[CANCER RESEARCH 42, 4771-4775, November 1982]0008-5472/82/0042-OOOOS02.00

Sensitivity of Cultured Human Osteosarcoma and Chondrosarcoma Cells toRetinole Acid1

Rafael Them2 and Reuben Lotan3

Department ol Biophysics, The Weizmann Institute of Science, Rehovot 76100, Israel

ABSTRACT

The ability of retinoic acid (RA) to inhibit the growth of threecell lines (Te85, Hs781, and Hs791) derived from humanosteosarcomas and two cell lines (Hs705 and Hs819) derivedfrom human chondrosarcomas was studied in culture. Theexposure to 1CT5 M RA resulted, within 4 days, in changes inboth cell morphology and cell growth. RA-treated cells ap

peared flat and spread on the substratum more than untreatedcells, their exponential growth rates decreased, and their saturation densities were markedly reduced. All these effectscould be reversed by removal of RA from the growth medium.The various cell lines exhibited differential susceptibility to thegrowth-inhibitory effect of RA. The most sensitive was theHs705 Chondrosarcoma. The proliferation of these cells wasinhibited 50% by 10~9 M RA and was completely blocked by10~5 M RA. In contrast, the concentrations of RA required for50% inhibition of Hs791, Te85, Hs819, and Hs781 were 10~7,2 x 10~7, 2.5 x 10~7, and 2 x 10~6 M, respectively. Only the

Te85 and the Hs781 osteosarcoma cells and cells derived froma Chondrosarcoma biopsy were able to form colonies in asemisolid medium, and this growth was dramatically inhibitedby RA. These results demonstrate that RA can suppress inthese mesenchymal tumor cells the expression of morphological and growth properties frequently associated with transformed cells.

INTRODUCTION

An increasing number of studies has demonstrated thatretinoids, a group of natural and synthetic vitamin A analogs,can exert profound effects on fundamental cellular processessuch as the growth and the differentiation of normal, transformed, and tumor cells in vivo and in culture (for recentreviews, see Refs. 3, 6, 10, 23, 30, and 42). The remarkableability of retinoids to inhibit tumor cell growth (8, 9, 17, 22-27,

32) and to enhance the differentiation of certain malignant cells(5, 10, 23, 25, 26, 41) has led to their consideration aspotential noncytotoxic antitumor agents (3, 23, 30, 31).

Although initial studies with several transplantable tumors ofexperimental animals failed to demonstrate any inhibitory effects of RA4 on tumor growth (2), subsequent studies with other

tumor systems yielded positive results. Thus, significant therapeutic effects of retinoids have been demonstrated againstmouse tumors such as a melanoma (11), a mammary carci-

1Supported by a grant from the United States-Israel Binational Science

Foundation, Jerusalem, Israel.2 Present address: Department of Orthopaedic Surgery, Kaplan Hospital,

Rehovot, affiliated with the Medical School of the Hebrew University and Had-assah, Jerusalem, Israel.

3 To whom requests for reprints should be addressed.4 The abbreviation used is RA, ¿S-all-frans-retinoic acid.

Received November 23. 1981; accepted August 5, 1982.

noma (36), a pulmonary carcinoma (16), and a sarcoma (7)and against rat tumors such as a Chondrosarcoma (33, 42) anda hepatocellular carcinoma (32). The growth of a human bronchial carcinoma transplanted in nude mice was also inhibitedby retinoids (20). Favorable results have been obtained in thetreatment of human basal-cell carcinoma and squamous cellcarcinoma (4, 30, 35). Furthermore, clinical trials with 13-c/s-

retinoic acid against squamous epithelial precancers and cancers suggest significant activity (30).

The most dramatic in vivo antitumor effect of retinoids,among those described thus far, is the inhibition of rat Chondrosarcoma growth since the retinoids not only inhibited tumorgrowth but also caused regression of established tumors attolerated doses (33, 42). The mechanism by which the retinoidsinhibit Chondrosarcoma growth in vivo is unknown. However,it has been found that treatment of chondrosarcoma-bearing

rats with retinoids decreased glycosaminoglycan synthesis bythe tumor cells (38). This finding has led to the suggestion thatthe loss of extracellular matrix by the treated tumor cells mayhave rendered them susceptible to immunological rejection(42). The possibility that retinoids may exert direct growth-

inhibitory effects on the Chondrosarcoma cells has not beeninvestigated.

We (22-27) and others (8, 9) have demonstrated the abilityof certain retinoids to inhibit anchorage-dependent growth andanchorage-independent growth of various transformed and

tumor cells. However, there were several tumor cell lines thegrowth of which was not inhibited by retinoids (9, 17, 23). Invitro evaluation of the sensitivity of human tumor cells toanticancer drugs appears to give some indication of their invivo response (37). Given the previously demonstrated in vivosensitivity of a rat Chondrosarcoma to retinoids, the presentstudy was undertaken to assess the effect of RA on the growthand clonogenicity of several cell lines derived from humanchondrosarcomas and osteosarcomas. Here, we report that allthe 5 cell lines tested are sensitive to RA; their exponentialgrowth rate is reduced, their saturation density is lowered, andtheir ability to form colonies in semisolid medium is suppressed.

MATERIALS AND METHODS

Cells. The human tumor cell lines derived from osteosarcomas (40)and chondrosarcomas were generously supplied by Dr. Walter A.Nelson-Rees of the Naval Biosciences Laboratory, School of Public

Health, University of California, Berkeley, Calif. The designation of thecell lines and some details on their source are given in Table 1.

Cell Culture Procedures. All cells were grown in plastic tissueculture dishes (Miniplast, Ein Shemer, Israel) in Dulbecco's modifiedEagle's minimum essential medium (Grand Island Biological Co., GrandIsland, N. Y.) containing 10% (v/v) heat-inactivated (56°, 30 min) fetal

bovine serum (Bio-Lab, Jerusalem, Israel), nonessential amino acids,penicillin, and streptomycin. The cells were incubated at 37° in a

humidified atmosphere consisting of 6% CO2:94% air. Growth media

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R. Thein and R. Lotan

Table 1Description of human osteosarcoma-derived and chondrosarcoma-derived cell lines

Source of tumor cells

History of patient

Cell linedesignationOsteosarcomasTe85Hs781Hs791ChondrosarcomasHs705Hs819TypeoftumorPrimary

tumor, distalfemurPrimarytumor, anteriortibiaLung

metastasis from a primary tumor in distalfemurPrimary

tumor,ribPrimarytumor, tibiaAge1312202860SexFMMMMRaceCaucasianCaucasianCaucasianCaucasianCaucasian

were replaced every 72 hr with fresh medium. To harvest cells, cellswere detached after a 5-min exposure to a solution containing 0.1%

trypsin (1:250; Difco Laboratories, Inc., Detroit, Mich.) and 2 mM EDTAin calcium- and magnesium-free phosphate-buffered saline, pH 7.2,

containing, per liter H2O, the following: KH2PO„,0.25 g; Na2HP(V7H2O, 2.16 g; KCI, 0.20 g; and NaCI, 8.00 g. The cells were thensuspended by repeated pipeting either in growth medium (for subculture) or in an isotonic solution (Isoton II; Coulter Electronics, Ltd.,Luton, England) for cell counting. The cells were counted using anelectronic particle counter (Model ZBI; Coulter Electronics). Cell viabilities were determined from the proportion of cells excluding 0.1%trypan blue.

Cell growth in semisolid medium (anchorage-independent growth)

involved suspending cells in growth medium containing 0.5% (w/v)agarose (Sea Plaque; Marine Colloids Division, FMC Corp., Rockland,Maine) and placing 1-ml aliquots in a series of 35-mm dishes on top of

a layer formed by 1 ml of agarose that had been allowed to gelpreviously. After cooling at 4°for 10 min to solidify the cell-containingagarose, the dishes were placed in the 37°incubator. After 72 hr, and

every 72 hr thereafter, the cultures were fed by pipeting 1 ml growthmedium on top of the agarose. After 13 days, the medium was removedand 1 ml of a solution containing 1 mg of the tetrazolium salt 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride per ml in

0.9% NaCI solution was placed over the agarose. After 24 hr ofadditional incubation, the colonies stained bright red were countedunder a microscope at x 40. Only colonies containing >10 cells werescored.

Treatment of Cells with RA. RA, the generous gift of Dr. Fritz F.Frickel of BASF Aktiengesellschaft (Ludwigshafen, Federal Republicof Germany), was stored in crystalline form under N2 at -70°. Before

each experiment, RA was dissolved in dimethyl sulfoxide and storedfor up to 3 weeks under N2 at —¿�70°.RA was added to the growth

medium of cells that had been seeded 24 hr earlier by direct dilution1:1000 of stock solutions. Control cultures received dimethyl sulfoxide(final concentration, 0.1%).

All procedures involving RA were performed under subdued light.The cultures were fed medium with RA every 72 hr. When the effect ofRA on growth in agarose was investigated, it was added at 10~5 M to

the cell-containing agarose solution and subsequently to the liquid

medium used for refeeding.

RESULTS

RA-induced Changes in Cell Morphology. All of the cellsdescribed in Table 1 exhibited an altered morphology and areduced growth rate following their exposure to 1CT5 M RA for

several days. RA enhanced cell flattening and spreading on thesubstratum as well as a reduction in cell overlapping. Examplesof this response are given in Fig. 1. The growth-inhibitory

effects of RA were further investigated as described below.

RA-induced Inhibition of Cell Proliferation. The growthcurves of 3 osteosarcoma-derived and 2 chondrosarcoma-de

rived cell lines cultured in the absence and in the presence of10~5 M RA are shown in Charts 1 and 2, respectively. The

number of cells in cultures exposed to RA was similar to thenumber in control cultures for the first 3 days; however, furthertreatment resulted in a reduction in the rate of exponentialgrowth in all of the osteosarcoma cells (Chart 1) and in theHs819 cells (Chart 2B). In contrast, the growth of the Hs705chondrosarcoma cells was almost completely blocked in thepresence of RA irrespective of the initial cell density (Chart2A). Upon further incubation, both the control and the RA-

treated cultures reached a confluent state (Charts 1 and 2).

4 8 I2 I6 4 8 I2 I6 4 8 I2 I6Days in Culture

Chart 1. Growth curves of osteosarcoma cell lines cultured in the absence orpresence of 1(T5 M RA. Cells were plated at 1.5 x 104cells/dish in a series of60-mm dishes, and after 24 hr, one half of them received RA whereas the otherhalf (control cultures) received dimethyl sulfoxide (0.1 %). At the indicated times,cells from duplicate cultures were detached, collected, and counted. Mediumwas changed every 72 hr. The differences in cell number between duplicateswere <10%. Similar results were obtained in 3 independent experiments.

IO t-:(A)Hs705

uOi_a>

IO4

~i—i—i—i—i—i(B)Hs8l9

CONTROL

4 8 I2 I6 4 8 I2 I6

Days in CultureChart 2. Growth curves of chondrosarcoma cell lines cultured in the absenceor presence of 10~5 M RA. The experimental procedure was similar to that

described in Chart 1 except that the Hs705 cells were plated at 2 different initialdensities (2 and 5x10" cells/dish) and the Hs819 were plated at 2 x 10"

cells/dish.

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Effects of RA on Osteosarcoma and Chondrosarcoma Cells

However, the saturation densities of the treated cultures weremuch lower than those of the control cultures (Chart 3).

The viability of cells exposed to 1CT5 M RA (90%) and the

plating efficiencies (80 to 90%) were not lower than those ofuntreated cells. Another indication for the lack of cytotoxicityof the RA was the reversible nature of the growth-inhibitoryeffect of RA. Cells treated for 12 days with 10~5 M RA resumed

the control growth rate within 5 days after they were replatedat low density in new culture dishes (Chart 4). The Hs705 cellsrequired a longer recovery period than the Hs791 cells.

Dose Dependence of RA-induced Growth Inhibition. Whenthe cells were cultured in the presence of RA concentrations inthe range 10~'° M to 10~5 M, their growth was inhibited in a

dose-dependent mode (Chart 5). It should be noted that the

experiment was terminated before the control cells reachedconfluence; therefore, the inhibition measured was of the exponential growth and does not merely reflect differences insaturation densities.

Although all the cells were inhibited by RA even at concentrations lower than 10~5 M, there were considerable differences

in their sensitivities. The Hs705 cells were the most sensitiveand their growth was inhibited by 50% at 10~9 M RA, whereasHs791, Te85, Hs819, and Hs781 required 10~7, 2 x 1CT7,2.5 x 10~7, and 2 x 10~6 M RA, respectively, for 50%

inhibition.

16

124-~•iCD

Control -

Treated¡1

L ruHS781 Te85 Hs791 Hs705 Hs619

Chart 3. Saturation densities of untreated and RA-treated cells. Cells wereplated in 50-mm dishes, and after 24 hr, one half of them received 10 5 M RA.

The cultures were fed every 72 hr, and when the cells formed a confluentmonolayer, duplicate cultures were harvested and counted. This procedure wasrepeated every 72 hr until no further increase in cell number was observed in 3successive cell counts. The final cell number was divided by the surface area ofthe dishes (19.6 sq cm) to give the saturation densities.

i IO -

E IO -

5 IO I5 20 5 IO I5 20 25 30

Days in Culture

Chart 4. Reversibility of RA-induced growth inhibition. Cells were grown inthe absence or presence of 10~5 M RA for 12 days and then detached andreplated in 60-mm dishes at 4 to 6 x 10s cells/dish. Cells that had been grown

in the absence of RA were further fed medium without RA (control). One half ofthe cultures that had been initially grown with RA were further fed medium with10~5 M RA whereas the other half of the RA-treated cells were refed medium

without RA. The cells were refed every 4 days. At the indicated times, duplicatecultures were harvested and counted.

IOO

80

60

40

20

IO- IO" IO"

RETINOIC ACID (M)

Chart 5. Dose-response relationship of RA-induced growth inhibition. Cellswere plated at 1.5 x 104 cells/dish in 60-mm dishes. After 24 hr, duplicate

cultures received the indicated concentrations of RA or dimethyl sulfoxide (0.1 %).The cultures were fed every 72 hr and harvested and counted after 9 days beforethe control cultures reached confluence. Similar results were obtained in 2additional independent experiments.

Table 2

Inhibition of colony formation in semisolid medium by RA

CelllineTe85Hs781HC"No.

ofcells1CT3)4104101040No.of colonies after 14 days ofgrowth"Control16,

24104,74128,89142,

16195,71330,

400RA0,

04,00,00,

00.00,

0The experimental procedure is given in "Materials and Methods." The

numbers represent colonies counted in duplicate dishes. Similar results wereobtained in 2 independent experiments.

" A cell line derived from a biopsy of a 66-year-old Caucasian female diag

nosed as Chondrosarcoma Grade III. The fresh malignant tissue was dissociatedmechanically, and single cells were plated in 0.5% agarose. A single colony waspicked up aseptically 2 weeks later and cultured in a tissue culture dish. Whenthe dish became confluent, the cells were detached and plated in 0.5% agarosein the absence or presence of RA and incubated for 14 days.

RA-induced Inhibition of Anchorage-independent Growth.The osteosarcoma-derived cell lines Te85 and Hs781 were theonly cells, among those tested, that could grow in semisolidmedium under the conditions of our assay and using up to 2x 105 cells/35-mm dish.

Since none of the Chondrosarcoma lines exhibited anchorage independence, we derived clonogenic cells from a freshbiopsy of a Chondrosarcoma. As can be seen in Table 2, RAdramatically inhibited the formation of colonies in 0.5% agaroseby the osteosarcoma and Chondrosarcoma cells.

DISCUSSION

Our data show clearly that human osteosarcoma and Chondrosarcoma cells are responsive to RA in culture. All the celllines of these types that we have examined, including tumorcells derived from a fresh Chondrosarcoma biopsy, were significantly affected by RA. The exposure of these cells to RAresulted in marked changes in growth and morphology; therate of exponential growth was reduced, the cells assumed aflat appearance, due to enhanced cell spreading and decreased overlapping; and their saturation density was lowered.All the tumor cell lines that were capable of forming colonies inagarose have lost this anchorage independence after RA treatment. Similar responses to RA have been observed previouslywith several cultured transformed rodent fibroblasts (1, 8, 17,34). However, certain virally transformed fibroblasts (e.g.,SV3T3 and MSV3T3) and the human fibrosarcoma HT1080

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R. Thein and R. Lotan

cells were not susceptible to RA (9, 17, 27). Although there ispresently no information on direct effects of RA on normalhuman chondroblasts or osteoblasts, it may be relevant todiscuss previous reports on the effects of retinoids on normalmesenchymal cells in rodents. Studies with mesenchymal cellsfrom prechondrogenic mouse limb buds and with culturedmouse chondrocytes indicated that RA did not inhibit cellproliferation in low-density cultures (14, 39) in contrast to our

observations with human chondrosarcoma cells. The responses of cultured normal human skin fibroblasts to RA werealso distinct from the responses of the chondrosarcoma andosteosarcoma cells. Whereas RA reduced the exponentialgrowth rate in the normal as well as the tumor cells, there wasno change in the morphology of the treated skin fibroblasts orin their saturation density as compared to untreated cells (21 ).Thus, it seems as if the responses of the tumor cells aredifferent from those of the above normal cells.

Several in vitro and in vivo studies have shown that RA canmodulate chondrogenesis and osteogenesis (12, 14, 15, 28,29). The differentiation of prechondrogenic mesenchymal limbbud cells to chondrocytes was inhibited by RA (12, 14), andthe production of glycosaminoglycans by cultured mouse chondrocytes (39) and by rat chondrosarcoma cells (33, 38) wasreduced. An additional direct effect of RA on these cells is anenhancement of proteoglycan release presumably via the induction of the synthesis of a metal-dependent proteinase (18,

19). The results presented here imply that RA suppresses inthe human osteosarcoma and chondrosarcoma tumor cells theexpression of cellular properties which are often associatedwith the transformed phenotype. Since the growth-inhibitory

effects of RA were observed at concentrations that can beachieved pharmacologically in cancer patients (13), our resultssuggest that certain chondrosarcomas and osteosarcomasmay be responsive to treatment with retinoids in humans.

ACKNOWLEDGMENTS

We are grateful to Dr. Walter A. Nelson-Rees for the cells used in this study.These cells were produced with support from the National Cancer Institute, ViralCarcinogenesis Program, under the auspices of the Office of Naval Researchand the Regents of the University of California. Our thanks are also extended toDafna Lotan for excellent assistance.

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Effects of RA on Osteosarcoma and Chondrosarcoma Cells

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-C*^ -

l~

3ZFZZ

1d

.

1bFig. 1. Photomicrographs of osteosarcoma Hs781 cells (a and a') and Chondrosarcoma Hs819 cells (£>and b'i cultured for 12 days in the absence (a and t>) or

presence of 10"s M RA (a' and £>').Phase contrast, x. 120.

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1982;42:4771-4775. Cancer Res   Rafael Thein and Reuben Lotan  Chondrosarcoma Cells to Retinoic AcidSensitivity of Cultured Human Osteosarcoma and

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