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c cells in culture
R. Hayashi and C. Balny (Editors), High Pressure Bioscience and Biotechnology 9 1996 Elsevier Science B.V. All rights reserved.
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Effect of hydrostatic pressure on the proliferation and morphology of the mouse BALB/c cells in culture T. Naganuma a,b, T. Mizukoshi c, K. Tsukamoto c, R. Usami c and K. Horikoshi b,c a Faculty of Applied Biological Science, Hiroshima University, 1-4-4 Kagamiyama, Higashi-hiroshima, 739, Japan b Deep Star Program, Japan Marine Science and Technology Center, 2-15 Natsushima-cho, Yokosuka, 237, Japan c Faculty of Engineering, Toyo University, 2100 Kujirai-nakanodai, Kawagoe, 350, Japan Abstract
The mouse BALB/c cells were cultured under hydrostatic pressures of 0.1 to 60 MPa. Proliferation activity declined with the increase of hydrostatic pressure. Sharp decline was observed at the pressure increase from 20 to 30 MPa. Cell viability was lost between 40 and 50 MPa. Cell morphology was affected by hydrostatic pressure. Cells were shortened, collapsed, or coagulated by increased pressure. Cytoskeletal F-actin was also affected by pressure. F-actin lost organization at 50 MPa, and collapsed at 60 MPa. Close association in the pressure-induced loss of proliferation activity, morphology and cytoskeletal organization was confirmed. 1. I N T R O D U C T I O N Effects of hydrostatic pressure on cytological processes have been studied with cultured cells, oocytes, embryos and protozoans [1, 2]. The studies focused mainly on cell viability and proliferation, embryogenesis, polymerization-depolymerization of cytoskeletal filaments, cytoplasmic viscosity and sol-gel transformation, etc. However, the topics were studied independently with different biological sources. Among the cytological topics, cytoskeleton and related-motility seem to have been a primary interest [2]. Microtubule has been intensively studied, probably because of availability and easy determination of assemblydisassembly. Actin filament (F-actin) was mainly studied with invertebrate
74 and yeast cells [2, 3]. The studies with vertebrates are few [2, 4], and the mammalian cells used for the studies were of epithelium origin [2]. This study aims at the coordinative pressure-induced changes in cell proliferation, cell morphology and cytoskeletal F-actin organization. This communication reports the results from a mammalian cell line of fibroblast origin, the mouse BALB/c cells.
2. M A T E R I A L S AND METHODS 2.1. Cell culture under pressure The mouse BALB/c CL.7, which is a normal embryonic fibroblast cell line (ATCC TIB 80) [5] was purchased from ATCC and maintained at 35~ in RPMI 1640 medium (GIBCO BRL) added with 5% (v/v) fetal bovine serum (GIBCO BRL). After several subculturings, the cells were seeded in fresh RPMI 1640 medium (GIBCO BRL) at a density of 103 cells/cm 2 in test tube-like culture flasks (Leyton tubes; Coastar, Cambridge, Massachusetts). The Leyton tube is about 10 cm long and 1 cm wide, and has a flat side for horizontal placement. A plastic cover slip (9 x 55 mm) is placed inside a tube, which the cells attach to and grow on. Immediately after attachment to the slip, the mouse cells were incubated at the pressures (MPa) of 0.1 (atmospheric), 10, 20, 30, 40, 50 and 60 in pressurizing vessels for 24 hours at 35~ The tubes were filled with the medium so that no air space is left.
2.2. Cell proliferation measurement Cell proliferation activity was measured as the increase in cell abundance during the 24-hr incubation. Cell abundance was estimated based on mitochondrial dehydrogenase activity. The dehydrogenase activity was determined by colorimetry of the formazan yielded from the sodium salt of 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl-2H-tetrazolium-5-carboxyanilide, or XTT (Sigma) [6]. The XTT colorimetric incubation was done in RPMI 1640 without phenol red (GIBCOBRL) at 35~ at the atmospheric pressure. The results were expressed as percentage to the maximum activity (colorimetric value). 2.3. Cell m o r p h o l o g y observation Immediately after decompression, the cells were fixed for 30 min at room temperature in a fixation buffer containing 4% paraformaldehyde and 400 mM sucrose in 15 mM Na-HEPES (pH 7.6) [7]. The cell appearance was observed at this stage by phase-contrast microscopy. To increase the cell membrane permeability, the cells were further incubated in ethanol/glacial acetic acid (95:5) at -20~ for 5 min [8], rinsed in deionized distilled water, air-dried, and stored at-80~ Then the cytoskeletal F-actin was stained with 30 units/ml rhodamine phalloidin (Molecular Probes,
75 Inc., Eugene, Oregon) in PBS for 30 min at room temperature, and observed by epifluorescence microscopy.
3. RESULTS AND DISCUSSION
3.1. Cell proliferation activity There was a consistent decrease of growth activity with the increase of pressure (Figure, left column ). A sharp decrease was seen between the pressures of 20 and 30 MPa, where about 60% activity was lowered down to 18%. There was low but positive proliferation at 50 and 60 MPa. However, the proliferation could be false positive, because extracellular (leached) dehydrogenases might cause false positive XTT measurements. Thus the cell proliferation is thought to be positive only up to 40 MPa. This was confirmed by the fact that the 40 MPa-incubated cells recovered full proliferation activity after decompression, while the 50 MPa-incubated cells lost viability. Therefore, there were two breaks in cell proliferation activity. One was the sharp decline between 20 and 30 MPa; and the other was the loss of cell viability (recoverability of the growth) between 40 and 50 MPa. The loss of proliferation activity is, in other words, the blockage of cell division. Earlier observation of the blockage pressure ranged from 20 to 80 MPa, mostly from 30 to 40 MPa [1], with which our observation is in good agreement. 3.2. Cell m o r p h o l o g y The decrease of proliferation activity by pressure was associated with the change of the cell morphology (Figure, middle column ). Typically, the cultured mouse cells are elongated and >100 #m long at the atmospheric pressure. The cell morphology, as well as proliferation activity, was not visually affected at 10 MPa. The cells began to lose elongated morphology and shrink at 20 MPa. The cells became spherical ("rounding-up" [9,10]) at 30 MPa, and even smaller at 40 MPa. The "rounding-up" pressure was previously reported 2040 to 50 MPa for Amoeba cells [1, 10] and 50-70 MPa for human cells [9]. At 50 MPa, the cells were partially collapsed, and coagulated at 60 MPa. The coagulation may suggest the changes in the nature of the the cell membrane surface. The molecular structure of cell membrane is known to be affected by "physiological" pressure of 0.1-100 MPa [11].
3.3. Cytoskeletal F-aetin Cell morphology is largely based on the organization cytoskelton such as actinfilaments (F-actin). Changes in the formation and distribution of Factin under pressure was also observed by epifluorescence microscopy (Figure, right column ).
76 The types of F-actin organization that are generally observed are: stress fibers and the focal contacts; peripheral fibers; and cortical fibers. These organization was still intact at 10 MPa. Stress fiber F-actin was first affected at 20 MPa and retarded in supporting the elongated cell morphology. At 30 MPa, F-actin became accumulated in the peripheral region of the cells; stress fibers were visually disordered, and losing focal contacts. This was responsible for the "rounding-up" of the cells. Stress fibers were completely disordered at 40 MPa, as also shown with in the green monkey cells [2], while peripheral fibers were still organized. However, even the peripheral fibers began to be disturbed at 50 MPa, which might be associated with the loss of cell viability. Similar F-actin disorganization at 50 MPa was observed in yeast cells [3]. At 60 MPa, stress fibers, focal contacts and peripheral fibers were all collapsing. Even cortical fibers seemed to be disordered and F-actin collapsed at 60 MPa. Because the F-actin, as well as microtubules [2], is known to be the most dynamic bio-macromolecule that shows the "dynamic stability" through the balance between polymerization and depolymerization. Also, F-actin has essential functions in various cellular processes such as motility, muscle force generation, cell division and morphology suppot. The cells in culture are suitable for the observation of F-actin organization, and easy to maintain and manipulate. Therefore the cells in culture can serve as a model system to study the pressure effects on the kinetics and dynamism of cellular and biochemical processes.
Figures (next page)
(Left) Effect of hydrostatic pressure on the proliferation of the mouse BALB/c cells in culture.
(Middle column) Effect of hydrostatic pressure on the appearance of the mouse BALB/c cells in culture, observed by phase-contrast microscopy. Scale bar, 50 ktm.
(Right Column) Effect of hydrostatic pressure on the cytoskcletal F-actin (stained with rhodamine phalloidin) of the mouse BALB/c cells in culture, observed by epifluorescencc microscopy. Scale bar, 50 gin.
77
78 4. REFERENCES
1 F. H. Johnson, H. Eyring and M.J. Polissar, The Kinetic Basis of Molecular Biology, John Wiley & Sons, Inc., New York, 1954. 2 H.W. Jannasch, R.E. Marquis and A.M. Zimmerman (eds.), Current Perspectives in High Pressure Biology, Academic Press, London, 1987. 3 H. Kobori, M. Sato, Z.H. Feng, A. Tameike, K. Hamada, S. Shimada and M. Osumi, Program and Abstracts of International Conference on High Pressure Bioscience and Biotechnology, Kyoto (1995) 60. 4 R.R. Swezey and G.N. Somero, Biochemistry, 21 (1982) 4496. 5 P. Patek, J. Collins and M. Cohn, Nature 276 (1978) 510. 6 N.W. Roehm, G.H. Rodgers, S.M. Hatfield and A.L. Glasebrook, J. Immunol. Methods, 142 (1991) 257. 7 P. Forscher and S.J. Smith, J. Cell Biol., 197 (1988) 1505. 8 A. Sarzinski-Powitz (1992) In" Nonradioactive In Situ Hybridization Application Manual, Boehringer Mannheim, Indianapolis (1992) 44. 9 J.V. Landau, Exp. Cell Res., 23 (1961) 538. 10 J. V. Landau and L. Thibodeau, Exp. Cell Res., 27 (1962) 591.
73
Effect of hydrostatic pressure on the proliferation and morphology of the mouse BALB/c cells in culture T. Naganuma a,b, T. Mizukoshi c, K. Tsukamoto c, R. Usami c and K. Horikoshi b,c a Faculty of Applied Biological Science, Hiroshima University, 1-4-4 Kagamiyama, Higashi-hiroshima, 739, Japan b Deep Star Program, Japan Marine Science and Technology Center, 2-15 Natsushima-cho, Yokosuka, 237, Japan c Faculty of Engineering, Toyo University, 2100 Kujirai-nakanodai, Kawagoe, 350, Japan Abstract
The mouse BALB/c cells were cultured under hydrostatic pressures of 0.1 to 60 MPa. Proliferation activity declined with the increase of hydrostatic pressure. Sharp decline was observed at the pressure increase from 20 to 30 MPa. Cell viability was lost between 40 and 50 MPa. Cell morphology was affected by hydrostatic pressure. Cells were shortened, collapsed, or coagulated by increased pressure. Cytoskeletal F-actin was also affected by pressure. F-actin lost organization at 50 MPa, and collapsed at 60 MPa. Close association in the pressure-induced loss of proliferation activity, morphology and cytoskeletal organization was confirmed. 1. I N T R O D U C T I O N Effects of hydrostatic pressure on cytological processes have been studied with cultured cells, oocytes, embryos and protozoans [1, 2]. The studies focused mainly on cell viability and proliferation, embryogenesis, polymerization-depolymerization of cytoskeletal filaments, cytoplasmic viscosity and sol-gel transformation, etc. However, the topics were studied independently with different biological sources. Among the cytological topics, cytoskeleton and related-motility seem to have been a primary interest [2]. Microtubule has been intensively studied, probably because of availability and easy determination of assemblydisassembly. Actin filament (F-actin) was mainly studied with invertebrate
74 and yeast cells [2, 3]. The studies with vertebrates are few [2, 4], and the mammalian cells used for the studies were of epithelium origin [2]. This study aims at the coordinative pressure-induced changes in cell proliferation, cell morphology and cytoskeletal F-actin organization. This communication reports the results from a mammalian cell line of fibroblast origin, the mouse BALB/c cells.
2. M A T E R I A L S AND METHODS 2.1. Cell culture under pressure The mouse BALB/c CL.7, which is a normal embryonic fibroblast cell line (ATCC TIB 80) [5] was purchased from ATCC and maintained at 35~ in RPMI 1640 medium (GIBCO BRL) added with 5% (v/v) fetal bovine serum (GIBCO BRL). After several subculturings, the cells were seeded in fresh RPMI 1640 medium (GIBCO BRL) at a density of 103 cells/cm 2 in test tube-like culture flasks (Leyton tubes; Coastar, Cambridge, Massachusetts). The Leyton tube is about 10 cm long and 1 cm wide, and has a flat side for horizontal placement. A plastic cover slip (9 x 55 mm) is placed inside a tube, which the cells attach to and grow on. Immediately after attachment to the slip, the mouse cells were incubated at the pressures (MPa) of 0.1 (atmospheric), 10, 20, 30, 40, 50 and 60 in pressurizing vessels for 24 hours at 35~ The tubes were filled with the medium so that no air space is left.
2.2. Cell proliferation measurement Cell proliferation activity was measured as the increase in cell abundance during the 24-hr incubation. Cell abundance was estimated based on mitochondrial dehydrogenase activity. The dehydrogenase activity was determined by colorimetry of the formazan yielded from the sodium salt of 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl-2H-tetrazolium-5-carboxyanilide, or XTT (Sigma) [6]. The XTT colorimetric incubation was done in RPMI 1640 without phenol red (GIBCOBRL) at 35~ at the atmospheric pressure. The results were expressed as percentage to the maximum activity (colorimetric value). 2.3. Cell m o r p h o l o g y observation Immediately after decompression, the cells were fixed for 30 min at room temperature in a fixation buffer containing 4% paraformaldehyde and 400 mM sucrose in 15 mM Na-HEPES (pH 7.6) [7]. The cell appearance was observed at this stage by phase-contrast microscopy. To increase the cell membrane permeability, the cells were further incubated in ethanol/glacial acetic acid (95:5) at -20~ for 5 min [8], rinsed in deionized distilled water, air-dried, and stored at-80~ Then the cytoskeletal F-actin was stained with 30 units/ml rhodamine phalloidin (Molecular Probes,
75 Inc., Eugene, Oregon) in PBS for 30 min at room temperature, and observed by epifluorescence microscopy.
3. RESULTS AND DISCUSSION
3.1. Cell proliferation activity There was a consistent decrease of growth activity with the increase of pressure (Figure, left column ). A sharp decrease was seen between the pressures of 20 and 30 MPa, where about 60% activity was lowered down to 18%. There was low but positive proliferation at 50 and 60 MPa. However, the proliferation could be false positive, because extracellular (leached) dehydrogenases might cause false positive XTT measurements. Thus the cell proliferation is thought to be positive only up to 40 MPa. This was confirmed by the fact that the 40 MPa-incubated cells recovered full proliferation activity after decompression, while the 50 MPa-incubated cells lost viability. Therefore, there were two breaks in cell proliferation activity. One was the sharp decline between 20 and 30 MPa; and the other was the loss of cell viability (recoverability of the growth) between 40 and 50 MPa. The loss of proliferation activity is, in other words, the blockage of cell division. Earlier observation of the blockage pressure ranged from 20 to 80 MPa, mostly from 30 to 40 MPa [1], with which our observation is in good agreement. 3.2. Cell m o r p h o l o g y The decrease of proliferation activity by pressure was associated with the change of the cell morphology (Figure, middle column ). Typically, the cultured mouse cells are elongated and >100 #m long at the atmospheric pressure. The cell morphology, as well as proliferation activity, was not visually affected at 10 MPa. The cells began to lose elongated morphology and shrink at 20 MPa. The cells became spherical ("rounding-up" [9,10]) at 30 MPa, and even smaller at 40 MPa. The "rounding-up" pressure was previously reported 2040 to 50 MPa for Amoeba cells [1, 10] and 50-70 MPa for human cells [9]. At 50 MPa, the cells were partially collapsed, and coagulated at 60 MPa. The coagulation may suggest the changes in the nature of the the cell membrane surface. The molecular structure of cell membrane is known to be affected by "physiological" pressure of 0.1-100 MPa [11].
3.3. Cytoskeletal F-aetin Cell morphology is largely based on the organization cytoskelton such as actinfilaments (F-actin). Changes in the formation and distribution of Factin under pressure was also observed by epifluorescence microscopy (Figure, right column ).
76 The types of F-actin organization that are generally observed are: stress fibers and the focal contacts; peripheral fibers; and cortical fibers. These organization was still intact at 10 MPa. Stress fiber F-actin was first affected at 20 MPa and retarded in supporting the elongated cell morphology. At 30 MPa, F-actin became accumulated in the peripheral region of the cells; stress fibers were visually disordered, and losing focal contacts. This was responsible for the "rounding-up" of the cells. Stress fibers were completely disordered at 40 MPa, as also shown with in the green monkey cells [2], while peripheral fibers were still organized. However, even the peripheral fibers began to be disturbed at 50 MPa, which might be associated with the loss of cell viability. Similar F-actin disorganization at 50 MPa was observed in yeast cells [3]. At 60 MPa, stress fibers, focal contacts and peripheral fibers were all collapsing. Even cortical fibers seemed to be disordered and F-actin collapsed at 60 MPa. Because the F-actin, as well as microtubules [2], is known to be the most dynamic bio-macromolecule that shows the "dynamic stability" through the balance between polymerization and depolymerization. Also, F-actin has essential functions in various cellular processes such as motility, muscle force generation, cell division and morphology suppot. The cells in culture are suitable for the observation of F-actin organization, and easy to maintain and manipulate. Therefore the cells in culture can serve as a model system to study the pressure effects on the kinetics and dynamism of cellular and biochemical processes.
Figures (next page)
(Left) Effect of hydrostatic pressure on the proliferation of the mouse BALB/c cells in culture.
(Middle column) Effect of hydrostatic pressure on the appearance of the mouse BALB/c cells in culture, observed by phase-contrast microscopy. Scale bar, 50 ktm.
(Right Column) Effect of hydrostatic pressure on the cytoskcletal F-actin (stained with rhodamine phalloidin) of the mouse BALB/c cells in culture, observed by epifluorescencc microscopy. Scale bar, 50 gin.
77
78 4. REFERENCES
1 F. H. Johnson, H. Eyring and M.J. Polissar, The Kinetic Basis of Molecular Biology, John Wiley & Sons, Inc., New York, 1954. 2 H.W. Jannasch, R.E. Marquis and A.M. Zimmerman (eds.), Current Perspectives in High Pressure Biology, Academic Press, London, 1987. 3 H. Kobori, M. Sato, Z.H. Feng, A. Tameike, K. Hamada, S. Shimada and M. Osumi, Program and Abstracts of International Conference on High Pressure Bioscience and Biotechnology, Kyoto (1995) 60. 4 R.R. Swezey and G.N. Somero, Biochemistry, 21 (1982) 4496. 5 P. Patek, J. Collins and M. Cohn, Nature 276 (1978) 510. 6 N.W. Roehm, G.H. Rodgers, S.M. Hatfield and A.L. Glasebrook, J. Immunol. Methods, 142 (1991) 257. 7 P. Forscher and S.J. Smith, J. Cell Biol., 197 (1988) 1505. 8 A. Sarzinski-Powitz (1992) In" Nonradioactive In Situ Hybridization Application Manual, Boehringer Mannheim, Indianapolis (1992) 44. 9 J.V. Landau, Exp. Cell Res., 23 (1961) 538. 10 J. V. Landau and L. Thibodeau, Exp. Cell Res., 27 (1962) 591.