No. 3, 1999
supernatant collected was assayed immediately. Folate deaminase activity was assayed following the initial rate of change of absorbance of folic acid solution at 325 nm in a spectrophotometer (UV200, Hitachi). Samples were measured in 100 folic acid, 0.1 imidazole-HCl (pH 7.2) at 23C with respect to a reference containing similar solution without the enzyme. One unit of folate deaminase activity was defined as 1.0 nmol of folic acid converted to product (2-hydroxy 2-deamino folic acid) per minute. Colony blots Control and cisplatin treated Dictyostelium cells were plated as a small droplet on nutrient agar plates seeded with E. coli, the plates were then incubated at 22C. Upon appearance of plaques, 25 mm Millipore filters [Millipore (I), 0.45 m] were placed on top of the plaques for 3 min and then immediately frozen in liquid nitrogen. The filters were then stained with 0.2% ponceau ‘S’ (Sigma, U.S.A.) and post-fixed with 3% TCA. Phagocytosis assay Phagocytosis was measured by administering Fluorescein isothiocynate (FITC) labelled bacteria as substrate particles according to the method describe by Vogel (1987). Preparation of FITC labelled bacteria. FITCbacteria were prepared by suspending exponentially grown E. coli (K-802) in 50 m sodium phosphate, pH 9.2 (OD420 =20) and by the addition of FITC (isomer I, Sigma) stock solution (10 mg/ ml) to give a final FITC concentration of 0.1 mg/ ml. After 3 h incubation at 37C on a rotary shaker at 100 rpm, bacteria were collected and washed six times with P-buffer to remove unlabelled FITC from bacterial cell suspension. Phagocytosis assay. Control and cisplatin-treated Dictyostelium cells were suspended in P-buffer along with FITC-labelled bacteria in a 1:200 ratio, they were then incubated on a rotary shaker at 100 rpm. One millilitre samples were withdrawn at 10 min intervals for 1 h, phagocytosis was stopped by dilution of the sample with 4 ml of chilled P-buffer (pH 6.2). Uningested bacteria were removed by washing Dictyostelium cells three times in P-buffer. Washed cells were resuspended in sodium phosphate buffer (pH 9.2) and cell numbers were determined. The cells were lysed by the addition of Triton X-100 (0.2% final concen-
Cell Biology International, Vol. 23, No. 3, 1999
229
Fig. 1. Folic acid chemotaxis by control and cisplatin treated Dictyostelium cells. C, Control, T1, 100 g/ml dose, T2, 200 g/mldose. h, Time in hours after plating on folic acid-agar.
tration). The fluorescence intensity was measured using a fluorescence spectrophotometer (RF-540, Shimadzu) with exitation and emission wave lengths of 470 and 520 nm respectively. The number of phagocytosed bacteria was determined by comparison with a standard curve. A standard curve was obtained by lysing a known number of FITC-bacteria in 50 m sodium phosphate buffer (pH 9.2) containing 1% SDS solution by heating for 2 min at 90C and then measuring the fluorescence intensity as described above.
The supernatant was discarded and the pellet containing cytoskeletal proteins was lyophylized. Cytoskeletal proteins isolated from a fixed number of cells by the above methods were boiled for 1 min in 1 ml sample buffer. Ten microlitres of sample were loaded on to a 6–16% polyacrylamide gel and were electrophoresed at a constant voltage of 120 volts (Laemmli, 1970). The proteins were stained with 0.025% Coomassie brilliant blue R-250 in 40% methanol and 7% acetic acid for 2 h, then destained in destaining solution containing 50% methanol and 10% acetic acid.
Isolation and analysis of cytoskeletal proteins Cytoskeletal proteins were isolated as Triton X-100 insoluble fractions according to the method described by Maeda (1988). Dictyostelium cells were suspended at a density of 1107 cells/ml in 5 ml of lysis solution containing 1% Triton X-100, 10 m KCl, 10 m imidazole, 2 m sodium azide, 5 m EGTA in 8.5 m phosphate buffer (pH 6.2). Cell suspension was kept at room temperature for 10 min with occasional agitation, the samples were then centrifuged for 4 min at 8000g (4C).
RESULTS Folic acid chemotaxis In the small population assay, Dictyostelium amoebae move out of the droplet in the direction of the folic acid gradient generated by the action of folate deaminase released by Dictyostelium cells into the surroundings. As folate deaminase released by Dictyostelium cells inactivates the folic acid
230
Cell Biology International, Vol. 23, No. 3, 1999
FDA activity in units/5 × 106 cells
6.0
5.0
4.0
3.0
2.0
1.0
0
2
4
6 8 Time (h)
10
12
Fig. 2. Folate deaminase activity in control and cisplatin treated Dictyostelium cells. Each value is the mean (..) of three independent experiments. , Control; , 100 g/ml; , 200 g/ml.
near the droplet, a higher concentration of folic acid is found away from the droplet and cells therefore move in the direction of the increasing concentration of folic acid. Cisplatin-treated Dictyostelium cells showed dose dependent inhibition in folic acid chemostaxis. As shown in Fig. 1, by 3 h of plating on folic acid-agar plates a fair number of control cells moved out of the droplet. In cisplatin-treated cells, fewer cells moved shorter distances. By 6 h, control cells completely spread out of the droplet. Cells treated with 100 g/ml of cisplatin also appeared to spread by 6 h, but the cell number was considerably less than control. In cells treated with 200 g/ml of cisplatin, by 6 h only a few cells moved shorter distances compared the distance travelled by control and cells treated with 100 g/ml dose. The extracellular folate deaminase of Dictyostelium cells facilitates the establishment of a folic acid gradient. As shown in Fig. 2, cisplatin treatment inhibited the folate deaminase activity to different extents depending upon the dose. Both 100 and 200 g/ml dose showed maximum inhibition at 4 h after the treatment, as compared to control. Colony blots Dictyostelium cells are plated on an E. coli lawn where a plaque will form representing the feeding amoebae. As cells feed on bacteria at the periphery of the plaque, the colony expands in diameter. As
Fig. 3. Colony blots of control and cisplatin treated cells. (1) Control, (2) 100 g/ml dose, (3) 200 g/ml dose. H, Time in hours from the time of plating on E. coli seeded nutrient-agar.
shown in Fig. 3, plaques started to appear both in control and cisplatin treated cells by 24 h of plating. In cisplatin treated cells, the size of the plaques and the rate at which they grew was less when compared to control cells. Phagocytosis assay Cisplatin treated Dictyostelium cells showed reduced rates of FITC-labelled bacterial uptake in a dose-dependent manner. As shown in Fig. 4, there is approximately 25 and 45% inhibition of phagocytosis in cells treated with 100 and 200 g/ml cisplatin respectively. Analysis of cytoskletal proteins The SDS-PAGE analysis of cytoskeletal proteins revealed association of more actin and myosin with cell cortex. The effect of cisplatin on the increasing association of actin with cell cortex was found to be dose dependent. As shown in Fig. 5, the higher dose (200 g/ml) showed more actin with cell cortex than in the lower dose. DISCUSSION Phagocytosis is an essential biological process in eucaryotes. It serves as the sole method of food
Cell Biology International, Vol. 23, No. 3, 1999
231
Number of bacteria phagocytosed/cell
90
kDa 205
80
(1)
(2)
(3)
(4)
(5) My
70 60
116
50 40
66
30 20 10
0
10
20
30 40 Time (h)
50
60
45
Ac
Fig. 4. Uptake of FITC-labelled bacteria by control and cisplatin treated Dictyostelium cells. Each value is the mean (..) of three independent experiments. , control; , 100 g/ml; , 200 g/ml.
acquisition in some single celled protozoa and functions as an important part of immune defense mechanisms in metazoa. The phagocytotic process is a complex phenomena where several cellular events take place in a coordinated fashion in order to facilitate the uptake of substrate particles by a phagocyte (reviewed by Lewis and O’Day, 1996). Folic acid chemotaxis towards bacteria enable Dictyostelium cells to recognize prey. The present results indicate the impairment in folic acid chemotaxis in cisplatin-treated cells. The colony blots (Fig. 3) show the slow plaque clearing ability of cisplatin-treated cells, indicating their low phagocytotic ability. Protein synthesis inhibitors, like cycloheximide, anisomycin and puromycin (Gonzalez and Satre, 1991) and caffeine (Gonzalez et al., 1990), and antibodies directed against certain common carbohydrate epitopes of a major cell surface glycoprotein (Chia and Luna, 1989), inhibit phagocytosis in Dictyostelium discoideum. Our unpublished results also show a net inhibition in protein synthesis and growth of cisplatin treated Dictyostelium cells. Endocytosis is an energydependent process which is completely inhibited by 10 m azide in Dictyostelium cells (Rossomando et al., 1981). Both chemotaxis and phagocytosis represent the dynamic state of the cell and a rapid reorganization of the cytoskeletal network takes place following either chemotactic stimulation and/or binding of the prey to a phagocyte. Cislatin causes uncoupling of oxidative phosphoryl-
Fig. 5. SDS-PAGE analysis of cytoskeletal proteins. Lanes (1) and (2) low and high molecular weight markers respectively. K Da, Molecular weight in Kilo Daltons. Lane (3) Control, lane (4) 100 g/ml dose, lane (5) 200 g/ml dose. Ac, Actin. My, Myosin.
ation and calcium efflux from the mitochondria (Aggarwal, 1993). The higher levels of cytosolic calcium results in increased association of actin with the cell cortex (Maeda, 1988). The present results indicate an increased association of actin with the cell cortex following cisplatin treatment (Fig. 5), which could be due to increased cytosolic calicum. Unterweger and Schlatterer (1995) reported the inhibition of pseudopod formation and chemotaxis in Dictyostelium amoebae following the introduction of calcium ions into the cytosol. Caffeine releases calcium from intracellular stores leading to the perturbation of calcium homeostasis (Abe et al., 1988) and the inhibition endocytosis in Dictyostelium. Cisplatin effectively cross links the sulfhydryl rich groups in membrane proteins (Peyrot et al., 1983). The cell surface receptors that mediate the recognition of prey (Vogel et al., 1980; Chia and Luna, 1989) could be the likely target of cisplatin which leads to the inhibition of phagocytosis. Studies by Fumarulo et al. (1985) indicate sulfhydryl groups as being the possible cisplatin target to bring a net inhibitory effect in chemotaxis and phagocytotic functions in guinea-pig polymorphonuclear leukocytes. Once the prey is
232
internalized, the endocytic cargo is channeled from acidified endosomes to a lysosomal like compartment, where digestion of nutrients takes place. Undigested material is returned to the cell surface through near neutral postlysomal vacuoles (Padh et al., 1993). Endocytotic circuit requires the function and association of a number of proteins associated with endocytotic vesicles (Adessi et al., 1995). Cisplatin forms adducts with various cellular components and is known to cross-link several proteins. The receptor modifying and cross linking ability of cisplatin may affect the endocytic circuit resulting in the inhibition of phagocytosis in Dictyostelium cells. Thus, cisplatin appears to interact with the cytoskeleton and cell membrane leading to impairment in chemotaxis and subsequently phagocytotic functions.
ACKNOWLEDGEMENTS T.B.K.R. is thankful to the University Grants Commission, New Delhi for extending financial assistance in the form of a Research Fellowship during the tenure of this work. We are thankful to Dr R. van Driel for providing useful information for the assay of folate deaminase activity. REFERENCES A T, M Y, L T, 1988. Transient increase of the intracellular calcium concentration during chemotactic signal transduction in Dictyostelium discoideum cells. Differ 39: 90–96. A C, C A, V M, R T, K G, S M, G J, 1995. Identification of major proteins associated with Dictyostelium discoideum endocytic vesicles. J Cell Sci 108: 3331–3337. A SK, 1993. A histochemical approach to the mechanism of action of cisplatin and its analogs. J Histochem Cytochem 44: 1053–1073. A JM, W DJ, 1970. Metabolism of the slime mould Dictyostelium discoideum grown in axenic culture. Biochem J 119: 175–182. B RL, V D R, 1980. Control of folate deaminase activity of Dictyostelium discoideum by cyclic AMP. FEBS Lett 119: 249–253. B JM, L RB, 1983. Endocytosis: a review of mechanisms and plasma membrane dynamics. Biochem J 210: 1–13. B JT, H EM, S W, W BK, 1979. Evidence for a second chemotactic system in the cellular slime mould, Dictyostelium discoideum. J Bacteriol 102: 682–687. B MO, H IC, H CC, 1979. Relationship between sister chromatid exchange and mutagenicity, toxicity, and DNA damage. Nature 282: 318–320.
Cell Biology International, Vol. 23, No. 3, 1999
B LJN, Y KJ, L SJ, E JM, 1993. Mutagenicity and genotoxicity of the major DNA adduct of the antitumor drug cis-diammine dichloroplatinum (II). Biochemistry 32: 982–988. C CP, L EJ, 1989. Phagocytosis in Dictyostelium discoideum is inhibited by antibodies directed primarily against common carbohydrate epitopes of major cell-surface plasma membrane glycoprotein. Exp Cell Res 181: 11–26. F RJ, 1992. Cisplatin and platinum analogues; recent advances. Curr Opin Oncol 4: 1073–1079. F R, R S, P R, C M, G D, A M, 1985. In vitro cisplatin effects on phagocyte functions. Int J Cancer 35: 777–780. G C, G K, S M, 1990. Caffeine an inhibitor of endocytosis in Dictyostelium discoideum amoebae. J Cell Physiol 144: 408–415. G C, S M, 1991. Endocytosis in Dictyostelium discoideum amoebae: inhibition by cycloheximide and other inhibitors of protein synthesis. J Cell Sci 99: 535–543. L UK, 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685. L KL, O’D DH, 1996. Phagocytosis in Dictyostelium discoideum: Nibbling, eating and cannibalism. J Eucary Microbio 43: 65–69. L CL, L AF, G AM, 1979. The disposition and distribution of platinum following parentol administration to animals of cis-dichloride diammine platinum (II). Cancer Treat Rep 63: 1485–1492. M Y, 1988. Changes of endocytic activities during the cell cycle of Dictyostelium cells. Dev Growth Diff 30: 15–24. P H, H J, L M, S TL, 1993. A post lysosomal compartment in Dictyostelium discoideum. J Biol Chem 268: 6742–6747. P P, H EM, B JT, 1972. Folic acid as second chemotactic substance in the cellular slime moulds. Nature 237: 181–182. P U, B C, C A, B D, CM AM, S JC, 1983. Action of hydrolyzed cisplatin and some analogues on microtubule protein polymerization in vitro. Cancer Treat Rep 67: 641–646. R TBK, C S, 1997. Cisplatin inhibits pinocytosis in Dictyostelium discoideum. Cell Biol Int 21: 237–241. R JJ, T AJ, 1979. The mechanism of action and antitumor platinum compounds. Prog Nucl Acid Res Molec Biol 22: 71–133. R EF, J EG, V B, S DR, 1981. Inhibition of a nutrient-dependent pinocytosis in Dictyostelium discoideum by the amino acid analogue Hadacidin. J Cell Biol 91: 227–231. S BK, C CF, 1979. Interaction of anti tumor drugs with human erythrocyte ghost membranes and mastocytoma P815: a spin label study. Biochem Biophys Res Commun 86: 1051–1057. S TP, 1989. From signal to pseudopod. How cells control cytoplasmic assembly. J Biol Chem 264: 18,261– 18,264. T D, P NC, D JA, M BC, 1979. Cis-platinum (II) diammine dichloride causes mutation, transformation, and sister chromatid exchanges in cultured mammalian cells. Mutat Res 66: 267–275. U N, S C, 1995. Introduction of calcium buffers into the cytosol of Dictyostelium discoideum amoebae alters cell morphology and inhibits chemotaxis. Cell Calcium 17: 97–110.
Cell Biology International, Vol. 23, No. 3, 1999
V G, 1987. Endocytosis and recognition mechanisms in Dictyostelium discoideum. Methods Cell Biol 28: 129–137. V G, T L, S H, S, 1980. Mechanism of phagocytosis in Dictyostelium discoideum: Phagocytosis is
233
mediated by different recognition sites as disclosed by mutants with altered phagocytotic properties. J Cell Biol 86: 456–465.