Critically review the p53 tumour suppressor response pathway as a therapeutic target A dissertation presented by Nishachand Vohreh Student

Number. : S8735320FJ !" No. : 0#03$%%# "atch: ""S&' '0'2A For the "Sc ()ons* in "iomedica+ Sciences in the ni,ersity o- "rad-ord &i,ision o- "iomedica+ Sciences Schoo+ o- .i-e Sciences ni,ersity o- "rad-ord "rad-ord

Critically review the p53 tumour suppressor response pathway as a therapeutic target Abstract /53 is tumour suppressor is o-ten mutated in cancers. 0his +eads to the suppression o- the abi+ity o- p53 to induce apoptosis and ce++ cyc+e arrest in the presence o- a dama1ed 1ene o- en,ironmenta+ stresses. As such2 e3cessi,e ce++u+ar 1ro4th is seen. 0he response path4ay o- p53 has +on1 been a tar1et -or dru1 disco,ery. "y acti,atin1 the path4ay usin1 dru1s can be a 4ay to reacti,ate the 4ho+e path4ay and stop cancers -rom spreadin1 or 1ro4 e,en -urther. )ere2 dru1s can be c+assi-ied into those that acti,ate 4i+d type p53 that has be e3cessi,e+y de1raded by its ne1ati,e re1u+ator 5&52 or inacti,ated by ,ira+ oncoprotein and those that restore the -unction omutant p53 into 4i+d type p53. Ne4 strate1ies ha,e been disco,ered +i6e 7yc+otherapy 4hich tries to reduce the e--ect o- traditiona+ chemotherapy. 1. Introduction 5any o- human cancers are +in6ed 4ith the mutation o- the tumour suppressor 1ene p53 4hose -unction as a tumour suppressor protein (0S/* 4hich 4as disco,ered in '#7# 4hen a host protein8s -unction (no4 6no4n as p53* 4as seen to be a+tered by an onco1enic ,irus a-ter bindin1 to it (Linzer and Levine, 19 9! (Craw"ord and Lane, 19 9!. 9n -act2 ha+- o- a++ tumours contain a 0S/ that is inacti,ated by point mutations (#ogelstein, $. et al., %&&9!. A +ot o- research has been carried out -rom then ti++ no4 to determine the -unction2 structure2 etc o- this protein. "asica++y2 acti,ated p53 is made up o- -our identica+ monomers -ormin1 a tetramer. :ach monomer 4i++ consist o- the -o++o4in1 domains: a* transacti,ation b* pro+ine rich domain c* &NA

bindin1 domain and d* o+i1omerisation and re1u+atory domain ('rives and Lepten(o, 19))!.

p53 is a+so 6no4n as the ;1uardian o- the 1enome8 (*ao, $. et al., %&1&!. 9t protects &NA -rom ;attac6s8 such as dama1e2 &NA brea6s2 u+tra<,io+et (u,* radiation2 stress and onco1enes. /538s abi+ity to suppress tumours is because o- its abi+ity to acts as a se=uence speci-ic transcription -actor induced by &NA dama1e. As such2 it re1u+ates e3pression o- di--erent ce++u+ar 1enes to modu+ate ,arious ce++u+ar processes (+igure %!. 5utant p53 inacti,ates the tetramers containin1 4i+d<type (>0* p53 ma6in1 the protein non<-unctiona+ (+inlay, ,.C. et al., 19)9!. !nce the tumour suppressor 1ene product is inacti,ated2 bindin1 to the e--ector protein is b+oc6ed and as such pre,ents inhibition. )o4e,er2 it doesn8t mean that p53 doesn8t 1et inacti,ated by any other 4ay. 9n cancers containin1 a >0 p532 Vira+ (:%* and ce++u+ar (5&52* onco1enes can -unctiona++y inacti,e p53 as 4e++. (-ainaut, '. et al., 199 !.

7urrent+y2 many researchers ha,e embar6ed on the ?ourney to create a tar1eted cancer therapy based on the research done on the p53 path4ays. Some 4ays inc+ude the acti,ation o- >0 p53 to restore its transcriptiona+ acti,ity usin1 chemo<radiation2 1ene therapy and sma++ mo+ecu+es@ the rescue o- mutant p53 usin1 synthetic peptides and sma++ mo+ecu+es@ the inhibition o>0 p53 to pre,ent ad,erse e--ects o- radiation and the use o- cyc+otherapy to tar1et the tumour ce++s speci-ica++y by a++o4in1 them to pro+i-erate and usin1 an inhibitor a1ainst those ce++s.

Aesearch ha,e sho4n the importance o- restorin1 the -unction o- p53 in order to tri11er o-- apoptosis and ce++ cyc+e arrest (#entura et al., %&& ! (.ue et al., 1993!. 0his sho4s the importance o- creatin1 such a tar1eted therapy in cancer treatment in the -uture. 0his artic+e 4i++ 1o on to discuss in detai+ the ,arious methods used as cancer therapeutics not -or1ettin1 a 1+ance on the p53 acti,ation path4ay.

%. /he tumour suppressor protein, p53 2.1. Structure of p53 9n humans2 p53 protein o- mo+ecu+ar 4ei1ht 536&a and contains a 20<6b 1ene +ocated on the sma++ arm o- chromosome '7. 0he core domain o- the protein is rather unstab+e. 9t has a me+tin1 temperature o- $$o7 and a short ha+- +i-e o- # minutes 4hen it8s at norma+ body temperature (Lam0 and Craw"ord, 19)9!.

2.2. Functions of the tumour suppressor protein, p53 /53 -unctions throu1h a response path4ay (+igure %! once bein1 tri11ered by dama1e2 &NA brea6s2 u+tra<,io+et (u,* radiation2 stress and onco1enes2 p53 is acti,ated. 0hrou1h the acti,ation2 p53 tar1et 1enes are acti,ated and apoptosis2 ce++ cyc+e arrest and inhibition o- an1io1enesis occur. For e3amp+e2 i- a &NA brea6s occur2 it pre,ents those ce++s -rom di,idin1 ti++ the prob+em is so+,ed other4ise a pro1rammed ce++ death (apoptosis* is a++o4ed to occur. (*ao, $. et al., %&1&!. 0his is done to ensure tumour 1ro4th is suppressed.

!ne e3amp+e cou+d be p538s response to &NA dama1e that causes doub+e strand brea6s (&S"s* in &NA. A05 (ata3iate+an1iectasia mutated*2 a protein

6inase is acti,ated 4hich then acti,ates 7)B2 6inase. "oth A05 and 7)B2 then phosphory+ated p53 acti,atin1 one o- the tar1et 1ene p21 4hich +eads to C' phase ce++ arrest (+ritsche, 1., et al., 1993!. 0his in,o+,es an increase in p53 protein +e,e+s and chan1es in the protein throu1h modi-ications +i6e phosphory+ation ($anin, 2. et al., 1998).

2.3. Mutations of p53: Loss and Gain of Function 5ost common+y p538s -unction is +ost by a +oss o- chromosome re1ion containin1 a sin1+e a++e+e and a mi+d mutation in the other. )o4e,er2 in cancer o- the +i,er2 +un12 b+adder2 co+on and brain2 both the a++e+es in p53 are +ost. A+so2 a missense mutation in one o- the a++e+e is enou1h to cause inacti,ation (-ainaut, '. et al., %&&9!. Such mutations resu+ts in de-ecti,e -o+din1 o- the protein.

0hese mutations -a++ into t4o cate1ories. !ne is 4hen the structura+ con-ormation o- the protein is chan1ed disruptin1 its stabi+ity(structura+ mutants* and the other is &NA contact mutations. 0his is 4hen the mutation inter-eres 4ith the residues needed -or &NA contact ($ulloc(, ,.3. et al., %&&&!. Sites that are 6no4n to be important -or stabi+iDin1 p53 are phosphory+ated or acety+ated in the mutant -orm. 0his causes a compromise in p538s &NA bindin1 capabi+ity and a+so its response path4ay ($ai, L. et al., %&&4!.

Not on+y does p53 +ooses it -unction2 it a+so causes a 1ain o- -unction (C!F* 4hich a++o4s abnorma+ ce++ pro+i-eration. )ere the mutant proteins become dominant<ne1ati,e 4here the mutants pre,ent interaction bet4een p53 and its -ami+y members. nab+e to cause apoptosis2 the mutant p53 1ains the -unction o- a++o4in1 tumour ce++s to e,ade si1na+s -or apoptosis (Loten and 2achs, 1995! by suppressin1 the e3pression o- Fas/AP 1enes in,o+,ed in apoptosis (5alcenstein, ,. et al., %&&3!. A+so2 mutated p53 can cause disrupt norma+ ce++ cyc+e arrest -unction +eadin1 to the accumu+ation o- such 1enes 4ithout any contro+ (6ual0erto, , et al., 199)! un+i6e its norma+ counterpart. Studies on di--erent mutated -orms o- p53 can assist in dru1 disco,ery to reacti,ate the mutant -or so it can attain its norma+ 4i+d type -unction. 2.!. p53 "ene domain and its mutations 9n a++ cancers2 p53 is non<-unctiona+. A+most ha+- o- them are resu+ted -rom a mutation in the 1ene that transcribes into a protein2 as such2 inacti,atin1 the p53 response path4ay. (-ainaut, '. et al., %&&9!. 0he p53 1ene has $ -unctiona+ domains: a* transacti,ation domain interacts 4ith transcription -actors inc+udin1 p538s ne1ati,e re1u+ator 5&52. 9- a mutation occurs at this re1ion2 it 4ou+d not cause a drastic con-ormationa+ chan1e in the protein (Lapten(o and 'rives, %&&4!. b* pro+ine rich domain is in,o+,ed in the stabi+ity o- p53 as 4ithout it p53 is more prone to de1radation by 5&522 a+so tri11ers apoptosis and 1ro4th arrest (2a(amura, 7. et al., 199 ! c* &NA bindin1 domain is in,o+,ed in &NA bindin1. 0his domain binds stron1+y to the reco1nition e+ements on p53 tar1et 1enes and causes the

transcription o- the 1enes to occur ('rives, C. et al., 1999!. /oint mutations occurrin1 in the &"& usua++y cause the protein to be unab+e to bind to its reco1nition e+ements on the ad?acent 1enes as such pre,entin1 their acti,ation. 0his is 4here most point mutations ta6e p+ace causin1 a 4ho+e con-ormation a+teration. At codon 273 -or e3amp+e 4i++ decrease the abi+ity o- p53 to bind by remo,in1 the contact bet4een p53 and the &NA reco1nition e+ements. d* the o+i1omerisation and re1u+atory domain at the 7<terminus(70&* -unctions as a ne1ati,e re1u+atory domain in,o+,ed in pre,ention oan1io1enesis. 0his re1ion is sub?ected to phosphory+ation and acety+ation. 0he 70& is a hi1h+y basic domain. 0he +ysine residues in the 70& contributes to the stabi+ity o- p53 as it is sub?ected to 5&52 mediated ubi=uitinisation ($rus(in, ,. et al., 1991!. A de+etion in the 70& pre,ents p53 to -orm tetramers2 the -orm it is 4hen acti,ated.

2.5. #a$s to inacti%ate p53 0here are other 4ays to inacti,ate the p53 acti,ation path4ay other than mutations. Viruses a+so can pre,ent the acti,ation o- p53 4hen they in-ect a ce++. 0he ,iruses produce proteins that 4i++ o,ercome the de-ense o- the in-ected ce++s and in this 4ay inacti,ate p53 predisposin1 the ce++ to become cancerous (Lallemand, C. et al., %&& !.

2.&. 'he re(ationship )et*een 5&52 and p53

(,dapted "rom8 2hangary, 2., and 9ang, 2. (%&&9!. 2mall:molecule inhi0itors o" the 171%:p53 protein: protein interaction to reactivate p53 "unction8 a novel approach "or cancer therapy. Annual Reviews Pharmacological Toxicology 49, %%3:%;1.

+igure 18 /he 171%:p53 "eed0ac( loop.

>hen p538s re1u+ator 5&52 mu+tip+es in the ce++2 more o- p53 than norma+ 1et de1raded reducin1 the amount o- p53 a,ai+ab+e -or tumor suppression (#olgestein, $. et al., %&&)!. 5&52 encoded by the mdm2 1ene is a potent inhibitor o- transacti,ation o- p53 (*ozieres, 2.7. et al., %&&&!. 5dm2 has a p53 bindin1 poc6et at its N terminus. 5dm2 inhibits p53 throu1h bindin1 to p53 at the transacti,ation domain b+oc6in1 the abi+ity o- p53 to acti,ate transcription. (1omand, <. et al., 199%!.

5dm2 can ne1ati,e+y re1u+ate p53 transcriptiona+ acti,ity by bindin1 to and occ+udin1 the p53 transcriptiona+ acti,ation domain. 9t is a+so in,o+,ed in the nuc+ear e3port o- p53 4here p53 acts as a transcription -actor and acts as an ubi=uitin :3 +i1ase (Fi1ure '* that de1rades p53 that occurs in may steps in,o+,in1 :'2 :2 and :3. "asica++y2 :' protein 4i++ bibd to a ubi=uitin protein acti,atin1. 0hen2 :2 accepts it and trans-ers it to :3

4hich causes the bondin1 o- the ubi=uitin to p53 4hich is then tar1eted by proteosomes -or de1radation (*odriguez. 1.2. et al., %&&&!.

As o,er e3pression o- mdm2 is -ound in many tumours2 acti,ation o- p53 throu1h the inhibition o- 5dm2<p53 interaction and inhibitin1 its :3 +i1ase acti,ity is a promisin1 strate1y to restore 4i+d type p538s acti,ity (#assilev, /.L. et al., %&&;!. >hen 5dm2 +e,e+s increase2 it 4i++ cause the inacti,ation o- p53 apoptotic and ce++ cyc+e arrest -unctions simi+ar to the -ate o- p53 once mutated (+a(harzadeh, 2.2., et al., 1991!. A piece oe,idence to proo- 5dm28s -unction as a ne1ati,e re1u+ator o- p53 4as sho4n. 0he 5utant mouse -ibrob+asts that 4ere nu++ o- mdm2 sho4s p53 dependent apoptosis and dep+eted 1ro4th rate. As such2 5dm2 cou+d be seen as an onco1ene as 4hen o,er e3pressed it compromised p538s apoptotic -unction -or e3amp+e (*ozieres, 2.7. et al., %&&&!.

3. '53=s activation pathway

(,dapted "rom8 $ai, L. et al., (%&&4!. p538 2tructure, +unction and /herapeutic ,pplications. <. Cancer 1ol. %(;!, 1;1: 153.!

+igure %: Stresses enDymatic acti,ity (6inases and acety+ases* modi-y p53 and its ne1ati,e re1u+ator 5&52 increase acti,ated p53 p53 binds to re1u+atory re1ions otar1et 1enes causin1 1ro4th2 apoptosis and pre,ention o- an1io1enesis. A ne1ati,e -eedbac6 +oop bet4een 5&52 and p53 inhibits the processes abo,e. 5&52 is a+so under the contro+ o- p'$AAF. 9t binds 4ith 5&52 to b+oc6 p53 de1radation by inhibitin1 its :3 ubi=uitin +i1ase acti,ity.

3.1. +e(( c$c(e re"u(ation !nce p53 detects any de-ect in &NA2 an enDymatic cascade o- e,ents starts and it 4i++ pre,ent that particu+ar &NA -rom continuin1 in the ce++ cyc+e unti+ it is repaired. 9n response to &NA brea6s2 A05 (ata3iate+an1iectasia mutated* 6inase is acti,ated 4hich on turns acti,ate 7)B2. "oth then phosphory+ate p53 and p53 4ith up re1u+ate p2' mANA and protein +e,e+s ($anin, 2. et al., 199)!. 0his 4as +ater pro,ed that 4ith a p21 de+etion &NA dama1e in mice did not cause any 1ro4th arrest (7eng, C. et al., 199)!.

3.2. Apoptosis ,nduction >hen &NA dama1es cannot be repaired2 they 4i++ be tar1et -or apoptosis rather than bein1 a++o4ed to enter the ce++ cyc+e. /53 re1u+ates pro<apototic 1enes +i6e -A., Puma, / .A and Fas. 9n a mode+ o- +ymphan1io1enesis2 "AE 4as seen as a 6ey 1ene to tri11er apoptosis. 0he interaction bet4een "AE and p53 encoura1es mitochondria to re+ease cytochrome 7 -or the apoptosis process (Chipu(, <.>. et al., %&&;!.

3.3. ,nhi)ition of An"io"enesis >hen norma+ ce++s become ma+i1nant2 they 1ain an1io1enic abi+ty to sustain their sur,i,a+ as the amount o- p53 0S/' starts decreasin1.

0hrombospondin ' (0S/'* is a potent inhibitor o- this process. /53 4i++ re1u+ate the promoter se=uence o- 0S/' and an1io1enesis is inhibited. 9n the brain ce++s2 ho4e,er2 a di--erent inhibitor is present. "rain speci-ic an1io1enesis inhibitor ("A9'* is a+so re1u+ated by p53 in response to any ma+i1nant acti,ity (3ishimori, -. et al., 199 !.

;. 2trategies to activate wt p53 0he main aim -or therapy -or tumours harbourin1 a dys-unctiona+ 4i+d type p53 is to restore its -unction. 9n such cases2 p53 is seen to be inacti,ated by 5&52 or ,ira+ onco<protein.

!.1. +hemoradiation p53 mutations are associated 4ith resistance to chemoradiation. 0he success o- this therapy seems to be dependent on the status o- p53 a+thou1h not so+e+y. 9n cases o- 4t p53 cancers2 chemoradiation acts as an ;attac68 to the system to acti,ate it to cause apoptosis. )o4e,er2 o,er e3pression o- 5&52 4i++ hinder in this process. 7ertain cancers +i6e cancers o- the breast2 co+on and head and nec6 cancers seems to be resistance a1ainst such treatment due to the presence o- mutant p53 in them. ($urdelya, L.6. et al., %&&4! &ue to the ,ariabi+ity o- p53 status in cancer2 chemoradiation ha,e been proposed to patients a-ter under1oin1 sur1ery or in combination 4ith a 1enoto3icFnon<1enoto3ic dru1.

!.2. Gene therap$ usin" adeno%irus 01ecom)inant 2uman Ad3p53 4rAd3 p5356to introduce functiona( *i(d t$pe p53

rAd<p53 is a rep+ication de-ecti,e adeno,irus ,ector e3pressin1 the 4i+d type tumour suppressor 1ene. sin1 this adeno,irus as a ,ector2 -unction copies o- p53 cou+d be introduced into ce++ de-icient o- p53. sin1 adeno,iruses as a ,ector2 p53 encapsu+ated in an adeno,irus is in?ected and as such tumour ce++s 4i++ be in-ected. 0he 1ene then 4ou+d be re+ease into the en,ironment to distribute p53 protein into tumour ce++s de1radin1 them. Such treatment 4as sho4n to decrease tumour pro1ression in +un1 carcinoma. 0he resu+ts sho4ed ce++ cyc+e arrest2 and apoptosis (*oth, <.,. et al 1993!. 9n '##%2 Jac6 Aoth and his co++ea1ues 4as the -irst to sho4 this in a c+inica+ tria+. 7urrent+y2 under the brand name o- 9ntro1en2 this ,ector is in its /hase '<3 tria+ phase.

A-ter this2 many tria+s 4ere carried out. 0he most success-u+ one bein1 the appro,a+ o- Cendicine2 a rAd<p53 in 7hina on 2003 -or the treatment ohead and nec6 s=uamous ce++ carcinoma. A+thou1h2 this may seem as a brea6 throu1h in the -ie+d o- cancer therapy2 =uestions do arise on 4hy 7hina 4as a++o4ed to do such. !ne reason cou+d be the a,ai+abi+ity o- a +ar1e poo+ o- patients to carry out tria+s on and the 4i++in1ness o- the pub+ic to come -orth. 0his re+ati,e+y simp+e procedure appea+s the thousands in 7hina 4ho don8t 1et access to the medica+ options a,ai+ab+e to those in the >estern 4or+d. A+so2 the S F&A became more re+uctant to appro,e 1ene therapy a-ter a mishap occurred durin1 one o- the tria+s in '### 6i++in1 an '8 year o+d a-ter a massi,e immune response occurred a1ainst the adeno,irus ,ector. ('earson, 2 et al., %&&;!.

.ater2 in -urther testin1 o- Cendicine in 7hina2 it 4as reported that in combination 4ith chemotherapy or irradiation yie+d a 1reater therapeutic potentia+ in the treatment o- +un1 cancer. )o4e,er2 this didn8t 4or6 on ad,ance sta1es o- tumour. )ere2 1ene therapy causes an o,er e3pressed p53 4hich +eads to increase induction o- se,era+ apoptotic 1enes +i6e -a7 (6uan, ?.2. et al., %&&9!. A+so2 rAd<p5 trans-er o- modi-ied p53 in combination 4ith radiotherapy sho4ed an increase in anti<cancer acti,ity in ce++s that o,er e3pressed 5&52 ( p53 most+y inacti,ated* (@oom, 9.2. et al., %&1%!.

Se,era+ impro,ements are ho4e,er2 sti++ needed to impro,e the e--icacy othis therapy in the -ie+ds o- cancer ce++ 6i++in1 and ce++u+ar upta6e. A trip+e mosaic adeno,irus ,ector that -unctioned as a tar1eted tumour ce++ 6i++er has been created by 0an1 et a+ in 200# c+ear+y enhanced the treatment. )ere they created a adeno,irus ,ector containin1 po+y<tysin -or better tar1etin12 herpes<simp+e3 ,irus type ' thymidine 6inase -or enhancement o- cancer ce++ 6i++in1 and red -+uorescent protein to trac6 and ,isua+iDe the ,ector in a patient. 0reatment o- cisp+atin in con?unction 4ith 1ene therapy sho4ed promisin1 resu+ts as 4e++. (5hao, 9.5. et al., %&&9! A ,ery promisin1 strate1y indeed it is to increase 6i++in1 e--ect by restorin1 the 4i+d type abi+ity in tumours.

!.3. Gene therap$ to e(iminate mutant p53 containin" ce((s usin" /8.3 915

A norma+ adeno,irus contains a 1ene2 :'"2 that causes inacti,ation o- the tumor suppressor protein p53. )o4e,er2 4hen a mutated -orm o- the adeno,irus nu++ o- :'" is in?ected into tumour ce++s de-icient o- p532 it 4as ab+e to rep+icate in the ce++s and subse=uent+y +yse them. ($ischo"" et al., 1994*. !ne such ,irus is named as !NEG<!'5. 9nitia++y2 it 4as thou1ht2 !NGE<0'5 is dependent on the status o- p53 in tumour ce++s. 5ore recent+y2 it 4as pro,ed that a+thou1h !NEG<0'5 4as ab+e to rep+icate in both mutant and 4i+d type containin1 ce++s2 more rep+ication 4as seen in the mutant p53 ce++s.

9n combination 4ith radiation therapy2 the ,ira+ therapy cou+d +yse more ce++s rather than 4hen a+one. An interestin1 point to note is that norma+ ce++s pro,ed to be resistant to !NGE<0'5 (*oguls(i, @.*. et al %&&&!. 0his combination therapy 4as ear+ier document usin1 mouse tumour 3eno1ra-ts'. )ere2 !NGE<0'5 4as de+i,ered intra,enous+y to1ether 4ith chemotherapy usin1 cisp+atin2 5<-+uro<uraci+. >hen compared 4ith those mice in 4hich !NGE<0'5 4as 1i,en a+one2 the combined therapy pro,ed to be o- hi1her e--icacy (-eise, C. et al 199 !.

0a6en to1ether2 these studies pro,ed that radiation can be success-u++y administered 4ith ,ira+ therapy. 0his can be so as these treatments didn8t inter-ere in ,irus rep+ication as the e--ects o- radiation is +imited to the radiated ce++. 0he siDe o- the ,irus is -ar too sma++ to be a--ected by any radiation app+ied. As such2 both the ,ira+ therapy and radiation comp+ements each other (*oguls(i, @.*. et al %&&&!.
'

A Eeno1ra-t in e3perimenta+ studies is an anima+ 4here a -orei1n ce++ has been 1ra-ted onto the anima+ to study its response.

!.!. Sma(( Mo(ecu(es inhi)itors of the p533mdm2 interaction a5 /ut(ins 9t has been a di--icu+t tas6 to de,e+op sma++ mo+ecu+es to inhibit +ar1e protein interaction. )o4e,er2 e,er since the crysta+ structure o- 5dm2 4as seen bound to p538s side chain phe'# trp23 and +eu2% at the N terminus in a deep hydrophobic poc6et it 4as much easier. A +ibrary o- synthetic chemica+s 4ere screened -or a potentia+ candidate and Nut+ins 4ere -ound in the -irst e,er in ,i,o e3periements usin1 Nut+ins. 9t 4as ab+e to disp+ace p53 protein -rom it bindin1 4ith 5dm2 causin1 the acti,ation o- the p53 path4ay and suppression o- tumours in cancer ce++s. (#assilev, /.L. et al., %&&;!. 9t 4as ab+e to disp+ace p53 protein -rom it bindin1 4ith 5dm2 causin1 the acti,ation o- the p53 path4ay and suppression o- tumours in cancer ce++s. !ut o- the ana+o1ues2 Nut+in 3a pro,ed to be one that bound to p53 4ith the hi1hest a--inity. A point to note 4as that2 Nut+ins on+y inhibited the ce++s +ines that contained a dys-unctiona+ 4i+d<type p53.

Further research 4as done on Nut+ins to pro,e its syner1istic a--ects 4ith other the 6no4n chemotherapeutic dru1s. A+thou1h use-u+2 chemotherapy resu+ts in harm-u+ side e--ects. sin1 Nut+ins 4ith cisp+atin not on+y reduce tumour 1ro4th but a+so protected 6idney ce++s a1ainst dama1e (<iang, 1. et al., %&&4!. )ere2 a ne4 -unction o- Nut+ins 4as disco,ered. 9t cou+d b+oc6 cisp+astin induced "a3 acti,ation pre,entin1 the re+ease ocytochrome 7. 0here-ore2 Nut+in 3 pre,ented apoptosis o- hea+thy 6idney ce++s durin1 cisp+atin treatment. Simi+ar+y2 tests 4ere done on 7hronic

.ymphoid .eu6emia (7..* ce++ +ines usin1 F+udarbine as the chemotherapy dru1 (,ndree", 1. et al., %&&4!.

5ost recent+y2 Nut+ins 4ere used a1ainst 1+ioma ce++ +ines in an e--ort to impro,e patients sur,i,a+ rate in C+iob+astoma 5u+ti-orme 4hich is a type o- a11ressi,e adu+t brain tumours (#illalonga:'lanells, *. et al., %&11!. Nut+in 3<a 4as ab+e to induce p53 dependent ce++ arrest in the C' phase and a+so induced senescence in those tumour ce++s 4hich harbour a 4i+d< type p53 to pre,ent them -rom rep+icatin1. 0o1ether 4ith radiation therapy2 more ce++s in cu+ture sho4ed increase responsi,eness to Nut+in 3< a. )5-en:odia:epene3diones, ';P521252 0his mo+ecu+e here a+so binds 4ith the p53<bindin1 poc6et o- 5&52. 9t induced p53 tar1et 1enes in cu+ture ce++ +ines and a+so decreases the pro+i-eration (6ras0erger, $.L. et al., %&&5!. A year a-ter its initia+ disco,ery2 the mo+ecu+e 0&/52'252 4as synthesiDed (@o0lish, -.@. et al., %&&4!. 0hey too decreased ce++ pro+i-eration especia++y in 4i+d type p53 tumour ce++ +ines. 5&52 4as dissociated -rom p53 4hen 0&/52'252 4as administered ?ust 4ithin '5minutes. A+so2 an increase in p2'4a-'Fcip' 4as seen 4hich can be re+ated to inducin1 o- ce++ cyc+e arrest. 0his mo+ecu+es has be pro,en to syner1ise 4ith a 6no4n dru12 do3orubicin to decrease tumour 1ro4th in mouse 3eno1ra-t mode+s. "asica++y2 it 4or6s by increasin1 the transcription o- p53 tar1et 1enes and reduce pro+i-eration otumour ce++s. A++ to1ether2 these studies su11est the potentia+ o- sma++ mo+ecu+es in the treatment o- 4i+d<type p53 retainin1 tumours. c5 M,3Series

59 series mo+ecu+es are spiro<o3indo+es in nature. 9t 4as desi1ned to decipher its mechanism and therapeutic potentia+ a1ainst co+on ce++ +ines. 9ts deri,ati,e2 59<$3 b+oc6s ce++u+ar 5&52<p53 interaction in norma+ and cancer ce++s 4ith 4i+d type p53. 0his caused the induction o- ce++ cyc+e arrest by up<re1u+atin1 p2'. Another deri,ati,e2 59<2'# 4as more potent causin1 a comp+ete inhibition o- tumour 3eno1ra-ts and sho4ed 1reater bio<a,ai+abi+ity2.

)o4e,er2 in norma+ ce++s apoptosis do not occur (2hangary, 2., et al., %&&)!. A+thou1h not pro,en2 this mechanism may be use-u+ in protectin1 norma+ ce++ -rom the dama1e o- chemotherapeutic a1ents i- used in combination. >hen used 4ith chemotherapy2 the on+y documented e,idence is that there 4as increase sensiti,ity o- the adenocarcinoma ce++ +ines used to chemotherapy (2un, 2.-. et al., %&&)!. !.5. Sma(( mo(ecu(es that acti%ate p53 0he procedure to 1o a+on1 disco,ery +ies in basic screenin1 o- mo+ecu+es that anti<tumour in nature. From these mo+ecu+es2 any interestin1 mo+ecu+es 4ou+d then be synthesiDe -urther in an e--ort to decipher ho4 that mo+ecu+e acti,ate p53. a5 1eacti%ation and ,nduction of 'umour Apoptosis 41,'A5: ;irect($ interact *ith ;/A A90A 4as identi-ied usin1 a ce++ pro+i-eration assay on ce++s di--erin1 in its p53 status. A90A 4as ab+e to bind to p53 to induce its accumu+ation in tumour ce++s. 9n ,itro studies usin1 )70 ''% co+on cancer ce++ +ines2 A90A inhibited 1ro4th more e--ecti,e+y in those ce++s containin1 a 4i+d<type p53
2

"io<a,ai+abi+ity re-ers to the amount o- an administered dose o- dru1 that enters unchan1ed into the systemic circu+ation.

(Issaeva, 3. et al., %&&;!. 0he e3act mechanism on ho4 A90A acti,ates is sti++ under debate 4hen a Nuc+ear 5a1netic Aesonance study sho4ed that A90A mi1ht not direct+y bind to p53 (@raAews(i, 1. et al., %&&5!. .ater2 A90A 4as sho4n to be a speci-ic inducer o- the apoptotic response in tumour ce++s (>nge, 1. et al., %&&9!. 9t causes 5&52 re+eased -rom p53 to induce de1radation o- p2'. As such2 no ce++ cyc+e arrest occurs.

A+so2 pro<apoptotic 1enes +i6e -a7 and Fas 4ere most+y up<re1u+ated compared to those that induce ce++ cyc+e arrest 4hen e3posed to p53 acti,atin1 stresses. A90A cou+d a+so pre,ent the interaction o- p53 and :%< assosiated protein2 b+oc6in1 p53 ubi=uitination (5hao, C.?. et al., %&1&!. As seen in cer,ica+ cancers2 p53 is inacti,ated 4hen it is tar1et by )/V encoded :% protein. sin1 co+on cancer ce++ +ines2 the interaction bet4een :%<assosiated protein and p53 starts to diminish. )o4e,er2 4hen :% stop interactin1 4ith p532 it +ea,es the space -or 5&52 to interact. 0hus2 a point to note 4hen usin1 this techni=ue -or treatment is to inhibit the 5&52 interaction as 4e++. )5 <<=>:12 :;isrupt Mitosis 0his compound 4as disco,ered 4hi+e screenin1 -or p53 acti,ators. 9nitia++y screens yie+ded the compound JJ78:' 4hich 4as +ater optimiDed to -orm JJ78:'2 (2taples, B.7. et al., %&&)!. 0his pro,ed to be more potent in arrestin1 ce++s in mitosis and impairin1 po+ymeriDation o- tubu+in 3 in ce++s. At ,ery sma++ amounts2 JJ78:'2 4as ab+e to sho4 anti<tumour acti,ity by itse+-. >hen compared 4ith a 6no4n poison that causes tub+in depo+ymerisation2 the mechanism seems to be simi+ar. 0here-ore2 it 4as
3

A notab+e structure in,o+,in1 microtubu+es(tubu+in* is the mitotic spind+e used by most eu6aryotic ce++s to se1re1ate their chromosomes correct+y durin1 ce++ di,ision

deri,ed that by tubu+in depo+ymerisation JJ78:'2 4as ab+e to reacti,ate p53. 0he e3act mechanism is current+y un6no4n but indirect+y acti,ation p53 4as achie,ed. JJ78<'2 is current+y in c+inica+ tria+s in SA -or patients 4ith hemato+o1ica+ ma+i1nancies. c5 'eno%ins: ,nhi)ition of Sirutins 4re"u(ator of p535 0hese mo+ecu+es 4ere deri,ed -rom a screen to -ind mo+ecu+es that acti,ate p538s transcription. 0he ana+o1ues o- 0eno,ins2 0eno,in<' and 0eno,in<%2 4ere ab+e to induce ce++ cyc+e arrest and apoptosis in a p53 dependent manner. Amon1 them2 0eno,in<% 4as more potent in reduce tumour siDe as a +one a1ent (Lain, 2. et al., %&&)!. 9t 4as deri,ed that these mo+ecu+es act throu1h inhibitin1 Sirutin8s (Sir0'and Sir02* protein deacety+ase acti,ity (#aziri, -. et al., %&&1!. 0hey cause the deacty+ation o- p53 at +ysine residues causin1 p53 to +ose its stabi+ity ans deacti,ate.

9t is a -act that acety+ation o- p53 at +ysine 4i++ enhance &NA bindin1 abi+ity.As such2 these mo+ecu+es can be 6no4n as a ne1ati,e re1u+ator op53. 0here-ore2 i- 0eno,ins 4ere ab+e to inhibit Sir0' and Sir022 there shou+d be an increase in p53 +e,e+s and acety+ation. 0his 4as ?ust 4hat happened 4hen breast cancer ce++ +ines 4ere treated 4ith 0eno,in<% (Lain, 2. et al., %&&)!. 5. 2trategies to rescueCreactivate 1utant p53 5utant p53 is unab+e to per-orm its -unction as the 1uardian o- the 1enome due to its incorrect -o+din1 resu+tin1 -rom mutations. Strate1ies to correct this -unction ha,e been on the 4ay these -e4 years. 5.1. S$nthetic peptides a5 +;-3

7&"3 is short synthetic peptide that 4as desi1ned to bind speci-ica++y to p53 and pre,ent p53 -rom denaturation (Issaeva, 3. et al., %&&3!. 7&"3 interacted 4ith p538s &NA bindin1 domain and induced the re-o+din1 ot4o hot spots mutants name+y )is<273 and his '75 4ith a decrease in the amount o- denatured protein. A+so2 in the tumour ce++s2 p53 dependent induction o- p53 tar1et 1enes 4as noticed 4ith a partia+ induction oapoptosis. A+thou1h 7&"3 didn8t -u++y restore the bio+o1ica+ acti,ity op532 it sti++ did bind to p53 and rescue it. 7&"3 needs 4or6 to be done on it and cou+d be a +ead to desi1nin1 anti<cancer dru1s. )5 P53 +3termina( Peptide 4p53+5 /537 peptide 4as ab+e to restore -unction to p53 &NA contact mutants speci-ica++y and a+so induced apoptosis in those cancer ce++ +ines that contain p53 4ith contact mutants. )o4e,er2 nu++ e--ects 4ere seen in those ce++ +ines that had a con-ormation chan1e p53 mutant in them. A /537 peptide -used to the transduction domain 0A0 4as synthesiDed (2ynder, >.L. et al., %&&)!. sin1 this -used mo+ecu+es2 p53 e3pression 4as increased in cancer ce++ +ines. >hen anima+ 3eno1ra-ts o- peritonea+ carcinomatosis 4ere treated 4ith the -used peptide2 the +i-espan o- these anima+s increased and 1enerate anima+s that 4ere tota++y rid o- the tumour. 5.2. Sma(( Mo(ecu(es a5 +P3313?> 7/3'3#8 4as emer1ed 4hen screenin1 -or possib+e a1ents that cou+d restore a 4i+d<type 53 epitope onto mutant p53 &NA bindin1 domain restorin1 p53 -unction. A+so2 7/<3'3#8 b+oc6s ubi=uitination and de1radation o- p53 in most+y ce++s e3cept those e3pressin1 :% +i6e those cancers caused by )uman /api++oma Virus (9ang, 9. et al., %&&;!. A

point to note is that2 this does not brea6 the interaction bet4een 5&52 and p53 a+thou1h ubi=uitination 4as inhibited. 9nhibition 4as a+so seen in 3eno1ra-t anima+ mode+s o- -ami+ia+ adematous po+yposis (*ao,, #.C. et al., %&&)!. /romotion o- p53 bindin1 to its response e+ements 4as a+so -ound. "ut2 the e3act mechanism on ho4 7/<3'3#8 act upon the mis< -o+ded proteins in mutant protein to restore it remains debatab+e. 9t is possib+e that this mo+ecu+e does not bind to p53 but interca+ated 4ith &NA instead (/anner and $ar0eris, %&&;!. 5ore research sti++ need to be conducted usin1 7/<3'3#8 as a +ead -or -uture compounds that stabi+iDes mutant p53. )5 P53 Reacti%ation and nduction of !assi%e Apoptosis4P1,MA315 /A95A 4as identi-ied in a screen -or compounds that restore mutant p538s acti,ity ($y(ov, #.<.3. et al., %&&%!. /A95A induced apoptosis throu1h the restoration o- p538s transacti,ation -unction. Aestoration o- speci-ic &NA bindin1 abi+ity and the acti,e con-ormation 4as seen in )is<'75 mutants in human +ymphob+astoid ce++ +ines. )o4e,er2 a nu++ e--ect 4as seen in /he<'7% mutants in rena+ carcinoma ce++ +ines. 5ore e,idence on the mechanism 4as disco,ered +ater sho4in1 /A95A does no acti,ate 4i+d<type p53. 9nstead2 /A95A decomposes into a compound that 4i++ -orm co,a+ent bonds to modi-y thio+ adducts$ on mutant p532 restorin1 the &NA bindin1 abi+ity (Lam0ert, <.1.*. et al., %&&9!. /A95A<'5:02 ana+o1 o- /A95A 4as induce apoptosis and inhibit tumour in 3eno1ra-ts mode+s o- S79& mice in syner1y 4ith cisp+atin2 a chemotherapeutic dru1. )o4e,er2 due to a possib+e 1ain o- -unction mutation2 this syner1y 4as seen more e--ecti,e in ce++s 4ithout p53 ($y(ov, #.<.3. et al., %&&5!.
$

A thio+ adduct is a piece o- thio+ residue co,a+ent+y bonded to a cancer<causin1 chemica+. 0his process cou+d be the start o- a cancerous ce++2 or carcino1enesis

c5 @((ipticine :++ipticine is an a+6a+oid iso+ated -rom p+ants. 9ts sho4s hi1h e--icacy -or many cancer types 4ith minima+ to3icity and comp+ete +ac6 ohaemato+o1ica+ to3icity (2ti0orova, 1. et al., %&&1!. :++ipticine cou+d restore the &NA bindin1 and transcription -unction in both 6inds omutants i.e. &NA contact or con-ormationa+ chan1e. 9t 4or6s by inducin1 the correct -o+din1 to mutant p53 chan1in1 it -rom mutant to 4i+d type ('eng, ?. et al., %&&3!. 9t 4as sho4n to cause ce++ cyc+e arrest2 induce apoptosis2 increase amount o- 4i+d type o523 and rescue p53 mutants. )o4e,er2 :++ipticine a+so contribute to p275 de1radation. Such considerations ha,e to be ta6en note o- 4hen -urther de,e+opin1 this mo+ecu+e a as anti<cancer dru1. d5P53313 /53<A3 restores se=uence speci-ic &NA bindin1 to A'75 and A273 mutation in p53 (9einmann, L. et al., %&&)!. 0hese mutated -orms o- p53 4ere -ound to be hi1h+y e3pressed in 1+ioma ce++ +ines. )ere2 /53<A3 induced ce++s not to pro+i-erate by p53 dependent 1ro4th arrest more speci-ica++y than a pre,ious+y identi-ied mutant p53 restorin1 dru12 /A95A. 5.3. PhiAan9>3 /hiBan083 is a carbaDo+e% deri,ati,e that 4as speci-ica++y desi1ned to interact 4ith the G2207 p53 (p53G2207* mutants that creates a ca,ity in the core domain ($oec(ler, +.1. et al., %&&)!. 0his cou+d pro,ide a tar1et re1ion -or dru1 desi1n a1ainst cancer 4ith this mutation. >hen /hiBan083
5 %

9ncrease +e,e+s o- the p27 protein typica++y cause ce++s to arrest in the C' phase o- the ce++ cyc+e. 7arbaDo+e is an aromatic heterocyc+ic or1anic compound. 9t has a tricyc+ic structure2 consistin1 o- t4o si3<membered benDene rin1s -used on either side o- a -i,e<membered nitro1en<containin1 rin1.

-ormed a comp+e3 4ith p53G22072 the me+tin1 temperature o- the mutant increases stabi+isin1 it. 0his -unction cou+d be a possib+e tar1et -or the mutant cancers that reduce p538s me+tin1 temperature (appro3imate+y 752000 o- ne4 cancer cases*. A+thou1h this mutation is not -re=uent2 it seems to be simi+ar to the "7A<A". trans+ocation in chronic mye+o1enous +eu6aemia. 0i++ date2 insu--icient data is a,ai+ab+e therapeutica++y. )o4e,er2 it is ,ery possib+e that usin1 /hiBan083 as +ead2 anti<cancer dru1s cou+d be de,e+oped to restore p53 -unction in G2207 mutations. 4. Cyclotherapy 0he basis behind the idea o- 7yc+otherapy 4as to create a specia+ise -orm o- chemotherapy that cou+d protect norma+ ce++ 4hi+e 6i++in1 the ma+i1nant ones. 7yc+otherapy in,o+,es the administration o- t4o dru1s. 0he -irst dru1 shou+d be a non<1enoto3ic dru1 that can re,ersib+y arrest norma+ ce++s (CarvaAal, 7., et al., %&&5! in the ce++ cyc+e phase a++o4in1 the ma+i1nant to continue cyc+in1. 0he ne3t dru1 is needed to 6i++ these cyc+in1 ce++s. 7yc+otherapy 4ou+d pre,ent the side e--ects -rom chemotherapy (2ur, 2. et al., %&&9!. )o4e,er2 the di--icu+ty here +ies in the synthesis o- the second dru1 that is on+y to3ic to ma+i1nant ce++s. . Conclusion 7ancer is comp+e3 diseases that can arise -orm mutation o- the p53 1ene or the deacti,ation o- it causin1 a hitch in the p53 response path4ay. nderstandin1 the p53 path4ay has pro,ed to be a mi+estone in cancer therapy. Bno4in1 the modu+ators o- p53 throu1h this path4ay has +ed to the disco,ery o- many mo+ecu+es to treat cancer by either acti,atin1 4i+d<type p53 or restorin1 -unction o- mutants. 5a?ority o- the mo+ecu+es are sti++ either c+inica+ or +aboratory tria+s. !ne therapy2 ho4e,er2 has been appro,ed on+y in 7hina. Vira+ therapy sti++ is a

debatab+e topic in many countries. &i--erent ru+es 1o,ern them and thus the appro,a+ o- any dru1 ta6es a +on1 time.

*>+>*>3C>2 $ai, L., and 5hu, 9.6. (%&&4!. p538 2tructure, +unction and /herapeutic ,pplications. "ournal o# $anver !olecules %&4), 1;1:153. $a(er, 2.<., +earon, >.*., 3igro, <.1., -amilton, 2.*., 'reisinger, ,.C., <essup, <.1., #an/uinen, '., Led0etter, 7.-., $ar(er, 7.+., 3a(amura, ?., 9hite, *., and #olgelstein, $. (19)9!. Chromosome 1 7eletions and p53 6ene 1utations in Colorectal Carcinomas. 'cience, (ew 'eries %44&49)1), %1 :%%1. $oec(ler, +.1., <oerger, ,.C., <aggi, 6., *uther"ord, /.<., #eprintsev, 7.$. and +ershy, ,.*. (%&&)!. /argeted rescue o" a desta0ilized mutant o" p53 0y an in silico screened drug. Procee*ings o# the (ational Aca*emy o# 'ciences 1)+&,)), 1&34&:1&345. $urdelya, L.6., @omarova, >.,., -ill, <.>., $rowder, /., /aranova, 3.7., 1avra(is, L., 7iCorleto, '.>., +ol(man, <., and 6udov, ,.#. ( %&&4!. Inhi0ition o" p53 *esponse in /umor 2troma Improves >""icacy o" ,nticancer /reatment 0y Increasing ,ntiangiogenic >""ects o" Chemotherapy and *adiotherapy in 1ice. $ancer Research --, 9354:9341. $ulloc(, ,.3., -enc(el, <., and +ersht, ,.*. (%&&&!. Duantitative analysis o" residual "olding and 73, 0inding in mutant p53 core domain8 de"inition o" mutant states "or rescue in cancer therapy. .ncogene 19, 1%;5:1%54 $anin, 2., 1oyal, L., /aya, 2.?., ,nderson, C.9., Chessa, L., 2morodins(y, 3.I., 'rives, C., *eiss, ?., 2hiloh, ?., and 5iv, ?. (199)!. >nhanced 'hosphorylation o" p53 0y ,/1 in *esponse to 73, 7amage. 'cience %81&+,8,), 1-/401-//.

$ischo"", <.*., @irn, 7.-., 9illiams, ,., -eise, C., -orn, 2., 1una, 1., 3g, L., 3ye, <.,., 2ampson:<ohannes, ,., +attaey, ,., and 1cCormic(, +. (1994!. ,n adenovirus mutant that replicates selectively in p53:de"icient human tumor cells. 'cience %/4&+%8-), 3 3:3 4. $y(ov, #.<.3., 5ache, 3., 2tridh, -., 9estman, <., $ergman, <., 2elivanova, 6., and 9iman, @.6. (%&&5!. '*I1,:11>/ synergizes with cisplatin to induce tumor cell apoptosis. .ncogene %4, 3;);:3;91. $y(ov, #.<.3., Issaeva, 3., 2hilov, ,., -ultcrantz, 1., >lena, '., Chuma(ov, '., 9imnan, @.6., and 2elivanova, 6. (%&&%!. *estoration o" the tumor suppressor "unction to mutant p53 0y a low:molecular:weight compound. (ature !e*icine 8&,), %)%:%)). CarvaAal, 7., /ovar, C., ?ang, -., #u, $./., -eim0roo(, 7.C, and #assilev, L./. (%&&5!. ,ctivation o" p53 0y 171% antagonists can protect proli"erating cells "rom mitotic inhi0itors. $ancer research -+&+), 191):19%;. Chipu(, <.>., @uwana, /., $ouchier:-ayes, L., 7roin, 3.1., 3ewmeyer, 7.7., 2chuler, 1., and 6reen, 7.*. (%&&;!. 7irect activation o" $aE 0y p53 mediates mitochondrial mem0rane permea0ilization and apoptosis. 'cience ,),&+--)), 1&1&:1&1;. Chen, .., @o, L.<., <ayaraman, L., and 'rives, C. (1994!. p53 levels, "unctional domains, and 73, damage determine the eEtent o" the apoptotic response o" tumor cells. 1enes an* 2evelo3ment 1)&19), %;3):%;51. 7eng, C., 5hang, '., -arper, <.9., >lledge, 2.<., and Leder '. (1995!. 1ice lac(ing p%1CI'1C9,+1 undergo normal development, 0ut are de"ective in 61 chec(point control. $ell 8%&4), 4 5:4);. +riedler, ,., -ansson, L.B., #eprintsev, 7.$., +reund, 2.1.#., *ippin, /.1., 3i(olova, '.#., 'roctor, 1.*., *udiger, 2., and +ersht, ,.*. (%&&%!. , peptide that 0inds and sta0ilizes p53 core domain8 Chaperone strategy "or rescue o" oncogenic mutants. P(A' 99&%), 93 :9;%. +inlay, C.,., -inds, '.9., and Levine, ,. (19)9!. /he p53 'roto:Bncogene Can ,ct as a 2uppressor o" /rans"ormation. $ell +/, 1&)3:1&93. +a(harzadeh, 2.2., /rus(o, 2.'., and 6eorge, 7.L. (1991!. /umorigenic potential associated with enhanced eEpression o" a gene that is ampli"ied in a mouse tumor cell line. 4!5. "ournal 1)&-), 1545:1549. +ritsche, 1., -aessler, C., and $randner, 6. (1993!. Induction o" nuclear accumulation o" the tumor:suppressor protein p53 0y 73,:damagingagents. .ncogene 8&%), 3& :31). 6ras0erger, $.L., Lu, /., 2chu0ert, C., 'ar(s, 7.<., Carver, /.>., @o0lish, -.@., Cummings, 1.7., La+rance, L.#., 1il(iewicz, @.L., Calvo, *.*., 1aguire, 7., Lattanze, <., +ran(s, C.+., 5hao, 2, *ammachandren, @., $yle0yl, 6.*., 5hang, 1., 1anthey, C.L., 'etrella, >.C., 'antoliano, 1.9.,

7ec(man, I.C., 2purlino, <.C., 1aroney, ,.C., /omczu(, $.>., 1olloy, C.<., and $one, *.+. (%&&5!. 7iscovery and Cocrystal 2tructure o" $enzodiazepinedione -71% ,ntagonists /hat ,ctivate p53 in Cells. "ournal o# !e*icinal $hemistry 48, 9&9:91%. Lane, 7.'., and Craw"ord, L.#. (19 9!. / antigen is 0ound to a host protein in 2?;&:trans"ormed cells. (ature %/8, %41 :%43. 6uan, ?.2., Liu, ?., 5ou, -e, D., La, 5., ?ang, L., and -u, ?. (%&&9! ,denovirus:mediated wild:type p53 gene trans"er in com0ination with 0ronchial arterial in"usion "or treatment o" advanced non:small:cell lung cancer, one year "ollow:up. "ournal o# 6he7iang 8niversity '$ 4($4 5 1)&1+), 331:3;&. 6ual0erto, ,., ,ldape, @., @oza(iewicz, @., and /lsty, /.7. (199)!. ,n oncogenic "orm o" p53 con"ers a dominant, gain:o":"unction phenotype that disrupts spindle chec(point control. Procee*ings o# the (ational Aca*emy o# 'ciences 9+&9), 5144:51 1.

-anahan, 7., and 9ein0er, *. (%&&&!. /he -allmar(s o" Cancer. $ell 1)), 5 : & -ainut, '., 2oussi, /., 2homer, $., -ollstein, 1., 6reen0latt, 1., -ovig, >., -arris, C.C., and 1ontesano, *. (199 !. 7ata0ase o" p53 gene somatic mutations in human tumors and cell lines8 updated compilation and "uture prospects. (ucleic Aci*s Research %+&1), 151:15 . -eise, C., 2ampson:<ohannes, ,., 9illiams, ,., 1cCormic(, +., #on -o"", 7.7., and @irn, 7.-. (199 !. B3?.:&15, an >1$ gene:attenuated adenovirus, causes tumor:speci"ic cytolysis and antitumoral e""icacy that can 0e augmented 0y standard chemotherapeutic agents. (ature !e*icine 3, 439: 4;5. Issaeva, 3., +riedler, ,., $ozco, '., 9iman, @.6., +ersht, ,.*., and 2elivanova, 6. (%&&3!. *escue o" mutants o" the tumor suppressor p53 in cancer cells 0y a designed peptide. P(A' 1))&%,), 133&3:133& . Issaeva, 3., $oz(o, '., >nge, 1., 'rotopopova, 1., #erhoe", L.6.6.C., 1asucci, 1., 'ramani(, ,., and 2elivanova, 6. (%&&;!. 2mall molecule *I/, 0inds to p53, 0loc(s p53F-71:% interaction and activates p53 "unction in tumors. (ature !e*icine 1)&1%), 13%1:13%). <iang, 1., 'a0la, 3., 1urphy, *.+., ?ang, /., ?in, ..1., 7egenhardt, @., 9hite, >., and 7ong, 5. (%&& !. 3utlin:3 'rotects @idney Cells during Cisplatin /herapy 0y 2uppressing $aEC$a( ,ctivation. "ournal o# 5iological $hemistry %8%&4), %434:%4;5. @o0lish, -.@., 5hao, 2., +ran(s, C.+., 7onatelli, *.*., /ominovich, *.1., La+rance, L.#., Leonard, @.,., 6ushie, <.1., 'ar(s, 7.<., Calvo, *.*.,

1il(iewicz, @.L., 1arugan, <.<., *a0oisson, '., Cummings, 1.7., 6ras0erger, $.L., <ohnson, 7.L., Lu, /., 1olloy, C.<., and 1aroney, ,.C. (%&&4!. $enzodiazepinedione inhi0itors o" the -dm%8p53 compleE suppress human tumor cell proli"eration in vitro and sensitize tumors to doEoru0icin in vivo. !olecular $ancer Thera3eutics +, 1-)01-9. @raAews(i, 1., Bzdowy, '., 7G2ilva, L., *othweiler, H., and -ola(, /.,. (%&&5!. 31* indicates that the small molecule *I/, does not 0loc( p53: 171% 0inding in vitro. (ature !e*icine 11&11), 1134:113 . @oAima, @., @onopleva, 1., 1cDueen, /., BG$rien, 2., 'lun(ett, 9., and ,ndree"", ,. (%&&4!. 1dm% inhi0itor 3utlin:3a induces p53:mediated apoptosis 0y transcription dependent and transcription:independent mechanisms and may overcome ,tm:mediated resistance to "ludara0ine in chronic lymphocytic leu(emia. 5loo* 1)8&,), 993:1&&&. @oom, 9.2., 'ar(, 2.?., @im, 9., @im, 1., @im, <.2., @im, -., Choi, @.I., ?un, C.B., and 2eong, <. (%&1%!. Com0ination o" *adiotherapy and ,denovirus:1ediated p53 6ene /herapy "or 171%:BvereEpressing -epatocellular Carcinoma. "ournal o# Ra*ation Research +,, %&%:%1&. Lallemand, C., $lanchard, $., 'almieri, 1., Le0on, '., 1ay, >., and /ovey, 1,6. (%&& !. 2ingle:stranded *3, viruses inactivate the transcriptional activity o" p53 0ut induce 3B.,:dependent apoptosis via post:translational modi"ications o" I*+:1, I*+:3 and C*>$. .ncogene %-&,), 3%):33). Lam0, '., and Craw"ord, L. (19)9!. Characterization o" the -uman p53 6ene. !ollecular an* $ellular 5iology -&+), 13 9:13)5. Lam0ert, <.1.*., 6orzov, '., #eprintsev, 7.$., 2oderIvist, 1., 2eger0ac(, 7., $ergman, <., +ersht, ,.*., -ainaut, '., 9iman, @.6., and $y(ov, #.<.3. (%&&9!. '*I1,:1 *eactivates 1utant p53 0y Covalent $inding to the Core 7omain. $ancer $ell 1+, 3 4:3)). Luo, <., Li, 1., /ang, ?., Lasz(ows(a, 1., *oeder, *.6., and 6u, 9. (%&&;!. ,cetylation o" p53 augments its site:speci"ic 73, 0inding 0oth in vitro and in vivo. P(A' 1)1&8), %%59:%%4;. Lallemand, C., $lanchard, $., 'almieri, 1., Le0on, '., 1ay, >., and /ovey, 1,6. (%&& !. 2ingle:stranded *3, viruses inactivate the transcriptional activity o" p53 0ut induce 3B.,:dependent apoptosis via post:translational modi"ications o" I*+:1, I*+:3 and C*>$. .ncogene %-&,), 3%):33). Lapten(o, B., and 'rives, C. (%&&4!. /ranscriptional regulation 0y p538 one protein, many possi0ilities. $ell 2eath an* 2i##erentiation 1,, 951:941. Lain, 2., -ollic(, <.<., Camp0ell, <., 2taples, B.7., -iggins, 1., ,ou0ala, 1., 1cCarthy, ,., ,ppleyard, #., 1urray, @.>., $a(er, L., /hompson, ,., 1athers, <., -ollamd, 2.<., 2tar(, 1.<.*., 'ass, 6., 9oods, <., Lane, 7.'., and 9estwood, 3.<. (%&&)!. 7iscovery, In #ivo ,ctivity, and 1echanism o" ,ction o" a 2mall:1olecule p53 ,ctivator. $ancer $ell 1,&+), ;5;:;43.

1omand, <., 5am0etti, 6.'., Blson, 7.C., 6eorge, 7., and Levine, ,.<. (199%!. /he mdm:% Bncogene 'roduct +orms a CompleE with the p53 'rotein and Inhi0its p53:1ediated /ransactivation. $ell -9, 1%3 :1%;5. 3ishimori, -., 2hiratsuchi, /., Hrano, /., @imura, ?., @iyono, @., /atsumi, @., ?oshida, 2., Bno, 1., @uwano, 1., 3a(imura, ?., and /o(ino, /. (199 !. , novel 0rain speci"ic '53 target gene, $,I1, containing /hrom0oposdin type 1 repeats inhi0its eEperimental angiogenesis. .ncogene 1+&18), %1;5: %15&. 'eng, ?, Li, C., Chen, L., 2e0ti, 2., and Chen, <. (%&&3!. *escue o" mutant p53 transcription "unction 0y ellipticine. .ncogene %%, ;; ):;;) . 'earson, 2., <ia, -., and @andachi, @. (%&&;!. China approves "irst gene therapy. (ature 5iotechnology %%&1), 3:;. *ao, C.#., 2wamy, 1.#., 'atlolla, <.1.*., and @opelovich, L. (%&&)!. 2uppression o" +amilial ,denomatous 'olyposis 0y C':3139), a /'53 1odulator, in ,'CminCJ 1ice. $ancer Research -8&18), 4 &: 4 5. *oguls(i, @.*., +reytag, 2.B., 5hang, @., 6il0ert, <.7., 'aielli, 7.L., @im, <.-., -eise, C.C., and @im, 7.-. (%&&&!. In #ivo ,ntitumor ,ctivity o" B3?.:&15 Is In"luenced 0y p53 2tatus and Is ,ugmented 0y *adiotherapy. $ancer Research -), 1193:1194. *odriguez, 1.2., 7esterrol, <.1.'., Lain, 2., Lane, 7.'., and -ay, *./. (%&&&!. 1ultiple C:/erminal Lysine *esidues /arget p53 "or H0iIuitin: 'roteasome:1ediated 7egradation. !olecular $ell 5iology %)&%%), );5):);4 *ao, $., Leeuwen, I.1.1.#., -iggins, 1., Camp0ell, <., /hompson, ,.1., Lane, 7.'., and Lain, 2. (%&1&!. >valuation o" an ,ctinomycin 7C#.:4)& aurora (inase inhi0itor com0ination in p53:0ased cyclotherapy. .ncotarget 1&/), 439:45&. *ippin, /.1., $y(ov, #.<.3., +reund, 2.1.#, 2elevanova, 6., 9iman, @.6., and +ersht, ,.*. (%&&%!. Characterization o" the p53:rescue drug C':3139) in vitro and in living cells. .ncogene %1, %119:%1%9. *ozieres, 2.7., 1aya, *., Bren, 1., and Lozano, 6. (%&&&!. /he loss o" m*m% induces p53 mediated apoptosis. .ncogene 19, 1491:149 . 2a(amuro, 7., 2a00atini, '., 9hite, >., and 'rendergast, 6.C. (199 !. /he polyproline region o" p53 is reIuired to activate apoptosis 0ut not growth arrest. .ncogene 14, )) :)9). 2un, 2.-., 5heng, 1., 7ing, @., 9ang, 2., and 2un, ?. (%&&)!., small molecule that disrupts 1dm%:p53 0inding activates p53, induces apoptosis and sensitizes lung cancer cells to chemotherapy. $ancer 5iology Thera3y /&-), );5:5%.

2ynder, >.L., 1eade, $.*., 2aenz, C.C., and 7owdy, 2.+. (%&&;!. /reatment o" /erminal 'eritoneal Carcinomatosis 0y a /ransduci0le p53:,ctivating 'eptide. P9o' .(4 %&%), &1)4:&193. 2taples, B.7., -ollic(, <.<., Camp0ell, <., -iggins, 1., 1cCarthy, ,.*., ,ppleyard, #., 1urray, @.>., $a(er, L., /hompson, ,., *onseauE, 2., 2lawin, ,.1.5., Lane, 7.'., 9estwood, 3.<., and Lain, 2. (%&&)!. Characterization, chemical optimization and anti:tumour activity o" a tu0ulin poison identi"ied 0y a p53:0ased phenotypic screen. $ell $ycle /&%1), 3;1 :3;% . 2elivanova, 6., and 9iman, @.6. (%&& !. *eactivation o" mutant p538 molecular mechanisms and therapeutic potential. .ncogene %-, %%;3:%%5;. 2trom, >., 2athe, 2., @omarov, '.6., Chernova, B.$., 'avlovs(a, I., 2hyshynova, I., $osy(h, 7.,., $urdelya, L.6., 1ac(lis, *.1., 2(aliter, *., @omarova, >.,., and 6ud(ov, ,.#. (%&&4!. 2mall Fmolecule inhi0itor o" p53 0inding to mitochondria protects mice "rom gamma radiation. (ature $ancer 5iology 1, 1:9. 2ur, 2., 'agliarini, *., $unz, +., *ago, C., 7iaz, L.,., @inzler, @.9., #ogelstein, $., and 'apadopoulos, 3. (%&&9!. , panel o" isogenic human cancer cells suggests therapeutic approach "or cancers with inactivated p53. P(A' 1)-&1)), 394;:3949. 2ti0orova, 1., 'olAa(ova, <., 1artin(ova, >., $ore(:7ohals(a, L., >c(schlager, /., @ize(, *., and +rei, >. (%&11!. >llipticine cytotoEicity to cancer cell lines F a comparative study. nter*isci3linary Toxicology 4&%), 9): 1&5. 2hangary, 2., 7ing, @., Dui, 2., 3i(olovs(a:Coles(a, 5., $auer, <.,., Lui, 1., 9ang, 6., Lu, ?., 1c>achern, 7., $ernard, 7., $rad"ord, C.*., Carey, /.>., and 9ang, 2. (%&&)!. *eactivation o" p53 0y a speci"ic 171% antagonist (1I:;3! leads to p%1:mediated cell cycle arrest and selective cell death in colon cancer. !olecular $ancer Thera3eutics /&-), 1533:15;%. /ang, ?., 9u, -., Hgai, -., 1atthews, D.L., and Curiel, 7./. (%&&9!. 7erivation o" a /riple 1osaic ,denovirus "or Cancer 6ene /herapy. P9o' .(4 4&1%), 1:1&. /anner, 2., and $ar0eris, ,. (%&&;!. C':3139), a putative p53:sta0ilizing molecule tested in mammalian cells and in yeast "or its e""ects on p53 transcriptional activity. "ournal o# (egative Results in 5io!e*icine ,&+), 118-0119-. #assilev, L./., #u, $./., 6raves, $., CarvaAal, 7., 'odlas(i, +., +ilipovic, 5., @ong, 3., @ammlott, H., Lu(acs, C., @lein, C., +otouhi, 3., and Lui, >.,. (%&&;!. In #ivo ,ctivation o" the p53 'athway 0y 2mall:1olecule ,ntagonists o" 171%. 'cience ,),&+-+9), );;:);).

#entura, ,., @irsch, 7.6., 1cLaughlin, 1.>., /uveson, 7.,., 6rimm, <., Lintault, L., 3ewman, <., *ecze(, >.>., 9eissleder, *., and <ac(s, /. (%&& !. *estoration o" p53 "unction leads to tumour regression in vivo. (ature 44+, 441:445. #illalonga:'lanells, *., Coll:1ullet, L., 1artinez:2oler, +., Castan, >., ,ce0es, <.<., 6imenez:$ona"e, '., 6ill, <., and /ortosa, ,. (%&11!. ,ctivation o" p53 0y 3utlin:3a Induces ,poptosis and Cellular 2enescence in -uman 6lio0lastoma 1ulti"orme. P9o' .(4 -&4), e1)5)). #aziri, -., 7essain, 2.@., >aoton, .3., Imai, 2.I., +rye, *.,., 'andita, /.@., 6uarente, L., and 9ein0erg, *.,. (%&&1!. h2I*%2I*/1 +unctions as an 3,7:7ependent p53 7eacetylase. $ell 1)/, 1;9:159 9enge, 9., /a(imoto, *., *astineAad, +., and >l:7iery, 9.2. (%&&3!. 2ta0ilization o" p53 0y C':3139) Inhi0its H0iIuitination without ,ltering 'hosphorylation at 2erine 15 or %& or 171% $inding. !olecular an* $ellular 5iology %,&-), %1 1:%1)1. 9einmann, L., 9ischhusen, <., 7emma, 1.<., 3aumann, H., *oth, '., 7asmahapatra, $., and 9eller, 1. (%&&)!. , novel p53 rescue compound induces p53:dependent growth arrest and sensitises glioma cells to ,po%LC/*,IL:induced apoptosis, $ell 2eath 2i##erentiation 1+&4), 1): %9. ?u, 2., Din, 7., 2hangary, 2., Chen, <., 9ang, 6., 7ing, @., 1c>achern, 7., Dui, 2., 3i(olovs(a:Coles(a, 5., 1iller, *., @ang, 2., ?ang, 7., and 9ang, 2. (%&&9!. 'otent and Brally ,ctive 2mall:1olecule Inhi0itors o" the 171%: p53 Interaction. "ournal o# !e*ical $hemistry +%&%4), 9 &: 9 3. 5hao, 9.5., 9ang, <.@., Li, 9., and 5hang, ..L. (%&&9!. Clinical research on recom0inant human ,dp53 inAection com0ined with cisplatin in treatment o" malignant pleural e""usion induced 0y lung cancer. $hinese 7ournal o# $ancer %8&1%),);:) . 5ache, 3., Lam0ert, <.1.*., *o(aeus, 3., 2hen, <., -ainaut, '., $ergman, <., 9iman, @.6., and $y(ov, #.<.3. (%&&)!. 1utant p53 targeting 0y the low molecular weight compound 2/I1,:1. !olecular .ncology %, &:)&. 5hao, C.?., 2ze(ely, L., $ao, 9., and 2elivanova, 6. (%&1&!. *escue o" p53 +unction 0y 2mall:1olecule *I/, in Cervical Carcinoma 0y $loc(ing >4: 1ediated 7egradation. $ancer Research /)&8), 33 %:33)1.