OESTROGEN-INDUCED PROTEIN IN RAT MAMMARY TUMOUR AND UTERUS N.
MAIRESSE, J. C. HEUSON, P. GALAND AND G. LECLERCQ
Biology Unit, Institute of Interdisciplinary Research, Free University of Brussels, 115 Waterloo, 1000-Brussels, Belgium and *Service de Médecine et Laboratoire d'Investigation Clinique, Institut J. Bordet, 1000-Brussels, Belgium
(Received 19 April 1977) We wish to report evidence for the early induction by oestradiol-17\g=b\ of a protein in mammary tumours induced in Sprague\p=n-\Dawley rats by 7,12-dimethylbenz(a)anthracene (DMBA). The protein appears to be analogous to the oestrogen-induced protein first discovered in the rat uterus by Notides & Gorski (1966) and widely known as 'induced protein' (IP). Induction of IP by oestradiol-17\g=b\has been demonstrated in rodents in several tissues of the female genital tract that are targets for oestrogens (Katzman, Larson & Podratz, 1971; Katzenellenbogen & Leake, 1974; Dupont-Mairesse & Galand, 1975) and correlated with the rate of nuclear
accumulation of oestrogen receptors (Katzenellenbogen & Gorski, 1972; Katzenellenbogen, 1975). Therefore, the search for induction of IP in DMBA-induced mammary tumours containing oestrogen receptors was of interest, not only from the general viewpoint of oestrogen action but also for the understanding of hormone dependence. Mammary tumours were induced in female Sprague-Dawley rats by gastric instillation of DMBA (Eastman Organic Chemicals, Rochester, New York) as described previously by Leclercq & Heuson (1973). Carcinomatous tumours were recovered from intact (metoestrous or dioestrous) and ovariectomized rats. The concentration of cytoplasmic oestrogen receptors (Leclercq & Heuson, 1973) ranged between 33 and 106 fmol/mg protein. Hormonal stimulation was achieved by incubating tumour fragments in the presence of 3x 10"8M-oestradiol-17j3. This offers the advantage over stimulation in vivo that groups of randomly selected fragments of treated and control tissues can be used, thus reducing inter¬ ference of tissue heterogeneity in the results. Synthesis of IP was measured by the doublelabelling method of Katzenellenbogen & Gorski (1972) (see details in the legend of Fig. 1). The search for synthesis of IP in ten tumours revealed a peak in the 3H: 14C ratio in the region of the gel corresponding to the mean position of the uterine IP (RF 0-75). Tire magnitude of induction of IP in the tumours varied between 12 and 30%, compared with 30-86% in the uteri of the same animals. Values lower than 15% were considered to be in¬ conclusive (Fig. 1), because with tumours, such values were often found associated with minor unspecific peaks. Experiments in which the stimulation was performed in vivo (10µg oestradiol, administered intraperitoneally) gave equivocal results. Indeed, basal values were too variable (probably because of tissue heterogeneity) to permit any valid quantitation of peaks eventually found in the IP region. Hormone independence of mammary tumours possessing the receptor system could result from interruption of oestrogen-triggered metabolic pathways. If interruption was located at the IP level, measurement of the latter, with due improvement of the detection procedure, would become a valuable criterion of oestrogen dependence. =
P. Galand is Martre de Recherches of the Belgian Fonds National de la Recherche Scientifique. This work was supported by the Fonds Cancer of C.G.E.R. and of the Ministère de la Politique Scientifique within the framework of the Association Euratom, Universities of Brussels and Pisa. The authors are grateful to Mrs D. Leemans for the preparation of the manu¬
script.
1. Isotope ratios of soluble proteins from 7,12-dimethylbenz(a)anthracene-induced mammary tumours in Sprague-Dawley rats after separation by gel electrophoresis. Tumour samples were incubated in Krebs-Ringer buffer medium with added [3H]leucine ( µ / ) and oestradiol-17(3 (3 X 10"8mol/l) dissolved in propylene glycol (final concentration l%v/v) or for reference, in medium containing [!4C]leucine (5 MCi/ml) and propylene glycol alone. After 2h, 100 Mg cytosol proteins from stimulated and reference samples were mixed and submitted to co-electrophoresis on polyacrylamide gel (Katzenellenbogen & Gorski, 1972). The peak in the 3H:14C ratio was used to detect 'induced protein' (IP). The amount of IP synthesized was estimated by the increase in the 3H:"*C ratio in this area of the gel, compared with the mean ratio in all other regions. The count rates for 3H and ,4C were 100 and 50 c.p.m. above background, respectively, (b) and (/) show inconclusive tests (3H:14Cless than 15 % above the basal value); (a), (c), (d), (e) and (g) show positive responses (3H:14C 30, 19, 25, 17 and 25% above the basal value respectively). The shaded area indicates the mean position of IP, which, relative to the front of migration, has a mobility of 0-75. (a)-(f), Separate experiments with fragments from six different tumours; (g), pooled fragments from ten tumours taken from seven rats.
Fig.
REFERENCES
Dupont-Mairesse, N. & Galand, P. (1975). Endocrinology 96, 1587-1591. Katzenellenbogen, B. S. (1975). Endocrinology 96, 289-297.
Katzenellenbogen, B. S. & Gorski, J. (1972). Journal ofBiological Chemistry 274, 1299-1305. Katzenellenbogen, B. S. & Leake, R. E. (1974). Journal of Endocrinology 63,439-449. Katzman, P. ., Larson, P. L. & Podratz, K. C. (1971). In The sex steroids. Molecular mechanisms, pp. 107-147. Ed. K. W. McKerns. New York: Appleton Century Crofts. Leclercq, G. & Heuson, J. C. (1973). European Journal of Cancer 9, 675-680. Notides, A. & Gorski, J. (1966). Proceedings of the National Academy of Sciences of the U.S.A. 56, 230-235.
MAIRESSE, J. C. HEUSON, P. GALAND AND G. LECLERCQ
Biology Unit, Institute of Interdisciplinary Research, Free University of Brussels, 115 Waterloo, 1000-Brussels, Belgium and *Service de Médecine et Laboratoire d'Investigation Clinique, Institut J. Bordet, 1000-Brussels, Belgium
(Received 19 April 1977) We wish to report evidence for the early induction by oestradiol-17\g=b\ of a protein in mammary tumours induced in Sprague\p=n-\Dawley rats by 7,12-dimethylbenz(a)anthracene (DMBA). The protein appears to be analogous to the oestrogen-induced protein first discovered in the rat uterus by Notides & Gorski (1966) and widely known as 'induced protein' (IP). Induction of IP by oestradiol-17\g=b\has been demonstrated in rodents in several tissues of the female genital tract that are targets for oestrogens (Katzman, Larson & Podratz, 1971; Katzenellenbogen & Leake, 1974; Dupont-Mairesse & Galand, 1975) and correlated with the rate of nuclear
accumulation of oestrogen receptors (Katzenellenbogen & Gorski, 1972; Katzenellenbogen, 1975). Therefore, the search for induction of IP in DMBA-induced mammary tumours containing oestrogen receptors was of interest, not only from the general viewpoint of oestrogen action but also for the understanding of hormone dependence. Mammary tumours were induced in female Sprague-Dawley rats by gastric instillation of DMBA (Eastman Organic Chemicals, Rochester, New York) as described previously by Leclercq & Heuson (1973). Carcinomatous tumours were recovered from intact (metoestrous or dioestrous) and ovariectomized rats. The concentration of cytoplasmic oestrogen receptors (Leclercq & Heuson, 1973) ranged between 33 and 106 fmol/mg protein. Hormonal stimulation was achieved by incubating tumour fragments in the presence of 3x 10"8M-oestradiol-17j3. This offers the advantage over stimulation in vivo that groups of randomly selected fragments of treated and control tissues can be used, thus reducing inter¬ ference of tissue heterogeneity in the results. Synthesis of IP was measured by the doublelabelling method of Katzenellenbogen & Gorski (1972) (see details in the legend of Fig. 1). The search for synthesis of IP in ten tumours revealed a peak in the 3H: 14C ratio in the region of the gel corresponding to the mean position of the uterine IP (RF 0-75). Tire magnitude of induction of IP in the tumours varied between 12 and 30%, compared with 30-86% in the uteri of the same animals. Values lower than 15% were considered to be in¬ conclusive (Fig. 1), because with tumours, such values were often found associated with minor unspecific peaks. Experiments in which the stimulation was performed in vivo (10µg oestradiol, administered intraperitoneally) gave equivocal results. Indeed, basal values were too variable (probably because of tissue heterogeneity) to permit any valid quantitation of peaks eventually found in the IP region. Hormone independence of mammary tumours possessing the receptor system could result from interruption of oestrogen-triggered metabolic pathways. If interruption was located at the IP level, measurement of the latter, with due improvement of the detection procedure, would become a valuable criterion of oestrogen dependence. =
P. Galand is Martre de Recherches of the Belgian Fonds National de la Recherche Scientifique. This work was supported by the Fonds Cancer of C.G.E.R. and of the Ministère de la Politique Scientifique within the framework of the Association Euratom, Universities of Brussels and Pisa. The authors are grateful to Mrs D. Leemans for the preparation of the manu¬
script.
1. Isotope ratios of soluble proteins from 7,12-dimethylbenz(a)anthracene-induced mammary tumours in Sprague-Dawley rats after separation by gel electrophoresis. Tumour samples were incubated in Krebs-Ringer buffer medium with added [3H]leucine ( µ / ) and oestradiol-17(3 (3 X 10"8mol/l) dissolved in propylene glycol (final concentration l%v/v) or for reference, in medium containing [!4C]leucine (5 MCi/ml) and propylene glycol alone. After 2h, 100 Mg cytosol proteins from stimulated and reference samples were mixed and submitted to co-electrophoresis on polyacrylamide gel (Katzenellenbogen & Gorski, 1972). The peak in the 3H:14C ratio was used to detect 'induced protein' (IP). The amount of IP synthesized was estimated by the increase in the 3H:"*C ratio in this area of the gel, compared with the mean ratio in all other regions. The count rates for 3H and ,4C were 100 and 50 c.p.m. above background, respectively, (b) and (/) show inconclusive tests (3H:14Cless than 15 % above the basal value); (a), (c), (d), (e) and (g) show positive responses (3H:14C 30, 19, 25, 17 and 25% above the basal value respectively). The shaded area indicates the mean position of IP, which, relative to the front of migration, has a mobility of 0-75. (a)-(f), Separate experiments with fragments from six different tumours; (g), pooled fragments from ten tumours taken from seven rats.
Fig.
REFERENCES
Dupont-Mairesse, N. & Galand, P. (1975). Endocrinology 96, 1587-1591. Katzenellenbogen, B. S. (1975). Endocrinology 96, 289-297.
Katzenellenbogen, B. S. & Gorski, J. (1972). Journal ofBiological Chemistry 274, 1299-1305. Katzenellenbogen, B. S. & Leake, R. E. (1974). Journal of Endocrinology 63,439-449. Katzman, P. ., Larson, P. L. & Podratz, K. C. (1971). In The sex steroids. Molecular mechanisms, pp. 107-147. Ed. K. W. McKerns. New York: Appleton Century Crofts. Leclercq, G. & Heuson, J. C. (1973). European Journal of Cancer 9, 675-680. Notides, A. & Gorski, J. (1966). Proceedings of the National Academy of Sciences of the U.S.A. 56, 230-235.
