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Response to prolactin and ovarian steroids of normal mammary epithelial cell cultures
Molecular and Cellular Endocrinology, 0 Elsevier/North-Holland
RESPONSE TO PROLACTIN MAMMARY
8 (1977)
95-103
Scientific Publishers, Ltd.
AND OVARIAN
STEROIDS OF NORMAL
EPITHELIAL CELL CULTURES *
Roberto L. CERIANI ** and E.W. BLANK Lahoratoire de Physioiogie de la Lactation, C.N.R.Z., Jouy-en-Josas, France, and Bruce Lyon Memorial Research Laboratory, Children’s Hospital Medical Center, 51st and Grove Streets, Oakland, California 94609, U.S.A.
Received 14 February 1977; accepted 12 April 1977
Mouse mammary epithelial cells in confluent primary monolayer cultures retain responsiveness to the specific hormones that induce mammary growth in vivo. Simultaneous stimulation by prolactin, progesterone and estrogen, in the presence of 5% fetal calf serum, is required to induce an increase in both thymidine uptake into DNA and in cell replication (as judged by mitotic indexes) over the hormone-free control. This increase in mitogenic response could not be elicited in either mouse fibroblasts or in mouse mammary tumor cells, the latter known to be hormone insensitive. Keywords;
hormonal control; breast epithelium; cell replication.
Most of our knowledge in the area of hormone stimulation of epithelial cell replication in the mammary gland has been obtained from in vivo experiments (for review see Ceriani, 1974). However, the main drawbacks of the in vivo system are the heterogeneity of the tissue (even in lactating glands, 30% or more of the cells are not epitheliai in nature, as reported by Nicoll and Tucker, 1965) the uncertainty about hormone concentrations at the cellular level, and the possible simultaneous action of other hormones and/or factors with the hormone under study in inducing cell replication. Organ culture has also been used as an alternative for these studies, as discussed by Forsyth (1971). However, it has inherent deficiencies in the study of mammary epithelial cell replication in that: (a) the round of cell replication induced by hormones is short-lived (as in the case of insulin stimulation described by Topper, 1970) (b) the tissue remains viable for only a short time, as shown by Dilley * This research was partly supported by a Population Council Postdoctoral Fellowship to R.L.C., and in part by Contract No. NOI CB 33906 from the National Cancer Institute, National Institutes of Health, DHEW, awarded to S. Abraham. ** Present address: Bruce Lyon Memorial Research Laboratory, Children’s Hospital Medical Center, 5 1st and Grove Streets, Oakland, California 94609, U.S.A. 95
96
R. L. Ceriani and E. W. Blank
(1971) (c) the cell heterogeneity permits only histological and histochemical studies and precludes large-scale biochemical separations. For these reasons, monolayer cultures of mouse mammary epithelial cells (MMEC), which provide a more manageable, homogenous and controlled system, were used in this study. In it, the concomitant effect of prolactin and ovarian steroids in inducing cell replication of MMEC is demonstrated. This mitogenic effect was usually associated only with prolactin in the previous in vivo experiments of Simpson and Schmidt (1971), and in the organ culture studies of Dilley (1971). The defined cell culture conditions described herein permitted the establishment of such double hormonal requirement, in the presence of serum, for mouse mammary epithelium cell replication.
MATERIALS
AND METHODS
Mouse mammary tissue was dissected from pregnant (15-18 days) BALB/c mice. Monodisperse suspensions of epithelial cells were obtained by collagenase plus hyaluronidase, followed by pronase digestion of the tissue in accordance with Weipjes and Prop (1970) the only exception being that a 10 pm Nitex filter (TETKO, Inc., Monterey Park, California) was used for cell filtration. This method of cell dispersion yielded cell suspensions that were enriched up to 90% or more for MMEC (Thompson et al., 1976). MMEC obtained by this method retained epithelial morphology after plating and for the time period of the experiments. In addition, this MMEC population had a very small percentage of polyploid nuclei, since they were plated at a high density (3.5 X 105/cm2) (Das et al., 1974). [3H]Thymidine uptake was measured in MMEC, freshly dispersed, by the method described above and plated at a density of 3.5 X 10’ cells/cm’ in 2.5 ml of Waymouth’s medium supplemented by 3.5 mg/lOO ml penicillin G and 5 mg/lOO ml streptomycin plus 20% fetal calf serum (FCS), and incubated for 72 h at 37°C in a humidified atmosphere of 95% air plus 5% C02, in plastic Petri dishes (Falcon) 3.2 cm in diameter. At day 3 after plating the cells were already in a confluent monolayer, and, at this moment, the medium was changed to Waymouth’s medium plus 5% FCS. After another 3 days the medium was changed again to Waymouth’s medium plus 5% FCS, with or without hormones (this was considered to be 0 time for the labeling experiments). Each group consisted of three 3.2 cm diameter plates. 3-h pulses were performed at times indicated by placing the cells in fresh medium plus 1 nCi/ml of [3H] thymidine. After the pulse, the plates were washed with cold Ca2+- and Mg2+-free solution (CMF) (Weipjes and Prop, 1970). Then ihe cells were scraped off the plates and washed 3 times with 5% TCA, 3 times with acetone, and the pellet was then redissolved in I .O N NaOH. Half the sample was neutralized and used for radioactivity counts, the other half was used for fluorimetric determinations of DNA in accordance with Hinegardner (1971). Results were expressed as cpm/,ug DNA.
Hormones and mammary epithelial cell cultures
91
Fetal fibroblasts were obtained from fetuses of midpregnant mice, and the mouse epithelial mammary tumor cells from a transplantable tumor that arose in a CsH fostered BALB/c mouse (Bern and Nandi, 1961, showed this type of mouse mammary tumor to be hormone insensitive and Turkington, 1974, demonstrated that it lacks prolactin receptors). For cell dispersion, tumor or fetal tissues were dissected, minced, and then digested with 0.25% trypsin (Difco) plus 0.02% EDTA, in CMF for 30 min; large pieces were allowed to settle, the cell suspension floating above separated, and the latter, containing dispersed cells, was centrifuged (6Og for 5 mm) and its pellet washed once in CMF. The cells were then resuspended in Waymouth’s medium, counted and seeded at a density of 2 X lO’/cm* for the fibroblasts, and 5 X 105/cm2 for the tumor cells in plastic Petri dishes 3.2 cm in diameter. Both cell preparations were cultured in 2.5 ml of Waymouth’s medium plus 3.5 mg penicillin G and 5 mg streptomycin per 100 ml medium, in 10% FCS, in a humidifed atmosphere of 95% air plus 5% CO,, at 37’C; media were changed every 3 days. 3 days after the cells attained confluency, media were changed to Waymouth’s plus 5% FCS, and the cells left in culture for another 3 days. At the end of this period the media were changed to Waymouth’s plus 5% FCS either alone (controls) or plus hormones. 3-h pulses with [3H]thymidine were performed as for the MMEC. Each group consisted of 3 separate plates. The intracellular concentration of [3H]thymidine in MMEC was measured at the same time intervals when [3H] thymidine uptake was measured. For this purpose, MMEC were pulsed with [3H]thymidine for 3 h, then the cells were subjected to 2 fast washes, with gentle swirling, with cold CMF. After this, the cells were scraped, and the cell suspension made up to 0.1% in Tween 20 in CMF (Atlas Powder Co., ‘Wilmington, Delaware) to lyse the cells. Following this, the samples were made up to 5% TCA, let stand for 1 h at room temperature, and then centrifuged. The supernatant fraction was saved. The pellet was washed again twice with 5% TCA, and the washes saved. The supernatant fraction after TCA precipitation plus the 2 washes was consolidated, [3H]thymidine activity was measured, and DNA was estimated in the pellets in accordance with Hinegardner (1971). Results were expresses as cpm of tritium found in the supernatant fraction (that were considered to correspond to labeled thymidine) per pg of DNA of MMEC in the pellet. To determine the effect of hormones on mitotic indexes, monolayers of confluent MMEC, fibroblasts or mammary tumor cells were prepared as for the thymidine incorporation studies. 3 days after confluency (the cells now in 5% FCS) the media were changed to Waymouth’s plus 5% FCS with or without hormones, and at times indicated, their media were substituted by the same plus 0.12 ng/ml colchitine (Colcemid) for 6 h. At the end of the colchicine incubation the plates were washed with phosphate buffered saline and then the cells were fixed with Bouin’s fixative and stained with Wright’s stain. At least 5 X IO3 nuclei were counted, and mitoses per 1 X 1O3 cells estimated in triplicates. The hormones and hormone concentrations used were: prolactin (NIH-PS-8) 0.2 pg/ml; progesterone (Calbiochem), 1 pg/ml; estradiol (17/3-estradiol, Calbio-
98
R.L. Cerimi and E. W. Blank
them), 0.001 E.rg/ml. Prdactin was dissolved in 0.0001 N NaOH, and steroid hormones were first diluted into absolute ethanol, then this stock solution was diluted in media, where the final ethanol concentration never exceeded 0.001%.
RESULTS After medium with or without hormones plus 5% FCS was added to the confluent MMEC, a 4-fold increase in thymidine uptake into 5% trichloroacetic acid (TCA~precipitable material occurs within 24 h (fig. I>. Thymidine uptake increases under similar circumstances have already been reported for other cells by Todaro et al. (1965), by Weibel and Baserga (1969), by Becker and Levitt (1968), and by Baserga et al. (1971). In contrast, this wave of thymidine uptake is not apparent in
Hormone Combination Time
(hours):
:
c I23 O-3
Cl
23 24-27
C
I23
48-51
Fig. I. Effect of hormones on [ 3H 1tby~lidine incorporation into DNA by MMEC in confluent monolayers. Results are expressed as cpm/pg DNA. Hormone combinations: C = no hormone; 1 = prolactin (NIH-PSI, 0.2 fig/ml); 2 = progestetone (Calbiochem, 1 Irg/ml) plus estradiol (17p-estradiol, Calbiochem, 0.001 pg/ml); 3 = prolactin (90.2 &ml) plus progesterone (1 fig/ ml) plus estradiol (0.001 erg/ml). Each point represents the average of 6 samples corresponding to 2 experiments of 3 samples each, performed with different MMfC preparations; each sample corresponded to determinations performed on the cells monolayered in a 3.2 em diameter plastic Petri dish. Vertical bars represent the standard error of mean (SEM).
Hormones and mammary epithelial cell cultures
99
nonconfluent cells. The latter incorporated [3H]thymidine at a sustained level for several days after medium change. That [3H]thymidine was incorporated into DNA in the present studies is suggested by the fact that the radioactivity in the TCA-precipitable material of the MMEC was redissolved by 10% TCA digestion at 4O”C, overnight. Confluent monolayers of MMEC responded to prolactin plus progesterone plus estrogen, added to the medium-change to trigger DNA replication, with an increase in uptake of [3H] thymidine into TCA-precipitable material over the hormone-free control, between 24 and 27 h. This effect of prolactin plus estrogen plus progesterone was even noticeable when compared to the estrogen plus progesterone stimulated group (fig. 1). (This latter group was usually lower in value than the hormonefree, and often significantly lower.) Prolactin alone did not induce this increased response; rather, its effect on thymidine uptake was inhibitory. In addition, MMEC exposed for 24 h to progesterone alone, estrogen alone, progesterone plus prolactin, and estrogen plus prolactin, and then pulsed with thymidine for 3 h, gave values of 511.2 f 127.8, 835.2 + 61.7, 670.2 f 50.1 and 962 f. 113.2 cpm/pg DNA, respectively, all of them below their hormone-free control (1125 + 76.5) (each group represents values from 3 plates; f indicates standard error of mean). Further, at 48 h, the increase in thymidine uptake shown by addition of prolactin to progesterone plus estradiol is still apparent (fig. 1). These hormonal effects could not be elicited if MMEC were stimulated by hormones in the presence of FCS in higher concentrations than 5%. With FCS concentrations of 10% and 20% without hormones, [3H]thymidine incorporation was more than 60% higher than at 5% FCS without hormones. This was the level obtained for the estrogen plus progesterone plus prolactin stimulated group above the 5% FCS control without hormones. The difference in intracellular concentration of TCA-soluble [3H] thymidine after 3 h incubations of the hormone-treated cells or their controls, at any of the given times, was not statistically significant. This similar content of intracellular [3H] thymidine in every experimental cell group suggests that the increase in [3H] thymidine uptake after hormone stimulation is not a result of altered intracellular thymidine pools. To corroborate these results showing an increase in thymidine uptake by cells exposed to prolactin plus estrogen plus progesterone, experiments were carried out where number of mitoses per 1 X lo3 cells after mitotic arrest with colchicine was determined in control and hormone-stimulated groups (table 1). These experiments were performed in MMEC preparations prepared on different days than the ones used for the [3H] thymidine incorporation experiments. Here again, the media supplemented with prolactin plus the ovarian steroids induced higher mitotic indexes over any partial combination of the three hormones, each of the hormones alone or the control group. In contrast to the responsiveness in terms of [3H]thymidine uptake, shown by the MMEC to the triple hormone combination (estrogen plus progesterone plus prolactin), both fibroblasts and tumor cells were unresponsive (table 2). In parallel
R. L. Ceriatzi arzd E. W. Blank
Hormones and mammary epithelial cell cultures
experiments, after stimulation with the triple hormone combination, mitotic indexes over the hormone-free control could be evidenced fetal fibroblasts or the mammary tumor cells (table 1).
101
no increase in for either the
DISCUSSION Normal monolayers of mouse mammary gland epithelial cells (MMEC) retaining in vivo characteristics are valuable in cell differentiation studies and as a point of reference for their transformed counterparts in cell biology investigations. Conditions for both cell culture of hormone-responsive MMEC, and for eliciting response of MMEC to hormones, are described herein. It is shown that the number of MMEC entering replication could be modulated by appropriate hormones under such conditions, therefore proving that MMEC retain their mechanism of hormonal response after cell dispersion and 7 days of monolayer culture. The hormone-induced increase in [ 3H] thymidine uptake into TCA-precipitable material (presumably DNA) of MMEC results from specific stimulation by the same hormones that are thought to be responsible for mammary growth in vivo, i.e. ovarian steroids and prolactin. Cell replication, as shown by the mitotic arrest studies, was also stimulated in the MMEC by these hormones, lending further credibility to the thymidine uptake studies. The values of mitotic indexes of MMEC after prolactin plus ovarian steroid stimulation (17.0%, table 1) are in the range of values for percentage of cells entering mitosis in the midpregnant mouse mammary gland reported by Bresciani (1971). In vivo and organ culture experiments, including those of Lyons et al. (1958) and of Ichinose and Nandi (1964, 1966) seem to rule out any action of ovarian steroids when used in the absence of other hormones in promoting mammary growth. In contrast, Norgren (1967) has been the only one to date to report stimulatory action on the growth of the mammary gland by these steroids. In this report Norgren shows that ovarian steroids cause stunted growth of ducts in the mammary gland of hypophysectomized rabbits. Further, the growth-promoting effect of prolactin alone on the in vivo mammary gland has been reported by Simpson and Schmidt (1971) by Meites and Kragt (1964), and by Cole and Hopkins (1962). However, this induction of mammary growth directed by prolactin alone (with the reservation that it was induced in vivo and that other stimulatory factors could not be ruled out) was always restricted and could not be compared to mammary growth found in midpregnancy. The growth obtained was mainly ductal with scant alveolar development. Midpregnant type of development is seen after in vivo prolactin injection only when ovarian steroids are also injected in situ in the mammary gland,, as was shown by Nagasawa and Yanai (1971). Apparently, prolactin requires the cooperative action of other hormones to induce mammary cell replication in vitro. Simultaneous stimulation by ovarian steroids and prolactin, in the presence of serum, was required here to increase the level of DNA synthesis in the MMEC
102
R.L. Ceriani and E. W. Blank
monolayers. Possibly prolactin renders the MMEC sensitive to ovarian steroids via an increase in steroid binding capacity, as was shown by Leung and Sasaki (1973) for mammary glands in organ culture. Thus, information must be present in the genome of mouse mammary cells for a prolactin-plus-ovarian-steroid-mediated cell replication, as there is for specific protein synthesis as shown by Ceriani (1976) for MMEC. Therefore, mammary epithelium replication in vivo can be envisaged as occurring at a constant basal level maintained by serum and/or other stimuli and with hormones acting as modulating agents, as proposed by Bresciani (1968, 1971). This basal level of cell replication would be controlled by density-dependent inhibition of growth (DDIG) and hormones; the latter would have as their role to counter the former. Nonconfluent MMEC in vitro maintain a high level of thymidine uptake and cell replication for several days after medium change. In contrast, confluent ones will respond with a low and short-lived level of cell replication after medium change, showing that they, as well as the tissue cells, are subjected to DDIG. After appropriate hormone stimulation, DDIG restrictions were superseded in our MMEC cultures and increases of up to 80% above basal thymidine incorporation and mitotic rate were obtained. These levels of mammary epithelial cell replication are comparable to those obtained after serum stimulation of the mouse mammary gland in organ culture by Majumder and Turkington (197 1). The stimulator-y action of hormones on thymidine uptake of monolayered MMEC was obtained here at a concentration of 5% FCS, but not at higher concentrations. At high serum concentrations MMEC in monolayers replicated possibly at their maximum as a result of intrinsic serum factors and/or high enough concentrations of the hormones carried in it. A comparable situation is found when casein synthesis is induced in MMEC monolayers by hormones. The action of the latter could only be demonstrated in the absence of serum by Ceriani (1976). Further, Majumder and Turkington (1971) have shown that epithelial cells in mouse mammary gland explants increase their thymidine uptake only as a result of stimulation by 5% FCS concentrations and above. This would suggest that the concentration of FCS used in this study is in the borderline for stimulation, and thus it would be at this level that any cooperative action of hormones could be demonstrated. The possibility that the observed hormonal stimulation of thymidine uptake and mitotic indexes in MMEC could be related to the unusual conditions of the culture system and, therefore, could be present in all cultured mouse cells, was ruled out. Monolayers of mouse fetal fibroblasts and of epithelial cells from a hormone-insensitive transplantable mouse mammary tumor did not respond to the hormonal stimulation effective on MMEC. At best, prolactin and the ovarian steroids proved to be inhibitory of cell replication in the nonmammary cell monolayers. The cell type specificity of the hormonal stimulation of MMEC is thus proved.
ACKNOWLEDGEMENT I thank the late Dr. R. Denamur for advice and encouragement.
Hormones and mammary epithelial cell culhtres
103
REFERENCES Baserga, R., Rovera, G. and Farber, J. (1971) In Vitro 7, 80-87. Becker, Y. and Levitt, F. (1968) Exp. Cell. Res. 51,27-33. Bern, H.A. and Nandi, S. (1961) Prog. Exp. Tumor Res. 2,90-144. Bresciani, F. (1968) Cell Tissue Kinet. 1,51-6.3. Bresciani, 1:. (1971) In: Basic Actions of Sex Steroids on Target Organs (Karger, Basel) pp. 130-159. Ceriani, R.L. (1974) J. Invest. Dermatol. 63,93-108. Ceriani, R.L. (1976) .I. Exp. 2001. 196, 1-12. Cole, R.D. and Hopkins, T.R. (1961) Endocrinology 71,395-398. Das, N.K., Hosick, H.L. and Nandi, S. (1974) J. Natl. Cancer Inst. 52,849-855. Dilley, W.G. (1971) Endocrinology 88,514-517. Forsyth, I.A. (1971) J. Dairy Res. 3,419-444. Hinegardner, R.T. (1971) Anal. Biochem. 39, 197-201. Ichinose, R.R. and Nandi, S. (1964) Science 145,496-497. Ichinose, R.R. and Nandi, S. (1966) J. Endocrinol. 35,331-340. Leung, B.L. and Sasaki, G.H. (1973) Biochem. Biophys. Res. Commun. 55,1180-1187. Lyons, W.R., Li, C.H. and Johnson, R.E. (1958) Recent Prog. Horm. Res. 14,219-254. Majumder, G.P. and Turkington, R.W. (1971) Endocrinology 88, 1506-1510. Meites, J. and Kragt, C.L. (1964) Endocrinology 75,565-570. Nagasawa, H. and Yanai, R. (1971) Endocrinol. Jpn. 18,53-56. Nicoll, C.S. and Tucker, H.A. (1965) Life Sci. 4,993-1001. Norgren, A. (1967) Acta Univ. Lund. Sect. II, 11,2. Simpson, A.A. and Schmidt, G.H. (1971) J. Endocrinol. 51,265-270. Todaro, G.J., Lazar, G.K. and Green, H. (1965) J. Cell. Comp. Physiol. 66,325-334. Thompson, K., Ceriani, R.L., Wong, D. and Abraham, S. (1976) J. Natl. Cancer Inst. 57, 167-172. Topper, Y.J. (1970) Recent Prog. Horm. Res. 26,287-308. Turkington, R.W. (1974) Cancer Res. 34,758-763. Weibel, F. and Baserga, R. (1969) J. Cell. Physiol. 74, 191-202. Weipjes, G.J. and Prop, F.J.A. (1970) Exp. Cell Res. 61,451-454.
RESPONSE TO PROLACTIN MAMMARY
8 (1977)
95-103
Scientific Publishers, Ltd.
AND OVARIAN
STEROIDS OF NORMAL
EPITHELIAL CELL CULTURES *
Roberto L. CERIANI ** and E.W. BLANK Lahoratoire de Physioiogie de la Lactation, C.N.R.Z., Jouy-en-Josas, France, and Bruce Lyon Memorial Research Laboratory, Children’s Hospital Medical Center, 51st and Grove Streets, Oakland, California 94609, U.S.A.
Received 14 February 1977; accepted 12 April 1977
Mouse mammary epithelial cells in confluent primary monolayer cultures retain responsiveness to the specific hormones that induce mammary growth in vivo. Simultaneous stimulation by prolactin, progesterone and estrogen, in the presence of 5% fetal calf serum, is required to induce an increase in both thymidine uptake into DNA and in cell replication (as judged by mitotic indexes) over the hormone-free control. This increase in mitogenic response could not be elicited in either mouse fibroblasts or in mouse mammary tumor cells, the latter known to be hormone insensitive. Keywords;
hormonal control; breast epithelium; cell replication.
Most of our knowledge in the area of hormone stimulation of epithelial cell replication in the mammary gland has been obtained from in vivo experiments (for review see Ceriani, 1974). However, the main drawbacks of the in vivo system are the heterogeneity of the tissue (even in lactating glands, 30% or more of the cells are not epitheliai in nature, as reported by Nicoll and Tucker, 1965) the uncertainty about hormone concentrations at the cellular level, and the possible simultaneous action of other hormones and/or factors with the hormone under study in inducing cell replication. Organ culture has also been used as an alternative for these studies, as discussed by Forsyth (1971). However, it has inherent deficiencies in the study of mammary epithelial cell replication in that: (a) the round of cell replication induced by hormones is short-lived (as in the case of insulin stimulation described by Topper, 1970) (b) the tissue remains viable for only a short time, as shown by Dilley * This research was partly supported by a Population Council Postdoctoral Fellowship to R.L.C., and in part by Contract No. NOI CB 33906 from the National Cancer Institute, National Institutes of Health, DHEW, awarded to S. Abraham. ** Present address: Bruce Lyon Memorial Research Laboratory, Children’s Hospital Medical Center, 5 1st and Grove Streets, Oakland, California 94609, U.S.A. 95
96
R. L. Ceriani and E. W. Blank
(1971) (c) the cell heterogeneity permits only histological and histochemical studies and precludes large-scale biochemical separations. For these reasons, monolayer cultures of mouse mammary epithelial cells (MMEC), which provide a more manageable, homogenous and controlled system, were used in this study. In it, the concomitant effect of prolactin and ovarian steroids in inducing cell replication of MMEC is demonstrated. This mitogenic effect was usually associated only with prolactin in the previous in vivo experiments of Simpson and Schmidt (1971), and in the organ culture studies of Dilley (1971). The defined cell culture conditions described herein permitted the establishment of such double hormonal requirement, in the presence of serum, for mouse mammary epithelium cell replication.
MATERIALS
AND METHODS
Mouse mammary tissue was dissected from pregnant (15-18 days) BALB/c mice. Monodisperse suspensions of epithelial cells were obtained by collagenase plus hyaluronidase, followed by pronase digestion of the tissue in accordance with Weipjes and Prop (1970) the only exception being that a 10 pm Nitex filter (TETKO, Inc., Monterey Park, California) was used for cell filtration. This method of cell dispersion yielded cell suspensions that were enriched up to 90% or more for MMEC (Thompson et al., 1976). MMEC obtained by this method retained epithelial morphology after plating and for the time period of the experiments. In addition, this MMEC population had a very small percentage of polyploid nuclei, since they were plated at a high density (3.5 X 105/cm2) (Das et al., 1974). [3H]Thymidine uptake was measured in MMEC, freshly dispersed, by the method described above and plated at a density of 3.5 X 10’ cells/cm’ in 2.5 ml of Waymouth’s medium supplemented by 3.5 mg/lOO ml penicillin G and 5 mg/lOO ml streptomycin plus 20% fetal calf serum (FCS), and incubated for 72 h at 37°C in a humidified atmosphere of 95% air plus 5% C02, in plastic Petri dishes (Falcon) 3.2 cm in diameter. At day 3 after plating the cells were already in a confluent monolayer, and, at this moment, the medium was changed to Waymouth’s medium plus 5% FCS. After another 3 days the medium was changed again to Waymouth’s medium plus 5% FCS, with or without hormones (this was considered to be 0 time for the labeling experiments). Each group consisted of three 3.2 cm diameter plates. 3-h pulses were performed at times indicated by placing the cells in fresh medium plus 1 nCi/ml of [3H] thymidine. After the pulse, the plates were washed with cold Ca2+- and Mg2+-free solution (CMF) (Weipjes and Prop, 1970). Then ihe cells were scraped off the plates and washed 3 times with 5% TCA, 3 times with acetone, and the pellet was then redissolved in I .O N NaOH. Half the sample was neutralized and used for radioactivity counts, the other half was used for fluorimetric determinations of DNA in accordance with Hinegardner (1971). Results were expressed as cpm/,ug DNA.
Hormones and mammary epithelial cell cultures
91
Fetal fibroblasts were obtained from fetuses of midpregnant mice, and the mouse epithelial mammary tumor cells from a transplantable tumor that arose in a CsH fostered BALB/c mouse (Bern and Nandi, 1961, showed this type of mouse mammary tumor to be hormone insensitive and Turkington, 1974, demonstrated that it lacks prolactin receptors). For cell dispersion, tumor or fetal tissues were dissected, minced, and then digested with 0.25% trypsin (Difco) plus 0.02% EDTA, in CMF for 30 min; large pieces were allowed to settle, the cell suspension floating above separated, and the latter, containing dispersed cells, was centrifuged (6Og for 5 mm) and its pellet washed once in CMF. The cells were then resuspended in Waymouth’s medium, counted and seeded at a density of 2 X lO’/cm* for the fibroblasts, and 5 X 105/cm2 for the tumor cells in plastic Petri dishes 3.2 cm in diameter. Both cell preparations were cultured in 2.5 ml of Waymouth’s medium plus 3.5 mg penicillin G and 5 mg streptomycin per 100 ml medium, in 10% FCS, in a humidifed atmosphere of 95% air plus 5% CO,, at 37’C; media were changed every 3 days. 3 days after the cells attained confluency, media were changed to Waymouth’s plus 5% FCS, and the cells left in culture for another 3 days. At the end of this period the media were changed to Waymouth’s plus 5% FCS either alone (controls) or plus hormones. 3-h pulses with [3H]thymidine were performed as for the MMEC. Each group consisted of 3 separate plates. The intracellular concentration of [3H]thymidine in MMEC was measured at the same time intervals when [3H] thymidine uptake was measured. For this purpose, MMEC were pulsed with [3H]thymidine for 3 h, then the cells were subjected to 2 fast washes, with gentle swirling, with cold CMF. After this, the cells were scraped, and the cell suspension made up to 0.1% in Tween 20 in CMF (Atlas Powder Co., ‘Wilmington, Delaware) to lyse the cells. Following this, the samples were made up to 5% TCA, let stand for 1 h at room temperature, and then centrifuged. The supernatant fraction was saved. The pellet was washed again twice with 5% TCA, and the washes saved. The supernatant fraction after TCA precipitation plus the 2 washes was consolidated, [3H]thymidine activity was measured, and DNA was estimated in the pellets in accordance with Hinegardner (1971). Results were expresses as cpm of tritium found in the supernatant fraction (that were considered to correspond to labeled thymidine) per pg of DNA of MMEC in the pellet. To determine the effect of hormones on mitotic indexes, monolayers of confluent MMEC, fibroblasts or mammary tumor cells were prepared as for the thymidine incorporation studies. 3 days after confluency (the cells now in 5% FCS) the media were changed to Waymouth’s plus 5% FCS with or without hormones, and at times indicated, their media were substituted by the same plus 0.12 ng/ml colchitine (Colcemid) for 6 h. At the end of the colchicine incubation the plates were washed with phosphate buffered saline and then the cells were fixed with Bouin’s fixative and stained with Wright’s stain. At least 5 X IO3 nuclei were counted, and mitoses per 1 X 1O3 cells estimated in triplicates. The hormones and hormone concentrations used were: prolactin (NIH-PS-8) 0.2 pg/ml; progesterone (Calbiochem), 1 pg/ml; estradiol (17/3-estradiol, Calbio-
98
R.L. Cerimi and E. W. Blank
them), 0.001 E.rg/ml. Prdactin was dissolved in 0.0001 N NaOH, and steroid hormones were first diluted into absolute ethanol, then this stock solution was diluted in media, where the final ethanol concentration never exceeded 0.001%.
RESULTS After medium with or without hormones plus 5% FCS was added to the confluent MMEC, a 4-fold increase in thymidine uptake into 5% trichloroacetic acid (TCA~precipitable material occurs within 24 h (fig. I>. Thymidine uptake increases under similar circumstances have already been reported for other cells by Todaro et al. (1965), by Weibel and Baserga (1969), by Becker and Levitt (1968), and by Baserga et al. (1971). In contrast, this wave of thymidine uptake is not apparent in
Hormone Combination Time
(hours):
:
c I23 O-3
Cl
23 24-27
C
I23
48-51
Fig. I. Effect of hormones on [ 3H 1tby~lidine incorporation into DNA by MMEC in confluent monolayers. Results are expressed as cpm/pg DNA. Hormone combinations: C = no hormone; 1 = prolactin (NIH-PSI, 0.2 fig/ml); 2 = progestetone (Calbiochem, 1 Irg/ml) plus estradiol (17p-estradiol, Calbiochem, 0.001 pg/ml); 3 = prolactin (90.2 &ml) plus progesterone (1 fig/ ml) plus estradiol (0.001 erg/ml). Each point represents the average of 6 samples corresponding to 2 experiments of 3 samples each, performed with different MMfC preparations; each sample corresponded to determinations performed on the cells monolayered in a 3.2 em diameter plastic Petri dish. Vertical bars represent the standard error of mean (SEM).
Hormones and mammary epithelial cell cultures
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nonconfluent cells. The latter incorporated [3H]thymidine at a sustained level for several days after medium change. That [3H]thymidine was incorporated into DNA in the present studies is suggested by the fact that the radioactivity in the TCA-precipitable material of the MMEC was redissolved by 10% TCA digestion at 4O”C, overnight. Confluent monolayers of MMEC responded to prolactin plus progesterone plus estrogen, added to the medium-change to trigger DNA replication, with an increase in uptake of [3H] thymidine into TCA-precipitable material over the hormone-free control, between 24 and 27 h. This effect of prolactin plus estrogen plus progesterone was even noticeable when compared to the estrogen plus progesterone stimulated group (fig. 1). (This latter group was usually lower in value than the hormonefree, and often significantly lower.) Prolactin alone did not induce this increased response; rather, its effect on thymidine uptake was inhibitory. In addition, MMEC exposed for 24 h to progesterone alone, estrogen alone, progesterone plus prolactin, and estrogen plus prolactin, and then pulsed with thymidine for 3 h, gave values of 511.2 f 127.8, 835.2 + 61.7, 670.2 f 50.1 and 962 f. 113.2 cpm/pg DNA, respectively, all of them below their hormone-free control (1125 + 76.5) (each group represents values from 3 plates; f indicates standard error of mean). Further, at 48 h, the increase in thymidine uptake shown by addition of prolactin to progesterone plus estradiol is still apparent (fig. 1). These hormonal effects could not be elicited if MMEC were stimulated by hormones in the presence of FCS in higher concentrations than 5%. With FCS concentrations of 10% and 20% without hormones, [3H]thymidine incorporation was more than 60% higher than at 5% FCS without hormones. This was the level obtained for the estrogen plus progesterone plus prolactin stimulated group above the 5% FCS control without hormones. The difference in intracellular concentration of TCA-soluble [3H] thymidine after 3 h incubations of the hormone-treated cells or their controls, at any of the given times, was not statistically significant. This similar content of intracellular [3H] thymidine in every experimental cell group suggests that the increase in [3H] thymidine uptake after hormone stimulation is not a result of altered intracellular thymidine pools. To corroborate these results showing an increase in thymidine uptake by cells exposed to prolactin plus estrogen plus progesterone, experiments were carried out where number of mitoses per 1 X lo3 cells after mitotic arrest with colchicine was determined in control and hormone-stimulated groups (table 1). These experiments were performed in MMEC preparations prepared on different days than the ones used for the [3H] thymidine incorporation experiments. Here again, the media supplemented with prolactin plus the ovarian steroids induced higher mitotic indexes over any partial combination of the three hormones, each of the hormones alone or the control group. In contrast to the responsiveness in terms of [3H]thymidine uptake, shown by the MMEC to the triple hormone combination (estrogen plus progesterone plus prolactin), both fibroblasts and tumor cells were unresponsive (table 2). In parallel
R. L. Ceriatzi arzd E. W. Blank
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experiments, after stimulation with the triple hormone combination, mitotic indexes over the hormone-free control could be evidenced fetal fibroblasts or the mammary tumor cells (table 1).
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no increase in for either the
DISCUSSION Normal monolayers of mouse mammary gland epithelial cells (MMEC) retaining in vivo characteristics are valuable in cell differentiation studies and as a point of reference for their transformed counterparts in cell biology investigations. Conditions for both cell culture of hormone-responsive MMEC, and for eliciting response of MMEC to hormones, are described herein. It is shown that the number of MMEC entering replication could be modulated by appropriate hormones under such conditions, therefore proving that MMEC retain their mechanism of hormonal response after cell dispersion and 7 days of monolayer culture. The hormone-induced increase in [ 3H] thymidine uptake into TCA-precipitable material (presumably DNA) of MMEC results from specific stimulation by the same hormones that are thought to be responsible for mammary growth in vivo, i.e. ovarian steroids and prolactin. Cell replication, as shown by the mitotic arrest studies, was also stimulated in the MMEC by these hormones, lending further credibility to the thymidine uptake studies. The values of mitotic indexes of MMEC after prolactin plus ovarian steroid stimulation (17.0%, table 1) are in the range of values for percentage of cells entering mitosis in the midpregnant mouse mammary gland reported by Bresciani (1971). In vivo and organ culture experiments, including those of Lyons et al. (1958) and of Ichinose and Nandi (1964, 1966) seem to rule out any action of ovarian steroids when used in the absence of other hormones in promoting mammary growth. In contrast, Norgren (1967) has been the only one to date to report stimulatory action on the growth of the mammary gland by these steroids. In this report Norgren shows that ovarian steroids cause stunted growth of ducts in the mammary gland of hypophysectomized rabbits. Further, the growth-promoting effect of prolactin alone on the in vivo mammary gland has been reported by Simpson and Schmidt (1971) by Meites and Kragt (1964), and by Cole and Hopkins (1962). However, this induction of mammary growth directed by prolactin alone (with the reservation that it was induced in vivo and that other stimulatory factors could not be ruled out) was always restricted and could not be compared to mammary growth found in midpregnancy. The growth obtained was mainly ductal with scant alveolar development. Midpregnant type of development is seen after in vivo prolactin injection only when ovarian steroids are also injected in situ in the mammary gland,, as was shown by Nagasawa and Yanai (1971). Apparently, prolactin requires the cooperative action of other hormones to induce mammary cell replication in vitro. Simultaneous stimulation by ovarian steroids and prolactin, in the presence of serum, was required here to increase the level of DNA synthesis in the MMEC
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monolayers. Possibly prolactin renders the MMEC sensitive to ovarian steroids via an increase in steroid binding capacity, as was shown by Leung and Sasaki (1973) for mammary glands in organ culture. Thus, information must be present in the genome of mouse mammary cells for a prolactin-plus-ovarian-steroid-mediated cell replication, as there is for specific protein synthesis as shown by Ceriani (1976) for MMEC. Therefore, mammary epithelium replication in vivo can be envisaged as occurring at a constant basal level maintained by serum and/or other stimuli and with hormones acting as modulating agents, as proposed by Bresciani (1968, 1971). This basal level of cell replication would be controlled by density-dependent inhibition of growth (DDIG) and hormones; the latter would have as their role to counter the former. Nonconfluent MMEC in vitro maintain a high level of thymidine uptake and cell replication for several days after medium change. In contrast, confluent ones will respond with a low and short-lived level of cell replication after medium change, showing that they, as well as the tissue cells, are subjected to DDIG. After appropriate hormone stimulation, DDIG restrictions were superseded in our MMEC cultures and increases of up to 80% above basal thymidine incorporation and mitotic rate were obtained. These levels of mammary epithelial cell replication are comparable to those obtained after serum stimulation of the mouse mammary gland in organ culture by Majumder and Turkington (197 1). The stimulator-y action of hormones on thymidine uptake of monolayered MMEC was obtained here at a concentration of 5% FCS, but not at higher concentrations. At high serum concentrations MMEC in monolayers replicated possibly at their maximum as a result of intrinsic serum factors and/or high enough concentrations of the hormones carried in it. A comparable situation is found when casein synthesis is induced in MMEC monolayers by hormones. The action of the latter could only be demonstrated in the absence of serum by Ceriani (1976). Further, Majumder and Turkington (1971) have shown that epithelial cells in mouse mammary gland explants increase their thymidine uptake only as a result of stimulation by 5% FCS concentrations and above. This would suggest that the concentration of FCS used in this study is in the borderline for stimulation, and thus it would be at this level that any cooperative action of hormones could be demonstrated. The possibility that the observed hormonal stimulation of thymidine uptake and mitotic indexes in MMEC could be related to the unusual conditions of the culture system and, therefore, could be present in all cultured mouse cells, was ruled out. Monolayers of mouse fetal fibroblasts and of epithelial cells from a hormone-insensitive transplantable mouse mammary tumor did not respond to the hormonal stimulation effective on MMEC. At best, prolactin and the ovarian steroids proved to be inhibitory of cell replication in the nonmammary cell monolayers. The cell type specificity of the hormonal stimulation of MMEC is thus proved.
ACKNOWLEDGEMENT I thank the late Dr. R. Denamur for advice and encouragement.
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