Vol. 173, No. 3,1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1239-1247
December 31, 1990
LOSS OF GROWTH INHIBITORY ACTIVITY OF TGF-13 TOWARD NORMAL HUMAN MAMMARY EPITHELIAL CELLS GROWN WITHIN COLLAGEN GEL MATRIX
Kazuhide Takahashi, Katsuo Suzuki, and Tetsuo Ono Department of Biochemistry, Kanagawa Cancer Center Research Institute, 54-2 Nakao-cho, Asahi-ku, Yokohama 241, Japan Received October 22, 1990
SUMMARY. TGF-13 at concentrations in the range from 0.1 to 10 ng/ml gave significant growth inhibition of nonmalignant human mammary epithelial cells (HMEC) but not of malignant HMEC grown in monolayer cultures in serum-free medium. However, no growth inhibition of the nonmalignant cells was observed when the cells were cultivated within a type-I collagen gel matrix either adhering to a plastic substratum or floating on the medium. Within floating collagen gels, both nonmalignant and malignant HMEC formed a cell mass having radial extensions, and TGF-13 at 1 or 10 ng/ml prevented the formation of extensions only in the nonmalignant HMEC. ©1990Academic Press,
Inc.
TGF-I~, a known bifunctional growth regulator (1), was originally defined by its ability to induce the anchorage-independent growth of nontransformed cells (2). The effects of TGF-I] on cell growth vary depending on cell type. In general, cells of mesenchyme origin, such as normal rat kidney fibroblasts (3) and AKR-2B mouse embryo cells (4), are stimulated by TGF-13 Conversely, the growth of epithelial ceils, such as mouse and human keratinocytes (5,6), human bronchial epithelial cells (7), and human mammary epithelial cells (8), is generally inhibited by the factor. Such growth inhibitory effects of TGF-I3 on normal ceils differ from its effects on their malignantly transformed counterparts (7,8). The mammary gland, which grows and forms a branched ductal structure within fat tissues during adolescence in the female, provide a complex and yet versatile model for studies of hormonal responsiveness and morphogenesis of mammary epithelial cells. The early pioneering work of Emerman and Pitelka (9) showed that
1239
0006-291X/90 $1.50 Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 173, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
mammary epithelial cells embedded in collagen gels used as an extracellular matrix in place of fat tissues could proliferate and morphologically develop to produce three-dimensional structures resembling the mammary gland in situ. Using this collagen gel culture system, we examined the effects of TGF-13 on the cell growth as well as the morphological development of nonmalignant and malignant HMEC and compared the results with those obtained with TGF-13 treatment of the cells in monolayer culture.
MATERIALS AND I~rHtOBS Tissue collection and monolayer c u l t u r e . Fresh tissue fragments of human breast carcinomas were obtained from mastectomy specimens. Histologically nonmalignant tissue fragments were obtained from areas away from the primary lesion in the same mastectomy specimens (10,11). The tissue fragments were minced into smaller pieces and placed onto plastic culture dishes containing a small amount of serum-free MCDB 170 medium supplemented with 5 ng/ml EGF, 5 ~g/ml insulin, 5 ktg/ml human transferrin, 1.4 ~M hydrocortisone, 0.1 mM ethanolamine, 0.1 mM phosphoethanolamine, 1 ~tg/ml ovine prolactin, and 25 nM prostaglandin E1 (12). Epithelial cells that grew out from the tissue fragments were harvested with 0.25% (w/v) trypsin-0.1% (w/v) E D T A and subcultured in the serumfree medium. For experiments involving monolayer cultures of HMEC, the cells were harvested and seeded on plastic culture dishes. Human platelet TGF-[~ (Takara Suzo Co., Kyoto) was added at 2 days after the start of the culture, and the cells were successively cultivated for another 6 days. Number of cells was counted with a hemocytometer, and statistical analysis was performed by Student's t-test. Collagen gel culture. Experiments using collagen gels were carried out according to the method previously described (13). A monolayer culture of HMEC was harvested and a 10-~tl drop of the cell suspension containing 10 4 viable cells was placed onto a base layer of 0.1% (w/v) type-I collagen gel previously formed in each well of a 24-well plastic p l a t e . After the culture plates had been kept for 2 h at 37 °C, the cells in each well were overlaid with 0.1% collagen gel and then flooded with 1 ml of serum-free medium. After cultivation of the cells for 2 days, the collagen gels were detached from or left attached to the platic plates and the medium was then replaced with fresh medium containing or lacking TGF-~. M e a s u r e m e n t of DNA content. Cells grown within collagen gels were washed once with PBS, and the gels were then minced into small fragments with scissors. After digestion of the gel fragments with 40 U/ml collagenase for 1 h at 37 °C with vigorous shaking, the cells were fixed with chilled (-20°C) ethanol and air-dried. DNA content was measured spectrofluorometrically (Ex at 415 nm, Em at 1240
Vol. 173, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
505 nm) after incubation of for 45 min at 60°C (14).
the cells with 3,5-diaminobenzoic acid
~ T S Effects of TGF-[~ When HMEC
on monolayer cultures of HMEC
were seeded on plastic culture dishes, no apparent
increase
in the cell number could be observed until after 2 days after
the start
of the culture (Fig. 1). This is in agreement with a previous
observation of HMEC seeded on plastic dishes (11), and is due to an incomplete
adhesion
of the
cells
onto
the
surface
of the plastic
substratum in spite of the fact that the cells are viable. Consequently, in the present study TGF-I3 was added to each culture at 2 days after initiation of cultures to avoid the possible effects of the factor on the initial attachment of the cells to the substratum. After culture of nonmalignant HMEC in monolayers for 6 days in the absence of TGF-I3 , number of cells increased to 4.31 x 104 p e r culture and 10
(Table 1). When TGF-[~
was added to the cultures at 0.1, 1.0,
ng/ml, the cell number per culture was reduced to 2.86 x
10 4 , 1.69 x 104 , and 1.47 x 104 , respectively. The decrease in cell number in the presence of TGF-[~ at the three doses used was statistically significant
(P
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1239-1247
December 31, 1990
LOSS OF GROWTH INHIBITORY ACTIVITY OF TGF-13 TOWARD NORMAL HUMAN MAMMARY EPITHELIAL CELLS GROWN WITHIN COLLAGEN GEL MATRIX
Kazuhide Takahashi, Katsuo Suzuki, and Tetsuo Ono Department of Biochemistry, Kanagawa Cancer Center Research Institute, 54-2 Nakao-cho, Asahi-ku, Yokohama 241, Japan Received October 22, 1990
SUMMARY. TGF-13 at concentrations in the range from 0.1 to 10 ng/ml gave significant growth inhibition of nonmalignant human mammary epithelial cells (HMEC) but not of malignant HMEC grown in monolayer cultures in serum-free medium. However, no growth inhibition of the nonmalignant cells was observed when the cells were cultivated within a type-I collagen gel matrix either adhering to a plastic substratum or floating on the medium. Within floating collagen gels, both nonmalignant and malignant HMEC formed a cell mass having radial extensions, and TGF-13 at 1 or 10 ng/ml prevented the formation of extensions only in the nonmalignant HMEC. ©1990Academic Press,
Inc.
TGF-I~, a known bifunctional growth regulator (1), was originally defined by its ability to induce the anchorage-independent growth of nontransformed cells (2). The effects of TGF-I] on cell growth vary depending on cell type. In general, cells of mesenchyme origin, such as normal rat kidney fibroblasts (3) and AKR-2B mouse embryo cells (4), are stimulated by TGF-13 Conversely, the growth of epithelial ceils, such as mouse and human keratinocytes (5,6), human bronchial epithelial cells (7), and human mammary epithelial cells (8), is generally inhibited by the factor. Such growth inhibitory effects of TGF-I3 on normal ceils differ from its effects on their malignantly transformed counterparts (7,8). The mammary gland, which grows and forms a branched ductal structure within fat tissues during adolescence in the female, provide a complex and yet versatile model for studies of hormonal responsiveness and morphogenesis of mammary epithelial cells. The early pioneering work of Emerman and Pitelka (9) showed that
1239
0006-291X/90 $1.50 Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 173, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
mammary epithelial cells embedded in collagen gels used as an extracellular matrix in place of fat tissues could proliferate and morphologically develop to produce three-dimensional structures resembling the mammary gland in situ. Using this collagen gel culture system, we examined the effects of TGF-13 on the cell growth as well as the morphological development of nonmalignant and malignant HMEC and compared the results with those obtained with TGF-13 treatment of the cells in monolayer culture.
MATERIALS AND I~rHtOBS Tissue collection and monolayer c u l t u r e . Fresh tissue fragments of human breast carcinomas were obtained from mastectomy specimens. Histologically nonmalignant tissue fragments were obtained from areas away from the primary lesion in the same mastectomy specimens (10,11). The tissue fragments were minced into smaller pieces and placed onto plastic culture dishes containing a small amount of serum-free MCDB 170 medium supplemented with 5 ng/ml EGF, 5 ~g/ml insulin, 5 ktg/ml human transferrin, 1.4 ~M hydrocortisone, 0.1 mM ethanolamine, 0.1 mM phosphoethanolamine, 1 ~tg/ml ovine prolactin, and 25 nM prostaglandin E1 (12). Epithelial cells that grew out from the tissue fragments were harvested with 0.25% (w/v) trypsin-0.1% (w/v) E D T A and subcultured in the serumfree medium. For experiments involving monolayer cultures of HMEC, the cells were harvested and seeded on plastic culture dishes. Human platelet TGF-[~ (Takara Suzo Co., Kyoto) was added at 2 days after the start of the culture, and the cells were successively cultivated for another 6 days. Number of cells was counted with a hemocytometer, and statistical analysis was performed by Student's t-test. Collagen gel culture. Experiments using collagen gels were carried out according to the method previously described (13). A monolayer culture of HMEC was harvested and a 10-~tl drop of the cell suspension containing 10 4 viable cells was placed onto a base layer of 0.1% (w/v) type-I collagen gel previously formed in each well of a 24-well plastic p l a t e . After the culture plates had been kept for 2 h at 37 °C, the cells in each well were overlaid with 0.1% collagen gel and then flooded with 1 ml of serum-free medium. After cultivation of the cells for 2 days, the collagen gels were detached from or left attached to the platic plates and the medium was then replaced with fresh medium containing or lacking TGF-~. M e a s u r e m e n t of DNA content. Cells grown within collagen gels were washed once with PBS, and the gels were then minced into small fragments with scissors. After digestion of the gel fragments with 40 U/ml collagenase for 1 h at 37 °C with vigorous shaking, the cells were fixed with chilled (-20°C) ethanol and air-dried. DNA content was measured spectrofluorometrically (Ex at 415 nm, Em at 1240
Vol. 173, No. 3, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
505 nm) after incubation of for 45 min at 60°C (14).
the cells with 3,5-diaminobenzoic acid
~ T S Effects of TGF-[~ When HMEC
on monolayer cultures of HMEC
were seeded on plastic culture dishes, no apparent
increase
in the cell number could be observed until after 2 days after
the start
of the culture (Fig. 1). This is in agreement with a previous
observation of HMEC seeded on plastic dishes (11), and is due to an incomplete
adhesion
of the
cells
onto
the
surface
of the plastic
substratum in spite of the fact that the cells are viable. Consequently, in the present study TGF-I3 was added to each culture at 2 days after initiation of cultures to avoid the possible effects of the factor on the initial attachment of the cells to the substratum. After culture of nonmalignant HMEC in monolayers for 6 days in the absence of TGF-I3 , number of cells increased to 4.31 x 104 p e r culture and 10
(Table 1). When TGF-[~
was added to the cultures at 0.1, 1.0,
ng/ml, the cell number per culture was reduced to 2.86 x
10 4 , 1.69 x 104 , and 1.47 x 104 , respectively. The decrease in cell number in the presence of TGF-[~ at the three doses used was statistically significant
(P
