0013-7227/79/1051-0099$02.00/0 Endocrinology Copyright © 1979 by The Endocrine Society
Vol. 105, No. 1 Printed in U.S.A.
Characterization of Cultured Bovine Adrenocortical Cells and Derived Clonal Lines: Regulation of Steroidogenesis and Culture Life Span* MICHAEL H. SIMONIAN,t PETER J. HORNSBY, CHARLES R. ILL, MICHAEL J. O'HARE, AND GORDON N. GILL Department of Medicine, Division of Endocrinology, University of California, San Diego, School of Medicine La Jolla, California 92093; and the Ludwig Institute for Cancer Research, Royal Marsden Hospital (M.J.O'H.), Surrey, England
is the principal source of cholesterol for steroidogenesis. Fibroblast growth factor (FGF) is required for proliferation of both mass cultures and clonal lines throughout the finite life span in culture. Removal of FGF from later passage cells results in a rapid increase in generation time. In the continuous presence of FGF, the life span of the clonal line ACl is the same as that of uncloned cultures. FGF also has a weak steroidogenic effect, with dose-response characteristics identical to those for FGFstimulated growth. The clonal line ACl, which has lost responsiveness to ACTH, thus retains regulation of steroidogenesis and growth by FGF, angiotensin II, cholera toxin, prostaglandin Ei, and monobutyryl cAMP similar to that of mass uncloned cultures. These mass cultures and cloned lines represent well defined homogeneous populations of endocrine cells suitable for study of growth and differentiated function throughout a finite life span. (Endocrinology 105: 99, 1979)
ABSTRACT. Steroid production by cultured bovine adrenocortical cells and derived clones has been characterized by high pressure liquid chromatography. Although bovine adrenocortical cell cultures produce 17a-hydroxy-A'4,3-ketosteroids characteristic of the zona fasciculata-reticularis, the ability to ll/?-hydroxylate steroids is rapidly lost in culture. Thus, in response to steroidogenic factors, later passage cultures and clonal lines produce progesterone, 17a-hydroxyprogesterone (17a-hydroxypregn-4-ene-3,20-dione), 20a-dihydroprogesterone (20a-hydroxypregn-4-en-3-one), 17a-hydroxy-20a-dihydroprogesterone (17a,20a-dihydroxypregn-4-en-3-one), and 11-deoxycortisol (17a,21-dihydroxypregn-4-ene-3,20-dione). Although mass cultures respond to ACTH throughout their life span of 55-65 generations in culture, clonal lines uniformly lack responsiveness to ACTH. Steroidogenic activity, which is low in mass cultures and clones in the absence of steroidogenic factors, is progressively increased in response to angiotensin II, cholera toxin, and monobutyryl cAMP. In stimulated cells, low density lipoprotein
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bovine adrenocortical cell cultures which proliferate with a finite life span of 55-65 generations (5, 6). Because fibroblast growth factor (FGF), which is a potent mitogen for these cells, allows growth from low cell density, it has been possible to isolate clonal lines of bovine adrenocortical cells. These mass cultures and cloned lines represent homogeneous populations of endocrine cells suitable for investigation of factors controlling both growth and differentiated function. In the present studies, the steroidogenic properties of one of the cloned lines have been defined and compared to the steroidogenic properties of mass uncloned cultures. Factors regulating both steroidogenesis and proliferation throughout the clonal life span of 55-65 generations have been investigated.
HE USE of tissue culture has greatly facilitated investigation of hormonal control mechanisms in endocrine cells. Studies using tumor cell lines such as rat pituitary GH cells (1), mouse pituitary AT cells (2), and mouse adrenal Y-l cells (3), have helped to identify a number of regulatory events in hormone biosynthesis and secretion in homogeneous cell populations. While these tumor cell lines proliferate indefinitely, primary cultures of most normal endocrine cells have not been demonstrated to be homogeneous and have very limited replicative potential (4). Studies of control of both differentiated function and cell proliferation have necessarily been limited. We have recently developed functional Received October 31, 1978. Address requests for reprints to: Dr. Gordon N. Gill, Department of Medicine, M-013, University of California, San Diego, La Jolla, California 92093. * This work was supported by NIH Research Grant AM-13149 from the NIAMDD and NIH Research Grant AG-00936 from the National Institute on Aging. t Bank of America—Giannini Foundation Fellow.
Materials and Methods Pituitary and brain FGF were generous gifts from Dr. Denis Gospodarowicz, University of California, San Francisco (7, 8). Human low density lipoprotein (LDL) was a generous gift from Dr. David Weinstein, University of California, San Diego. An99
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SIMONIAN ET AL.
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giotensin II was purchased from Bachem, Inc. (Torrance, CA); ACTH (ACTHar) was obtained from Armour Pharmaceutical (Chicago, IL); cholera toxin was from Becton, Dickinson, and Co. (Rutherford, NJ); and N6-monobutyryl cAMP was from Sigma Chemical Co. (St. Louis, MO). Methanol, acetonitrite, and dichloromethane were of the highest purity available. Cloning and growth of bovine adrenocortical cells To select clones, confluent primary cultures (third generation) were plated at low density (4 cells/cm2) in F-12 medium containing 10% fetal calf serum (Irvine Scientific, Irvine, CA) and 100 ng/ml FGF. The medium was changed every other day. Visible colonies were isolated by the procedure of Puck (9). Thirty clones were grown to confluence and designated adrenocortical clone 1, 2, etc. (AC1, AC2, etc.). The life span of AC1 cells was determined by subculturing confluent or quiescent cultures into 10-cm dishes at a density of ~2 X 104 cells/ dish. Cells were subcultured and maintained in Coon's modification of Ham's F-12 medium containing 10% fetal calf serum and 40 ng/ml FGF. The medium was changed every 2 days. The generation time was calculated from the initial cell number at 18 h after subculture and the final cell number at confluence or quiescence. Cell number was quantitated by removing cells from culture dishes by incubation with bicarbonate-buffered saline (10) containing 0.05% trypsin and 0.02% ethylenediamine tetraacetic acid for 1-2 min at 37 C. Cells were diluted and immediately counted in a model ZF Coulter counter (Coulter Electronics, Inc., Hialeah, FL). Steroid production Cells were grown in 3.5-cm dishes (Falcon Plastics, Oxnard, CA) containing 2 ml growth medium. Ultraviolet light-absorbing steroids secreted by cultured cells were identified and quantitated by reversed phase high pressure liquid chromatography, as described by O'Hare et al. (11). Medium was extracted with dichloromethane and washed with 0.1 M NaOH to remove saponifiable lipid. Dichloromethane extracts were evaporated under nitrogen and redissolved in 40% ethanol or the primary solvent. An aliquot of each sample was injected into a DuPont 830 chromatograph and separated using either a Nucleosil 5Cis octadecylsilane (ODS) or Lichrosorb-ODS (5 jum) column (id, 100 X 5 mm). Separation was effected by a concave exponential gradient of either methanol-water or acetonitrile-water (11). A4,3-Ketosteroids were detected by their absorbance at 240 nm. When medium from cultures treated with [7-3H]pregnenolone (New England Nuclear, Boston, MA; 17.2 Ci/mmol; 5 /iCi/plate) was analyzed, the fractions were collected from the chromatograph and radioactivity was quantitated by liquid scintillation counting. Radiolabeled steroids produced from [3H]pregnenolone and secreted by cultured cells were also identified by thin layer chromatography (12). Cell cultures (3.5-cm dishes) were treated with [7-3H]pregnenolone (New England Nuclear; 17.2 Ci/mmol; 5 juCi/plate) for 24 h. The medium was then collected and extracted with dichloromethane; dichloromethane extracts were evaporated under nitrogen. Steroids were redissolved in a minimal volume of dichloromethane and separated on silica gel plates (Silica gel 60, E. Merck, Rahway, NJ) using a toluene-
Enclo • 1979 Vol 105 • No 1
acetone (115:35) solvent system. The radiolabeled steroids were detected by a radiochromatogram scanner. Fluorogenic steroid production was quantitated by ethanolic sulfuric acid'fluorescence (13). Results are expressed as micrograms of cortisol, which was used as the standard. Fluorogenic steroid production in cultured bovine adrenocortical cells is, however, due principally to 20a-dihydroprogesterone (DHP)1 and 17a-hydroxy-20a-dihydroprogesterone (17aOH-DHP). On a mass basis, DHP andl7aOH-DHP are 5- and 7-fold, respectively, more fluorescent than cortisol.
I
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Preparation of' LDL-deficient serum LDL-deficient serum was prepared using a recently developed method which involves adsorbing hydrophobic substances, including lipoproteins, to Cabosil powder gel (ICN Chemical and Radioisotope Division, Irvine, CA) (Weinstein, D., personal communication). Cabosil (2%, wt/vol) was added to fetal calf serum and stirred for 24 h at 4 C. The mixture was then centrifuged at 20,000 X g for 30 min and the supernatant was filtered twice through a 0.22-/xm Millex filter (Millipore Corp., Bedford, MA). The resulting Cabosil-treated fetal calf serum contained (0 LU
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Vol. 105, No. 1 Printed in U.S.A.
Characterization of Cultured Bovine Adrenocortical Cells and Derived Clonal Lines: Regulation of Steroidogenesis and Culture Life Span* MICHAEL H. SIMONIAN,t PETER J. HORNSBY, CHARLES R. ILL, MICHAEL J. O'HARE, AND GORDON N. GILL Department of Medicine, Division of Endocrinology, University of California, San Diego, School of Medicine La Jolla, California 92093; and the Ludwig Institute for Cancer Research, Royal Marsden Hospital (M.J.O'H.), Surrey, England
is the principal source of cholesterol for steroidogenesis. Fibroblast growth factor (FGF) is required for proliferation of both mass cultures and clonal lines throughout the finite life span in culture. Removal of FGF from later passage cells results in a rapid increase in generation time. In the continuous presence of FGF, the life span of the clonal line ACl is the same as that of uncloned cultures. FGF also has a weak steroidogenic effect, with dose-response characteristics identical to those for FGFstimulated growth. The clonal line ACl, which has lost responsiveness to ACTH, thus retains regulation of steroidogenesis and growth by FGF, angiotensin II, cholera toxin, prostaglandin Ei, and monobutyryl cAMP similar to that of mass uncloned cultures. These mass cultures and cloned lines represent well defined homogeneous populations of endocrine cells suitable for study of growth and differentiated function throughout a finite life span. (Endocrinology 105: 99, 1979)
ABSTRACT. Steroid production by cultured bovine adrenocortical cells and derived clones has been characterized by high pressure liquid chromatography. Although bovine adrenocortical cell cultures produce 17a-hydroxy-A'4,3-ketosteroids characteristic of the zona fasciculata-reticularis, the ability to ll/?-hydroxylate steroids is rapidly lost in culture. Thus, in response to steroidogenic factors, later passage cultures and clonal lines produce progesterone, 17a-hydroxyprogesterone (17a-hydroxypregn-4-ene-3,20-dione), 20a-dihydroprogesterone (20a-hydroxypregn-4-en-3-one), 17a-hydroxy-20a-dihydroprogesterone (17a,20a-dihydroxypregn-4-en-3-one), and 11-deoxycortisol (17a,21-dihydroxypregn-4-ene-3,20-dione). Although mass cultures respond to ACTH throughout their life span of 55-65 generations in culture, clonal lines uniformly lack responsiveness to ACTH. Steroidogenic activity, which is low in mass cultures and clones in the absence of steroidogenic factors, is progressively increased in response to angiotensin II, cholera toxin, and monobutyryl cAMP. In stimulated cells, low density lipoprotein
T
bovine adrenocortical cell cultures which proliferate with a finite life span of 55-65 generations (5, 6). Because fibroblast growth factor (FGF), which is a potent mitogen for these cells, allows growth from low cell density, it has been possible to isolate clonal lines of bovine adrenocortical cells. These mass cultures and cloned lines represent homogeneous populations of endocrine cells suitable for investigation of factors controlling both growth and differentiated function. In the present studies, the steroidogenic properties of one of the cloned lines have been defined and compared to the steroidogenic properties of mass uncloned cultures. Factors regulating both steroidogenesis and proliferation throughout the clonal life span of 55-65 generations have been investigated.
HE USE of tissue culture has greatly facilitated investigation of hormonal control mechanisms in endocrine cells. Studies using tumor cell lines such as rat pituitary GH cells (1), mouse pituitary AT cells (2), and mouse adrenal Y-l cells (3), have helped to identify a number of regulatory events in hormone biosynthesis and secretion in homogeneous cell populations. While these tumor cell lines proliferate indefinitely, primary cultures of most normal endocrine cells have not been demonstrated to be homogeneous and have very limited replicative potential (4). Studies of control of both differentiated function and cell proliferation have necessarily been limited. We have recently developed functional Received October 31, 1978. Address requests for reprints to: Dr. Gordon N. Gill, Department of Medicine, M-013, University of California, San Diego, La Jolla, California 92093. * This work was supported by NIH Research Grant AM-13149 from the NIAMDD and NIH Research Grant AG-00936 from the National Institute on Aging. t Bank of America—Giannini Foundation Fellow.
Materials and Methods Pituitary and brain FGF were generous gifts from Dr. Denis Gospodarowicz, University of California, San Francisco (7, 8). Human low density lipoprotein (LDL) was a generous gift from Dr. David Weinstein, University of California, San Diego. An99
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 17 November 2015. at 02:18 For personal use only. No other uses without permission. . All rights reserved.
SIMONIAN ET AL.
100
giotensin II was purchased from Bachem, Inc. (Torrance, CA); ACTH (ACTHar) was obtained from Armour Pharmaceutical (Chicago, IL); cholera toxin was from Becton, Dickinson, and Co. (Rutherford, NJ); and N6-monobutyryl cAMP was from Sigma Chemical Co. (St. Louis, MO). Methanol, acetonitrite, and dichloromethane were of the highest purity available. Cloning and growth of bovine adrenocortical cells To select clones, confluent primary cultures (third generation) were plated at low density (4 cells/cm2) in F-12 medium containing 10% fetal calf serum (Irvine Scientific, Irvine, CA) and 100 ng/ml FGF. The medium was changed every other day. Visible colonies were isolated by the procedure of Puck (9). Thirty clones were grown to confluence and designated adrenocortical clone 1, 2, etc. (AC1, AC2, etc.). The life span of AC1 cells was determined by subculturing confluent or quiescent cultures into 10-cm dishes at a density of ~2 X 104 cells/ dish. Cells were subcultured and maintained in Coon's modification of Ham's F-12 medium containing 10% fetal calf serum and 40 ng/ml FGF. The medium was changed every 2 days. The generation time was calculated from the initial cell number at 18 h after subculture and the final cell number at confluence or quiescence. Cell number was quantitated by removing cells from culture dishes by incubation with bicarbonate-buffered saline (10) containing 0.05% trypsin and 0.02% ethylenediamine tetraacetic acid for 1-2 min at 37 C. Cells were diluted and immediately counted in a model ZF Coulter counter (Coulter Electronics, Inc., Hialeah, FL). Steroid production Cells were grown in 3.5-cm dishes (Falcon Plastics, Oxnard, CA) containing 2 ml growth medium. Ultraviolet light-absorbing steroids secreted by cultured cells were identified and quantitated by reversed phase high pressure liquid chromatography, as described by O'Hare et al. (11). Medium was extracted with dichloromethane and washed with 0.1 M NaOH to remove saponifiable lipid. Dichloromethane extracts were evaporated under nitrogen and redissolved in 40% ethanol or the primary solvent. An aliquot of each sample was injected into a DuPont 830 chromatograph and separated using either a Nucleosil 5Cis octadecylsilane (ODS) or Lichrosorb-ODS (5 jum) column (id, 100 X 5 mm). Separation was effected by a concave exponential gradient of either methanol-water or acetonitrile-water (11). A4,3-Ketosteroids were detected by their absorbance at 240 nm. When medium from cultures treated with [7-3H]pregnenolone (New England Nuclear, Boston, MA; 17.2 Ci/mmol; 5 /iCi/plate) was analyzed, the fractions were collected from the chromatograph and radioactivity was quantitated by liquid scintillation counting. Radiolabeled steroids produced from [3H]pregnenolone and secreted by cultured cells were also identified by thin layer chromatography (12). Cell cultures (3.5-cm dishes) were treated with [7-3H]pregnenolone (New England Nuclear; 17.2 Ci/mmol; 5 juCi/plate) for 24 h. The medium was then collected and extracted with dichloromethane; dichloromethane extracts were evaporated under nitrogen. Steroids were redissolved in a minimal volume of dichloromethane and separated on silica gel plates (Silica gel 60, E. Merck, Rahway, NJ) using a toluene-
Enclo • 1979 Vol 105 • No 1
acetone (115:35) solvent system. The radiolabeled steroids were detected by a radiochromatogram scanner. Fluorogenic steroid production was quantitated by ethanolic sulfuric acid'fluorescence (13). Results are expressed as micrograms of cortisol, which was used as the standard. Fluorogenic steroid production in cultured bovine adrenocortical cells is, however, due principally to 20a-dihydroprogesterone (DHP)1 and 17a-hydroxy-20a-dihydroprogesterone (17aOH-DHP). On a mass basis, DHP andl7aOH-DHP are 5- and 7-fold, respectively, more fluorescent than cortisol.
I
>
Preparation of' LDL-deficient serum LDL-deficient serum was prepared using a recently developed method which involves adsorbing hydrophobic substances, including lipoproteins, to Cabosil powder gel (ICN Chemical and Radioisotope Division, Irvine, CA) (Weinstein, D., personal communication). Cabosil (2%, wt/vol) was added to fetal calf serum and stirred for 24 h at 4 C. The mixture was then centrifuged at 20,000 X g for 30 min and the supernatant was filtered twice through a 0.22-/xm Millex filter (Millipore Corp., Bedford, MA). The resulting Cabosil-treated fetal calf serum contained (0 LU
z o
