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Induction of dna synthesis in cultured rabbit uterine cells by estradiol and inhibition of the estrogen response
J. sreroid Biochem. Vol. 21, No. 2. pp. 135-136, Printed in Great Britain. All rights reserved
1984 Copyright 0
0022-4731/84 $3.00 + 0.00 1984 Pergamon Press Ltd
INDUCTION OF DNA SYNTHESIS IN CULTURED RABBIT UTERINE CELLS BY ESTRADIOL AND INHIBITION OF THE ESTROGEN RESPONSE L. E. GERSCHENSON, JACK GORSKI* and D. M. PRESCOTT? Department of Pathology, School of Medicine, University of Colorado Health Sciences Center, Denver, CO 80262, *Department of Biochemistry, University of Wisconsin, Madison, WI 53706, TDepartment of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, CO 80309, U.S.A. (Received 14
October
1983)
Summary-Addition of 17/3-estradiol to primary cultures of rabbit uterine cells resulted in the induction of DNA synthesis. This phenomenon is present only in cells cultured at low density. Culture medium taken
from high density cultures inhibited it, suggesting that those cells produce a soluble estrogen-inhibiting factor.
Hormones have a major role in regulating cell proliferation in many tissues. One example is the induction by 17/?-estradiol of proliferation of cultured rabbit uterine epithelial cells reported by Gerschenson et a1.[2]. In the same paper it was also reported that the same rabbit endometrial cells cultured at high density produce a factor, probably a protein, that inhibits the induction of 17/?-estradiol of DNA synthesis. We have repeated part of these experiments and report here confirmation of induction of DNA synthesis by 17fi-estradiol in cultured uterine cells and inhibition of this response by medium taken from high density cultures of uterine cells. The methods and materials in general were as described by Gerschenson et a1.[2]. Primary cultures of rabbit uterine cells were plated in 35 mm diameter Falcon plastic dishes at low density (2.5 x lo4 cells/2 ml medium per dish) and high density (1.875 x lo6 cells/2 ml medium per dish). To obtain conditioned medium, the cells were incubated for 24 h from day 3-4 of culture, then rinsed gently with sterile saline and culture medium was added. After another 24 h incubation, the medium was changed and the cultures incubated for another day. These conditioned media from the low and high density cultures were collected, centrifuged at 1,OOOrpm at room temperature for 10 min, and the supernatants pooled and stored at -20°C. Another 30 low density cultures (1.25 x lo4 cells/2 ml medium per 35 mm dia dish) of primary uterine cells were initiated later for testing the previously collected media. The thirty cultures were divided into six groups of five, the medium removed and replaced with 2 ml of medium containing 0.5 pCi/mI of [-‘H]thymidine (sp. act. 62 Ci/mmol, New England Nuclear) plus other components as follows: Group I 0 + 2 ~1 ethyl alcohol; Group II 0.4 ml medium from culture + 2 ~1 ethyl alcohol;
low
density
Group III 0.4ml medium from high density culture + 2 ~1 ethyl alcohol; Group IV 0 + 2~1 estradiol solution (IO-‘M final concentration in ethyl alcohol; Group V 0.4ml medium from low density culture + 2 ~1 estradiol solution ( 10e8 M final concentration) in ethyl alcohol; Group VI 0.4ml medium from high density culture + 2 ~1 estradiol solution ( 10m8M final concentration) in ethyl alcohol. The cultures were incubated of 24 h, rinsed with saline solution and fixed with methanol for 2 min and coated with Kodak NTB2 autoradiographic emulsion. The autoradiographs were developed after three days at 4°C and scored blind by two independent observers for labeling indices or percentage of cells with labeled nuclei. Seven hundred to 1,100 cells were scored in each of five dishes per group. The average labeling indices (number of labeled cells/100 cells) for each group with standard errors were as follows: Group Group Group Group Group Group
I 0.22 f 0.13; II 0.02 + 0.02; III 0.27 f 0.10; IV 5.65 + 1.93; V 6.70 f 1.98; VI 0.54 f 0.04.
These results show the following: (1) 17/?-estradiol stimulates induction of DNA synthesis (the labeling index for Group IV is at least 20 x the labeling indices for Groups I, II or III) (2) Medium from low density cultures permits the estradiol response [the labeling indices for Groups IV and V are not significantly different] (3) Medium from high density cultures inhibits the estradiol response of cells plated at low density (compare Group VI with Groups IV and V). It is interesting to point out the quantitative differences between the present data and that pre135
136
L. E. GERSCHENWN ef al.
viously published [2]. The labeling indices of the control groups were lower and the effect of estradiol by itself, as well as the inhibition brought about by the media conditioned with high density cultures, were several-fold higher. Therefore, the results shown in this paper demonstrate the phenomena previously described in a more dramatic way. The reasons for the quantitative changes can be explained on the basis of (a) the already observed inherent quantitative differences among our experiments from one preparation of primary cultures to the next; and/or (b) the lower number of cells plated for the cultures used here to assay the conditioned media. The latter has been observed experimentally to result in a more marked estradiol effect accompanied by lower labeling indices in the absence of the steroid hormone, and can be explained through cell selection, resulting in a higher number of quiescent cells in the cultures, which has been shown previously to result in a lower labeling index, higher responsiveness to estrogen stimulation, and marked inhibition of the estrogen effect by its inhibitors [ 1, 21.
The inhibitor in medium from high density cultures was previously reported to block specifically the estradiol response and is probably a protein [2]. Purification and characterization of this protein and characterization of its mode of action are crucial issues that must now be addressed.
The validity of some of the data in the paper by Gerschenson et al. (1981) has been questioned. The new data reported in this paper attest to the validity of the original results and the correctness of the original conclusions.
REFERENCES
E. A., Yang J. and Gerschenson L. E., Connor Andersson M.: Hormonal regulation of proliferation in two populations of rabbit endometrial cells in culture. Life Sci. 24 (1979) 1337-1344. Gkrschenson‘ L. R., Depaoli J. R. and Murai J. T.: Inhibition of estrogen-induced proliferation of cultured rabbit uterine epithelial cells by a cell density-dependent factor produced by the same cells. J. steroid Biochem. 14 (1981) 959-969.
1984 Copyright 0
0022-4731/84 $3.00 + 0.00 1984 Pergamon Press Ltd
INDUCTION OF DNA SYNTHESIS IN CULTURED RABBIT UTERINE CELLS BY ESTRADIOL AND INHIBITION OF THE ESTROGEN RESPONSE L. E. GERSCHENSON, JACK GORSKI* and D. M. PRESCOTT? Department of Pathology, School of Medicine, University of Colorado Health Sciences Center, Denver, CO 80262, *Department of Biochemistry, University of Wisconsin, Madison, WI 53706, TDepartment of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, CO 80309, U.S.A. (Received 14
October
1983)
Summary-Addition of 17/3-estradiol to primary cultures of rabbit uterine cells resulted in the induction of DNA synthesis. This phenomenon is present only in cells cultured at low density. Culture medium taken
from high density cultures inhibited it, suggesting that those cells produce a soluble estrogen-inhibiting factor.
Hormones have a major role in regulating cell proliferation in many tissues. One example is the induction by 17/?-estradiol of proliferation of cultured rabbit uterine epithelial cells reported by Gerschenson et a1.[2]. In the same paper it was also reported that the same rabbit endometrial cells cultured at high density produce a factor, probably a protein, that inhibits the induction of 17/?-estradiol of DNA synthesis. We have repeated part of these experiments and report here confirmation of induction of DNA synthesis by 17fi-estradiol in cultured uterine cells and inhibition of this response by medium taken from high density cultures of uterine cells. The methods and materials in general were as described by Gerschenson et a1.[2]. Primary cultures of rabbit uterine cells were plated in 35 mm diameter Falcon plastic dishes at low density (2.5 x lo4 cells/2 ml medium per dish) and high density (1.875 x lo6 cells/2 ml medium per dish). To obtain conditioned medium, the cells were incubated for 24 h from day 3-4 of culture, then rinsed gently with sterile saline and culture medium was added. After another 24 h incubation, the medium was changed and the cultures incubated for another day. These conditioned media from the low and high density cultures were collected, centrifuged at 1,OOOrpm at room temperature for 10 min, and the supernatants pooled and stored at -20°C. Another 30 low density cultures (1.25 x lo4 cells/2 ml medium per 35 mm dia dish) of primary uterine cells were initiated later for testing the previously collected media. The thirty cultures were divided into six groups of five, the medium removed and replaced with 2 ml of medium containing 0.5 pCi/mI of [-‘H]thymidine (sp. act. 62 Ci/mmol, New England Nuclear) plus other components as follows: Group I 0 + 2 ~1 ethyl alcohol; Group II 0.4 ml medium from culture + 2 ~1 ethyl alcohol;
low
density
Group III 0.4ml medium from high density culture + 2 ~1 ethyl alcohol; Group IV 0 + 2~1 estradiol solution (IO-‘M final concentration in ethyl alcohol; Group V 0.4ml medium from low density culture + 2 ~1 estradiol solution ( 10e8 M final concentration) in ethyl alcohol; Group VI 0.4ml medium from high density culture + 2 ~1 estradiol solution ( 10m8M final concentration) in ethyl alcohol. The cultures were incubated of 24 h, rinsed with saline solution and fixed with methanol for 2 min and coated with Kodak NTB2 autoradiographic emulsion. The autoradiographs were developed after three days at 4°C and scored blind by two independent observers for labeling indices or percentage of cells with labeled nuclei. Seven hundred to 1,100 cells were scored in each of five dishes per group. The average labeling indices (number of labeled cells/100 cells) for each group with standard errors were as follows: Group Group Group Group Group Group
I 0.22 f 0.13; II 0.02 + 0.02; III 0.27 f 0.10; IV 5.65 + 1.93; V 6.70 f 1.98; VI 0.54 f 0.04.
These results show the following: (1) 17/?-estradiol stimulates induction of DNA synthesis (the labeling index for Group IV is at least 20 x the labeling indices for Groups I, II or III) (2) Medium from low density cultures permits the estradiol response [the labeling indices for Groups IV and V are not significantly different] (3) Medium from high density cultures inhibits the estradiol response of cells plated at low density (compare Group VI with Groups IV and V). It is interesting to point out the quantitative differences between the present data and that pre135
136
L. E. GERSCHENWN ef al.
viously published [2]. The labeling indices of the control groups were lower and the effect of estradiol by itself, as well as the inhibition brought about by the media conditioned with high density cultures, were several-fold higher. Therefore, the results shown in this paper demonstrate the phenomena previously described in a more dramatic way. The reasons for the quantitative changes can be explained on the basis of (a) the already observed inherent quantitative differences among our experiments from one preparation of primary cultures to the next; and/or (b) the lower number of cells plated for the cultures used here to assay the conditioned media. The latter has been observed experimentally to result in a more marked estradiol effect accompanied by lower labeling indices in the absence of the steroid hormone, and can be explained through cell selection, resulting in a higher number of quiescent cells in the cultures, which has been shown previously to result in a lower labeling index, higher responsiveness to estrogen stimulation, and marked inhibition of the estrogen effect by its inhibitors [ 1, 21.
The inhibitor in medium from high density cultures was previously reported to block specifically the estradiol response and is probably a protein [2]. Purification and characterization of this protein and characterization of its mode of action are crucial issues that must now be addressed.
The validity of some of the data in the paper by Gerschenson et al. (1981) has been questioned. The new data reported in this paper attest to the validity of the original results and the correctness of the original conclusions.
REFERENCES
E. A., Yang J. and Gerschenson L. E., Connor Andersson M.: Hormonal regulation of proliferation in two populations of rabbit endometrial cells in culture. Life Sci. 24 (1979) 1337-1344. Gkrschenson‘ L. R., Depaoli J. R. and Murai J. T.: Inhibition of estrogen-induced proliferation of cultured rabbit uterine epithelial cells by a cell density-dependent factor produced by the same cells. J. steroid Biochem. 14 (1981) 959-969.