JOURNAL OF CELLULAR PHYSIOLOGY 150:304311 (1992)

Parathyroid Hormone-Related Protein Production by Primary Cultures of Mammary Epithe1ial CelIs S.L. FERRARI, R. R I Z Z O L I , * AND J.P. B O N J O U R Division of Clinical Pathophysiology, Department of Medicine, University Hospital of Geneva, 72 I 1 Geneva 4 , Switzerland Parathyroid hormone-related protein (PTHrP) plays a major role in the pathogenesis of malignant hypercalcemia, but has also been found in fetal and adult nonneoplastic tissues. Among them, lactating mammary gland was shown to produce PTHrP, and high levels of PTHrP were measured in milk. However, the regulation of PTHrP production by breast cells is still unknown. Primary cultures of mammary cells isolated from rat lactating glands were grown on collagen gels in an insuliniepidermal growth factor (EGF)-supplemented medium. Under these conditions, mammary cells displayed an epithelial phenotype and their number increased more than twofold after 1 week in culture. At that time, the cells were capable of producing immunoreactive PTHrP (range: 25 to 1SO pgil0' cells X 24 h) and PTH-like bioactivity, as indicated by a 60% increase in cyclic adenosine monophosphate (CAMP)production induced by mammary epithelial cell conditioned medium in the PTH-responsive osteoblast-like UMR-106 cell line. When cell proliferation was hindered by lowering plating density, by removing medium supplements, or by adding transforming growth factor (TGFI-P, a well-known autocrine inhibitor of mammary epithelial cell growth. PTHrP production was increased. In contrast, the omission of EGF or addition of specified anti-EGF antibodies decreased PTHrP production. In conclusion, primary cultures of mammary epithelial cells isolated from lactating rat were shown for the first time to produce PTHrP in vitro. This production was higher in the presence of EGF and could be modulated by cell growth rate.

Parathyroid hormone-related protein (PTHrP) is a PTHrP production and the state of cell proliferation newly discovered 141 amino acid protein sharing and differentiation. Moreover, a better knowledge of strong homology with parathyroid hormone (PTH) in the factors influencing the production of PTHrP by the amino-terminal amino acid sequence as well as bioac- breast epithelium might provide information for the tivity (Martin et Suva, 1989; Goltzman et al., 1989). understanding of the frequent association between Among the various malignant tumors associated with breast carcinoma and PTHrP synthesis. We investihypercalcemia (Broadus et al., 1988), breast carcinoma gated whether mammary epithelial cells would synthewas soon recognized to synthesize PTHrP (Stewart size and release PTHrP in vitro, and the influence of et al., 1987; Burtis et al., 1987). Very recently, Southby culture conditions on the production of PTHrP in this et al. (1990) reported the presence of immunoreactive system. Thus, the effect of cell seeding density and of PTHrP in more than 50% of the breast tumors exam- factors known to modulate the development of breast ined, even in the absence of overt hypercalcemia. Be- epithelium, i.e. epidermal growth factor (EGF) (Tonelli sides it production by neoplastic tissues, PTHrP was and Sorof, 1980; McGrath et al., 1985) and transformalso described to be synthesized during fetal develop- ing growth factor (TGF)-P (Daniel et al., 1989; Ethier ment (Moniz et al., 1990), as well as in adult non-tu- and Van de Velde, 1990), were studied. In summary, we developed primary cultures of mammoral tissues. Thus, PTHrP can be produced by the placenta (Abbas et al., 19901, where it could be a physi- mary epithelial cells isolated from lactating rats and ological regulator of calcium transport (Martin et Suva, grown in a n insulin/EGF-supplemented medium. 19891, and by lactating mammary gland (Thiede and These cells were capable of producing immunoreactive Rodan, 1988). High amounts of PTHrP were found in and bioactive PTHrP, a t a rate that was modulated by milk (Budayr et al., 1989). However, the various deter- the seeding density and culture conditions modifying minants of the regulation, if any, of PTHrP production the rate of cell proliferation. by non-neoplastic cells are still poorly defined. Since the mammary gland appears to synthesize PTHrP during lactation, a state characterized by marked cell proliferation and functional changes, i t should be a suit- Received June 12. 1991; accepted September 16,1991. able model to investigate the relationship between "To whom reprint requestsicorrespondence should be addressed. (01992 WILEY-LISS, INC

PTHrP PRODUCTION BY MAMMARY EPITHELIAL CELLS

MATERIALS AND METHODS Rat mammary epithelial (RME) cell isolation and culture On day 5 post-partum, 3- to 9-month-old lactating female Wistar rats were sacrificed by cervical dislocation. Epithelial organoids were isolated from whole mammary glands following standard procedures (McGrath, 19871, with slight modifications. Briefly, enzymatic digestion of minced mammary tissue was performed in medium 199 (Flow Laboratory, Irvine, Scotland), containing 100 IUiml penicillin and 100 pgiml streptomycin, 6.6% fetal calf serum (FCS) (GIBCO, Paisley, Scotland), 200 U/ml type I collagenase (Worthington Corp, Bedford, MA), and 500 UFUiml type 1hyaluronidase (Sigma Chemical Co., St. Louis, MO) at 32°C for 2.5 h. Further incubation in 0.02% deoxyribonuclease 1 (DNAse 1, Sigma) and filtration through Nitex cloth (Nybold, mesh size 150 pm, Swiss Silk Bolting Cloth Mfg., Zurich, Switzerland) preceded a 10 min isopyknic centrifugation of the dispersed mammary fragments through a preformed 42% Percoll density gradient (Pharmacia Fine Chemicals, Piscataway, NJ). Epithelial organoids were recovered from the Percoll density fraction higher than 1.055 giml and resuspended in Dulbecco’s modified Eagle’s minimum essential medium:F12 medium [DMEM:F12 ( l : l ) , GIBCO] supplemented with 10% FCS and insulin (5 pgiml) (bovine insulin, Sigma). Epithelial organoids were plated a t various densities in 6 multiwell 35 mm diameter plates (Costar, Cambridge, MA) on 1.5 mm thick type 1 collagen gels (Vitrogen, Collagen Corp., Palo Alto, CA), and left for 48 h for attachment. Medium was then replaced with DMEM:F12 (1:l) medium containing 2% FCS, 5 pgiml insulin, and 10 ngiml EGF (Collaborative Research, Bedford, MA), unless otherwise indicated, and changed every other day for 6 to 12 days. In order to modulate cell growth rate, supplements were omitted after 6 days in culture, during 24 h prior to collection of the conditioned medium (CM). Alternatively, from day 3 after seeding, TGF-f3 was added to insuliniEGFsupplemented medium a t a concentration of 3 ngiml, up to the time of CM collection, which was performed on day 8 in culture. Finally, to evaluate the requirement for EGF, this was replaced by M dexamethasone (Sigma) and 2 pgiml ovine prolactin (Sigma), a combination that is also known to promote mammary epithelial cell growth (Pasco e t al., 1982; McGrath et al., 1985). Characterization of RME cells Epithelial organoids recovered from Percoll density fractions higher or lower than 1.055 giml were grown separately on glass coverslips for 5 days in insulin/ EGF-supplemented growth medium as described above. At that time, glass coverslips were washed in phosphate-buffered saline (PBS), fixed in acetone a t -20°C for 5 min, prior to a 45 min incubation a t room temperature with polyclonal rabbit anti-keratin antiserum (diluted 1:6 in PBS) (Bio-Science, Emmenbriicke, Switzerland) or mouse anti-a-smooth muscle actin monoclonal antibodies (diluted 1:300 in PBS) (kindly provided by Dr. A.P. Sappino, Division of Oncology, University Hospital of Geneva), a s described by

305 CM (plltube)

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0.75

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1000

PTHrP(1-34)[pg/lube]

Fig. 1. Serial dilutions of 24 h conditioned medium (CM) containing 1% FCS from RME cells cultured in insuliniEGF-supplemented medium (closed circles), or in insulinidexamethasoneiprolactinsupplemented medium (closed triangles). The radioimmunoassay standard curve was obtained using synthetic human parathyroid hormone-related peptide (hPTHrP)-(1-34) (open squares). B = bound tracer with competitor. Bo = bound tracer without competitor.

Combette et al. (1990). Incubation was continued with fluorescein isothiocyanate (F1TC)-conjugated anti-rabbit or anti-mouse IgG (diluted 1:40 and l:lO, respectively) (Nordic, Tilburg, The Netherlands) a t room temperature for 45 min. In controls, the first antibody was omitted.

PTHrP production PTHrP production by RME cells was measured in 1% FCS-containing medium conditioned by the RME cells for 12 to 48 h. CM were immediately centrifuged to eliminate cell debris and stored at -80°C until analysis. PTHrP immunoreactivity in RME cell CM was estimated by competitive radioimmunoassay (RIA) performed under non-equilibrium conditions as follows: 200 pl of CM and 100 pl barbital-acetate buffer (BAB), pH 8.6, containing 0.1% BSA (bovine serum albumin, RIA grade, Sigma), or standards diluted in 200 pl of the same medium, were incubated in duplicate a t 4°C with 100 pl of rabbit anti-human PTHrP-(1-34) [hPTHrP-(134) antibodies (Peninsula Lab. Inc., Belmont, CA)] for 24 h. Synthetic hPTHrP-(1-34) (kindly provided by Drs. B. Kemp and T.J. Martin, Melbourne, Australia) was radiolabeled by a chloramine-T procedure and purified on C18-SepPak cartridge column. Approximately 10,000 cpm of the radiolabeled tracer was added in a volume of 100 p1 for a n additional 24 h at 4°C. Sheep anti-rabbit IgG antiserum was used for the separation of the bound and free fractions. Standard synthetic hPTHrP-(1-34) ranged between 1.6 and 200 pgitube. Under these conditions, the lower detection limit of our assay was 2 pg PTHrPitube, non-specific binding lower than 1.5%, and half-maximal displacement occurred with about 20 pg/tube. One percent FCS-containing unconditioned medium incubated with the collagen gel alone, with or without the various hormone supplements, did not displace the bound tracer. Serial dilu-

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FERRARI ET AL

tions of CM samples from RME cells provided values that paralleled the standard curve (Fig. 1).Similar dilution curves were obtained using rat milk or CM from cultured rat Leydig tumor cells, which are known to produce PTHrP.

Adenylate cyclase-stimulating activity PTH-like bioactivity was assessed by measuring the cyclic adenosine monophosphate (CAMP) stimulation induced by CM from RME cells or various mammary cell lines in the PTH-responsive osteoblast-like cell line UMR-106 (Rizzoli and Bonjour, 1989). UMR-106 cells were seeded a t a density of 1.3 x lo4 cells/cm2 in 2 cm2 wells and cultured in Eagle’s minimal essential medium (MEM) supplemented with 10% FCS for 6 days. At confluence, CAMPproduction was measured in intact cells prelabeled with [”]-adenine (1.2 pCiiwel1) in MEM with 2% FCS a t 37°C for 2 h. After a 10 min incubation at 37°C in 150 pl HEPES-buffered 1%FCScontaining MEM with 1 mM isobutylmethylxanthine, 300 p1 of conditioned or control media were added for another 10 min incubation. Supernatant was then discarded and intracellular [3H]-cAMP extracted a t 4°C with 5% trichloracetic acid containing a s carrier 2 mM adenosine, 2 mM adenosine triphosphate (ATP), 2 mM CAMP, and 2 mM adenosine monophosphate (AMP). After neutralization with KOH, [‘HI-cAMP was isolated by sequential chromatography on Dowex AG 50 W-X4 and alumina (Salomon, 1979). Recovery of CAMP was assessed by adding 2,000 cpm [l*C]-cAMP and was found to be 70-85%. The results were expressed as cpmi dish.

than 80%, as estimated by trypan blue exclusion. Plating density varied from 0.5 x lo3organoidsi35 mm culture dish (low plating density), to 1.5 x lo3 organoidsi dish (high plating density), with a n intermediate group a t 1 x lo3 organoidsidish (intermediate plating density). After 48 h in culture, the percentage of seeded organoids adherent to the collagen gels was greater than 50%,whatever the plating density. Plating density had a major influence on the rate of cell proliferation. In the presence of insulin and EGF, we observed a phase of rapid growth occurring from day 6 after seeding only in high plating density cultures, which reached confluence by day 9. Removal of growth medium supplements from high plating density cultures, or lowering cell seeding density, prevented this rapid growth phase. As a consequence, cell number did not change substantially over a 12 day period in these cultures (data not shown).

Phenotypical characterization of RME cells In order to ascertain the epithelial phenotype of the mammary cells isolated and cultured, the cells were examined by phase-contrast and immunofluorescent microscopy. Phase-contrast microscopy of RME cells cultured under the conditions defined above revealed three major morphological cell types: 1) rare isolated clusters of small cuboidal cells with a low propensity to diffuse; 2) abundant large flattened cells spreading on the surface of the gels; and 3) thin elongated cells slowly growing in the depth of the gels that gave a stellate appearance to early colonies. In high plating density cultures, large flattened epithelioid cells fiCell growth nally formed a confluent monolayer, whereas thinner Cell growth was assessed by estimating total number elongated cells built a three-dimensional branching of cell nuclei per dish a t various times in culture (Eth- pattern forming ductular-like structures (Fig. 2a). ier et al., 1987). Collagen gels were dissolved in 25% Fluorescent immunostaining of high plating density (vo1:vol) glacial acetic acid, and nuclei subsequently cultures grown for 5 to 10 days on glass coverslips indiisolated according to the method of Butler (1984). cated that organoids, as well as more than 95% of the Briefly, the cell pellet was resuspended in 500 pl 0.01 cells growing out of organoids isolated from the Percoll M Hepes buffer, pH 7.4, with 1.5 mM MgC1, for 5 min density fraction higher than 1.055 giml (both the small prior to the addition of 100 pl of a lysis solution contain- cuboidal and the large flattened cells), were positively ing 3%glacial acetic acid (vo1:vol)and 5% ethylhexade- stained with anti-cytokeratin antibodies (Fig. 2b,c). On cyldimethylammonium bromide (w:vol) (Fluka Chem- the contrary, neither showed any positive staining with ica, Buchs, Switzerland). Test tubes were shaken anti-a-smooth-muscle actin antibodies. In contrast, frequently for 10 to 12 min. At that time all the cells only about 50% of cells growing from the Percoll denwere lysed, as confirmed by microscopical examination. sity fraction lower than 1.055 g/ml showed positive Finally, 9 ml of a 0.05% formaldehyde in isotonic saline anti-keratin immunostaining, mostly because of the solution was added, and aliquots of suspended nuclei presence of numerous cytokeratin-negative, spindlecounted with a Coulter Counter (Coulter Electronics shaped, fibroblast-like cells (Fig. 2d). This fraction was Ltd, Beds., Luton, England). consequently considered as a mixed mammary cell population, and discarded for subsequent investigations of Statistics PTHrP production by breast epithelial cells. Results were obtained from a t least triplicate deterPTHrP production by RME cells minations repeated in two to eight separate series of experiments. Values are expressed as mean ? SEM. A We investigated whether RME cells produced PTHrP two-tailed Student’s t-test for unpaired values was used by measuring immunoreactive PTHrP released in the and P < 0.05 was taken as the minimal level of statisti- 24 h 1% FCS-containing CM at day 7 after plating. As cal significance. shown in Figure 1,serial dilutions of RME cell CM gave values of immunoreactive PTHrP that paralleled the RESULTS RIA standard curve, whatever the growth medium Rat mammary epithelial cells isolation used. and growth Time course experiments of PTHrP accumulation in A typical isolation yielded 2.5 x lo4 organoids per RME cell CM showed that PTHrP was already detectrat (range 1.5 x lo4 to 4 x lo4),with a viability higher able after 12 h of incubation, and peaked at 24 h, with-

PTHrP PRODUCTION BY MAMMARY EPITHELIAL CELLS

307

Fig. 2. a: Phase-contrast micrographs ( x 100) of insuliniEGF-supplemented high plating density cultures at day 8 after seeding, showing spreading of large epithelioid cells, as well as ductular-like structures; bd:Micrographs (X400) of cytokeratin fluorescent immunostaining showing positive staining in small cuboidal cells (b),and large flattened epithelioid cells (c). but lack of fluorescence in spindle-shaped, fibroblast-like cells (d, arrow).

out any further apparent accumulation of the peptide up to 48 h (data not shown). Thus, a 24 h incubation time was used in subsequent experiments. Moreover, the 2.5 times higher PTHrP concentration measured in CM a t 24 h a s compared to 12 h was not accompanied by a n equivalent increase of cell number. The bioactivity of PTHrP produced by RME cells was evaluated by assessing PTH-like CAMP-stimulating activity in 24 h CM. RME cells CM induced a mean 61.3 _t 9% increase in cAMP production by the PTHresponsive osteoblast-like cell line UMR-106 (n = 8 separate experiments performed in quadruplicate, P = 0.02). For comparison, 24 h CM from HC-11 mouse mammary epithelial cells, grown in the same insulin/ EGF-supplemented medium as RME cells (Ball et al., 1988),&-from MDA-MB-231 human breast carcinoma cells. did not contain anv activitv that stimulated production in UMR-io6 cells'"whereas HBL-lOO human breast Only CAMPproduction (33.5 _t 11.5%,n.s.1 (Fig. 3). Influence of culture conditions on PTHrP production Many cellular processes are modulated by the State of cell proliferation and/or differentiation. This could also

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Fig. 3, PTH-like bioactivity in 24 h conditioned medium (CM) from various mammary epithelial cell lines as well as primary cultures of rat mammary epithelial (RME)cells. HC-11, mouse mammary epithelial cells; MDA-MB-231, human breast carcinoma cells; HBL-100, human breast epithelial cells. PTH-like CAMP-stimulating activity of each CM was measured in PTH-responsive UMR-106 osteoblast-like cells, and expressed as percent of basal cAMP production. Results are mean -t sEM of two to eight experiments performed in quadruplicate. * P < 0.05.

FERRARI ET AL.

308

TABLE 1. Influence of dating density on PTHrP production by RME cells' Plating density Low Intermediate Hieh

PTHrP production (pg/105 cells x24 h)

Range of PTHrP concentrations (pg/ml CM)

157.0 k 2.0 97.0 k 14.5** 25.0 k 1.5***

49.0 f 5.5 to 117.0 f 11.5 106.0 f 13.5 to 135.0 25.5 21.0 i 1.0 t o 64.0 3.5

**

IThe results are mean f SEM of 3-4 separate experiments using RME cells seeded at different densities, and cultured in insulin/epidermal growth factor (EGP)-supplementedmediumfor 7 days. Immunoreactive PTHrP was measured in 24 h CM and normalized for cell number. CM, conditioned medium; PTHrP, parathyroid hormone-related protein; RME, rat mammary epithelial. The range of PTHrP concentrations shows, for each plating density used, the extreme mean values k SEM of triplicate determinationsperformedinseparateexperiments.**P