Are MG-63 and HOS TE85 human osteosarcoma cell lines representative models of the osteoblastic phenotype?

Bone, Vol. 15, No. 6, pp. 585-591. 1994 Copyright 6 1994 Elsevier Science Ltd Printed in the USA. All tights reserved

Pergamon

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Are MG-63 and HOS TE85 Human Osteosarcoma Cell Lines Representative Models of the Osteoblastic Phenotype? J. CLOVER*

and M. GOWENf

Bone Research Unit, Bath Institute for Rheumatic Diseases, Trim Bridge, Bath BAI IHD, and University of Bath BAl IHD, Avon, UK Address for correspondence and reprints: J. Clover, Glaxo Group Research,

Building 40, Greenford

Road, Greenford,

Middlesex

UB6 OHE, UK.

TE85 cells were not very representative of bone ceU cultures and therefore neither cell line is ideally suited to studying these aspects of osteoblast function.

Abstract The aim of this study was to perform a systematic comparison of two widely used osteosarcoma cell lines and ascertain their relevance as experimental models for investigating osteoblast function. We have therefore compared growth, differentiated cell function, i&grin expression and adhesive profdes of MG-63, HOS TESS, and human bone derived cells. Both &eosarcoma cell lines proliferated more rapidly than osteoblast-like cells with HOS cells exhibiting the shortest doubling time. HOS cells expressed higher levels of alkaline phosphatase than MG-63 cells under basal conditions but only MG-63 cells showed the increased enzyme activity following 1,25diiydroxyvitamin D, (1,25(0&D,) administration, which is characteristic of bone derived cells. Osteocalcin was not detected in supernatants from any cells under basal conditions but levels produced by MG-63 cells on addition of 1,25(OH),D, were comparable with those of o&oblast-like cells. oil, 012,013,a5, CUV,and Bl hntegrin subunits were detected on all cells and there was no staining for aL, c&4, $2, and p3. 013 and pl were the major subunits detected on MG-63, HOS, and bone derived cells but relative concentrations of other Q subunits were dependent on ceU type; cx4 and a6 subunits could only be detected on osteosarcoma cell lines. Short term, serum-free ceU adhesion assays showed that the three ceU types adhered in a saturable manner to collagen I, flbronectin, and lamhdn. Maximal adhesion of 6smma cell lines and human bone derived cells was observed after 2 hours and similar extracellular matrix protein coating concentrations were required by the three ceU types to achieve optimum binding: maximal adhesion to collagen I and Ubronectin occurred at 0.1-0.5 pg/ cm”, and maximal adhesion to lami& occurred at 5-10 pg/ cm2. These studies indicate that both osteosarcoma cell lines provide appropriate models for studying integrin subunit expression and cell adhesion. In addition, MG-63 cells are also suitable for investigating regulation and production of osteocalcin by human osteoblast-like cells. Proliferation and alkaline phosphatase activity exhibited by both MG-63 and HOS

Key Words: MG-63-HOS-Osteoblast-Bone-IntegrinAdhesion.

Introduction

Advances in bone cell research have been hampered by the availability of suitable in virro model systems. In vitro studies of osteoblast behaviour utilise either osteoblast-like cells derived from animal and human bones (Beresford et al. 1984, 1986; Gehron Robey & Termine 1985) or osteogenic osteosarcoma cell lines that may also be of animal or human origin (Rodan et al. 1988; Thavarajah et al. 1985). Widespread use of all of these culture systems has frequently resulted in divergent responses to the same osteotropic agents being observed. One explanation for the variability is differences in responsiveness of osteoblast-like cells of different species. Furthermore, it is not always possible to extrapolate effects on osteosarcoma cell cultures with those of bone cell cultures because osteosaroma cells possess abnormal growth characteristics (hence their transformed state) and bone cell cultures contain heterogeneous cell populations comprised of cells at several stages of maturation. Despite these limitations, human osteosarcoma cell lines can contribute to our understanding of osteoblast function because they initially represent clonal populations derived from specific stages of the osteoblast lineage. However, in order that these cell lines produce data of physiological relevance, it is important that different osteosarcoma cell lines are extensively characterized and the cell line whose particular function most closely resembles that exhibited by human osteoblast-like cells is chosen for investigation. Cell lines that possess different characteristics can then be used for comparative study to ascertain the significance of a particular cellular function. The aim of this.study was to compare the functions of two human osteosarcoma cell lines, namely MG-63 and HOS TE8.5 cells, with human osteoblast-like cells to assess their relevance as experimental models for studying particular aspects of osteoblast function. MG-63 cells were originally derived from an osteogenic sarcoma of a lCyear-old male (Heremans et al. 1978)

*Resentaddress: Glaxo Group Research, Building 40, Greenford Road, Greenford, Middlesex UB6 OHE, UK. Wresent address: Smith Kline Beecham Pharmaceuticals, Department of Cell Science, 709 Swedeland Road, P.O. Box 1539, King of Prussia, PA 19406, USA. 585

586

and HOS TE85 cells were from a sarcoma of a 13.year-old female (McAllister et al. 1971). MG-63 cells are considered to show a number of features typical of an undifferentiated osteoblast phenotype. This includes the synthesis of collagen types I and III, a low basal expression of alkaline phosphatase which is increased following 1,25dihydroxyvitamin D, (I .25(OH),D,) administration, and production of osteocalcin in the presence of 1,25(OH),D, (Franceschi & Young 1990; Lajeunesse et al. 1990). These cells have been used as an experimental model to study a variety of different osteoblast functions such as adhesion (Dedhar et al. 1987; Franceschi et al. 1987; Heino & Massague 1989), extracellular matrix (ECM) synthesis (Bassols & Massague 1988; Franceschi et al. 1988), alkaline phosphatase activity (Boyan et al. 1989; Franceschi et al. 1985; Franceschi & Young, 1990), and osteocalcin production (Lajeunesse et al. 1990). HOS cells have not been as widely used as MG-63 cells despite their higher basal expression of alkaline phosphatase. These cells have been previously employed for studying the effects of estrogen (Komm et al. 1988) and for the localization of estrogen and androgen receptors (Benz et al. 1991; Komm et al. 1988). Other experiments concerning the isolation and purification of insulin-like growth factor (IGF) binding proteins (Hassager et al. 1991; Mohan & Baylink 1991; Lempert et al. 1991) have also been performed. The complicated process of bone remodeling results from the coordinated actions of osteoblasts and osteoclasts and is regulated systemically by hormones and locally by soluble and insoluble mediators. The ECM may be considered as an insoluble local mediator that plays an important role in regulating cell function. For example, it has been shown to exert a number of different effects in non-bone cell types (Kosher & Church 1975; Lee et al. 1985) and is also concerned with the presentation of cytokines and other growth factors to neighboring cells (Nathan & Sporn 1991). Information contained within the ECM is translated to the cell in part by the integrins, which are a family of transmembrane proteins composed of noncovalently linked a and B subunits. In this study, we have sought to ascertain an osteosarcoma cell line for studying specific aspects of human osteoblast function. We have therefore compared growth, differentiated cell function, integrin expression, and adhesive profiles of MC-63, HOS TE85 and human bone derived cells. Differentiated cell function was assessed by measuring alkaline phosphatase activity and osteocalcin production, markers that are representative of early and late stages of osteoblast maturation. Adhesive properties were assessed by comparing binding of the three cell types to collagen I, fibronectin, and laminin. Collagen I is the most abundant component of the bone matrix and fibronectin is present around osteoblasts during osteogenesis (Weiss & Reddi 1980, 1981). Laminin is a major component of endothelial cell basement membranes. Because angiogenesis is a prerequisite for bone formation (Froidart & Reddi 1980; Trueta 1963) and osteoprogenitor cells are in contact with basement membranes of the invading vascular system, it has been suggested that this ECM component may play a role in regulating bone cell function (Vukicevic et al. 1990). Results show that neither cell type displays characteristics identical to that observed in human osteoblast-like cells. However, both osteosarcoma cell lines provide appropriate models for studying integrin subunit expression and cell adhesion. In addition, MG-63 cells are also suitable for investigating regulation and production of osteocalcin by human osteoblast-like cells. Proliferation and alkaline phosphatase activity exhibited by both MG-63 and HOS TE85 cells were not very representative of bone cell cultures and therefore neither cell line is ideally suited to studying these aspects of osteoblast function.

J. Clover and M. Gowen: Osteosarcoma cell lines and osteoblast phenotype Materials and Methods Materials

MG-63 cells were obtained from the European collection of animal cultures (Salisbury, UK) and HOS TE85 cells were purchased from the American Type Culture Collection (Rockville, MD). Monoclonal antibody (MAb) T5217 recognizes the al (CDw49a) subunit of integrin (Y1B 1 and was generously donated by Dr. Hemler. MAbs raised against a3 (CDw49c), a6 (CDw49t), and Bl (CD29) subunits were purchased commercially from Bioquote Ltd. (West Yorkshire, UK), Serotec (Oxfordshire, UK), and Coulter Instruments (Beds, UK) respectively. MAbs directed against a2 (CDw49b), u4 (CDw49d) and a.5 (CDw49e), CWL(CDlla), and B2 (CD18) subunits were obtained from Immunotech (Birmingham, UK) and a MAb directed against the crM (CD1 lb) subunit was purchased from Dakopatts (High Wycombe, UK). MAb 23C6 which recognizes the o,V (CD51) subunit was generously donated by Dr. Horton (London, UK) and MAb C22 which recognizes the B3 subunit (CD61) was raised in our laboratory (James et al. 1991). All MAbs were obtained from ascites of hybridoma nulnu mice or tissue culture supematants. All FITC-conjugated immunoglobulins were obtained from Sigma Chemical Co. (Dorset, UK) and standardized FITC-coated latex beads were purchased from Flow Cytometry Standards Corp. (Research Triangle Park, NC). Human plasma fibronectin and mouse EHS laminin were obtained from Sigma Chemical Co. Collagen type I was isolated from rat tails as described previously (Strom & Michalopoulos 1982). Osteocalin products were obtained from Biogenesis (Boumemouth, UK) and ‘251 was purchased from Amersham (Bucks, UK). Protein dye reagent was obtained from Biorad Laboratories (Hertfordshire, UK) and remaining chemicals were obtained from Sigma. Tissue culture plastics were purchased from Costar (Northumberland, UK) and culture media were obtained from Gibco (Middlesex, UK). Human bone cell culture

Explants of trabecular bone were cultured as described previously (Beresford et al. 1984). Briefly, trabecular bone fragments approximately 3 mm3, dissected from femoral heads following orthopaedic surgery, were washed extensively in PBS and transferred into 9-cm tissue culture grade Petri dishes. Explants were cultured in Eagle’s minimum essential medium (MEM) containing 10% heat inactivated fetal calf serum (FCS), 100 III/ml of penicillin, 100 pg/ml of streptomycin, and 2 mM L-glutamine (growth medium), in a humidified atmosphere of 5% CO, and 95% air at 37°C. Culture medium was replaced once a week and confluency was usually achieved after 4-6 weeks. The cells obtained have been characterized previously and possess an osteoblastic phenotype. (Beresford et al. 1984, 1986; MacDonald et al. 1984). This includes the synthesis of alkaline phosphatase, osteocalcin, and type I collagen and the production of CAMP in response to PTH. MG-63

and HOS culture

Cells were maintained in MEM supplemented with 10% FCS, 100 IU/ml of penicillin, 100 kg/ml streptomycin, and 2 mM L-glutamine. Prol$erution

Confluent cultures of MC-63, HOS, and human osteoblast-like cells were plated into 24-well plates at 10,000 cells/cm’ in

J. Clover and M. Gowen: Osteosarcoma

cell

lines and osteoblast phenotype

growth medium (1 ml/well). At intervals over the next 6 days, the medium was removed, cells were washed with PBS (1 ml), and trypsin/EDTA was added (250 t&well). After cell detachment (5 min/37”C for osteosarcoma cell lines and 10-15 mitt/ 37°C for bone derived cells), trypsin was neutralized by adding 750 @well growth medium, cells were resuspended in PBS (200 111)and counted in a hemocytometer. Ten fields were counted from a total of eight wells for each time point. D$ferentiated

cell function

MG-63, HOS, and human osteoblast-like cells were seeded into 24-well plates at a density of 20,000 cells/cm’ (1 ml/well). After 24 hours, the medium was removed, cells were washed in serumfree medium, and MEM + 3% charcoal stripped FCS containing vitamin K (lo-* M) and vitamin C (50 ).r,g/ml) was replaced (500 pi/well). Some of the cells also received lo-* M 1,25(OH),D,, a dose which produces maximal induction of osteocalcin (Beresford et al. 1984) and good stimulation of alkaline phosphatase (Beresford et al. 1986). After a further 72 hours, supematants were removed and stored at - 20°C for osteocalcin assay, and cells were trypsinized and assessed for alkaline phosphatase activity: this enzyme has been shown to be resistant to the trypsinization procedures used for this experiments (not shown). Alkaline phosphatase

assay

The cellular contents of each well were divided in a ratio of 2: 1

and assayed for alkaline phosphatase activity (Bereford et al. 1986) and DNA (West et al. 1985). The larger aliquot was therefore resuspended in 200 p,l Tween-20 (0.1% in deionized water) and alkaline phosphatase activity was assessed calorimetrically using p-nitrophenylphosphate as the substrate. The smaller aliquot was resuspended in 100 pl EDTA (10 n&l, pH 12.3) and incubated for 20 min (37°C) to ensure cell lysis. Ten microliters KH,PO, was then added to reduce the pH to 7.0 followed by 16OO~lassaybuffer(lOmMNaCl, lOmMTris, lOmMEDTA, pH 7.4) and 500 pl Hoescht 33258 (final cont. 80 pg/ml). Fluorescence was then recorded using a fluorimeter (excitation wavelength 354 nm, emission wavelength 473 nm) and cellular DNA was determined from a standard curve. All treatments were performed in triplicate and enzyme activity was expressed as micromoles p-nitrophenollyg DNA/hour.

587 (1%) and human AB serum (1:5 dilution). After 90 min on ice with occasional agitation, cells were washed in dilution buffer and resuspended in the appropriate FITC-conjugated secondary antibody. After a further 30 min, cells were washed again, fixed in 1% paraformaldehyde in PBS, and passed through a Becton Dickinson Facstar Plus. The mean fluorescence intensity of all samples were then analyzed using a region set to 5% of the negative control and results were converted to mean antibody sites/cell using standardized FITC-coated latex beads (Le Bouteiller et al. 1983).

Attachment assay

Flat-bottomed plates (96-well) were coated overnight at 4°C with ECM glycoproteins, diluted in PBS. Plates were then rinsed in PBS (3 X 100 pl) and nonspecific binding to uncoated plastic was blocked by incubation for 1 hour with BSA (1 mg/ml in PBS, 100 t&well). Plates were rinsed again (3 X 100 pl) and cells were seeded into wells at 50,000 cells/cm2 in serum-free MEM (100 p&well). Therefore, cells were detached by trypsinization and enzyme activity neutralized by the addition of MEM + 10% FCS. Cells were then washed twice in serum-free. MEM to remove traces of serum, counted, and seeded into precoated wells. Cells were allowed to adhere at 37°C for up to 5 hours and then nonadherent cells were removed by washing with PBS (1 x 100 @well). Attached cells were lysed by the addition 200 $/well deionized H,O and adhesion was assessed by protein assay (Bradford 1976).

Results Proliferation

Growth curves obtained for MG-63, HOS TE85, and human osteoblast-like cells (Fig. 1) show that both osteosarcoma cell lines proliferated considerably more rapidly than bone cell cultures, with HOS cells exhibiting the shortest doubling time (21.6 hours).

1OOOOOO Osteocalcin assay

Osteocalcin released into supematants was measured by specific competitive radioimmunoassay (Beresford et al. 1984) using a polyclonal anti-bovine osteocalcin antibody and iodinated bovine osteocalcin diluted in assay buffer (0.1 M NaCl, 0.025 M EDTA, 0.01 M Tris, 0.1% Tween-20, 0.25% bovine serum albumin, pH 7.4). Antibody-osteocalcin complexes were separated from free iodinated tracer using a secondary antibody (goat anti-rabbit gammaglobulin) in the presence of 5% polyethylene glycol (MW 8000). Values for osteocalcin released into the culture media were obtained using a standard curve constructed using an osteocalcin standard. All treatments were performed three times and each sample was assayed in triplicate. FACS analysis

Suspensions of MG-63, HOS, and human osteoblast-like cells were incubated with saturating concentrations of anti-integrin MAb or IgG (negative control), diluted in PBS containing FCS

1

looo!

.,

0

.,.,.,.,., 1

2

3

4

5

6

day Fig. 1. Growth and doubling time for MG-63 (Cl), HOS TE85 (0), and human osteoblast-like cells (4). Cells were seeded into 24-well plates at 10,000 cells/cm’ and, at intervals over the next 6 days, cells were detached and counted in a hemocytometer. Values for each time point represent means and standard errors from eight wells. Doubling times: MG-63 cells, 24 hours; HOS cells, 21.6 hours; human osteoblast-like cells, 125 hours.

588

Differentiated

J. Clover and M. Gowen: Osteosarcoma

expression in response to I ,2S(OH)ZD,

Table II. lntegrin subunit expression man osteoblast-like cells

There was a large variation in basal and stimulated levels of alkaline phosphatase (Table I). Human osteoblast-like cells exhibited a high basal expression that was further induced following 1,25(OH),D, administration. MG-63 cells expressed levels that were greater than tenfold lower than those exhibited by osteoblast-like cells under basal conditions, but expression was increased approximately twofold following treatment with Induction of alkaline phosphatase by 1,25(OH),D,. 1,25(OH),D, is a well-documented response and has been described previously for human bone derived cells (Beresford et al. 1986) and MG-63 cells (Franceschi et al. 198.5; Franceschi & Young 1990; Lajeunesse et al. 1990). HOS cells exhibited a higher basal expression of alkaline phosphatase than MG-63 cells but levels were not induced by 1,25(OH),D,. This result is in agreement with previous observations (Benz et al. 1991) and has also been observed in other osteos~oma cell lines including OHS-4 and SAOS-2 (Fournier & Price, 1991). Osteocalcin was not produced in significant amounts by any cell type under basal conditions but was induced in bone cell cultures and MG-63 cells following 1,25(OH),D, administration; no osteocalcin was detected in supernatants from 1,25(OH),D,-treated HOS cells (Table I). Stimulation of osteocalcin synthesis by 1,25(OH),D, in MG-63 cells and osteoblastlike cells has been previously described (Beresford et al. 1984: Foumier & Price, 1991; Franceschi & Young, 1990; Lajeunesse et al. 1990). Integrin subunit expression lntegrin subunits expressed by MG-63, HO& and human bone derived cells were detected and quantified by FACS analysis (Table II). (~1, (~2, (~3, a5, nV, and fil could be detected on all cell types but there was no staining for crL. aM, p2, and p3 subunits. Negative staining patterns did not result from impaired functionality; aL, aM, and p2 subunits could be identified on freshly prepared lymphocytes and p3 subunits could be observed on osteoclasts (not shown). (~3 and /!l were the most highly expressed subunits in MG-63, HOS and bone derived cells, but relative concentrations of other a subunits was dependent on cell type. For example, cxl, (r2, and a5 subunits were detected at higher levels on bone cell cultures and a3 subunits were more strongly expressed by both osteosarcoma cell lines. The main differences occurred with expression of c~4 and a6 subunits. which were detected on MG-63 and HOS cells but not on bone derived cells. Adhesive properties Integrin subunit profiles exhibited by MG-63, HOS. and human bone derived cells suggested that potential ligands used for at-

Table I. Alkaline phospha~se

Alkaline phosphatase fpmol p-nit~p~enol/~g Osteocalcin (ng/ml)

activity and osteocalcin

DNA/hour)

pr~uction

cell lines and osteoblast

Integrin subunit detected

mAb

n 1 (CDw49a)

T52/?

a2 (CDw49b)

Cl9

a3 (CDw49c)

PB15

a4 (CDw49d)

HP211

a5 (CDw49e)

SAM1

a6 (CDw4Yf)

GoH3

UV fCD.51)

23C6

pl (CD29)

4B4

aL (CD1 la) aM (CDllb) p2 (CD18) p3 (CD61)

fOT16 R841 IOT18 c22

by MC-63,

phenotype

HOS TE85, and hu-

MG-63 cells

HOS cells

18,407” (767) 65,730’ (2434) 276,055’ (6900) 35,652’ (574) 19,428’ (771) 21,091 ( 1272) 13,822b (240) 573,787” (21,878)

50,859" (6067) 6675’ (775) 255,905’ (20,306) 20,048” (821) 27, 147h (4196) 3 1,097” (2537) 25,793b (2045) 386,258* (29,561)

15,561 (1701) 556,947 (26,167)

negative

negative

negative

negative

negative

negative

Osteoblast-like cells 101,023 (60773 101,866 (6707) 127,184 (5134) negative 47,544 (3735) negative

Fluorescence values obtained from FACS analysis were converted into receptor sites/cell and data obtained from a minimum of nine different samples were pooled to produce means and standard errors for integrin subunit sites/cell. Values obtained for OsteosarComa cell lines and osteoblast-like cells were compared, and the significance of any differences in integrin subunit sites/cell were assessed by unpaired Student’s r-test. “p < 0.05; bp < 0.01: “p < O.OOl_

tachment to the ECM include collagen I, fibronectin, and laminin. Therefore, short term, serum-free cell adhesion assays were performed using these ECM proteins and the adhesive properties of the three cell types were compared. Time course experiments showed that MG-63, HOS, and human osteoblast-like cells adhered to collagen I, fibronectin, and laminin. The three cell types exhibited very similar binding properties and maximal adhesion to all ECM proteins was achieved after 2 hours (not shown). In experiments where cells were allowed to attach for 2 hours to surfaces coated with increasing condensations of each ECM protein, all cell types again exhibited similar binding properties (verified by unpaired Student’s t-test). In each case, cells bound in a saturable manner with optimum adhesion to collagen I and fibronectin occurring at 0.1-0.5 p,glcm2, and maximal adhesion to laminin occurring at 5-10 p.g/cm* (Fig. 2A-C). At optimal coating concentrations, 9@--100% of cells attached to all matrices, compared with 20-40% in uncoated wells.

by MC-63,

HOS TE85, and human osteoblast-like

celIs

Treatment

MC-63 cells

HOS cells

Control I ,25(0HfZD,

0.025 (0.002)” 0.046 (0.002)”

0.113 (0.007)” 0.102 (0.015)

0.347 (0.030) 0.634 (0.035)

Control I .25(OH),D,

0 9.0 (0.82)”

0 0

0 9.76 (0.202)”

Osteoblast-like

cells

Values represent means and standard errors from three samples each of which was assayed in triplicate (i.e., n = 9) and are ~presentative of three different experiments. Values obtained for osteosarcoma cell lines and osteoblast-like cells were compared t 1,25D,and the significance of any differences in differentiated cell function were assessed by unpaired Student’s r-test. “p < 0.001.

J. Clover and M. Gowen: Osteosarcoma cell lines and osteoblast phenotype

100 % maximal

attachment

80

Collagen I (&cm*

% maximal attachment

Fibronectin

)

(&cm*

)

I go

Laminin (&cm*

)

Fig. 2. Adhesion of MG-63 (O), HOS TESS (0), and human osteoblast-like cells (4) to ECM glycoproteins: concentration dependence. Cells (50,000/cmZ) were allowed to attach to wells coated with collagen I (A), fibronectin (B), and laminin (C) (0.001-20 kg/cm’). After 2 hours, 37”C, cell attachment was assessed by protein assay. Points shown represent means and standard etmrs from six wells. Values are representative of at least three separate experiments.

Diiussion

This study compares

the osteoblastic

features of two osteosar-

coma cell lines, MG-63 and HOS TE85, with cultured human bone derived cells. Three of the main parameters commonly used to assess osteoblast function are proliferation, alkaline phosphatase activity, and osteocalcin production. Growth curves obtained for MG-63, HOS, and human bone derived cells showed that HOS cells proliferated the most rapidly, exhibiting a doubling time of 21.6 hours. Cellular alkaline phosphatase was also dependent on cell type. Bone cell cultures displayed the highest activity, which could be further enhanced by 1,25(OH),D,. HOS cells produced higher levels of alkaline phosphatase than MG-63 cells under basal conditions, but MG-63 cells behaved more similarly to human osteoblast-like cells in their response to 1,25~(0H)~D,. Osteocalcin could be detected in supematants from human bone derived cells and MG-63 cells following 1,25(OH),D, administration; no osteocalcin could be detected in supematants from 1,2S(OH),D,-treated HOS cells. In view of the likely importance of cell-matrix interactions in the local control of osteoblast function, we have also compared integrin expression and adhesive properties of MG-63, HOS, and human osteoblast-like cells. Integrin subunit profiles were similar in that a 1, a2, a3,a5, aV, and B1 subunits were identified on all cell types. In each case, a3 and pl were the most highly expressed subunits which can combine to form a3p1, a receptor which is commonly found in cells from most tissues and binds collagen fibronectin, and laminin (Humphries 1990). The most striking difference in subunit expression was observed with a4

and a6 subunits, which were detected on MG-63 and HOS cells but not on bone cell cultures. Expression of these subunits by osteosarcoma cell lines could result from an adaptation to long term culture conditions and may not have originally been detected on these cells. However, invasion of tumor cells require attachment to components of the basement membrane, degradation of the basement membrane and finally, migration to the underlying stroma. The laminin receptor, a6E 1, has previously been shown to be overexpressed in highly invasive cells (Dedhar & Saulnier 1990) and a4@1 is clearly associated with migration (Chan et al. 1992). Therefore, expression of ~14and a6 by MG63 and HOS cells could reflect the metastatic potential of osteosarcoma cells. Another variation between osteosarcoma cell lines and bone cell cultures concerned the relative concentrations of individual a subunits expressed. For example, al, cr2, and a5 subunits were detected at higher levels in bone cell cultures and a3 subunits were more. strongly expressed by both osteosarcoma cell lines. al 91 and a2Bl form receptors for collagen and laminin whereas a5pl encodes a high affinity receptor for fibronectin (Hynes 1992). A similar modulation of integrin subunit expression following oncogenic transformation has been reported elsewhere. For example, Plantefaber and Hynes (1989) demonstrated a reduction in expression of three different a subunits with oncogenic transformation, whereas expression of a3 subunits was slightly increased or unchanged. The quantitative differences in integrin subunit profiles did not grossly affect the ability of MG-63, HOS, or human bone derived cells to interact with collagen I, tibronectin, or laminin.

590

However, given the variations in oV subunit expression, it would perhaps have been interesting to compare adhesion of the three cell types to vitronectin and fibrinogen, two of the other potential ligands used by the heterodimer oVB1 (Hynes 1992). The similar ECM coating concentrations required by all cell types for maximum attachment to collagen I, fibronectin, and laminin could be explained by ligand redundancy. lntegrin profiles suggest that the three cell types expressed three collagen receptors (al B 1, a2p 1, a3B I), at least two fibronectin receptors (u3B1, aSB1, cwVBl), and at least three laminin receptors (alB1, a2B1, cu3Bl). Therefore, a change in the expression of one of these receptors could easily be masked by another integrin molecule binding to the same ligand. The situation is made more complex by differences in ligand affinity. For example, the heterodimer a5Bl is considered to be of a higher affinity nature than a3Bl (Plantefaber & Hynes 1989). Therefore, it is the overall cell surface expression of integrin subunits that determines the adhesive properties of a given cell type. The integrins are concerned with regulating cell phenotype by transmitting information between the external environment and the interior of the cell. In addition to controlling cell adhesion, the integrins can also regulate many other diverse aspects of cell phenotype including gene induction (Dhawan et al. 199 1; Seftor et al. 1992; Werbet al. 1989), phagocytosis (Graham et al. 1989; Savill et al. 1990), gel contraction (Schiro et al. 1991), and migration (Chan et al. 1992). Therefore. it is possible that the differences in integrin subunit expression exhibited by MC-63, HOS, and osteoblast-like cells are associated with other phenotypic effects yet to be established. In conclusion, proliferation, differentiated cell function, and integrin expression exhibited by osteosarcoma cell lines are different from those observed in human osteoblast-like cells. One explanation for these discrepancies concerns the heterogeneity of bone cell cultures. Integrin subunit expression, together with alkaline phosphatase and osteocalcin detected in bone cell cultures, represent a mean expression of the variously differentiated cells present. For example, histochemical staining has revealed that only some cells within bone cell cultures express this alkaline phosphatase (or osteocalcin) even after treatment with 1,25(OH),D, (Thavarajah et al. 1985). Similar heterogeneous staining patterns were also observed in situ (Clover et al. 1992) for integrin subunit expression. If osteosarcoma cells are derived from clonal populations of cells from the osteoblastic lineage, then cell lines with different phenotypic characteristics are likely to arise. Heterogeneity of bone cell cultures is unlikely to solely account for the different characteristics observed between osteosarcoma cell lines and bone derived cells. The process of malignant transformation together with long term culture conditions will affect the regulatory constraints exposed to a cell and this in turn will alter cell phenotype (Albelda & Buck 1990). For example, there are known to be changes in integrin subunit expression accompanying malignant transformation: some B 1 integrins are no longer expressed on RSV transformed cells (Plantefaber & Hynes 1989) and a downregulation of some of the Bl integrins has been reported in basal cell or squamous carcinoma of the skin (Peltonen et al. 1989). For these experiments it can be seen that there are numerous differences between osteosarcoma cell lines and human osteoblast-like cells. However, providing this is taken into consideration, these cells can be used as valuable tools for investigating specific aspects of bone cell function. The integrin profiles of osteosarcoma cell lines and bone cell cultures suggest that either MG-63 or HOS cells would provide suitable models for studying the expression of those integrin subunits detected on osteoblastlike cells. In addition, attachment properties exhibited by the

J. Clover and M. Gowen: Osteosarcoma

cell lines and osteoblast

phenotype

three cell types suggest that both MG-63 and HOS cells would be appropriate for investigating one of the potential consequences of any changes in integrin expression. MG-63 cells would also be appropriate for studying the regulation and production of osteocalcin by osteoblasts. In contrast, proliferation and alkaline phosphatase activities exhibited by MG-63 cells and HOS cells were not very representative of bone cell cultures. Therefore, neither of these cell lines is suitable for investigating these aspects of osteoblast function.

Acknowledpwnrs:

The authors thank Dr. Hemler and Dr. Horton for generous monoclonal antibody donations. This work was supported by Glaxo Group Research, UK and the Nuffield Foundation. their

References Alhelda,

S. M.;

Buck, C. A. lntegrins

J. 4:2868-2880: Bass&

A.; Massague,

I. Transformmg

and structure of extracellular Chem.

cans J. B&l. Benr,

D. J.: Haussler

affinity

263:303%3045;

1. N.. Gallagher, by

Brresford.

M.

A.: Speelman.

regulation

cells. Endocrinology

hone

parathyrold

5:229-234:

in

cells

hormone

Boyan.

J. N.: Gallagher,

B. D

(ROS

; Schwartz.

1712.8,

Biol.

Chrm.

Bradford,

M.

vitro.

1991.

R. G. G. Productton

Effects

responsive

MC-63

R. G. G.

Mrrub.

119:177&1785;

L. F.; Swain,

alkaline

and MC3T3)

261:11X7%-1

and growth

cartilage

the principle

of protein-dye

cellular

functions

domains.

mediated

S

cell lines J.

cells in culture.

of microgram

binding.

by different

Cell 68:1051-1060;

Anal.

Bio-

R. A.; Gowen.

M.

Integrin

; Argraves.

W. S.: Suzuki,

Human

osteosaxoma

peptide

overproduce

subunit expression

S.; Ruoslahti,

E.;

cells resistant to detachment the lihronectin

receptor.

T. S.; Hem-

VLA

integrin

a

1992.

teohlasts and osteoclasts in siru and in culture. J. Ceil Sci. Dedhar.

of I .25-

in osteoblast

1976.

E. Distinct

Dodds,

I

1986.

L. D. Localisation

phosphatase

D, type

1886; 1989.

of protein utilising

subunit cytoplasmic I.:

Dis.

phosphatase,

Chan. B. M. C.; Kassner P. D.: Schiro. J. A.: Byers, R. H.; Kupper,

Clover,

Bone

I ,25 Dihydroxyvitamin

Effects of alkaline

Endocrinolqqy

Z.: Bonewald.

D,

72~248-254;

ler. M.

of

I ,25(OH),D,.

of

M. A rapid and sensitive method for the quantitation

quantities chrm.

acid levels in human

128:2723-2730;

and glucocorticoids.

; Russell,

J. A

and proliferation.

dihydroxyvitamin

B. S. High

and transforn-

1984.

and human bone derived cells in viva: collagen

B.; Komm,

of ul (I)-procollagen

J. A.; Poser, J W.: Russell,

human

24,25(OH),D,, Res.

sulphate proteogly-

1988

M. R.; Thomas.

osteosarcoma

osteocalcin Rel.

factor p regulates the expressmn

chondroitinidermatan

factor p steady state messenger ribonucletc

osteoblast-like Beresford.

growth

matrix

binding and androgenic

mg growth

FASEB

and other cell adhesion molecules.

1990.

by human OS-

103:267-271,

1992.

Pierschhacher,

M. D.

by arg-gly-asp

J. Cell

Biol.

containing

105:1175-1182;

1987 Dedhar.

S.. Saulmer,

transformed

R. Alterations J. Cell

ceptor complexes. Dhawan.

I.: Lichtier,

pro-u]

Chem.

Foumier.

Biol.

mRNA

Franceschi.

of growth,

Chem.

262:4165-4171; R. T.;

Zerlauth,

Romano.

263:1893%18945;

Franceschi.

R. T

;

Young,

dihydroxyvitamin Res. Froldart.

D,

spe-

in a human osteosar-

1985. P. R. Regulation

of cel-

D,. J. Biol.

synthesis by la25-dihydroxyvitamin

1987. P. R.;

Park,

K. Y.

Regulation

of type 1 collagen

D, in human osteosarcoma

cells. J. Biol.

1988. J. Regulation

of

alkaline

D, and ascorbic acid in bone

5:1157-1167;

1990.

J. M.:

A. H. lmmunofluorescent

Reddi,

cell line OHS-4.

la,25-dihydroxyvitamm

123:401-409;

synthesis by 1,25-dihydroxyvitamin Chem.

G.

and fihronectin

C. J.; Peter, T. C.; Romano,

lular adhesion and fibronectin

Franceschi,

of a new osteosarcoma

morphology

Physiol.

R. T.; Linson,

J

in mouse tibrohlasts.

1991.

R. T.; James, W. M.;

coma cell line. J. Cell Francaschl.

re-

1991.

114:577-583;

cific regulation

on chemically

and collagen

S. R. Cell adhesion regulates

and transcription

B.; Price, P. A. Characterisation

J. CeN Biol.

of laminin

1990.

D. W.: Farmer,

stability

266:847%-8475:

subunit expression

enhancement

110:481-489:

; Rowe,

A. C

(I) collagen

Biol.

in mtegrin

human cells: Specific

phosphatase

by

derived cells. J. Bone

localisation

1.25. Miner.

of type IV collagen

J. Clover and M. Gowen: Osteosarcoma

cell lines and osteoblast

phenotype

and laminin during endochondral bone differentiation and regulation by pituitary growth hormone. Dev. Biol. 75:130-136; 1980. Gehron Robey, P.; Termine, 1. D. Human bone cells in virro. Calcif. Tin. Im. 3’1:45%460; 1985. Graham, I. L.; C&sham, H. D.; Brown, E. J. An immobile subset of plasma membrane CDlIb/CDlS (Mac-l) is involved in phagocytosis of targets recognised by multiple receptors. J. fmmunol. 1422352-2358; 1989. Hassager, C.; Spencer, E. M.; Fitzpatrick. L. A.; Riggs, B. L.; Conover, C. A. Insulin-like growth factor binding protein secretion in osteoblast-like cells is cell line specific [abstract]. J. Bone Miner. Res. L(Suppl. l):Sl46; 1991. Heino, J.; Massague, J. Transforming growth factor p switches the pattern of integrins expressed in MC-63 human osteosarcoma cells and causes a selective loss of adhesion to laminin. J. Biol. Chem. 264:218&21811; 1989. Heremans, H.; Billiau, A.; Cassiman, J. J.; Mulier, J. C.; Desomer, P. In vitro cultivation of human tissues II. Morphology and virological characteristics of three cell lines. Oncology 35:246-252; 1978. Humphries, M. J. The molecular basis and specificity of receptor-ligand interactions. J. Cell Sci. 97:585-592; 1990. Hynes, R. 0. Integrins: Versatility. modulation and signalling in cell adhesion. CeN 69:l l-25; 1992. James, I. E.; Walsh, S.; Dodds, R. A.; Gowen, M. Production and characterisadon of osteoclast selective monoclonal antibodies that distinguish between multi-nucleated giant cells derived from different human dssues. .I. Histochem. Cytochem. 39:905-914; 1991. Komm, B. S.; Terpening, C. M.; Benz, D. J.; et al. Oestrogen binding, receptor mRNA, and biological response in osteoblast-like osteosarcoma cells. Science 241:81-83; 1988. Kosher, R. A.; Church, R. L. Stimulation of in vitro chondrogenesis by procollagen and collagen. Nature 258:327-330; 1975. Lajeunesse, D.; Frondoza, C.; Schoffield, B.; Sacktor, B. Osteocalcin secretion by human osteosarcoma cell lines MG-63. J. Bone Miner. Res. 5:915-922; 1990. Le Bouteiller, P. P.; Mishal, Z.; Lemonnier, R. A.; Kourilsky, F. M. Quantiticadon by flow cytofluorimetq of HLA class I molecules at the surface of murine cells transformed by cloned HLA genes. J. Immunol. Meth. 61:301-315; 1983. Lee, E. Y.-H.; Lee, W.-H.; Kaetzel, C. S.; Parry. G.; Bissell, M. J. Interactions of mouse mammary epithelial cells and collagen substrata: Regulation of casein gene expression and secretion. Proc. Natl. Acad. Sci. USA 82:141%1423; 1985. L-empert, U. G.; Bautista, C.; Strong, D. D.; Baylink, D.; Mohan, S. Purification of a novel human bone derived insulin-like growth factor binding protein (huBP-IGFBP): a potential candidate for fixing IGF II in bone [abstract]. J. Bone Min. Res. 6(Suppl. l):S250; 1991. MacDonald, B. R.; Gallagher, J. A.; Ahnfelt-Ronne, I.; Beresford, I. N .; Gowen, M.; Russell, R. G. G. Effects of bovine parathyroid hormone and 1.25. dihydroxyvitamin D, on production of prostaglandins by cells derived from human bone. FEBS Lea. 169:49-52; 1984. McAllister, R. M.; Gardner, M. B.; Greene, A. E.; Bradt, C.; Nichols, W. W.; Landing, B. H. Cultivation in vitro of cells from a human osteosarcoma. Cancer 2239742; 197 1.

591 Mohan, S.; Baylink, D. Purification of IGF binding proteins from serum free conditioned medium from HOS TE85 osteosarcoma cells [abstract]. J. Bone Min. Res. 6(Suppl. l:)S141; 1991. Nathan, C.; Spom, M. Cytokiies in context. J. Cell Biol. 113:981-986; 1991. Peltonen, J.; Larjava, H.; Jaakola, S.; et al. Localisation of integrin receptors for fibmnectin, collagen and laminin in human skin. J. Clin. Invest. 84:1916 1923; 1989. Plantefaber, L. C.; Hynes, R. 0. Changes in integrin receptors on oncogenically transformed cells. Cell 56:281-290, 1989. Rodan, G. A.; Heath, J. K.; Yoon, K.; Noda, M.; Rodan, S. 8. Diversity of the osteoblast phenotype. Cell and molecular biology of vertebrate hard tissues. Ciba Foundorion Symp. 136:78-91; 1988. Savill, J.; Dramfield, I., Hogg, N.; Haslett, C. Vitronectin receptor mediated phagocytosis of cells undergoing apoptosis. Nature 343:17&173; 1990. Schiro, J. A.; Chan, B. M. C.; Roswit, W. T.: et al. Integrin a2Pl (VLA-2) mediates reorganisation and contraction of collagen matrices by human cells. Cell 67:403-410; 1991. Seftor, R. E. B.; Seftor, E. A.; Gehlson, K. R.; et al. Role of the a,& integrin in human melanoma cell invasion. Proc. Nat[. Acad. Sci. USA 89:1557-1561; 1992. Strom, S. C.; Michalopoulos, G. Collagen as a substrate for cell growth and differentiation. Cunningham, L. W.; Frederiksen, D. W., eds. Methods in enzymology. StrucUral and conwacrile proteins. Parr A. Vol. 82. New York: Academic Pres; 1982; 544-555. Thavarajah, M.; Evans, D. B.; Russell, R. G. G.; Kanis, 1. A. Immunocytochemical demonstration of osteocalcin in human bone derived cells [abstract]. Calcf. Es. Int. 383516510; 1985. Trueta, J. The role of the vessels in osteogenesis. J. Bone Join? Surg. 45B:402411; 1963. Vukicevic, S.; Luyten, F. P.; Kleinman, H. K.; Reddi, A. H. Differentiation of cannalicuhu cell processes in bone cells by basement membrane matrix components. Regulation by distinct domains of laminin. Cell 63437-445; 1990.

Weiss, R. E.; Reddi, A. H. Synthesis and localisation of fibronectin during collagenous matrix mesenchymal cell interaction and differentiation of cartilage and bone in viva. Proc. Nad. Acad. Sci. USA 77:2074-2078; 1980. Weiss, R. E.; Reddi, A. H. Appearance cartilage and bone marrow. J. Cell Werb, Z.; Tremble, P. hi.; Behrendsten, transduction through the fibronectin ysin gene expression. J. Cell Biol.

of fibronectin during the differentiation Biol.

88~630-636;

of

1981.

0.; Crowley, E.; Damsky, C. H. Signal receptor induces collagenase and stromol-

109:877-889; 1989. West, D. C.; Sattar, A.; Kumar, S. A simplified in situ solubilisation procedure for the determination of DNA and cell number in tissue cultured mammalian cells. Anal.

Biochem.

147:289-295;

1985.

Dare Received: Date Revised: Date Accepted:

June 30, 1993 January 7, 1994

February

1, 1994