cytotechnology volume 12 issue 1-3 1993 [doi 10.1007%2fbf00744674] susan mcdonnell; barbara...

8/10/2019 Cytotechnology Volume 12 issue 1-3 1993 [doi 10.1007%2Fbf00744674] Susan McDonnell; Barbara Fingleton -- R

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Cytotechnology 12: 367-3 84, 1993.

9 KluwerAca dem ic Publishers. Prin ted in the Netherlands.

R o l e o f m a t r i x m e t a l lo p r o t e i n a s e s i n i n v a s i o n a n d m e t a s ta s i s: b i o lo g y

d i a g n o s i s a n d i n h i b i t o r s

S u s a n M c D o n n e l l a n d B a r b a r a F i n g l e t o n

Schoo l o f B io log ica l Sc iences D ub l in C i ty U n ivers i ty D ub l in I r e land

K e y w o r d s : invas ion , me ta l lop ro te inas es , me tas ta s i s , T IM P S

bs t ra c t

The p roces s e s o f tumour inv as ion and s ubs e quen t me tas ta s i s a r e the mos t l e tha l a s pec t s o f cance r. W hi l s t

many f ac to r s a r e invo lved , the ma t r ix me ta t lop ro te inas es (M M P s ) have been imp l ica ted a s key - r a te l imi t ing

enzy me s in the invas ive p roces s . Th i s f ami ly cons i s t ing o f e igh t mem ber s o f simi la r s truc tu re , can be rough ly

d iv ided in to th r ee g roups bas ed on s ubs t r a te s pec i f ic i ty . A l l a re s ec re ted in a la t en t fo rm and r equ i r e

p ro teo ly t i c c l e avag e fo r ac t iva t ion . The exp res s ion o f thes e enzym es i s r egu la ted a t the t r ans c r ip tiona l l eve l

by a va r i e ty o f g row th f ac to r s and oncogene s . The y a r e a l s o r egu la ted a t the p ro te in l eve l by a f ami ly o f

s pec i f i c inh ib ito r s ca l l ed the t i s sue inh ib ito r s o f me ta l lop ro te inas es (T IM P s ) . S tud ies in hum an tum our

s amples have s how n a pos i t ive co r r e la t ion be tw een me ta l lop ro te inas e exp res s ion and me tas ta t i c po ten t i a l .

T h e l e v e l s o f m e t a l l o p r o t e i n a s e e x p r e s s i o n h a v e b e e n m a n i p u l a t e d u s i n g m o l e c u l a r b i o l o g y t e c h n i q u e s i n

s eve ra l ce l l l ines and s how n a s imi la r co r r e la t ion . Thes e r e s u l t s s ugges t tha t an under s tand ing o f

meta l lop ro te inas e exp res s ion and p ro teo ly t i c ac t iv i ty may l ead to the deve lopmen t o f e f f ec t ive the rapeu t i c

agen t s w i th the po ten t i a l to r educe the inc idence o f me tas ta t ic cance r .

A b b r e v i a t i o n s : M M P - - ma t r ix me ta l lop ro te inas e ; TIM P - - t i s s ue inh ib i to r o f me ta l lop ro te inas e ; rTIM P - -

r e c o m b i n a n t t i ss u e i n h ib i t or o f m e t a ll o p r o te i n a se ; T P A - - 1 2 - O - te t r ad e c a n o y l p h o rb o l - 1 3 - a ce t at e ; T R E - - T P A

res pon s ive e lemen t ; E G F - - ep ide rm al g row th f ac to r ; TG F -~ - - t r ans fo rming g row th f ac to r [3; EL1S A - -

e n z y m e - l i n k e d i m m u n o s o r b e n t a s s a y ; b p - - b a s e pa ir ; P C R - - p o l y m e r a s e c ha i n r e a c ti o n ; R T - P C R - - r e v e r s e

t r ans c r ip t ion po lymeras e cha in r eac t ion .

In t roduc t i on

O v e r t h e p a s t d e c a d e t h e f i e l d s o f c a n c e r b i o l o g y ,

m o l e c u l a r b i o l o g y a n d g e n e t i c s h a v e p r o v id e d p r o o f

tha t cance r i s , i n e s s ence , a gene t i c d i s eas e . A t the

m o l e c u l a r l e v e l w e c a n n o w s a y t ha t c a n c e r f o r t h e

m o s t p a r t i s c a u s e d b y a n a c c u m u l a t i o n o f s o m a t i c

muta t ions . A cco rd ing to V oge l s t e in and K inz le r

(1993) , th r ee to s ix mu ta t ions ap pea r to be r equ i r ed

to fo rm a cance r . Th i s 'mu l t i -h i t' hypo thes i s ,

invo lv ing bo th ac t iva t ion o f an oncogene and lo s s

o f tum our s uppres s o r gene ac t iv i ty , has been

c a r e f u l l y d o c u m e n t e d b y V o g e l s t e i n ' s g r o u p i n

co lon cance r (F ea ron and V oge l s t e in , 1990 ; V oge l -

s t e in and K inz le r, 1993). T he dev e lopm en t o f co lon

tumou rs appea r s to be in i t ia t ed by m u ta t ion o f the

A P C t u r n o u t s u p p r e s so r g e n e p r o d u c t w h i c h r e su l ts

i n th e f o r m a t io n o f b e n ig n a d e n o m a s . M u t a t i o n o f


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i nvade , and tha t they may a l s o be invo lved in

p e n e t r a ti o n t h r o u g h t h e b a s e m e n t m e m b r a n e d u r i n g

t h e e x t r a v a s a t i o n o f t u m o u r c e l l s f r o m b l o o d

v e s s e l s S l o a n e a nd H o n n , 1 9 8 4 ; D a n o e t a l .

1985) . A l though a l l o f thes e p ro te inas es in t e r ac t in

a complex manner tha t u l t ima te ly l eads to the

p roces s o f invas ion i t has been s ugges ted tha t

in i ti a l ex t r ace l lu la r m a t r ix deg rada t ion i s due to the

meta l lop ro te inas es L io t t a , 1991a).

T h e m e t a l l o p r o te i n a s e s a r e b e l i e v e d t o b e t h e

no rmal , ph ys io log ica l med ia to r s o f ma t r ix deg rada -

t ion u s ua l ly r e f e r r ed to a s ma t r ix me ta l lo -

p ro te inas es , M M P s ) . They a r e s ec r e ted p ro te in s ,

p lac ing the m in the p roper loca t ion fo r ex t r ace l lu la r

ma t r ix deg rada t ion , and the i r enzymat ic ac t iv i t i e s

a r e mos t po ten t a t pH va lues c lo s e to neu t r a l i ty

M at r i s i an , 1992) . There i s qu i t e a l a rge amoun t o f

ev idence tha t s uppor t s a c r i t i ca l r o le fo r me ta l -

lop ro te inas es in invas ion and m e tas t a s is . R i fk in and

c o l l e a g u e s M i g n a t t i e t a I . 1986) demo ns t r a t ed tha t

a p ro te inas e cas cade i s r equ i r ed fo r the invas ion o f

me lano m a ce l l s and tha t the me ta l lop ro te inas es p lay

a ma jo r ro le in th i s p roces s . A dd i t iona l ev idence

has come f rom s tud ies w i th the inh ib i to r s o f

M M P s , the t i s s ue inh ib i to r o f me ta l lop ro te inas es

T I M P s ) . A n i n v e r s e c o r re l a ti o n b e t w e e n T I M P

leve l s and the invas ive po ten t i a l o f mur ine and

h u m a n c e l l s h a s b e e n o b s e r v e d H i c k s

e t a l .

1984;

H a l a k a e t a l . 1983) . D enhard t and co l l eagues

K h o k h a e t a l . 1989) have u s ed an an t i s ens e

a p p r o a c h t o d e m o n s t r a te a r ol e f o r M M P s i n

tumour invas ion . I n thes e exper imen t s ce l l s tha t

w e r e n o t n o r m a l l y i n v a s i v e b e c a m e i n v a s i v e w h e n

t h e y n o l o n g e r p r o d u c e d T I M P a f t e r t r a n s f e c t i o n

w i th an an t i s ens e TIM P cons t ruc t . I n add i t ion ,

A l v a r e z e t a l . 1990) have demons t r a t ed tha t

r e p e a t e d i n je c t i o n s o f r e c o m b i n a n t h u m a n T I M P

inh ib i t s lung me tas t a s es fo l low ing in t r avenous

in jec t ion o f

H a - r a s

t r ans f ec ted r a t embryo ce l l s . I t

i s p r es u me d tha t the ac t ion o f the TIM P s in thes e

s tud ies i s to inh ib i t ma t r ix deg rada t ion by me ta l lo -

p ro te inas es . T he a im o f th i s r ev iew i s to examine

t h e e x p r e s s i o n o f m e m b e r s o f th i s f a m i l y o f p ro -

t e inas es in human tumour s amples and eva lua te

c r i t i ca l ly the i r r o le in tumour invas ion and me ta -

stasis .

369

a t r i x m e t a l lo p r o t e i n a s e s

M M P ) f a m i l y

S t r u c t u r e a n d f u n c t i o n

T h e M M P s a r e a m u l t i g e n e f a m i l y o f m a t r i x

deg rad ing z inc me ta l lop ro te inas es tha t deg rade a t

l eas t one com pone n t o f the ex t r ace l lu la r ma t r ix.

M o l e c u l a r c l o n i n g o f th e v a r i o u s f a m i l y m e m b e r s

has r evea led cons ide rab le amino ac id cons e rva t ion

and r a i s es the pos s ib i l i ty tha t the genes a ros e by

dup l i ca t ion o f a s ing le p r imord ia l gene . S o f a r in

h u m a n s , e i g h t m e m b e r s o f t h e M M P f a m i l y h a v e

been iden t i f i ed and cha rac te r i zed Tab le 1 ). The

M M P s h a v e b e e n k n o w n b y v a r io u s n a m e s t h r o u g h

i s o la t ion by d i f f e r en t g roups , bu t f o r the pu rpos es

o f th is r e v i e w t h e d e s i g n a ti o n r e c o m m e n d e d b y t h e

I n te r n at io n a l U n i o n o f B i o c h e m i s t r y a n d M o l e c u l a r

B i o l o g y w i l l b e u se d . T h e r e f o r e t h e 7 2 k D a a n d 9 2

kD a type IV co l l agenas es w i l l be r e f e r r ed to a s

ge la t inas e A and ge la t inas e B r es pe c t ive ly , s imi la r-

l y P U M P - l , p u t a ti v e m e t a l l o p r o te i n a s e ) w il l b e

refer red to as matr i lys in .

A na lys i s o f the amino ac id s equenc es r evea l s

tha t thes e p ro te in s con ta in s eve ra l d i s t inc t dom ains

t h a t a r e c o n s e r v e d a m o n g t h e v a r i o u s m e m b e r s

Fig . 1). The f i r s t of these is the lead er seq uen ce

-17 amino ac id s ) w h ich t a rge t s the mo lecu le fo r

s ec re t ion , i s s ubs equen t ly r emoved and i s the r e fo re

no t p r es en t in the l a t en t enz ym e W i lhe lm

e t a l .

1987). The s econd domain i s the p ropep t ide ~ 80

amino ac id s ) , w h ich has been s how n to be c l eaved

w h e n M M P s a r e a c t i v a t e d a n d w h i c h c o n t a i n s t h e

h i gh l y c o n s e rv e d s e q u e n c e P R C G V / N P D G r a nt e t

a l . 1987; S te t ler -S tevenson e t a l . 1989a ; N agas e

e t a l . 1 9 9 0 ) p r e s e n t i n a l l m e m b e r s o f t h e M M P

fami ly s ee F ig . 1 ) . The a va i l ab le da ta s ugges t tha t

th i s r eg ion i s invo lved in ma in ta in ing the enzyme

in a la tent s ta te s ince mutat ions in th is region resul t

in an enzyme tha t no longer r equ i r es p ro teo ly t i c

ac t iva t ion M at r i s i an e t a l . 1991). I n v i t r o this

ac t iva t ion can be ach ieved by a va r i e ty o f agen t s ,

inc lud ing o rganomercu r i a l s , ox idan t s , s u l fhyd ry l

a lky la t ing agen t s and , in s ome cas es , p ro teo ly t i c

c l e a v a g e b y tr y p s in o r p l a s m i n W i l h e lm e t a l .

1987; S te t ler -S tevenson e t a l . 1989a ; H e e t a l .

1989; Spr ingman e t a L 1990) . Th i s s pec t rum o f

ac t iva to r s s ugges t s tha t a con fo rmat iona l change i s


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Table 1

Proper t i es of human mat r ix meta l loprote inase

M M P No. ca) Nam e ~b) La ten t M r kDa) De grad es

A c t i ve

1 Inters t it i a l col lagenase 55 45

8 Neut rophi l col lagenase 75 58

2 G e l a t i na s e -A 72 66

Gela t inase-B 92 86

3 St romelys in-1 57 45

10 St rome lys in-2 57 44

7 Ma t r i lys in 28 19

Fibr i l l a r col lagens

Gela t in

Proteoglycan

As in ters ti t i a l col lagenase

Denatured col lagens

Col lagen IV, V, VII , X

Elas t in

As gela t inase-A

Proteoglycan

Col lagen I I , IV, XI

Gela t ins , l aminin ,

F i b r one c t i n

As s t romelys in-1

As s t romelys in-1

Elas t in

En t a c t i n

11 S t r om e l ys in - 3 51 44 U nk now n

At present the fami ly cons i s t s of e ight membe rs , the i r or ig ina l MM P class i f i ca tion a) i s g iven, fo l lowed by the na me recom-

men ded by the In ternat ion al Un ion of Biochem is t ry and Mo lecular B iology b) . The ext race l lu lar mat r ix subs t ra tes l i s ted are

representa t ive and are no t necessar i ly comprehens ive .

r equ i r ed fo r ac t iva t ion and tha t a th io l bond i s

i n v o l v e d . T h e c u r r e n t v i e w o f M M P a c t iv a t io n i s

tha t the N - te rm ina l pa r t o f the m o lecu le i s fo lded

a r o u n d i n t h e l a t e n t e n z y m e s o t h a t t h e c y s t e i n e

r e s i d u e i n t h e c o n s e r v e d P R C G V / N P D r e g i o n

complexes w i th a z inc mo lecu le . A c t iva t ion r e s u l t s

w h e n a c o n f o r m a t i o n c h a n g e d i s s o c i a t e s t h e c y s -

t e ine f rom the z inc a tom and r ep laces i t w i th w a te r ,

th i s has been r e f e r r ed to by V an War t and co l -

l eagues a s the cys te in e s w i tch mec han i s m (S p r ing -

m a n e t a l . 1990) . The ac t iva ted me ta i lop ro te inas e

i s then capab le o f au top ro teo lys i s . I n v i v o the

e n d o g e n o u s a c t i v a t o r i s t h o u g h t t o b e p l a s m i n

w h i c h h a s b e e n s h o w n t o a c t i v a t e M M P s i n a c o -

cu l tu re s ys tem o f ke ra t inocy tes and f ib rob las t s (H e

e t a l . 1989) . I n v i t r o o the r enzymes s uch as

ca theps in G and neu t roph i l e l a s t a s e have been

s how n to ac t iva te s t romelys in -1 and ge la t inas e A

(O kada and N akan i s h i , 1989) . I n s t romelys in -3

there is an addi t ional inser t of 10 amino acids

b e t w e e n t h e p r o p e p t i d e a n d t h e c a t a l y t i c d o m a i n

(Bas s e t e t a l . 1990) . The th i rd domain i s the

c a t a l y t i c d o m a i n ( - 1 7 0 a m i n o a c i d s ) w h i c h c o n -

t a i n s t h e c o n s e r v e d s e q u e n c e H E F / L / I G H , p o s -

tu la ted to be the z inc -b ind ing domain . The fou r th

dom ain w h ich i s p r es en t in al l m em ber s o f the

M M P s e x c e p t m a t r i l y s i n s h a r e s h o m o l o g y t o

hemopex in and v i t ronec t in (H un t e t a l . 1987) and

i s l inked to the ca ta ly t i c domain by a P ro - r i ch

s equence o f 5 - -10 amino ac id s . I t w as o r ig ina l ly

pos tu la ted to be invo lved in s ubs t r a te s pec i f i c i ty

bu t , a s can be s een f rom Tab le 1 , ma t r i ly s in s ub -

s t ra te specif ic i ty is s imilar to s t romelys in-1 and -2 .

Th i s domain i s r equ i r ed fo r the s pec i f i c b ind ing

and c leavage o f f ib r i ll a r co l l agen in hum an in te r -

s t it ia l co l l agenas e bu t no t s t romelys in -1 (C la rk and


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371

M a t n l y s i n

PRCGVPD

I i : i : i i t

St rome lys in- 1

St romelys in-2

S t r o m e l y s i n - 3

In t er s t i t i a l Col l agenase

N e u t r o p h i l C o l l a g e n a se

G e l a t i n a s e A

G e l a t i n a s e B 9 ~ 9 ~

n

S IG N A L S E Q U E N C E

P R O P E P T I D E

CATALYTIC DOMAIN

H E M O P E X I N D O M A I N

G E L A T I N B I N D I N G D O M A I N O F F IB R O N E C T IN

C O L L A G E N B I N D I N G D O M A IN

F i g 1 D o m a i n s t r u c t u re o f t h e M M P f a m i l y m e m b e r s . A l l m e m b e r s o f t h e M M P f a m i ly c o n t a i n a t le a s t th r e e p ro t e i n d o m a i n s : a p r e

d o m a i n e n c o d i n g t h e l e a d e r s e q u e n c e t h a t ta r g e ts t h e e n z y m e s f o r s e c r et io n , a p r o d o m a i n w h i c h i s r e m o v e d w h e n t h e e n z y m e b e c o m e s

a c t i v a t e d , a n d t h e c a t a l y t ic d o m a i n w h i c h c o n t a i n s t h e z i n c b i n d i n g re g i o n . A l l m e m b e r s e x c e p t m a t r i ly s i n c o n t a i n a d o m a i n t h a t s h a r e s

h o m o l o g y w i t h h e m o p e x i n . G e l a t i n a s e A a n d B c o n t a i n a n a d d i t io n a l f ib r o n e c t i n d o m a i n , a n d g e l a t in a s e B c o n t a i n s a n a d d i t i o n a l

c o l l a g e n b i n d i n g d o m a i n . A d d i t i o n a l i n f o r m a t i o n o n t h e i r m o l e c u l a r w e i g h t s a n d s u b s t r a t e s p e c i f i c i ti e s i s g i v e n i n T a b l e 1 . E a c h d o m a i n

i s d e s i g n a t e d b y p a r t i c u l a r s h a d i n g a s i n d i c a t e d i n t h e f i g u r e .

Cawston 1989 ; Murphy

e t a l .

1992). The gelatin-

ases A and B bo th con ta in an add i t i ona l domain

which con ta ins t h r ee r epea t s o f a sequence homo-

logous to t h e ge l a t in b ind ing dom ain o f f i b ronec t in

Col l ier e t a l . 1988 ; Wi lhe lm e t a l . 1989), this

d o m a i n m a y m e d i a t e t h e s e q u e s t e r i n g o f t h e s e

enzy me s wi th in the ex t r ace l lu l a r ma t rix . F ina lly , a t

t he C- t e rmina l end o f t he ge l a t inase B ca t a ly t i c

dom ain the r e i s an add i t iona l domain tha t has some

h o m o l o g y t o c o l la g e n W i l h e l m e t a l . 1989).

B r o a d l y s p e a k i n g t h e M M P s m a y b e d i v i d e d

into 3 subclasses wi th respect to thei r subst ra te

spec i f i c i t y ; t he type I co l l agenases , t he type IV

co l l agenases , and the s t r omelys ins see Tab le 1 fo r

add i t i ona l de t a il s on mo lecu la r we igh t s and sub -

s t r a t e s ) . The co l l agenase subc lass has two mere -

bers: in terst i t ia l col lagenase and neut rophi l col -

l agenase . Bo th these enzymes c l eave the a lpha

chains of types I , I I and I I I col lagen a t a s ingle

si te . In terst i t ia l col lagenase i s produced by f ibro-

b l as t s and macrophages in pa r t i cu l a r , wh i l e t he

express ion of neut rophi l col lagen ase i s rest r ic ted to

ce l ls o f the neu t roph il l ineage Has ty

e t a l .

1990a).

The co l l agenase type IV subc lass con ta ins two

member s : ge l a t inase A and ge l a t inase B . Bo th o f

these enzym es deg rade dena tu r ed co l l agens ge l -

a t in s ) and a r e spec i f i c f o r t he deg rada t ion o f t ype

I V b a s e m e n t m e m b r a n e c o l l a g e n . E x p r e s s i o n o f

ge l a t inase A i s w idesp read and i s f r equen t ly e l -

eva t ed in ma l ignan t t umour s , ge l a t inase B was

t r ad it i ona lly t hough t o f a s t he m acroph age ge l a t in -

ase bu t i ts exp ress ion has been desc r ibed in m a l ig -


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nan t ce l l s , neu t roph i l s , cy to t rophob las t s and ke ra -

t i nocy tes Co l l i e r

e t a l .

1988 ; Wi lhe lm

e t a l .

1989).

The th i rd subc l ass i s t he s t romelys in subcIass

w h i c h c o n t a i n s t h r ee m e m b e r s : t w o h i g h ly h o m o -

logous enzymes , s t romelys in -1 and-2 , and ma t r i l y -

s in . A poss ib l e fou r th member , s t romelys in -3 , has

b e e n c l o n e d B a s s a t e t a l . 1991) but i t s subst ra te

spec i f i c i t y has no t ye t been de t e rmined . Th i s

subc l ass has t he wides t subs t ra t e spec i f i c i t y and

degrades p ro t eog lycans and g lycopro t e ins such as

f ibronect in and laminin . This subclass has a lso

b e e n s h o w n t o c l e a v e t y p e I V c o l la g e n a n d d e g r a d e

e l as t i n w i th ma t r i l y s in be ing the mos t po t en t

W i l h e l m

e t a l .

1987 ; Murphy

e t a l .

1991).

Mat r i l y s in has recen t ly been shown to deg rade

e n t a c t i n , a b a s e m e n t m e m b r a n e p r o t e i n w h i c h

br idges l amin in and type IV co l l agen S i res

e t a l .

1993) . Ano the r recen t ly desc r ibed cDNA encodes

a mur ine me ta l l oe l as t a se wi th a domain s t ruc tu re

tha t i s s im i l a r t o t he co l l agenases and s t romelys ins

Shap i ro e t a l . 1992). S t romelysin-1 was orig inal ly

c loned us ing sub t rac t ive hybr id i za t ion f rom a v i rus

t rans fo rm ed ra t ce l l l i ne Mat r i s ian e t a l . 1985),

however , i t i s no t w ide ly expressed no rmal ly bu t

can be i nduced read i ly by g rowth fac to rs , t umour

p r o m o t e r s a n d o n c o g e n e s i n m e s e n c h y m a l c e ll s

such as f ibroblasts M atri s ian

e t a l .

1985; Kerr

e t

a l .

1988 , McDonne l l

e t a l .

1990a). S t romelysin-2

a n d m a t r il y s i n w e r e o r i g i n al ly c l o n e d f r o m h u m a n

t u m o u r s a m p l e s M u l l e r

e t a l .

1988), and have

b e e n f o u n d in a n u m b e r o f tu m o u r t y p e s B a s s at

e t

a I .

1990; Pajouh

e t a l .

1991 ; McDonne l l

e t a l .

1991) and a l so i n no rmal ce l l s i nc lud ing macro -

p h a g e s B u s i e k e t a l . 1992) and the endomet r ium

R o d g e r s e t a I . 1993).

R e g u l a t i o n o f t he M M P s

T h e e x p r e s s i o n o f t h e s e e n z y m e s i s v e r y h i g h l y

regu la t ed and occu rs a t man y d i f fe ren t l eve ls .

Regu la t i on occu rs a t t he t ransc r ip t i ona l l eve l by

gene express ion and a t the p ro t e in l eve l by p ro t eo -

ly r ic ac t i va t ion o r by com plex ing to TIMPs see

sec t ion on inh ib ito rs ) wh ich a re a l so i ndepend en t ly

regu la t ed . S tud ies ove r t he pas t seve ra l yea rs have

show n tha t M M Ps a re regu la t ed bo th pos i t ive ly and

nega t ive ly a t t he t ransc r ip t i ona l l eve l by a va r i e ty

o f g rowth fac to rs , cy tok ines , oncogenes and tum our

p romote rs .

There i s ev idence tha t pos i t i ve regu la t i on o f

st romelysin-1 and in ters t i t ia l col lagenase genes i s

m e d i a t e d b y t h e a c t i o n o f p r o t o o n c o g e n e s o n

spec i f i c DNA sequences wi th in t he p rox imal 210

bases o f t hese p romote rs . These genes con ta in a

T R E T P A r es p o n s iv e e l e m e n t ) o r A P - 1 a c t iv a t o r

p ro t e in - l ) b ind ing s i t e wh ich i s recogn ized and

t r a n sa c t iv a t e d b y t h e p r o d u c t o f t h e p r o t o o n c o g e n e s

c - f o s

and

c - j u n

A n g e l

e t a l .

1987; Kerr

e t a l .

1988; Schonthal

e t a l .

1988 ; McDonne l l

e t a l .

1990b). T hey a l so con ta in a PEA -3 e l em en t wh ich

recogn izes t he

c - e t s

o n c o g e n e W a s y l y k

e t a I .

1990, 1991). Mutat ional analysis has shown that

the AP-1 a lone , o r i n combina t ion wi th PEA-3

regu la t e t he basa l l eve l s and induc ib i l i t y o f t hese

genes by a va r i e ty o f agen ts Ange l e t a l . 1987;

W a s y l y k e t a l . 1991 ; Aub le and Br inckerhof f ,

1991) . We have p rev ious ly demons t ra t ed t ha t t he

induc t ion o f ra t s t romelys in by e p ide rm al g rowth

fac to r EGF ) requ i res t he i nduc t ion o f c - f o s and c -

j u n

pro toon conge ne p roduc t s and the ac t iva t ion o f

p ro t e in k inase C Mc Donn e l l

e t a l .

1990b). Induc-

t ion of in ters t i t ia l col lagenase by

H a - r a s v - m o s v -

s r c

and TPA a l so requ i res

c - f o s

i nduc t ion and the

T R E S c h o n t h a l

e t a I .

1988) . Tumour nec ros i s

factor-o~ TNF-c~) induct ion of in ters t i t ia l col lagen -

ase a l so co r re l a t e s w i th i nduc t ion o f

c - j u n

and

r e q u ir e s t h e T R E B r e n n e r

e t a l .

1989). Platelet

de r ived g rowth fac to r Ker r e t a l . 1988), nerve

g r o w t h f a c t o r M a c h i d a e t a l . 1989) and in ter-

leukin-1 Frisch and Ru ley, 1987; S i rum and

Br inckerhof f , 1989) i nduc t ion o f s t romelys in may

a l so ac t t h rough a s imi l a r pa thway . The p romote r

reg ion o f t he ge l a t inase A F r i sch

e t a l .

1990) is

ve ry d i f fe ren t f rom those o f s t romelys in -1 and -2

and co l l agenase , mos t no t ab ly by the abs ence o f an

obv ious TATA box and AP-1 s i t e . The ge l a t i nase

B g e n e c o n t a in s T A T A a n d A P - l - l i k e m o t if s b u t it

i s not c lear that they are act ing as t rue t ranscrip-

t iona l e l emen t s Huh ta l a

e t a l .

1990).

Recen t s tud ies Ker r e t a l . 1990) have shown

that the t rans form ing growth factor-]3 TGF-[3)

inh ib i t s t he EGF induc t ion o f s t romelys in gene

express ion th rough an ups t ream sequence re fe r red


7/18

to as the TGF-[3 inhib i tory e lement (TIE) . In teres t -

ing ly , the F os p ro te in i s p r es en t in the p ro te in

c o m p l e x t h a t r e c o g n i z e s t h e T I E e l e m e n t ( K e r r e t

a l . , 1 9 9 0 ) . R a h m s d o r f a n d c o l l e a g u e s h a v e a l s o

s ugges ted tha t g lucoc o r t i co id inh ib it ion o f co l-

l agenas e gene exp res s ion i s med ia ted by the in t e r -

ac t ion o f the g lucoco r t i co id r ecep to r and the

F o s / J u n p r o t e i n c o m p l e x e s ( J o n a t e t a l . , 1990).

Thus i t appea r s p robab le tha t p ro tooncogene

induc t ion b y g row th f ac to r s can be invo lved in bo th

pos i t ive and ne ga t ive r egu la t ion o f me ta l -

lop ro te inas e gene exp res s ion .

In te r es t ing ly , T IM P -1 and me ta l lop ro te inas es

appea r to be r ec ip roca l ly r egu la ted in s ome s ys -

t ems , TG F - [3 caus es an inc reas e in TIM P -1 mRN A

in human f ib rob las t s (O vera l l

e t a l . ,

1989), Ad-

d i t io n a l ly t h e m o u s e T I M P p r o m o t e r a l s o c o n ta i n s

an A P -1 s i t e and can be t r ans c r ip t iona l ly ac t iva ted

by m any o f the s ame agen t s tha t ac t iva te co l -

l agenas e o r s t romelys in (Campbe l l e t a l . , 1991). In

conc lus ion w e can s ay tha t the u l t ima te ma t r ix -

deg rad ing ac t iv i ty o f a me ta l lop ro te inas e i s the r e -

fo re s ub jec t to r egu la t ion a t mu l t ip l e l eve l s and

m u s t m e e t a n u m b e r o f r e q u i re m e n t s b e f o r e p r o t e o -

ly s i s occu r s : the p r es ence o f an induce r and ab -

s ence o f an inh ib i to r i s r equ ir ed fo r t r ans c rip t ion o f

t h e m R N A ; e n z y m e m u s t b e a c t i v a t e d a n d t h e

b a l a n c e b e t w e e n p r o t e a s e a n d i n h i b it o r m u s t f a v o u r

p ro teo lys i s .

Tissue inhibito rs of metalloproteinases TIM Ps)

The t i s s ue inh ib i to r s o f me ta l lop ro te inas es (TIM P s )

a re a mu l t igene f ami ly (S te t l e r -S tevens on e t a I . ,

1990) w h ich a t p r es en t cons i s t s o f tw o m em ber s ,

TIM P - I and TIM P -2 . There i s an ove ra l l 38

i d e n t i t y b e t w e e n t h e t w o h u m a n T I M P s a t t h e

amino ac id s equence l eve l , a l though the deg ree o f

s imi la r ity i s h ighe r a t 68 . Bo th con ta in 12 cys -

te ine res idues a t v i r tual ly ident ical pos i t ions c lear ly

ind ica t ing tha t the tw o p ro te in s a r e f rom the s ame

fami ly . Thes e tw o p ro te in s b ind noncova len t ly to

ac t ive me ta l lop ro te inas es in a 1 :1 mo la r r a t io and

s pec i f i ca l ly inh ib it t he i r enzym at ic ac t iv i ty . T IM P -1

a l s o f o rm s a c o m p l e x w i t h t h e i n a c ti v e p r o f o r m o f

g e l a ti n a s e B ( W i l h e lm e t a l . , 1989) , w h i l e TIM P -2

373

f o r m s a c o m p l e x s p e c i f i c a l l y w i t h t h e p r o f o r m o f

ge la t inas e A (S te t l e r -S tevens on

e t a l . ,

1989b) .

T1MP-1 is a g lycoprote in

M r

30 K da) and i t s

c D N A a n d a m i n o a c i d s e q u e n c e h a s b e e n r e p o r t e d

( D o c h e r t y

e t a l . ,

1985 ; Carmichae l

e t a , . ,

1986),

a l though pu r i f i ca tion and cha rac te r i za t ion o f the

p ro te in f rom rabb i t bone t i s s ue had ea r l i e r been

ach ieved by Caw s ton (1981) , and f rom va r ious

other sources a lso (Welgus and S tr ickl in , 1983;

M u r p h y e t a l . , 1981) . Ear ly s tudies inves t igat ing

t h e l i n k b e t w e e n T I M P - 1 e x p r e s s i o n a n d i n v a s i v e

ab i l i ty in ce l l l i ne mode l s , s how ed tha t T IM P -1

leve l s w ere dec reas ed 10 - -20 fo ld in h igh ly invas ive

as compared to the l eve l in no rmal o r poo r ly

invas ive ce l l s (H icks

e t a l . ,

1984) . The hypo thes i s

tha t dec reas ed TIM P leve l s r e s u l t ed in inc r eas ed

invas ive /me tas t a t i c p roper t i e s , w as fu r the r inves -

t iga ted u s ing an t i s ens e RN A to induce r educ t ion o f

TIM P leve l s in a non - invas ive mur ine ce l l l i ne .

U s ing an amnion invas ion as s ay , the ce i l s w h ich

h a d r e d u c e d T I M P e x p r e s s i o n w e r e s h o w n t o

bec om e invas ive in com par i s on to con~ xo l ce l l s

( K h o k h a e t a l . , 1989).

W i t h t h e a v a i l a b il it y o f r e c o m b i n a n t T I M P

(Carmichae l e t a l . , 1986) , numerous inves t iga to r s

have looked a t the po ten t i a l t he r apeu t i c u s e o f

TIM P -1 as an an t i - invas ive agen t . Recombinan t

T1M P ( rT1M P ) has been s how n to inh ib i t t he

invas ive ab i l i ty o f tumour ce l l s in bo th

i n v i v o

and

i n v i t r o as s ays . A dd i t ion o f 5 ~ g /ml rTIM P res u l t ed

in comple te inh ib i t ion o f the invas ive ab i l i ty o f a

mur ine me lanoma ce l l l i ne B16-F 10 in an amnion

invas ion as s ay . Th i s a s s ay meas u res the ab i l i ty o f

ce l l s to pene t r a t e an amnio t i c membrane i s o la t ed

f r o m h u m a n p l a c e n ta ( S c h u l t z e t a l . , 1988) .

To inves t iga te the i n v i v o e f f e c t o f r T I M P , a

h igh ly me tas t a t i c r a t embryo ce l l l i ne ( t r ans f ec ted

w i th H a - r a s ) k n o w n a s H R A S 1 w a s i n j e c t e d i n t o

the t a il ve in o f nude mice . T he ab i l i ty o f thes e ce l l s

to co lon ize the lungs w as inh ib i t ed by up to 83

fo l low ing r epea ted in jec tions o f rTIM P (A lva rez

e t

a l . ,

1990). Sy s tem ic adm inis t ra t ion of r 'I71MP has

a l s o been us ed s ucces s fu l ly in the t r ea tmen t o f

co l l agen - induced a r th r i t i s in mice (Carmichae l e t

a l . , 1989).

T h e s e c o n d m e m b e r o f t h e T I M P f a m i l y , T I M P -

2 i s a nong lycos y la ted p ro te in (M 23 kD a) w h ich


8/18

374

was i so l a t ed and c loned more r ecen t ly and by

severa l d i f f e r en t g roups DeC le r ck

e t a l .

1989;

G o l d b e r g

e t a l .

1989; S te t ler -Stevenson

e t a l .

1989b ; Boone

e t a l .

1990). T he ac t iv ity o f TIMP-2

has a l so bee n we l l cha r ac t e r i zed . It r eac t s s to ich io -

metr ical ly wi th act ive in terst i t ia l col lagenase and

a l so p r even t s t he ac t iva tion o f t h is enz ym e f rom i ts

5 2 k D a p r o f o r m to 4 2 k D a a c t iv e fo r m D e C I e r c k

e t a l . 1991a) . TIMP-2 has been pu r i f i ed f rom a

com plex i t f o rms w i th the inac t ive fo rm o f ge l at in -

a s e A W a r d e t a l . 1991). The b ind ing o f TIMP -2

to th i s ge l a t inase does no t p r even t t he ge l a t inase

beco min g ac t iva t ed thus sugges t ing tha t t he TIMP-

2 is bound to a s i te o ther than the act ive s i te . I t i s

t hough t t ha t t he TIMP-2 p r even t s au toca t a ly t i c

ac t iva t ion o f t he ge l a t inase How ard

e t a l .

1991).

A l s o t h e T I M P - 2 w h i l e c o m p l e x e d to g e l at in a s e A

has been shown to r e t a in inh ib i to ry ac t iv i ty f o r

o the r me ta l lop ro te inases Ko lke nbrock

e t a l .

1991).

The inh ib i to ry ac t iv i ty o f TIM P-2 has been show n

to be g r ea t e r f o r t he ge l a t inase A than tha t o f

T I M P - 1 H o w a r d e t a l . 1991) a l though both

TIMP-1 and TIMP-2 can a l so inh ib i t a l l member s

o f t he o the r me ta l lop ro te inase subc lasses Mat r i s -

ian, 1990).

The inh ib i to ry ac t iv i ty o f r ecombinan t TIMP-2

rTIMP -2) on the invas ion o f smoo th m usc le ce l l

mu l t i l aye r s by human f ib rosa r coma ce l l s HT-1080

h a s b e e n d e m o n s t r a t e d b y D e C l e r c k a n d c o l le a g u e s

D e C l e r c k

e t a l .

1991b) . Pur i f ied TIMP-2 has a lso

been show n to inh ib i t the ab i l it y o f HT-1080 ce l ls

to i nvade a ma t r ige l me mb rane Alb in i

e t a l .

1991) . In th is s tudy ant i -gela t inase A ant ibodies

we re used to show that i t i s speci f ical ly th is pro-

t e inase tha t i s r e spons ib l e f o r t he invas ive p roper -

t ies of these cel l s .

Whi l e t hese s tud ies u s ing pu r i f i ed and r ecom-

b inan t TIMPs have been qu i t e success fu l and a r e

though t t o be func t ion ing th rough inh ib i t i on o f

meta l lop ro te inase ac t iv i ty , t hey shou ld be in t e r -

p r e t e d w i t h s o m e c a u t i o n a s T I M P s h a v e b e e n

show n to ha ve som e g row th - f ac to r - li ke ac t iv ity

S te t l e r -S tevenson

e t a l .

1992) . Potent ia l ly , drugs

mimick ing the cys t e ine - con ta in ing pep t ide o r

p ropep t ide r eg ion cou ld be dev e loped as t he rapeu t -

i c agen t s f o r t he inh ib i t i on o f i nvas ion and me ta -

s t as i s. As ye t none o f t he MM Ps have been c rys t a l -

l i zed , once th i s has been ach ieved , i t may be

possib le to design agents to b ind to the act ive s i te

and thereby inhibi t thei r ac t iv i ty .

The ex i s t ence o f o the r i nh ib ito r s o f m e ta l -

lop ro te inases has a l so been demons t r a t ed . ~2 -

macrog lobu l in , a n onspec i f i c p ro t ease inh ib i to r has

poten t act iv i ty but due to i t s large s ize 750 kD a) ,

l ack o f spec i f i c i t y and i r r eve r s ib l e i nh ib i to ry

mechan i sm, i t s ac t iv i ty i s mos t l i ke ly conf ined to

p ro teases i n t he b loods t r eam W elgus and S t r i ck lin ,

1983) . O the r sec r e t ed inh ib i to r s w h ich a r e o f

smal l e r s i ze t han the TIMPs have been t e rmed

I M P s . T h e y a p p e ar t o s h a re s o m e s e q u e n c e h o m o -

logy wi th TIMPs bu t a r e i ndependen t ly r egu la t ed

B a n d a

e t a l .

1992) . Thei r s igni f icance

i n v i v o

has

ye t t o be de t e rmined .

x p r e s s i o n a n d lo c a li z a ti o n o f M M P s i n h u m a n

t n m o u r s

The express ion o f va r ious member s o f t he me ta l -

lop ro te inase f ami ly by tumour ce l l l i nes has l ong

been assoc i a t ed wi th m e tas t a ti c po ten t ia l . Som e o f

the ea r l i e s t exper imen t s conduc ted by Lio t t a and

h i s co l l eagues demons t r a t ed a pos i t i ve co r r e l a t ion

be tween type IV co l l agenase ac t iv i ty and the

invas ive po ten t ia l o f va r ian t s o f t he m ur ine m e lano-

ma ce l l l ine B16 Lio t t a

e t a l .

1980) . Recent

exper imen t s S r eena th

e t a l .

1992) have shown a

simi lar cor re la t ion in ra t embryo cel l l ines t rans-

fo rmed wi th r a s . However , i n t hese exper imen t s

cor re la t ion was seen wi th s t romelysin-1 and -2 , and

no t wi th ge l a tinase A o r B . M M P ex press ion i s a lso

assoc ia t ed wi th a r ange o f no rmal even t s such as

t rophob last i nvas ion du r ing p r egna ncy La la and

Graham, 1990) , endomet r i a l changes du r ing the

r ep roduc t ive cyc l e Rodger s e t a l . 1993) , d i f feren-

t i at i on o f mo nonu c lea r phagocy tes W elgus

e t a l .

1992) as wel l as o ther d isease s ta tes , pr incipal ly

ar thr i t i s Hasty

e t a l .

1990b).

S ince the i so l a t ion and c lon ing o f t he human

cDN As fo r t hese p ro t e inases M ul l e r

e t a l .

1988;

L e v y e t a l . 1991) , i t has been possib le for resear -

che r s t o examine human tumour t i s sue fo r me ta l -

lop ro te inase expression . As can be seen f ro m Tab le

2 , an ex tens ive num ber o f s tud ies have been ca r r i ed


9/18

ou t in the pas t f ew yea r s examin ing a va r i e ty o f

t i s s ues , inc lud ing b reas t , co lon , p ros ta te , head and

neck , pu lmonary , and oes ophagea l . Tab le 2 , s um-

mar izes mos t o f thes e s tud ies , and a l s o s how s the

var ious t echn iques u s ed .

The exp res s ion o f me ta l lop ro te inas es in human

t u m o u r s h a s b e e n b e e x a m i n e d b y a v a r i e t y o f

t echn iques . Ea r ly s tud ies u s ed no r the rn b lo t t ing

M u l l e r e t a l . 1991) w h ich r equ i r ed a l a rge amoun t

o f t i s s ue and i s o la tion o f in tac t RN A . Rec en t

s tud ies have u s ed

i n s i t u

hybr id iza t ion , a ve ry

s ens i t ive and s pec i f i c me thod w h ich a l low s loca l i -

za t ion o f the m R N A to s pec i f i c ce l l types . A l -

though , th i s t echn ique can be t echn ica l ly d i f f i cu l t ,

the u s e o f non rad ioac t ive p robe s has mad e i t

p o s s i b l e t o b e m o r e r o u t in e l y u s e d K o m m i n o t h ,

1992) . H ow ever , i t has been s ugges ted by s ome

inves t iga to r s tha t fo r r e t ro s pec t ive s tud ies , p ro te in -

375

b a s e d t e c h n i q u e s s u c h a s i m m u n o h i s t o c h e m i s t r y

s hou ld be u s ed as a r ch iva l ma te r i a l i s un l ike ly to

have been co l l ec ted in RN as e f r ee cond i t ions . The

m R N A o f th e m e t a l l o p r o te i n a s e c a n a l s o b e q u a n -

t i t a t ed u s ing a mod i f i ca t ion o f the P CR reac t ion ,

r eve r s e t r ans c rip t ion P C R RT -P CR ) , th is techn ique

has been r epo r ted a s be ing ab le to de tec t the

m R N A f r o m a s in g le e m b r y o R a p p o l e e e t a l .

1988). O ne o f the advan tages o f thi s me thod i s tha t

t h e R N A d o e s n o t n e e d t o b e f u l l y i n t a c t a n d c a n

be ampl i f i ed d i rec t ly f rom a rch iva l ma te r i a l S h i -

b a t a

e t a l .

1988).

A n t ibod ies bo th mo noc lona l and po lyc lon a l ) a r e

now ava i l ab le to many o f the me ta l lop ro te inas es .

T h u s t h e t e c h n i q u e o f i m m u n o h i s t o c h e m i s t r y h a s

made i t pos s ib le to loca l i ze exp res s ion o f the

p ro te in to s pec i f i c ce l l types . A number o f s tud ies

h a v e u s e d b o t h i m m u n o h i s t o c h e m i s t r y t o l o c a l i z e

T a b l e 2 .

E x p r e s s i o n o f M M P s i n h u m a n t u m o u r s

L o c a t io n c a r c in o m a M M P M e t h o d a R e f e r e n c e

B r e a s t S t r o m e l y s i n - 3 N B , I S H

G e l a t i n a s e B I H C

G e l a t i n a s e B E L I S A

C o l o n

G a s t r i c

H e a d a n d N e c k

M a t r i l y s i n N B , I S H , I H C

G e l a t i n a s e A N B , I H C

G e l a t i n a se A I H C

M a t r i l y si n N B , I S H , I H C

S t r o m e l y s in - 2 N B , I S H , I H C

S t r o m e l y s i n - 3 N B , I S H , I H C

I n t e r s ti t i a l c o l l a g e n a s e N B , I S H , I H C

I n t e r s ti t i a l c o l l a g e n a s e I S H

O e s o p h a g e a l G e l a t i n a se A I H C

S t r o m e l y s i n - 1 I H C

P r o s t a t e M a t r i l y s i n N B , I S H

G e l a t i n a se A N B

P u l m o n a r y

B a s s e t e t a l . 1 9 9 0

M o n t e a g u d o

e t a l .

1 9 9 0

Z u c k e r e t a l . 1 9 9 3

M c D o n n e l l e t a l . 1991

L e v y e t a l . 1991

P o u l s o m

e t a l .

1 9 9 2

M c D o n n e l l

e t a l .

1991

M u l l e r e t a l . 1991 h, 11993

M u l l e r

e t a l .

1 9 9 3

M u l l e r e t a l . 1991 b, 199 3

G r a y e t a l . 1 9 9 2

S h i m a e t a l . 1 9 9 2

S h i m a

e t a l .

1 9 9 2

P a j o u h

e t a l .

1991

P a j o u h e t a l . 1991

S t r o m e l y s i n - 3 N B , I S H U r b a n s k i e t a l . 1 9 9 2

G e l a t i n a s e A a n d B N B , I S H U r b a n s k i e t a l . 1 9 9 2

I n t e r s t it i a l c o l l a g e n a s e N B , I S H U r b a n s k i

e t a l .

1 9 9 2

T h i s t a b l e s u m m a r i z e s s o m e o f t h e s t u d ie s t h a t h a v e b e e n u n d e r t a k e n i n t h e p a s t fe w y e a rs , e x a m i n i n g t h e e x p r e s s i o n o f M M P s i n

h u m a n t u m o u r s p e c i m e n s . V a r i o u s d i f fe r e n t t e c h n i q u e s h a v e b e e n u s e d t o e x a m i n e t h e i r e x p re s s io n . a ) A b b r e v i a t i o n s us e d : N B - -

n o r t h e r n b l o t t i n g , I H C - - i m m u n o h i s t o c h e m i s t r y , a n d I S H - - i n s i t u h y b r i d i z a t i o n . b ) T h e s t u d i e s c o n d u c t e d i n 1 9 9 ] o n l y u s e d

n o r t h e r n b l o t t i n g f o r a n a l y s i s .


10/18

376

the p ro t e in and

i n s i t u

hybr id i za t ion to l oca l i ze t he

m R N A ( M c D o n n e l l

e t a l .

1991; Mul ler

e t a l .

1 9 9 3 ) . R e c e n t l y a n u m b e r o f E L I S A s h a v e b e e n

es t ab l i shed w h ich a l l ow d i rec t quan t if i ca t ion o f t he

l e v e l s o f M M P s e x p r e s s e d . A n E L I S A f o r t h e

de t ec t ion o f ge l a t i nase B in p l a sma showed con-

cen t ra t i ons o f ge l a t i nase B f rom normal hea l thy

sub jects to be 9 _+ 11 ng/lal and in patients w ith

gas t ro in t es t i na l cance rs t he p l a sma ge l a t i nase B

level was increased to 18 + 23 ng/pl and to 21 +_

22 ng/ la l in breast cancer pat ients (Zucker e t a l .

1993) . Ano the r ELISA fo r t he quan t i f i ca t i on o f

s t r o m e l y s i n h a s b e e n d e v e l o p e d a n d u s e d f o r

measu r ing the l eve l s o f s t romelys in i n a r th r i t i c

pa t i en t s (Oba ta

e t a l .

1992).

Unfo r tuna t e ly , i t i s beyond the scope o f t h i s

rev i ew to exa min e in de t a i l a ll o f t he s tud ies l i s ted

in Tab le 2 , i n s t ead we w i ll focus on a few spec i f i c

e x a m p l e s . T h e c o e x p r e s s io n o f s e v e r a l m e m b e r s o f

t h e M M P g e n e f a m i l y a p p e a r s t o b e a g e n e r a l

cha rac t e r i s t i c o f human ca rc inomas , and has been

obse rv ed in b reas t (Basse t e t a l . 1990 ; M on teagu do

e t a I . 1990) , co lon (Levy e t a l . 1991 ; McDonne l l

e t a l . 1991) and prosta te (Pajouh e t a l . 1991).

There a re a l so a num ber o f t is sues wh ere pa r ti cu l a r

me ta l l op ro t e inases a re exp ressed in p re fe rence t o

o the rs . S t romelys in -3 has been shown to be ex -

press ed in 100 (30 of 30) of a l l invasive breast

cance rs ana lysed so fa r (Basse t

e t a l .

1990). The

s t r o m e l y s i n - 3 c D N A w a s c l o n e d f r o m a e D N A

l ib ra ry cons t ruc t ed f rom a b reas t cance r su rg i ca l

spec imen ra the r t han an es t ab l i shed b reas t cance r

ce l l l i ne , i n o rde r t o have bo th neop las t i c and

s t romal ce l l com ponen t s . In t e res t i ng ly , in t h i s s t udy

s t romelys in -3 mRNA express ion i s re s t r i c t ed t o t he

s t romal ce l l s immed ia t e ly su r round ing the i s l ands

o f ma l ignan t ep i the l i a l ce l l s o f t he i nvas ive com-

ponen t s o f t he t umours . I t has been sugges t ed tha t

s t romelys in -3 i s one o f t he s t rom a-der ived fac to rs

tha t have long b een pos tu l a t ed t o p l ay an impor t an t

ro l e i n p rog ress ion o f ep i the l i a l ma l ignanc i es . In

th is s tudy, the gela t inase A, s t romelysin-1 , s t ro-

melys in -2 and ma t r i l y s in were expressed in bo th

mal ignan t and ben ign tumours , w h i l e the ge l a t inase

B and in t e rs ti t ia l co l l agenase w ere de t ec t ed in on ly

the b reas t ca rc inom as bu t no t t o t he sam e ex t en t a s

s t romelys in -3 .

A m o r e r e c e n t s t u d y ( M u l l e r

e t a l .

1993) has

shown tha t s t romelys in -3 i s exp ressed in 95 (106

o f 1 11 ) o f h e a d a n d n e c k s q u a m o u s c e l l c a r c in o m a s

e x a m i n e d . T h e s t r o m e l y s i n - 3 m R N A a n d p r o t e i n

was spec i f i ca l ly de t ec t ed in s t romal ce l l s imm e-

d ia t e ly su r round ing invas ive can ce r ce l l s ( s imi l a r t o

b reas t ca rc inomas) . They a l so obse rved tha t t u -

m o u r s w i th h i g h l e v e ls o f s t r o m e l y s in - 3 R N A w e r e

more l i ke ly t o exh ib i t h igh loca l i nvas iveness ,

sugges t ing tha t s t romelys in -3 m ay co n t r ibu t e to t he

p r o g r e s s i o n o f h e a d a n d n e c k c a r c i n o m a s . T h e

leve ls o f s t rom elys in -2 and in t e rs ti t ia l co l l agen ase

were a l so e l eva t ed in head and neck squamous

ca rc inomas (Mul l e r

e t a l .

1991; Mul ler

e t a l .

1 9 9 3 ) . H o w e v e r , t h e s t r o m e l y s i n - 2 m R N A w a s

loca l i zed spec i f i ca ll y to t he neop las t i c ce l l s , and the

in t ers t it i al co l l agenase mR NA was loca l i zed to bo th

s t romal and neop las t i c ce l ls (Mu l l e r

e t a l .

1993).

In con t ras t t o t he above s tud i es , ma t r i l y s in

express ion has been found to be re s t r i c t ed t o

main ly neop las t ic ce ll s in co lon , s t omac h (M cDo n-

nel l e t a l . 1991) and prosta te (Pajouh e t a l . 1991)

ca rc inomas . We have recen t ly ex t ended ou r i n i t i a l

obse rva t ions on ma t r i l y s in express ion in co lon

ca rc inom as by inves t i ga t ing the s t age o f co lo rec t a l

t umo ur p rog ress ion assoc i a t ed wi th ma t r i l y s in gene

express ion . Th i s was don e by exam in ing a se r ie s o f

co lon l e s ions rang ing f rom smal l adenomas to

ca rc inomas . P re l imina ry da t a sugges t s t ha t ma t r i l y -

s in express ion a r i ses i n a reas o f ben ign po lyps

d e s ti n e d to b e c o m e m a l ig n a n t c a r c i n o m a s ( N e w e l l

e t a l .

1993). S im i l a r ly ge l a t i nase A has be en fou nd

b y i m m u n o h i s t o c h e m i c a l m e t h o d s t o b e e x p r e s s e d

spec i f i ca l l y i n neop las t i c ce l l s i n b reas t (Mon-

t eagudo e t a l . 1990) and in co lon (Levy e t a l .

1991) carc inomas.

I t appea rs f rom a numb er o f s tud i es tha t ce r t a in

meta l lop ro t e inases may be expressed in a t i s sue

spec i f i c manner , s t romelys in -3 i n s t romal ce l l s o f

b reas t ca rc inomas (Basse t

e t a l .

1990) and matr i ly-

s in in epi thel ia l ce l l s of colon and prosta te (Pajouh

e t a l .

1991 ; McDonne l l

e t a l .

1991). A s imi lar

t i s sue spec i f i c i t y i s a l so seen in cyc l ing human

e n d o m e t r i u m , o n e o f t h e f e w e x a m p l e s o f n o r m a l

t i s sue i n wh ich me ta l l op ro t e inase express ion has

b e e n o b s e r v e d ( R o d g e r s e t a l . 1993). M atr i lysin i s

p resen t i n t he ep i the l i a l ce l l s o f p ro l i fe ra t i ve and


11/18

mens t rua l endomet r ium, w h i l e s t romelys in -1 i s s een

i n t h e s t r o m a l c o m p o n e n t d u r i n g t h e s a m e c y c l i c

in te rva l . The pos s ib i l i ty tha t t i s s ue - s pec i f i c e l e -

m e n t s m a y c o n t r o l t he e x p r e s s i o n o f m e t a l lo -

p ro te inas e w i l l be an in te r es t ing top ic fo r fu tu r e

research .

A s men t ioned p rev ious ly , the f ina l p ro teo ly t i c

ac t iv i ty o f me ta l lop ro te inas e w i l l be dependen t no t

on ly on the i r exp res s ion bu t a l s o on the i r ac tiva t ion

w hich i s l a rge ly con t ro l l ed by the i r inh ib i to r s , t he

TIM P s . A r ecen t s tudy (U rbans k i

e t a l .

1992) has

e x a m i n e d n o t o n l y t h e e x p r e s s io n o f m e t a ll o -

p ro te inas es in pu lmonary ca r c inomas bu t a l s o the i r

inh ib i to r s . Bo th TIM P genes w ere exp res s ed in a l l

no rmal and ma l ignan t t i s s ue s amples ana lys ed . Tw o

ma jo r c l a s s es o f TIM P -2 t r ans c r ip ts o f 3 .5 kb and

1 .0 kb w ere obs e rved . I n a lmos t a l l cas es the 3 .5

kb s pec ies w as the mos t abundan t w i th the 1 .0 kb

f o r m b e i n g b a r e l y v i si b l e f o r s o m e t u m o u r R N A . I n

s i t u h y b r i d i z a t i o n s h o w e d T I M P - 1 a n d T I M P - 2

t ra n s c ri p ts e x p r e s s e d p r e d o m i n a t e l y o v e r t u m o u r

s t romal ce l l s . Thes e f ind ings s uppor t the idea tha t

t h e T I M P s m a y p l a y a r o le i n h u m a n n e o p l a s i a d u e

to thei r ab i l i t ies to inhib i t the act ive forms of

meta l lop ro te inas es . I t i s impor tan t f o r fu tu r e w ork

to look no t on ly a t the exp res s ion o f me ta l lo -

prote inases but a lso thei r inhib i tors .

I n v i t r o m o d d s

M os t o f the da ta p r es en ted in the above s ec t ion

s h o w s a c o r r e la t i o n b e t w e e n M M P e x p r e s s i o n a n d

tumour fo rmat ion , s ome s tud ies even s how co r -

r e l a t ion w i th ex ten t o f me tas t a s i s (M ul le r

e t a l .

1 99 3). T h e l e v e l s o f M M P s h a v e b e e n m a n i p u la t e d

b y m o l e c u l a r b i o l o g y t e c h n iq u e s i n a n u m b e r o f

ce l l cu l tu r e ce l l l i nes . Thes e s tud ies s ugges t tha t the

exp res s ion o f thes e p ro te in s can con t r ibu te to the

invas ive and m e tas t a t i c ab i l i ty o f ma l ignan t ce l l s .

I n the fo l low ing s ec t ion w e w i l l l ook a t 3 s tud ies

w h ich have a t t emp ted to look d i r ec t ly a t t he e f f ec t

o f s t romelys in and ma t r i ly s in on the invas ive

b e h a v i o u r o f t u m o u r c e l l l in e s.

S tud ies u s ing the c l a s s i ca l tw o- s t age mode l o f

c h e m i c a l c a r c i n o g e n e s i s i n m o u s e s k i n d e m o n -

s t r a ted a p os i t ive co r r e l a tion be tw een s t romelys in -1

377

m R N A e x p r e s s i o n a n d t u m o u r p r o g r e s s io n ( M a t ri s -

ian e t a l . 1986) . Th i s p ro toco l cons i s t en t ly p ro -

duces ben ign pap i l lomas a f t e r t r ea tmen t w i th a

s in g l e d o s e o f d i m e t h y lb e n z ( a ) a n th r a c e n e ( D M B A ) ,

f o l l o w e d b y r e p e a t e d a p p l i ca t io n o f t u m o u r p r o m o -

te r 12 -O - te t r adecanoy l pho rbo l -13 -ace ta t e (TP A ) .

A t 25 - -30 w eeks fo l low ing in i t i a l t r ea tmen t , 5 - -7

o f thes e an ima l s deve lo p s quam ous ce l l ca r c in -

o m a s . A n a l y s i s o f t h e m R N A e x t r a c te d f r o m t h e s e

tumour s r evea led tha t s t romelys in -1 t r ans c r ip t s

w ere de tec ted in 73 o f the ca r c inom as and in

on ly 6 pap i l lomas (M at r i s ian

e t a l .

1986) . The

mur ine pap i l loma ce l l l i ne S P -1 w as t r ans f ec ted

w i t h a v e c t o r e x p r e s si n g s t r o m e l y s i n c D N A ( M a -

tris ian

e t a l .

1991) under contro l of a s t rong v ira l

p romote r . F o r thes e s tud ies , a cD N A con ta in ing a

s ing le bas e -pa i r mu ta t ion r e s u l t ing in a va l ine to

g lyc ine change a t pos i t ion 92 w as u s ed . Th i s

muta t ion c aus ed au toac t iva t ion o f the s t romelys in -1

p ro tein , and r e s u l ted in ove r 90 o f the p ro te in

be ing s ec re ted in the ac t ive fo rm (P a rk e t a l .

1991) . The t rans fected SP-1 cel ls were tes ted in an

i n v i t r o

i nvas ion as s ay and s how ed up to a 2 .2 - fo ld

increase in thei r invas ive potent ia l . In th is

i n v i t r o

i nvas ion as s ay ce l l s a re s eeded on to m a t r ige l - coa ted

f i lt e r s con ta in ing 10 tam po res in a M em bran e

Invas ion Cu l tu r e S ys tem (M ICS ) o r ig ina l ly deve l -

o p e d b y H e n d r i x a n d c o l l e a g u e s ( H e n d r i x

e t a l .

I987 ) . F o l low ing 72 -h incuba t ion , ce l I s tha t have

comple te ly invaded the ma t r ige l - coa ted f i l t e r s a r e

co l l ec ted by t rea tmen t w i th t r yps in -ED TA , s t a ined

w i th hematoxy l in and coun ted . Thes e r e s u l t s s how

tha t exp res s ion o f s t romelys in -1 con t r ibu tes to the

invas ive ab i l i ty o f thes e ce l l s , Expres s ion o f s t ro -

me lys in -1 i s a l a t e even t in tumour p rog res s ion

w hich co r r e l a t e s w i th the conver s ion o f a tumour

f rom the ben ign to the ma l ignan t s t a t e .

B o w d e n a n d c o l l e a g u e s h a v e o b s e r v e d t h a t

14 /18 p ros ta t e adenoca rc inomas exp res s ma t r i ly s in

m R N A ( P aj o uh

e t a l .

1991) . Us ing

i n s i t u

hybr id i -

za t ion th i s mRN A w as loca l i zed to ep i the l i a l ce l l s

and no t to s t romal o r in f l ammato ry ce l l s . Th i s da ta

s ugges t s tha t ma t r i ly s in may be invo lved in the

invas ion and m e tas t a s is o f p ros ta t e adenoca rc i -

nomas . T o t e s t t h i s hypo thes i s , a hum an p ros ta t e

cance r ce l l l i ne D U -1 45 w h ich does no t : exp res s

mat r i ly s in and i s nonmetas ta t i c w as t r ans f ec ted


12/18

378

w i th a p la s m id con ta in ing the fu ll l eng th ma t r i ly s in

c D N A P o w e l l e t a l . 1993). U s ing a s eve re com -

b i n e d i m m u n o d e f i c i e n t S C I D ) m o u s e m o d e l o f

tumour ce l l invas ion the ma t r i ly s in t r ans fec ted ce l l

l ines w ere obs e rved to invade th rough the d ia -

p h r a g m m o r e e f f e c t i v e l y th a n c o n t r o l t r a n s fe c t e d

ce l l l ines . Thes e s tud ies s ugges t a func t iona l ro le

fo r m a t r i ly s in in the in i t ia l invas ion o f p ros ta te

cancer .

In a p rev ious s tudy w e have s how n tha t ma t r i ly -

s in i s exp res s ed in 8 /10 gas t r i c ca rc inomas and 6 /8

c o l o n c a r c i n o m a s e x a m i n e d M c D o n n e l l

e t a l .

1991) . U s ing

i n s i t u

hybr id iza t ion and an a f f in i ty

pu r i f i ed an t ibody , the exp res s ion o f ma t r i ly s in

m R N A a n d p r o t e i n w a s l o c a l i z e d t o t u m o u r c e l l s

a n d w a s n o t d e t e c t e d i n s t r o m a l o r l y m p h o c y t i c

c e l ls . W e h a v e c o n d u c t e d a s e ri e s o f e x pe r i m e n t s t o

de te rmine w he the r ma t r i ly s in exp res s ion i s caus a l ly

re la ted to co lon tumo ur ce l l invas ion and me tas ta s i s

by gene t i ca l ly a l t e r ing the exp res s ion o f ma t r i ly s in

i n t w o c o l o n d e r i v e d c e l l l i n e s . T h e S W 4 8 0 a n d

S W 6 2 0 c e l l li n e s p r o v i d e a n id e a l m o d e l s y s t e m to

tes t th i s hypo thes i s . The S W480 ce l l s a r e de r ived

f rom a p r imary ca rc inoma , do no t exp res s ma t r i ly -

s in RN A o r p ro te in , and a r e r e l a t ive ly non invas ive

in an

i n v i t r o

i n v a s i o n a s s a y . T h e S W 6 2 0 c e l ls a r e

d e r i v e d f r o m a l y m p h n o d e m e t a s t a s i s f r o m t h e

s ame pa t i en t a s the S W480 ce l l l ine , exp res s

mat r i ly s in RN A and p ro te in , and a r e s ign i f i can t ly

m o r e i n v a s i v e i n a n i n v i t r o invas ion as s ay com-

p a r e d t o t h e S W 4 8 0 c e l ls N e w e l l e t a l . 1993).

T h e S W 4 8 0 c e l l s h a v e b e e n t r a n s f e c t e d w i t h a

p las mid con ta in ing the fu l l l eng th ma t r i ly s in cD N A

a n d t h e n e o m y c i n r e s i s t a n c e g e n e . T h i s c D N A h a s

b e e n m u t a t e d i n t h e h i g h l y c o n s e r v e d P R C G V P D

reg ion s o tha t ma t r i ly s in p ro te in i s s ec re ted in an

ac t ive fo rm. The t r ans fec ted ce l l s d i s p layed va r ious

deg rees o f invas ivene s s , r ang ing f rom a 1 .5 fo ld to

4 fo ld inc reas e in invas ive ab i l i ty w hen compared

to con t ro l ce l l s t r ans fec ted w i th neomyc in on ly

N e w e l l e t a l . 1 9 9 3 ). W e n o w h o p e t o t r a ns f e c t th e

S W620 ce l l s w i th a p la s mid con ta in ing the ma t r i ly -

s in cD N A in the an t i s ens e o r i en ta t ion , thus e l imi -

nat ing express ion of matr i lys in in th is cel l l ine .

T h e s e e x p e r i m e n t s w i l l d e t er m i n e w h e t h e r m a t r il y -

s in exp res s ion p lays a func t iona l ro le in the in -

v a s i v e a n d m e t a s t a t ic a b i li t y o f c o l o n c a r c i n o m a

ce l l l ines . Th i s ev idence , in combina t ion w i th the

s tu d i es o f V o g e l s t e in a n d c o w o r k e r s w h o h a v e

charac te r i zed man y o f the gene t i c a l t e ra t ions w h ich

occu r in co lon ca rc inogenes i s , may r es u l t in a more

t h o ro u g h u n d e r st a n d in g o f t h e g e n e t i c e v e n t s w h i c h

lead to ma t r i ly s in exp res s ion and co lon tu rnou t

p rog res s ion .

S tud ies in w h ich the l eve l s o f m e ta l lop ro te inas es

h a v e b e e n m a n i p u l a t e d b y m o l e c u l a r b i o l o g y

techn iques s ug ges t tha t the exp res s ion o f thes e

p ro te in s can con t r ibu te to the invas ive and me tas -

t a t ic ab i l i ty o f ma l ignan t ce l l s . H o w e ver , ev ide nce

o b t a i n e d f r o m e x a m i n i n g t h e e x p r e s s i o n i n h u m a n

tumor s amples ind ica tes tha t l eve l s o f more than

o n e m e m b e r o f th i s f a m i l y s h o u l d b e m a n i p u l a t e d

a t any one t ime . F o r example , w i th cu r r en t t ech -

n iques i t s hou ld be po s s ib le to t r ans fec t ma t r i ly s in -

exp res s ing ce l l s w i th o the r me ta l lop ro te inas es and

then look to s ee i f the invas ive ab i l i ty inc reas es

synergis t ical ly .

A n t i m e t a s t a t i c g e n e n m 2 3

N o r e v i e w o f i n v a s i o n a n d m e t a s t a s i s w o u l d b e

c o m p l e t e w i t h o u t s o m e d i s c u s s i o n o n m e t a s t a s i s

s uppres s o r genes . A s men t ioned b r i e f ly in the

in t roduc t ion , the ma l ignan t pheno type i s the cu l -

mina t ion o f a s e r i e s o f gene t i c changes tha t in -

vo lves bo th ac t iva t ion o f oncog enes and lo s s o f

tumour s uppres s o r genes . I n i t i a l ev idence fo r the

ex i s t ence o f genes w i th me tas ta s i s s upp res s ing

ac t iv it i e s cam e f rom ce l l f u s ion exper im en ts S ide -

bo t tom and C la rk , 1983) . The lo s s o f the me tas ta t i c

pheno type f rom hybr id s r e s u l t ing f rom the fu s ion

o f a no rmal ce l l and a me tas ta t i c tumour ce l l

ind ica ted the ex i s t ence o f gene s ) in the no rm al ce l l

tha t cou ld r eve r s e /ove rcome the me tas ta t i c p roper -

t i e s o f the tum our ce l l s . The hyb r id s w ere tumour i -

gen ic ind ica t ing tha t the gene s ) l inked to m e tas -

t a s is w a s independen t o f thos e invo lved in ce l l

g row th con t ro l.

A pos s ib le cand ida te fo r a me tas ta s i s s upp res s o r

gene w as f i r s t i s o la ted u s ing d i f f e r en t ia l hy b r id iza -

t i o n w i t h m R N A s e x t r a c t e d f r o m h i g h a n d l o w

metas ta t i c va r i an t s o f a mur ine me lanoma ce l l l ine

K - 1 3 7 5 S t e e g

e t a l .

1 9 8 8 ) . T h e i s o l a t e d c D N A


13/18

w as then us ed in N or the rn b lo t s to s how tha t i t s

exp res s ion inve r s e ly co r r e l a t e s w i th me tas t a t i c

po ten t i a l . I n fu r the r s tud ies to t a l RN A w as i s o la t ed

f rom in f i l t r a t ing duc ta l ca r c inomas and f ib roadeno-

mas (ben ign t i s s ue ) o f b r eas t and p robed fo r nm23

e x p r e s s i o n ( B e v i l a c q u a e t a l . 1989) . The resul t

c o r r e l a t e d w i t h l e v e l o f l y m p h n o d e i n v o l v e m e n t ,

a c o m m o n l y u s e d m a r k e r i n b r e a s t c a n c e r . S i g -

n i f i can t ly a l l pa t i en t s w i th lymph node invo lvemen t

s h o w e d l o w e r l e v e l s o f n m 2 3 t h a n t h e f i b ro a d e n o -

ma pa t i en t s . I n s amples f rom pa t i en t s w i thou t

l y m p h n o d e i n v o l v e m e n t , a ra n g e o f n m 2 3 e x p r e s -

s ion l eve l s w ere s een and i t w as s ugges ted tha t

s o m e o f t h e h i g h e r l e v e l s m a y b e d u e t o c o n t a m i -

na t ing no rmal t i s s ue . S tud ies s uch as thes e and a

s imi la r one co r r e l a t ing low nm23 exp res s ion w i th

r educe d d i s eas e f r ee in t e rva l and ove ra l l s u rv iva l in

b reas t cance r pa t i en t s (H ennes s y e t a l . 1991) ,

s ugges t the va lue o f nm23 as a p rognos t i c ind i -

ca to r . Befo re i t s u s e fu lnes s can be t r u ly a s ce r t a ined ,

l a rge s ca le p ros pec t ive and r e t ro s pec t ive s tud ies a r e

neces s a ry . A s no ted ea r l i e r t echn iques s uch as RT-

P C R , i n s i t u hybr id iza t ion and immunoh i s to -

chem is t ry m ay be s u i t ab le fo r r e tro s pec t ive s tud ies.

A n i m p o r t a n t q u e s t io n c o n c e r n i n g r e d u c e d n m 2 3

leve l s i s w he the r th i s caus es me tas t a s i s o r i s

a s s oc ia t ed w i th i t . To ans w er th i s ques t ion , t r ans -

f ec t ions o f h igh ly m e tas t a t ic K -137 5 mu r ine me la -

n o m a c e l l s w i t h n m 2 3 w e r e p e r f o r m e d ( L e o n e

e t

a l .

1991) . S tab le h igh -exp res s ing c lones w ere

i s o l a t e d a n d e x a m i n e d f o r t u m o u r i g e n i c i t y a n d

metas ta t i c po ten t i a l by in jec t ion in to mice . The

p e r c e n t a g e o f m i c e t h a t d e v e l o p e d p r im a r y t u m o u r s

a f t e r in j ec t ion w i th r im23 c lones w as l e s s w hen

compared w i th mice in jec ted w i th con t ro l ce l l s .

A l s o , tumour s i ze w as , on ave rage 2 .7 t imes

s mal l e r in the nm23 g roup than the con t ro l g roup .

Th i s s tudy p rov ided c l ea r ev idence tha t nm23 i t s e l f

can s upp res s ce r t a in tumour igen ic and me tas t a t i c

p roces s es in the s ys tems examined .

A fu r the r s tudy inves t iga ted nm23 a l l e l i c de le -

t ions in co lo rec ta l ca r c inoma pa t i en t s (Cohn

e t a l .

1991) . The D N A o f 21 pa t ien t s f r ee o f me tas t a s is

w a s a n a l y z e d a n d f o u n d t o b e h e t e r o z y g o u s u s i n g

S ou the rn ana lys i s. O f thes e , 11 w ere found to have

an nm23 a l l e l i c de le t ion in tumour D N A . A f te r a

f o l l o w - u p t i m e o f 2 5 m o n t h s , t h e o c c u r r e n c e o f

3 7 9

metas ta s es deve lopmen t w as s ign i f i can t ly h ighe r in

the g roup w i th nm23 de le t ion a s compared to the

nonde le ted g roup . A more r ecen t s tudy has u s ed

R T - P C R t o e x a m i n e m o r e c l o s e l y t h e l i n k b e t w e e n

nm23 gene de le t ion and me tas t a s i s in co lo rec ta l

c a n c e r ( W a n g e t a l . 1993). U s ing p r ime r s f l ank ing

the en t i r e nm23 cod ing r eg ion , 533 bas e pa i r ( bp )

s equences w e re ampl i f ied . O f the 13 pa i r ed s am ples

(no rmal and tumour ) , 11 gave the expec ted s i ze

f r agmen t , one s ample had a 533 bp band in bo th

tumour and no rmal s amples and a s econd s mal l e r

470 bp band w as a l s o s een in the tu rnou t s ample .

The f ina l s ample s how ed 533 bp band in the

n o r m a l a n d 3 5 0 b p b a n d i n t h e t u m o u r . S e q u e n c e

ana lys i s s how ed the 533 f r agmen t s in a l l cas es to

be iden t i ca l to the 533 bp pub l i s hed da ta s equence .

T h e 4 7 0 b p w a s e q u i v a l e n t t o t h e n m 2 3 s e q u e n c e

w i th a 64 bp de le t ion w hereas the 350 bp f r agmen t

d i d n o t m a t c h a n y k n o w n s e q u e n c e .

A s ye t no de f in i t ive p roo f o f the no rmal b io -

chem ica l f unc t ion o f nm2 3 i s ava i l ab le . P os tu la -

t ions a s to i t s pos s ib le func t ion a r e bas ed on i t s

7 7 h o m o l o g y t o t h e D r o s o p h i l a d e v e l o p m e n t a l

g e n e a w d a n d i ts > 5 0 h o m o l o g y t o N D P k i n a s es

(Wal le t e t a l . 1990) . Mutat ions in the a w d gene in

D r o s o p h i l a l e a d s to a n u m b e r o f d e v e l o p m e n t a l

a l t er a t ions in t i s s ue morp ho log y and d i f f e r en t i a t ion

( D e a r o l f e t a l . 1988). The s im ilar i ty of nm 23 to

a w d

w ould s ugges t tha t i t i s i nvo lved in no rmal

t i s s ue deve lopmen t and tha t a l t e r a t ions in i t s

exp res s ion w ou ld r e s u l t i n abe r r an t deve lopmen t

pe rhaps con t r ibu t ing in the p rog res s ion tow ards

m a l i g n a n c y . T h e N D P k i n a s e s a r e a g r o u p o f

enzymes w hos e func t ion i s to ca ta lyze the t r ans f e r

o f a phos pha te g roup f rom nuc leos ide t r iphos pha tes

( N T P s ) t o n u c l e o s i d e d i p h o s p h a t e s ( N D P s ) . T w o

areas in w h ich they a r e pa r t i cu la r ly impor tan t and

w h e r e a b n o r m a l b e h a v i o u r m a y l e a d t o m a l i g n a n c y

are in mic ro tubu le fo rmat ion and G -p ro te in func -

t ion (L io t t a e t a l . 1991).

M ic ro tubu les a r e impor tan t am ongs t o the r th ings

in re l a t ion to mo t i l i ty and a r e nec es s a ry fo r co r r ec t

mito t ic sp indle format ion - - a l tera t ions in thei r as -

s e m b l y / d i s a s s e m b l y d u e t o i n c o r r e c t N D P k i n a s e

ac t iv i ty cou ld the re fo re in f luence the mo t i l e ab i l i ty

of a cel l and/or mitos is .

G -p ro te in s a r e ac t ive in s igna l t r ans duc t ion


14/18

380

relaying information from first messengers such as

hormones or growth factors through effectors to

second messengers within the cell. Their correct

function is dependent on their ability to replace

bound GDP with GTP and then to hydrolyze the

GTP to GDP in a cyclic manner that is equivalent

to a switching on/off mechanism Linder and

Gilman, 1992). NDP kinases and possibly nm23)

apparently have a role in the switching on event

and alterations in NDP kinase activity could result

in a particular G-protein being permanently switch-

ed on or off. This is important when it is realized

that G proteins regulate either by stimulation or

inhibition a wide range of ceil functions. It is also

possible that rim23 has other biochemical functions

which have not yet been elucidated.

Conclus ion

Evidence presented in this review strongly suggests

that the expression of members of the metallo-

proteinase family of proteolytic enzymes is as-

sociated with the progression of a tumour to a

malignant, invasive and metastatic state. The

induction of the metalloproteinase genes may be as

a result of the activation of oncogenes or the loss

of mmour suppressor genes in the turnout cells.

The specific metalloproteinase that is induced in a

tumour cell may be dictated by the tissue in which

the tumour arises. These results suggest that an

understanding of metalloproteinase expression and

proteolytic activity may lead to the development of

effective therapeutic agents with the potential to

reduce the incidence of metastatic cancer.

cknowledgement s

The authors would like to express their thanks to

the Health Research Board and the Irish Science

and Technology Agency EOLAS) for their con-

tinued support and funding. Many thanks also to

Geraldine Grant and Keara Halt who took time off

busy schedules to read this manuscript and make

helpful suggestions.

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