Advances in Molecular Biology (2): 67-70, 2008 Istanbul Kultur University, Printed in Turkey www.advmolbiol.org
Ras o n c o g e n e s p o l y m o r p h i s m
patients
Fatma Öztürk
1, Cihan Daloğlu
1,
Leyla Açık
1*, Mehmet Kılıç
2,
Mahmut Koç
21 Gazi University, S c i e n c e a n d Letters Faculty, D e p a r t m e n t of Biology, 0 6 5 0 0 A n k a r a - T u r k e y 2 N u m u n e Hospital, A n k a r a - T u r k e y
Abstract
M o l e c u l a r m u t a t i o n s t o p r o t o - o n c o g e n e s e q u e n c e s m a y b e i n v o l v e d i n t h e p a t h o g e n e s i s o f h u m a n t h y r o i d n e o p l a s m . P r o b l e m s o n o n c o g e n e s a n d t u m o r s u p r e s s o r g e n e s a c t i v a t i o n i n c e l l c i r c l e c o u l d c a u s e t u m o r . M a n y o n c o g e n e s a n d t u m o r s u p p r e s s i n g g e n e s e x i s t i n v a r y i n g p e r c e n t a g e s i n v a r i o u s t y p e s of t h y r o i d c a n c e r s . Ras, Gsp, Ret or Trk o n c o g e n e s c a n b e i n v o l v e i n t h y r o i d t u m o r s . M e m b e r s of t h e Ras g e n e f a m i l y (H-ras, K-ras, and N-ras) a r e s i g n a l t r a n s f e r r i n g p r o t e i n s . T h e s e g e n e s c o d e s f o r 2 1 k D a G T P b i n d i n g p r o t e i n s . W e s t u d i e d 2 4 t h y r o i d c a n c e r a n d 7 7 c o n t r o l f o r ras g e n e p o i n t m u t a t i o n s i n t w o d i f f e r e n t c o d o n s ( 1 2 a n d 1 3 ) u s i n g a r e s t r i c t i o n f r a g m e n t l e n g t h p o l y m o r p h i s m t e c h n i q u e . A c c o r d i n g t o e n z y m e d i g e s t i n g , n o c-K-ras g e n e c o d o n 12/13 a n d N - r a s g e n e c o d o n 1 2 p o i n t m u t a t i o n w e r e o b s e r v e d i n a n y o f t h e s a m p l e s w e s t u d i e d . K e y w o r d s : ras g e n e , thyroi d c a n c e r , m u t a t i o n , R F L P * Corresponding Author:Gazi University, Science and Letters Faculty, Department of Biology, 06500 Ankara - Turkey Email address: leylaacik@gmail.com
Received: September 1, 2008; Accepted: October 5, 2008
in T u r k i s h t h y r o i d cancer
Introduction
M u t a t i o n s in the g e n e s controlling cell g r o w t h a n d differentiation are c o n s i d e r e d to be the m a i n c a u s e of c a n c e r (Bos, 1 9 8 9 ) . T h e m o s t c o m m o n c a n c e r i n T u r k e y is lung c a n c e r . T h y r o i d c a n c e r is not v e r y c o m m o n i n g e n e r a l ; h o w e v e r s o m e p e o p l e are a f f e c t e d in Black S e a a n d C e n t r a l region of T u r k e y . T h e n u m b e r of t h y r o i d c a n c e r patients in T r a b z o n is 6 in 2 0 0 2 a n d 22 in 2 0 0 3 (Tuncer, 2 0 0 7 ) . This t y p e of c a n c e r is characteristic of a g r o u p of m a l i g n a n t t u m o r s related to thyroid g l a n d . T h e r e are different t y p e s of thyroid c a n c e r w h i c h are papillary, follicular, medullar a n d anaplastic (Donis-Keller, 1 9 9 3 ) . Radiation e x p o s u r e , c h r o n i c goiter, family history, a n d e n v i r o n m e n t a l factors c o u l d c a u s e t h y r o i d c a n c e r (De Groot, 1 9 9 6 ) .
T h e t h y r o i d g l a n d is located in the throat, b e l o w the larynx. It is m a d e of t w o lobes. T h e t h y r o i d g l a n d s e c r e t e s v e r y i m p o r t a n t h o r m o n e s that regulate s o m a n y m e t a b o l i c p r o c e s s e s , s u c h a s g r o w t h a n d e n e r g y e x p e n d i t u r e . W o m e n p o p u l a t i o n slightly m o r e a f f e c t e d t h a n m e n . A p p r o x i m a t e l y 10 % of t h y r o i d c a n c e r s are p r e s e n t in patients y o u n g e r t h a n 21 y e a r s of a g e , r e p r e s e n t i n g 3 % of all c a n c e r s of c h i l d r e n a n d a d o l e s c e n t s , w i t h p r e d o m i n a n c e in f e m a l e s 2:1 in relation to m a l e s . T h y r o i d c a n c e r s g o u p in this a g e are usually papillary (90 % ) , bilateral, multifocal a n d bigger in size c o m p a r e d to adults ( M o n t e etal., 2 0 0 7 ) . A n a p l a s t i c c a r c i n o m a i s t h e m o s t a g g r e s s i v e a n d m a l i g n a n t f o r m o f t h y r o i d c a n c e r . T h e m o s t c o m m o n t y p e of thyroid c a r c i n o m a is papillary c a r s i n o m . T h e m o s t f r e q u e n t m u t a t i o n s in papillary c a r c i n o m a s are point m u t a t i o n s o f the B R A F , Ras g e n e s a n d RET/PTC r e a r r a n g e m e n t (Nikiforov, 2 0 0 8 ) . T h e R a s - R a f - M e k - M A P kinase-signaling p a t h w a y is a c t i v a t e d i n h u m a n c a r c i n o m a s , p a r t i c u l a r l y i n a d e n o c a r c i n o m a s . T h e p a t h w a y i s r e s p o n s i b l e for t r a n s m i t i o n of m i t o g e n i c signal to t h e n u c l e u s . T h e constitutive activation of t h e p a t h w a y is t h o u g h t to r e a s o n of u n c o n t r o l l e d cell division ( F u k u s h i m a et al., 2 0 0 3 ) . T h e r e are three h u m a n RAS g e n e s w h i c h are N-Ras, H-N-Ras, a n d K-Ras. T h e ras g e n e f a m i l y e n c o d e m e m b r a n e - a s s o c i a t e d g u a n i n e n u c l e o t i d e - b i n d i n g
68
Ras o n c o g e n e s p o l y m o r p h i s m i n T u r k i s h t h y r o i d c a n c e r p a t i e n t s Ozturk et al. T a b l e 1. Primer used in PCR P r i m e r N u c l e o t i d e s e q u e n c e s 5 ' - 3 ' c-K-ras (first r o u n d a m p l i f i c a t i o n ) F : G A A G C T T A T G T G T G A C A T G T C T A R : A G G C A C T C T T G C C T A C G T C A c - K - r a s c o d o n 12 (Hph\) F : C C T G G T G A A A A T G A C T G A A T R : A G G C A C T C T T G C C T A C G T C A c - K - r a s c o d o n 1 3 ( H a e l I I ) F : G C C T G C T G A A A A T G A C T G A A R : C G T C A A G G C A T C T T G C C T A G G N - r a s (first r o u n d a m p l i f i c a t i o n ) F : T A A A G T A C T G T A G A T G T G G C T R : T C A C C T C T A T G G T G G G A T C A T N - r a s c o d o n 12 (BstNl) F : C A A A C T G G T G G T G G T T G G A C C A R : A G T G G T C C T G G A T T A G C T G G A T p r o t e i n s ( p 2 1r a s) ( G i n e s a et al., 2 0 0 3 ) . M u t a t i o n s in c o d o n 12, 1 3 , o r 6 1 o f g e n e o f g u a n i n e n u c l e o t i d e -b i n d i n g p r o t e i n s , c o n v e r t t h e s e g e n e s i n t o a c t i v e o n c o g e n e s ( B o s , 1 9 8 9 ) In o r d e r to f i n d b i o m a r k e r s for t h y r o i d c a n c e r , it is n e c e s s a r y t o s t u d y t h e m e c h a n i s m o f c a n c e r p r o g r e s s i o n . M u t a t i o n s i n c e l l u l a r ras g e n e h a v e b e e n r e p o r t e d i n v a r i o u s t h y r o i d c a n c e r t y p e s . I n t u m o r s , t h e p o i n t m u t a t i o n s w h i c h h a v e b e e n i d e n t i f i e d h a v e b e e n l o c a l i z e d to c o d o n 12, 13 or 61 of t h r e e ras g e n e s ( L e o n et al., 1 9 8 7 ) . I n this s t u d y , w e e v a l u a t e t h e f r e q u e n c y o f RAS m u t a t i o n s i n t h y r o i d c a r c i n o m a b y P C R - R F L P m e t h o d .Materials and methods
D N A i s o l a t i o n I n this s t u d y t o t a l 111 D N A s a m p l e s w e r e i s o l a t e d f r o m 3 4 p a t i e n t s w i t h h i s t o l o g i c a l l y v e r i f i e d t h y r o i d c a n c e r ( A n k a r a N u m u n e E d u c a t i o n a n d I n v e s t i g a t i o n H o s p i t a l i n T u r k e y ) a n d 7 7 c o n t r o l g r o u p b l o o d s a m p l e s . D N A i s o l a t i o n f r o m b l o o d s a m p l e s w a s i s o l a t e d a s d e s c r i b e d b y M a n i a t i s e t al. ( 1 9 8 9 ) . 3 0 m l R B C l y s e s b u f f e r w a s a d d e d t o 9 m l b l o o d s a m p l e a n d s h a k e d g e n t l y . T h e m i x t u r e w a s i n c u b a t e d o n i c e f o r 2 0 m i n a n d c e n t r i f u g e t e d a t 4 0 0 0 r p m for 2 0 m i n a t 40C . T h e s u p e r n a t a n t w a s r e m o v e d 2 5 m l R B C l y s e s b u f f e r w a s a d d e d a n d t h i s p r o c e s s w a s r e p e a t e d till all t h e r e d c e l l s w e r e r e m o v e d . 2 0 u g / m l p r o t e i n a s e K , 1 0 % S D S (final c o n c e n t r a t i o n 0.5 % ) a n d 2 , 5 v o l u m e S T E w e r e a d d e d a n d i n c u b a t e d o v e r n i g h t a t 5 6 ° C i n a w a t e r b a t h . 1:1 p h e n o l : c h l o r o f o r m : I s o a m y l a l c o h o l ( 2 5 : 2 4 : 1 ) w a s a d d e d a n d s h a k e d b y h a n d f o r 1 0 m i n . T h e n t h e m i x t u r e w a s i n c u b a t e d o n i c e f o r 2 0 m i n a n d c e n t r i f u g e t e d a t 4 0 0 0 r p m for 2 0 m i n a t 40C . U p p e r p h a s e w a s t r a n s f e r r e d i n t o a n e w t u b e , 1:10 v o l u m e 2 M s o d i u m a c e t a t e ( p H 5 . 2 ) a n d 9 5 % e t h a n o l ( 2 f o l d o f t o t a l v o l u m e ) w e r e a d d e d a n d s h a k e d g e n t l y until t h e D N A w a s p r e c i p i t a t e d , i n c u b a t e d o v e r n i g h t a t - 2 00C . D N A w a s c e n t r i f u g e t e d a t 4 0 0 0 r p m for 2 0 m i n a t 40C . S u p e r n a t a n t w a s r e m o v e d ; D N A w a s w a s h e d i n 5 0 0 u l 7 0 % e t h a n o l a n d d i s s o l v e d i n 0.5-1 m l T E - b u f f e r o v e r n i g h t a t 3 7 ° C i n a w a t e r b a t h . P C R A m p l i f i c a t i o n a n d R F L P T h e p r o c e d u r e t o d e t e c t a n y m u t a t i o n a t c o d o n 1 2 a n d c o d o n 1 3 o f t h e c - K - r a s g e n e a n d N - r a s g e n e h a s b e e n d e s c r i b e d b y C a p e l l a e t al. ( 1 9 9 6 ) . D N A s e q u e n c e s o f t h e first c o d i n g e x o n o f t h e c - K - r a s g e n e w e r e a m p l i f i e d u s i n g t h e p r i m e r s 5 ' G A A G C T T A T G T G T G A C A T G T C T A 3 ' a n d 5 ' A A G G A T C C T G C A G T A A T A T G C A 3 ' for 1 5 c y c l e s ( 9 2 oC for 1 m i n a n d 4 s ; 5 0 oC for 3 5 s ; 7 2 oC for
1 m i n a n d 2 5 s ; P C R 1). T h e n 1 m l o f t h e a m p l i f i e d p r o d u c t w a s r e a m p l i f i e d b y m e a n s o f n e s t e d P C R u s i n g p r i m e r s i n T a b l e 1 for 3 0 c y c l e s ( 9 2 oC f o r 1 5 s ; 5 0 oC f o r 1 5 s ; 7 2 oC for 3 0 s ; P C R 2 ) . P C R w a s p e r f o r m e d ( B i o m e t r a , G e r m a n y ) u s i n g 1 m g g e n o m i c D N A , 2 . 5 U Taq p o l y m e r a s e ( F e r m e n t a s ) 0.2 m M e a c h d N T P , 1 0 m M T r i s - H C l ( p H 8 . 8 ) , 5 0 m M K C l , 2 m M M g C l 2 , 0.1 % T r i t o n X - 1 0 0 , a n d 2 0 p m o l p r i m e r s i n t o t a l v o l u m e 5 0 u l . C o d o n 1 2 a n d 1 3 m u t a t i o n s w e r e d e t e c t i o n s b y d i g e s t i n g 7 0 b p a m p l i f i e d f r a g m e n t w i t h H p h I a n d HaeIII, r e s p e c t i v e l y . A s i m i l a r a p p r o a c h w a s u s e d to d e t e c t m u t a t i o n s a t c o d o n 1 2 o f t h e N-ras g e n e . T h e A d v a n c e s i n M o l e c u l a r B i o l o g y
Ras o n c o g e n e s p o l y m o r p h i s m i n T u r k i s h t h y r o i d cancer p a t i e n t s
Ozturk et al.
69
1 2 3 4 6 6 7 8 9 10 M
F i g u r e 1. RFLP (HaeIII) of PCR products of codon12 for the F i g u r e 2. RFLP (HphI) of PCR products of codon13 for the
c-c-K-ras gene. M- DNA marker (100bp), lane1-5 control sam- K-ras gene. M- DNA marker(100bp), lane1-5 control samples, ples, lane 6-9 thyroid cancer samples, lane 10 uncut samples. lane 6-10 thyroid cancer samples, lane 11 uncut samples.
first r o u n d a m p l i f i c a t i o n w a s p e r f o r m e d i n t h e s a m e c o n d i t i o n s a s for t h e c-K-ras g e n e u s i n g t h e p r i m e r s d e s c r i b e d i n T a b l e 1 . C o d o n 1 2 m u t a t i o n s w e r e d e t e c t i o n s b y d i g e s t i n g 8 1 b p a m p l i f i e d f r a g m e n t w i t h B s t n I . A 10-ul a l i q u o t of a m p l i f i e d m a t e r i a l is t a k e n for r e s t r i c t i o n e n z y m e d i g e s t i o n u s i n g Hph\, H a e I I I and B s t N I ( G e n e M a r k ) a n d r e s p e c t i v e b u f f e r for 2 h r a t 3 7 ° C i n a total v o l u m e o f 2 0 u l . A f t e r e n z y m a t i c d i g e s t i o n , t h e d i g e s t e d f r a g m e n t s w e r e s u b j e c t e d t o e l e c t r o p h o r e s i s i n a 1 2 % p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s a n d s t a i n e d w i t h e t h i d i u m b r o m i d e (Fig 1-3).
Results and discussion
I n t h i s s t u d y , w e e x a m i n e d 111 s a m p l e s w i t h t h y r o i d t u m o r s a n d w i t h o u t t u m o r s . D N A s a m p l e s w e r e a n a l y z e d for m u t a t i o n s a t c o d o n s 1 2 / 1 3 o f c - K - r a s g e n e a n d c o d o n s 1 2 o f N-ras u s i n g N e s t e d P o l y m e r a s e C h a i n R e a c t i o n a n d R e s t r i c t i o n F r a g m e n t P o l y m o r p h i s m ( P C R - R F L P ) . S e l e c t i v e a m p l i f i c a t i o n o f t h e r e g i o n a r o u n d t h e s t u d i e d c o d o n s o f t h e ras o n c o g e n e w a s p e r f o r m e d b y P C R . A t t w o s t a g e s P C R r e a c t i o n , a m p l i f i c a t i o n p r o d u c t s s i z e w e r e 8 1 b p for c -K-ras g e n e a n d 7 1 b p for N-ras g e n e . T h e A m p l f i c a t i o n
M 1 2 3 4 5 6 ~ 8 o 10 11 12 13 14 If 16 K 18
I
S
71 bp
F i g u r e 3. RFLP (BstNI) of PCR products of codon12 for the N-ras gene. M- DNA marker(100bp), lane1-8 control samples, lane 9¬
17 thyroid cancer samples, lane 18 uncut samples.
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Ras o n c o g e n e s p o l y m o r p h i s m in Turkish t h y r o i d cancer patients Ozturk et al.
p r o d u c t s w e r e d i g e s t e d w i t h restriction e n z y m e s H p h 1 a n d HaeIII for c-K-ras (Figure 1-2) a n d Bstn\ for N-ras g e n e (Figure 3 ) . A c c o r d i n g t o e n z y m e d i g e s t i n g , n o m u t a t i o n a t c o d o n 1 2 a n d c o d o n 1 3 w a s o b s e r v e d . Similarly, B o u r a s et al. (1998) w e r e f o u n d a n y point m u t a t i o n s at t h e s a m e c o d o n for 128 t h y r o i d tissue s a m p l e s .
M u t a t i o n s in all t h r e e families of ras o n c o g e n e s h a v e b e e n d e t e c t e d in both b e n i g n a n d m a l i g n a n t t h y r o i d t u m o r s (Du Villard et al., 1 9 9 5 ; M a n e n t i et al., 1 9 9 4 ) . M u t a t i o n s of the ras (Ha-, Ki-, N-ras) proto¬ o n c o g e n e h a v e b e e n r e p o r t e d in 2 0 - 6 0 % of t h y r o i d t u m o r s , particularly in follicular t y p e s ( N a m b a et al., 1 9 9 0 ; W r i g h t et al. 1989) a n d m o r e f r e q u e n t l y in iodine-deficient a r e a s (Shi et al., 1 9 9 1 ) . For this r e a s o n , ras m u t a t i o n s h a v e b e e n s u g g e s t e d a s a n i m p o r t a n t p r o g n o s t i c m a r k e r for t h y r o i d c a n c e r ( K a r g a et al., 1 9 9 1 , Shi et al., 1 9 9 1 ; N a m b a et al., 1 9 9 0 , Shi et al., 1 9 9 1 ) . T h e r e a s o n w e c o u l d not find a n y m u t a t i o n s c o u l d be that the n u m b e r of patients s t u d i e d is low. We n e e d to i n c r e a s e the n u m b e r of patients for future s t u d i e s .
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