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Growth in suspension culture of rat pituitary cells which produce growth hormone and prolactin
Experimental Cell Research 64 (1971) 125-128
GROWTH WHICH
IN SUSPENSION PRODUCE
CULTURE
GROWTH
F. C. BANCROFT’
OF RAT PITUITARY
HORMONE
AND
and A. H. TASHJIAN,
CELLS
PROLACTIN
Jr
Pharmacology Department, Harvard School of Dental Medicine, and Department of Pharmacology, Harvard Medical School, Boston, Mass. 02115, USA
SUMMARY The GH, strain of rat pituitary tumor cells has been adapted to growth in suspension culture. The cells have been grown in this fashion for a year and have maintained a doubling time of 42-48 h. Theq’ have continued to produce both growth hormone and prolactin. When the cells were exposedto hydrocortisone (3 x 1O-BM), GH production was stimulated by about 7-fold, and prolactin production was decreased by about 3-to 5-fold, while the cell doubling time was unaffected.
In recent years a variety of strains of neoplastic cells capable of performing differentiated functions has been established in monolayer culture by Sato and co-workers [l-7]. Studies of the control of the expression of differentiated function by these cells would be facilitated if the cells could be grown in suspension culture as well. One of these cell strains (GH,) is derived from a rat pituitary tumor, and has been shown to produce both GH [2, 7j and prolactin [8]. Studies of the control of the production of these protein hormones by GH, cells have revealed that HC stimulates GH production [9] and inhibits prolactin production [8]. We report here studies on GH, cells which have 1 Present address: Deuartment of Biolo&al Sciences. Columbia University;New York, N. Y: 10027, USA: Abbreviations used: GH, growth hormone; HC, hydrocortisone sodium succinate.
been grown in suspension culture for a year. In this condition of growth the cells continue to produce both GH and prolactin. In addition, the effects of HC on the production of these hormones are similar to those observed when the cells are grown in monolayer culture. MATERIALS
AND METHODS
Plastic tissue culture dishes were obtained from Falcon Plastics (Los Angeles, Calif.). Plastic bacterial dishes, which‘are not-treated for’ growth of animal cells in monolayer, were also obtained from Falcon. The cells (GH,) used in these experiments have been described in detail previously [7-91. Until the present experiments were performed they were grown in monolayer in complete Ham FlO medium [7, 91. In suspension culture the cells were grown at 36.5”C in Minimum Essential Medium (Spinner) [lo] (Grand Island Biological Co., Grand Island, N. Y.), supplemented with 15 % horse serum and 2.5 % fetal calf serum (complete spinner medium) in spinner bottles purchased either from Metaloglass, Inc. (Boston, Mass). or Bellco Glass, Inc. (Vineland, N. J.). Hydrocortisone sodium succinate (Solu-Cortef) was obtained from the Upjohn Co. (Kalamazoo, Mich.) Exptl Cell Res 64.
126 F. C. Bancroft & A. H. Tashjian Jr Viokase was purchased from the Viobin Corp. (Monticello, Ill.). Cell number was determined from cell counts performed in duplicate using a hemocytometer. The method of Lowry et al. [II] was used to measure the protein content of cells which had been washed with Minimum Essential Medium (Spinner) balanced salts. GH and prolactin concentrations in the growth medium were measured immunologically by the method of microcomplement fixation, as described previously [2, 81. The reproducibility of the immunological assays as performed in these experiments was+ 15 %. Nine months after the GH, cells were established in suspension culture, they were shown to be free of pleuropneumonia-like organisms by Dr Iolanda E. Low (Harvard Medical School).
RESULTS AND DISCUSSION The GH, cells were introduced into suspension culture as follows. Cells growing in monolayer in complete Ham FlO medium were removed with Viokase and placed in bacterial Petri dishes in complete spinner medium. In these dishes the cells aggregated to form floating clusters, each containing about 50100 cells. They were maintained in this condition for 1 week, after which they were placed in a 50 ml spinner flask in complete spinner medium, at a density of about 2 x lo5 cells/ml. Within a day the large clusters had dispersed into smaller clusters containing about 5-10 cells each, and within a week the majority of these clusters had completely dispersed into individual cells. (It has since been found that GH, cells which have been removed from monolayer culture with Viokase will begin growing immediately when introduced directly into suspension culture.) Cell number and GH and prolactin concentrations in the growth medium were followed beginning 10 days after the cells were first placed in suspension culture. The cells were observed to grow exponentially from 2 x lo5 to 8 x lo5 cells/ml with a doubling time of about 48 h. Growth hormone and prolactin concentrations increased about loExptl Cell Res 64
1
2.8 2.4
-
2.0
-
1.6 -
1.2-
f
I.O-
:
0.8-
(
0.6-
:
0.4
0.2-
-
2
1
JI
J 5
Fig. 1. Abscissa: days; oukr ordinate: A -A, ,ug prolactin/ml; inner ordinate: (left) O-O, cells x 10-5/ml;(right) l - l , ,ug GH/ml. Growth of GH, cells in suspension, and production of GH and Prolactin.
fold during this experiment, indicating that both hormones were being produced by the cells. GH, cells have been maintained almost continuously in suspension culture for 1 year. Two-and-one-half months after their establishment in suspension culture they were frozen at -196°C in 10% glycerol. Two weeks later the cells were thawed and returned directly to suspension culture, whereupon they began to grow. Growth of GH, cells and the concentrations of GH and prolactin in the medium were followed in an experiment performed 9 months after the cells were established in suspension culture (fig. 1). The cells grew with a population doubling time of about 46 h. GH and prolactin concentrations increased continuously during the experiment,
Rat pituitary
cells which produce growth hormone and prolactin
indicating that both hormones were being produced by the GH, cells. We have reported previously the stimulation by HC of GH production by GH, cells in monolayer culture [9]. In experiments carried out 9; months after the cells were established in suspension culture, we investigated whether HC had a similar effect on cells growing in suspension (fig. 2). To compare results with those obtained previously [9], it is necessary to express GH production in the same units, i.e., ,ug GH/mg cell protein/24 h. Therefore, cell growth was followed by measuring both cell protein and cell number. GH production over a 24 h interval was then estimated from the difference between the GH concentrations measured at the beginning and end of the interval, divided by the average cell protein concentration during the interval. HC (3 x 1O-6 M) was observed to stimulate GH production. The maximum stimulation, about 7-fold, was observed by 60 h, when the stimulated rate was about 70 pugGH/mg cell protein/24 h (fig. 2). These values are slightly higher than those observed when GH, cells growing exponentially in monolayer culture are exposed to HC [9]. In addition, in suspension culture maximum stimulation by HC appears to be achieved more rapidly than in monolayer [9]. We have recently observed that HC decreases the rate of prolactin production by GH, cells in monolayer culture by about 50% [8]. It is seen in fig. 2 that HC, after a lag of about 48 h, decreased prolactin production by GH, cells in suspension culture by 3- to 5-fold. HC does not appear to affect the growth rate of GH, cells in suspension (fig. 2). This is in contrast to what is observed in monolayer culture, where HC (3 x 10~~ M) increased the population doubling time (measured by following cell protein) by about 50% [9].
CELL
127
PROJEIN
~
b
/
4
c
PROL ACT/N
c --
120
Fig. 2. Abscissa: hours; ordinate: (a) pg cell protein/ mg; (b) pg GH/mg cell protein/24 h; (c) pg prolactin/mg cell protein/24 h. x - x , - HC; O-O, + HC. Effects of HC on growth, and GH and prolactin production of GH, cells in suspension.At zero time a suspension culture was divided equally between two spinner bottles. One bottle (+ HC) receivedHC at a final concentration of 3 x 1O-6M, and the other ( - HC) received an equal volume of saline. Additions were repeatedon the 2nd and 4th day of the experiment. Aliquots were removed every 24 h for assay of cell number, cell protein, and GH and prolactin concentration. GH and prolactin production were calculated as describedin the text. The vertical bars on eachGH and prolactin point represent the uncertainty in the difference betweentwo values each having an uncertainty of i 15%.
Exptl Cell Res 64
128 F. C. Bancrojt & A. H. Tashjian Jr It is noteworthy that while cell protein increased with a doubling time of about 60 h (fig. 2), the doubling time for cell number measured in the same experiment was about 42 h. This difference must of course arise from a decrease in the amount of protein per cell as cell density increases. The former quantity was in fact observed to drop from 2.4 x IO-’ to 1.6 x IO-’ mg cell protein/cell during the course of this experiment. The latter value is in good agreement with that previously measured for GH, cells in the early stationary phase of growth in monolayer culture (1.7 x lo-’ mg cell protein/cell)
PIThe ability to grow GH, cells in suspension culture, and to bring about effects of HC on GH and prolactin production similar to those observed in monolayer, should facilitate further biochemical studies of the control of the production of these protein hormones. Furthermore, HC has now been observed to have similar effects on protein hormone production by GH, cells in three different growth states: in both the exponential and stationary phases of growth in monolayer [8, 91 and, as reported here, during growth in suspension. Thus these effects are seen to be an inherent property of the GH, cells,
Exptl Cd Res 64
and not merely a reflection state of growth.
of a particular
The authors wish to thank Miss Norma J. Barowsky, Mrs Adele K. Gallucci. and Mrs Lethia S. Gilliard for expert assistance. We are also most grateful to Dr Lawrence Levine. Graduate Denartment of Biochemistry, Brandeis University, for his help with the immunoassays; and to Dr Iolanda K. Low, Harvard Medical School, for examining the GH, cells for PPLO contamination. This investigation was supported in part by research grants from the National Institutes of Health (AM-11011) and the American Cancer Society, (Massachusetts Division, Award No. P-374) and a General Research Support Grant (RR-05318).
REFERENCES 1. Sato, G H & Yasumura, Y, Trans NY acad sci 28 (1966) 1063. 2. Tashjian, A H Jr, Yasumura, Y, Levine, L, Sato, G H & Parker, M L, Endocrinology 82 (1968) 342. 3. Shin,‘S-I, Endocrinology 81 (1967) 440. 4. Benda, P, Lightbody, J, Sato, G H, Levine, L & Sweet, W, Science 161 (1968) 370. 5. Richardson, U I, Tashjian, A H Jr & Levine, L, J cell biol 40 (1969) 236. 6. Augusti-Tocco. G & Sato. G. Proc natl acad sci US-64 (1969) 311. ’ ’ 7. Yasumura, Y, Tashjian, A H Jr & Sato, G H, Science 154 (1966) 1186. 8. Tashjian, A H Jr, Bancroft, F C & Levine, L, J cell biol 47 (1970) 61. 9. Bancroft, F C, Leve,in L & Tashjian, A H Jr, J cell biol 43 (1969) 432. 10. Eagle, H, Science 130 (1959) 432. 11. Lowry, 0 H, Rosebrough, N F, Farr, A L & Randall, R J, J biol them 193 (1951) 265. Received July 27, 1970
GROWTH WHICH
IN SUSPENSION PRODUCE
CULTURE
GROWTH
F. C. BANCROFT’
OF RAT PITUITARY
HORMONE
AND
and A. H. TASHJIAN,
CELLS
PROLACTIN
Jr
Pharmacology Department, Harvard School of Dental Medicine, and Department of Pharmacology, Harvard Medical School, Boston, Mass. 02115, USA
SUMMARY The GH, strain of rat pituitary tumor cells has been adapted to growth in suspension culture. The cells have been grown in this fashion for a year and have maintained a doubling time of 42-48 h. Theq’ have continued to produce both growth hormone and prolactin. When the cells were exposedto hydrocortisone (3 x 1O-BM), GH production was stimulated by about 7-fold, and prolactin production was decreased by about 3-to 5-fold, while the cell doubling time was unaffected.
In recent years a variety of strains of neoplastic cells capable of performing differentiated functions has been established in monolayer culture by Sato and co-workers [l-7]. Studies of the control of the expression of differentiated function by these cells would be facilitated if the cells could be grown in suspension culture as well. One of these cell strains (GH,) is derived from a rat pituitary tumor, and has been shown to produce both GH [2, 7j and prolactin [8]. Studies of the control of the production of these protein hormones by GH, cells have revealed that HC stimulates GH production [9] and inhibits prolactin production [8]. We report here studies on GH, cells which have 1 Present address: Deuartment of Biolo&al Sciences. Columbia University;New York, N. Y: 10027, USA: Abbreviations used: GH, growth hormone; HC, hydrocortisone sodium succinate.
been grown in suspension culture for a year. In this condition of growth the cells continue to produce both GH and prolactin. In addition, the effects of HC on the production of these hormones are similar to those observed when the cells are grown in monolayer culture. MATERIALS
AND METHODS
Plastic tissue culture dishes were obtained from Falcon Plastics (Los Angeles, Calif.). Plastic bacterial dishes, which‘are not-treated for’ growth of animal cells in monolayer, were also obtained from Falcon. The cells (GH,) used in these experiments have been described in detail previously [7-91. Until the present experiments were performed they were grown in monolayer in complete Ham FlO medium [7, 91. In suspension culture the cells were grown at 36.5”C in Minimum Essential Medium (Spinner) [lo] (Grand Island Biological Co., Grand Island, N. Y.), supplemented with 15 % horse serum and 2.5 % fetal calf serum (complete spinner medium) in spinner bottles purchased either from Metaloglass, Inc. (Boston, Mass). or Bellco Glass, Inc. (Vineland, N. J.). Hydrocortisone sodium succinate (Solu-Cortef) was obtained from the Upjohn Co. (Kalamazoo, Mich.) Exptl Cell Res 64.
126 F. C. Bancroft & A. H. Tashjian Jr Viokase was purchased from the Viobin Corp. (Monticello, Ill.). Cell number was determined from cell counts performed in duplicate using a hemocytometer. The method of Lowry et al. [II] was used to measure the protein content of cells which had been washed with Minimum Essential Medium (Spinner) balanced salts. GH and prolactin concentrations in the growth medium were measured immunologically by the method of microcomplement fixation, as described previously [2, 81. The reproducibility of the immunological assays as performed in these experiments was+ 15 %. Nine months after the GH, cells were established in suspension culture, they were shown to be free of pleuropneumonia-like organisms by Dr Iolanda E. Low (Harvard Medical School).
RESULTS AND DISCUSSION The GH, cells were introduced into suspension culture as follows. Cells growing in monolayer in complete Ham FlO medium were removed with Viokase and placed in bacterial Petri dishes in complete spinner medium. In these dishes the cells aggregated to form floating clusters, each containing about 50100 cells. They were maintained in this condition for 1 week, after which they were placed in a 50 ml spinner flask in complete spinner medium, at a density of about 2 x lo5 cells/ml. Within a day the large clusters had dispersed into smaller clusters containing about 5-10 cells each, and within a week the majority of these clusters had completely dispersed into individual cells. (It has since been found that GH, cells which have been removed from monolayer culture with Viokase will begin growing immediately when introduced directly into suspension culture.) Cell number and GH and prolactin concentrations in the growth medium were followed beginning 10 days after the cells were first placed in suspension culture. The cells were observed to grow exponentially from 2 x lo5 to 8 x lo5 cells/ml with a doubling time of about 48 h. Growth hormone and prolactin concentrations increased about loExptl Cell Res 64
1
2.8 2.4
-
2.0
-
1.6 -
1.2-
f
I.O-
:
0.8-
(
0.6-
:
0.4
0.2-
-
2
1
JI
J 5
Fig. 1. Abscissa: days; oukr ordinate: A -A, ,ug prolactin/ml; inner ordinate: (left) O-O, cells x 10-5/ml;(right) l - l , ,ug GH/ml. Growth of GH, cells in suspension, and production of GH and Prolactin.
fold during this experiment, indicating that both hormones were being produced by the cells. GH, cells have been maintained almost continuously in suspension culture for 1 year. Two-and-one-half months after their establishment in suspension culture they were frozen at -196°C in 10% glycerol. Two weeks later the cells were thawed and returned directly to suspension culture, whereupon they began to grow. Growth of GH, cells and the concentrations of GH and prolactin in the medium were followed in an experiment performed 9 months after the cells were established in suspension culture (fig. 1). The cells grew with a population doubling time of about 46 h. GH and prolactin concentrations increased continuously during the experiment,
Rat pituitary
cells which produce growth hormone and prolactin
indicating that both hormones were being produced by the GH, cells. We have reported previously the stimulation by HC of GH production by GH, cells in monolayer culture [9]. In experiments carried out 9; months after the cells were established in suspension culture, we investigated whether HC had a similar effect on cells growing in suspension (fig. 2). To compare results with those obtained previously [9], it is necessary to express GH production in the same units, i.e., ,ug GH/mg cell protein/24 h. Therefore, cell growth was followed by measuring both cell protein and cell number. GH production over a 24 h interval was then estimated from the difference between the GH concentrations measured at the beginning and end of the interval, divided by the average cell protein concentration during the interval. HC (3 x 1O-6 M) was observed to stimulate GH production. The maximum stimulation, about 7-fold, was observed by 60 h, when the stimulated rate was about 70 pugGH/mg cell protein/24 h (fig. 2). These values are slightly higher than those observed when GH, cells growing exponentially in monolayer culture are exposed to HC [9]. In addition, in suspension culture maximum stimulation by HC appears to be achieved more rapidly than in monolayer [9]. We have recently observed that HC decreases the rate of prolactin production by GH, cells in monolayer culture by about 50% [8]. It is seen in fig. 2 that HC, after a lag of about 48 h, decreased prolactin production by GH, cells in suspension culture by 3- to 5-fold. HC does not appear to affect the growth rate of GH, cells in suspension (fig. 2). This is in contrast to what is observed in monolayer culture, where HC (3 x 10~~ M) increased the population doubling time (measured by following cell protein) by about 50% [9].
CELL
127
PROJEIN
~
b
/
4
c
PROL ACT/N
c --
120
Fig. 2. Abscissa: hours; ordinate: (a) pg cell protein/ mg; (b) pg GH/mg cell protein/24 h; (c) pg prolactin/mg cell protein/24 h. x - x , - HC; O-O, + HC. Effects of HC on growth, and GH and prolactin production of GH, cells in suspension.At zero time a suspension culture was divided equally between two spinner bottles. One bottle (+ HC) receivedHC at a final concentration of 3 x 1O-6M, and the other ( - HC) received an equal volume of saline. Additions were repeatedon the 2nd and 4th day of the experiment. Aliquots were removed every 24 h for assay of cell number, cell protein, and GH and prolactin concentration. GH and prolactin production were calculated as describedin the text. The vertical bars on eachGH and prolactin point represent the uncertainty in the difference betweentwo values each having an uncertainty of i 15%.
Exptl Cell Res 64
128 F. C. Bancrojt & A. H. Tashjian Jr It is noteworthy that while cell protein increased with a doubling time of about 60 h (fig. 2), the doubling time for cell number measured in the same experiment was about 42 h. This difference must of course arise from a decrease in the amount of protein per cell as cell density increases. The former quantity was in fact observed to drop from 2.4 x IO-’ to 1.6 x IO-’ mg cell protein/cell during the course of this experiment. The latter value is in good agreement with that previously measured for GH, cells in the early stationary phase of growth in monolayer culture (1.7 x lo-’ mg cell protein/cell)
PIThe ability to grow GH, cells in suspension culture, and to bring about effects of HC on GH and prolactin production similar to those observed in monolayer, should facilitate further biochemical studies of the control of the production of these protein hormones. Furthermore, HC has now been observed to have similar effects on protein hormone production by GH, cells in three different growth states: in both the exponential and stationary phases of growth in monolayer [8, 91 and, as reported here, during growth in suspension. Thus these effects are seen to be an inherent property of the GH, cells,
Exptl Cd Res 64
and not merely a reflection state of growth.
of a particular
The authors wish to thank Miss Norma J. Barowsky, Mrs Adele K. Gallucci. and Mrs Lethia S. Gilliard for expert assistance. We are also most grateful to Dr Lawrence Levine. Graduate Denartment of Biochemistry, Brandeis University, for his help with the immunoassays; and to Dr Iolanda K. Low, Harvard Medical School, for examining the GH, cells for PPLO contamination. This investigation was supported in part by research grants from the National Institutes of Health (AM-11011) and the American Cancer Society, (Massachusetts Division, Award No. P-374) and a General Research Support Grant (RR-05318).
REFERENCES 1. Sato, G H & Yasumura, Y, Trans NY acad sci 28 (1966) 1063. 2. Tashjian, A H Jr, Yasumura, Y, Levine, L, Sato, G H & Parker, M L, Endocrinology 82 (1968) 342. 3. Shin,‘S-I, Endocrinology 81 (1967) 440. 4. Benda, P, Lightbody, J, Sato, G H, Levine, L & Sweet, W, Science 161 (1968) 370. 5. Richardson, U I, Tashjian, A H Jr & Levine, L, J cell biol 40 (1969) 236. 6. Augusti-Tocco. G & Sato. G. Proc natl acad sci US-64 (1969) 311. ’ ’ 7. Yasumura, Y, Tashjian, A H Jr & Sato, G H, Science 154 (1966) 1186. 8. Tashjian, A H Jr, Bancroft, F C & Levine, L, J cell biol 47 (1970) 61. 9. Bancroft, F C, Leve,in L & Tashjian, A H Jr, J cell biol 43 (1969) 432. 10. Eagle, H, Science 130 (1959) 432. 11. Lowry, 0 H, Rosebrough, N F, Farr, A L & Randall, R J, J biol them 193 (1951) 265. Received July 27, 1970