Cancer Letters, 6 (1979) 99--105 © Elsevier/North-HollandScientificPublishersLtd.
99
SIMULTANEOUS PRODUCTION OF CASEIN AND MAMMARY TUMOR V I R U S IN M O U S E M A M M A R Y EPITHELIAL CELLS GROWN ON FLOATING COLLAGEN GELS JUMPEI ENAMI*, JASON Y A N G * * and S. NANDI** * Cancer Research Laboratory and Department of Zoology, University of California, Berkeley, California 94720 (U.S.A.)
(Received 22 June 1978) (RevisedversionreceivedIi August 1978) (Accepted 17 August 1978)
SUMMARY Simultaneous production of casein and mammary t u m o r virus (MTV) was analyzed in monolayer cultures of mammary epithelial cells from pregnant BALB/cfC3H mice. A comparison of the 2 celI culture substrata, plastic culture dish and floating collagen gel, showed that the latter supported a much higher degree of simultaneous casein and MTV production in the presence of insulin, cortisol and prolaetin in serum-free culture medium, Importance of floating collagen gel was further shown by delaying the flotation of gels. When the release of gels was delayed, there were concomitant delays in the increase of casein and MTV production. These results indicate that hormones, nature of substratum and flotation regulate the degree of differentiation of mam'~nary epithelial cells in vitro.
INTRODUCTION Differentiation of mammary epithelial cells in vitro has been studied extensively in the past using organ culture techniques [for review, see 2]. Similar studies using cell culture systems have met with only limited success [ 1,5,12]. Primary cultures of mammary epithelial cells maintained as monolayers on tissue culture dishes rapidly lose their differentiated chaxacteristic and no longer respond to hormones. However, it has recently been show~ in * Current address: Department of Physiolol~, Dokkyo University School of Medicine, Mibu, Tochigi 321-02 (Japan), ** To whom requests for reprints should be addressed. *** To whom all Correspondenceshould be addressed. Abbreviation.: MTV, mammary tumor virus.
100
our laboratory that m a m m a r y epithelial cells cu.~tured on floating collagen gels respond to 'thelactogenic combination of'hormones to express a program of m~.~nmary cell differentiation which includes both morphological [8] and biochemical [7] changes that are strikingly pazallel to the events that take place in rico. The availability ,of MTV-infected strains of mice permits the studies of both m a m m a r y differentiation and viral replication under various physiological :states.in the present study, we applied the floating collagen gel technique for the cultivation of nozmal marnma~- epithelial cells isolated t~om chronically MTV-infected BALB/cfC3H mice and analyzed the simultaneous prod~ction of casein and MTV in response to hormones. MATERIALS AND M E T H O D S
Disso,~.iation and cultivation of mammary epithelial cells Ep!ithelial cells were dissociated from mammary glands o f MTV-infected 10- to 14-day nullipatous pregnant BALB/cfC3H/Crgl mice. Finely minced mammary glands were mixed 'with 0.1% eollagenase (CLS III, 120--150 U/rag, Worthington Corp.) in Hanks' balanced salt solution and incubated for I h at 3T'C in a gyratory shaker with a shaking speed o f 120--150 rev./min. Ten milliliters o f collagenase solwLion were u ~ d for each gram o f tissue. After incubation, the solution was passed through Nitex cloth (mesh size, 150 pro; Tobler, Ernst and Traber, Inc. Elmsford, New York). A rubber policeman was used to ensure the filtration. The filtrate was further incubated for 30 rain and the cells were collected b y eentrifugation at 80 × g for 3 rain. The cells were ~hen suspended in 0.05% pronase (45,000 U/g, B grade, Calbiochem) and incubated for 45 rain at 37°C with shaking at 70 rev./min. At the e n d o f the incubation pe:dod 1/10 volume o f fetal calf serum was added and the cells were collected by centrifugation at 80 × g f o r 3 rain. Serum was added directly to the cell pellet and the pellet was broken b y shaldng. Cell suspension was diluted with incubation medium, ~vashed twice with centrifugation at 80 × g for 1 rain and finally suspended in the incubation medium. Cell number was estimated b y a modification o f the m e t h o d o f B.ryant e¢ el. [ 3]. One volume of cell suspension was mixed with 9 vols. o f 0.02% crystal violet in 0.1 M citric acid and vortexe
99
SIMULTANEOUS PRODUCTION OF CASEIN AND MAMMARY TUMOR V I R U S IN M O U S E M A M M A R Y EPITHELIAL CELLS GROWN ON FLOATING COLLAGEN GELS JUMPEI ENAMI*, JASON Y A N G * * and S. NANDI** * Cancer Research Laboratory and Department of Zoology, University of California, Berkeley, California 94720 (U.S.A.)
(Received 22 June 1978) (RevisedversionreceivedIi August 1978) (Accepted 17 August 1978)
SUMMARY Simultaneous production of casein and mammary t u m o r virus (MTV) was analyzed in monolayer cultures of mammary epithelial cells from pregnant BALB/cfC3H mice. A comparison of the 2 celI culture substrata, plastic culture dish and floating collagen gel, showed that the latter supported a much higher degree of simultaneous casein and MTV production in the presence of insulin, cortisol and prolaetin in serum-free culture medium, Importance of floating collagen gel was further shown by delaying the flotation of gels. When the release of gels was delayed, there were concomitant delays in the increase of casein and MTV production. These results indicate that hormones, nature of substratum and flotation regulate the degree of differentiation of mam'~nary epithelial cells in vitro.
INTRODUCTION Differentiation of mammary epithelial cells in vitro has been studied extensively in the past using organ culture techniques [for review, see 2]. Similar studies using cell culture systems have met with only limited success [ 1,5,12]. Primary cultures of mammary epithelial cells maintained as monolayers on tissue culture dishes rapidly lose their differentiated chaxacteristic and no longer respond to hormones. However, it has recently been show~ in * Current address: Department of Physiolol~, Dokkyo University School of Medicine, Mibu, Tochigi 321-02 (Japan), ** To whom requests for reprints should be addressed. *** To whom all Correspondenceshould be addressed. Abbreviation.: MTV, mammary tumor virus.
100
our laboratory that m a m m a r y epithelial cells cu.~tured on floating collagen gels respond to 'thelactogenic combination of'hormones to express a program of m~.~nmary cell differentiation which includes both morphological [8] and biochemical [7] changes that are strikingly pazallel to the events that take place in rico. The availability ,of MTV-infected strains of mice permits the studies of both m a m m a r y differentiation and viral replication under various physiological :states.in the present study, we applied the floating collagen gel technique for the cultivation of nozmal marnma~- epithelial cells isolated t~om chronically MTV-infected BALB/cfC3H mice and analyzed the simultaneous prod~ction of casein and MTV in response to hormones. MATERIALS AND M E T H O D S
Disso,~.iation and cultivation of mammary epithelial cells Ep!ithelial cells were dissociated from mammary glands o f MTV-infected 10- to 14-day nullipatous pregnant BALB/cfC3H/Crgl mice. Finely minced mammary glands were mixed 'with 0.1% eollagenase (CLS III, 120--150 U/rag, Worthington Corp.) in Hanks' balanced salt solution and incubated for I h at 3T'C in a gyratory shaker with a shaking speed o f 120--150 rev./min. Ten milliliters o f collagenase solwLion were u ~ d for each gram o f tissue. After incubation, the solution was passed through Nitex cloth (mesh size, 150 pro; Tobler, Ernst and Traber, Inc. Elmsford, New York). A rubber policeman was used to ensure the filtration. The filtrate was further incubated for 30 rain and the cells were collected b y eentrifugation at 80 × g for 3 rain. The cells were ~hen suspended in 0.05% pronase (45,000 U/g, B grade, Calbiochem) and incubated for 45 rain at 37°C with shaking at 70 rev./min. At the e n d o f the incubation pe:dod 1/10 volume o f fetal calf serum was added and the cells were collected by centrifugation at 80 × g f o r 3 rain. Serum was added directly to the cell pellet and the pellet was broken b y shaldng. Cell suspension was diluted with incubation medium, ~vashed twice with centrifugation at 80 × g for 1 rain and finally suspended in the incubation medium. Cell number was estimated b y a modification o f the m e t h o d o f B.ryant e¢ el. [ 3]. One volume of cell suspension was mixed with 9 vols. o f 0.02% crystal violet in 0.1 M citric acid and vortexe
