Alteration by Malignant Transformation of the Calcium Requirements for Cell Proliferation In Vitro" 2 S. H. H. Swierenga, J. F. Whitfield, and D. J. Gillan 3 ABSTRACT-Cells from the thigh muscles of normal fetal rats proliferated rapidly and indefinitely in a medium containing adult rat "plasma" and a normal free·calcium concentration, but they could not proliferate in calcium·deficient plasma medium. As the animals grew older, the cells became increasingly less able to proliferate even in normal (high-calcium) plasma medium, though they retained the potential to proliferate in a more conventional medium containing fetal bovine serum. By contrast, neoplastic adult cells from malignant rhabdomyosarcomas (induced by NiaS2 ) proliferated rapidly and indefinitely in both normal and lowcalcium plasma medium.-J Natl Cancer Inst 57: 125-129, 1976.
The calcium ion controls the proliferation of a wide variety of cells (chicken fibroblasts; human peripheral lymphoblasts; mouse BALB/c and Swiss albino 3T3 cells; rat bone marrow cells; heart cells, hepatocytes, thymic lymphoblasts) by triggering a part of the preDNA synthetic reaction sequence (1-13). The possibility of calcium's proliferative control system being altered in neoplastic transformation is suggested by the ability of chicken fibroblasts infected in vitro with the Rous sarcoma virus to proliferate rapidly in a low-calcium medium containing homologous (chicken) "plasma" that does not support DNA synthesis or multiplication of uninfected cells (1). To begin exploring this possibility, we compared the proliferation of normal cells from rat skeletal muscles and corresponding neoplastic cells from Ni 3 S2-induced rhabdomyosarcomas and mixed rhabdomyoibrosarcomas in a calcium-deficient medium containing homologous rat plasma. MATERIALS AND METHODS
Tumor induction and characteristics.-Primary tumors were induced by implantation of 10 mg washed Ni 3 S2 (suspended in 0.1 ml PBS containing 2,000 IU procaine penicillin G and 2.5 mg dihydrostreptomycin sulfate) into the right thigh muscles (14) of specific-pathogenfree, male 200-g Sprague-Dawley rats that had been bred in this laboratory. The same volume (0.1 ml) of suspending fluid only was injected im into the left thigh of each animal. As expected from previous studies (14, 15), the readily transplantable malignant tumors that appeared 5 months later in the right thighs of 80% of the animals were diagnosed by Dr. K. Sarkar of the Ottawa University Medical School as rhabdomyosarcomas or mixed rhabdomyofibrosarcomas. Secondary malignant tumors invariably appeared in the right thighs of young adult male rats only 1-3 weeks after injection of 106 cells from primary tumors or cultures of primary tumors. Culture media.-Cells from normal muscle tissue or tumors were cultivated in either a plasma or serum medium consisting of 90% (vol/vol) medium M-150 (16) and 10% (vol/vol) heat-inactivated (at 56° C for 30 min) FBS (Grand Island Biological Co., Grand Island, N.Y.) or in rat plasma prepared in this laboratory. For preparation of rat plasma, blood was removed VOL. 57, NO. I, JULY 1976
125
without anticoagulants from the dorsal aortae of adult (300 g) male rats with an ice-cold Venotube (Abbott Laboratories, Montreal, Quebec, Canada) fitted with a #18 needle. The first few drops of blood were discarded to avoid contamination with products from injured tissue. The remainder was collected in unstoppered, "siliconized" Vacutainer tubes (Becton, Dickinson & Co., Rutherford, N.J.) that had been precooled to 0° C. The blood cells were sedimented by centrifugation (l,OOOXg for 30 min) at 0° C, and the supernatant plasma was transferred to cold siliconized tubes. Like the commercial FBS, the plasma was then heated to 56° C for 30 minutes, sterilized by filtration through a Nalgene (Nalge, Rochester, N.Y.) filter (0.2-J-L pore size), and then frozen. The combination of clean bleeding (with minimal wounding), ice-cold containers, and siliconized surfaces prevented clotting throughout the procedure. This plasma, which did not clot during subsequent use, presumably contained fibrinogen (but not platelets) and a full complement of the clotting factors, some of which might have been inactivated by the heat treatment. The average calcium concentration of complete medium containing 10% rat plasma (vol/vol) was 1.3 mM, and of medium containing 10% (vol/vol) FBS, 1.4 mM. In such complete media, most of the calcium (95%) is ionic or readily ionized (hence physiologically available) in an atmosphere of 5% CO 2-95% air because of the tenfold dilution of the calcium-binding serum proteins. To prepare low-calcium medium, each batch of complete medium (containing either plasma or FBS) ~as titrated with a G.K. Turner Model III fluorometer WIth the specific calcium chelator EGT A (Eastman Organic Chemicals, Rochester, N. Y.), to contain a nonchelated or ionizable calcium concentration of 0.01 or 0.02 mM, according to the very sensitive method of Borle and Briggs (17). This procedure could not have significantly affected the concentration of other ions such as Mg2+. In addition, Ca-EGTA itself does not affect DNA synthesis or proliferation in vitro, as shown in (2, 8-11). The media were stored in Teflon bottles to prevent calcium interaction with the container. The calcium concentration in each stock medium was reassayed immediately before use. Culture procedures.-The trypsin method of Basrur and Gilman (18) was used to isolate cells for primary cultures from firm, nonnecrotic regions of primary or secondary (transplanted) tumors and from the thigh muscles of 16- to 20-day-old fetal rats. We prepared ABBREVIATIONS USED: PBS=phosphate-buffered saline; FBS=fetal bovine serum; EGT A=[ ethylenebis(oxyethylenenitrilol]tetraacetic acid. Received September 22, 1975; accepted January 5, 1976. Issued as National Research Council of Canada (NRCC) No. 15271. a Animal and Cell Physiology Group, Division of Biological Sciences, NRCC, Ottawa, Ontario, Canada KIA OR6. 1
2
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SWIERENGA, WHITFIELD, AND GILLAN
secondary tumor cell cultures (i.e., first subcultures) without using trypsin by gently shaking dense primary cultures to dislodge all clumps and seeding the suspended cells in fresh plasma medium. Cell cultures from the normal left thigh muscles of the 5-month-old, 400- to 500-g tumor-bearing rats were most effectively prepared by the method of Swierenga et al. (8). Tissue fragments were maintained in plasma medium (which, however, allowed only slow multiplication of such adult cells) until the cells growing from the margins ofthe explants formed monolayers. The monolayers were dispersed with 0.25% trypsin (l :250; Difco Laboratories, Inc., Detroit, Mich.) and reseeded in fresh medium as secondary cultures. Experimental cell cultures were always started in medium containing 1.3 mM calcium. Twenty-four hours later, this normal, high-calcium medium was removed from some of the dishes and replaced with the appropriate low-calcium medium after the cells had been thoroughly rinsed with calcium- and magnesium-free PBS (19). The extracellular free-calcium concentration could be subsequently raised to the normal value by the addi-
200
Fetal Cells
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tion of an appropriate volume of a solution of 50 mM CaCI2 • Cell counting and autoradiography.-The cultures were rinsed with calcium- and magnesium-free PBS and the gcells suspended by a I-hour exposure to either 0.25% trypsin plus 1.3 mM EGT A in divalent, cation-free PBS (for cells grown in high-calcium medium) or to 0.25% trypsin (in PBS) alone (for cells maintained in low-calcium medium). Such prolonged trypsinization was needed for the complete dispersion of cell clumps into their constituent single cells. The cells were finally suspended in Isoton R fluid (Coulter Electronics, Hialeah, Fla.) before being enumerated with a Coulter Model F cell counter. The effects of calcium and serum factor(s) on DNA synthesis were determined autoradiographically with cultures grown on plastic cover slips (Flow Laboratories, Rockville, Md.) in 35-mm petri dishes. The cultures were exposed for 30 minutes to 10 /LCi [3H]thymidine/ ml (New England Nuclear Corp., Boston, Mass.) with a specific activity of 20 Ci/mmole. Such pulse-labeled cells were rinsed with PBS, fixed in a solution consisting of 9 parts phosphate-buffered neutral formalin and 1 part glacial acetic acid, washed (at room temperature) with a
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DAYS IN CULTURE TEXT-FIGURE I.-Examples of multiplication of cells from fetal and adult thigh muscles in high (1.3 mM) or low (0.02 mM) levels of extracellular (nonchelated) calcium in a medium containing lO% (vol/vol) rat plasma and 90% (vol/vol) medium M-I50. All cells were plated in high-calcium medium on day 0 and exposed 24 hours later to low-calcium medium. Closed and open symbols denote cell populations from different animals. Points and vertical lines are meanS±SE of values from four dishes. NATL CANCER INST
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The calcium ion controls the proliferation of a wide variety of cells (chicken fibroblasts; human peripheral lymphoblasts; mouse BALB/c and Swiss albino 3T3 cells; rat bone marrow cells; heart cells, hepatocytes, thymic lymphoblasts) by triggering a part of the preDNA synthetic reaction sequence (1-13). The possibility of calcium's proliferative control system being altered in neoplastic transformation is suggested by the ability of chicken fibroblasts infected in vitro with the Rous sarcoma virus to proliferate rapidly in a low-calcium medium containing homologous (chicken) "plasma" that does not support DNA synthesis or multiplication of uninfected cells (1). To begin exploring this possibility, we compared the proliferation of normal cells from rat skeletal muscles and corresponding neoplastic cells from Ni 3 S2-induced rhabdomyosarcomas and mixed rhabdomyoibrosarcomas in a calcium-deficient medium containing homologous rat plasma. MATERIALS AND METHODS
Tumor induction and characteristics.-Primary tumors were induced by implantation of 10 mg washed Ni 3 S2 (suspended in 0.1 ml PBS containing 2,000 IU procaine penicillin G and 2.5 mg dihydrostreptomycin sulfate) into the right thigh muscles (14) of specific-pathogenfree, male 200-g Sprague-Dawley rats that had been bred in this laboratory. The same volume (0.1 ml) of suspending fluid only was injected im into the left thigh of each animal. As expected from previous studies (14, 15), the readily transplantable malignant tumors that appeared 5 months later in the right thighs of 80% of the animals were diagnosed by Dr. K. Sarkar of the Ottawa University Medical School as rhabdomyosarcomas or mixed rhabdomyofibrosarcomas. Secondary malignant tumors invariably appeared in the right thighs of young adult male rats only 1-3 weeks after injection of 106 cells from primary tumors or cultures of primary tumors. Culture media.-Cells from normal muscle tissue or tumors were cultivated in either a plasma or serum medium consisting of 90% (vol/vol) medium M-150 (16) and 10% (vol/vol) heat-inactivated (at 56° C for 30 min) FBS (Grand Island Biological Co., Grand Island, N.Y.) or in rat plasma prepared in this laboratory. For preparation of rat plasma, blood was removed VOL. 57, NO. I, JULY 1976
125
without anticoagulants from the dorsal aortae of adult (300 g) male rats with an ice-cold Venotube (Abbott Laboratories, Montreal, Quebec, Canada) fitted with a #18 needle. The first few drops of blood were discarded to avoid contamination with products from injured tissue. The remainder was collected in unstoppered, "siliconized" Vacutainer tubes (Becton, Dickinson & Co., Rutherford, N.J.) that had been precooled to 0° C. The blood cells were sedimented by centrifugation (l,OOOXg for 30 min) at 0° C, and the supernatant plasma was transferred to cold siliconized tubes. Like the commercial FBS, the plasma was then heated to 56° C for 30 minutes, sterilized by filtration through a Nalgene (Nalge, Rochester, N.Y.) filter (0.2-J-L pore size), and then frozen. The combination of clean bleeding (with minimal wounding), ice-cold containers, and siliconized surfaces prevented clotting throughout the procedure. This plasma, which did not clot during subsequent use, presumably contained fibrinogen (but not platelets) and a full complement of the clotting factors, some of which might have been inactivated by the heat treatment. The average calcium concentration of complete medium containing 10% rat plasma (vol/vol) was 1.3 mM, and of medium containing 10% (vol/vol) FBS, 1.4 mM. In such complete media, most of the calcium (95%) is ionic or readily ionized (hence physiologically available) in an atmosphere of 5% CO 2-95% air because of the tenfold dilution of the calcium-binding serum proteins. To prepare low-calcium medium, each batch of complete medium (containing either plasma or FBS) ~as titrated with a G.K. Turner Model III fluorometer WIth the specific calcium chelator EGT A (Eastman Organic Chemicals, Rochester, N. Y.), to contain a nonchelated or ionizable calcium concentration of 0.01 or 0.02 mM, according to the very sensitive method of Borle and Briggs (17). This procedure could not have significantly affected the concentration of other ions such as Mg2+. In addition, Ca-EGTA itself does not affect DNA synthesis or proliferation in vitro, as shown in (2, 8-11). The media were stored in Teflon bottles to prevent calcium interaction with the container. The calcium concentration in each stock medium was reassayed immediately before use. Culture procedures.-The trypsin method of Basrur and Gilman (18) was used to isolate cells for primary cultures from firm, nonnecrotic regions of primary or secondary (transplanted) tumors and from the thigh muscles of 16- to 20-day-old fetal rats. We prepared ABBREVIATIONS USED: PBS=phosphate-buffered saline; FBS=fetal bovine serum; EGT A=[ ethylenebis(oxyethylenenitrilol]tetraacetic acid. Received September 22, 1975; accepted January 5, 1976. Issued as National Research Council of Canada (NRCC) No. 15271. a Animal and Cell Physiology Group, Division of Biological Sciences, NRCC, Ottawa, Ontario, Canada KIA OR6. 1
2
J
NATL CANCER INST
126
SWIERENGA, WHITFIELD, AND GILLAN
secondary tumor cell cultures (i.e., first subcultures) without using trypsin by gently shaking dense primary cultures to dislodge all clumps and seeding the suspended cells in fresh plasma medium. Cell cultures from the normal left thigh muscles of the 5-month-old, 400- to 500-g tumor-bearing rats were most effectively prepared by the method of Swierenga et al. (8). Tissue fragments were maintained in plasma medium (which, however, allowed only slow multiplication of such adult cells) until the cells growing from the margins ofthe explants formed monolayers. The monolayers were dispersed with 0.25% trypsin (l :250; Difco Laboratories, Inc., Detroit, Mich.) and reseeded in fresh medium as secondary cultures. Experimental cell cultures were always started in medium containing 1.3 mM calcium. Twenty-four hours later, this normal, high-calcium medium was removed from some of the dishes and replaced with the appropriate low-calcium medium after the cells had been thoroughly rinsed with calcium- and magnesium-free PBS (19). The extracellular free-calcium concentration could be subsequently raised to the normal value by the addi-
200
Fetal Cells
Adult Cells
tion of an appropriate volume of a solution of 50 mM CaCI2 • Cell counting and autoradiography.-The cultures were rinsed with calcium- and magnesium-free PBS and the gcells suspended by a I-hour exposure to either 0.25% trypsin plus 1.3 mM EGT A in divalent, cation-free PBS (for cells grown in high-calcium medium) or to 0.25% trypsin (in PBS) alone (for cells maintained in low-calcium medium). Such prolonged trypsinization was needed for the complete dispersion of cell clumps into their constituent single cells. The cells were finally suspended in Isoton R fluid (Coulter Electronics, Hialeah, Fla.) before being enumerated with a Coulter Model F cell counter. The effects of calcium and serum factor(s) on DNA synthesis were determined autoradiographically with cultures grown on plastic cover slips (Flow Laboratories, Rockville, Md.) in 35-mm petri dishes. The cultures were exposed for 30 minutes to 10 /LCi [3H]thymidine/ ml (New England Nuclear Corp., Boston, Mass.) with a specific activity of 20 Ci/mmole. Such pulse-labeled cells were rinsed with PBS, fixed in a solution consisting of 9 parts phosphate-buffered neutral formalin and 1 part glacial acetic acid, washed (at room temperature) with a
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DAYS IN CULTURE TEXT-FIGURE I.-Examples of multiplication of cells from fetal and adult thigh muscles in high (1.3 mM) or low (0.02 mM) levels of extracellular (nonchelated) calcium in a medium containing lO% (vol/vol) rat plasma and 90% (vol/vol) medium M-I50. All cells were plated in high-calcium medium on day 0 and exposed 24 hours later to low-calcium medium. Closed and open symbols denote cell populations from different animals. Points and vertical lines are meanS±SE of values from four dishes. NATL CANCER INST
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