Localization and production of proteoglycans by HT1080 cell lines with altered N-ras expression

145

Cancer Letters, 53 (1990) 145- 150 Elsevier Scientific Publishers Ireland Ltd.

Localization and production of proteoglycans lines with altered N-ras expression

by HTl080

cell

J. Timar” and H. Patersonb ‘1st Institute of Pathology and Experimental and Thester

Beatty Research

Laboratories,

Cancer Research, The Semmelweis Medical University, Budapest Institute of Cancer Research, London (U.K.)

(Hungary)

(Received 29 March 1990) (Revision received 25 June 1990) (Accepted 26 June 1990)

Summary

Introduction

The alterations in the production of proteoglycans in relation to the expression of the malignant phenotype - controlled by the level of expression of actioated N-ras gene - was studied in HTI080 human fibrosarcoma cells and in its revertant variants reu.lc and reu.lOa. A decreased production of radiolabelled PGs especially HSPGs was observed in HT1080 cells, compared to the reuertanr lines. By immunofluorescence, the HSPG epitopes were localized mainly into the putative endoplasmic reticulum and/or Golgi zone in HT1080 cells, and to the diffuse cytoplasmic and membrane localization in the reuertant lines. It is suggested, that the altered expression of PGs represents an important aspect of the transformed phenotype of HT1080 fibrosarcoma cells.

Proteoglycans (PG) are ubiquitous components of the extracellular matrix (ECM) with a wide range of structural and functional heterogeneity [ 11. In several systems the biosynthesis of the sugar chains of PGs, the glycosaminoglycans (GAG), is diminished in transformed cells [2,3]. In CHO cells tumour proliferation correlated positively with the capacity to synthesize heparan sulphate (HS), but negatively with the expression of chondroitin (CS)/dermatan sulphate (DS) proteoglycan [4,5]. In our study we have used the HT1080 human fibrosarcoma cell line [6] together with its flat revertants lc and 10a [7]. It has been shown in this system, that manifestation of the malignant phenotype is directly controlled by the level of expression of activated N-ras gene [7]. We have thus been able to compare production of PGs in a well-characterized human cell line, originating from a spontaneously occuring tumour, with that of non-tumorigenic revertant sublines derived from it.

Keywords: human proteoglycans. Correspondence

ment

fibrosarcoma

to: J. Timar,

of Radiation

Oncology,

cells; N-ras;

Cancer Biology Division, DepartWayne State University, 431

Chemistry Bldg., Detroit, MI 48202, U.S.A. Abbreviations: PG, proteoglycan; CSPG, chondroitin sulphate proteoglycan; HSPG, heparan sulphate proteoglycan; CS. chondroitin sulphate; HS, heparan sulphate; DS, dermatan sulphate; GAG, glycosaminoglycan: ECM, extracellular matrix; sGAG, sulphated glycosaminoglycan; TCA, trichloroacetic acid; CPC. cetthyl-pyrimidium chloride; HP, heparin.

0304-3835/90/$03.50 Published and Printed

0

1990 Elsevier Scientific Publishers

in Ireland

Materials and methods Cell lines HT1080 human fibrosarcoma cell line [6] was from Flow Labs. Irvine, Scotland. The cultures were maintained on plastic tissue culture dishes in DMEM supplemented with 10% fetal Ireland Ltd

calf serum at 37OC in 10% CO,. The cells were passaged by trypsinization. Flat revertants of the HT1080 cell line rev.lc and rev.lOa [7] were cultured similarly to the parent HTlOSO. Production of sulphated glycosaminoglycans (sGAG) Cell lines were plated into 25 cm2 Falcon dishes at a cell density of 5 x 104/cm2 in 10 ml complete culture medium and were labeled by 30 pCi/ml Na,SO, (spec. act. 2.29 GBq/ mmol, Amersham) for 48 h at 37OC. Then the medium containing the extracellular GAG fraction was collected. The cells were removed from the dishes by 1 ml 0.25% trypsin/PBS at 20°C for 2 min, the action of which was stopped by adding 20 ~1 serum. The cells were collected, centrifuged and the supernatant was added to the medium while the cells were kept in 1 ml PBS. The medium and the cellular fractions were extensively digested by pronase E (Serva) for 24 h. To isolate the sGAGs, these fractions were p-eliminated [8] in 0.5 M NaOH for 3 h at 4OC to remove the core peptides. After neutralization with cc. acetic acid (by adding l/40 of the sample volume) the samples were adjusted to 10% TCA at 4OC. This mixture was centrifuged at 500 g for 25 min and was reprecipitated with ethanol to remove the small mol. wt. components. The precipitate was washed with ethanol again, air-dried and dissolved in 1 ml distilled water. The sGAGs were precipitated by 1 ml 1% (w/v) CPC [9]. This sediment was further purified by dissolving in 1 ml mixture of 2 M NaC1/96% ethanol (100: 15 v/v) and precipitated by ethanol again. The sediment was air-dried, dissolved in 200 ~1 distilled water and considered as sGAG fraction. Radioactivity in isolated sGAG fraction was measured in a toluene-based scintillation coctail by Beckman LSlOOC spectrometer. The ratio of HS to CS-DS was determined upon the sensitivity of the sGAG fraction to heparitinase (Sigma) digestion which was performed at 37OC for 24 h (1 pg/ml in 0.1 M sodium acetate, pH = 6.9) [lo]. The digested

HS-derived mono- and di-saccharides were separated from the heparitinase resistant sGAGs (CS and DS) by gel-permeation chromatography using Sephadex G-25. The fractions containing the macromolecular GAGS were collected according to the elution position of dextran blue (20 kDa) while the low mol. wt. oligosaccharide-containing ones were collected according to the elution position of phenol-red. The radioactivity in these fractions was then counted. Indirect immunofluorescence of the proteoglycan antigens FW16 rabbit polyclonal antibody was raised against pig skin fibroblast membrane HSPG (mol. wt. = 60 kDa) and was provided by M.F. Watt, ICRF, London, U.K. MK172 mouse monoclonal IgGl was produced against human articular cartilage CSPG recognizing core protein epitope situating nearby the glycosylation site [ 111. The cells were grown on 1 cm glass coverslips at a density of 5 x 104/cm2, were washed in PBS and fixed in 1 ml cc. methanol for 15 min. After 3 washings in PBS 10% normal sheep serum was applied for 10 min to block the non-specific binding sites. The primary mono- or polyclonal antibodies were applied for 1 h at room temperature (working dilution in PBS was 1: 100 and 1:50, respectively) then the coverslips were washed 3 times in PBS and an appropriate FITC-labelled secondary antibody (anti-mouse or anti-rabbit) was used to localise the primary. In negative controls the primary antibody was omitted. After the immunoreaction the coverslips were washed in PBS then in distilled water and mounted in Mowiol (Sigma). The slides were viewed in Letz epifluorescence microscope or in a confocal laser microscope (BRL) . Results The proliferation of the revertant cell lines was much slower than those of the HT1080 cells within 48 h (Fig. 1). The 35S-labeling for 48 h showed that the production of sGAGs is

147

FW16 antibody detecting HSPG antigen showed different localizations in cell lines. In HT1080 the pericellular zone displayed the majority of the epitopes, while the ceil membrane was less positive (Fig. Za,b). In rev.lc the cell membrane and at much lesser extent the cytoplasm were positive (Fig. 3a,b), while in rev. 10a the cytoplasm contained more antigen than the cell membrane (Fig. 4). MK172 antibody recognizing CSPG epitope was present in a significant amount in rev.lc only, especially in the pericellular zone (Fig. 5) and no reaction was observed in case of the other lines.

Cell

24

4%

h

Fig. 1.

Discussion

almost lo-fold lower in HT1080 cells than in the revertant lines, with the exception of the extracellular sGAG fraction of rev.lOa (Table 1). The amount of HS in the sGAG was determined after heparitinase digestion and separation of the digested (HS) and resistant (CS-DS) fractions by gel-permeation chromatography. The HS and CS-DS were present in equal amounts in sGAG fractions of HT1080 while in both revertants a shift was observed reflecting a more dominant HS production (Table 1).

The HT1080 cells contain normal and mutant N-t-as genes, which control their transformed phenotype [71. In the revertant cell lines (rev. lc and rev.lOa) the dosage of the mutant N-ras gene is reduced [7] and the contact inhibition is re-established as their proliferation curves showed. The biochemical studies demonstrated, that HT1080 cells produce much less sGAG, than the revertant ones, based upon the incorporation of the radiosulphate. It is interesting, that in rev.lOa the GAG-products are accumulated intracellularly, while in rev. lc they were secreted. Immunolocalization of membrane-directed HSPG-epitopes revealed, that in the low

Proliferation of HT1080 cells and its revertant lines in vitro. Data are means of four parallel samples. 0 = HT1080, 0 = rev.lc, 0 = rev.lOa.

Table 1. Cell line

HTlOSO rev. l.c. rev. 10a.

35S-incorporation

into sulphated

GAGS of HT1080 and revertant

cells.

Cellular sGAG compartment

Extracellular sGAG compartment

sGAG

HS/CS-DS ratio

sGAG

HS/CS-DS ratio

5498 f 1395 30808 f 8327 34046 + 10225

1.21 + 0.08 2.30 -+ 0.05 2.40 + 0.06

3249 + 794 37609 2 12143 4710 k 2029

1.27 + 0.09 2.13 k 0.07 2.33 2 0.03

Data are expressed as cpm/106 cells and represent means + S.D. sGAG = sulphated glycosaminoglycans, HS = heparan sulphate, phate.

CS = chondroitin

sulphate,

DS = dermatan

sul-

Fig. 4.

Localization of HSPG epitopes by FW16 in rev.lOa copy. Note the strong diffuse cytoplasmic reaction. 600 x

Fig. 5. rescence

cells. Method:

same as Fig. 2a). Confocal

Localization of cartilage CSPG epitopes by MK172 in rev. lc cells. Indirect immunolabelling, microscopy. Note the reaction in the vicinity of the nuclear membrane. 1800 X

sGAG-producer HT1080 cells, these epitopes accumulated in the putative ER and/or Golgi zone, while in the high sGAG-producer revertants in the cell membrane and diffusely in the cytoplasm. The two times higher HS/CS-DS ratio in revertants supports the conclusion that the increase in the production of HSPGs in the

laser micros-

conventional

fluo-

revertant cells is more pronounced than those of the CSPGs. The limited positive reaction of MK172-detectable CSPG antigen in HT1080 cells and in its revertants suggests that other CSPG types could be produced by these cell lines. Our results are consistent with those findings

Localization of HSPG epitopes by FW16 antibody in HT1080 cells. Immunofluorescence, indirect labelling. (a) Fig. 2. Confocal laser microscopy. Serial optical sections (0.5 pm), bottom left: free cell surface, top right: plastic. Note the clear positivity in the perinuclear zone as well as at the cell membrane. 600 x (b) Conventional fluorescence microscopy. Note the intense reaction in the putative ER and Golgi region. 1800 x Localization of HSPG epitopes by FW16 in rev.lc cells. Method: same as Fig. 2. (a) Confocal laser microscopy Fig. 3. as in case of Fig. 2a. Note the diffuse cytoplasmic and strong membrane reaction. 600 x (b) Conventional fluorescence microscopy. There is a clear positivity on cell projections. 1800 x

150

that the production of GAGS (especially of HS) in transformed cells is reduced compared to their normal counterpart [12]. We postulate that this could be due to the impaired glycosylation as well as to the altered biosynthesis and/or transport of the HSPG molecule. The HSPGs in the cell membrane were shown to function as anchors in the ECM and to have close association with the cytoskeletal constituents [13]. HS and its structural arialogue heparin (HP) have been implicated in the regulation of cell proliferation. HS and HP were shown to inhibit cell proliferation of normal cells [14] and to stimulate the growth of some transformed cells [4,14]. There has been speculations on the possible mechanisms whereby HSPGs may transduce ECM-mediated signals essential for growth control [15,16]. The altered effect of HSPG sugar chains HS/HP on cell proliferation of different cell types may be caused by the altered signal transduction mechanisms. The N-ras gene product the ~21 protein, is actually a member of that signal transduction system [ 17,181. Therefore we conclude, that the impaired production and altered localization of PGs especially HSPGs, probably represent important aspects of transformation in HTlO80 cells and that these parameters - at least partially - are controlled by the expression of the N-ras oncogene.

4

5

6

7

a

9

10

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Bloomfiled, F. and Dexter, T.M (1988) Heparan sulphate bound growth factors : a mechanism for stromal cell mediated hemopoesis. Nature, 332, 376-378. Folkman, J.. Klagsburn, M., Sasse, J., Wadzinski, M.,

Acknowledgement This work was supported by the British Council (UK) and by the International Cultural Institute (Hungary) in the frame of the Hungarian-British cooperative research programme (J . Timar) .

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