Growth requirements and neoplastic transformation of two types of normal human breast epithelial cells derived from reduction mammoplasty

Summary A chemically defined culture medium was developed to support the growth of two distinctly different types of normal human breast epithelial cells (HBEC) derived from reduction mammoplasty. Type I cells expressed luminal epithelial cell markers and were deficient in gap junctional intercellular communication (GJIC), whereas Type II cells expressed basal epithelial cell markers and were efficient in GJIC. In this study, we examined and compared the growth factor and hormone requirements of these two types of cells and a series of cell lines that were obtained by sequential transfection with SV40 DNA (extended lifespan, nontumorigenic), treatment with 5-bromodeoxyuridine (BrdU)/black light (immortal and weakly tumorigenic), and infection of a virus carrying the neu oncogene (highly tumorigenic). Growth of Type I cells was inhibited by withdrawing epidermal growth factor (EGF), hydrocortisone (HC), or insulin (INS) from the culture media, but was enhanced by fetal bovine serum (FBS) supplementation. Growth of Type II cells was inhibited by withdrawal of EGF, HC, or INS from the media, and was inhibited by FBS supplementation. Withdrawal of human transferrin (HT) or 17β-estradiol (E₂) from the media did not alter the growth of Type I or Type II cells. SV40 transfected Type I cell lines still required EGF, HC, or INS for optimal growth. However, the highly tumorigenic cell line did not show a growth dependence on EGF, HC, or INS but did appear to require HT and 3,3′,5-triiodo-D.L. thyronine (T₃) for optimal growth. In addition, FBS stimulated the growth of these cell lines. Thus, this study shows that Type I HBEC are distinctly different from Type II HBEC in growth response to FBS and that neoplastically transformed Type I cells could become growth factor and hormone independent.     

Anti-epidermal growth factor receptor antibodies inhibit the autocrine-stimulated growth of MDA-468 human breast cancer cells

The response of malignant and nonmalignant human breast cell lines to the growth inhibitory effects of monoclonal antibodies against the epidermal growth factor (EGF) receptor was studied. A series of human breast cell lines, which express EGF receptor, were used: MDA-468, MDA-231, and Hs578T human breast cancer cells and the transformed human mammary epithelial cell lines 184A1N4 and 184A1N4-T that have been benzo[a]pyrene immortalized and further transformed with SV40T, respectively. Four antibodies of two different classes were tested: 225 immunoglobulin G (IgG), 108.4 IgG, 96 immunoglobulin M (IgM), and 42 IgM. All four antibodies inhibited the anchorage-dependent and -independent, EGF-stimulated growth of 184A1N4 and 184A1N4-T cells, respectively, and this growth inhibition could be reversed by the addition of increasing concentrations of EGF. In contrast, the antibodies inhibited the anchorage-dependent and -independent growth of MDA-468 cells in the absence of exogenous EGF suggesting that the antibodies were acting to block access of an endogenously produced ligand to the EGF receptor. In the presence of antibody and increasing concentrations of EGF, MDA-468 cell growth was first stimulated then inhibited as the EGF concentration increased, thus, uncovering the growth stimulatory potential of low concentrations of EGF in these cells. Data is presented that indicates MDA-468 cells secrete a transforming growth factor with autocrine growth stimulatory capabilities. The growth of MDA-231 and Hs578T cells, which contain activated ras oncogenes, was not inhibited by the antibodies and the growth of these cell lines was not stimulated by EGF. Of the cell lines studied only MDA-468 cells appear to possess an autocrine growth stimulatory capacity.     

Effect of sustained serum prolactin elevation on breast epithelial and myoepithelial cell proliferation

Oral administration of the dopamine antagonist perphenazine (0.01% in drinking water) to adult female Sprague-Dawley rats led to a three- to fourfold increase in serum prolactin by the first time point sampled (day 2) and a sustained fourfold elevation from day 4 of treatment to the end of the experiment (day 54). In response, five- to sixfold (day 7) and three- to fourfold (day 4) peak elevations in the epithelial cell metaphase indices were seen in the breast lobular and ductular compartments respectively. Both indices fell to basal levels on day 14 but returned to a second, but diminished, peak on day 27. By day 54, the mitotic activity of the epithelium had fallen to just above basal levels in both compartments. A similar mitotic response occurred in the myoepithelial cells, clearly indicating that these must be considered an important cell kinetic component during breast stimulation. Breast epithelial cell number increased 13-14 fold in the lobular but only two- to threefold in the ductular compartments in response to perphenazine administration. Again, similar responses were seen in the myoepithelial cell population. The major proliferative response therefore occurred within the lobular as opposed to the ductular compartment. A considerable discrepancy was shown between the cell number at each time point and that predicted on the assumption of constant cell death rate. We conclude that a growth desensitizing mechanism exists in the rat breast which limits breast growth in the presence of a sustained trophic hormone stimulation. Furthermore, we suggest that this limitation in breast growth is brought about by a mechanism which involves increased cell death in addition to decreased mitotic activity.     

Effect of sustained serum prolactin elevation on breast epithelial and myoepithelial cell proliferation

Abstract.
Oral administration of the dopamine antagonist perphenazine (0·01% in drinking water) to adult female Sprague-Dawley rats led to a three- to fourfold increase in serum prolactin by the first time point sampled (day 2) and a sustained fourfold elevation from day 4 of treatment to the end of the experiment (day 54). In response, five- to sixfold (day 7) and three- to fourfold (day 4) peak elevations in the epithelial cell metaphase indices were seen in the breast lobular and ductular compartments respectively. Both indices fell to basal levels on day 14 but returned to a second, but diminished, peak on day 27. By day 54, the mitotic activity of the epithelium had fallen to just above basal levels in both compartments. A similar mitotic response occurred in the myoepithelial cells, clearly indicating that these must be considered an important cell kinetic component during breast stimulation. Breast epithelial cell number increased 13-14 fold in the lobular but only two- to threefold in the ductular compartments in response to perphenazine administration. Again, similar responses were seen in the myoepithelial cell population. The major proliferative response therefore occurred within the lobular as opposed to the ductular compartment. A considerable discrepancy was shown between the cell number at each time point and that predicted on the assumption of constant cell death rate. We conclude that a growth desensitizing mechanism exists in the rat breast which limits breast growth in the presence of a sustained trophic hormone stimulation. Furthermore, we suggest that this limitation in breast growth is brought about by a mechanism which involves increased cell death in addition to decreased mitotic activity.     

Effects of growth factors on an intestinal epithelial cell line: transforming growth factor beta inhibits proliferation and stimulates differentiation

The effects of epidermal growth factor transforming growth factor beta (TGF beta) and other growth factors on the proliferation and differentiation of a cell line derived from rat intestinal crypt epithelium (IEC-6) were defined. Incorporation of [3H]-thymidine was stimulated 1.4-2.4 fold by insulin, insulin like growth factor (IGF), platelet derived growth factor (PDGF), epidermal growth factor (EGF) and 2% fetal calf serum (FCS) respectively. Additive stimulation was observed when FCS was supplemented by insulin,IGF-I or PDGF but not EGF. Incorporation of [3H]-thymidine by IEC-6 was strongly inhibited by TGF beta with greater than 80% inhibition of incorporation at concentration approximately equal to 2.0 pM. IEC-6 cells bound 4.1 +/- 0.15 X 10(4) molecules TGF beta/cell and appeared to have only a single class of high affinity receptors (Kd approximately equal to 0.5 pM). TGF beta inhibition was unaffected by the presence of insulin or IGF-I suggesting it inhibits proliferation at a step subsequent to that at which these growth factors stimulate [3H]-thymidine incorporation. TGF beta also reduced the stimulation induced by FCS by 65%. In contrast EGF reduced TGF beta inhibition by 60%. IEC-6 cells demonstrated the appearance of sucrase activity after greater than 18 hours treatment with TGF beta. These findings suggest that TGF beta may inhibit proliferative activity and promote the development of differentiated function in intestinal epithelial cells.     

Human breast epithelial cells in serum-free collagen gel primary culture: growth, morphological, and immunocytochemical analysis

Human breast epithelial cells derived from various sources (fibroadenoma, reduction mammoplasty, and mastectomy tissues from premenopausal patients) have been cultured in collagen gel matrix using serum-free medium. Response to various additives has been analyzed for growth-promoting effect when added to a basal medium containing insulin, cholera toxin, and BSA. A consistent observation has been the effect of EGF and cortisol in growth stimulation of human breast epithelial cells, while separately, each additive elicited only a small response. Under this condition, employing EGF and cortisol combinations, these cells gave rise to organized colonies consisting of clusters of cells, usually spherical, without any duct-like extensions. Ultrastructural and immunocytochemical studies, using a panel of monoclonal and polyclonal antibodies, have shown that cell types and features that can be identified in the original breast tissue can also be delineated in the progeny populations. The topographical feature, consisting of lumina surrounded by a single inner layer of epithelial cells and an outer layer of basal/myoepithelial cells, can be re-created in the collagen gel system starting from small clumps of cells.     

Differential response of cultured rabbit articular chondrocytes (RAC) to transforming growth factor beta (TGF-beta)-evidence for a role of serum factors

We show that addition of TGF-beta (0.01-10 ng/ml) to proliferating rabbit articular chondrocytes in presence of low level of fetal calf serum (FCS, 2%) results in a sustained decrease of cell number and DNA synthesis up to 72 h. In contrast, incubation with high serum concentration (10% FCS) induces a transient increase of cell number after 48 h without elevation of DNA synthesis. Moreover, when the factor is added in 10% FCS-containing medium, a differential effect is observed at 48 h (either increase or decrease of cell number) depending on the serum level (2 or 10%) present between 24 and 48 h. Recruitment of cells in late S-phase occurred under TGF-beta-treatment in both 2 and 10% FCS. These arrested cells may then be released by further exposure to 10% FCS-containing medium. The data show that factor(s) from the serum modulate(s) the action of TGF-beta on chondrocyte proliferation. Addition of epidermal growth factor (EGF) to the cultures in presence of 2% FCS mimicks the effects observed with 10% serum, suggesting that the serum component(s) involved in the mechanism could be of EGF type.     

Transforming growth factor-beta,basic fibroblast growth factor,and platelet-derived growth factor-BB interact to affect proliferation of clonally derived porcine satellite cells

Transforming growth factor beta-1 (1GF-β) stimulated porcine satellite cell proliferation in basal serum-free medium by 25%, but inhibited growth in serumcontaining medium by 58%. The effect of TGF-β on cell proliferation in serumfree medium was examined in combination with the following human recombinant growth factors: platelet-derived growth factor-BB (PDGF), basic fibroblast growth factor (FGF), insulin-like growth factor I (IGF-I), and epidermal growth factor (EGF). TGF-β inhibited PDGF-stimulated proliferation, enhanced FGF-stimulated proliferation, and had no effect on proliferation stimulated by IGF-I. The response of satellite cells to EGF and TGF-β in serum-free medium was not different than TGF-β alone. TGF-β depressed proliferation stimulated by the following combinations of two growth factors: PDGF and IGF-I, PDGF and EGF, PDGF and FGF, and IGF-I and EGF. In combination with IGF-I and FGF, TGF-β did not affect proliferation. TGF-β inhibited proliferation stimulated by the combination of PDGF, EGF, and IGF-I, but had no effect on proliferation stimulated by combinations of three growth factors that included FGF. FGF stimulated proliferation in Minimum Essential Medium containing 10% porcine serum (MEM-10% PS) by 13% above control. When the combination of TGF-β and FGF was added to MEM-10% PS, a 78% increase in proliferation was observed. Polyclonal antihuman PDGF-AB (this form neutralizes PDGF-AA, AB, and BB) reduced proliferation in MEM-10% PS by 44%. The combination of TGF-β and anti-PDGF-AB reduced proliferation by 59%, indicating the effects were not additive. These data indicate that: (1) FGF and TGF-β interact to increase proliferation of clonally derived porcine satellite cells, and (2) the inhibitory effect of TGF-β on proliferation of clonally derived porcine satelite cells can be primarily attributed to a reduction in the mitogenic effects of PDGF. © 1993 Wiley-Liss, Inc.     

Regulation of rat ovarian cell growth and steroid secretion

A cultured rat ovarian cell line (31 A-F(2)) was used to study the effect of growth factors (epidermal growth factor [EGF] and fibroblast growth factor [FGF]), a survival factor (ovarian growth factor [OGF]), a hormone (insulin), and an iron-binding protein (transferring) on cell proliferation and steroid production under defined culture conditions. EGF and insulin were shown to be mitogenic (half-maximal response at 0.12 nM and 0.11 muM, respectively) for 31A-F(2) cells incubated in serum-free medium. EGF induced up to three doublings in the cell population, whereas insulin induced an average of one cell population doubling. FGF, OGF, and transferrin were found not to have any prominent effect on cell division when incubated individually with 31A-F(2) cells in serum-free medium. However, a combination of EGF, OGF, insulin, and transferrin stimulated cell division to the same approximate extent as cells incubated in the presence of 5 percent fetal calf serum. EGF or insulin did not significantly affect total cell cholesterol levels (relative to cells incubated in serum-free medium) when incubated individually with 31A-F(2) cells. However, cell cholesterol levels were increased by the addition of OGF (250 percent), FGF (370 percent), or a combination of insulin and EGF (320 percent). Progesterone secretion from 31A-F(2) cells was enhanced by EGF (25 percent), FGF (80 percent), and insulin (115 percent). However, the addition of a mitogenic mixture of EGF, OGF, insulin, and transferrin suppressed progesterone secretion 150 percent) below that of control cultures. These studies have permitted us to determine that EGF and insulin are mitogenic factors that are required for the growth of 31A-F(2) cells and that OGF and transferrin are positive cofactors that enhance growth. Also, additional data suggest that cholesterol and progesterone production in 31A-F(2) cells can be regulated by peptide growth factors and the hormone insulin.     

Human breast epithelium in vitro: The re-expression of structural and functional cellular differentiation in long-term culture

Summary Fragments of human breast epithelium, devoid of all stromal and basal lamina components, which maintain their in vivo topological organisation can be cultured for up to 28 days within a reconstituted rat-tail-derived collagen matrix. These organoids initially undergo a loss of structural and 3-dimensional organisation, typified by loss of lumina formed by epithelial cells, and myosin from myoepithelial cells. Their subsequent reorganisation is dependent on the presence of serum, insulin, hydrocortisone, and cholera toxin in tissue culture medium. After this preliminary phase, a reduction in the concentration of serum, insulin, hydrocortisone, and cholera toxin is necessary to allow the structural differentiation of epithelial and myoepithelial cells. The myoepithelial cells also regain their ability to produce the basal lamina component laminin. The use of bovine-dermal collagen as the matrix, rather than rat-tail-derived collagen is shown to result in more stable organisation and differentiation of the organoids. The successful use of single-cell pellets (derived by trypsinisation of the organoids) in place of organoids in such cultures illustrates that there is no requirement for pre-existing cell/ cell contact or topological organisation of cells prior to embedding within the collagen matrix.     

Regulation of c-fos expression in primary culture of guinea pig glandular epithelial cells stimulated by growth factors and estradiol

The c-fos expression was investigated in primary culture of guinea pig glandular epithelial cells. These cells were made quiescent by serum deprivation and stimulated with fetal calf serum (FCS, 15%), 17 beta-estradiol (E2 10(-8) mol/l) alone or in combination with epidermal growth factor (EGF, 100 ng/ml) and insulin (I, 10 micrograms/ml). Low levels of c-fos mRNA were detectable in quiescent cells and were not increased in cells stimulated with either E2, EGF, I, or EGF plus I. On the contrary, the c-fos mRNA were early and transiently increased by FCS or E2 plus EGF plus I (4.5 and 9.5 fold induction, respectively). This effect was independent of de novo protein synthesis since it was not abolished in the presence of cycloheximide. It appears that E2 acts in a multiple step process including the stimulation by EGF plus insulin.     

Fibroblast and epidermal growth factors modulate proliferation and neural cell adhesion molecule expression in epithelial cells derived from the adult mouse tongue

Lingual epithelial cells, including those of the taste buds, are regularly replaced by proliferative stem cells. We found that integrin beta(1), a keratinocyte stem cell marker, was expressed at the basal layer and taste buds of adult mouse tongue epithelium. We purified and cultured integrin beta(1)-positive cells (termed KT-1 cells), whose growth was stimulated by epidermal growth factor (EGF) and basic fibroblast growth factor (FGF-2). FGF-2 stimulation induced translocation of the FGF type I receptor (FGFR1) into nuclei, suggesting that the growth-stimulating effect of FGF-2 was mediated through FGFR1. EGF and FGF-2 also regulated cell surface expression of the neural cell adhesion molecule (N-CAM) in KT-1 cells. Anti-N-CAM antibody immunoreactivity was restricted to the gustatory epithelium and the nerves in the tongue epithelium, giving rise to the possibility that KT-1 may contain gustatory epithelial cells. KT-1 cells may thus be useful for analyzing the factors that regulate the growth and differentiation of lingual and gustatory epithelial cells in vitro.     

Differential effects of transforming growth factor-beta and epidermal growth factor on the cell cycle of cultured rabbit articular chondrocytes

We examined the effect of transforming growth factor (TGF-beta) on the proliferative rate and cell cycle of cultured rabbit articular chondrocytes using cell counting, cytofluorometry, and [3H]-thymidine incorporation. In the presence of 2% or 10% FCS (fetal calf serum), TGF-beta at 0.01, 0.1, 1, and 10 ng/ml had an inhibitory effect on cell proliferation after 24 h exposure with a dose dependence only for 2% FCS. Flow cytometric analysis of cell DNA content at that time showed that a high proportion of cells were arrested in late S-phase (SQ or G2Q) in either 2% or 10% FCS-containing medium. In both cases, a disappearance of the cell blockage occurred between 24 and 48 h after TGF-beta addition. However, whereas a stimulation of cell proliferation rate was observed at that time in cultures containing 10% FCS, a dose-dependence inhibition of cell growth was detected, in contrast, for 2% FCS-treated cells. Presence of TGF-beta during the last 24 h was not necessary to release the arrested cells. Furthermore, platelet-poor plasma at 10% produced the same effects as FCS, suggesting that platelet-derived factors, such as platelet-derived growth factor (PDGF), could not be responsible for the release of blocked cells in this case. We compared the effect of TGF-beta to that of epidermal growth factor (EGF), used at an optimal concentration (10 ng/ml). In both slowly growing (2% FCS) and proliferating chondrocytes (10% FCS), EGF caused a significant increase of cell proliferation as early as 24 h. No arrest in late S-phase but an augmentation of the percentage of cells in S- and G2M-phases were observed. When combined, TGF-beta and EGF did not induce synergistic effect on the chondrocyte proliferation, as estimated by cell counting. [3H]-thymidine labeling showed that the factors induced identical maxima of incorporation but the peak occurred earlier for TGF-beta than for EGF (approximately 6 h versus 12 h, respectively). Although both factors induce similar cell-number increases at 48 h in 10% FCS-containing medium, these proliferative effects were due to different actions on the cell cycle. The present study indicates that TGF-beta induces first a recruitment of chondrocytes in noncycling SQ- or G2Q-blocked cells. The, the release of these cells may produce either apparent stimulation of cell proliferation if sufficient levels of an unknown serum factor are present (10% FCS) or an inhibition of growth rate when only reduced amounts of this factor are available (2% FCS).(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of estrogen, epidermal growth factor, and transforming growth factor-alpha on the growth of human breast epithelial cells in primary culture.

Since 17 beta-estradiol (E2)-stimulated growth in human breast cancer cell lines has been shown to be accompanied by increased production of epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) and their receptor, we investigated the effects of E2 and these growth factors on the growth of human breast epithelial cells (HBEC) in primary culture. HBEC from normal, benign, and malignant tissues were cultured in serum-free medium [DME:F12(1:1), 5 mg/ml BSA, 10 ng/ml cholera toxin, 0.5 micrograms/ml cortisol, 10 micrograms/ml insulin] in the presence and absence of E2, EGF, and TGF-alpha. Tritiated-thymidine ([3H]TdR) incorporation into DNA was used as a measure of cell growth. E2 did not stimulate growth of any of the cultures at all concentrations examined (10(-9) to 10(-6) M). In contrast, EGF ranging from 1 to 100 ng/ml consistently increased the growth of cells of all three breast tissue types in a dose-dependent manner. The EGF stimulation was inhibited by MAb 528, a monoclonal antibody against the EGF receptor. TGF-alpha was equally or more effective in stimulating proliferation, although its dose-response range was different than that of EGF. E2 and EGF together acted in a synergistic manner in 50% of the samples examined. These studies suggest that E2 can exert effects on HBEC growth via modulation of the cells' response to EGF.     

Correlation of receptors for growth factors on mouse embryonal carcinoma cells with growth in serum-free, hormone-supplemented medium

High affinity receptors for insulin, transferrin, epidermal growth factor (EGF) and a multiplication-stimulating activity (MSA) have been identified and partially characterized on a mouse embryonal carcinoma cell line, OTT-6050, using various ¹²⁵I-ligands. With the exception of MSA receptors which bound both MSA and insulin, the receptors for EGF, insulin and transferrin exhibited specificity of binding for their respective ligands. There is a correlation between the saturation of these receptors and the concentration of growth factors necessary for optimal growth of OTT-6050 cells in serum-free medium supplemented with insulin (or MSA), transferrin, EGF, fibroblast growth factor (FGF) and Pedersen fetuin on culture surfaces treated with polylysine or various types of collagen. Cells cultured in this medium exhibit growth rates equivalent to that observed with cells maintained in medium containing 5% fetal calf serum (FCS). These results suggest that relatively undifferentiated mouse embryonal carcinoma cells or endoderm cells possess receptors for various growth factors and that their presence on these cells is correlated with the ability of these cells to mitogenically respond to these growth factors.     

Three novel hormone-responsive cell lines derived from primary human breast carcinomas: functional characterization

Human breast cancer primary cultures are useful tools for the study of several aspects of cancer biology, including the effects of chemotherapy and acute gene expression in response to different hormonal/chemotherapy treatments. The present study reports the conditions for primary culture of breast cancer samples from untreated patients and the most effective collagenization method to dissociate human samples consisting in an overnight incubation with 1 mg/ml types II or IV collagenase and further incubation in DMEM:F12 (1:1) medium supplemented with glutamine, bovine insulin, penicillin-streptomycin, HEPES, estradiol, cortisol (F), tri-iodothyronine (T(3)), transferrine (TR), and 10% fetal calf serum (FCS). These conditions proved to be appropriate for both primary culture and the development of stable cell lines. Of the seven cell lines obtained, three fast growing and estrogen receptor (ER)+/progesterone receptor (PgR)+/EGF receptor (EGFR)+ have been characterized. The cells are able to grow both in soft agar and in nude mice, and express cytokeratins, all parameters characteristic of malignant epithelial cell lines. The cells also exhibit an increased proliferation rate in the presence of estradiol, progesterone, and EGF, suggesting the presence of the corresponding receptors. The mRNA expression of type alpha- and beta-ER as well as EGFR, was confirmed by RT-PCR. In conclusion, the novel cell lines described, arose from primary tumors and are sensitive to estradiol, progesterone, and EGF. This not only expands the repertoire of breast cancer cells available as potentially useful tools for examining most parameters in breast cancer "in vitro", but also provides unique new models to explore the complex regulation by steroids as well as growth factors in such cells.     

Regulation of vimentin expression in cultured human mammary epithelial cells

Using five different monoclonal antibodies to vimentin, we have examined the expression of vimentin in cryostat sections and serum-free cultures of normal human breast tissue. In cryostat sections, myoepithelial cells as well as stromal cells showed immunoreactivity to vimentin, irrespective of the antibody used. In contrast, luminal epithelial cells were negative for vimentin, but positive for keratin K18. In culture, myoepithelial cells showed immunoreactivity to vimentin from their first appearance in monolayer. Moreover, a fraction of luminal epithelial cells expressed vimentin in addition to keratin K18. We found a clear, reversible correlation between proliferation, determined by incorporation of [3H]-TdR, and induction of vimentin in the luminal epithelial cells. Thus, in growth-stimulated cultures on a medium containing cholera toxin (CT), epidermal growth factor (EGF), transferrin (Tf), hydrocortisone (H) and insulin (I), the fraction of vimentin-positive luminal epithelial cells increased, while it decreased within 14 days from approximately 36% to 3% on a medium containing CT and EGF, only. We therefore conclude: (1) vimentin is constantly expressed in myoepithelial cells in situ and in vitro, and (2) expression of vimentin in luminal epithelial cells in vitro is not a result of monolayer cultivation as such, but rather associated with the increased growth rate seen in culture.     

A novel calcium signalling response in the breast cancer cell line MDA-468

In the human breast carcinoma cell line MDA-468 addition of epidermal growth factor (EGF) is growth inhibitory. Calcium signalling was investigated in this cell line using the calcium sensitive fluorescent probe Indo-1. Addition of EGF to MDA-468 cells resulted in a novel biphasic calcium response. In the first phase of the response EGF raised calcium to levels significantly above basal. This was followed by a prolonged fall in calcium to levels significantly lower than original basal levels. The G-protein activator aluminum fluoride (AlF), stimulated a rise in calcium which was not proceeded by a fall below basal levels. Conversely addition of PMA, an activator of protein kinase C (PKC), induced a fall in calcium from basal without a prior increase. Down regulation of PKC eliminated the response to PMA, however the biphasic nature of the EGF response was maintained. Pretreatment of the cells with pertussis toxin did not alter the response to EGF nor to AlF. We conclude that in the MDA-468 cell in which EGF is growth inhibitory: 1) EGF results in a biphasic calcium response which ultimately leads to reduction below baseline levels, 2) a rise in calcium itself is not sufficient to account for the subsequent fall below basal levels, 3) G-proteins may be involved in the initial phase of the EGF response, 4) activation of PKC can also reduce intracellular calcium, however the response to EGF is not dependent on this pathway.     

Receptor-mediated vectorial transcytosis of epidermal growth factor by Madin-Darby canine kidney cells

Transcellular transport of a variety of ligands may be an important mechanism by which regulatory substances reach their site of action. We have studied the transcellular transport of two 6,000-mol-wt proteins, epidermal growth factor (EGF) and insulin, across polarized Madin-Darby canine kidney (MDCK) cells grown on dual-sided chambers on a nitrocellulose filter substrate. When grown on these chambers, MDCK cells are polarized and express distinct basal and apical surfaces. MDCK cells are capable of unidirectional transport of EGF from the basal-to-apical direction, 50% of bound EGF transported in 2 h. Transport was inhibited by the addition of unlabeled EGF in a dose-dependent manner. Anti-EGF receptor Ab, which inhibited binding, also inhibited transport. No transport in the apical-to-basal direction is noted. Insulin transport is not observed in either direction. Transport correlates with the presence of ligand-specific receptors on the cell surface. Hence, EGF receptors (Ro = 48,000, Kd = 3.5 X 10(-10) M) are found only on the basal surface of the MDCK cells and neither surface expresses insulin receptors. Characterization of the EGF receptors on MDCK cells, as assessed by affinity, molecular mass, and anti-receptor antibody binding reveals that this receptor has similar characteristics to EGF receptors previously described on a variety of cells. Hence, the EGF receptor can function as a transporter of EGF across an epithelial cell barrier.