Pruebas de Campo

7.1 INTRODUCTION

P la s m in o g e n a c tiv a to r in h ib ito r 1 (P A I-1 ) is th e m a jo r p h y sio lo g ical in h ib ito r o f tPA in plasm a (C h m ielew sk a e t al.

1983) and tissues. PAI-1 can react w ith both the single and tw o c h ain form s o f tPA to form an e n zy m atica lly in ac tiv e com plex. PAI-1 which acts as an alternative substrate to tPA is c leav ed at an A r g g ^ ^ '^ ^ e t g ^ ? p e p tid e b ond re le a s in g the carboxy term inal com plex (review s- Sprengers and K luft, 1987, A ndreasen et al., 1990).

PA I-1 b e lo n g to the se rin e p ro te a se fa m ily o f in h ib ito rs (SE R PIN S) w hich also include PA I-2 and p ro tease nexin. The tw o PA Is w ere originally nam ed endothelial type and p lacen tal type according to th eir sites o f iso latio n . H ow ever, as both types w ere later found in several types o f tissu e they w ere term ed PAI-1 and P A I-1 -2 respectively. PAI-1 can inhibit not only tPA , but also uPA.

tPA has high affinity for fibrin and its activity is elevated when bound to fib rin , fu rth erm o re tPA is p ro tected from in h ib itio n by PA I-1 w hen bound to fib rin (C h m ielew sk a et a l., 1988). M any n e o p la stic c e lls are know n in d u c e fib rin d e p o sitio n (Zacharski et al., 1986). This may protect cell bound tPA which is probably involved in tissue invasion at secondary sites from

becom ing in activ ated and also increase the p ro teo ly tic activ ity at the h o st-tu m o u r in terface.

PAI-1 is a glycoprotein w ith a m olecular w eight o f 54000. In the EC M it is found bound to vitro n ectin ; the bound form of PAI-1 has g reater stab ility than the free form (W im an et al., 1988). It has been also suggested that the bound form o f PAI-1 m ay be arran g ed in a c o n fo rm atio n al form th at p re se n t the A rg-M et bond for optim al in teractio n w ith tPA (C arrell et al., 1991). In cultured cells PAI-1 is secreted in an activ e form which is then rapidly converted to an inactive latent form . This latent form can be reactivated by dénaturants such as SDS and guanidium chloride (H eckm an and L oskutoff, 1985).

A lth o u g h in itia lly th o u g h t to be m ain ly e n d o th e lia l sp ecific m any d iffe re n t ty p es o f c e lls sy n th e siz e PA I-1 in c lu d in g hum an hepatocytes in cu ltu re (Sprengers et al., 1985), several hepatom a cell lines (C olem an et al., 1982), granulosa cells (Ny et al., 1986), M JZJ m elanom a cells (W agner et al., 1986) as well as endothelial cells (Pannekoek et al., 1986).

As the fast acting principal reg u lato r o f tPA and thus plasm in, PA I-1 has a sig n ific a n t ro le in th ro m b o ly tic e v en ts u n d er norm al p h y sio lo g ical conditions. D ecreased p lasm a fib rin o ly sis due to increased expression o f PAI-1 and not due to low ered tPA lev e ls has been im p lic a te d as an im p o rta n t fa c to r in throm bosis and coronary arterial disease (Param o et al., 1985).

7.2. M odulation of PAI-1 bv extrinsic ag en ts.

A m ultitude o f extrinsic agents have been show n to m odulate PAI-1 m RNA and its activity in a variety o f cells. These include b acterial en d o to x in w hich in d u ces bo th m essag e and p ro tein levels in m ouse endothelial cells, tum our necrosis facto r (TN F) w hich e le v ates PA I-1 a c tiv ity and m R N A lev e ls in hum an endothelial cells (van den Berg et al., 1988) and in terleu k in 1 (IL -1 ) an d b a c te r ia l lip o p o ly s a c h a r id e in c u ltu r e d r a t endothelial cells (Em eis and K ooistra, 1986). IL-1 also elevates PAI-1 antigen and m RNA in hum an en d o th e lia l cell cu ltu res (S chleef et al., 1988). H ow ever, T N F was found to have no affect on PAI-1 in a num ber o f hum an cell lines (G eorg et al.,

1989) and in prim ary cu ltu res o f hum an and ra t h ep ato cy tes (van H insberg et al., 1988).

T hese w orkers have shown that m any o f these ex trin sic agents in c lu d in g d e x a m e th a s o n e re g u la te PA I-1 a t th e le v e l o f transcription. O ther groups have suggested the p o ssib ility th at PAI-1 m ay be regulated through stab ility , as both the rat and the hum an PAI-1 transcripts contain A U -rich elem ents in th eir 3' UTR. The rat PAI-1 contain 2 AUUUA pentam ers w ithin this region. In the hum an there are 2 form s o f PAI-1 (N y et al., 1986, P an n ek o ek et al., 1986), a 3.2 kb form w hich have 2 A REs and a 2.4 kb form w hich lacks one o f the AU elem ents. B oth form s are derived from sam e hnR N A through d ifferen tial pro cessin g and p o ly ad en y latio n (B osm a e t al., 1988). Som e w orkers found the 2.4 kb to be m ore stable (M ed calf et al..

1988), w hilst others show ed th at its the 3.2 kb PA I-1 th at is m ore stable (van den Berg et al., 1988). Cytokines such as TNF and IL-1 are know n to enhance sta b ility o f m R N A s w ith 3’ AREs and both o f these agents have been show n to increase the steady state level o f 3.2 kb PAI-1 in hum ans. It is p o ssib le that this increase is due to an increase in stability, though it is also possible that the 3.2 kb form is p referen tially processed in the nucleus.

SI DNA d ig estio n assays on the p ro m o ter o f the ra t PA I-1 identified 8 protein footprints (Johnson et al., 1992). Five of the footprints were shown to bind to know n TFs. T here w ere 3 sites for SPI like proteins, one CTF/N FI and one that overlapped a SPI and a C T F/N FI site. D efin ite binding p ro tein s fo r 3 footprints could not be identified.

7.3 . E xpression of PAI-1 in tum our cells.

T h ere is c o n tra stin g e v id e n ce fo r th e e x p re ssio n o f PA I-1 protein and mRNA in neoplastic cells. It w ould be obvious to expect suppression of PAI-1 activity in tum our cells as it w ould fu rth er enhance PA activ ity and thus in crease the p ro teo ly tic properties o f the cells.

In c o n trast to this expected b eh av io u r there is ev id en ce th at som e tu m o u rs ex p ress h ig h er PA I-1 a c tiv ity sim u lta n e o u sly w ith increased PA levels. Study by Quax et al. (1990) on 22 hum an tum our cell lines of various origins that expressed uPA, tPA or both, found that m any o f these cells also expressed the

m ain inhibitors of PA concurrently. 20 out o f the 22 expressed tPA protein and 16 out of the 22 had eith er PAI-1 or PA I-2. M ore in terestin g ly there was a c o rrelatio n betw een ex p ressio n o f PA s and th eir in h ib ito rs, that is the cells w ith the h ig h est levels PA also generally produced highest levels o f inhibitors.

In tran sp lan tab le m urine lew is lung carcinom a K irsten sen et.al. (1 9 8 9 ) fo u n d th at c e n tra l areas o f th e tu m o u rs e x p re sse d elev ated levels o f both uPA and PA I-1, w h ilst the p erip h eral tissu e w hich show ed in v asio n and lo c a l tissu e d e stru c tio n ex p ressed only uPA p ro tein . It w as su g g ested th at PA I-1 w ith in the tu m o u r m ay a ct to p re v e n t p la sm in m e d iate d d estruction of the tum our's own structure.

W o rk by o th e r g ro u p s h a v e in d ic a te d th a t o n c o g e n ic transform ation leads to suppression of PAI-1 activity. Cohen et al. (1989) d isco v ered th at o n cogenic tran sfo rm a tio n o f rat 1 fibroblast cells w ith V -ras or EJ-ras leads to a dow n regulation of the high PAI-1 levels found in these cells. H ow ever there was a m odest but con co m itan t rise in PA I-2 m RNA in these cells.

T ran sfo rm atio n o f other cell lines w ith v-m yc and v -src w as also found to su p p ress PA I-1 lev e ls, in d ic a tin g th a t PA I-1 e x p re ssio n c o u ld be re g u la te d by se v e ra l o n c o g e n e s w ith different m odes of action. T hat is myc is a nuclear factor, v-src is a non-receptor tyrosine kinase and ras is cytoplasm ic G Tpase.

7.4. The activitv of PAI-1 in norm al tissue.

A correlation betw een PA and its inhibitor is also seen in som e o f the rat tissu es exam ined by L ucore et al. (1988) w here it was found that those tissue expressing high levels o f PA also co n tain ed elev ated PA I-1 lev els. T hese d ata su g g est th at in n o rm al p h y sio lo g ic a l ev en ts the to ta l PA a c tiv ity m ay be reg u lated through co u n teractin g PA levels w ith PAI-1 activ ity th u s c o n fin in g PA a c tiv ity to targ e t sites and p re v e n tin g u n co n tro lled p ro teo ly sis and tissue dam age. T h erefo re it has been proposed that it is the im balance in this reg u latio n that lead to a b n o rm al p ro te o ly tic a c tiv ity such as th o se fo u n d during tissu e in v asio n and m etastasis o f tum ours. In th o se cells w here both agents are elevated sim ultaneously the change in the to tal PA a ctiv ity is p ro b ab ly due to d isp ro p o rtio n a te change in one protein com pared to the other.

It w as d em o n strated in the e arlie r w ork th at tPA m R N A is expressed at a higher level in the gliom a cell line than in the norm al cell line. This elevation was show n to be prim arily due to the enhanced stability of the tPA mRNA in A 15A 5. H ow ever the d ifferen ce in the m essage levels does not acco u n t fo r the e v en g re a te r d iffe re n c e in the fib rin o ly tic a c tiv ity fo u n d betw een the cell lines. T herefore it was decided to investigate the expression of PAI-1 m RNA in these cell lines to determ ine w h e th e r a lte re d e x p re ss io n o f PA I-1 c o u ld a c c o u n t fo r differences in the PA activity.

7.5. RESULTS

7.5.1 The transcriptional regulation of PAI-1 m RNA

The level o f transcription of the PAI-1 m RNA in the tw o cell lin e s w as m e a su re d to d e te rm in e w h e th e r P A I-1 w as transcribed at a low er rate in A15A5 than in ARBO C9.

T he rate o f tran scrip tio n was m easured using the n u clear run on assay as d escribed in ch ap ter 4. The nu clei w ere iso lated from cells grow ing in 15% serum , and 4 hours after change of medium.

F ig u re 7.1 show s the lev el o f tran scrip tio n o f PA I-1 in the A15A5 and the ARBO C9 cell lines. The signals w ere analysed d e n sito m etrica lly and stan d ard ised to rat rep e titiv e sequences. T he resu lts in d icated that PAI-1 is tran scrib ed at an alm o st identical rate in A15A5 and in ARBO C9. The figure also show the rate o f tran scrip tio n o f tPA co m p ared to P A I-1 . T he co n stitu tiv e transcription of PAI-1 was found to be low er than tPA in both cells which suggest that PAI-1 m RNA levels m ay be identical in both cell lines. How ever, it is possible that PAI-1 is also regulated at a post-transcription level as found w ith tPA in A 15A 5. T hat is, despite the com parable level o f tran scrip tio n of tPA betw een the cell lines the stability of the tPA m RNA in A15A5 is m uch higher

t P A P A I - 1 R a t r e p. t P A P A I - 1 R a t r e p. PAI - 2 i £ A PAI-1 i £ A PAI -