[CANCER RESEARCH 48, 6103-6108. November I. 1988]

Differentiation-associated Changes in the Expression of Chondroitin SulfateProteoglycan in Induced U-937 Cells1

Svein O. Kolset,2 Irene Ivhed, Aud 0vervatn, and Kenneth Nilsson

Institute of Medical Biology, University of Tromse fS. O. K., A. 0.J, N-9000 Troms«,Norway and Institute of Pathology, University of Uppsala, [I. I., K. N.j,S-751 85 Uppsala, Sweden

ABSTRACT

A monoblastic cell line U-937 (clone 4), was induced to differentiatealong the monocytoid lineage by 12-O-tetradecanoylphorbol-13-acetate(TPA), retinoic acid (RA), and vitamin I). (VIM. By immunochemicaland morphological criteria the cells were found to differentiate intomacrophage-like cells in the presence of all three inducers. The expression of proteoglycans was investigated in control cultures and in cellsdifferentiated in the presence of both TPA, RA, and VD3. The cells werelabeled with |'"S|sulfalr and cell and medium-associated 35S-macromole-

cules were either solubili/od ¡nsodium dodecyl sulfate or subjected toproteolytic digestion. By use of chondroitinase ABC digestions and deam-inative cleavage at pH 1.5 it was demonstrated that all cell culturesincorporated |35S|sulfate exclusively into chondroitin sulfate proteoglycan

(CSPG). The expression of CSPG was found to decrease with differentiation to 60% in the presence of TPA, 67% in RA, and 40% in VD3 ofcontrol cultures on a cellular basis. The CSPG synthesized was consistently recovered from the medium fractions, whereas free glycosaminogly-can (GAG) chains were found in the cell fraction in all the cell cultures.GAG chains from both control and TPA-, RA-, and VDj-induced cultureswere found to be exclusively of the chondroitin 4-sulfate type. However,the CSPGs from RA- and VDj-treated cells were found to differ inmolecular size from those of control and TPA-induced cultures, as judgedby Sepharose CL-6B gel chromatography. This difference in macromo-lecular properties following the induced differentiation of the monoblasticcells into macrophage-like cells was found to reside in expression ofCSPGs (in the presence of RA and VD3) with smaller GAG chains.Control cells and TPA-induced cells synthesized CSPGs with GAGchains of approximate M, of 30,000, contrasted by approximate V/rof17,000 and 16,000 in RA- and VD3-induced cells, respectively. Accordingly, all three agents used in this study were found to induce differentiation of the U-937-4 cells and a decrease in the expression of CSPG, butonly RA and VD3 were found to influence the structure of the proteoglycans synthesized.

INTRODUCTION

The U-937 cell line is derived from a patient with a histiocyticlymphoma (1), with monoblast characteristics (2, 3). The cellscan be induced to further maturation along the monocyte differentiation pathway in the presence of TPA3 (3), RA (4), or

vitamin Dj (5). By use of these three different agents variablephenotypic aspects of monocytic cells are inducible. Thus TPA-treated cells are adherent, have typical macrophage morphologyand appear to share functional markers with activated normalmonocytes. RA and VD^-induced cells, in contrast, displayindividual morphological and functional characteristics, distinct from those of TPA (6, 7). As the U-937 cells, regardless

Received 12/9/87; revised 7/15/88: accepted 7/19/88.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported by grants from the Norwegian Cancer Society and

the Swedish Cancer Society.•To whom requests for reprints should be addressed, at University of Troms0,

Institute of Medical Biology. P. O. Box 977. N-9001 TromsB. Norway.3The abbreviations used are: TPA. 12-O-tetradecanoylphorbol-13-acetate;

SDS, sodium dodecyl sulfate; RA, retinoic acid: VD3, la,25-dihydroxycholecal-ciferol; CSPG. chondroitin sulfate proteoglycan; PC, proteoglycan; GAG, gly-cosaminoglycan; HPLC, high-performance liquid chromatography: PBS. phosphate buffered saline.

of inducer, become irreversibly blocked in the G l-phase of thecell cycle like normal monocytic cells the findings suggestalternative pathways of terminal monocytic differentiation. Furthermore, 7-interferon has been demonstrated to increase theexpression of IgE receptors in U-937 cells (8) and the capacityto function as effector cells in antibody dependent cellularcytotoxicity (9), but does not affect cell growth (8), and maythus represent a way of inducing a nonterminal differentiation.

Proteoglycans are acidic macromolecules with GAG chainsattached to a protein core. They have an ubiquitous distributionin nature, and have been shown to be important as structuraland functional elements in tissues (10), as potent regulators ofblood coagulation (11, 12), and to be involved in phenomenasuch as regulation of cell growth and tumor cell killing (13).Changes in the structure of proteoglycans have been demonstrated in conjunction with differentiation of mast cells (14)and monocytes (15). In the latter cell system the monocyteswere found to synthesize only chondroitin 4-sulfate, whereasthe in v//ra-differentiated macrophages synthesized a mixtureof chondroitin 4-sulfate and chondroitin-4,6-disulfate (15, 16).It has also been demonstrated that human macrophages obtained from the peritoneal cavity express oversulfated chondroitin sulfate proteoglycan in vitro (17), identical in structureto the CSPG isolated from macrophages differentiated in vitro(15). These changes in CSPG structure may be a correlate tothe differentiation of cells in the monocyte-macrophage lineage.The possibility that stimulated monocytes and macrophagesexpress oversulfated CSPG cannot, however, be excluded (16).

To elucidate further the possible relationship between monocytic differentiation and changes in CSPG structure, the proteoglycan biosynthesis in U-937 cells differentiated by a panelof different inducers was investigated. The results show thatthe cells differ in the acquirement of phenotypic markers, andalso in the expression of proteoglycans, depending upon thetype of inducer exposed to the cells.

MATERIALS AND METHODS

Materials. Sephadex G-50 and Sepharose CL-6B were obtained fromPharmacia Fine Chemicals, Uppsala, Sweden. [35S]SuIfate was boughtfrom Institut! for Energiteknikk, Kjeller. Norway, and ['Hjthymidineand ['Hjacetic anhydride were obtained from the Radiochemical Centre,Amersham, Buckinghamshire, England. Retinoic acid, DNP-alanine,bacterial chondroitinase ABC (E.C. 4.2.2.4), dextran blue and 12-O-tetradecanoylphorbol-13-acetate were all from Sigma Chemical Co., St.Louis, MO. l«,25-Dihydroxycholecalciferol was a gift from Roche,Basel, Switzerland. Sodium dodecyl sulfate was purchased from BDHChemicals Ltd., Poole. Dorset, England. VD3 and RA were dissolvedin 100% ethanol and diluted at least 1000-fold in the culture medium,to give a final concentration less than 0.1%. TPA was stored at —20°C

in acetone at a concentration of 200 ng/ml.Cells. The U-937 clone 4 (U-937-4) was maintained in RPMI 1640

supplemented with 10% heat-inactivated fetal calf serum, 50 Mg/mlstreptomycin and 100 IU of penicillin per ml (all from GIBCO Laboratories, Grand Island Biological Co., Glasgow, Scotland). Severalclones of the U-937 cell line are now available, and in the present studyU-937 clone 4 has been used. This particular cell line has been shown

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PROTEOGLYCANS IN DIFFERENTIATING MONOCVTES

to be more monocyte-like than the parent U-937 line (4. 18). Differentiation of U-937-4 into macrophage-like cells was accomplished byexposing the cells to 1.6 x IO"7M TPA, IO"6M RA, or IO"8M VD, for72 h. TPA doses in the range of 10~"-10~4 M have been tested on U-937 cells. Concentrations of 1.6 x 10~8 to 1.6 x 10~7 M have been

shown in previous reports from one of our laboratories to inducedifferentiation of the cells and to change the subcellular localization ofprotein kinase C (19), and to decrease susceptibility of U-937 cells toNK cell-mediated cytolysis, and to increase the ability of U-937 cellsto participate in antibody-dependent cellular cytotoxicity. No suchchanges were observed at concentrations below 1.6 x 10~8M (20). Dueto the efficiency of 1.6 x IO"7 M TPA to induce functional changes in

the U-937 cells this dose was chosen for further studies. The use of10~7 M and higher concentrations of TPA in studies on U-937 cells

have also been reported by others (21).The monoclonal antibodies OKM 1 and OKT 4 were obtained from

Ortho Diagnostics. Sollentuna, Sweden. The monoclonal antibodyHLA-DR was bought from Becton and Dickinson, Grenoble, France.Second-stage antibodies for immunofluorescence, sheep anti-mouse Ig-fluorescein isothiocyanate conjugated, was obtained from Swedish Bacteriological Laboratory, Solna, Sweden.

The proliferation of U-937-4 in the absence and presence of inducersafter 3 days of culture was measured by incubating the cells with 0.1tiC\ of ['Hjthymidine per ml (specific activity, 2 Ci/mmol) under

standard conditions for 60 min. The cells were then harvested onMillipore filters (0.45 urn), washed twice with PBS, and precipitatedwith lOTc trichloroacetic acid and ciliar ml. The filters were counted ina bctacounter after addition of scintillation fluid.

Biosynthetic Labeling. U-937-4 cells were cultured in the absenceand presence of inducers for 3 days and then labeled for 20 h with [15S]-

sulfate (50 ¿iCi/ml).The conditioned media were then harvested andcentrifuged at 1000 rpm for 10 min. The medium fractions and thecorresponding pelleted cells were solubili/ed in PBS containing 1%SDS, boiled for 3 min and frozen before further analyses.

Proteoglycan Structure. [15S]Sulfate-labeled material from medium

and cell fractions was subjected to Sephadex G-50 gel chromatographyto separate "S-labeled macromolecules from free [35S]sulfate. The

columns were equilibrated and run in PBS with 0.1% SDS. Markersfor void ( y„)and total ( V,) volumes were dextran blue and DNP-alanine. respectively. The F0-material was collected and the content ofradioactivity used as a measure of PG and GAG biosynthesis in therespective cell cultures (see also "Results").

Alternatively, medium and cell fractions were subjected to proteolyticdigestions and Sephadex G-50 gel chromatography (in l M NaCI) aspreviously described (15). The V0fractions were dialyzed against distilled water, lyophili/ed, and used for GAG-structure analyses.

"S-Labeled material from medium and cell fractions were subjectedto chondroitinase ABC digestions (15) and Sephadex G-50 gel chromatography in 0.2 M NHjHCO3. The K-fractions containing materialsusceptible to chondroitinase ABC digestions was lyophilized and subjected to HPLC using a Lichrocart-NH2-column (250 x 4 mm; Merck,Darmstadt, West Germany) run in 0.05 M sodium acetate buffer, pH5.0 as starting buffer. The column was eluted with a gradient extendingfrom 0 to 0. l M Na2SO4 in the same buffer as above using a SpectraPhysics HPLC pump system (Spectra Physics, San José,ÇA).Standards used were 35S-labeled ADi-4-S from monocyte cultures (15), and3H-labeled ADÌ-4.6-DÌSfrom squid cartilage chondroitin sulfate E (a

kind gift from Dr. N. Seno, Ochanomizu University, Tokyo, Japan).Chondroitin sulfate E was labeled with ['H]acetic anhydride according

to the method of H00k et al. (22).'5S-Macromolecules from medium and cell fractions were analyzed

by Sepharose CL-6B gel chromatography and the molecular size of theGAG chains liberated from PG by alkali treatment determined aspreviously described (23).

RESULTS

Differentiation of U-937-4 in the Presence of Inducers. TheU-937-4 cell line was cultured in the presence of 1.6 x 10~7 MTPA, 10~8M VD, or 10~6 M RA for 3 days. All three inducers

cause the cells to become adherent and to inhibit their proliferation to a great extent, as can be seen in Table 1. The effectof TPA was found to vary to some extent, from a small decreasein cell number to an increase of approximately 50% of thestarting concentration after 3-day incubation. The TPA-treatedcells remaining after 3 days incubation were, however found tobe viable and functional by several criteria (see below). AfterRA and VDHreatment the cells were found to increase innumber by 70 and 100%, respectively. The untreated controlcells were found to increase their cell number by 440% (Table1). The DNA synthesis, as measured by [3H]thymidine incor

poration on Day 3 was, however, almost completely inhibitedin the presence of all three inducers, as can also be seen inTable 1.

The stage of differentiation of U-937-4 cells cultured in theabsence and presence of inducers was investigated by immunofluorescence using the monoclonal antibody OKM 1, raisedagainst an antigen expressed by highly differentiated cells inthe monocytoid lineage. As can be seen in Table 2, TPA-,RA-, and VDj-treated cells were all found to be positive in thisrespect, the latter being more highly positive than the twoformer. The OKT 4 antigen has been shown to be expressed byimmature monocytes, but not by the more differentiated species,which was demonstrated also in this study; only untreated U-937-4 cells were found to be positive for this antigen. Thehistocompatibility antigen HLA-DR, in contrast, was expressedby all cultures, with the one exception that the TPA-treatedcultures were found to be more positive than the other three(Table 2). Furthermore, by morphological criteria the cellsexposed to inducers of differentiation were all found to displaya morphology typical for monocyte-macrophage-like cells, as isevident in Table 2. Further corroborative evidence for thisconclusion was obtained by microscopic inspection of untreated

Table 1 Proliferation of U-937 cells in the absence and presence ofTPA, RA, and VD,

Cells were seeded at a density of 0.2 x ]()" ml and incubated for 3 days with

and without inducers. At Day 3 the cells were counted in Biirker chambers andreconstituted at a density of 0.2 x 106/ml. [3H|Thymidrne incorporation wasmeasured after a 60-min pulse. Background of 302 cpm is subtracted. Each valueis the mean of triplicates.

I'HJThymidine

incorporationInducerTPA

RAVD3Cell

number(xlO'/ml)0.88

0.22°

0.340.40Viability(%)96

829392cpm

xIO"32.91

0.370.250.11%of

control12.9

8.53.9

" In three separate counting experiments the cell numbers were 150.000/220,000 and 300,000 after 3-day exposure of TPA.

Table 2 Characterization of U-937 cells cultured in the absence and presence ofTPA, RA, and VD,

Cells were cultured in the absence and presence of inducers for 3 days.Immunofluorescence"Inducer

OKM1TPA

++RA+++

VD3 ++++OKT

4+(68)'HLA-DR

Morphology*+++

(48) ++++ (64) +++ (46) ++

" The number of immunofluorescence-positive cells was scored visually byusing a Leitz fluorescence microscope: —,negative; +, to ++++, positive to verystrong positive fluorescence intensity, respectively.

* Morphology of Gremsa-stained cells. Positive reaction graded from ++ to

+++: increased cell size, increased number of vacuoles, increased ratio cytoplasm/nucleus, increased adherence and phagocytic activity.

' Figures in brackets are percentage of fluorescence-positive cells. In each test

a minimum of 150 individual cells were counted.

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and inducer-treated cells after cytospin centrifugation andGiemsa staining. As can be seen in Fig. 1 the cells exposed toinducers are larger, contain a larger number of vacuoles, displaycell surface protrusions, and have noncentrally located nuclei(Fig. 1, B, C, and D), as compared to untreated control cells(Fig. 1/0-

It may therefore be concluded that treatment of U-937-4 cellswith TPA, RA, or VD3 induces differentiation of these mon-oblastic cells into monocyte/macrophage-like cells. The im-munofluorescence and microscopic data, do however, indicatethat U-937 cells treated with the different inducers may represent different stages (or pathways) of monocyte differentiation.

Glycosaminoglycan Structure. U-937-4 cells cultured either

in absence or presence of differentiation inducers were labeledwith [MS]sulfate for 20 h from Day 3 to 4 of the incubationperiod. "S-Labeled material obtained by proteolytic digestion

and gel Chromatograph) was subjected to chondroitinase ABCdigestion and Sephadex G-50 gel chromatography to separatematerial resistant and susceptible to this enzymatic treatment.However, only depolymerized material could be detected bythis method, from both medium and cell fractions in all the cellcultures tested (result not shown). The disaccharides, appearingin the Ft-fractions, were further analyzed by HPLC, and were

found to contain almost exclusively the ADi-4S-type for 35S-labeled medium-material from all the cell cultures tested (notshown). Differentiation of U-937-4 cells in the presence ofeither TPA, RA, or VD.i does not, accordingly, lead to theexpression of disulfated disaccharide units, as has been demonstrated in normal monocytes during their differentiation intomacrophages in vitro (15).

35S-Labeled material from both medium and cell fractionsfrom U-937-4 cell cultures is on the basis of the results presented above incorporated only into chondroitin sulfate, irrespective of treatment with differentiation inducers or not. Theincorporation of [15S]sulfate into SDS-soluble macromolecules

was therefore used as a measure of the capacity of the differentcultures to synthesize PG/GAG. Control cells incorporatedapproximately the same amount of [35S]sulfate into cell and

medium fractions both in the initial phase and the exponentialphase of growth (not shown). When the cells were differentiatedin the presence of TPA, RA, and VD, the content of 35S-PG/GAG in the cultures (cell and medium fractions/IO6 cells) were

60,67 and 40%, respectively, of the amount found in the controlcultures. A significant decrease in 15S incorporation could be

observed already after 4-h incubation with TPA. Furthermore,labeling the cells for 20 h, either from the onset of the experi-

Fig. 1. Light microscopy micrographs of U-937-4 cells. A. control; B, TPA-induced: C RA-mduccd; and />. VDj-induccd. Bar, 5 urn.

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ment, from Day 1 or 2, in the presence of TPA revealedapproximately the same degree of decrease in 35Sincorporation.

Macromolecular Properties. The macromolecular propertiesof the chondroitin sulfate synthesized by monoblastic or mac-rophage-like U-937-4 cells was investigated by subjecting medium fractions solubili/ed in SDS to Sepharose CL-6B gelchromatography. As can be seen in Fig. 2 only negligibledifferences could be observed between material from controland TPA-treated cells, eluting with peak Km values of 0.14 and0.12, respectively. However, the 35S-macromolecules from RA-and VD3-treated cells were significantly smaller in molecularsize than the two former, peak Km values of 0.28 and 0.29,respectively. Chromatography of identical material after alkalitreatment induced a shift in the elution patterns for all fourfractions tested (see Fig. 3), demonstrating that the chondroitinsulfate in the respective medium fractions were of proteoglycannature. All cell fractions were, however, found to have elutionpatterns closely similar to those of the corresponding alkali-liberated GAG chains from the medium CSPGs with somematerial eluting in positions between those of intact CSPGsand the corresponding GAG chains (result not shown). Thealmost exclusive presence of CSPG in the medium fractions isin accordance with what has been demonstrated previously inthe U-937 cells (24). From Fig. 3 it is evident that the smallermolecular size of the medium CSPGs synthesized by RA- andVD,-treated cells (see Fig. 2) is mainly due to the substitutionof smaller GAG chains, as evidenced by the retarded elutionpatterns for these two fractions compared to those from controland TPA-treated cells. By the methods employed in this studyit has not been possible to establish whether the decrease in themolecular size of the two distinct CSPGs could also reside in asmaller number of GAG chains being attached to each peptidecore. The approximate molecular size of the GAG chainsliberated from the respective medium CSPGs was determinedon the basis of the ATavof the peak elution fractions comparedto those of chondroitin sulfate standards (23). The molecularsize of the GAG chains in the control is approximately 30,000,as is also the case for the GAG chains from the TPA-treated

1.0-

1.0-

30 40 50 60 30 40Fraction Number

Fig. 2. Sepharose CL-6B gel chromatography of medium 3!S-macromoleculesfrom U-937-4 cultures. Media from cultures labeled with [35S]sulfate in the

absence and presence of differentiation inducers were solubilized in SDS. Free["Sjsulfate was removed by gel chromatography.

40 50Fraction Number

Fig. 3. Sepharose CL-6B gel chromatography of "S-GAG chains from CSPGsobtained from the media of U-937-4 cells cultured in the absence and presence ofdifferentiation inducers (see also Table 3).

Table 3 Approximate molecular size of[35S]glycosaminoglycan chains fromU-937 cultures

35S-CSPG from conditioned media of U-937 cultures were treated with alkaliand subjected to Sepharose CL-6B gel chromatography.

Cell cultures Approximate A/,*

Control1.6 x l<r7MTPAIO-'MRA1(T8M VD,

0.420.420.530.55

30,00030,00017,00016,000

°The K„values displayed are for the peak elution fractions displayed in Fig.

3.* The approximate M, of the 35S-GAG chains was calculated on the basis of a

standard curve of A., of chondroitin sulfate standards versus log M, (23).

cells. After differentiation with RA or VD3 the M, of the GAGchains was found to decrease to 17,000 and 16,000, respectively(see Table 3).

DISCUSSION

The relationship between changes in the cellular phenotypeand changes in the expression of proteoglycans has been thesubject of a large number of studies. In the mast cell system,rat mucosal mast cells have been demonstrated to synthesizeoversulfated chondroitin sulfate (25, 26), whereas connectivetissue mast cells contain heparin (27, 28). This difference inPG content of two distinct mast cell populations may reflectdifferences in differentiation status, as has been indicated bydifferentiation of bone marrow-derived mast cells in vitro correlated to the expression of heparin proteoglycan (14). Furthermore, phorbol ester- or retinoic acid-induced differentiation ofHL-60 cells led to a decrease in GAG synthesis without anystructural differences detected (29, 30). In contrast, transformation or diabetes has been shown to be associated with adecrease in the sulfation of heparan sulfate (31, 32).

In the monocyte system it has previously been demonstratedthat differentiation in vitro of human monocytes was correlatedto an increased sulfation of the CSPG synthesized (15). Humanperitoneal macrophages were also found to express oversulfated

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PROTEOGLYCANS IN DIFFERENTIATING MONOCYTES

CSPG when cultured in vitro (17). Recently, it has been demonstrated that stimulation of monocytes after cultivation of thecells for one day in vitro resulted in the expression of oversul-fated CSPG.4 The distinction between differentiation and stim

ulation in the monocyte/macrophage system may be difficultto draw, and it may be perceived that changes in CSPG structuremay be implied both in the stimulation of monocytes andmacrophages and the differentiation of monocytes into macrophages. In the present study, however, it was not possible todetect any differences in the composition of disaccharides ofGAG chains from control and differentiated cultures. All cultures were found to synthesize exclusively chondroitin 4-sulfate,in accordance with what has been demonstrated for freshlyisolated monocytes (15) and U-937 "wild-type" (24). No in

crease in the sulfate density of the CSPGs can, accordingly, beseen following differentiation of the monoblastic cells intomacrophage-like cells in vitro. If the expression of disulfateddisaccharide units is regarded as a parameter of monocytedifferentiation (and not stimulation) the inducer-treated U-937-4 cells cannot, by this criteria, be regarded as completely differentiated monocytes.

By gel chromatography of medium CSPGs and the corresponding GAG chains it was possible to establish that differentiation in the presence of RA or VD3 resulted in the expression of PGs substituted with GAG chains of smaller M, thanwhat was expressed by control or TPA-induced cells. By im-munochemical and morphological criteria it was clearly established (Tables 1 and 2) that all three agents induced differentiation of U-937-4 cells into macrophage-like cells, with somenotable differences between the different inducers. Furthermore, the content of PG/GAG was lower in all cultures exposedto differentiation inducers when compared to untreated controlcultures. By using three different inducers of differentiation inthe U-937-4 cell line it has been possible to detect differencesbetween these agents, with respect to their effects on the structure of CSPG synthesized and released into the culture medium.This distinct difference in CSPG structure may reflect thepossibility that the cells differentiated in the presence of RAand VD3 on the one hand, and TPA on the other, may representalternative phenotypes of mature monocytic cells. The recentobservation that the U-937 "wild-type" synthesize CSPG con

taining GAG chains with molecular weight of approximately60,000 (24), versus Mr 30,000 in the U-937-4 cells, suggest thatchanges in this particular phenotype may be used as a parameterfor differentiation of cells in the monocyte-macrophage lineage.The results presented for the RA- and VD3-differentiated cellsfurther supports this notion. The molecular size of the GAGchains from RA and VD3-treated cells are in close agreementwith what has previously been reported for normal humanmonocytes (24, 33). In addition, macrophages differentiatedfrom neonatal blood monocytes in vitro express CSPG withlarger GAG chains than the adult in vitro macrophage cultures(34). If monocytes isolated from cord blood is considered torepresent a less mature population of cells than the adultcounterpart the higher molecular weight of the GAG chainsproduced by the former cell culture may be a reflection of sucha difference. The lacking effect of TPA on this particularphenotypic parameter cannot be explained in terms of poordifferentiation of the cells (Table 1 and 2), but may result fromTPA affecting other intracellular signal pathways important forthe modulation of PG biosynthesis than RA and VD3 do. Thedecrease in 35Sincorporation after treatment with RA and VD3

can be accounted for by the decrease in molecular weight of theGAG chains. TPA would, accordingly, be the only agent inducing a lower PG/GAG synthesis in U-937 cells. The differencesin the effects of the three inducers employed may provide a toolfor the study of regulation of CSPG expression in this cellsystem in conjunction with cellular differentiation.

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17. Kolset, S. O. Oversulfated chondroitin sulfate proteoglycan in cultured human peritoneal macrophages. Biochem. Biophys. Res. Commun., 139: 377-382, 1986.

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1988;48:6103-6108. Cancer Res   Svein O. Kolset, Irene Ivhed, Aud Øvervatn, et al.   Chondroitin Sulfate Proteoglycan in Induced U-937 CellsDifferentiation-associated Changes in the Expression of

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