- Email: [email protected]
Cholera enterotoxin stimulation of cAMP in cultured adrenal tumor cells
436
Preliminary notes
comparable with that of the established cells analysed. The values of TS determined here are considerably shorter than determined by Malamud [3] for Rana pipiens cells in monolayer culture (22.3 h). It was suggested that this time could be related to the amount of DNA in the Rana pipiens cells. As doubling the number of chromosomes in Xenopus laevis cells does not result in a comparable increase in T,, however, it is possible that there are other reasons for the long TS observed by this author. From the results presented here, it is clear that Xenopus laevis cells in vitro are capable of as rapid DNA synthesis at 25°C as many mammalian cells in vitro at 37°C; similar amounts of DNA are being synthesised in both cases. The other cell cycle phases are also comparable with mammalian cells. If the mammalian relationship between in vivo and in vitro values discussed by Mitchison [l] applies, then the Xenopus laevis cell cycle in vivo may also be similar to mammalian systems. I am grateful to Dr M. Balls for much heluful discussion Gf this work, which was supported-by a grant from the Science Research Council of the United Kingdom.
References 1. Mitchison, J M, The biology of the cell cycle. Cambridge University Press, Cambridge (1971). 2. Flickinger, R A, Freedman, M L & Stambrook, P J, Devel biol 16 (1967) 457. 3. Malamud, D, Exptl cell res 45 (1967) 277. 4. Reddan, J R & Rothstein, H, J cell physiol 67 (1966) 307. 5. Grillo. R S. Oncology 25 (1971) 347. 6. Zalik,‘S E & Yamada, T, J exptl zoo1 165 (1967) 385. 7. Chibon, P, Compt rend hebd seances acad sci 8. 9.
10. 11.
s&r D sci nat 267 (1968) 203. Rafferty, K A, Biology of amphibian tumors (ed M Mizell) p. 52. Springer-Verlag, New York (1969). Godsell, P M. Submitted for publication. Watanabe, I & Okada, S, J cell bio132 (1967) 309. Okada, S, J cell biol 34 (1967) 915.
Received May 5, 1974 Exptl
Cell Res 87 (1974)
Cholera enterotoxin stimulation cultured adrenal tumor cells CATHERINE
of CAMP in
N. KWAN and R. M. WISHNOW,’
Departments of Medicine and Microbiology, University of California, Irvine, and Veterans Administration Hospital, Long Beach, Calif. 90801, USA Summary. Cholera enterotoxin (CT) increased the concentration of adenosine 3’-5’-cyclic monophosphate (CAMP) in monolayer cultures of adrenal tumor cells after a 60 min lag phase in contrast to the rapid effect of adrenocorticotropin (ACTH). The change in intracellular CAMP was accompanied by the release of steroids into the culture medium and a reversible alteration of monolayer morphology.
Cholera enterotoxin (CT) increasesadenosine 3’-5’-cyclic monophosphate (CAMP) in gut mucosa leading to changes in ion transport and fluid accumulation within the intestinal lumen [14]. Recently, several laboratories have shown that cholera enterotoxin stimulates steroidogenesisand alters the morphology of cultured adrenal tumor cells [5-71. The present communication demonstrates that CT increases CAMP after a 60 min lag phase, leading to increased steroidogenesis in cultured adrenal cells. Materials and Methods CeN cultures. Y-l cells, cloned from a mouse adrenal cortex tumor, were obtained from the American Type Culture Collection, Rockville, Md. The cells were maintained in monolayer culture in Eagle’s minimum essential medium (MEM) with Earle’s salts, supplemented with 12.5 % horse serum, 2.5 % fetal calf serum. and 1.0 mM L-alutamine without antibiotics (GIBdo, Grand 1sland;N.Y.). Cells were grown in 60 x 15 mm Petri dishes (Falcon Plastics) at 37°C in a humidified atmosphere of 5 % CO, in air. Time course of ACTH and CT induction of steroidogenesis and CAMP determinations. Growing cultures
(approx. lo6 cells/plate) were washed twice with phosphate-buffered saline. Then Eagle’s MEM without serum containing cholera enterotoxin (50 rig/ml) or adrenocorticotropin (ACTH) (10 mu/ml) was added for 5, 10, 15, 30, 40, 60, 90, 120, or 205 min of incubation. The medium was removed and assayed for 1 Please address requests for reprints to: Dr Rodney M. Wishnow, Medical Research Programs (151), Veterans Administration Hospital, Long Beach, Calif. 90801, USA.
Preliminary notes steroid content. Four ml of cold redistilled ethanol was added to the cell monolayers and the cells were mechanically removed from the plates. The cell suspension was heated to 100°C for three 1 min periods, and the pellet was removed by centrifugation at 400 g. 3H CAMP (5 x 1O-5pmoles/ml) was added to the ethanol and used to follow the recovery of CAMP, which routinely was 85-90% [8]. Ethanol was removed by evaporation to dryness and the cell extract was redissolved in 1.0 ml of 0.05 M sodium acetate (pH 4.0). CAMP in a 50 ,ul aliquot was determined by the protein binding method of Gilman [9]. The pellet was dissolved in 0.1 N NaOH and protein was determined by the method of Lowry et al. [lo]. Adrenal monolayer morphology was observed under a phase contrast microscope (Nikon MS). Steroid assay and protein determination. The steroids in the incubation medium were estimated fluorimetrically, using a modification of the procedure of Vernikos-Danellis et al. [ll]. This method has been described previously 1121. Cell cultures were washed twice with 0.9 % saline, and then lysed with 2.0 ml of 0.1 N NaOH and protein determined by the method of Lowry et al. [lo]. The following reagents were used: porcine ACTH (Armour). cholera enterotoxin 1131.(nreoared under contract for NIAID by Dr R. A. &&el&ein, Southwestern Medical School). and 3H CAMP (24 \ Gil, mmole) (New England Nuclear).
Results Time course of CAMP and steroid production in response to maximal doses of ACTH or CT.
In both intact adrenal tumor cells and isolated membrane fragments, ACTH activates adenyl cyclase leading to increased CAMP and steroidogenesis [14, 151. Fig. 1 shows the time course of CAMP and steroid production in adrenal tumor cells after the addition of maximal dosesof ACTH (10 mu/ ml) or CT (50 rig/ml). Ten minutes after the addition of ACTH to adrenal tumor cells, the CAMP concentration reached a maximum of 480 pmoles/mg protein. The rapid increase in intracellular CAMP in response to ACTH was followed by a slow decreaseover the next 3 h. The increase in adrenal CAMP was accompanied by an increase in steroids released into the incubation medium. In contrast, after CT was added to adrenal cells, a 60 min lag occurred before increased CAMP concentration or steroidogenesis could be detected.
431
a
b 20
60
100
140
180
220
time (min): ordinate: (a) CAMP (pmoles/mg protein); (d) steroids~g/mg‘ protein); O-O, ACTH (10 mu/ml); O-O, CT (50 rig/ml). Time course of CAMP and steroid production in response to ACTH or CT. Eagle’s MEM without serum but containing 10 mu/ml ACTH or 50 rig/ml CT was added to adrenal monolayer cultures and incubated at 37°C. After 5, 10, 15; 30, 40, 60, 90, 120, and 205 min, the medium was removed and assayed for steroid content. CAMP was measured in the cell pellet and expressed as pmoles/mg cell protein. Fin. 1. Abscissa:
Morphological change associated with induction of steroidogenesis. The addition of
10 mu/ml ACTH causes adrenal cells to undergo a morphological alteration from a flattened, irregular to a spherical shape [16]. Phase contrast microscopy showed that 5 min after the addition of ACTH, adrenal cells began to ‘round-up’ and by 15 min all the cells were rounded-up. Six hours later the cells began to regain their normal morphology and by 10 h all the cells appeared normal. CT required 60 min to cause rounding-up, and the cells remained spherical 24 h later [5-71. But by 48 h, 50 % of the cells regained their normal morphology. Exptl
Cell Res 87 (1974)
438
Preliminary notes
Discussion
CT stimulates steroidogenesisin adrenal tumor cells after a 60 min lag and through a membrane receptor different from the ACTH receptor [7]. Our results demonstrate that steroidogenesisis delayed because of a 60 min latent period before CT increases intracellular CAMP levels compared with the immediate response to ACTH. Steroidogenesis and CAMP concentration may vary with different tumor sublines but these experiments were repeated at least four times with the same relative results. Cuatrecasas has suggested that the long latent period before CT increaseslipolysis in isolated fat cells is due to slow penetration of toxin from its membrane receptor site to the inner cell membrane where adenyl cyclase activation takes place [I 71.In adrenal cells, ACTH caused an immediate increase in intracellular CAMP concentration followed by a slow decrease,compared with the slow increase in CAMP observed in response to CT. The long lag phase and slow rate of increase in CAMP concentration may be a result of CT activating adrenal adenyl cyclase by a mechanism different from ACTH. The reversible morphological alteration in responseto ACTH or CT could be due to attachment to a membrane receptor, production of CAMP, or increased steroidogenesis. This work was supported (in part) by Cancer Research Funds of the University of California and by the Medical Research Programs, Veterans Administration Hospital, Long Beach, Calif., USA.
References 1. Schafer, D E, Lust, W D, Sercar, B & Goldberg, N 0, Proc natl acad sci US 67 (1970) 851. 2. Sharp, G W G & Hynie, S, Nature (Lond) 229 (1971) 266. 3. Kimberg, D V, Field, M, Johnson, J, Henderson, A & Gershon, E, J clin invest 50 (1971) 1218. 4. Pierce, N F, Greenough, W B & Carpenter, C C J, Bacterial rev 35 (1971) 1. 5. Donta S T, King, M & Sloper, K, Nature new biol 243 (1973) 246. Exptl Cell Res 87 (1974)
6. Wolff, J, Temple, R & Cook, G H, Proc natl acad sci US 70 (1973) 2741. 7. Wishnow, R M & Feist, P, J infect dis. In press. 8. Tsang, C P W, Lehotay, D C & Murphy, B E P, J clin endocrinol metab 35 (1972) 809. 9. Gilman, A G, Proc natl acad sci US 67 (1970) 305. 10. Lowry, 0 H, Rosebrough, N J, Farr, A L & Randall, R J, J biol them 193 (1951) 265. 11. Vernikos-Danellis, J, Anderson, E & Trigg, L, Endocrinology 79 (1966) 624. 12. Wishnow, R M, Feist, P & Glowalla, M B, Arch biochem biophys 161 (1974) 275. 13. Finkelstein, R A & LoSpalluto, J J, J infect dis 121, suppl. (1970) S63. 14. Schimmer, B P, J biol them 247 (1972) 3134. 15. Taunton, 0 D, Roth, J & Pastan, I, J biol them 244 (1969) 247. 16. Yasumura, Y, Am zoo1 8 (1968) 285. 17. Cuatrecasas, P, Biochemistry 12 (1973) 3567. Received April 8, 1974
Purification of the specific cell-aggregating factor from embryonic neural retina cells D. R. McCLAYland A. A. MOSCONA, Departments Biology and Pathology and the Committee on Development Biology, University of Chicago, Chicago, Ill. 60637, USA
of
The mechanisms of cell recognition and selective cell adhesion are of obvious importance in morphogenesis, differentiation, growth and neoplasia. Studies on cell aggregation in vitro suggestedthe working hypothesis that cell recognition and morphogenetic cell adhesion are mediated by interactions of specific cell-ligands localized on the cell surface and between cells [l-3]. This hypothesis led to the discovery in our laboratory of specific cell-aggregating factors with ligand-like activity [l-9]. These factors were obtained from live cells, first from embryonic neural retina cells [l, 3-61, then from sponge cells [2, 3, 7, 81 and subsequently, from embryonic cerebrum cells [9, lo]. The preparation of these factors was enabled by the fact that dispersed cells release into the culture medium turnover products of the 1 Present address: Department of Zoology, Duke University, Durham, N.C. 27706, USA.
Preliminary notes
comparable with that of the established cells analysed. The values of TS determined here are considerably shorter than determined by Malamud [3] for Rana pipiens cells in monolayer culture (22.3 h). It was suggested that this time could be related to the amount of DNA in the Rana pipiens cells. As doubling the number of chromosomes in Xenopus laevis cells does not result in a comparable increase in T,, however, it is possible that there are other reasons for the long TS observed by this author. From the results presented here, it is clear that Xenopus laevis cells in vitro are capable of as rapid DNA synthesis at 25°C as many mammalian cells in vitro at 37°C; similar amounts of DNA are being synthesised in both cases. The other cell cycle phases are also comparable with mammalian cells. If the mammalian relationship between in vivo and in vitro values discussed by Mitchison [l] applies, then the Xenopus laevis cell cycle in vivo may also be similar to mammalian systems. I am grateful to Dr M. Balls for much heluful discussion Gf this work, which was supported-by a grant from the Science Research Council of the United Kingdom.
References 1. Mitchison, J M, The biology of the cell cycle. Cambridge University Press, Cambridge (1971). 2. Flickinger, R A, Freedman, M L & Stambrook, P J, Devel biol 16 (1967) 457. 3. Malamud, D, Exptl cell res 45 (1967) 277. 4. Reddan, J R & Rothstein, H, J cell physiol 67 (1966) 307. 5. Grillo. R S. Oncology 25 (1971) 347. 6. Zalik,‘S E & Yamada, T, J exptl zoo1 165 (1967) 385. 7. Chibon, P, Compt rend hebd seances acad sci 8. 9.
10. 11.
s&r D sci nat 267 (1968) 203. Rafferty, K A, Biology of amphibian tumors (ed M Mizell) p. 52. Springer-Verlag, New York (1969). Godsell, P M. Submitted for publication. Watanabe, I & Okada, S, J cell bio132 (1967) 309. Okada, S, J cell biol 34 (1967) 915.
Received May 5, 1974 Exptl
Cell Res 87 (1974)
Cholera enterotoxin stimulation cultured adrenal tumor cells CATHERINE
of CAMP in
N. KWAN and R. M. WISHNOW,’
Departments of Medicine and Microbiology, University of California, Irvine, and Veterans Administration Hospital, Long Beach, Calif. 90801, USA Summary. Cholera enterotoxin (CT) increased the concentration of adenosine 3’-5’-cyclic monophosphate (CAMP) in monolayer cultures of adrenal tumor cells after a 60 min lag phase in contrast to the rapid effect of adrenocorticotropin (ACTH). The change in intracellular CAMP was accompanied by the release of steroids into the culture medium and a reversible alteration of monolayer morphology.
Cholera enterotoxin (CT) increasesadenosine 3’-5’-cyclic monophosphate (CAMP) in gut mucosa leading to changes in ion transport and fluid accumulation within the intestinal lumen [14]. Recently, several laboratories have shown that cholera enterotoxin stimulates steroidogenesisand alters the morphology of cultured adrenal tumor cells [5-71. The present communication demonstrates that CT increases CAMP after a 60 min lag phase, leading to increased steroidogenesis in cultured adrenal cells. Materials and Methods CeN cultures. Y-l cells, cloned from a mouse adrenal cortex tumor, were obtained from the American Type Culture Collection, Rockville, Md. The cells were maintained in monolayer culture in Eagle’s minimum essential medium (MEM) with Earle’s salts, supplemented with 12.5 % horse serum, 2.5 % fetal calf serum. and 1.0 mM L-alutamine without antibiotics (GIBdo, Grand 1sland;N.Y.). Cells were grown in 60 x 15 mm Petri dishes (Falcon Plastics) at 37°C in a humidified atmosphere of 5 % CO, in air. Time course of ACTH and CT induction of steroidogenesis and CAMP determinations. Growing cultures
(approx. lo6 cells/plate) were washed twice with phosphate-buffered saline. Then Eagle’s MEM without serum containing cholera enterotoxin (50 rig/ml) or adrenocorticotropin (ACTH) (10 mu/ml) was added for 5, 10, 15, 30, 40, 60, 90, 120, or 205 min of incubation. The medium was removed and assayed for 1 Please address requests for reprints to: Dr Rodney M. Wishnow, Medical Research Programs (151), Veterans Administration Hospital, Long Beach, Calif. 90801, USA.
Preliminary notes steroid content. Four ml of cold redistilled ethanol was added to the cell monolayers and the cells were mechanically removed from the plates. The cell suspension was heated to 100°C for three 1 min periods, and the pellet was removed by centrifugation at 400 g. 3H CAMP (5 x 1O-5pmoles/ml) was added to the ethanol and used to follow the recovery of CAMP, which routinely was 85-90% [8]. Ethanol was removed by evaporation to dryness and the cell extract was redissolved in 1.0 ml of 0.05 M sodium acetate (pH 4.0). CAMP in a 50 ,ul aliquot was determined by the protein binding method of Gilman [9]. The pellet was dissolved in 0.1 N NaOH and protein was determined by the method of Lowry et al. [lo]. Adrenal monolayer morphology was observed under a phase contrast microscope (Nikon MS). Steroid assay and protein determination. The steroids in the incubation medium were estimated fluorimetrically, using a modification of the procedure of Vernikos-Danellis et al. [ll]. This method has been described previously 1121. Cell cultures were washed twice with 0.9 % saline, and then lysed with 2.0 ml of 0.1 N NaOH and protein determined by the method of Lowry et al. [lo]. The following reagents were used: porcine ACTH (Armour). cholera enterotoxin 1131.(nreoared under contract for NIAID by Dr R. A. &&el&ein, Southwestern Medical School). and 3H CAMP (24 \ Gil, mmole) (New England Nuclear).
Results Time course of CAMP and steroid production in response to maximal doses of ACTH or CT.
In both intact adrenal tumor cells and isolated membrane fragments, ACTH activates adenyl cyclase leading to increased CAMP and steroidogenesis [14, 151. Fig. 1 shows the time course of CAMP and steroid production in adrenal tumor cells after the addition of maximal dosesof ACTH (10 mu/ ml) or CT (50 rig/ml). Ten minutes after the addition of ACTH to adrenal tumor cells, the CAMP concentration reached a maximum of 480 pmoles/mg protein. The rapid increase in intracellular CAMP in response to ACTH was followed by a slow decreaseover the next 3 h. The increase in adrenal CAMP was accompanied by an increase in steroids released into the incubation medium. In contrast, after CT was added to adrenal cells, a 60 min lag occurred before increased CAMP concentration or steroidogenesis could be detected.
431
a
b 20
60
100
140
180
220
time (min): ordinate: (a) CAMP (pmoles/mg protein); (d) steroids~g/mg‘ protein); O-O, ACTH (10 mu/ml); O-O, CT (50 rig/ml). Time course of CAMP and steroid production in response to ACTH or CT. Eagle’s MEM without serum but containing 10 mu/ml ACTH or 50 rig/ml CT was added to adrenal monolayer cultures and incubated at 37°C. After 5, 10, 15; 30, 40, 60, 90, 120, and 205 min, the medium was removed and assayed for steroid content. CAMP was measured in the cell pellet and expressed as pmoles/mg cell protein. Fin. 1. Abscissa:
Morphological change associated with induction of steroidogenesis. The addition of
10 mu/ml ACTH causes adrenal cells to undergo a morphological alteration from a flattened, irregular to a spherical shape [16]. Phase contrast microscopy showed that 5 min after the addition of ACTH, adrenal cells began to ‘round-up’ and by 15 min all the cells were rounded-up. Six hours later the cells began to regain their normal morphology and by 10 h all the cells appeared normal. CT required 60 min to cause rounding-up, and the cells remained spherical 24 h later [5-71. But by 48 h, 50 % of the cells regained their normal morphology. Exptl
Cell Res 87 (1974)
438
Preliminary notes
Discussion
CT stimulates steroidogenesisin adrenal tumor cells after a 60 min lag and through a membrane receptor different from the ACTH receptor [7]. Our results demonstrate that steroidogenesisis delayed because of a 60 min latent period before CT increases intracellular CAMP levels compared with the immediate response to ACTH. Steroidogenesis and CAMP concentration may vary with different tumor sublines but these experiments were repeated at least four times with the same relative results. Cuatrecasas has suggested that the long latent period before CT increaseslipolysis in isolated fat cells is due to slow penetration of toxin from its membrane receptor site to the inner cell membrane where adenyl cyclase activation takes place [I 71.In adrenal cells, ACTH caused an immediate increase in intracellular CAMP concentration followed by a slow decrease,compared with the slow increase in CAMP observed in response to CT. The long lag phase and slow rate of increase in CAMP concentration may be a result of CT activating adrenal adenyl cyclase by a mechanism different from ACTH. The reversible morphological alteration in responseto ACTH or CT could be due to attachment to a membrane receptor, production of CAMP, or increased steroidogenesis. This work was supported (in part) by Cancer Research Funds of the University of California and by the Medical Research Programs, Veterans Administration Hospital, Long Beach, Calif., USA.
References 1. Schafer, D E, Lust, W D, Sercar, B & Goldberg, N 0, Proc natl acad sci US 67 (1970) 851. 2. Sharp, G W G & Hynie, S, Nature (Lond) 229 (1971) 266. 3. Kimberg, D V, Field, M, Johnson, J, Henderson, A & Gershon, E, J clin invest 50 (1971) 1218. 4. Pierce, N F, Greenough, W B & Carpenter, C C J, Bacterial rev 35 (1971) 1. 5. Donta S T, King, M & Sloper, K, Nature new biol 243 (1973) 246. Exptl Cell Res 87 (1974)
6. Wolff, J, Temple, R & Cook, G H, Proc natl acad sci US 70 (1973) 2741. 7. Wishnow, R M & Feist, P, J infect dis. In press. 8. Tsang, C P W, Lehotay, D C & Murphy, B E P, J clin endocrinol metab 35 (1972) 809. 9. Gilman, A G, Proc natl acad sci US 67 (1970) 305. 10. Lowry, 0 H, Rosebrough, N J, Farr, A L & Randall, R J, J biol them 193 (1951) 265. 11. Vernikos-Danellis, J, Anderson, E & Trigg, L, Endocrinology 79 (1966) 624. 12. Wishnow, R M, Feist, P & Glowalla, M B, Arch biochem biophys 161 (1974) 275. 13. Finkelstein, R A & LoSpalluto, J J, J infect dis 121, suppl. (1970) S63. 14. Schimmer, B P, J biol them 247 (1972) 3134. 15. Taunton, 0 D, Roth, J & Pastan, I, J biol them 244 (1969) 247. 16. Yasumura, Y, Am zoo1 8 (1968) 285. 17. Cuatrecasas, P, Biochemistry 12 (1973) 3567. Received April 8, 1974
Purification of the specific cell-aggregating factor from embryonic neural retina cells D. R. McCLAYland A. A. MOSCONA, Departments Biology and Pathology and the Committee on Development Biology, University of Chicago, Chicago, Ill. 60637, USA
of
The mechanisms of cell recognition and selective cell adhesion are of obvious importance in morphogenesis, differentiation, growth and neoplasia. Studies on cell aggregation in vitro suggestedthe working hypothesis that cell recognition and morphogenetic cell adhesion are mediated by interactions of specific cell-ligands localized on the cell surface and between cells [l-3]. This hypothesis led to the discovery in our laboratory of specific cell-aggregating factors with ligand-like activity [l-9]. These factors were obtained from live cells, first from embryonic neural retina cells [l, 3-61, then from sponge cells [2, 3, 7, 81 and subsequently, from embryonic cerebrum cells [9, lo]. The preparation of these factors was enabled by the fact that dispersed cells release into the culture medium turnover products of the 1 Present address: Department of Zoology, Duke University, Durham, N.C. 27706, USA.