A C T A U N I V E R S I T A T I S L O D Z I E N S I S
F O L IA B IO C H IM 1C A E T B IO P H Y S IC A 14, 1999
Janusz Błasiak, Beata Sm olarz, D agmara Piestrzeniew icz, Ja cek P yte l
THE 4G/5G POLYMORPHISM IN THE PROMOTER OF THE PLASMINOGEN ACTIVATOR INHIBITOR-1 (PAI-1)
GENE IN SUBJECTS WITH CANCER
U ro k in a se plasm inogen activ ato r system includes p ro teo ly tic enzym es th a t m ay c o n trib u te to cancer cell invasion by d eg rad in g the s u rro u n d in g extracellu lar m atrix an d disso ciate cell-cell o r cell-m atrix atta ch m en ts. T h ere is su b s ta n tia l evidence in m an y ty p es o f cancer th a t the an tig en c o n te n t o f one c o m p o n e n t o f th e system - plasm in o g en a ctiv ato r inhibitor-1 (P A I-1) in p rim a ry ca n c er tissue e x tra cts a re o f s tro n g p ro g n o stic value: high level o f PAI-1 in tu m o r p re d ic t p o o r p ro g n o sis fo r the p a tie n t. M o re o v er, it was d e m o n s tra ted th a t the level o f PAI-1 in m eta stasis is significantly higher c o m p ared to p rim a ry tu m o rs. A n in sertio n /d eletio n p o ly m o rp h ism in the p ro m o te r o f th e PAI-1 gene has been described th a t relates to p la s m a PA I-1 levels. W e studied th is p o ly m o rp h ism in the subjects w ith cancer. B lood w as tak en fro m 53 p a tie n ts (16 b re a s t can cers, 12 co lo re cta l c an cers, 9 g a stric can cers, 9 m elan o m as an d 7 head a n d neck cancers) an d 53 m atch ed co n tro ls. T h e frequencies o f the 4 G an d 5G alleles in p a tie n ts were 0.53 an d 0.47, respectively, c o m p ared w ith 0.43 an d 0.57 in co n tro ls. T h e genotype d istrib u tio n differed significantly betw een the tw o g ro u p s - the 4 G /5 G gen o ty p e w as observed m o re freq u e n tly in p a tie n ts w ith cancer th a n in the co n tro ls. F u rth e r studies are needed to check w h e th er th e p rev alen ce o f th e 4 G /5 G g e n o ty p e m ay in flu en ce a n in d iv id u a l’s p lasm in o g en a ctiv atio n system cap acity a n d th ere b y c o n trib u te in a sm all w ay to th e cancer risk profile.
Key W ords: Plasm inogen a ctiv ato r inhibitor-1 (PA I-1), PAI-1 gene, gene p o ly m o r p h ism , cancer
IN T R O D U C T IO N
T h e u ro kinase plasm inogen activation system is n o t only responsible for the rem oval o f fibrin from the circulation , b u t is also believed to play a role in o th er biological processes, such as o v u latio n , em bryogenesis, in tim a pro liferatio n , atherosclerosis, d eg rad atio n o f th e extracellu lar m atrix , tum origenesis an d m etastasis [5, 25, 31]. T he system includes the u ro k in ase
ty p e p lasm in o g en a c tiv a to r (u-PA ), the specific p lasm in o g en a c tiv a to r inh ib ito rs PAI-1 and PA1-2 and the urokin ase recep to r (u -P A R ). P A I-1, an ap p roxim ately 50-k.D glycoprotein belonging to the serine p ro tease in h ib ito r superfam ily, is the m ajor physiological inhibitor o f the system. Its biosynthesis is regulated by a n u m b er o f horm ones, cytokines, grow th facto rs, tu m o r- -p ro m o tin g p h o rb o l esters and o th er agents [2, 14, 15].
C an cer cell invasion and m etastasis are m u ltifacto rial processes th a t include adherence to the basem ent m em b ran e, secretion o f proteo ly tic enzym es and cell m ig ratio n into vessels an d lym phatic nodes follow ed by ex tra v asatio n a t d ista n t sites [22]. It has been show n th a t plasm in bo und to tu m o r cells significantly increases the d eg rad atio n o f basem ent m em b ran e [4], so P A I-1, like the rem aining co m p o n en ts o f the plasm inogen activ ation system , can be involved in tu m o r invasion and m etastasis. T h e level o f PAI-1 was positively correlated with a m ore invasive tu m o r cell p h en o ty p e o f m an y types o f cancers, so it can be related to prognosis [3],
PAI-1 was show n first to be a prognostic m ark e r in b o th node-negative and node-positive breast cancer [12, 13, 17]. It was also show n th a t in gastric cancer, high level o f PAI-1 correlated with shorten ed p a tie n t survival [11, 28]. In lung ad e n ocarcino m a, PAI-1 was fo un d to be an in d ependen t in d ic a to r o f pro g n o sis [30], H ig h levels o f PAI-1 p re d ic ted sh o rten e d survival in patien ts with advanced o v arian cancer [21]. T h e feasibility o f alterin g tu m o r m etastasis by d isru p tin g plasm inogen activ ato rs fun ctio n by PAI-1 was d em o n strate d in m u rin e m odels o f p ro sta te ca rcin o m a and in tra o c u la r m e la n o m a [1, 32], T u m o r necrosis fa c to r a increased th e p ro d u c tio n o f PAI-1 in eight h u m an colon carcin o m a cell lines [33],
C hanges in PAI-1 biosynthesis is usually proceeded by changes in PAI-1 gene tran sc rip tio n and m R N A level [2, 23]. G ene variability could co n trib u te to the level o f the PAI-1 biosynthesis [16]. E igh t different p o ly m o rp hism on th e PAI-1 gene have been described: (a) tw o (CA)„ rep eat polym orphism s, one in the p ro m o ter and one in the in tro n 4 [7, 24], (b) an H in d lll restrictio n fragm ent length polym orphism [18], (1991); (c) a n insertion (5 G )/d eletio n (4G ) po ly m o rp h ism a t p o sitio n -6 7 5 o f th e PA I-1 gene p ro m o ter [6]; (d) tw o G —*A su b stitu tio n s a t positions -8 44 an d + 9 7 8 5 ; (e) tw o polym orphism s in the 3’ un tran slated region (T —vG su b stitu tio n at position 11 053 an d 9-nucleotide insertion/deletion located between nucleotides + 11 320 and + 1 1 345 in a threefold rep eated sequence) [16], In view o f th e possibly significant role o f PAI-1 fo r tu m o r spreading, it is im p o rta n t questio n , w hether these po lym orphism s can ac co u n t fo r the d evelop m ent a n d /o r the progression o f cancer.
In the p resent w o rk the frequencies o f the v ariants o f th e co m m o n in sertion (5 G )/deletion (4G ) polym orphism at po sitio n -6 7 5 o f th e PAI-1 pro m o ter (4G /5G polym orphism , Fig. 1) in subjects w ith cancer as com pared w ith health y co n tro ls was studied.
G G A C A C G T A G T C A G
GGGGG
F ig. 1. S chem atic rep re se n tatio n o f the PAI-1 gene show ing the lo ca tio n o f the 4 G /5 G p o ly m o rp h ic site and its flan k in g sequences
M A T E R IA L S A N D M E T H O D S
Chemicals
R N A se, p ro tein a se K , T rito n X -100 and E D T A w ere from Sigm a C hem icals (St. L ouis, M O ). T aq polym erase was ob tained from P ro m eg a (M ad iso n , W I, U SA ). Specific prim ers were purchased in A R K Scientific G m b H Biosystem s (D a rm stad t, G erm any). F o u r d eox yrib on ucleo tid es tri p h o sp h ates were from B oehringer (M annheim , G erm any).
All o th er reagents were o f the highest purity available.
Subjects
T h e study included blood from 53 healthy volunteers an d 53 p atien ts w ith various cancers - 16 breast cancers, 12 colorectal cancers, 9 g astric cancers, 9 m elan om as and 7 head and neck cancers. All p atien ts had biopsy-verified m alig n an t tum ors. Blood was collected a t the D e p a rtm e n t o f O ncology, M edical A cadem y o f Ł ódź from p atients enrolled to surgery. T h e c o n tro l subjects were age- and sex m atched.
D N A extraction
B lood was m ixed w ith equal volum e o f the buffer com prising 1% T rito n X -100, 2 % sarcosyl, 0.8 M urea, 20 m M E D T A , 0.4 M N aC l, 200 m M
T ris, pH 8.0 and R N A se A was added to a final co n c en tratio n o f 100 /xg/ml. A fter 2 h incubation at 55°C proteinase K was added to a final co ncentration o f 125 /¿g/ml and the sam ple was incubated as previously and extracted once w ith phenol and twice w ith chloroform .
Determination o f PAI-1 genotype
PAI-1 4 G /5 G p ro m o ter genotype was established for each subject by polym erase chain reaction (P C R ) am plification o f genom ic D N A using the allele specific prim ers (Fig. 1): insertion 5G allele: 5’-G T C T G G A C A C G T G G G G G -3 ’, deletion 4G allele: 5’-G T C T G G A C A C G T G G G G A -3 ’, each in a separate P C R reactio n to gether w ith th e co m m o n d o w n stream p rim er 5’-T G C A G C C A G C C A C G T G A T T G T C T A G -3 ’ and a c o n tro l upstream prim er 5’-A A G C T T T T A C C A T G G T A A C C C C T G G T -3 ’ to verify the occurrence o f D N A am plification in the absence o f the allele on the genom ic D N A [10]. T h e P C R was carried o u t in a P erkin-E lm er therm al cycler, m odel G ene A m p P C R System 2400 in a final volum e o f 25 /il co ntain in g 10 ng D N A , 13 pm ol o f specific prim ers, 1 m M d N T P s and 1 U T aq polym erase to g eth er w ith 2.5 /il l O x T a q buffer. T he P C R cycle conditions were 9 4 “C for 60 s, 54°C for 30 s th en 72°C for 40 s for 35 cycles. T h e am plified D N A frag m en ts were sep arated by 5% polyacrylam ide gel electrophoresis an d , afte r staining w ith ethidium brom ide, viewed under ultraviolet light. E ach subject was classified into one o f the three possible genotype groups: 4 G /4 G , 4 G /5 G o r 5G /5G .
Statistical analysis
A llele frequency w as estim ated by gene c o u n tin g an d an alyzed by Xz test. T h e test was also used to com p are the observed n um b ers o f each PAI-1 genotype w ith those expected for a p o p u la tio n in H a rd y - -W einberg equilibrium .
R E S U L T S A N D D IS C U S S IO N
T h e d istrib u tio n o f the 4 G /5 G genotypes in p atien ts w ith cancers and co n tro ls is displayed in T ab le 1.
T h e genolype d istrib u tio n differed significantly betw een the tw o groups (T ab le 1) - the 4G /5 G genotype was observed alm ost twice m o re frequ ently in p atien ts th an in the contro l; on the o th er h an d , the 5 G /5 G genotype was a b o u t 5 tim es less frequent th an in the co n tro l. T he frequencies o f the 4 G and 5G alleles in p atients did n o t differ significantly from controls.
T a b l e 1 D istrib u tio n o f 4 G /5 G genotypes and frequencies o f the 4 G a n d 5G alleles in
p a tie n ts with cancer and c o n tro ls
P atie n ts“ N u m b er F req u en cy C o n tro ls“ N u m b e r F req u en cy 4 G /4 G genotype 7 0.14 11 0.22 4 G /5 G genotype 42 0.79" 23 0.42 5 G /5G genotype 4 0.07h 19 0.36 4 G allele 56 0.53 45 0.43 5G allele 50 0.47 61 0.57 0 n = 53; b p < 0 .0 5 .
W ith the presented d a ta it was show n, th a t th ere w ere d ifferent d ist rib u tio n s o f 4 G /5 G genotypes in sm all p o p u latio n s o f p atien ts with cancers a n d controls.
Id ea th a t PAI-1 is an etiological facto r in som e diseases, first o f all o f ca rd io v ascu lar type [18], caused th a t hu m an PAI-1 p ro m o te r was searched fo r alteratio n s th a t m ay affect the level o f plasm a PAI-1 activity [9], Evidence th a t genetic factors can be im p o rta n t for PAI-1 activity com es from clinical studies. Initially, a 3’ H incilll restriction frag m en t length polym orp hism an d an intro n ic dinucleotide (C A ) repeat were associated w ith elevated PAI-1 levels [6]. M o re recently 4 G /5 G po lym o rp hism has been related to circulating level o f PAI-1 - levels are the highest in subjects w ith th e 4 G /4 G g enotype an d generally a b o u t o n e -th ird hig h er th a n subjects hom ozygous for the 5G allele [7, 9, 29],
A lth o u g h levels o f PAI-1 are correlated significantly w ith env iro n m en tal fa c to rs [19, 27] the v aria n ts c a n n o t be excluded. T h e genetic 4 G /5 G p o lym orphism seems to be especially interesting because subjects w ith the 4 G allele have higher plasm a level o f PAI-1 th a n individuals w ith the 5G allele [8] and the 4G allele was m ore co m m on in p atien ts w ho survived an m y o card ial infarction [9],
T h e association o f the 4 G /5 G polym orphism o f th e PAI-1 p ro m o te r w ith altered levels o f plasm a PAI-1 activity suggests a d ifferential binding o f protein s regu lating the tran scrip tio n al activity o f th e gene. It was show n
th a t b o th alleles b o und a com m on factor, while the 5G allele b o un d an ad d itio n al facto r [9], In an in vitro studies it was also d e m o n strate d th a t th e 4G and 5G sites differentially b oun d en h an cer/rep resso r [7, 9], th a t suggests a functional role o f the 4 G /5 G polym orphism .
Elevated PAI-1 m ay c o n trib u te also to vascular disease in diabetes m ellitus. P im a Ind ian s have a low incidence o f card io v ascu lar disease desp ite o f having a high prevalence o f n o n -in su lin -d ep c n d en t d iab ete s m ellitus which in this p o p u latio n is n o t associated w ith elevated PAI-1 activity [26], In P im a In d ian s p o p u latio n the frequencies were 23.0% for 4 G /4 G , 49.8% for 4 G /5 G and 27.2% for 5 G /5G co m pared to 35.4% , 50.8% an d 13.8% , respectively, previously reported in C au casian with n o n -insulin-dependent diabetes m ellitus. T he difference in the frequencies o f genotypes m ay indicate functional difference in the PAI-1 gene in this two p o p u latio n s associated w ith reduced card io v ascu lar risk.
O btain ed results can be treated as a prelim inary re p o rt, b u t th ey suggest th at the problem o f the possible correlation between genetic constitution o f the PAI-1 gene and possibly o th er genes o f the plasm inogen ac tiv a to r system is w o rth fu rth e r studying. O u r study involved a relatively sm all n u m b er o f subjects w ith cancer. M oreover, the p o p u latio n was heterogeneous in age, sex an d the type o f cancer. F u rth e r studies should consider also these aspects o f research. T he other problem to discuss is the association between genotype and circulating PAI-1 level. A s m entioned above such an association has been established, bu t it m u st be taken into acco u n t th a t this re latio n sh ip ca n be hold only fo r healthy individuals. In a patho lo gical state, like in cancer, variation in the level o f PAI-1 m ay, at least in p art, be attributed to undefined influences acting on PAI-1 a t th e tim e o f the disease.
A lth o u g h th ere was no difference in allele frequency betw een subjects w ith cancer and con tro ls, th e prevalence o f the 4 G /5 G gen otype m ay influence an individual’s plasm inogen activ atio n system cap acity and thereby c o n trib u te in a small way to the cancer risk profile, p ro b a b ly by in teractio n w ith o th e r genetic factors.
F u rth e r research are needed to elucidate the relation ship betw een the PAI-1 gene, circulating PAI-1 levels and cancer.
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W p ły n ęło d o R ed ak cji D e p a rtm e n t o f M o le cu lar
F o lia b iochim ica et biop h y sica G en etics
24.04.1998 U n iv ersity o f Ł ó d ź
D e p a rtm e n t o f O ncology M ed ical A c ad e m y o f Ł ó d ź
Janusz Błasiak, B eata S m o la n , Dagm ara Pieslrzeniewicz, Jacek P ytel
P O L IM O R F IZ M 4 G /5 G O B S Z A R U P R O M O T O R O W E G O G E N U IN H IB IT O R A A K T Y W A T O R Ó W P L A Z M IN O G E N U T Y P U 1 (P A I-1 )
U C H O R Y C H N A N O W O T W O R Y Z Ł O Ś L IW E
U ro k in a zo w y u k ład aktyw acji plazm in o g en u zaw iera enzym y p ro teo lity czn e m o g ące b ra ć u d z ia ł w inw azji k o m ó re k rak o w y ch p o p rz e z d e g rad a cję m acierzy z e w n ą trz k o m ó rk o w e j i przeryw aniu połączeń pom iędzy k o m ó rk am i i m acierzą. W yniki znaczącej liczby ek sperym entów w sk azu ją, że zaw arto ść przeciw ciał d la jed n e g o ze sk ład n ik ó w u k ład u ak ty w acji p lazm in o g en u in h ib ito ra a k ty w ato ró w plazm in o g en u ty p u 1 (PA I-1), w e k strak tac h z frag m en tó w p ierw o tn y c h guzów n o w o tw o ro w y ch m oże m ieć d u że znaczenie prognostyczne: w ysoki p o z io m PA I-1 źle ro k u je d la p acjen ta. Z ad e m o n s tro w an o także, że po zio m PAI-1 w m etastazie je s t wyższy niż w guzach pierw o tn y ch . P olim orfizm insercyjno/delecyjny 4 G /5 G w o bszarze p ro m o to ro w y m genu P A I-1 m o że być zw iązan y z o soczow ym p o z io m e m P A I-1 . W p ra c y b a d a n o ten po lim o rfizm w e krw i 53 p a cjen tó w cierpiących n a ró żn e ro d zaje n o w o tw o ró w (16 p rz y p ad k ó w ra k a su tk a , 12 jelita g rubego, 9 ż o łąd k a, 9 czern iak ó w i 7 n o w o tw o ró w głow y i szyi) o raz 53 o d p o w ied n io d o b ra n y ch o so b n ik ó w zdrow ych. Częstości alleli 4 G i 5G u c h o ry ch były od p o w ied n io : 0,53 i 0,47, p o d c za s gdy w grupie k o n tro ln ej w arto ści te w ynosiły 0,43 i 0,57. R o z k ła d y g en o ty p ó w w b a d an y ch g ru p a ch były zn acząco różne - u c h o ry ch g e n o ty p 4 G /5 G w ystępow ał ze zw iększoną częstością niż w grupie k o n tro ln e j. K onieczne są dalsze b a d a n ia d la oceny częstości w ystępow ania d an eg o g en o ty p u p o lim o rfizm u 4 G /5 G , ja k ró w n ież innych p o lim o rfizm ó w , genu PAI-1 i stopniem ry zy k a z ap ad n ięcia n a ch o ro b ę n o w o tw o ro w ą.
