M ETHYLATIO N OF THE 5 ' CpG ISLANDS AND
BLADDER CANCER PATHOGENESIS
Cevdet Kaya / Levent Türkeri / Atıf Akdaş
M .D ., D e p a rtm e n t o f U ro lo gy, S c h o o l o f M e d ic in e , M a rm a ra U n iv e rs ity , Is ta n b u l, T urkey.
ABSTRACT
Deregulation of cell cycle that results in
uncontrolled cellular proliferation is the basis of
neoplastic
process.
Bladder
tumors
are
heterogenous in their behavior, and it is very
difficult to predict the clinical course in many
patients. In order to alleviate this problem,
attention has been focused on mutations in
various cell cycle regulators and their association
with tumor behavior. Mutations of the p16/CDKN2
gene, encoding a cyclin-dependent kinase
inhibitor, are common in bladder cancer. In
contrast to other tumor suppressor genes, the two
most common mechanisms for loss of p16/CDKN2
function are homozygous deletion and loss of
transcription associated with hyperméthylation of
the 5'CpG island region. Recently, it is found that
méthylation of p16/CDKN2 is potentially reversible
with exposure to demethylating agents, such as 5-
aza-2'-deoxycytidine, which is a well-established
inhibitor of DNA méthylation, which may open up
new ways to effective tumor management.
K ey W ords:
M é th y la tio n , B la d d e r ca n ce r, C ell c y c leIN TR O D UCTIO N
In an a tte m p t to id e n tify th e u n d e rly in g m e c h a n is m s of n e o p la s ia m a n y s tu d ie s h a v e s u rv e y e d tu m o rs and tu m o r c ell lin e s fo r th e p re s e n c e of m u ta tio n s in g e n e s e n c o d in g c ell c y c le -re la te d p ro te in s (1). A s a re s u lt of th e s e s tu d ie s , m u ta tio n s in c ell c y c le g e n e s c o n s titu te th e m o s t c o m m o n g e n e tic in tu m o r ce lls. In fact, a lm o s t all o f th e tu m o rs h a v e m u ta tio n s in o n e of th e g e n e s in v o lv e d in c o n tro llin g p ro g re s s io n tro u g h the cell c y c le . M a lig n a n t c e lls m a y a c q u ire in d e p e n d e n c e
from re g u la to ry s ig n a ls th a t a re n o rm a lly re q u ire d fo r a c o n tro lle d cell c y c le p ro g re s s io n (2).
It has sh o w n th a t D N A m e th y la tio n Is e sse n tia l for no rm a l e m b ry o n ic d e v e lo p m e n t b u t a lte ra tio n s in DNA m e th y la tio n a re a lso v e ry c o m m o n in c a n c e r ce lls and c a p a b le o f d ire c tly m o d ify in g c a rc in o g e n e s is . C u rre n t in te re s t ha s fo c u s e d on th e p o te n tia l o f a b norm al m e th y la tio n e v e n ts in s ile n c in g tu m o r s u p p re s s o r g e n e s , an d c a u s in g th e ir p ro g re s s iv e e p ig e n e tic in a ctiva tion . R e c e n t s tu d ie s h ave sh o w n th a t it is p o s s ib le to re a c tiv a te th e s e d o rm a n t g e n e s by in h ib ito rs o f D N A m e th y la tio n a nd p o te n tia lly restore g ro w th co n tro l o f ce lls (3).
T h is re v ie w fo c u s e s on th e im p o rta n c e o f D NA m e th y la tio n p a rtic u la rly in p 1 6 /C D K N 2 in b la d d e r c a rc in o g e n e s is .
Cell Cycle, Cyclin-Dependent Kinases
and Inhibitors
C ell c ycle is a c a re fu lly p ro g ra m m e d p ro c e s s in w hich ju s t a fte r d ivisio n , th e cell th a t is d e s tin e d to divid e a g a in e n te rs a s ta g e c a lle d G 1 . A lte rn a tiv e is a resting p h a se c a lle d GO. A c ell in G1 n e x t m o v e s into S phase, d u rin g w h ic h tim e D N A re p lic a tio n o c c u rs , by a m e c h a n is m w h e re in e ach D N A stra n d s e rve s as a te m p la te fo r its o w n re p lic a tio n . A t th e end of S phase, a n o th e r g a p p h a s e (G 2) b e g in s , w h ich th e n le a d s to a ctual m ito s is (M ). E x tra c e llu la r s ig n a llin g m o lecules, such as p e p tid e g ro w th fa c to rs , b ind to s p e cific cell- s u rfa c e g ro w th -fa c to r re c e p to rs to m o d u la te th e cell cycle . N o rm a l c e lls re s p o n d to th e s e e x tra c e llu la r g ro w th s ig n a ls p rin c ip a lly d u rin g th e G1 p h a se . T h e se fa c to rs in d u c e re s tin g c e lls (GO) to e n te r th e cell cycle. H o w e v e r, s o m e fa c to rs a re n e c e s s a ry fo r th e p ro g re s s io n of c e lls fro m G1 p h a s e into S p h a s e (D N A s y n th e s is ). O n c e c e lls reach th e G 1 /S tra n s itio n and
Cevdet Kaya, et al
b e g in D N A s y n th e s is , th e y b e c o m e la rg e ly u n re s p o n s iv e to e x tra c e llu la r s ig n a ls and p ro c e e d a u to n o m o u s ly th ro u g h D N A re p lic a tio n , G 2 , a nd m itosis.
O v e r th e p a s t fe w d e c a d e s , s tu d ie s b y m a n y g ro u p s h ave sh o w n th a t p a s s a g e o f c e lls th ro u g h th e cell c ycle d e p e n d s on th e a c tiv ity o f e n z y m e s kn o w n as "c y c lin -d e p e n d e n t k in a s e s (C d k s )'' a n a m e in d ic a tin g th a t th e y b e c o m e a c tiv e o n ly w h e n th e y a s s o c ia te w ith p ro te in p a rtn e rs c a lle d cyclin s.
C dks, ta k e p la c e at th e c o re o f th e c ell c y c le e n g in e and d riv e cell p ro life ra tio n fo rw a rd by p h o s p h o ry la tin g s p e c ific s u b s tra te s in a cell c y c le -d e p e n d e n t fa s h io n . In o rd e r to b e c o m e a c tiv e k in a se s, th e C d ks m u s t a s s o c ia te w ith c y c lin s as w e ll as u n d e rg o an a c tiv a tin g p h o s p h o ry la tio n . T h e re are tw o ty p e s o f p rim a ry G1 p h a s e c y c lin s ; D -typ e c y c lin s an d c y c lin E fa m ily (4). T h e D fa m ily o f c y c lin s a s s e m b le into h o lo e n z y m e s w ith th e k in a s e c a ta ly tic s u b u n its C d k4 an d C dk6, w h ile th e p rin c ip a l p a rtn e r o f c y c lin E is C dk2.
G1 p h a s e p ro g re s s io n is a ls o s u b je c t to n e g a tiv e re g u la tio n b y a re c e n tly d is c o v e re d g ro u p o f m o le c u le s , th e c y c lin -d e p e n d e n t k in a s e in h ib ito rs (C K Is). T h e m e c h a n is m w h e re b y th e y a c h ie v e th e ir fu n c tio n a p p e a rs to b e th e fo rm a tio n o f s ta b le c o m p le x e s th a t in a c tiv a te th e c a ta ly tic a lly o p e ra tiv e units. N o rm a l c o n tro l of c e llu la r g ro w th re q u ire s a b a la n c e b e tw e e n th e a c tiv a to rs of c y c lin s an d th e in h ib ito rs o f th e C D K s . O v e ra c tiv ity o f th e C D K s, w h e th e r by e x c e s s iv e p ro d u c tio n of c y c lin s o r loss of C K Is, ca n re s u lt in a b e rra n t g ro w th of ce lls le a d in g to ca n c e r. C K Is fa m ily m e m b e rs ca n b e s u b d iv id e d into tw o g ro u p s on th e b a sis o f s e q u e n c e h o m o lo g y . T he firs t an d p ro b a b ly b e s t- c h a ra c te r iz e d C K I fa m ily m e m b e r to be id e n tifie d w a s p21 (also k n o w n as W A FI, C ip1, o r S di), w h ic h in a c tiv a te s th e c y c lin E- C dk2 c o m p le x , th e c y c lin A -C d k 2 c o m p le x , a nd th e c yclin D1-, D2-, an d D 3 -C d k 4 c o m p le x e s (5). P21- W A F1 g e n e e n c o d e s a c y c lin d e p e n d e n t k in a s e in h ib ito r and m e d ia te s tu m o r s u p p re s s o r g e n e p 53- in d u ced c ell c y c le a rre st. O v e re x p re s s io n o f p 2 1 - W AF1 s u p p re s s e s p ro life ra tio n an d g ro w th o f tu m o r c e lls in vitro , as w e ll as in v iv o (6). A n o th e r m e m b e r of th is g ro u p is p 2 7 /K ip 1 , w h ic h in a c tiv a te s th e s a m e su b s e t o f c y c lin -C d k c o m p le x e s as p 2 1 . T h e g e n e e n c o d in g p21 m a p s to c h ro m o s o m e 6 p 2 1 .1 , and th e g e n e e n c o d in g p 27 m a p s to c h ro m o s o m e 12 p 1 2- 1 2 p 1 3 . T h e o th e r C K I s u b g ro u p in c lu d e s th re e m e m b e rs : p 1 6 (IN K 4 A /M T S 1 /C D K N 2 ) (7), p 1 5 (IN K 4 B /M T S 2 ) (4), a n d p1 8 (8). T h e p 1 6 g e n e h a s b e e n fo u n d to b e m u ta te d fre q u e n tly in a v a rie ty of h u m a n c a n c e rs . It is lo c a te d at 9p21 and e n c o d e s a 16-kD p ro te in ; w h ic h is an in h ib ito r o f c y c lin D -C d k 4 a n d c y c lin D -C d k 6
c o m p le x e s re q u ire d fo r R e tin o b la s to m a p ro te in p h o s p h o ry la tio n (9 ,1 0 ). L o s s o f p 1 6 b y 9p21 d e le tio n or m u ta tio n a llo w s c y c lin /C D K c o m p le x e s to p ro c e e d w ith p h o s p h o ry la tio n o f Rb w h ic h in a c tiv a te s th is g ene. In m a lig n a n t ce lls , th e c e ll c y c le c o n tro l p a th w a y g o v e rn e d by th e D -ty p e c y c lin s is th e o n e th a t is m o st c o m m o n ly m u ta te d in tu m o r c e lls . In n o rm a l ce lls , th e p o s itiv e e ffe c ts o f c y c lin D c o m p e x e s on c ell c y c le p ro g re s s io n a re c o u n te ra c te d b y th e p 1 6 /C D K N 2 fa m ily o f C d k -in h ib ito r p ro te in s .
Genetic Alterations and D N A
H yperm ethylation in Bladder Cancer
M o le c u la r an d c y to g e n e tic a b n o rm a litie s of 9p21 h a v e b e e n re p o rte d in s e v e ra l m a lig n a n c ie s a nd tu m o r- d e riv e d c ell lines, in c lu d in g m e la n o m a (21), g lio m a (12), le u k e m ia (13), lu n g c a n c e r (14), h e a d an d neck s q u a m o u s c e ll c a rc in o m a (2 5 ), s q u a m o u s c e ll c a rc in o m a o f b la d d e r (1 6 ) a n d tr a n s itio n a l c e ll c a rc in o m a of th e b la d d e r (17). It ha s re c e n tly b een d e m o n s tra te d th a t th e p 1 6 /C D K N 2 c e ll c y c le re g u la to r g e n e m a y be in a c tiv a te d b y m u ltip le m e c h a n is m s , in c lu d in g m u ta tio n , d e le tio n , a n d m e th y la tio n . C y to g e n e tic a n a ly s is o f b la d d e r c a n c e r c e lls has re v e a le d th a t th e y c o n ta in n o n ra n d o m c h o ro m o s o m a l a b n o rm a litie s , m a in ly m o n o s o m y o f c h ro m o s o m e 9 (18). It w a s s h o w n th a t loss o f c h ro m o s o m e 9 is th e m o s t fre q u e n t a lte ra tio n in u ro th e lia l tra n s itio n a l cell c a rc in o m a (19).It has p re v io u s ly b e e n s h o w n th a t a lle lic loss of c h ro m o s o m e 9 is p o s s ib ly an e a rly a n d c o m m o n e v e n t in b la d d e r c a rc in o g e n e s is , a n d a b s e n c e of b lo o d g ro u p a n tig e n e x p re s s io n is p re d ic tiv e in d e fin in g th e tu m o r b e h a v io r (20). In th e p re s e n c e of c h ro m o s o m e 9 loss, tu m o r re c u rre n c e a n d /o r p ro g re s s io n w a s o b s e rv e d in 8 3 % o f b la d d e r c a n c e rs . M o le c u la r g e n e tic s tu d ie s of b la d d e r tu m o rs h a v e s h o w n fu rth e r th a t LO H in c h ro m o s o m e 9 s e q u e n c e s is a fr e q u e n t o c c u rre n c e in p a p illa ry s u p e rfic ia l tu m o r s (2 1 ). L o s s o f h e te ro z y g o s ity on 9 p 2 1 , w h e re th e p 1 6 /C D K N 2 and th e p 1 5 -IN K 4 B c ell c y c le re g u la to r g e n e s a re lo ca te d , is a c o m m o n g e n e tic a lte ra tio n in b la d d e r c a n c e r (22).
O rlo w e t al (23) re p o rte d a n o v e ra ll fre q u e n c y of d e le tio n s a n d re a rra n g e m e n ts fo r th e p 1 6 an d p15 g e n e s in b la d d e r c a n c e r of a p p ro x im a te ly 18% e ach. T h e ir s tu d y d id n o t d e te c t a n y p o in t m u ta tio n s e ith e r in e x o n 2 o f th e p1 6 g e n e o r in e x o n s 1 a n d 2 o f th e p15 g e n e (7 5 % o f th e to ta l c o d in g s e q u e n c e ). T h is s tu d y a ls o re v e a le d th a t th e p 1 5 g e n e a n d p 1 6 g e n e , w h ich a re a d ja c e n t to o n e a n o th e r, w e re a lm o s t a lw a y s d e le te d to g e th e r. A lth o u g h , m o s t o f th e p 1 6 /C D K N 2 h o m o z y g o u s d e le tio n s a ls o in c lu d e th e p 1 5 -IN K 4 B lo cu s, no in tra g e n ic m u ta tio n s in p 1 5 -IN K 4 B h ave
beed re p o rte d to d a te . H o m o z y g o u s d e le tio n s have b e e d d e s c rib e d to o c c u r p re fe re n tia lly in lo w -g ra d e b la d d e r c a rc in o m a . It sh o u ld be e m p h a s iz e d th a t only Ta a nd T 1, b u t n o t Tis, le s io n s s h o w e d d e le tio n s of e ith e r p1 6 o r p15. In a re c e n t s tu d y (24), it w as d e m o n s tra te d th a t p 1 6 a lte ra tio n s o c c u r in d e p e n d e n t o f p5 3 m u ta tio n s . B e c a u s e p5 3 a lte ra tio n s c o m m o n ly o c c u r in T is b la d d e r tu m o rs (25), it can be a h y p o th e s is th a t b la d d e r c a rc in o g e n e s is m ig h t d e v e lo p th ro u g h tw o d is tin c t m o le c u la r p a th w a y s (26).
T h e d e s c rib e d tu m o r s u p p re s s o r g e n e s m ig h t be part o f c h e c k p o in t p a th w a y s e n s u rin g th e a c c u ra te tra n s m is s io n o f g e n e tic in fo rm a tio n d u rin g cell division. In th e c a s e of in a c tiv a tio n of th e s e g e n e s by m u ta tio n s, bo th p ro m o tin g c ell p ro life ra tio n a nd a c c u m u la tio n of g e n o m ic e rro rs lead to c lo n a l e v o lu tio n o f c a n c e r cells. D e le tio n s at 9p21 h a v e b e e n re p o rte d in m a n y hum an tu m o r ty p e s , in c lu d in g n e a rly 5 0 % o f b la d d e r c a n c e rs (7). S e v e ra l s tu d ie s s h o w h o m o z y g o u s d e le tio n s at gp21/C -D K N 2 in b oth u n c u lte re d an d c u ltu re d b la d d e r c a n c e rs (8), m a k in g C D K N 2 a s tro n g c a n d id a te tu m o r s u p re s s o r g e n e .
D N A Hyperm éthylation
R e ce n tly , m é th y la tio n , an e p ig e n e tic e vent, has been a s s o c ia te d w ith th e loss of C D K N 2 e x p re s s io n in s e v e ra l c a n c e rs (27). D N A m é th y la tio n in e u k a ry o tic D N A is a n o rm a l p o s tre p lic a tiv e p ro c e s s and o c c u rs at th e 5 ’ -p o s itio n o f c y to s in e re s id u e s in th e m a jo rity of C pG d in u c le o tid e s . T h is m o d ific a tio n is a ss o c ia te d w ith g e n e a c tiv ity a n d is e s s e n tia l fo r n o rm a l m a m m a lia n d e v e lo p m e n t. D is c re te re g io n s of C pG - rich s e q u e n c e s w ith o u s m é th y la tio n are c lu s te re d as C pG is la n d s . T h e s e is la n d s h a v e b een sh o w n to be o fte n a s s o c ia te d w ith p ro m o te r re g io n s of g e n e s (28).
A lte ra tio n s o f D N A m é th y la tio n p a tte rn s in th e s e re g io n s h a v e im p o rta n t re g u la to ry e ffe c ts on g ene e x p re s s io n . H y p e rm é th y la tio n of C pG isla n d s have b e e n s h o w n to be a s s o c ia te d w ith stru c tu ra l a lte ra tio n s in c h ro m a tin a nd tra n s c rip tio n a l re p re s s io n (29). C pG is la n d s a re c o m m o n ly a s s o c ia te d w ith h o u s e k e e p in g g e n e s a n d m a y re g u la te th e ir tra n s c rip tio n a l a ctivitie s. H o w e v e r, a lte ra tio n s in th e m é th y la tio n s ta tu s of C pG is la n d s d u rin g m a lig n a n t tra n s fo rm a tio n m a y be c o rre la te d w ith tra n s c rip tio n a l c h a n g e s in a n u m b e r of g e n e s a s s o c ia te d w ith g ro w th re g u la tio n , an d it is p o s s ib le th a t o th e r g e n e s in v o lv e d in cell c y c le co n tro l m a y a ls o b e in flu e n c e d by th e s e e p ig e n e tic m e c h a n is m s . A p p ro x im a te ly 1% o f c y to s in e s in v e rte b ra te D N A are m e th y la te d at C pG d in u c le o tid e s (30). T he p re s e n c e of 5 -m e th y lc y to s in e at C pG d in u c le o tid e s m a y c o n trib u te to tu m o rig e n e s is e ith e r by g e n e ra tin g p o in t m u ta tio n s
o r by a lte rin g g e n e e x p re s s io n (31). D e a m in a tio n of 5- m e th y lc y to s in e fo rm s th y m id in e and g e n e ra te s a G -T m is m a tc h w h ic h , if not re p a rie d , fu rth e r p ro d u c e s a A to U tra n s itio n . A c o n s id e ra b le v a ria tio n a lso ha s been re p o rte d in m u ta tio n a l ty p e at C pG site s am ong d iffe re n t ty p e s o f c a n c e r. N e v e rth e le s s , a b e rra n t m é th y la tio n o f p ro m o te r re g io n s re s u ltin g in in a c tiv a tio n o f tu m o r s u p p re s s o r g e n e e xp re s s io n has b een p ro p o s e d to be an im p o rta n t m e ch a n ism in c a n c e r p ro g re s s io n (32). In s o m e tu m o rs , m é th yla tio n a p p e a rs to s u p p le m e n t d e le tio n or m u tation, resulting in h o m o z y g o u s g e n e in a c tiv a tio n (33). The 5' region of C D K N 2 c o n ta in s n u m e ro u s C G d in u c le o tid e s (term ed "C p G is la n d ") th a t a re u n m e th y la te d in th e tra n s c rip tio n a lly a c tiv e g e n e (32).
T h e c h a n g e s in D N A m é th y la tio n th a t a cc o m p a n y c a rc in o g e n e s is h a v e b e e n s u m m a riz e d in m a n y stu d ie s. D e c re a s e s in th e o v e ra ll c o n te n t o f 5 -m C yt (34), d é m é th y la tio n o f s p e c ific loci (35). de novo m é th y la tio n o f C pG is la n d s (36), an d in c re a s e d levels of D NA m e th y ltra n s fe ra s e e n z y m e (37) h ave all been o b se rve d . T h e s e o b s e rv a tio n s c o lle c tiv e ly have show n th a t m é th y la tio n c h a n g e s a re h ig h ly c o n s is te n t fe a tu re s of c a rc io g e n e s is .
A v a rie ty o f te c h n iq u e s h a v e b e e n p re v io u s ly d e v e lo p e d th a t ca n be u sed to in v e s tig a te p a tte rn s of D NA m é th y la tio n , such as g e n o m ic s e q u e n c in g (38), P C R -b a s e d m é th y la tio n a n a ly s is (3 9 ), S o u th e rn b lo ttin g , an d m é th y la tio n s e n s itiv e A P -P C R (40). H o w e ve r, n o n e o f th e s e ca n be u sed to iso la te spe cific and u n k n o w n D N A s e q u e n c e s fro m g e n o m ic D N A s th a t are d iffe re n tia lly m e th y la te d b e tw e e n norm ai and tu m o r tis s u e s . G lo b a l c h a n g e s in D N A m é th yla tio n p a tte rn s are kn o w n to o c c u r d u rin g tu m o rig e n e s is , and g e n e s ile n c in g has b e e n a s s o c ia te d w ith m é th yla tio n of C pG isla n d s lo c a te d in, o r near, p ro m o te rs and 5' re g u la to ry re g io n s. W ith th e e x c e p tio n o f g e n e s on the in a ctive X c h o ro m o s o m e (41), A lu an d 11 se q u e n c e s (42), and s o m e im p rin te d g e n e s (43), C pG isla n d s are u s u a lly u n m e th y la te d in n o rm a l s o m a tic ce lls . In co n tra st, w id e s p re a d m é th y la tio n of C pG islans o ccu rs on a u to s o m a l g e n e s d u rin g o n c o g e n ic tra n s fo rm a tio n . A b e rra n t m é th y la tio n w ith in 5 ’ p ro m o te r region is a s s o c ia te d w ith a tra n s c rip tio n a l s ile n c in g of C D K N 2 in m any c a n c e r c ell lin e s as w e ll as in lung, b la d de r, and co lo n tu m o rs (27). T h is e v e n t is a s s o c ia te d w ith a tig h tly c o m p a c te d c h ro m a tin c o n fo rm a tio n aro u n d the C D K N 2 p ro m o te r (29). G o n z a le z et al (27) s h o w e d the m é th y la tio n s ta tu s a n d e x p re s s io n le v e ls o f th e p 1 6 /C D K N 2 tu m o r s u p p re s s o r g e n e and th e p15- IN K 4 B c ell c y c le re g u la to r in n o rm a l tiss u e s , cell lines, and b la d d e r and c o lo n c a n c e r; and th e y c o n c lu d e d th a t e x p re s s io n of th e p 1 6 /C D K N 2 , but not e xp re s s io n of th e p1 5 IN K 4B c ell c y c le re g u la to r, is c o n tro lle d by m é th y la tio n o f its 5' C pG island, and th a t de novo m é th y la tio n o f th is is la n d is a m e c h a n is m fo r
Cevdet Kaya, et al
p 1 6 /C D K N 2 in a c tiv a tio n in b la d d e r c a n c e r. T h e y in d ic a te d th a t th e 5' C pG is la n d o f th e p 1 6 /C D K N 2 T S G is fre q u e n tly m e th y la te d in th is tu m o r ty p e . T he high fre q u e n c y (6 7 % ) o f u n c u lte re d tu m o rs s h o w in g de n ovo m é th y la tio n o f th e p 1 6 /C D K N 2 5' C pG island c o u ld e x p la in th e lo w rate at w h ic h p1 6 h o m o z y g o u s d e le tio n s a n d in tra g e n ic m u ta tio n s a re fo u n d in b la d d e r ca n c e r.
T h e y p o in te d o u t th a t, in c o n tra s t to s q u a m o u s cell c a rc in o m a o f th e b la d d e r in w h ic h p 1 6 is fre q u e n tly d e le te d (15), de n ovo m e th y ia tio n o f th e 5' C pG island of p 1 6 /C D K N 2 m a y be a m o re c o m m o n m e c h a n is m of in a c tiv a tio n o f th is tu m o r s u p p re s s o r g e n e in tra n s itio n a l c ell c a rc in o m a (T C C ). T h e y a lso s h o w e d th a t th e m é th y la tio n o f th e 5 ’ C p G is la n d o f p 1 6 /C D K N 2 o c c u rre d n o t o n ly in b la d d e r tu m o rs an d b la d d e r cell lines b u t a ls o in n o rm a l c o lo n m u co sa . J a rra rd et al (44), s h o w e d th a t 14% o f p rim a ry p ro s ta te tu m o rs d e m o n s tra te d a b e rra n t m é th y la tio n . T h e y p o in te d to th e a b s e n c e o f p 1 6 /C D K N 2 m é th y la tio n in n o rm a l, B P H , a n d s e m in a l v e s ic le tis s u e s s u g g e s tin g th a t m é th y la tio n is an e v e n t a s s o c ia te d w ith n e o p la s tic tr a n s fo rm a tio n . R e c e n t e v id e n c e a c tu a lly d e m o n s tra te s th a t a b n o rm a l h y p e rm é th y la tio n of C pG is la n d s e x is ts in a v a rie ty o f h u m a n n e o p la s ia , c o m m o n ly in s olid tu m o rs , in c lu d in g tra n s itio n a l cell c a rc in o m a o f th e b la d d e r (6 7 % ), b re a s t c a rc in o m a (31% ), g lio m a (2 4 % ), and c o lo re c ta l c a rc in o m a (10- 4 0 % ). S o m e s tu d ie s a ls o s h o w e d th a t h y p e rm é th y la tio n o f th e p 1 6 /C D K N 2 p ro m o te r re g io n is a fre q u e n t a nd e a rly o c c u rrin g e v e n t d u rin g th e p ro c e s s o f n e o p la s tic p ro g re s s io n in u lc e ra tiv e co litis. (45). In th is s tu d y H sie h et al. p o in te d o u t th a t p 1 6 /C D K N 2 p ro m o te r m é th y la tio n is a c a n d id a te b io m a rk e r in th e s u rv e illa n c e o f p a tie n ts w ith u lc e ra tiv e c o litis a n d p 1 6 /C D K N 2 re p re s s io n b y p ro m o te r m é th y la tio n ha s a m a jo r ro le in th e p ro c e s s of n e o p la s tic p ro g re s s io n in th is g ro u p o f p a tie n ts.
DNA Hyperm éthylation Inhibition
T h e re a re s e v e ra l fe a tu re s o f th e a b n o rm a l m é th y la tio n p a tte rn s in tu m o r c e lls m a k in g th e m a ttra c tiv e ta rg e ts fo r th e ra p e u tic in te rv e n tio n . T h e g ro w th -re g u la tin g g e n e s a re p re s e n t in a s u p p re s s e d form , th a t m a k e s it th e o re tic a lly p o s s ib le to re s to re e x p re s s io n a nd h e n c e re in s ta te g ro w th co n tro l. In a d d ition to th is, g e n e s s ile n c e d by m é th y la tio n e rro rs a re v e ry s e n s itiv e to re a c tiv a tio n by in h ib ito rs o f D N A m é th y la tio n such as a z a c y to s in e n u c le o s id e s (30).T h e e p ig e n e tic in a c tiv a tio n o f p 1 6 /C D K N 2 g e n e e x p re s s io n is p o te n tia lly re v e rs ib le w ith e x p o s u re to d e m e th y la tin g a g e n ts , s u c h as 5 -a z a -2 '-d e o x y c y tid in e , w h ic h is a w e ll-e s ta b lis h e d in h ib ito r o f D N A
m é th y la tio n (4 6 ). T h is a g e n t, in h ib its 5- m e th y ltra n s fe ra s e , a n d in d u c e s C D K N 2 re e x p re s s io n in s e v e ra l tu m o r ty p e s , in c lu d in g b la d d e r c ell lines c o n ta in in g a b e rra n t m é th y la tio n . C a s te llo et al (29) d e m o n s tra te d th a t th is re e x p re s s io n is a s s o c ia te d w ith a re la x a tio n o f tig h tly c o m p a c te d c h ro m a tin in th e C D K N p ro m o te r re g io n . B e n d e r e t al. (3) s h o w e d th a t a fte r e x p o s in g s e v e n h u m a n tu m o r cell lin e s an d tw o h u m a n fib ro b la s t c ell s tra in s to th e d e m e th y la tin g a g e n t, 5 -a z a - 2 '- d e o x y c y tid in e (5 - A z a - C d R ), to d e te rm in e w h e th e r th e s ile n c in g o f g ro w th -re g u la to ry g e n e s b y d e n o v o m é th y la tio n in im m o rta liz e d cell lin e s c o u ld be re v e rs e d , p o s s ib ly re s to rin g g ro w th c o n tro l, th is a g e n t s u p p re s s e d c e llu la r g ro w th in all s e v e n tu m o r lin e s b u t n o t in fib ro b la s t s tra in s . T h e y c o n c lu d e d th a t 5 -A z a -C d R m a y s lo w th e g ro w th of tu m o r c e lls b y re a c tiv a tin g g ro w th -re g u la to ry g e n e s s ile n c e d by d e n o v o m é th y la tio n .
B e n d e r et al. a lso re p o rte d th a t p 1 6 in d u c tio n and g ro w th s u p p re s s io n b y 5 -A z a -C d R tr e a tm e n t a re h e rita b le a n d d o s e d e p e n d e n t, s u p p o rtin g fin d in g s by C a s te llo e t al. in w h ic h 5 -A z a -C d R m e d ia te d p 1 6 /C D K N 2 e x p re s s io n w a s h e rita b le in vitro . J a rra rd e t al. in an a tte m p t to e v a lu a te th e e ffe c t o f th is d e m e th y la tin g a g e n t o n tra n s c rip tio n , tre a te d P C 3, P P C 1, an d T S U -P R 1 , c ell lin e s w ith d e n s e 5' C pG is la n d m é th y la tio n , fo r 5 d a y s . R e e x p re s s io n o f th e p 1 6 /C D K N 2 tra n s c rip t b y R T -P C R , a n d a p a rtia l d é m é th y la tio n o f th e 5 ’ C p G is la n d in S o u th e rn a n a ly s is , m o rp h o lo g ic a lte ra tio n s in c u ltu re c o n s is tin g o f c e llu la r a g g re g a te s fo rm a tio n , a n d d e c re a s e d g ro w th ra te w e re n o te d in th is s tu d y (47).
H e rm a n et al. (33), at th e b a s is o f th e a s s o c ia tio n of h y p e rm é th y la tio n in th e 5' p ro m o te r re g io n w ith lack of tra n s c rip tio n o f th e n o rm a l m R N A , e x a m in e d c ell lines of b re a s t, p ro s ta te , a nd c o lo n c a n c e r fo r p 1 6 /C D K N 2 e x p re s s io n by R T -P C R . T h e y d e m o n s tra te d th a t no m e th y la te d c e ll lin e e x p re s s e d th e e x p e c te d p1 6 p ro d u c t. In c o n tra s t, n o rm a l fe m a le b re a s t tis s u e and th re e o f fo u r te s te d n o rm a l c o lo n s a m p le s h a d d e te c ta b le p 1 6 m e s s a g e . T o c o n firm th a t D N A m é th y la tio n b lo c k e d tra n s c rip tio n , th e y tre a te d th e c o lo n c a n c e r c ell lin e s w ith th e d e m e th y la tin g agent, a n t th e y n o tic e d th a t b oth c o lo n c a n c e r c ell lin e s had d e te c ta b le p (p16) 16 m R N A a fte r tre a tm e n t w ith 5- A z a -C d R . A nd a ls o th e y p o in te d th a t th e a b e rra n t D N A m é th y la tio n is e s s e n tia l fo r m a in ta in in g tra n s c rip tio n a l s ile n c in g . A ll th e s e re s u lts s u g g e s t c a n c e r th e ra p ie s in w h ic h g ro w th -re g u la to ry g e n e s (lik e p 16) s ile n c e d by d e n o v o m é th y la tio n a re re a c tiv e d b y 5 -A z a -C d R , e ffe c tiv e ly s u p p re s s in g th e g ro w th o f h u m a n tu m o r ce lls.
H o w e v e r, a c c o rd in g to J o n e s (47) in s p ite o f th e ir p o te n c y a n d s p e c ifity fo r in h ib itio n o f D N A m é th y la tio n , th e c h e m ic a l in s ta b ility o f 5 - a z a - n u c le o s id e s in
a q u e o u s s o lu tio n a n d p re s e n t s u s p ic io n a b o u t th e c a rc io g e n e c ity a n d m u ta g e n e c ity o f 5 -A z a -C d R m ake it u n lik e ly th a t th e s e d ru g s w ill fin d w id e s p re a d clinical use. T h e re fo re , th e re is a need to se a rch fo r o th e r in h ib ito rs o f th e D N A m e th y ltra n s fe ra s e .
Conclusion
A s th e g e n e s a n d p a th w a y s g o v e rn in g cell cycle c o n tro l a re b e tte r u n d e rs to o d , th e ir in v o lv e m e n t in h u m a n c a n c e r b e c o m e s c le a re r. H ow th e s e m o le c u le s in te ra c t w ith o n e a n o th e r to c o n tro l n o rm a l c ell p ro life ra tio n a nd role o f th e ir loss in c o n trib u tio n to tu m o rig e n e s is h a v e n o t b e e n u n d e rs to o d exa ctly, and m a y be th e m o s t im p o rta n t q u e s tio n w ill be w h e th e r th e y c o n s titu te u s e fu l th e ra p e u tic ta rg e ts in c a n c e r ce lls . C h a n g e s in th e D N A m e th y la tio n m a c h in e ry and a lte ra tio n s in D N A m e th y la tio n p a tte rn s and le ve ls are v e ry c o m m o n in c a n c e r ce lls , in c lu d in g b la d d e r ca n ce r. A h ig h ly c o n s is te n t fin d in g in c a n c e r cells is th a t th e C pG is la n d s o f g ro w th -re g u la to ry g e n e s a re often m e th y la te d , in c o n tra s t to th e situ a tio n in th e ir norm al c o u n te rp a rts . T h e s e c h a n g e s c o u ld p lay a d ire c t role in th e in d u c tio n o f p o in t m u ta tio n s th a t in a c tiv a te tu m o r s u p p re s s o r g e n e s . T h is m a y a lso be an im p o rta n t a s p e c t o f fu tu re th e ra p e u tic a p p ro a c h e s .REFERENCES
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1 9 8 5 ; 3 2 -6 1 .
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1 9 9 4 ; 5 4 : 1 1 5 6 -1 1 5 8 .
16. G o n z a le z -Z u lu e ta M, A ts u k o S, O h n e s e ıt PF, e t al. H igh fre q u e n c y o f c h ro m o s o m e 9 p a lle lic lo s s a n d CDRM 2 t u m o r s u p p r e s s o r g e n e a lt e r a t io n s in s q u a m o u s c e ll c a rc in o m a o f b la d d e r. J M ail Cl
1 9 9 5 ; 8 7 : 1 3 8 3 -1 3 9 2 .
1 7. S ta d le r WM, S h e rm a n J, B o h la n d e r SR, R o u ls to n D, D re y lin g M, R u k s ta lis D. H o m o z y g o u s d e le tio n s w ith in c h ro m o s o m a l b a n d s 9 p 2 1 -2 2 in b la d d e r c a n c e r. C a n c e r Res 1 9 9 4 ; 5 4 : 2 0 6 0 -2 0 6 3 . 18. G ib a s Z, P ro u t GR, C o n n o lly JG , P o n te s JE, S a n d b e rg AA. M o n ra n d o m c h ro m o s o m a l ch a n g e s in tr a n s itio n a l c e ll c a rc in o m a o f th e b la d d e r. C a n c e r Res 1 9 8 4 : 4 4 : 1 2 5 7 -1 2 6 4 .
19. R n o w le s MA, E ld e r PA, W illia m s o n M, C a irn s JP, S h a w ME, L a w MG. A llo ty p e o f h u m a n b la d d e r c a n c e r. C a n c e r Res 1 9 9 4 ; 5 4 : 5 3 1 -5 3 8 . 2 0 . T ü rk e ri LM, A k d a ş G, Ö z y ü re k M, A k d a ş A. The in flu e n c e o f c h ro m o s o m e 9 a n d b lo o d g ro u p a n tig e n a b n o r m a lit ie s o n t u m o r b e h a v io r in c a rc in o m a o f th e b la d d e r. Ü r o lo ji B ü lte n i 1 9 9 7 ; 8: 2 0 1 -2 0 5 . 2 1 . R a m b A, G ru is MA, W e a v e r-F e ld h a u s J, e t al. A c e ll c y c le re g u la to r p o te n tia lly in v o lv e d in g e n e s is o f m a n y tu m o r ty p e s . S c ie n c e 19 9 4 ; 2 6 4 : 4 3 6 -4 4 0 . 2 2 . C a irn s P, T o k in o R, E b y Y, S id r a n s k y D. H o m o z y g o u s d e le tio n s o f 9 p 2 1 in p r im a r y h u m a n b la d d e r tu m o r s d e te c te d b y c o m p a ra tiv e m u ltip le x p o ly m e ra s e c h a in re a c tio n . C a n c e r Res 1 9 9 4 ; 5 4 : 1 4 2 2 -1 4 2 4 .
2 3 . O rlo w I, L a c o m b e L, H a n n o n GJ, e t al. D e le tio n o f th e p l 6 a n d p i 5 g e n e s in h u m a n b la d d e r tu m o rs . J M atl C a n c e r In s t 1 9 9 5 ; 8 7 : 1 5 2 4 -1 5 2 9 .
2 4 . G ru is MA, W e a v e r-F e ld h a u s J, L iu Q, e t al. G e n e tic e v id e n c e in m e la n o m a a n d b la d d e r c a n c e rs th a t
Cevdet Kaya, et al
p I 6 a n d p 5 3 fu n c tio n in s e p a ra te p a th w a y s o f tu m o r s u p p re s s io n . A m J P a th o l 1 9 9 5 ; 1 4 6 : 1 1 9 9 -
1 2 0 6 .
2 5 . S a rk is /IS , D a lb a g n i G, C o rd o n C, e t al. A s s o c ia tio n o f p 5 3 n u c le a r o v e r - e x p r e s s io n a n d t u m o r p ro g re s s io n in c a rc in o m a in s itu o f th e b la d d e r. J U ro l 1 9 9 4 ; 1 5 2 (2 Pt I ) : 3 8 8 -3 9 2 .
2 6 . D a lb a g n i G, P re s ti J, R u te r V, P a ir WR, C o rd o n -C a rd o C. G e n e tic a lte ra tio n s in b la d d e r c a n c e r. L a n c e t 1 9 9 3 ; 3 4 2 : 4 6 9 -4 7 1 .
2 7 . G o n z a le z -Z u lu e ta M, B e n d e r CM, Yang /Î5 , e t al. M é th y la tio n o f th e 5 ' C pG is la n d o f th e p l6 /C D K M 2 t u m o r s u p p re s s o r g e n e in n o rm a l a n d tr a n s fo r m e d h u m a n tis s u e s c o rr e la te s w ith g e n e s ile n c in g . C a n c e r Res 1 9 9 5 ; 5 5 : 4 5 3 1 -4 5 3 5 .
2 8 . L i E, B e a rd C, J a e n is c h R. R o le f o r DMA m é th y la tio n in g e n o m ic im p rin tin g , n a tu r e 1 9 9 3 ; 3 6 6 : 3 6 2 -3 6 5 . 2 9 . P e in b e rg , AP, a n d V o g e ls te in B. fly p o m e th y la tio n
d is tin g u is h e s g e n e s o f s o m e h u m a n c a n c e rs fr o m t h e ir n o rm a l c o u n te rp a rts , n a tu r e 1 9 8 3 ; 3 0 1 :8 9 -9 1 . 3 0 . J o n e s PA. D n A m é th y la tio n e rro rs a n d c a n c e r.
C a n c e r Res 1 9 9 6 ; 5 6 , 2 4 6 3 - 2 4 6 7 .
3 1 . B ird AP. C p G -ric h is la n d s a n d th e fu n c tio n o f D n A m é th y la tio n , n a tu r e 1 9 8 6 ; 3 2 1 : 2 0 9 -2 1 3 .
3 2 . B a y lin SB, M a k o s M, Wu JJ, e t al. A b n o r m a l p a t t e r n s o f th e D n A m é t h y la t io n in h u m a n n e o p la s ia : P o te n tia l c o n s e q u e n c e s f o r t u m o r p ro g re s s io n . C a n c e r C e lls 1 9 9 1 : 3 : 3 8 3 -3 9 0 . 3 3 . H e rm a n JG , L a t if F, W eng YK, e t al. S ile n c in g o f th e
VP1L tu m o r-s u p p re s s o r g e n e b y D n A m é th y la tio n in re n a l c a rc in o m a . P ro c n a t l A c a d S c i USA 1 9 9 4 ; 9 1 : 9 7 0 0 -9 7 0 4 . 3 4 . F e in b e rg , AP, G e h rk e CW, K u o KC, E h rlic h M. R e d u c e d g e n o m ic 5 -m e th y lc y to s in e c o n t e n t in h u m a n c o lo n ic n e o p la s ia . C a n c e r Res 1 9 8 8 ; 4 8 : 1 159-1 161. 3 5 . P e in b e rg AP, V o g e ls te in B. f ly p o m e t h y la t io n d is tin g u is h e s g e n e s o f s o m e h u m a n c a n c e rs fr o m th e ir n o rm a l c o u n te rp a rts , n a tu r e 1 9 8 3 ; 3 0 1 : 8 9 - 9 2 . 3 6 . B a y lin SB, F e a ro n ER, V o g e ls te in B, e t a l. f ly p e r n m é th y la tio n o f th e 5 re g io n o f th e c a lc ito n in g e n e is a p r o p e r ty o f h u m a n ly m p h o id a n d a c u te m y e lo id m a lig n a n c ie s . B lo o d 1 9 8 7 ; 7 0 : 4 1 2 -4 1 7 .
3 7 . K a u itia in e n TL, J o n e s PA. D n A m e th y ltra n s fe ra s e le v e ls in t u m o r ig e n ic a n d n o n tu m o r ig e n ic c e lls in c u ltu re . J B io l C h e m 1 9 8 6 ; 2 6 1 : 1 5 9 4 -1 5 9 8 . 3 8 . S in g e r-S a m J, L e B o n JM . T a n g u a y RL, Riggs AD. A q u a n t it a t iv e F lp a ll-P C R a s s a y t o m e a s u r e m é th y la tio n o f D n A fr o m a s m a ll n u m b e r o f c e lls, n u c le ic A c id s Res 1 9 9 0 ; 18: 6 8 7 . 3 9 . S o u th e rn EM. D e te c tio n o f s p e c if ic s e q u e n c e s a m o n g DMA f r a g m e n ts s e p a r t e d b y g e l e le c tro p h o re s is . J M o l B io l 1 9 7 5 ; 9 8 : 5 0 3 -5 1 7 . 4 0 . G o n z a lg o ML, L ia n g G, S p r u c k 111 C fl, Z in g g JM, R id e o u t WM, J o n e s P. I d e n t if ic a t io n a n d c h a ra c te r iz a tio n o f d if f e r e n t ia lly m e th y la te d re g io n s o f g e n o m ic DMA b y m e th y la tio n - s e n s itiv e a r b itr a r ily p r im e d PCR. C a n c e r R e s e a rc h 1 9 9 7 ; 5 7 : 5 9 4 -5 9 9 . 4 1 . B ird AP. C p G -ric h is la n d s a n d th e f u n c tio n o f DMA
m é th y la tio n . M a tu re 1 9 8 6 ; 3 2 1 : 2 0 9 -2 1 3 .
4 2 . L iu WM, S c h m id CW. P ro p o s e d ro le s f o r DMA m é th y la tio n in A lu tr a n s c r ip tio n a l re p re s s io n a n d m u ta tio n a l in a c tiv a tio n , n u c le ic A c id s Res 1 9 9 3 ; 2 1 : 1 3 5 1 -1 3 5 9 .
4 3 . F e rg u s o n -S m ith AC, S a s a k i FI, C a tta n a c h BM, S u ra n i MA. P a re n ta l-o rig in -s p e c ific e p ig e n e tic m o d ific a tio n o f th e m o u s e FI 19 g e n e . M a tu re 1 9 9 3 ; 3 6 2 : 7 5 1 - 75 5 . 4 4 . J a rr a r d DF, B o v a GS, E w in g CM, e t a l. D e le tio n a l, m u ta tio n a l, a n d m é th y la tio n a n a ly s e s o f CDKM2 (p 16 /M T S 1 ) in p r im a r y a n d m e ta s ta tic p ro s ta te c a n c e r. G e n es, C h ro m o s o m e s C a n c e r 1 9 9 7 ; 19: 9 0 -9 6 . 4 5 . F lsieh CJ, K lu m p B, F lo lz m a n n K, B o rc h a rd F, G re g o r M, P o rc h e n R. H y p e rm é th y la tio n o f th e p 16 /IM K 4 A p r o m o t e r in c o le c to m y s p e c im e n s o f p a tie n ts w ith lo n g - s ta n d in g a n d e x t e n s iv e u lc e r a t iv e c o lit is . C a n c e r Res. 1 9 9 8 ; 5 8 : 3 9 4 2 - 3 9 4 5 . 4 6 . M e rio A, H e rm a n JG , M ao L, e t al. 5 1 CpG is la n d m é t h y la t io n is a s s o c ia te d w ith t r a n s c r ip t io n a l s ile n c in g o f th e t u m o r s u p p r e s s o r p i 6 /C D K M 2 /M T S I in h u m a n c a n c e rs . M atu re M ed 1 9 9 5 ; 1: 6 8 6 -6 9 2 . 4 7 . J o n e s PA. A lt e r in g g e n e e x p r e s s io n w ith 5 - a z a c y tid in e . C e ll 1 9 8 5 ; 4 0 : 4 8 5 -4 8 6 .
