Characterisation of fibronectin fragments and plasminogen activators released by RSV-transformed cells

Fibrinofygis (1987) 1, 1X3-187 c 1987 Longman Group UK Ltd

Characterisation of Fibronectin Fragments and Plasminogen Activators Released by RSV-transformed

Cells

G. De Petro, S. Barlati SUMMARY. In the present study, we followed qualitatively the release’of fihronectin/s (FNs) and of plasminogen activators (PAS) into the serum-free medium of normal and Rous sarcoma virus (RSV) transformed chicken embryo fibroblasts (CEF). It has been found by Western blotting analysis that RSV transformed cells release into the medium intact FN and FN peptides with a Mr ranging between 230 and 110 kDa while uninfected cells and cells infected by NY68 and PA1 (temperature sensitive, ts, mutants for transformation), grown at the restrictive temperature for transformation (41”(Z),release mainly intact FN. The zymographic analysis of the PAS released by normal and transformed cells showed that transformed cells release into the medium a set of PA forms with Mr ranging between 180 and 43 kDa while uninfected cells or cells infected with NY68 or PA1 grown at 41°C do not release or release only the 43 kDa form respectively. The analysis of the PAS present in the extracts of uninfected or PA1 and NY68 infected cells, grown at 35” or 41”C, revealed mainly the presence of the 43 kDa form. KEYWORDS.

Rous Sarcoma Virus. Fibronectin fragments. Plasminogen activator. Cell transformation.

neoplastic cells, in cells transformed by oncogenic viruses, chemicals and in many tumorigenic cell lines.” PA appears early upon cell transformation in shift-down experiment performed using RSV temperature-sensitive mutants for transformation” and the serum free culture medium of RSV-transformed cells is able to promote transformation in vitro,’ ’ and stimulates the PA activity of CEF and of RSV-CEF;12 in addition purified human t-PA is able to promote cell transformation.’ 3 In this report, we present data on the fragmentation of FN in RSV transformed CEF, in serum-free medium, and on the release of multiple forms of PAS in concomitance with the expression of the transformed phenotype.

It is known that fibronectin (FN) fragments exert in vitro biological functions not associated with the intact molecule.‘, 2 In particular it has been shown that: 1) trypsin fibronectin fragments augment opsoninindependent phagocytosis’ in human monocyte cultures; 2) the 30 kDa gelatin-binding FN fragment acts as a Transformation Enhancing Factor promoting the transformed phenotype of RSV-infected CEFs3 while the intact FN induces a transient reversion of the transformed phenotype of RSV-CEF4; 3) FN fragments obtained with endogenous proteases augment chemotactic activity’ of peripheral monocytes and cathepsin D-generated digest of fibronectin stimulate DNA synthesis in cultured fibroblasts’; 5) heparinbinding fragments of FN are inhibitors of endothelial cell growth.* It is known that the level of FN is reduced in transformed CEF and that this is due to a reduction in transcription and in translation and also to protein degradation.1,2 The fragmentation of FN extracellular matrix (ECM) is likely to be due to transformation-associated protease such as plasminogen activator (PA) and/or metalloendoproteases6*7v8 and occurs at cell contact sites where viral pp60 src is localised.? Among the proteases associated with transformed cells PAS seem to play a key role. PA synthesis and secretion are elevated in human G. De Petro, S. Barlati, Division of Biology sity of Brescia, I., 25124 Brescia, Italy.

and Genetics,

MATERIALS AND METHODS Cell, Viruses and Media Primary and secondary cultures of CEF were prepared as previously described.” In all experiments, only secondary cultures of CEF were used, either uninfected or infected by Rous sarcoma viruses. The strains of viruses used (all clonal isolates) were: 1) wt SR-RSV of A or D subgroup;” 2) PAl, subgroup D and 3) NY68, subgroup A, these two viruses being ts mutants for transformation.’ ‘, l4 Unless otherwise indicated, cultures of CEF were grown in Eagle’s

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Minimum Essential Medium (MEM) supplemented with 5% (vol/vol) newborn calf serum (Flow) and 10% (vol/vol) tryptose phosphate broth (TPB). Antibodies Rabbit antisera to human plasma fibronectin was a kind gift of Professor Antti Vaheri. Peroxidaseconjugated swine immunoglobulins to rabbit immunoglobulins were purchased from Dako (Denmark).

Activators

Released by RSV-transformed

Cells

3.3% (wt/vol) in the spacer gel and 8% (wt/vol) in the separating gels and 10% (wt/vol) beta-mercaptoethanol in Laemli’s sample buffer was used as reducing agent. Commercially available low-molecular-weight markers (Pharmacia, Uppsala, Sweden) supplemented with purified human plasma FN were used. The proteins were immunoblotted according to the method of Towbin as already describedI using anti-FN serum at dilution of 1:lOOO(vol/vol), and immunoperoxidase staining. Zymography

Conditioned Media and Cell Extracts Secondary CEF, either uninfected or massively infected, were seeded onto 1Ocm cultures plates (4 x lo6 cells/plate) in Eagle’s MEM supplemented as above, and incubated at 35°C or at 41°C in a humidified 5% COz atmosphere. After 5 or 6 days incubation at 35°C at least 80 to 90% of the cells in the infected cultures appeared to be morphologically transformed. The culture medium was then removed and, after extensive washing of the cultures with phosphate buffered saline (PBS), it was replaced without addition of serum and TPB (MEM). The 24 h conditioned medium (CM) was collected and divided in two aliquots. One was treated with 0.001 mol/L Phenyl-methane-sulfonyl-fluoride (PMSF) and, after centrifugation at 2000 rpm for IO min, used for the immunoblotting analysis of FN under reducing conditions. The other aliquot, also centrifuged, was used for the zymographic analysis of PAS after NaDodSOJpolyacrilamide gel electrophoresis under non-reducing conditions. The same procedures were utilised for the preparation of the CM obtained from infected or noninfected CEF cultured at 41°C. CEF infected with ts mutant for transformation were utilised for shift-up and shift-down experiments. The medium of massively transformed PA1 or NY68 infected cultures, grown at 35”C, was, after extensive washing with PBS, renewed with serum and TPB free medium. After 24 h of further incubation at 35°C the medium was collected and treated as above. After addition of MEM, the cultures were shifted to 41°C and the medium was collected and renewed again at 24 h intervals. The shift-down experiments were performed in the reverse way. All the harvest fluids were treated as described above. Cell extracts of uninfected and virus infected cells were prepared after extensive washing with cold PBS, recovering the cells by scraping them with a rubber policeman in 0.05% (wt/vol) NaDodSOA Aliquots of cell extracts were analysed for the presence of PA activities by zymography. Na-Dod-SO&Polyacrylamide Gel Electrophoresis and Immunoblotting of Proteins Polyacrylamide gel electrophoresis in the presence of NaDodS04 was performed according to Laemli using vertical slab gels. The acrylamide concentrations were

Zymography of proteins was carried out according to a modified technique. l3 Proteins were separated under non-reducing conditions utilising the gel electrophoresis conditions reported above and after electrotransfer to nitrocellulose, the filter was overlaid onto a casein plasminogen-agar plate; these plates were prepared with a mixture of 1% (wt/vol) Agarose in 0.1 mol/L Tris HCl buffer pH 8, containing 0.6% (wt/vol) low fat milk powder as a source of casein and 2% (vol/vol) chicken serum as a source of homologous plasminogen. Chicken serum was not added in control plates. The lysis band, developed in 24 h at 35°C in a humidified atmosphere were photographed using dark field light.

RESULTS Fibronectin Fragments Released by RSV-transformed Cells Chicken embryo fibroblasts, uninfected or infected with RSV-SR(A) and (D) and ts mutants for transformation (NY68, PAl) were cultured at 35°C and at 41°C and the serum-free medium collected and treated in different experimental conditions, as described in Material and Methods. 100 p1 conditioned medium was submitted to electrophoresis under reducing conditions and analysed by Immunoblotting using antiFN sera. The results, shown in Figure 1 indicate that control CEF release into the medium only intact FN either at 35°C (lane 1) or at 41°C (lane 3) while PAltransformed CEF (lane 2) release intact FN and a set of fragments (FNdp) with an apparent M.W. ranging between 230 and 145 kDa; at 41°C PAl-infected CEF release mainly intact FN and lower levels of fragments with M.W. of about 230 kDa (these may or may not be present). Lanes 5 to 8 show the patterns of FN released into the medium of PAl-infected CEF under shift-down (41+35”C) and in shift-up (35”+41”C) conditions. Following the shift-down experiment the level of fragments increases with time and a reversible pattern is evidenced under the shift-up conditions. It should be noted that the presence of fragments is not strictly associated with the expression of the transformed phenotype, since between 24 and 48 h after shift-up, fragments are still present (lanes 6 and 7) when the

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Fig. 1 Immunological characterisation of fibronectin and its fragments released by control or RSV-infected CEF, grown at 35” or at 41°C. A 90 ul aliquot of the serum-free medium harvest and treated as reported in Materials and Methods was resolved on 8% (wt/vol) Na-Dod-SO4 polyacrylamide gel electrophoresis under reducing conditions and analysed by Immunoblotting with anti-fibronectin antibodies. The daily collections of media were from: control CEF and PAl-infected CEF grown at 35” or at 41°C (lanes 1,2,3 and 4); PAl-transformed CEF grown at 35” and shifted to 41°C twenty-four hours from the beginning of the experiment (lanes 5,6,7 and 8): the same lanes observed in the opposite direction from 8 to 5 refer to media harvest in a shift-down experiment; from SR(A)RSV, SR(D)RSV, NY68 and PA1 transformed CEF at 35°C (lanes 9,10,11 and 12).

cells show a detransformed morphology. Lanes 9 to 12 show the patterns of FNdp released at 35°C into the medium of CEF transformed with SR(A), SR(D), NY68, PA1 respectively. CEF transformed by subgroup D viruses, (lanes 10 and 12) as compared to those infected by subgroup A (lanes 9 and 1l), release into the medium higher levels of FNdp with an apparent Mr of 160, 170 and 210 kDa. Traces of lower MW fragments may be observed (N 110 kDa). It should be pointed out that transformed cells do not release FN fragments with a M.W. < 110 kDa while anti-human FN sera are able to recognize lower M.W. FN fragments after digestion of purified intact chicken FN with proteolytic enzymes (data not shown).

Plasminogen Cells

Activator Released by RSV-transformed

Uninfected or massive-infected CEF with RSV-SR(A) NY68 or with RSV-SR(D)PAl were cultured at 35” or at 41°C; the 24 h serum-free conditioned medium was collected as for the analysis of FN with the exception that NaDodSOCPAGE electrophoresis was performed under non-reducing conditions. Figure 2 shows the zymographic analysis of PAS present in the culture medium from NY68 transformed CEF (lanes 1 to 4) at 35°C. Multiple forms of PA, with a Mr of about 180, 170, 90, 80, 67, 54, 50 and 43 kDa, were detected. The high M.W. forms disappear following

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Fig. 2 Zymograms of plasminogen activators present in serum-free media collected from NY68-infected CEF, transformed at 35°C (lanes 1,2,3,4 and 5) and detransformed at 41°C (lanes 6,7 and 8). In one experiment, confluent and transformed cells were kept at 35°C after washing with PBS and daily renewing of the serum-free media (lanes 1,2,3 and 4); in another one, confluent and transformed cells were kept at 35°C for the first renewal of the medium (lane 5).

detransformation of NY68 transformed-CEF at 41°C (lanes 6 to 8). Similarly, PAl-transformed CEF release into the culture medium high and low M.W. forms of PAS at 35°C (Fig. 3, lane l), but with slightly different M.W. (110, 88, 67 and 43 kDa); PAl-infected CEF at

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Fig. 3 Zymograms of secreted (lanes 1,2 and 3) and cell-bound (lanes 4,s and 6) plasminogen activators from confluent cultures oE PAl-transformed CEF grown at 35°C (lanes 1 and 4) PAl-infected CEF grown at 41°C (lanes 2 and 4) and control CEF grown at 35°C (lanes 3 and 6). Confluent cultures of control and PAl-infected CEF grown at 35°C or at 41°C were washed with PBS three times, added with serum-free medium. The samples of harvest fluids (lanes 1,2 and 3) collected after 24 h and the cell extracts (lanes 4,5 and 6) of the correspondent cultures were prepared as reported in Materials and Methods and analysed for the presence of secreted or cell-bound plasminogen activator activity with casein-plasminogen-agar zymography (see Materials and Methods).

41°C (lane 2), release only the lowest 43 kDa form as for NY68 infected cells. Control uninfected CEF do not show any soluble form of PA (lane 3), but only a cell associated 43 kDa form (lane 6) common to the cell-bound one of PAl-infected CEF at 35” and at 41°C (lanes 4 and 5). The zymographic analysis performed utilising control casein-agar plates did not reveal any plasminogen independent activity in any sample.

DISCUSSION

AND CONCLUSIONS

RSV-induced cell transformation causes a number of cellular changes including alterations in cell growth and shape, loss of pericellular matrix, enhancement of PA activity, appearance in the conditioned medium of TEF activity. ‘, lo, l1 This activity is also associated with purified proteins like human t-PA13 and gelatinbinding fragments of FN.3 Moreover we have previously shown induction of release of high M.W. FN fragments (160 kDa) after treatment of human embryo but not adult fibroblasts with phorbol esters.15 It should be emphasised that embryonic, contrary to adult fibroblasts, release PA activity into the culture medium.” It was therefore of importance to verify whether FN fragmentation could be considered an additional change induced by transformation and if this could be associated with variations in PA activity. Indeed previous studies have shown that seeding transformed cells on labelled FN induces its degradation6 which occurs at cell contact sites corresponding to the site of expres-

sion of pp60src9 Moreover a plasminogen independent degradation of FN is induced by RSV-transformed cells cultured on ECM derived from uninfected cells.798 All these data, together with the demonstration of a direct binding of PAS with immobilised FN,16 indicate FN as a possible substrate for PAS. Data reported in the present study show that RSV@R-A, SR-D, NY68, PAl) transformed CEF release into the serum-free medium intact FN and high level of FN fragments with M.W. ranging between 230 and 110 kDa: ts mutants of RSV induce high levels of fragments only at the permissive-temperature for transformation, 35°C while only intact FN is released by infected cells at 41°C or uninfected cells either at 35°C or at 41°C. In shift-up experiments, the disappearance of FN fragments occurs slowly and later after the reversion to the normal phenotype, as detected by morphological criteria. The wt SR-RSV (D) virus and its mutant PA1 induce a different pattern of FN fragmentation as compared to that induced by the wt subgroup (A) virus and its ts mutant NY68. This as if a relationship existed between the subgroup type of virus utilised and the pattern of FN fragments detected. Concerning secretion of PAS, NY68 and PAl-transformed CEF release different sets of high and low MW forms of PAS which seem also to be specific for the subgroup A or D of the SR-RSV strain: the 67 and 43 kDa forms are common to both systems. The 67 kDa might be related, in analogy with the mammalian system, to t-PA type activator.” The 43 kDa form is also present in the cell extracts of uninfected CEF of PAl-transformed CEF or PAlinfected CEF grown at 41°C and corresponds to u-PA type activator. lo The fact that FN fragments have been observed in the serum free media, depleted therefore from PG for several days, suggests that they are generated by a PG independent process. The lack of low MW FN fragments also indicate that the degradation of FN should occur via a new type of digestion since all known proteases degrade FN generating always also low MW peptides.“’ Possible candidates for the degradation of FN may be the high MW forms of PAS which appear following transformation while, the 43 kDa PA form should not play a relevant role in this process since it is released in the medium concomitantly with intact FN, when infected cells are incubated at the non-permissive temperature (41°C) for transformation. The forms of other PA released by cells with a transformed phenotype may either correspond to different yet unknown types of PA induced by RSV infection and/or to. the association of PA with other proteins i.e. FN or FN fragments or proteases inhibitors.“* l6 If activation of pp60 src induces release of new forms of PA (t-PA type) this might contribute, together with high MW FN fragments, to the generation of TEF activity in the conditioned media of transformed cells. It is therefore of importance to better purify and characterise the new forms of PAS detected, together with their possible direct role on FN fragmentation.

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ACKNOWLEDGEMENTS 7. We thank Professor Antti Vaheri for kindly supplying antifibronectin antibodies and Dr Elisa Gionti for kindly providing RSV-SR(A), SR(D) and NY68 viruses. We are grateful to Mrs Silvana Faitini, Bruna Arici and Mr Marino Chiari for skilful technical assistance and to Miss Fiammetta Franzoni for excellent secretarial assistance. This work was suppo!ted by grants awarded by CNR, Special Project ‘Oncologia’ and ‘Ingegneria Genetica e Basi Molecolari delle Malattie Ereditarie’, by the Italian Ministry of Education (M.P.I.) and Associazione Italiana Ricerca sul Cancro (A.1.R.C).

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Offprint orders to: Giuseppina De Petro, Divisione di Biologia e Genetica, U.P. Scienze Biomediche di Base, Via Valsabbina n. 19, Universitl di Brescia, 25124 (Italy). Tel. 39-30-398261.

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