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Evidence of 3β-hydroxysteroid dehydrogenase activity in several murine embryonal carcinoma cell lines
SHORT NOTE Evidence
of 3/3-Hydroxysteroid Embryonal
Dehydrogenase Activity Carcinoma Cell Lines
CLAUDE ‘IWZET,*~’ YVES J. BIGNON,t JEAN C. VENNAT,t JACQUES CHASSAGNE,t GINETTE GAILLARD,? ROBERT PLAGNE,?
in Several
MUSTAPHA PHILIPPE and DANIEL
Murine
AFANE,” CHOLLET,? BOUCHER*
*Laboratoire de physiologie et tquipe associke au CNRS, Facultt de Mkdecine, 28 place Henri Dunant, 63001 Clerrnont-Ferrand Cedex, and f’Centre Jean Perrin, Place Henri Dunant, 63011 Clermont-Ferrand Cedex, France
This report describes, for the first time to our knowledge, a possible steroidogenic activity in established murine embryonal carcinoma cell lines (PCC3, PCC4, F9), revealed by a 3j3-hydroxysteroid dehydrogenase activity (revelation of NADHz by staining, and RIA assessment of A4-androstenedione). The remarkable analogy between such totipotent cells and embryonal cells may suggest that this activity could be present before histologic organization of the embryonal testis. Nonmalignant embryonal cells such as tibroblasts (3/A/l/D-3) or myoblasts (T984) were also found to possess a 3B-hydroxysteroid dehydrogenase activity, thus suggesting that this enzyme is not specific to hormone-secreting cells, but the sign of a more general phenomenon. @ 1988 Academic FWS, II-K.
Teratocarcinomas are germ cell tumors containing a heterogeneous cell mixture which forms during the early stages of differentiated tissues [l-3] and other more undifferentiated cells called embryonal carcinoma (EC) cells 141. They are of interest because of their remarkable similarity to early embryonal cells [5], and thus may be used for embryogenic studies [6, 71. They originate from malignant tumors appearing spontaneously in mice, both male [8, 91 and female 110, 111. The frequency of these tumors can be increased by ectopic embryo grafting [12-141. Many established cell lines have been obtained after intraperitoneal transfers [15], followed by in vitro culture [16-201. These EC cells have a very high potential for differentiation; by intrablastocyst injection, chimeras have been obtained, in which almost all organs were constituted, in different proportions, of such EC-derived cells [21, 221. In the present work, we have been able to demonstrate that EC cells can move into steroidogenic cells. This was shown by establishing the presence of the 3&hydroxysteroid dehydrogenase enzyme (3/3HSD), a major enzyme in steroidogenesis in the cell cytosol. Materials
and Methods
Three embryonal carcinoma cell lines were used, PCC3 [19,23,24], PCC4 [19,23-261, and F9 [27], as well as two differentiated cell lines, the myoblastic clonal cell line C17SlD-T984 (T984) 128, 291 and the fibroblastic clonal cell line 3/A/l/D-3, derived from PCC3 [30]. These cells were cultivated with Dulbecco’s modified MEM (DME) containing 4.5 g/liter of glucose [31] obtained from Boehringer-Mannheim, supplemented with 10% fetal calf serum (FCS). Evidence of 3#I-HSD activiry by cell staining. The histologic characterization of 3/3-HSD is a blue staining of the cells possessing the enzyme, when dehydroepiandrosterone (DHA) is used as substrate Cell
culture.
’ To whom reprint requests should be addressed. 223
Copyright 0 1988 by Academic Ress, Inc. All rights of reproduction in any form reserved 0014-4827/88 $03.00
224 Short notes
Fig. 1. (a) PCC3 (x100), (X400).
(b) PCC4 (x100),
(c) F9 (x100),
(d) myoblasts (x400), (e) fibroblasts
[32, 331. The culture medium was replaced by 1 ml of DME supplemented with 0.5 mg/ml nicotinamide (Sigma), 3 mg/ml NAD (Sigma), 0.75 mg/ml tetrazolium nitroblue (Sigma), and 5 rig/ml DHA. The cells were incubated for 2 to 3 h at 37°C. 3t%HSD evidence by A4-androstenedione assessment (A4). Cell steroidogenesis was shown by assessment of A4 in the culture supematant after incubation with DHA. Adult Wistar rat interstitial testicular cells enriched in Leydig cells were used as positive control; Leydig cells were isolated by the technique described by Lefevre et al. [34] using a Percoll gradient. Both human lymphocytes and the culture medium itself, without FCS, were used as negative controls. Eight groups of cells were divided as follows: Leydig cells (lot l), PCC3 cells (lot 2), PCC4 cells (lot 3), F9 cells (lot 4), myoblasts (lot 5), fibroblasts (lot 6), lymphocytes (lot 7), culture medium alone (lot
‘3). For culture, 300,000 cells per well were incubated in Nunc culture multidishes at a temperature of 37°C. DME without FCS, containing 500 rig/ml DHA, was used. After 24 h, the supematant was frozen until the subsequent androstenedione experiment. A4 assessment was made by RIA with antibody Biomerieux after extraction in ether and chromatography on chromatholite A. Tritiated androstenedione was used as labeled hormone. A4-Androstenedione adsorption and release. A4 was assessed in culture supematants from four other lots: PCC3, PCC4, and F9 cell lines in a culture medium containing 10% FCS, but without DHA (PCC3*, lot 9; PCC4*, lot 10; and F9*, lot ll), and FCS alone (lot 12). Statistics. RIA was carried out by “single blind” methods. Comparative studies were analyzed with a Student’s t test.
Results 3/3-Hydroxysteroid
dehydrogenase staining. After eight different experiments,
97+6% of PCC3 (3724 cells), 80+14% of PCC4 (1815 cells), and 98+5% of F9 cells (2240 cells) were stained blue (Fig. 1) and showed a 3fi-HSD activity. The lower percentage obtained for PCC4 suggested the presence of two subpopulations, or a heterogeneity of these cells at a particular phase of the cell cycle. To test these possibilities a cloning experiment was carried out by isolating a single cell before culture. Twelve wells were positive for culture: every well contained the same two cell types in similar proportions (76f24%). This rejected the first hypothesis. We can suppose that the enzyme was functional or not, depending on the cycle phase of each cell. One hundred percent of the myoblasts (291 cells) and 97% of the fibroblasts (1000 cells) were stained (Fig. 1). The lymphocytes were not stained at ah. An incomplete Leydig cell purification, usual with the Percoll gradient, was responsible for the staining of about 80% of this group of cells.
Short notes
TABLE
1
A4 level in the different culture media induced by cell lines at a concentration 300,000 cellslml after an incubation of 24 h with 500 nglml DHA Androstenedione (ng/mU 1 2 3 4 5 6 7 8 9 10 I1 12
Leydig cells PCC3 PCC4 F9 Myoblasts Fibroblasts Lymphocytes Basal medium PCC3” PCC4” F9” Fetal calf serum”
225
29.95k4.45 0.55+0.13 0.65kO.06 1.06f0.11 0.49Yko.10 0.461kO.04 0.33kO.05 0.24+0.08 0.16f0.06 0.17kO.03 0.19+0.02 0.17+0.02
Note. The results are given with the formula mftxsl~n deviation; n number of repetitions). ’ Cultivated without DHA. b Comparison with the sensibility limits of 0.2 r&ml.
n
tb 82.14 7.01 18.14 20.72 4.96 11.26 6.85 1.31 1.35 2.00 1.06 3.00
of
ab
(m, mean; t, Student’s t; s, standard
A4-Androstenedione assessment. Every value obtained (Table l), except that of the culture medium was significantly higher than the sensitivity limit of the dosage method (0.2 rig/ml). The culture medium (lot 8) and FCS alone (lot 12) can be considered devoid of A4. The lymphocyte culture medium concentration (lot 7) was at the significance limit, when compared with that of the culture medium (~65~ IO-*). A cross-reaction with other steroids seemed unlikely. It is possible that the lymphocytes could have adsorbed serum hormone and then released it into the culture medium. The culture media of PCC3, PCC4, F9, myoblast and fibroblast cell lines contained significantly more A4 than negative control media (Table 1) (a
Our results showed that PCC3, F9, myoblasts, fibroblasts, and to a lesser extent, PCC4 were blue-colored when a staining reaction of 3/?-HSD was carried out. Furthermore, A4 was released by the cells studied, since this hormone was found in significant concentrations after 24 h of culture in PCC3, PCC4, F9, myoblast, and fibroblast supernatants, but not in the DME medium, and only at a very low level in lymphocyte culture medium. This hormone appeared to be
226 Short notes secreted by the cell lines under study because its absence in FCS, and in the different culture media from cells incubated with DHA, eliminated the possibility of cell release. This cell secretion was further evidence of the presence of functional 3/3-HSD, and confirmed the cytologic results. F9 seemed to be able to secrete more A4 than the other cell lines (PCC3, PCC4, myoblasts, and fibroblasts). It may be deduced from our results that EC cells may exhibit a steroidogenie activity: PCC3, PCC4, and F9 did so at a basal level, i.e., without hormonal induction. It is known that testis interstitial tissue is first seen in mouse embryos from Day 10 of pregnancy [35-371 and at Day 13 for the rat [38]. It has been shown in the pig that blastocysts are able to secrete estrogens [39,40] both in uiuo and in vitro since DHA is transformed into estrogens [41], in which case they contain 3B-HSD. The remarkable homology between embryonal cells and EC cells has been well documented. We have shown that this homology may extend to the presence of 3/l-HSD activity by these cells. Such activity in EC cells obtained without hormonal induction supposes, besides, that the first steroidogenie stages can be obtained independently of pituitary hormonal stimulation, but are linked to paracrine cell interactions. Our results showed the presence of a functional 3/GHSD enzyme in differentiated cells such as myoblasts and fibroblasts. This may be surprising at first, but 3/l-HSD enzyme-possessing cells are not limited to Leydig cells [42, 431: they are also found in alveolar macrophages [44], in adrenal gland cells [45], and in placenta [45, 461. These results raise the possibility of using EC cells as a model in the study of embryonal cell differentiation into steroidogenic cells. The authors are indebted to Madame H. Jakob for supplying the various cell lines necessary to their work and for the helpful advice which she has given throughout.
References 1. Pierce, G. B., and Vemey, E. L. (1961) Cancer 14, 1017. 2. Lehman, J. M., Speers, W. C., Schwarzendruber, D. E., and Pierce, G. B. (1974) J. Cell. Physiol. 84, 13. 3. Nicolas, J. F., Dubois, P., Jakob, H., Gaillard, J., and Jacob, F. (1975) Ann. Microbial. (Paris) 126A, 4.
5. 6. 7. 8. 9.
10. 11. 12. 13.
14. 15. 16. 17. 18. 19.
3.
Stevens, L. C. (1959) J. Natl. Cancer Inst. 23, 1249. Jacob, F. (1975) The Early Development of Mammals, Symposium II, p. 233, Cambridge Univ. Press. Martin, G. R. (1975) Cell 5, 229. Martin, G. R. (1980) Science 209, 768. Stevens, L. C., and Little, C. C. (1954) Proc. N&l. Acad. Sci. USA 40, 1080. Stevens, L. C. (1973) J. N&l. Cancer Inst. 50, 235. Stevens, L. C., and Vamum, D. S. (1974) Deu. Biol. 37, 369. Eppig, J. J., Kosak, L. P., Either, E. M., and Stevens, L. C. (1977) Nature (London) 269, 517. Stevens, L. C. (1964) Proc. Natl. Acad. Sci. USA 52, 654. Stevens, L. C. (1970) J. Nutl. Cancer Inst. 44, 923. Stevens, L. C. (1970) J. Exp. Zool. 174, 407. Stevens, L. C. (1970) Dev. Biol. 21, 364. Finch, B. W., and Ephrussi, B. (1967) Proc. Natl. Acad. Sci USA 57, 615. Rosenthal, M. D., Wishnow, R. M., and Sato, G. H. (1970) J. Natl. Cancer Inst. 44, 1001. Evans, M. J. (1972) J. Embyrol. Exp. Morphol. 28, 163. Jakob, H., Boon, T., Gaillard, J., Nicolas, J. F., and Jacob, F. (1973) Ann. Microbial. (Paris) 124B, 269.
Short
notes
227
20. MC Bumey, M. W. (1976) J. Cell. Physiol. 89, 441. 21. Brinster, R. L. (1974) J. Exp. Med. 140, 1049. 22. Mintz, B., and Illmensee, K. (1975) Proc. Natl. Acad. Sci. USA 72, 3585. 23. Guenet, J. L., Jakob, H., Nicolas, J. F., and Jacob, F. (1974) Ann. Microbial. (Paris) 125A, 135. 24. Nicolas, J. F., Avner, P., Gaillard, J., Guenet, J. L., Jakob, H., and Jacob, F. (1976) Cancer Res. 34, 4224.
Lo, C. W., and Gilula, N. B. (1980) Deu. Biol. 75, 78. 26. Lo, C. W., and Gilula, N. B. (1980) Dev. Biol. 75, 93. 27. Bemstine, E. G., Hooper, M. L., Grandchamp, S., and Ephrussi, B. (1973) Proc. Nat/. Acad. Sci. USA 70, 3899. 28. Jakob, H., Buckingham, M. E., Cohen, A., DuPont, L., Fizman, M., and Jacob, F. (1978) Exp. Cell Res. 114, 403. 29. Nicolas, J. F., Jakob, H., and Jacob, F. (1981) Functionally Differentiated Cell Lines, p. 185, A. R. Liss, New York. 30. Nicolas, J. F., Jakob, H., and Jacob, F. (1978) Proc. Nat/. Acad. Sci. USA 75, 3292. 31. Morton, H. J. (1970) In Vitro 6, 89. 32. Browning, J. Y., D’Agata, R., and Grotejan, H. E., Jr. (1981) Endocrinology 109, 667. 33. Steinberger, E., Steinberger, A., and Vilar, 0. (1966) Endocrinology 79, 406. 34. Lefevre, A., Saez, J., and Finaz, J. (1983) Hormone Res. 17, 114. 35. Russo, J., and De Rosas, J. C. (1971) Amer. J. Anat. 130, 461. 36. Baillie, A. H., and Griffiths, K. (1964) J. Endocrinol. 31, 63. 37. Baillie, A. H. (1965) J. Anat. 99, 507. 38. Pelliniemi, J. J., Paranko, J., Grund, S. K., Frojdman, K., Froidart, J. M., and Lakkala-Paranko, T. (1984) Ann. N.Y. Acad. Sci. 405. 39. Heap, R. B., Flint, A. P., and Gadsby, J. E. (1979) Brit. Med. Bull. 35, 129. 40. Heap, R. B., Flint, A. P., Hartmann, P. E., Gadsby, J. E., Staples, L. D., Ackland, Nicolas, Hamon, and Maureen (1981) J. Endocrinol89, suppl, 77P. 41. Perry, J. S., Heap, R. B., and Amoroso, E. C. (1973) Nature (London) 245, 45. 42. Baillie, A. H., and Griffiths, K. (1964) J. Endocrinol. 24, 9. 43. Baillie, A. H., and Grifliths, K. (1965) J. Endocrinol. 31, 207. 44. Milewich, L., Lipscomb, M., Whisenant, M., and Macdonald, P. (1983) J. Steroid Biochem. 19, 1611. 45. Baillie, A. H., Cameron, E. II. D., Griffiths, K., and Hart, D., MC. K. (1965) J. Endocrinol. 31, 25.
227. 46.
Ainsworth, L., and Ryan, K. J. (1967) Endocrinology
81, 13.
Received July 24, 1987 Revised version received September 30, 1987
Printed
in Sweden
of 3/3-Hydroxysteroid Embryonal
Dehydrogenase Activity Carcinoma Cell Lines
CLAUDE ‘IWZET,*~’ YVES J. BIGNON,t JEAN C. VENNAT,t JACQUES CHASSAGNE,t GINETTE GAILLARD,? ROBERT PLAGNE,?
in Several
MUSTAPHA PHILIPPE and DANIEL
Murine
AFANE,” CHOLLET,? BOUCHER*
*Laboratoire de physiologie et tquipe associke au CNRS, Facultt de Mkdecine, 28 place Henri Dunant, 63001 Clerrnont-Ferrand Cedex, and f’Centre Jean Perrin, Place Henri Dunant, 63011 Clermont-Ferrand Cedex, France
This report describes, for the first time to our knowledge, a possible steroidogenic activity in established murine embryonal carcinoma cell lines (PCC3, PCC4, F9), revealed by a 3j3-hydroxysteroid dehydrogenase activity (revelation of NADHz by staining, and RIA assessment of A4-androstenedione). The remarkable analogy between such totipotent cells and embryonal cells may suggest that this activity could be present before histologic organization of the embryonal testis. Nonmalignant embryonal cells such as tibroblasts (3/A/l/D-3) or myoblasts (T984) were also found to possess a 3B-hydroxysteroid dehydrogenase activity, thus suggesting that this enzyme is not specific to hormone-secreting cells, but the sign of a more general phenomenon. @ 1988 Academic FWS, II-K.
Teratocarcinomas are germ cell tumors containing a heterogeneous cell mixture which forms during the early stages of differentiated tissues [l-3] and other more undifferentiated cells called embryonal carcinoma (EC) cells 141. They are of interest because of their remarkable similarity to early embryonal cells [5], and thus may be used for embryogenic studies [6, 71. They originate from malignant tumors appearing spontaneously in mice, both male [8, 91 and female 110, 111. The frequency of these tumors can be increased by ectopic embryo grafting [12-141. Many established cell lines have been obtained after intraperitoneal transfers [15], followed by in vitro culture [16-201. These EC cells have a very high potential for differentiation; by intrablastocyst injection, chimeras have been obtained, in which almost all organs were constituted, in different proportions, of such EC-derived cells [21, 221. In the present work, we have been able to demonstrate that EC cells can move into steroidogenic cells. This was shown by establishing the presence of the 3&hydroxysteroid dehydrogenase enzyme (3/3HSD), a major enzyme in steroidogenesis in the cell cytosol. Materials
and Methods
Three embryonal carcinoma cell lines were used, PCC3 [19,23,24], PCC4 [19,23-261, and F9 [27], as well as two differentiated cell lines, the myoblastic clonal cell line C17SlD-T984 (T984) 128, 291 and the fibroblastic clonal cell line 3/A/l/D-3, derived from PCC3 [30]. These cells were cultivated with Dulbecco’s modified MEM (DME) containing 4.5 g/liter of glucose [31] obtained from Boehringer-Mannheim, supplemented with 10% fetal calf serum (FCS). Evidence of 3#I-HSD activiry by cell staining. The histologic characterization of 3/3-HSD is a blue staining of the cells possessing the enzyme, when dehydroepiandrosterone (DHA) is used as substrate Cell
culture.
’ To whom reprint requests should be addressed. 223
Copyright 0 1988 by Academic Ress, Inc. All rights of reproduction in any form reserved 0014-4827/88 $03.00
224 Short notes
Fig. 1. (a) PCC3 (x100), (X400).
(b) PCC4 (x100),
(c) F9 (x100),
(d) myoblasts (x400), (e) fibroblasts
[32, 331. The culture medium was replaced by 1 ml of DME supplemented with 0.5 mg/ml nicotinamide (Sigma), 3 mg/ml NAD (Sigma), 0.75 mg/ml tetrazolium nitroblue (Sigma), and 5 rig/ml DHA. The cells were incubated for 2 to 3 h at 37°C. 3t%HSD evidence by A4-androstenedione assessment (A4). Cell steroidogenesis was shown by assessment of A4 in the culture supematant after incubation with DHA. Adult Wistar rat interstitial testicular cells enriched in Leydig cells were used as positive control; Leydig cells were isolated by the technique described by Lefevre et al. [34] using a Percoll gradient. Both human lymphocytes and the culture medium itself, without FCS, were used as negative controls. Eight groups of cells were divided as follows: Leydig cells (lot l), PCC3 cells (lot 2), PCC4 cells (lot 3), F9 cells (lot 4), myoblasts (lot 5), fibroblasts (lot 6), lymphocytes (lot 7), culture medium alone (lot
‘3). For culture, 300,000 cells per well were incubated in Nunc culture multidishes at a temperature of 37°C. DME without FCS, containing 500 rig/ml DHA, was used. After 24 h, the supematant was frozen until the subsequent androstenedione experiment. A4 assessment was made by RIA with antibody Biomerieux after extraction in ether and chromatography on chromatholite A. Tritiated androstenedione was used as labeled hormone. A4-Androstenedione adsorption and release. A4 was assessed in culture supematants from four other lots: PCC3, PCC4, and F9 cell lines in a culture medium containing 10% FCS, but without DHA (PCC3*, lot 9; PCC4*, lot 10; and F9*, lot ll), and FCS alone (lot 12). Statistics. RIA was carried out by “single blind” methods. Comparative studies were analyzed with a Student’s t test.
Results 3/3-Hydroxysteroid
dehydrogenase staining. After eight different experiments,
97+6% of PCC3 (3724 cells), 80+14% of PCC4 (1815 cells), and 98+5% of F9 cells (2240 cells) were stained blue (Fig. 1) and showed a 3fi-HSD activity. The lower percentage obtained for PCC4 suggested the presence of two subpopulations, or a heterogeneity of these cells at a particular phase of the cell cycle. To test these possibilities a cloning experiment was carried out by isolating a single cell before culture. Twelve wells were positive for culture: every well contained the same two cell types in similar proportions (76f24%). This rejected the first hypothesis. We can suppose that the enzyme was functional or not, depending on the cycle phase of each cell. One hundred percent of the myoblasts (291 cells) and 97% of the fibroblasts (1000 cells) were stained (Fig. 1). The lymphocytes were not stained at ah. An incomplete Leydig cell purification, usual with the Percoll gradient, was responsible for the staining of about 80% of this group of cells.
Short notes
TABLE
1
A4 level in the different culture media induced by cell lines at a concentration 300,000 cellslml after an incubation of 24 h with 500 nglml DHA Androstenedione (ng/mU 1 2 3 4 5 6 7 8 9 10 I1 12
Leydig cells PCC3 PCC4 F9 Myoblasts Fibroblasts Lymphocytes Basal medium PCC3” PCC4” F9” Fetal calf serum”
225
29.95k4.45 0.55+0.13 0.65kO.06 1.06f0.11 0.49Yko.10 0.461kO.04 0.33kO.05 0.24+0.08 0.16f0.06 0.17kO.03 0.19+0.02 0.17+0.02
Note. The results are given with the formula mftxsl~n deviation; n number of repetitions). ’ Cultivated without DHA. b Comparison with the sensibility limits of 0.2 r&ml.
n
tb 82.14 7.01 18.14 20.72 4.96 11.26 6.85 1.31 1.35 2.00 1.06 3.00
of
ab
(m, mean; t, Student’s t; s, standard
A4-Androstenedione assessment. Every value obtained (Table l), except that of the culture medium was significantly higher than the sensitivity limit of the dosage method (0.2 rig/ml). The culture medium (lot 8) and FCS alone (lot 12) can be considered devoid of A4. The lymphocyte culture medium concentration (lot 7) was at the significance limit, when compared with that of the culture medium (~65~ IO-*). A cross-reaction with other steroids seemed unlikely. It is possible that the lymphocytes could have adsorbed serum hormone and then released it into the culture medium. The culture media of PCC3, PCC4, F9, myoblast and fibroblast cell lines contained significantly more A4 than negative control media (Table 1) (a
Our results showed that PCC3, F9, myoblasts, fibroblasts, and to a lesser extent, PCC4 were blue-colored when a staining reaction of 3/?-HSD was carried out. Furthermore, A4 was released by the cells studied, since this hormone was found in significant concentrations after 24 h of culture in PCC3, PCC4, F9, myoblast, and fibroblast supernatants, but not in the DME medium, and only at a very low level in lymphocyte culture medium. This hormone appeared to be
226 Short notes secreted by the cell lines under study because its absence in FCS, and in the different culture media from cells incubated with DHA, eliminated the possibility of cell release. This cell secretion was further evidence of the presence of functional 3/3-HSD, and confirmed the cytologic results. F9 seemed to be able to secrete more A4 than the other cell lines (PCC3, PCC4, myoblasts, and fibroblasts). It may be deduced from our results that EC cells may exhibit a steroidogenie activity: PCC3, PCC4, and F9 did so at a basal level, i.e., without hormonal induction. It is known that testis interstitial tissue is first seen in mouse embryos from Day 10 of pregnancy [35-371 and at Day 13 for the rat [38]. It has been shown in the pig that blastocysts are able to secrete estrogens [39,40] both in uiuo and in vitro since DHA is transformed into estrogens [41], in which case they contain 3B-HSD. The remarkable homology between embryonal cells and EC cells has been well documented. We have shown that this homology may extend to the presence of 3/l-HSD activity by these cells. Such activity in EC cells obtained without hormonal induction supposes, besides, that the first steroidogenie stages can be obtained independently of pituitary hormonal stimulation, but are linked to paracrine cell interactions. Our results showed the presence of a functional 3/GHSD enzyme in differentiated cells such as myoblasts and fibroblasts. This may be surprising at first, but 3/l-HSD enzyme-possessing cells are not limited to Leydig cells [42, 431: they are also found in alveolar macrophages [44], in adrenal gland cells [45], and in placenta [45, 461. These results raise the possibility of using EC cells as a model in the study of embryonal cell differentiation into steroidogenic cells. The authors are indebted to Madame H. Jakob for supplying the various cell lines necessary to their work and for the helpful advice which she has given throughout.
References 1. Pierce, G. B., and Vemey, E. L. (1961) Cancer 14, 1017. 2. Lehman, J. M., Speers, W. C., Schwarzendruber, D. E., and Pierce, G. B. (1974) J. Cell. Physiol. 84, 13. 3. Nicolas, J. F., Dubois, P., Jakob, H., Gaillard, J., and Jacob, F. (1975) Ann. Microbial. (Paris) 126A, 4.
5. 6. 7. 8. 9.
10. 11. 12. 13.
14. 15. 16. 17. 18. 19.
3.
Stevens, L. C. (1959) J. Natl. Cancer Inst. 23, 1249. Jacob, F. (1975) The Early Development of Mammals, Symposium II, p. 233, Cambridge Univ. Press. Martin, G. R. (1975) Cell 5, 229. Martin, G. R. (1980) Science 209, 768. Stevens, L. C., and Little, C. C. (1954) Proc. N&l. Acad. Sci. USA 40, 1080. Stevens, L. C. (1973) J. N&l. Cancer Inst. 50, 235. Stevens, L. C., and Vamum, D. S. (1974) Deu. Biol. 37, 369. Eppig, J. J., Kosak, L. P., Either, E. M., and Stevens, L. C. (1977) Nature (London) 269, 517. Stevens, L. C. (1964) Proc. Natl. Acad. Sci. USA 52, 654. Stevens, L. C. (1970) J. Nutl. Cancer Inst. 44, 923. Stevens, L. C. (1970) J. Exp. Zool. 174, 407. Stevens, L. C. (1970) Dev. Biol. 21, 364. Finch, B. W., and Ephrussi, B. (1967) Proc. Natl. Acad. Sci USA 57, 615. Rosenthal, M. D., Wishnow, R. M., and Sato, G. H. (1970) J. Natl. Cancer Inst. 44, 1001. Evans, M. J. (1972) J. Embyrol. Exp. Morphol. 28, 163. Jakob, H., Boon, T., Gaillard, J., Nicolas, J. F., and Jacob, F. (1973) Ann. Microbial. (Paris) 124B, 269.
Short
notes
227
20. MC Bumey, M. W. (1976) J. Cell. Physiol. 89, 441. 21. Brinster, R. L. (1974) J. Exp. Med. 140, 1049. 22. Mintz, B., and Illmensee, K. (1975) Proc. Natl. Acad. Sci. USA 72, 3585. 23. Guenet, J. L., Jakob, H., Nicolas, J. F., and Jacob, F. (1974) Ann. Microbial. (Paris) 125A, 135. 24. Nicolas, J. F., Avner, P., Gaillard, J., Guenet, J. L., Jakob, H., and Jacob, F. (1976) Cancer Res. 34, 4224.
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Received July 24, 1987 Revised version received September 30, 1987
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