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Alterations in electrophysiology of cultured BALBcfC3H mouse mammary epithelium associated with neoplastic transformation
Cancer Letters,
14 (1981) 237-242
237
Elaevier/North-Holland Scientific PublishersLtd.
ALTERATIONS IN ELECIltOPHYSIOLOGY OF CULTURED BALB/cfC3H MOUSE MAMMARY EPlTHELIUM ASSOCIATED WITH NEOPLASTIC TRANSFORMATION
CHESTER A. BISBEE Department of Zoology and Cancer Research Berkeley, CA 94720 (U.S.A.)
Laboratory,
University
of California,
(Received 4 May 1981) (Accepted 3 September 1981)
SUMMARY
Mammary tumor virus and neoplastic transformation affect prolactinmodulated electrophysiology of mouse mammary epithelium in vitro. Transepithelial resistances of both prelactating and neoplastic BALB/cfC3H mouse mammary epithelial cell cultures are lower than those seen in BALB/c prelactating cell cultures. Unlike the response to prolactin in BALB/c cultures, there are no effects of this hormone on transepithelial potential difference or resistance of BALB/cfC3H prelactating or neoplastic cultures. However, prolactin significantly increases short-circuit current in both BALB/c and BALB/cfC3H prelactating cell cultures and in BALB/cfC3H neoplastic cell cultures. The response of tumor cell cultures suggests that pregnancyindependent mouse mammary epithelium may not be entirely refractory to prolactin. INTRODUCTION
Cell membrane transport properties are modified as an initial response to changes in physiological state [8,17,23]. These modifications result in variations in intracellular ion concentrations and concentration ratios which, in turn, influence cellular metabolic processes [ 14,161 and gene expression [21,31]. Specifically, cellular ion metabolism is a controlling factor in processes as diverse as egg development [ 131, cell division [6,7,20] and hepatic gluconeogenesis [ 121. The neoplastic state is also correlated with changes in cellular ion metabolism. In general, tissue ion contents [5] and transcellular membrane potentials [6,7] are different in normal and neoplastic cells derived from the Address all correspondence to: Dr. Chester A. Biubee, Room 6N-307, Building 10, National Instituteeof Health, Betheada, MD 20206 (U.S.A.). 0304-3836/81/0000-0000/$02.75 o 1981 Ekevier/North-Holland Scientific Pub&here Ltd.
238
same tissue. In particular, membrane permeability to sodium [24] and the molecular structure of tissue water [ 151 are modified in neoplastic mammary epithelial cells. The extent of these correlations suggest that changes in ion metabolism are at least an integral part of the process of transformation. In epithelial tissues such as the mammary gland, hormone-controlled transepithelial ion transport partially determines intracellular ion concentrations [ 14,161 and consequently overall cellular metabolism. This study describes prolactin-controlled transepithelial electrophysiological properties associated with the neoplastic state and with the presence of mammary tumor virus in BALB/cfC3H mouse mammary epithelium. The data are compared with those previously documented for BALB/c mouse mammary epithelium [ 1,3] in an effort to delineate the modifications of ion metabolism that are associated with neoplastic transformation in the mammary gland. MATERIALS AND METHODS
Methods for primary cell culture and maintenance of the resultant monolayers on floating collagen gels have been described elsewhere [ 2,3,10,19]. Midpregnant (lo--17-day pregnant) glands were obtained from a BALB/ cfC3HCrgl line that has been separated from the parent BALB/C line since 1971, Spontaneous tumors were obtained from 3 non-pregnant, non-lactating BALB/cfC3HCrgl mice. Electrical measurements were performed in lucite Ussing chambers [29] on 3-lOday-old monolayer cultures using standard electrophysiological techniques [ 3,291. Student’s t-test (or a modification of the test for unequal variances) was used to determine statistically significant differences [27] between hormonal treatment groups. Non-significant differences (NS) have a P> 0.05. RESULTS
Table 1 shows the transepithelial electrophysiology of prelactating and neoplastic BALB/cfC3H mouse mammary epithelial cell cultures maintained on floating collagen gels. Results from spontaneous tumors from 3 mice are pooled to give the data in Table 1, since the results from different tumors were not different. In both states, an increase in shortcircuit current is the only statistically significant effect of prolactin observed. However, short-circuit current values from tumor-cell cultures are at most 20% of prelactating cell culture values. Neoplastic epithelium potential differences are only slightly lower than in prelactating epithelium. Resistances of tumor-cell cultures are not different from those of prelactating cell cultures. DISCUSSION
The presence of mammary tumor virus appears to have effects on the
239 TABLE 1 ELECTROPHYSIOLOGY OF BALB/cfCaHCrgl PRELACTATING MOUSE MAMMARY EPITHELIAL CELL CULTURES
AND NEOPLASTIC
Transepithelial potential difference PD (mV) (basal side ground)
Short-circuit I, (fiA/cm’)
Transepithelial resistance R (ohm cmz)
Prelactating cell cultures Insulin and cortisol (n=lO) Insulin, cortisol and prolactin (n= 8)
-7.1
* 2.3
25.8 f 2.4
281 * 101
-9.4 NS
+ 2.4
34.6 f 2.9 P< 0.05
266 f 57 NS
Neoplastic cell cultures Insulin and cortisol (n=18) Insulin, cortisol and prolactin (n=17)
-0.5
* 0.1
2.3 f 0.5
182 * 25
-1.1 NS
f 0.3
5.4 f 1.2 P< 0.001
172 * 25 NS
-
-..
Cultures were maintained in Waymouth’s medium supplemented with 10% calf serum and either insulin (10 Mg/ml) and cortisol(5 fig/ml) or insulin, cortisol, and prolactin (10 rg/ ml) [1,3]. Data from 3 separate spontaneous tumors showed no evident differences and so were combined in this table. Data are tabulated f S.E.M. NS = P > 0.05.
electrophysiology of cultured mouse mammary epithelium. Both BALB/ cfC3H and BALB/c [3] cell cultures treated with insulin and cortisol have similar shortcircuit currents, but BALBlcfC3H epithelium has only 55% of the resistance of BALB/c epithelium. Prolactin addition to BALB/cfC3H cell cultures causes an increase in short-circuit current as it does in BALB/c cell cultures [ 31, although the effect is much reduced in the former. Prolactin does not affect transepithelial potential difference or resistance in BALB/ cfC3H mammary epithelium, unlike the situation in BALB/c epithelium [ 31. These results suggest a reduction in prolactin sensitivity with expression of mammary tumor virus, although generally mice with expressed mammary tumor virus appear to be at least as sensitive to hormones as are BALB/c mice [ 211. Floating collagen gel cultures of prelactating mammary epithelium show similar prolactin-induced casein production when derived from BALB/c or BALB/cfC3H mice [9,11]. However, other explanations for these differences do not necessitate reduced prolactin sensitivity. Some of the differences seen in BALB/cfC3H mammary epithelium may be due to the presence in the monolayer of preneoplastic and/or neoplastic cells whose electrophysiological characteristics are only partially known (see below). Additionally, since the electrophysiological properties measured in this study are the sum of several active and passive transport processes [cf. 1,3], the apparent
240
decreased prolactin sensitivity may be the result of differential effects of prolactin on the component elements of shortcircuit current and resistance. Neoplastic BALB/cfC3H mammary epithelial cell cultures exhibit reduced shortcircuit currents when compared with either BALB/c or BALB/cfC3H prelactating cell cultures. This reduction in short-circuit current indicates that the active ion transport present in neoplastic cell cultures is different from what has been previously documented for BALB/c prelactating cell cultures [ 1,3]. The maintenance of prolactin sensitivity in tumorous epithelium is somewhat surprising since mammary tumor virus-induced tumors appear refractory to prolactin [18,30] and prolactin receptors are absent [25,26,28]. However, the apparent maintenance of prolactin sensitivity upon transformation could be due to the presence in the cultured neoplastic epithelium of hyperplastic alveolar nodule cells which are sensitive to prolactin [ 25,301. One of the consequences of the changes in ion transport observed in this study could be a change in mammary fluid ion content in glands with neoplastic, and possibly hyperplastic alveolar nodular epithelial cells. If similar modifications occur in human mammary neoplasia and preneoplasia, analysis of the ionic content of breast fluid could provide a clinical monitoring procedure. The necessary human samples can be easily obtained from at least 50% of the non-pregnant, non-lactating female population [ 41. Since changes in milk ion concentrations are detectable prior to any clinical manifestation of mastitis [22], a routine check of breast fluid in ion content may allow early detection of human mammary abnormalities that may otherwise be undetectable. ACKNOWLEDGEMENTS
I am grateful to Professor Howard A. Bern for the support and encouragement that he provided during this study. This work was supported by National Cancer Institute Grants CA05388 and CA09041. REFERENCES 1 Bisbee, CA. (1981) Prolactin effects on ion transport across cultured mammary epithelium. Am. J. Physiol., 240, CllO-Cl15. 2 Bisbee, C.A. (1979) Prolactin effects on ion transport across mammary epithelium in vitro. Ph.D. Dissertation, University of California, Berkeley. 3 Bisbee, C.A., Machen, T.E. and Bern, H.A. (1979) Mouse mammary epithelial cells on floating collagen gels: transepithelial ion transport and effects on prolactin. Proc. Natl. Acad. Sci. U.S.A., 76, 536-540. 4 Buehring, G.C. (1979) Screening for breast atypias using exfoliative cytology. Cancer, 43,1788-1799. 5 Cameron, I.L., Smith, N.K.R., Pool, T.B. and Sparks, R.L. (1980) Intracellular concentration of sodium and other elements as related to mitogenesis and oncogenesls in viva Cancer Res., 40,1493-1500. 6 Cone, Jr., C.D. (1974) The role of the surface electrical transmembrane potential in normal and malignant mitogenesis. Ann. N.Y. Acad. Sci., 238,420-435.
241 7 Cone, Jr., C.D. (1971) Unified theory on the basic mechanism of normal mitogenic control and oncogenesis. J. Theor. Biol., 30,151-181. 8 Crabbe, J. (1973) Insulin, glucagon and active sodium transport: from man to amphibia - and back. In: Transport Mechanisms in Epithelia, Alfred Benzon Symposium V, pp. 173-184. Editors: H.H. Ussing and N.A. Thorn. Academic Press, New York. 9 Emerman, J.T., Enami, J., Pitelka, D.R. and Nandi, S. (1977) Hormonal effects on intracellular and secreted casein in cultures of mouse mammary epithelial cells on floating collagen membranes. Proc. Natl. Acad. Sci. U.S.A., 74,4466-4470. 10 Emerman, J.T. and Pitelka, D.R. (1977) Maintenance and induction of morphological differentiation in dissociated mammary epithelium on floating collagen membranes. In Vitro, 13, 316-328. 11 Enami, J., Yang, J. and Nandi, S. (1979) Simultaneous production of casein and mammary tumor virus in mouse mammary epithelial cells grown on floating collagen gels. Cancer Letters, 6, 99-105. 12 Friedman, N. and Dambach, G. (1980) Antagonistic effect of insulin on glucagonevoked hyperpolarization. A correlation between changes in membrane potential and gluconeogenesis. Biochim. Biophys. Acta, 596, 180-185. 13 Grainger, J.L., Winker, M.M., Shen, S.S. and Steinhardt, R.A. (1979) Intracellular pH controls protein synthesis rate in sea urchin egg and early embryo. Dev. Biol., 68, 396--406. 14 Handler, J.S., Preston, A.S. and Orloff, J. (1972) Effect of aldosterone on the sodium content and energy metabolism of epithelial cells of the toad urinary bladder. J. Steroid Biochem., 3, 137-141. 15 Hazelwood, C.G., Chang, D.C., Medina, D., Cleveland, G. and Nichols, B.L. (1972) Distinction between the preneoplastic and neoplastic state of murine mammary glands. Proc. Natl. Acad. Sci. U.S.A., 69,1478-1480. 16 Ismail-Beigi, F. and Edelman, I.S. (1973) Effects of thyroid status on electrolyte distribution in rat tissues. Am. J. Physiol., 225, 1172-1177. 17 Ismail-Beigi, F. and Edelman, I.S. (1970) The mechanism of thyroid calorigenesis: role of active sodium transport. Proc. Natl. Acad. Sci. U.S.A., 67,1071-1078. 18 McGrath, C.M. (1971) Replication of mammary tumor virus in tumor cell cultures: dependence on hormone-induced cellular organization. J. Natl. Cancer Inst., 47, 455467. 19 Michalopoulos, G. and Pitot, H.C. (1975) Primary cultures of parenchymal liver cells on collagen membranes. Exp. Cell Res., 94, 70-78. 20 Moscatelli, D., Sanui, H. and Rubin, A.H. (1979) Effects of depletion of K’, Na’ or Ca’+ on DNA synthesis and cell cation content in chick embryo fibroblasts. J. Cell Physiol., 101, 117-128. 21 Nandi, S. and McGrath, C.M. (1973) Mammary neoplasia in mice. In: Advances in Cancer Research, Vol. 17, pp. 353-414. Editors: G. Klein and S. Weinhouse. Academic Press, New York. 22 Peaker, M. (1978) Ion and water transport in the mammary gland. In: Lactation, Vol. 4, pp. 437-462. Editor: B.L. Larson, Academic Press, New York, London. 23 Petersen, O.H. (1976) Electrophysiology of mammalian gland cells. Physiol. Rev., 56, 535-577. 24 Shen, S.S., Hamamoto, S.T., Bern, H.A. and Steinhardt, R.A. (1978) Alteration of sodium transport in mouse mammary epithelium associated with neoplastic transformation. Cancer Res., 38,1356-1361. 25 Shiu, R.P.C. (1979) Prolactin receptors in human breast cancer cells in long term tissue culture. Cancer Res., 39, 4381-4386. 26 Sluyser, M. (1979) Hormone receptors in mouse mammary tumors. Biochim. Biophys. Acta, 560, 509-529.
27 Sokai, RR. and Rohlf, F.J. (1969) Biometry. W.H. Freeman Co., San Francisco. 28 Turkington, R.W. (1974) ProIactin receptors in mammary carcinoma cells. Cancer Res., 34, 758-763. 29 Ussing, H.H. and Zerahn, K. (1951) Active transport of sodium as the source of electric current in the short-circuited isolated frog skin. Acta Physiol. Stand., 23, 110-127. 30 Welsch, C.W. and Nagasawa, H. (1977) Prolactin and murine mammary tumorigenesis: a review. Cancer Res., 37, 951-963. 31 Wuhrmann, P., Ineichen, H., Riesen-Willi, U. and Lezzi, M. (1979) Changes in nuclear potassium electrochemical activity and puffing of potassium-sensitive salivary chromosome regions during Chironomus development. Proc. Natl. Acad. Sci. U.S.A., 76, 806-808.
14 (1981) 237-242
237
Elaevier/North-Holland Scientific PublishersLtd.
ALTERATIONS IN ELECIltOPHYSIOLOGY OF CULTURED BALB/cfC3H MOUSE MAMMARY EPlTHELIUM ASSOCIATED WITH NEOPLASTIC TRANSFORMATION
CHESTER A. BISBEE Department of Zoology and Cancer Research Berkeley, CA 94720 (U.S.A.)
Laboratory,
University
of California,
(Received 4 May 1981) (Accepted 3 September 1981)
SUMMARY
Mammary tumor virus and neoplastic transformation affect prolactinmodulated electrophysiology of mouse mammary epithelium in vitro. Transepithelial resistances of both prelactating and neoplastic BALB/cfC3H mouse mammary epithelial cell cultures are lower than those seen in BALB/c prelactating cell cultures. Unlike the response to prolactin in BALB/c cultures, there are no effects of this hormone on transepithelial potential difference or resistance of BALB/cfC3H prelactating or neoplastic cultures. However, prolactin significantly increases short-circuit current in both BALB/c and BALB/cfC3H prelactating cell cultures and in BALB/cfC3H neoplastic cell cultures. The response of tumor cell cultures suggests that pregnancyindependent mouse mammary epithelium may not be entirely refractory to prolactin. INTRODUCTION
Cell membrane transport properties are modified as an initial response to changes in physiological state [8,17,23]. These modifications result in variations in intracellular ion concentrations and concentration ratios which, in turn, influence cellular metabolic processes [ 14,161 and gene expression [21,31]. Specifically, cellular ion metabolism is a controlling factor in processes as diverse as egg development [ 131, cell division [6,7,20] and hepatic gluconeogenesis [ 121. The neoplastic state is also correlated with changes in cellular ion metabolism. In general, tissue ion contents [5] and transcellular membrane potentials [6,7] are different in normal and neoplastic cells derived from the Address all correspondence to: Dr. Chester A. Biubee, Room 6N-307, Building 10, National Instituteeof Health, Betheada, MD 20206 (U.S.A.). 0304-3836/81/0000-0000/$02.75 o 1981 Ekevier/North-Holland Scientific Pub&here Ltd.
238
same tissue. In particular, membrane permeability to sodium [24] and the molecular structure of tissue water [ 151 are modified in neoplastic mammary epithelial cells. The extent of these correlations suggest that changes in ion metabolism are at least an integral part of the process of transformation. In epithelial tissues such as the mammary gland, hormone-controlled transepithelial ion transport partially determines intracellular ion concentrations [ 14,161 and consequently overall cellular metabolism. This study describes prolactin-controlled transepithelial electrophysiological properties associated with the neoplastic state and with the presence of mammary tumor virus in BALB/cfC3H mouse mammary epithelium. The data are compared with those previously documented for BALB/c mouse mammary epithelium [ 1,3] in an effort to delineate the modifications of ion metabolism that are associated with neoplastic transformation in the mammary gland. MATERIALS AND METHODS
Methods for primary cell culture and maintenance of the resultant monolayers on floating collagen gels have been described elsewhere [ 2,3,10,19]. Midpregnant (lo--17-day pregnant) glands were obtained from a BALB/ cfC3HCrgl line that has been separated from the parent BALB/C line since 1971, Spontaneous tumors were obtained from 3 non-pregnant, non-lactating BALB/cfC3HCrgl mice. Electrical measurements were performed in lucite Ussing chambers [29] on 3-lOday-old monolayer cultures using standard electrophysiological techniques [ 3,291. Student’s t-test (or a modification of the test for unequal variances) was used to determine statistically significant differences [27] between hormonal treatment groups. Non-significant differences (NS) have a P> 0.05. RESULTS
Table 1 shows the transepithelial electrophysiology of prelactating and neoplastic BALB/cfC3H mouse mammary epithelial cell cultures maintained on floating collagen gels. Results from spontaneous tumors from 3 mice are pooled to give the data in Table 1, since the results from different tumors were not different. In both states, an increase in shortcircuit current is the only statistically significant effect of prolactin observed. However, short-circuit current values from tumor-cell cultures are at most 20% of prelactating cell culture values. Neoplastic epithelium potential differences are only slightly lower than in prelactating epithelium. Resistances of tumor-cell cultures are not different from those of prelactating cell cultures. DISCUSSION
The presence of mammary tumor virus appears to have effects on the
239 TABLE 1 ELECTROPHYSIOLOGY OF BALB/cfCaHCrgl PRELACTATING MOUSE MAMMARY EPITHELIAL CELL CULTURES
AND NEOPLASTIC
Transepithelial potential difference PD (mV) (basal side ground)
Short-circuit I, (fiA/cm’)
Transepithelial resistance R (ohm cmz)
Prelactating cell cultures Insulin and cortisol (n=lO) Insulin, cortisol and prolactin (n= 8)
-7.1
* 2.3
25.8 f 2.4
281 * 101
-9.4 NS
+ 2.4
34.6 f 2.9 P< 0.05
266 f 57 NS
Neoplastic cell cultures Insulin and cortisol (n=18) Insulin, cortisol and prolactin (n=17)
-0.5
* 0.1
2.3 f 0.5
182 * 25
-1.1 NS
f 0.3
5.4 f 1.2 P< 0.001
172 * 25 NS
-
-..
Cultures were maintained in Waymouth’s medium supplemented with 10% calf serum and either insulin (10 Mg/ml) and cortisol(5 fig/ml) or insulin, cortisol, and prolactin (10 rg/ ml) [1,3]. Data from 3 separate spontaneous tumors showed no evident differences and so were combined in this table. Data are tabulated f S.E.M. NS = P > 0.05.
electrophysiology of cultured mouse mammary epithelium. Both BALB/ cfC3H and BALB/c [3] cell cultures treated with insulin and cortisol have similar shortcircuit currents, but BALBlcfC3H epithelium has only 55% of the resistance of BALB/c epithelium. Prolactin addition to BALB/cfC3H cell cultures causes an increase in short-circuit current as it does in BALB/c cell cultures [ 31, although the effect is much reduced in the former. Prolactin does not affect transepithelial potential difference or resistance in BALB/ cfC3H mammary epithelium, unlike the situation in BALB/c epithelium [ 31. These results suggest a reduction in prolactin sensitivity with expression of mammary tumor virus, although generally mice with expressed mammary tumor virus appear to be at least as sensitive to hormones as are BALB/c mice [ 211. Floating collagen gel cultures of prelactating mammary epithelium show similar prolactin-induced casein production when derived from BALB/c or BALB/cfC3H mice [9,11]. However, other explanations for these differences do not necessitate reduced prolactin sensitivity. Some of the differences seen in BALB/cfC3H mammary epithelium may be due to the presence in the monolayer of preneoplastic and/or neoplastic cells whose electrophysiological characteristics are only partially known (see below). Additionally, since the electrophysiological properties measured in this study are the sum of several active and passive transport processes [cf. 1,3], the apparent
240
decreased prolactin sensitivity may be the result of differential effects of prolactin on the component elements of shortcircuit current and resistance. Neoplastic BALB/cfC3H mammary epithelial cell cultures exhibit reduced shortcircuit currents when compared with either BALB/c or BALB/cfC3H prelactating cell cultures. This reduction in short-circuit current indicates that the active ion transport present in neoplastic cell cultures is different from what has been previously documented for BALB/c prelactating cell cultures [ 1,3]. The maintenance of prolactin sensitivity in tumorous epithelium is somewhat surprising since mammary tumor virus-induced tumors appear refractory to prolactin [18,30] and prolactin receptors are absent [25,26,28]. However, the apparent maintenance of prolactin sensitivity upon transformation could be due to the presence in the cultured neoplastic epithelium of hyperplastic alveolar nodule cells which are sensitive to prolactin [ 25,301. One of the consequences of the changes in ion transport observed in this study could be a change in mammary fluid ion content in glands with neoplastic, and possibly hyperplastic alveolar nodular epithelial cells. If similar modifications occur in human mammary neoplasia and preneoplasia, analysis of the ionic content of breast fluid could provide a clinical monitoring procedure. The necessary human samples can be easily obtained from at least 50% of the non-pregnant, non-lactating female population [ 41. Since changes in milk ion concentrations are detectable prior to any clinical manifestation of mastitis [22], a routine check of breast fluid in ion content may allow early detection of human mammary abnormalities that may otherwise be undetectable. ACKNOWLEDGEMENTS
I am grateful to Professor Howard A. Bern for the support and encouragement that he provided during this study. This work was supported by National Cancer Institute Grants CA05388 and CA09041. REFERENCES 1 Bisbee, CA. (1981) Prolactin effects on ion transport across cultured mammary epithelium. Am. J. Physiol., 240, CllO-Cl15. 2 Bisbee, C.A. (1979) Prolactin effects on ion transport across mammary epithelium in vitro. Ph.D. Dissertation, University of California, Berkeley. 3 Bisbee, C.A., Machen, T.E. and Bern, H.A. (1979) Mouse mammary epithelial cells on floating collagen gels: transepithelial ion transport and effects on prolactin. Proc. Natl. Acad. Sci. U.S.A., 76, 536-540. 4 Buehring, G.C. (1979) Screening for breast atypias using exfoliative cytology. Cancer, 43,1788-1799. 5 Cameron, I.L., Smith, N.K.R., Pool, T.B. and Sparks, R.L. (1980) Intracellular concentration of sodium and other elements as related to mitogenesis and oncogenesls in viva Cancer Res., 40,1493-1500. 6 Cone, Jr., C.D. (1974) The role of the surface electrical transmembrane potential in normal and malignant mitogenesis. Ann. N.Y. Acad. Sci., 238,420-435.
241 7 Cone, Jr., C.D. (1971) Unified theory on the basic mechanism of normal mitogenic control and oncogenesis. J. Theor. Biol., 30,151-181. 8 Crabbe, J. (1973) Insulin, glucagon and active sodium transport: from man to amphibia - and back. In: Transport Mechanisms in Epithelia, Alfred Benzon Symposium V, pp. 173-184. Editors: H.H. Ussing and N.A. Thorn. Academic Press, New York. 9 Emerman, J.T., Enami, J., Pitelka, D.R. and Nandi, S. (1977) Hormonal effects on intracellular and secreted casein in cultures of mouse mammary epithelial cells on floating collagen membranes. Proc. Natl. Acad. Sci. U.S.A., 74,4466-4470. 10 Emerman, J.T. and Pitelka, D.R. (1977) Maintenance and induction of morphological differentiation in dissociated mammary epithelium on floating collagen membranes. In Vitro, 13, 316-328. 11 Enami, J., Yang, J. and Nandi, S. (1979) Simultaneous production of casein and mammary tumor virus in mouse mammary epithelial cells grown on floating collagen gels. Cancer Letters, 6, 99-105. 12 Friedman, N. and Dambach, G. (1980) Antagonistic effect of insulin on glucagonevoked hyperpolarization. A correlation between changes in membrane potential and gluconeogenesis. Biochim. Biophys. Acta, 596, 180-185. 13 Grainger, J.L., Winker, M.M., Shen, S.S. and Steinhardt, R.A. (1979) Intracellular pH controls protein synthesis rate in sea urchin egg and early embryo. Dev. Biol., 68, 396--406. 14 Handler, J.S., Preston, A.S. and Orloff, J. (1972) Effect of aldosterone on the sodium content and energy metabolism of epithelial cells of the toad urinary bladder. J. Steroid Biochem., 3, 137-141. 15 Hazelwood, C.G., Chang, D.C., Medina, D., Cleveland, G. and Nichols, B.L. (1972) Distinction between the preneoplastic and neoplastic state of murine mammary glands. Proc. Natl. Acad. Sci. U.S.A., 69,1478-1480. 16 Ismail-Beigi, F. and Edelman, I.S. (1973) Effects of thyroid status on electrolyte distribution in rat tissues. Am. J. Physiol., 225, 1172-1177. 17 Ismail-Beigi, F. and Edelman, I.S. (1970) The mechanism of thyroid calorigenesis: role of active sodium transport. Proc. Natl. Acad. Sci. U.S.A., 67,1071-1078. 18 McGrath, C.M. (1971) Replication of mammary tumor virus in tumor cell cultures: dependence on hormone-induced cellular organization. J. Natl. Cancer Inst., 47, 455467. 19 Michalopoulos, G. and Pitot, H.C. (1975) Primary cultures of parenchymal liver cells on collagen membranes. Exp. Cell Res., 94, 70-78. 20 Moscatelli, D., Sanui, H. and Rubin, A.H. (1979) Effects of depletion of K’, Na’ or Ca’+ on DNA synthesis and cell cation content in chick embryo fibroblasts. J. Cell Physiol., 101, 117-128. 21 Nandi, S. and McGrath, C.M. (1973) Mammary neoplasia in mice. In: Advances in Cancer Research, Vol. 17, pp. 353-414. Editors: G. Klein and S. Weinhouse. Academic Press, New York. 22 Peaker, M. (1978) Ion and water transport in the mammary gland. In: Lactation, Vol. 4, pp. 437-462. Editor: B.L. Larson, Academic Press, New York, London. 23 Petersen, O.H. (1976) Electrophysiology of mammalian gland cells. Physiol. Rev., 56, 535-577. 24 Shen, S.S., Hamamoto, S.T., Bern, H.A. and Steinhardt, R.A. (1978) Alteration of sodium transport in mouse mammary epithelium associated with neoplastic transformation. Cancer Res., 38,1356-1361. 25 Shiu, R.P.C. (1979) Prolactin receptors in human breast cancer cells in long term tissue culture. Cancer Res., 39, 4381-4386. 26 Sluyser, M. (1979) Hormone receptors in mouse mammary tumors. Biochim. Biophys. Acta, 560, 509-529.
27 Sokai, RR. and Rohlf, F.J. (1969) Biometry. W.H. Freeman Co., San Francisco. 28 Turkington, R.W. (1974) ProIactin receptors in mammary carcinoma cells. Cancer Res., 34, 758-763. 29 Ussing, H.H. and Zerahn, K. (1951) Active transport of sodium as the source of electric current in the short-circuited isolated frog skin. Acta Physiol. Stand., 23, 110-127. 30 Welsch, C.W. and Nagasawa, H. (1977) Prolactin and murine mammary tumorigenesis: a review. Cancer Res., 37, 951-963. 31 Wuhrmann, P., Ineichen, H., Riesen-Willi, U. and Lezzi, M. (1979) Changes in nuclear potassium electrochemical activity and puffing of potassium-sensitive salivary chromosome regions during Chironomus development. Proc. Natl. Acad. Sci. U.S.A., 76, 806-808.