MEDIOS Y MATERIALES

A n o v e l m em b er o f th e LAP fam ily has been identified by several groups. It h a s been

designated M L -IA P by V u c ic et al. (2000), livin by K a s o f a n d G om es (2001), an d K IA P b y

L in et al. (2000). (T his p ro tein will b e referred to as liv in in th is thesis). L iv in en cod es a

p ro tein w ith a sing le B IR do m ain and a C O O H -term inal R IN G dom ain and it is rep o rted to

be appro x im ately 26 % hom ologous to survivin. E x p ressio n o f livin inhibits apoptosis,

w hereas a livin antisense co n struct has been show n to induce apoptosis. T h e livin antisense,

h ow ever, did no t reduce th e levels o f survivin, sugg estin g th a t th e con struct has a specific

effect on livin an d n o t on related IA P fam ily m em b ers (K a s o f an d G om es, 2001).

S im ilar to survivin, livin w as found to be cap able o f b in d in g to caspase-3 and - 7 an d it has

b een show n to inhibit th e p roteolytic p ro cessin g o f caspase-9 in vitro (K a so f an d G om es,

2001). L iv in ’s expression, like su rv iv in ’s, w as u nd etected in m an y no rm al a d u lt tissues, b u t

w as presen t in develo p m en tal tissue and several c an cer cell line, w ith h ig h e st levels in

m elan o m a cell lines. L iv in , how ever, seem s to h av e m o re restricted ex p ressio n th an

survivin w ith a n arro w er distribution (i.e. few er c a n ce r typ es) th an su rviv in in cancer

(K a so f an d G om es, 2001; V u cic et al., 2000),

In th e study rep o rted b y K a s o f and G om es in 2001, H e la cells w ere tra n sfe c te d w ith

ap o p to tic genes in th e P tracer v e c to r containing a G F P m ark er for ac ce ssin g viability.

T ransfection w ith pro -ap o pto tic genes, e.g. B ax, led to ~ 90 % reductio n in viability. C o ­

transfection o f B a x w ith eith er livin or survivin caused a 4-6 -fo ld increase in viability. It

w as fo u nd th at liv in show ed m ore anti-apoptotic activ ity th an survivin. S urvivin has been

show n prev io u sly to be less effective at inhibiting apoptosis th a t o th er IA P fam ily m em bers

(T am m et al., 1998). K a s o f et al. (2000) reported th at livin w as observed p red o m in an tly in

th e nucleus and in a filam entous pattern th ro u g h o u t th e cy top lasm and th a t su rv ivin w as

observed in th e sam e p attern, indicating th a t livin and su rv iv in h ave sim ilar su b cellu lar

localisation (K a so f e t al., 2001). In a separate study by A sh h ab et al. in 2001, tw o splice

variants o f liv in w ere identified i.e. livin a and P, rep resentin g th e lo n g er and shorter

D ifferences in ex p ressio n patterns o f livin a and w e re also reported. L iv in P, b u t n o t liv in a

w as fo u n d in fetal an d a d u lt k idney a n d in several o th er fetal tissu es, in d ic a tin g th a t it m a y

p lay a p h y sio log ical ro le d u rin g fetal developm ent. E x p re ssio n o f bo th v ariants is m o st

p ro m in en t in th e p lac e n ta (A shhab et al., 2001). F u rth erm o re, it w as fo u n d th a t liv in a , b u t

n o t P p ro tects cells from apoptosis induced by stauro spo rin e, b u t in contrast, apo p to sis

in itiated b y etop o sid e w as blocked only b y th e P isoform . T h e findings fro m th e s e tw o

1 .6 .6 Survivin expression, localisation and m echanism o f action

T he su b cellu lar localisation and function o f su rv iv in has resu lted in m u ch con tro versy a n d

has attracted a lot o f in terest in recen t years. Studies reveal th a t surv ivin is lo calised at th e

m ito tic spindle, binds caspases, a n d can thus p ro tect cells from apoptosis. S om e studies

h ave dem onstrated th a t survivin is expressed a t the G 2/M p h a se o f th e cell cycle (L i et al.,

1998 and K obayashi et al., 1999) and inhibition o f h u m an survivin has b een asso ciated

w ith cell cycle defects (L i e t al., 1998). In addition, su rv iv in ex p ression has b een fo u n d in

em b ryo n ic tissue and in proliferating hem atop oietic stem cells, as w ell as rep ro d u ctiv e

tissu e (K onno et al., 2000; E n d o h et al., 2001), co nfirm ing its cell cycle-specific function.

S urvivin m ay also p ro te c t cells from apoptosis b y b in d in g to caspases. T h e types o f

caspases involved, how ever, are still disputed (B anks et al., 2000; C o n w ay e t al., 2000;

O ’C on n o r e t al., 2000; Shin etal., 2001).

1.6.6.1 S u rv iv in as a c a s p a s e in h ib ito r

T here is m uch discrepancy reg ard in g th e role o f survivin as an in h ib itor o f c asp ase activity.

S urvivin has been p ro p o sed to interact physically w ith caspase-3 and -7 (C onw ay et al.,

2000; Shin et al., 2001; Suzuki et al., 2001). H ow ever, reco m b in an t survivin expressed as

an N -term in al h istid in e-tag ged pro tein was u nab le to b in d caspase-3 o r to in h ib it caspase-3

activ ity in vitro (V erdecia e t al., 2000). O ther reports also su g g est th at su rvivin does n o t

in h ib it caspase-3 activ ity (B anks eta l., 2000; R eed, 2001).

W h eth er th e interaction b etw een survivin and caspase-3 is direct o r n o t is still unclear.

H ow ever, th ere is evidence o f an in verse correlatio n b etw een th e tw o p roteins. O ver­

expression o f su rvivin in g astric tum ours and ad jacen t m u co sa is acco m p anied by a dow n-

regulation o f caspase-3, indicating th at these changes m ay p lay a role in the transfo rm ation

from norm al g astric m u cosa to g astric c an cer (K ania et al., 2003). In o v arian can cer cells,

exogenous expression o f procaspase-3 leads to up -reg u latio n o f w ild -ty p e su rviv in

expression, w h ile o v er-expression o f procaspase-3 alone does n o t in duce apoptosis. W hen

survivin expression is blocked b y a do m in an t n egativ e m utan t (T 34A ), apoptosis is

1 .6 .6 .2 Survivin as a regulator o f cell cycle

Survivin has b een identified as an interface b etw een cell-cycle p ro g ressio n and c o n tro l o f

apoptosis. It m ay b e req u ired to counteract a constitutive p athw ay th a t induces ap o ptosis

during m itosis. It is expressed prim arily during th e G 2 /M phase a n d asso ciates w ith

m icrotubules o f th e m ito tic spindle durin g m etap hase a n d w ith m id-b o dies during late

telophase. A sso ciatio n w ith p olym erised m icrotubu les is m ed iated by a C -term inal coiled-

coil dom ain, n o t th e B IR dom ain (Li et al., 1997).

Survivin appears to be closely linked to th e m ito tic process. In n o rm al tissu e it is o n ly

expressed a t detectab le levels at th e G 2/M p h ase o f th e cell cycle, su rv iv in p ro tein th en

m igrates to th e m icrotubules in a process sim ilar to ch rom o som al p assen g er protein s

(Skoufias e t al., 2000; Speliotes e t al., 2000; Suzuki et al., 2000, A dam s e t al., 2001).

Survivin has also b een classified as a chrom osom al p assen g er protein (T em m e et al., 2003)

th at interacts w ith A urora-B and IN C E N P (Skoufias e t al., 2000; U ren e t al., 2000;

W h eatley e ta l., 2001; B olton et al., 2 002) to fo rm a chrom osom al p assen g er co m plex th at

is p roposed to p lay a crucial role in th e execution o f cytokenesis. S urvivin localises A urora-

B to its substrates and enhances it kinase activ ity in vitro and in vivo (C hen e t al., 2003).

Survivin is also essential for chro m o som e alig n m en t an d check p oin t resp o n ses elicited b y

drugs th a t interfere w ith tension. C ells lackin g su rv ivin entered m ito sis w ith norm al

kinetics an d fo rm ed bipolar spindles, b u t w ere delayed in a p ro m etaph ase state. T h ey also

w ere u n ab le to alig n th eir ch rom osom es, failed to recru it A urora B to kinetocho res, an d

poly p loid at a v e ry h ig h frequency (L ens et al., 2003).

O ne o f th e critical requirem ents for su rvivin function is th e p h o sph orylatio n on T hr34 b y

the m itotic k in ase p3 4 cdc2-cyclin B1 (O ’C on no r et al., 2000), as show n w h en a

p hosphorylation -m im etic survivin m u tan t strongly inhibited p 5 3 -in d u ced apoptosis

(H offm an et al., 2002). T his m ethod o f p rev en tin g survivin p h o sp h orylation results in

caspase-9 -d ep en d ent apoptosis and an ti-can cer activity (M esri et al., 2001). F urtherm ore,

inhibition o f su rv iv in p ho sphorylation on T h r3 4 , by th e cyclin -d ep en d en t k inase inhibitor

flavopiridol, resulted in loss o f survivin ex p ressio n and en hanced tu m o u r cell apoptosis

induced b y an ti-can cer agents and it also su pp ressed tu m o u r grow th, w ith o u t toxicity, in a