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University of Southern California Dissertations and Theses
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Studies on the role of a novel protein, TMEM 56 in tumorigenic growth for MCF-7 cells
(USC Thesis Other)
Studies on the role of a novel protein, TMEM 56 in tumorigenic growth for MCF-7 cells
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S T U D I E S O N T H E R O L E O F A N O V E L P R O T E I N ,
T M E M 56 I N T U M O R I G E N I C G R O WT H F O R
M C F - 7 C E L L S
B y
D a i H o n g
A T h e s i s P r e s e n t e d t o t h e
F A C U L T Y O F T H E U S C G R A D U A T E S C H O O L
U N I V E R S I T Y O F S O U T H E R N C A L I F O R N I A
I n P a r t i a l F u l f i l l m e n t o f t h e
R e q u i r e m e n t s f o r t h e D e g r e e
M A S T E R O F S C I E N C E
( B i o c h e m i s t r y a n d M o l e c u l a r B i o l o g y )
M a y 2 0 1 4
C o p y r i g h t 2 0 1 4 D a i H o n g
i
A C K N O WL E D G E M E N T S
I w oul d l i ke t o e xpr e s s m y de e pe s t gr a t i t ude t o m y pr i nc i pl e i nve s t i ga t or , D r .
E br a hi m Z a ndi , f or hi s e xc e l l e nt gui da nc e , c a r i ng, pa t i e nc e , a nd pr ovi di ng m e w i t h a n
e xc e l l e nt a t m os phe r e f or doi ng r e s e a r c h. I w oul d ne ve r ha ve be e n a bl e t o f i ni s h m y
di s s e r t a t i on w i t hout t he gui da nc e of hi m .
I w oul d l i ke t o t ha nk m y a dvi s e r D r . T oke s , f or gui di ng m y s t udy f or t he pa s t t w o
ye a r s . S pe c i a l t ha nks go t o D r . M a r kl a nd, w ho w a s w i l l i ng t o pa r t i c i pa t e i n m y f i na l
de f e ns e c om m i t t e e .
I w oul d a l s o l i ke t o t ha nk t he l a b m e m be r s , T i ng, F i ki r , K i a a nd Y u w ho ha ve
gi ve n m e l ot s of i ns t r uc t i ons a nd he l p f or m y pr oj e c t a nd l i f e . I t w oul d ha ve be e n a l one l y
l a b w i t hout t he m .
M a ny t ha nks t o m y f r i e nds M e l ody a nd X i nya ng, t he y w e r e a l w a ys t he r e w i l l i ng t o
he l p m e , c he e r m e up a nd s t a nd by m e t hr ough t he good t i m e s a nd ba d.
I w oul d l i ke t o t ha nk m y pa r e nt s . E ve n t hough t he y w e r e t e n t hous a nd m i l e s a w a y ,
t he y a l w a ys pr ovi de d t r e m e ndous s uppor t t o m e a nd e nc our a g e d m e w i t h t he i r be s t
w i s he s .
L a s t l y, I of f e r m y r e ga r ds a nd bl e s s i ngs t o a l l of t hos e w ho s uppor t e d m e i n a ny
r e s pe c t dur i ng t he c om pl e t i on of t he pr oj e c t a nd m y l i f e i n U ni t e d S t a t e s .
i i
T A B L E O F C O N T E N T S
A C K N O W L E D G E M E N T S ……………………………………………………………… ..i
L I S T O F F I G U R E S ………………………………………………………………………i v
L I S T O F T A B L E S ………………………………………………………….......................v
A B B R E V I A T I O N ………………………………………………………………………..vi
A B S T R A C T …………………………………………………………………………… ..vi i
C H A P T E R O N E : I N T R O D U C T I O N …………………………………………..................1
C H A P T E R T W O : M A T E R I A L S A N D M E T H O D S …………………………..................9
2.1 C e l l c ul t ur e c ondi t i ons ……………………………………………………………… ..9
2.2 E s t a bl i s hm e nt of M C F - 7 s t a bl e c e l l l i ne s ………………………………… .................9
2.3 R e a l - t i m e R T - P C R f or T M E M 56 e xpr e s s i on ………………………………………10
2.4 C e l l pr ol i f e r a t i on a s s a y …………………………………………...............................10
2.5 C yt oS c a n W S T - 1 c e l l c yt ot oxi c i t y ……………………………….............................11
2.6 N ude m i c e i nj e c t i on i n vi vo …………………………………… ................................11
2.7 A T P a s e β I m m unohi s t oc he m i s t r y ………………………….......................................12
2.8 P r ot e i n e xt r a c t i on f r om t um or a nd w e s t e r n bl ot f or A T P a s e β …………… ...............12
C H A P T E R T H R E E : R E S U L T S …………………………………………........................14
3.1 R e l a t i ve T M E M 56 ba s a l e xpr e s s i on i n di f f e r e nt c e l l l i ne s …………………… ........14
3.2 E s t a bl i s hm e nt of M C F - 7 s t a bl e c e l l l i ne s …………………………………… ...........15
3.3 C ha r a c t e r i z a t i on of s t a bl e c e l l l i ne s …………………………………………………18
3.4 C e l l s ’ s e ns i t i vi t y t o pa c l i t a xe l ……………………………………………………… .20
3.5 T um or i ge ne s i s i n nude m i c e f or i n vi vo a na l ys i s ………………………...................21
C H A P T E R F O U R : C O N C L U S I O N A N D D I S C U S S I O N ………………………...........31
i i i
R e f e r e nc e s …………………………………………………………………………… .....35
i v
L I S T O F F I G U R E S
F I G U R E 1: T M E M 56 m ol e c ul a r f e a t ur e s ………………………………………………..3
F I G U R E 2: S c he m a t i c di a gr a m of t he F - A T P a s e ………………………………………...7
F I G U R E 3: R e l a t i ve T M E M 56 ba s a l l e ve l e xpr e s s i on i n M C F - 7, P C 3
a nd H E K 293T C e l l s ……………… ...............................................................15
F I G U R E 4: R e l a t i ve T M E M 56 e xpr e s s i on i n di f f e r e nt M C F - 7 s t a bl e c e l l l i ne s …….....17
F I G U R E 5: G F P - c ont r ol a nd G F P - T M c e l l s ’ m or phol ogy unde r opt i c a l
a nd f l uor e s c e nt m i c r os c ope ……………………………………………… ...18
F I G U R E 6: M C F - 7 s t a bl e c e l l l i ne s ’ pr ol i f e r a t i on i n nor m a l c ul t ur i ng c ondi t i on ……....19
F I G U R E 7: C e l l pr ol i f e r a t i on i n a s e r i e s of c onc e nt r a t i ons of pa c l i t a xe l ……………….20
F I G U R E 8: T um or vol um e f or m i c e i nj e c t e d w i t h M C F - 7 s t a bl e c e l l l i ne s
a c c or di ng t o t he da ys a f t e r i noc ul a t i on ……………………………….........22
F I G U R E 9: X e nogr a f t a s s a y on i m m une - de f i c i e nt m i c e ………………………………..23
F I G U R E 10: T um or vol um e s i n xe nogr a f t a s s a y ……………………………………….24
F I G U R E 11: R e l a t i ve T M E M 56 e xpr e s s i on i n t um or s f r om t he nude
m i c e i noc ul a t e d w i t h T M E M 56 knoc k dow n or
ove r - e xpr e s s e d M C F - 7 s t a bl e c e l l l i ne s …………………………………...26
F I G U R E 12: M or phol ogy of t um or s i s ol a t e d f r om nude m i c e i nj e c t e d
w i t h di f f e r e nt M C F - 7 c e l l l i ne s …………………………………………….27
F I G U R E 13: I m m unohi s t oc he m i s t r y f or A T P a s e β i n t um or s f or m e d by
f our M C F - 7 s t a bl e c e l l l i ne s ( 10 × a nd 40× ) …………………… ..........…....29
F I G U R E 14: A T P a s e β e xpr e s s i on i n t um or s a nd c e l l l i ne s …………………………….30
v
L I S T O F T A B L E S
T a bl e 1: L i s t of pr i m e r s us e d f or R T - P C R ……………………………………………….10
v i
A B B R E V I A T I O N S
A D P A de nos i ne - 5 ’ - t r i phopha t e
A T P A de nos i ne D i phos pha t e
A T P a s e A T P S ynt ha s e
D N A D e oxyr i bonuc l e i c A c i d
E R E s t r oge n R e c e pt or
G F P G r e e n F l uor e s c e nt P r ot e i n
H E K 293T H um a n E m br yoni c K i dne y 293 c e l l
H E R 2 H um a n E pi de r m a l G r ow t h F a c t or R e c e pt or 2
I K K I κ B ki na s e
L T Q - E T D L i ne a r t r a p qua dr upol e - E l e c t r on T r a ns f e r D i s s oc i a t i on
m D N A M i t oc hondr i a l D N A
P B S P hos pha t e B uf f e r e d S a l i ne
P C 3 P r os t a t e C a nc e r C e l l
pC M V C yt om e ga l ovi r us
P R P r oge s t e r one R e c e pt or
R O S R e a c t i ve O xyge n S pe c i e s
s hR N A S m a l l H a i r pi n R N A
T M E M 56 T r a ns m e m br a ne P r ot e i n 56
T N F a l pha T um or N e c r os i s F a c t or A l pha
v i i
A B S T R A C T
T r a ns m e m br a ne pr ot e i n T M E M 56 w a s f i r s t i de nt i f i e d a s a n I K K - a s s oc i a t e d
pr ot e i n by m a s s s pe c t r om e t r y i n D r . E br a hi m Z a ndi ’ s l a b. T hi s 263A A pr ot e i n w a s f ound
t o be l oc a l i z e d i n m i t oc hondr i a a nd i nt e r a c t s w i t h α a nd β s ubuni t of A T P a s e . I n s om e
l ung, he a d a nd ne c k c a nc e r c e l l s , T M E M 56 w a s s how n t o ha ve a c or r e l a t i on w i t h
m i t oc hondr i a l a bunda nc e . I n a ddi t i on, i t c oul d a c t a s a pot e nt i a l t um or s uppr e s s or i n H E K
293T c e l l s . T hi s t he s i s w a s ba s e d on t he c onc l us i on of pr e vi ous s t udi e s t o r e s e a r c h t he
f unc t i on of T M E M 56 pr ot e i n on t um or i ge ni c gr ow t h of M C F - 7 hum a n br e a s t c a nc e r c e l l
l i ne . S t a bl e c e l l l i ne s ( C O N - s hR N A , T M - s hR N A , G F P - c ont r ol , a nd G F P - T M M C F - 7 )
w e r e e s t a bl i s he d t o knoc k- dow n a nd ove r - e xpr e s s T M E M 56 e xpr e s s i on. X e nogr a f t a s s a y
i n i m m une - de f i c i e nt m i c e s how e d t ha t t um or s f or m e d by bot h G F P - T M a nd T M - s hR N A
M C F - 7 c e l l s w e r e s m a l l e r t ha n G F P - c ont r ol a nd C O N - s hR N A c e l l s , r e s pe c t i ve l y . T hi s
i ndi c a t e d t ha t a de vi a t i on f r om t he ba s a l e xpr e s s i on l e ve l of T M E M 56 w oul d i nhi bi t
t um or pr ogr e s s i on i n M C F - 7 c e l l s . I m m unohi s t oc he m i s t r y a nd W e s t e r n B l ot w i t h
A T P a s e β a nt i body s how e d t ha t ove r - e xpr e s s i ng or dow n- r e gul a t i ng T M E M 56 di d not
c ha nge A T P a s e β e xpr e s s i on , s ugge s t i ng t ha t T M E M 56 pr ot e i n w i l l not i nf l ue nc e
A T P a s e β l e ve l . I n s um m a r y, da t a i n t hi s s t udy s ugge s t t ha t t he di f f e r e nc e i n c e l l t ype i s
a n i m por t a nt de t e r m i na nt f or t he f unc t i on of T M E M 56 pr ot e i n. S pe c i f i c a l l y, i n M C F - 7
c e l l s , t he a bnor m a l T M E M 56 pr ot e i n l e ve l m i ght r e s ul t i n t he i nhi bi t i on of t um or
pr ogr e s s i on w i t hout i nf l ue nc i ng A T P a s e β e xpr e s s i on.
1
C H A P T E R O N E
I N T R O D U C T I O N
C om pl e t e pr ot e om e a na l ys i s s how s t ha t ove r 20% of a n or ga ni s m ’ s pr ot e i ns a r e
e m be dde d i n c e l l ul a r m e m br a ne s [ 1] . T he r e a r e t w o c a t e gor i e s of m e m br a ne pr ot e i ns - -
i nt e gr a l a nd pe r i phe r a l m e m br a ne pr ot e i ns , a nd t he y a r e c l a s s i f i e d ba s e d on t he i r
s t r uc t ur e a nd t he i r i nt e r a c t i on w i t h t he l i pi d bi l a ye r [ 2] . T r a ns m e m br a ne pr ot e i ns a r e
s pe c i f i c a l l y i nt e gr a l m e m br a ne pr ot e i ns t ha t s t r e t c h ove r t he e nt i r e bi ol ogi c a l m e m br a ne
[ 3] . T he i r pol ype pt i de c ha i ns t r a ve r s e t he l i pi d bi l a ye r a nd s pa n f r om one s i de of t he
m e m br a ne t o t he ot he r s i de of t he m e m br a ne . T w o ki nds of m e m br a ne - s pa nni ng dom a i ns
a r e f ound i n t r a ns m e m br a ne pr ot e i ns : one or m or e α he l i c e s or , l e s s c om m onl y , m ul t i pl e β
s t r a nds [ 3] . I n t he c a s e of t r a ns m e m br a ne pr ot e i ns t ha t s e r ve a s c ha nne l s , t he s ur f a c e of
t he pr ot e i n i s hydr ophobi c t o i nt e r a c t w i t h t he i nt e r i or l i pi d bi l a ye r , a nd t he c or e i s
hydr ophi l i c t o a l l ow pa s s a ge of w a t e r - s ol ubl e m ol e c ul e s [ 3] . T r a ns m e m br a ne pr ot e i ns
pl a y pi vot a l r ol e s i n c e l l ul a r a c t i vi t i e s s uc h a s c e l l - c e l l c om m uni c a t i ons , t r a ns m e m br a ne
s i gna l i ng, nut r i e nt t r a ns por t a t i on a nd m a i nt e na nc e of c e l l s t r uc t ur e [ 3] . T he y a c t a s gua r ds
t ha t pe r m i t or de ny t he t r a ns por t of s pe c i f i c s ubs t a nc e t hr ough t he bi ol ogi c a l m e m br a ne .
F ur t he r m or e , t he y c oul d i nt e r a c t w i t h c he m i c a l m e s s e nge r s t ha t f a c i l i t a t e t he
c o m m un i c a t i on w i t h i n a n d be t w e e n c e l l s t o pr o pa ga t e s i gna l s . M a n y bi ol o gi c a l pr oc e s s e s ,
l i ke t he m e t a bol i s m of gl uc os e a nd t he pr oduc t i on of f a t t y a c i ds , a r e t r i gge r e d by t he
a c t i va t i on of a pa r t i c ul a r t r a ns m e m br a ne pr ot e i n [ 3] . T r a ns m e m br a ne pr ot e i ns f unc t i on
va r i ous l y . F or e xa m pl e , T M E M 16A a nd T M E M 16B i n t he a noc t a m i n f a m i l y c a n a c t a s
c a l c i um - a c t i va t e d c hl or i de c ha nne l s [ 4] . A nd a bnor m a l a c c um ul a t i on of hum a n
t r a ns m e m br a ne T M E M 176A a nd 176B pr ot e i ns w i l l be a s s oc i a t e d w i t h c a nc e r pa t hol ogy
2
[ 5] .
I n t hi s t he s i s , w e a r e r e s e a r c hi ng a nove l ge ne t ha t e xpr e s s e s a t r a ns m e m br a ne
pr ot e i n i n t he m i t oc hondr i a . T hi s t r a ns m e m br a ne pr ot e i n i s c a l l e d T M E M 56. I t i s a
263A A pr ot e i n t ha t w a s f i r s t i de nt i f i e d i n D r . E br a hi m Z a ndi ’ s l a b a s a n I K K - a s s oc i a t e d
pr ot e i n by m a s s s pe c t r om e t r y . T he m ol e c ul a r w e i ght of t he pr ot e i n i s a ppr oxi m a t e l y
30,041 D a . I t ha s 6 he l i c a l t r a ns m e m br a ne r e gi ons e ve r y 21 a m i no a c i ds , s t a r t i ng f r om
7
L e u a nd one T L C dom a i n [ 6] . T M E M 56 s e que nc e da t a c a n be f ound f r om N C B I
R e f e r e nc e S e que nc e : N P _689700.1 a nd U ni P r ot I D : Q 96M V 1. F i gur e 1 a nd 2 s how s t he
pr ot e i n s e que nc e a nd t he pr e di c t e d t r a ns m e m br a ne r e gi on. P r e vi ous s t udi e s i ndi c a t e d t ha t
T M E M 56 pr ot e i n w a s l oc a l i z e d i n m i t oc hondr i a i nt e r a c t i ng w i t h F 1 c om pone nt s of
A T P a s e [ 6] . T he pr ot e i n l e ve l s w e r e c or r e l a t e d w i t h m i t oc hondr i a l a bunda nc e i n s om e
l ung, he a d a nd ne c k c a nc e r c e l l l i ne s [ 7] . I n a ddi t i on, t um or i ge ne s i s e xpe r i m e nt
s ugge s t e d t ha t t hi s pr ot e i n c a n a c t a s a pot e nt i a l t um or s uppr e s s or i n H E K 293T c e l l
( hum a n e m br yoni c ki dne y c e l l s ) [ 7] . A l l t he s e da t a s ugge s t e d t ha t T M E M 56 m a y pl a y a
vi t a l r ol e i n hum a n br e a s t c a nc e r c e l l s .
3
A
B
F i gu r e 1 T M E M 56 m ol e c u l ar f e at u r e s
T M E M 5 6 s e q u e n c e d a t a a r e s e a r c h e d f r o m N C B I R e f e r e n c e S e q u e n c e : N P _ 6 8 9 7 0 0 . 1 a n d U n i P r o t I D :
Q 9 6 M V 1 . A . T M E M 5 6 p r o t e i n s e q u e n c e B . P r e d i c t e d t r a n s m e m b r a n e r e g i o n f r o m U n i p r o t K B
d a t a b a s e
h t t p : / / w w w . u n i p r o t . o r g / u n i p r o t / Q 9 6 M V 1
B r e a s t c a nc e r , a t ype of a de noc a r c i nom a , or i gi na t e s i n t he c e l l s of t he br e a s t .
T he s e br e a s t c e l l s l a r ge l y r e f e r t o e pi t he l i a l c e l l s i n t he i nne r l i ni ng of m i l k duc t s a nd
l obul e s [ 8] . M or e t ha n 80% of br e a s t c a nc e r c a s e s w e r e de t e c t e d by a l um p t ha t f e e l
di f f e r e nt f r om t he r e s t of t he br e a s t t i s s ue [ 9] . S ym pt om s ot he r t ha n a l um p m a y i nc l ude
ni ppl e c ha ngi ng pos i t i on or s ha pe , t hi c ke ni ng di f f e r e nt f r o m t he ot he r br e a s t t i s s ue , one
br e a s t be c om i ng l a r ge r or l ow e r a nd c ons t a nt pa i n i n pa r t s of t he br e a s t [ 10] . B r e a s t
c a nc e r i s a m a l i gna nt t um or a nd c a n pr e s e nt a s a m e t a s t a t i c di s e a s e [ 1 1 ] . H um a ns a nd
4
ot he r m a m m a l s c a n a l l ha ve br e a s t c a nc e r s . I t oc c ur s a l m os t e nt i r e l y i n w om e n, but m e n
c a n a l s o ge t i t [ 12] . T he pr i m a r y r i s k f a c t or s f or br e a s t c a nc e r a r e s e x a nd a ge ; t he ol de r
t he a ge , t he hi ghe r t he r i s k [ 13] . O t he r pot e nt i a l r i s k f a c t or s t ha t pl a y i m por t a nt r ol e s
i nc l ude l a c k of br e a s t f e e di ng [ 14] , hi ghe r l e ve l s of c e r t a i n hor m one s [ 15] , unhe a l t hy
di e t a r y ha bi t a nd ge ne t i c s us c e pt i bi l i t y [ 16] . F or e xa m pl e , t he m ut a t e d B R C A 1 a nd
B R C A 2 ge ne c a n i nt e r f e r e w i t h D N A r e pa i r w hi c h c a us e a bnor m a l i t y of br e a s t c e l l s [ 16] .
L i ke a l l ot he r t ype s of c a nc e r s , br e a s t c a nc e r s t a r t s a s a r e s ul t of t he i nt e r a c t i on be t w e e n a
ge ne t i c a l l y s us c e pt i bl e hos t a nd a n e xt e r na l f a c t or . C e l l s be c om e c a nc e r ous w he n t he y
l os e t he i r a bi l i t y t o s t op di vi di ng, a t t a c h t o ot he r c e l l s , s t a y a t t he r i ght pl a c e , a nd di e a t a
pr ope r t i m e [ 17] . T o a gr e a t e xt e nt , t hi s r e s ul t s f r om m ut a t i ons of ge ne s t ha t a r e i nvol ve d
i n t he c e l l ’ s pr ot e c t i ve pa t hw a ys - - a popt os i s [ 17] . S o f a r , t he r a pi e s f or br e a s t c a nc e r
i nc l ude s ur ge r y , r a di a t i on , i m m unot he r a py a nd m e di c a t i ons ( hor m ona l t he r a py a nd
c he m ot he r a py) [ 18] . P a c l i t a xe l ha s e m e r ge d a s a n i m por t a nt dr ug i n t he c he m ot he r a py of
br e a s t c a nc e r [ 19] . I t i s a m i t ot i c i nhi bi t or t ha t c a n s t a bi l i z e m i c r ot ubul e s a nd i nt e r f e r e
w i t h nor m a l br e a k dow n of m i c r ot ubul e s dur i ng c e l l di vi s i on. A s a r e s ul t , t he pr ogr e s s i on
of m i t os i s i s bl oc ke d a nd pr ol onga t i on of m i t ot i c c he c kpoi nt s w i l l t r i gge r a popt os i s or
r e ve r s i on t o t he G pha s e w i t hout c e l l di vi s i on [ 20] . I n a ddi t i on , br e a s t c a nc e r ha s va r i ous
i nt r i ns i c phe not ype s a c c or di ng t o hor m one r e c e pt or e xpr e s s i on, H E R 2 ( hum a n e pi de r m a l
gr ow t h f a c t or r e c e pt or 2) a m pl i f i c a t i on, node i nvol ve m e nt , a ge a nd gr a de [ 21] . T he r e f or e ,
c he m ot he r a py t r e a t m e nt s w i t h t he a ddi t i on of nove l dr ugs m a r c hi ng i nt o c l i ni c s w i l l be a
pr om i s i ng f ut ur e . F or e xa m pl e , pa c l i t a xe l c a n be c om bi ne d w i t h doxor ubi c i n ( a n a nt i -
c a nc e r dr ug t ha t i nt e r a c t s w i t h D N A by i nt e r c a l a t i on a nd i nhi bi t t he s ynt he s i s of
m a c r om ol e c ul a r ) a nd c yc l ophos pha m i de ( a c he m i c a l t ha t i nt e r f e r e s w i t h D N A r e pl i c a t i on
5
by f or m i ng i nt e r s t r a nd D N A c r os s l i nks ) [ 21] . E ve n t hough s i gni f i c a nt a dva nc e s ha ve
be e n m a de i n t he pr e ve nt i on of br e a s t c a nc e r , i t s t i l l r e m a i ns t o be a c om pl e x di s e a s e
a f f e c t i ng m i l l i ons w or l dw i de [ 22] . I t ha s be e n r e por t e d t ha t br e a s t c a nc e r i s now t he m os t
c om m on c a nc e r di a gnos e d a m ong w om e n i n t he U ni t e d S t a t e s s e c ond t o s ki n c a nc e r [ 23] .
M C F - 7 c e l l i s one of t he m os t c om m on br e a s t c a nc e r c e l l us e d i n r e s e a r c h a nd i s
i s ol a t e d f r om a pl e ur a l e f f us i on pa t i e nt w i t h m e t a s t a t i c br e a s t c a nc e r i n 1970. I t i s t he
a c r onym of M i c hi ga n C a nc e r F ounda t i on- 7, r e f e r r i ng t o t he i ns t i t ut e i n D e t r oi t w he r e t he
c e l l l i ne w a s e s t a bl i s he d [ 24] . P r i or t o M C F - 7 c e l l , i t w a s a l m os t i m pos s i bl e f or
r e s e a r c he r s t o ge t a m a m m a r y c e l l l i ne t ha t w a s c a pa bl e of l i v i ng l onge r t ha n a f e w
m ont hs [ 25] . P a r t i c ul a r l y , M C F - 7 w a s t he f i r s t hor m one - r e s pons i ve br e a s t c a nc e r c e l l l i ne
w hi c h he l pe d m a ke a huge a dva nc e i n f i ndi ng a ppl i c a t i ons i n e xpe r i m e nt a l t he r a pe ut i c s
[ 24] . B r e a s t c a nc e r ha s he t e r oge ne i t y t ha t m a de t he c l a s s i f i c a t i on of i t s c e l l l i ne s ba s e d on
hi s t ol ogi c a l t ype , l ym ph node s t a t us , t um or gr a de a nd t he pr e s e nc e of pr e di c t i ve m a r ke r s
s uc h a s E R ( e s t r oge n r e c e pt or ) a nd/ or H E R 2( hum a n e pi de r m a l gr ow t h f a c t or r e c e pt o r )
[ 26] . A c c or di ng t o t he ge ne e xpr e s s i on pr of i l e a s w e l l a s t he i m m unohi s t oc he m i c a l
e xpr e s s i on of E R , P R ( P r oge s t e r one r e c e pt or ) a nd H E R 2, w e c a n c l a s s i f y t he br e a s t
c a nc e r c e l l l i ne s i nt o f i ve s ubt ype s : l um i na l A , l um i na l B , H E R 2, ba s a l a nd nor m a l [ 24] .
M C F - 7 c e l l s be l ong t o t he l um i na l A s ubt ype , a nd i t s i m m unopr of i l e i s i ndi c a t e d a s E R + ,
P R + / –, H E R 2 – [ 25] . T he a bi l i t y t o pr oc e s s e s t r a di ol vi a c yt opl a s m i c e s t r oge n r e c e pt or s
a nd gr ow t h i nhi bi t i on by T N F a l pha ( t um or ne c r os i s f a c t or a l pha ) a l l m a ke s t hi s c e l l l i ne
uni que t o r e s e a r c h [ 26] . A c c or di ng t o M e dl i ne - ba s e d s ur ve y , M C F - 7 a nd ot he r t w o br e a s t
c a nc e r c e l l l i ne s , M D A - M B - 231 a nd T - 47D , ha ve a c c ount e d f or m or e t ha n t w o- t hi r ds of
a l l a bs t r a c t s r e por t i ng s t udi e s [ 27] .
6
I n pr e vi ous s t udi e s f r om D r . Z a ndi ’ s l a b , pr ot e i ns t ha t c o- i m m unopr e c i pi a t e t d
w i t h T M E M 56 w e r e i de nt i f i e d by L T Q –E T D m a s s s pe c t r om e t r y . M os t l y , T M E M 56
pr ot e i n w a s pul l e d dow n w i t h A T P a s e α a nd β s ubuni t s [ 6] . T hi s r e s ul t s ugge s t e d t ha t
T M E M 56 pr ot e i n m a y r e gul a t e m i t oc hondr i a l A T P a s e . The c o- l oc a l i z a t i on of T M E M 56
pr ot e i n a nd A T P a s e F 1 c om pone nt s w a s s how n by i m m unof l uor e s c e nc e us i ng a nt i - F l a g
a nd a nt i - A T P a s e α a nt i bod i e s [ 6] . I n a ddi t i on, de c r e a s e d A T P s ynt ha s e a c t i vi t y i n
i s ol a t e d m i t oc hondr i a w a s obs e r ve d w he n T M E M 56 w a s knoc ke d dow n i n H E K 293T
c e l l s . A nd t he e xpr e s s i on of F L A G - T M E M 56 r e s ul t e d i n a n i nc r e a s e d A T P s ynt ha s e
a c t i vi t y us i ng i s ol a t e d m i t oc hondr i a i n vi t r o [ 6] . The s e da t a i l l us t r a t e d a f unc t i ona l
r e l a t i ons hi p be t w e e n T M E M 56 pr ot e i n w i t h A T P a s e F 1c om pone nt of m i t oc hondr i a . F -
A T P a s e ( a l s o c a l l e d A T P s ynt ha s e ) i s a di s t i nc t l y i m por t a nt e nz ym e i n t he i nne r
m e m br a ne of m i t oc hondr i a . F - A T P a s e c a n us e t he pr ot on gr a di e nt w hi c h i s ge ne r a t e d by
oxi da t i ve phos phor yl a t i on t o dr i ve t he s ynt he s i s of A T P [ 27] . F - A T P a s e i s a bi pa r t i t e
s t r uc t ur e w i t h i nt e gr a l m e m br a ne por t i on F 0 a nd pe r i phe r a l por t i on F 1. F 1 i s c om pos e d
of f i ve di f f e r e nt s ubuni t s , ( α β ) 3 , a nd a c t s a s a n A T P a s e . F 0 a nd F 1 t oge t he r f or m a
r ot a r y m ot or . T he r e i s a s ubs t r a t e - bi ndi ng s i t e on e a c h of t he α a nd β s ubuni t s . T he
s ubs t r a t e - bi ndi ng s i t e s on t he β s ubuni t s a r e c a t a l yt i c , w hi l e t hos e on t he α s ubuni t s a r e
r e gul a t or y [ 27] [ 28] . F - A T P a s e c a n c oupl e pr ot on t r a ns l oc a t i on t hr ough F 0 w i t h A T P
s ynt he s i s i n F 1. I n de t a i l , w he n t he pr ot ons bi nd t o t he s ubuni t s of t he F 0 dom a i ns , t he y
c a n c a us e pa r t s of t he A T P a s e t o r ot a t e . T hi s s pa c e va r i a t i on c a n i nduc e t he r ot a t i on of
s ubuni t , i n t ur n m a ki ng t he α a nd β s ubuni t s of t he F 1 dom a i n unde r go a s e que nc e of
c onf or m a t i ona l c ha nge s . A D P a nd P i bi nd t o t he β s ubuni t s a nd t he c onf or m a t i on c ha nge
7
i n F 1 l e a ds t o t he f or m a t i on of A T P f r om A D P a nd P i [ 27] [ 28] . T hus , F - A T P a s e i s
i ndi s pe ns a bl e t o t he s ynt he s i s of A T P .
F i gu r e 2 S c h e m at i c d i agr am of t h e F - A T P as e
F - A T P a s e c o n s i s t s o f a r o t a t i n g i o n p u m p ( F 0 ) c o u p l e d b y a s h a f t t o a n A T P a s e ( F 1 ) . I t a l l o w s i o n s
( u s u a l l y H
+
, s o m e t i m e s N a
+
) t o e n t e r t h e i n t e r - m e m b r a n e s p a c e a t h i g h c o n c e n t r a t i o n , t h e n d r i v e A T P
s y n t h e s i s w i t h e n z y m e r o t a t i o n .
h t t p : / / w w w . b i o l o g y e x a m s 4 u . c o m / 2 0 1 2 / 1 2 / p o l a r - b e a r s - m a i n t a i n - t h e i r - b o d y . h t m l
M a ny s t udi e s ha ve s how n t ha t m ut a t i ons i n m i t oc hondr i a l ge ne s a r e c om m on i n
c a nc e r c e l l s a nd t he y a l t e r t he m i t oc hondr i a l bi os ynt he t i c a nd bi oe ne r ge t i c s t a t e [ 29] .
M i t oc hondr i on i s a m e m br a ne - bound or ga ne l l e f ound i n m os t e uka r yot i c c e l l s [ 30] . I t
s e r ve s a s a s our c e of c he m i c a l e ne r gy w he r e m os t of t he c e l l ’ s s uppl y of a de nos i ne
t r i phos pha t e i s ge ne r a t e d [ 30] . I n a ddi t i on, m i t oc hondr i a a r e i nvol ve d i n bi o- a c t i vi t i e s
s uc h a s s i gna l i ng, c e l l di f f e r e nt i a t i on a nd c e l l de a t h [ 31] . N or m a l c e l l s us e m i t oc hondr i a l
oxi da t i ve phos phor yl a t i on t o pr oduc e e ne r gy , w he r e a s c a nc e r c e l l s l a r ge l y de pe nd on
8
a e r obi c gl yc ol ys i s ( t he W a r bur g e f f e c t ) t o ge ne r a t e e ne r gy [ 32] . M i t oc hondr i a l
dys f unc t i on c a n be c a us e d by t he m ut a t i ons e i t he r i n t he m i t oc hondr i a l D N A ( m D N A ) or
nuc l e a r ge ne s w hi c h e nc ode m i t oc hondr i a l pr ot e i ns [ 33] . T he di s or de r of m i t oc hondr i a
a nd i t s m D N A w i l l a l t e r i t s m e t a bol i c a c t i vi t i e s a nd i nhi bi t oxi da t i ve phos phor yl a t i on,
i nc r e a s e R O S ( R e a c t i ve O xyge n S pe c i e s ) pr oduc t i on, pr om ot e unc ont r ol l e d gr ow t h,
e nha nc e t um or i ge ne s i s a nd e ve n s uppor t a m e t a s t a t i c phe not ype [ 32] . m D N A m ut a t i ons
oc c ur a t a ve r y hi gh r a t e i n t um or s i nc l udi ng br e a s t c a nc e r .
T h e a i m of t hi s t he s i s w a s t o s t udy t he pot e nt i a l r ol e of T M E M 56 pr ot e i n i n
t um or i ge ni c gr ow t h of M C F - 7 hum a n br e a s t c a nc e r c e l l s . S t a bl e c e l l l i ne s w e r e
e s t a bl i s he d t o ove r - e xpr e s s a nd knoc k - dow n t he T M E M 56 e xpr e s s i on. A c c or di ng t o t he
r e s ul t s f r om i n vi vo t um or i ge ne s i s , c e l l c yt ot oxi c i t y a s s a y , c e l l pr ol i f e r a t i on a s s a y , R e a l -
t i m e R T - P C R a nd w e s t e r n bl ot , w e c onc l ude t ha t T M E M 56 pr ot e i n doe s not ha ve a
t um or s uppr e s s or f unc t i on i n M C F - 7 c e l l s . H ow e ve r , t he a bnor m a l T M E M 56 e xpr e s s i on
w i l l de c r e a s e t um or pr ogr e s s i on t o s om e e xt e nt w i t hout c ha ngi ng t he e xpr e s s i on of
A T P a s e β s ubuni t . T he r e l a t i ons hi p be t w e e n T M E M 56 pr ot e i n a nd A T P a s e β s t i l l ne e ds
t o be f ur t he r i nve s t i ga t e d .
9
C H A P T E R T WO
M A T E R I A L S A N D M E T H O D S
2.1 C e l l c u l t u r e c on di t i on s
T he br e a s t c a nc e r c e l l l i ne M C F - 7 ( a gi f t f r om D r . N our i N e a m a t i ’ s l a b , S c hool of
P ha r m a c y, U S C ) w a s c ul t ur e d i n H i gh G l uc os e D ul be c c o ’ s M odi f i e d E a gl e ’ s M e di um
( D M E M ) s uppl e m e nt e d w i t h 1% a nt i bi ot i c s ( pe ni c i l l i n a nd s t r e pt om yc i n) a nd 10% f e t a l
bovi ne s e r um . A l l c e l l s w e r e m a i nt a i ne d a t 37
o
C a nd 5% C O 2 i n 100m m × 20m m c or ni ng
pl a t e s . T he c e l l s w e r e s ubc ul t ur e d a t a r ound 80% c onf l ue nc e e ve r y 3 t o 4 da ys .
2.2 E s t abl i s h m e n t of M C F - 7 s t abl e c e l l l i n e s
F our di f f e r e nt M C F - 7 s t a bl e c e l l l i ne s : G F P - c ont r ol , G F P - T M , C O N - s hR N A a nd
228- s hR N A M C F - 7 w e r e c ons t r uc t e d by t r a ns f e c t i on . T he r e s pe c t i ve pl a s m i ds f or G F P -
c ont r ol , G F P - T M , C O N - s hR N A a nd T M - s hR N A M C F - 7 c e l l s w e r e pC M V 6- A C - G F P
ve c t or ( O r i G e ne T e c hnol ogi e s , R oc kvi l l e , M D , U S ) , pC M V 6- A C - G F P - T M ( O r i G e ne
T e c hnol ogi e s , R oc kvi l l e , M D , U S ) , m i s s i on non- t a r ge t s hR N A c ont r ol ve c t or ( S i gm a
A l dr i c h, S t . L oui s , M O , U S ) a nd T M E M 56 m i s s i on s hR N A pl a s m i d D N A ( O l i go N a m e :
T R C N 0000141547, S i gm a A l dr i c h, S t . L oui s , M O , U S ) . T M E M 56 w a s i ns e r t e d a t S gf 1
a nd M l u 1 r e s t r i c t i on s i t e of pC M V 6- A C - G F P ve c t or . C e l l s w e r e t r a ns f e c t e d a t 80%
c onf l ue nc e by us i ng L i pof e c t a m i ne ® L T X R e a ge nt ( L i f e t e c hnol ogi e s , G r a nd I s l a nd, N Y ,
U S ) .T he t r a ns f e c t i on pr oc e s s w a s a s f ol l ow s : 0.5ug of pl a s m i d w a s i nc uba t e d w i t h 100ul
P L U S r e a ge nt a nd O P T I - M E M , t he m i x w a s a dde d t o 100ul l i pof e c t a m i ne a nd M E M
m e di um a nd t he n l e f t a t r oom t e m pe r a t ur e f or 30 m i nut e s . A f t e r 30 m i nut e s , c e l l m e di um
w a s r e pl a c e d w i t h 1m l O P T I - M E M , a nd t he pl a s m i d- r e a ge nt - l i pof e c t a m i ne m i x w a s
a dde d t o t he c e l l pl a t e . T he c e l l s w e r e i nc uba t e d a t 37° C w i t h 5% C O 2 f or 5 t o 6 hour s ,
1 0
a nd s ubs e que nt l y , t he m e di um w a s c ha nge d t o nor m a l c ul t ur e m e d i um . A l l pl a s m i ds w e r e
t r a ns f e c t e d by us i ng t he s a m e pr ot oc ol a s a bove . A c c or di ng t o t he s e l e c t i on t a g on our
pl a s m i ds , 0.8ug/ m l pur om yc i n w a s us e d t o s e l e c t knoc k dow n c e l l l i ne s , a nd 400ng/ m l
G e ne t i c i n- 418 w a s us e d t o s e l e c t ove r - e xpr e s s e d c e l l l i ne s . A ppr oxi m a t e l y t w o w e e ks
l a t e r , t he c e l l s w e r e s or t e d t o be t he pur e one s . D r ug c onc e nt r a t i ons w e r e de c r e a s e d t o
0.1ug/ m l pur om yc i n a nd 50ng/ m l G e ne t i c i n- 418 f or m a i nt e na nc e . T he ove r - e xpr e s s e d
c e l l l i ne s c a n be e xa m i ne d unde r t he f l uor e s c e nc e m i c r os c ope a s t he y c ont a i ne d G F P t a g
ge ne .
2.3 R e al - t i m e R T - P C R f or T M E M 56 e xpr e s s i on
T ot a l R N A w a s e xt r a c t e d f r om M C F - 7 c e l l s f ol l ow i ng t he pr ot oc ol i n R N e a s y
M i ni K i t ( Q i a G e n, U S A ) . 75ng i s ol a t e d R N A f r om t he f our di f f e r e nt M C F - 7 s t a bl e c e l l
l i ne s : G F P - c ont r ol , G F P - T M , C O N - s hR N A a nd T M - s hR N A w e r e us e d t o do R e a l t i m e
R T - P C R . R e a ge nt s us e d i nc l ude E V A - G R E E N , one s t e p f a s t m a s t e r m i x a nd R T f a s t
e nz ym e w hi c h w e r e pur c ha s e d f r om Q ua nt a B i os c i e nc e .
F or w a r d pr i m e r ( 5 ’ t o 3’ ) R e ve r s e pr i m e r ( 3 ’ t o 5’ )
T M E M 56 T T T C C A G C C C G T T T G T G A A T C
A G C
C A T T G A G G C A A T C C G C A C G
A T G A A
B e t a - a c t i n G G C A C C C A G C A C A A T G A A G G C C G A T C C A C A C G G A G T A
T a b l e 1 L i s t of p r i m e r s u s e d f or R T - P C R
2.4 C e l l pr ol i f e r at i on as s ay
F i ve di f f e r e nt c e l l l i ne s ( M C F - 7, c on- s hR N A , T M - s hR N A , G F P - c ont r ol a nd
G F P - T M c e l l s ) w i t h a ppr oxi m a t e l y 3× 10
4
c e l l s w e r e i nc uba t e d i n 12- w e l l pl a t e . T he n t he
1 1
c e l l num be r w a s c ount e d by us i ng he m oc yt om e t e r a f t e r 24h, 48h , 72h a nd 96h f or e a c h
c e l l l i ne .
2.5 C y t oS c an W S T - 1 C e l l c y t ot oxi c i t y as s ay
C yt oS c a n W S T - 1 C e l l c yt ot oxi c i t y a s s a y i s a c ol or i m e t r i c a s s a y t ha t qua nt i f i e s
c e l l ul a r c yt ot oxi c i t y a nd pr ol i f e r a t i on . T he pr i nc i pl e of t he a s s a y i s ba s e d on t he r e duc t i on
of t he t e t r a z ol i um s a l t W S T - 1A t o f or m a z a n by c e l l ul a r de hydr oge na s e s . T he ge ne r a t i on
of t he da r k c ol or e d f or m a z a n c a n be m e a s ur e d a t 440nm a nd t he a bs or ba nc e di r e c t l y
c or r e l a t e w i t h c e l l num be r . A s s a y buf f e r w a s pr e pa r e d a c c or di ng t o t he ki t pr ot oc ol pr i or
t o us e . C e l l s f or e a c h of t he f our M C F - 7 s t a bl e c e l l l i ne s w e r e c ul t ur e d i n a 96- w e l l pl a t e
w i t h a ppr oxi m a t e l y 5× 10
4
c e l l s pe r w e l l i n a f i na l vol um e of 90ul . A f t e r i nc uba t i ng t he
c e l l s ove r ni ght , a s e r i e s of di f f e r e nt c onc e n t r a t i on s of 10ul pa c l i t a xe l ( 0.1n M , 1nM , 10nM ,
100nM , 1uM , 10uM ) w e r e a dde d t o t he w e l l s a nd t he c e l l s w e r e c ul t ur e d a t 37
0
C
i nc uba t or w i t h 5% C O 2 f or 48 hour s . E a c h c e l l l i ne a t a pa r t i c ul a r c onc e nt r a t i on w a s done
i n t r i pl i c a t e . B l a nk w e l l s w i t h c ul t ur e m e di um onl y w e r e a l s o pr e pa r e d . A t t he e nd of t he
t r e a t m e nt , 10ul W S T - 1/ C E C A s s a y buf f e r w a s a dde d t o e a c h w e l l . T he pl a t e w a s ge nt l y
s ha ke n t o m i x c he m i c a l s w i t h m e di um a nd t he c e l l s w e r e i nc uba t e d f or 30 m i n a t 37
0
C .
T he pl a t e w a s t he n s ha ke n ge nt l y f or 1 m i nut e . T he a bs or ba nc e w a s m e a s ur e d on
m i c r opl a t e r e a de r ( B M G L A B T E C H F L U O s t a r O m e ga , O r t e nbe r g, G e r m a ny) a t a
w a ve l e ngt h of 440nm a c c or di ng t o t he m a nuf a c t ur e r ’ s s pe c i f i c a t i ons .
2.6 N u de m i c e i n j e c t i on i n v i v o
N ude m i c e a r e l a bor a t or y m i c e w i t h ge ne t i c m ut a t i on t ha t r e s ul t s i n i nhi bi t e d
i m m une s ys t e m . F our M C F - 7 c e l l l i ne s : c on- s hR N A , T M - s hR N A , G F P - c ont r ol a nd
G F P - T M w e r e i nj e c t e d i nt o i m m une - de f i c i e nt m i c e ( J a c ks on L a bor a t or i e s ) t o m e a s ur e
1 2
t um or gr ow t h. T hr e e m i c e w e r e i nj e c t e d w i t h c on- s hR N A a nd T M - s hR N A on e a c h s i de ,
w hi l e t hr e e ot he r s w e r e i nj e c t e d w i t h G F P - c ont r ol a nd G F P - T M on e a c h s i de . T he m i c e
w e r e a na e s t he t i z e d i nt r a - pe r i t one a l l y us i ng X yl a z i ne a nd K e t a m i ne . O ne m i l l i on c e l l s
w e r e s us pe nde d i n 100ul P B S a nd m a t r i ge l a nd i nj e c t e d t o one s i de of t he m ous e
s ubc ut a ne ous l y. I nj e c t i ons w e r e done i n t hr e e m i c e t o m a ke a t r i pl i c a t e . T he c ondi t i on of
e a c h m ous e w a s c he c ke d a f t e r t he i nj e c t i on. T he m i c e w e r e w e l l m a i nt a i ne d i n s pe c i a l
hous i ng a nd m oni t or e d r e gul a r l y by a ni m a l r oom s t a f f . T um or s i z e ( l e ngt h a nd w i dt h)
w a s m e a s ur e d e ve r y 3 t o 4 da ys by c a l i pe r s a nd t he t um or vol um e w a s c a l c ul a t e d w i t h t he
f or m ul a ( w i dt h)
2
× l e ngt h/ 2. 50 da ys a f t e r i noc ul a t i on, t he m i c e w e r e s a c r i f i c e d by
c e r vi c a l di s l oc a t i on. T um or s w e r e t a ke n out a nd c ut i nt o t hr e e pi e c e s a nd s t or e d
s e pa r a t e l y i n f or m a l i n, c e l l f r e e z i ng m e di um a nd bl a nk t ube . A l l t he ope r a t i on s i nvol ve d
w e r e i n a c c or da nc e t o t he U S C a ni m a l e xpe r i m e nt gui de l i ne s .
2.7 A T P as e β I m m u n oh i s t oc h e m i s t r y
T i s s ue s e c t i ons w e r e c ut f r om t he t um or s e c t i ons t ha t w e r e s t or e d i n f or m a l i n. T he
t i s s ue s e c t i ons w e r e a na l yz e d by he m a t oxyl i n a nd e os i n s t a i ni ng ( H & E s t a i n) a nd
i m m unohi s t oc he m i s t r y w i t h a nt i - A T P a s e β a nt i body ( B D B i os c i e nc e s ) t o e xa m i ne t he
e xpr e s s i on of A T P a s e β s ubuni t .
2.8 P r ot e i n e xt r ac t i on f r om t u m or an d W e s t e r n bl ot f or A T P as e β
P r ot e i ns w e r e e xt r a c t e d f r om t um or s by us i ng R I P A l ys i s buf f e r ( 50 m M T r i s - H C l
pH 7.5, 150 m M N a C l , 1 m M P M S F , 1 m M E D T A , 1% T r i t on x- 100 , 1% S odi um
de oxyc hol a t e a nd 0.1% S D S ) . T he pr ot e i n c onc e nt r a t i on w a s m e a s ur e d f or e a c h t um or by
us i ng t he B r a df or d a s s a y . 6ug pr ot e i n f r om t he t um or s a nd c e l l l i ne s w a s l oa de d i nt o
12% S D S - ge l a nd t he W e s t e r n- bl ot w a s done a c c or di ng t o t he ge ne r a l pr ot oc ol . T he
1 3
pr i m a r y a nt i body us e d w a s a nt i A T P a s e β ( B D B i os c i e nc e s ) w i t h 1: 1000 di l ut i on. T he
s e c onda r y a nt i body us e d w a s α a nt i - m ous e ( B D B i os c i e nc e s ) w i t h 1: 10000 di l ut i on.
A l s o t he e x pr e s s i o n of β - a c t i n w i t h β - a c t i n pr i m a r y a n t i bo dy ( B D B i os c i e nc e s ) ( 1 : 150 00 )
w a s e xa m i ne d a s a c ont r ol .
1 4
C H A P T E R T H R E E
R E S U L T S
3.1 R e l at i v e T M E M 56 bas al e xpr e s s i on i n di f f e r e n t c e l l l i n e s
T he pr e vi ous s t udi e s on T M E M 56 pr ot e i n w e r e done i n H E K 293T c e l l s ( hum a n
e m br yoni c ki dne y c e l l s ) . I t w a s s how n t ha t T M E M 56 pr ot e i n w a s l oc a t e d i n
m i t oc hondr i a i nt e r a c t i ng w i t h α a nd β s ubuni t s of A T P a s e [ 6] . I t ha s a l s o be e n s how n t ha t
T M E M 56 m a y ha ve a r ol e a s a t um or s uppr e s s or a nd r e gul a t e m i t oc hondr i a l a bunda nc e
a nd t um or pr ogr e s s i on i n H E K 293T c e l l s . H ow e ve r , t he f unc t i on of T M E M 56 pr ot e i n
ha s not be e n i de nt i f i e d i n ot he r c e l l l i ne s . T o be gi n t hi s s t udy , t he ba s a l e xpr e s s i on of
T M E M 56 i n M C F - 7 c e l l s w e r e c om pa r e d t o t hos e of H E K 293T a nd P C 3 c e l l s . R e a l -
t i m e R T - P C R w a s done t o t e s t t he ba s a l e xpr e s s i on of T M E M 56 ge ne on a
t r a ns c r i pt i on a l l e ve l i n M C F - 7 c e l l s , P C 3 c e l l s ( hum a n pr os t a t e c a nc e r c e l l s ) a nd H E K
293T c e l l s . F i gur e 3 s how s t he r e l a t i ve T M E M 56 e xpr e s s i on l e ve l i n t he s e t hr e e c e l l
l i ne s . C om pa r e d t o P C 3 a nd H E K 293T c e l l s , M C F - 7 c e l l s ha d t he l ow e s t T M E M 56
ge ne e xpr e s s i on.
1 5
F i gu r e 3 R e l at i ve T M E M 56 b as al l e ve l e xp r e s s i on i n M C F - 7, P C - 3 an d H E K 293T
c e l l s
3.2 E s t abl i s h m e n t of M C F - 7 s t abl e c e l l l i n e s
I n or de r t o knoc k - dow n or ove r - e xpr e s s hum a n T M E M 56 pr ot e i n i n M C F - 7 c e l l s ,
s t a bl e c e l l l i ne s w e r e ge ne r a t e d. O ur s t a bl e c e l l l i ne s w e r e ge ne r a t e d vi a di r e c t i nt e gr a t i on
of t he t r a ns f e c t e d pl a s m i d i nt o t he t a r ge t c e l l s ge nom e w i t h a pos i t i ve s e l e c t i on m a r ke r - -
pur om yc i n or G 418. M C F - 7 c e l l s w e r e t r a ns f e c t e d w i t h T M E M 56 m i s s i on s hR N A
pl a s m i d w i t h pur om yc i n r e s i s t a nc e t o knoc k dow n T M E M 56 e xpr e s s i on, a nd t r a ns f e c t e d
w i t h pC M V 4- A C - G F P - T M ve c t or w i t h G 418 r e s i s t a nc e t o e xpr e s s T M E M 56. C O N -
s hR N A a nd G F P - c ont r ol w e r e a l s o c ons t r uc t e d a s c ont r ol s . T he gr a ph s how n i n F i gur e 4
i s t he R e a l - t i m e R T - P C R r e s ul t f or t he r e l a t i ve T M E M 56 e xpr e s s i on i n knoc k- dow n a nd
ove r - e xpr e s s e d M C F - 7 c e l l l i ne s . B e t a - a c t i n w a s us e d a s a n i nt e r na l c ont r ol . T he
1 6
e xpr e s s i on of T M E M 56 ha d s uc c e s s f ul l y l ow e r e d i n T M - s hR N A M C F - 7 c e l l s by 60%
a nd i nc r e a s e d i n G F P - T M M C F - 7 c e l l s 32 t i m e s c om pa r e d t o t he i r c or r e s pondi ng
c ont r ol s ( s how n i n F i gur e 4) . A s G F P - T M a nd G F P - c ont r ol c e l l s a l s o ha d G F P t a g s , w e
w e r e a bl e t o m e a s ur e a nd s e l e c t t he gr e e n pos i t i ve c e l l s by f l uor e s c e nc e m i c r os c ope .
A c c or di ng t o t he obs e r va t i ons , G F P - T M M C F - 7 c e l l s w e r e gr ow i ng s l ow l y a nd ke pt
dyi ng a f t e r t r a ns f e c t i on. T he r e f or e , s i ngl e c l one w a s pi c ke d a nd gr ow n. T he pi c t ur e s i n
F i gur e 5 s how t he f l uor e s c e nc e pe r c e nt a ge s i n G F P - c ont r ol M C F - 7 a nd G F P - T M M C F - 7
c e l l s unde r f l uor e s c e nt m i c r os c ope . T he gr e e n f l uor e s c e n c e pe r c e nt a ge f or G F P - T M
M C F - 7 w a s a ppr oxi m a t e l y 50% of t he t ot a l c e l l s .
1 7
A
B
F i gu r e 4 R e l at i ve T M E M 56 e xp r e s s i on i n d i f f e r e n t M C F - 7 s t ab l e c e l l l i n e s
R N A f r o m d i f f e r e n t c e l l l i n e s w a s e x t r a c t e d b y R N e a s y M i n i K i t . T h e R e a l - t i m e R T - P C R w a s d o n e
w i t h 7 5 n g R N A t e m p l a t e i n t r i p l i c a t e . A . R e l a t i v e T M E M 5 6 e x p r e s s i o n i n k n o c k - d o w n M C F - 7 c e l l
l i n e s . B . R e l a t i v e T M E M 5 6 e x p r e s s i o n i n o v e r - e x p r e s s e d M C F - 7 s t a b l e c e l l l i n e s .
1 8
F i gu r e 5 G F P - c on t r ol an d G F P - T M c e l l s ’ m or p h ol ogy u n d e r c on t r as t p h as e
m i c r os c op e
G F P - c o n t r o l a n d G F P - T M c e l l s w e r e s e l e c t e d b y 0.8ug/ m l pur om yc i n. H ow e ve r , G F P - T M c e l l s
ke pt dyi ng a nd l os t gr e e n f l uor e s c e nc e . A c c or di ng t o t hi s s i t ua t i on, s i ngl e c l one w a s
pi c ke d i n G F P - T M c e l l s . G F P - T M ha s a ppr oxi m a t e l y 50 % gr e e n f l uor e s c e nt pe r c e nt a ge .
3.3 C h ar ac t e r i z at i on of M C F - 7 s t abl e c e l l l i n e s
C e l l vi a bi l i t y a nd gr ow t h r a t e w e r e t e s t e d by pr ol i f e r a t i on a s s a y . C e l l num be r s
w e r e c ount e d by he m oc yt om e t e r a f t e r c ul t ur i ng f or 24 hour s , 48 hour s a nd 72 hour s
r e s pe c t i ve l y . F i gur e 6 s how s t he c e l l pr ol i f e r a t i on r a t e i n t he nor m a l i nc uba t i ng c ondi t i on.
T he gr ow t h r a t e w a s s i m i l a r be t w e e n t he t r a ns f e c t e d c e l l s a nd c ont r ol s a s s e e n f r om t he
gr a ph . T hi s i ndi c a t e d t ha t t he c ha r a c t e r i s t i c s of t he c e l l s ha d not c ha nge d e ve n t hough
T M E M 56 pr ot e i n e xpr e s s i on i n c e l l s ha s be e n knoc ke d dow n or ove r - e xpr e s s e d.
1 9
A
B
F i gu r e 6 M C F - 7 s t ab l e c e l l l i n e s ’ p r ol i f e r at i on i n n or m al c u l t u r i n g c on d i t i on
A . P r o l i f e r a t i o n o f C O N - s h R N A a n d T M - s h R N A M C F - 7 c e l l l i n e s
B . P r o l i f e r a t i o n o f G F P - c o n t r o l a n d G F P - T M M C F - 7 c e l l l i n e s
2 0
3.4 C e l l s ’ s e n s i t i v i t y t o pac l i t axe l
C yt ot oxi c i t y i s t he qua l i t y of be i ng t oxi c t o c e l l s . T he dr ug us e d f or t hi s
c yt ot oxi c i t y a s s a y w a s pa c l i t a xe l . P a c l i t a xe l i s a dr ug us e d i n br e a s t c a nc e r c he m ot he r a py
a s i t i nt e r f e r e s w i t h c e l l di vi s i on. W i t h i nc r e a s e d c onc e nt r a t i on of pa c l i t a xe l , t he vi a bi l i t y
of c a nc e r c e l l s de c r e a s e d s i gni f i c a nt l y . T he c yt ot oxi c i t y a s s a y w a s pe r f or m e d t o t e s t i f t he
ove r - e xpr e s s i on or dow n- r e gul a t i on of T M E M 56 c oul d c ha nge t he s e ns i t i vi t y of t he c e l l s
t o pa c l i t a xe l . T he gr a ph i n F i gur e 7 s how s t he c ha nge i n c e l l pr ol i f e r a t i on a s t he
c onc e nt r a t i on of pa c l i t a xe l c ha nge s . T he s l ope of t he c ur ve r e f l e c t s c e l l s ’ s e ns i t i vi t y t o
pa c l i t a xe l . B a s e d on t he c ur ve s s how n i n F i gur e 7, t he r e w a s no s i gni f i c a nt di f f e r e nc e i n
c e l l pr ol i f e r a t i on a f t e r t r e a t i ng w i t h pa c l i t a xe l i n t r a ns f e c t e d c e l l s a nd t he i r c or r e s pondi ng
c ont r ol s .
F i gu r e 7 C e l l p r ol i f e r at i on i n a s e r i e s of c on c e n t r at i on s of p ac l i t axe l
C e l l s w e r e t r e a t e d w i t h 0 n M , 0 . 1 n M , 1 n M , 1 0 n M , 1 0 0 n M , 1 u M a n d 1 0 u M , r e s p e c t i v e l y , f o r 4 8 h o u r s .
D y e s o l u t i o n w a s a d d e d a n d a b s o r b a n c e w a s e x a m i n e d a t 4 4 0 n m f o r e a c h c e l l l i n e .
2 1
3.5 T u m or i ge n e s i s i n n u de m i c e f or i n v i v o an al y s i s
S i nc e i t w a s pr e vi ous l y s how n t ha t t he r e duc i ng T M E M 56 e xpr e s s i on i n H E K
293T c e l l s r e s ul t e d i n i nc r e a s e d t um or i ge ni c i t y , i t w a s hypot he s i z e d t ha t T M E M 56 m a y
ha ve a s i m i l a r t um or s uppr e s s or f unc t i on i n M C F - 7 c e l l s gr ow n i n nude m i c e . A t o t a l of
s i x nude m i c e w e r e us e d, t hr e e of t he m w e r e i nj e c t e d w i t h knoc k- dow n T M E M 56 M C F -
7 c e l l s a nd t he ot he r t hr e e w e r e i nj e c t e d w i t h ove r - e xpr e s s e d T M E M 56 M C F - 7 c e l l s . F or
one m ous e , one s i de w a s i nj e c t e d w i t h t he s t a bl e c e l l l i ne s , w hi l e t he ot he r s i de w a s
i nj e c t e d w i t h c or r e s pondi ng c ont r ol s . F i gur e 8 s how s t ha t t he t um or gr ow t h r a t e . F or t he
knoc k dow n T M E M 56 M C F - 7 s t a bl e c e l l l i ne s , t he t um or f or m e d by T M - s hR N A c e l l s
w a s bi gge r t ha n C O N - s hR N A a t t he be gi nni ng. H ow e ve r , a f t e r l onge r t i m e pe r i od, C O N -
s hR N A t um or s gr e w m uc h f a s t e r t ha n T M - s hR N A . O ppos i t e phe nom e non w a s obs e r ve d
i n ove r e xpr e s s e d M C F - 7 s t a bl e c e l l l i ne s . T he gr ow t h r a t e s f or bot h t um or s w e r e hi gh
dur i ng t he f i r s t w e e k but t he n t he r a t e s l ow e d dow n i nt o a s t a t i ona r y pha s e . I n a ddi t i on,
t um or s f or m e d by G F P - T M gr e w ve r y s l ow l y .
2 2
F i gu r e 8 T u m or vol u m e f or m i c e i n j e c t e d w i t h M C F - 7 s t ab l e c e l l l i n e s ac c or d i n g t o
t h e d ays af t e r i n oc u l at i on
O n t he 50
t h
da y of i noc ul a t i on, t he m i c e w e r e s a c r i f i c e d by c e r vi c a l di s l oc a t i on.
T u m or s w e r e t a ke n out a nd s a ve d. F r om F i gur e 9, m i c e i nj e c t e d w i t h e i t he r G F P - T M
M C F - 7 c e l l s or 228- s hR N A ha s s how n l e s s t um or vol um e t ha n G F P - c ont r ol or C O N -
s hR N A r e s pe c t i ve l y . T u m or vol um e s w e r e c a l c ul a t e d a c c or di ng t o t he f or m ul a ( w i dt h)
2
× l e ngt h/ 2 f or e a c h c e l l l i ne . T he a ve r a ge t um or vol um e s how n i n F i gur e 10 r e ve a l e d t ha t
m i c e i nj e c t e d w i t h G F P - c ont r ol M C F - 7 c e l l s ha d on a ve r a ge 2.5 t i m e s l a r ge r t um or s t ha n
t he G F P - T M one s . S i m i l a r l y , C O N - s hR N A M C F - 7 c e l l s f or m e d on a ve r a ge 2.4 t i m e s
l a r ge r t um or s i n nude m i c e t ha n T M - s hR N A M C F - 7 c e l l s .
2 3
A B
C D
F i gu r e 9 X e n ogr af t as s ay on i m m u n e - d e f i c i e n t m i c e
A . N u d e m i c e i n j e c t e d w i t h k n o c k - d o w n M C F - 7 c e l l s . L e f t s i d e o f t h e m o u s e w a s i n j e c t e d w i t h
C O N - s h R N A , r i g h t s i d e w i t h T M - s h R N A
B . N u d e m i c e i n j e c t e d w i t h O v e r - e x p r e s s e d M C F - 7 c e l l s . L e f t i s G F P - c o n t r o l , r i g h t i s G F P - T M
C . I s o l a t e d t u m o r m o r p h o l o g y f o r m e d b y C O N - s h R N A a n d T M - s h R N A M C F - 7 c e l l l i n e s
D . I s o l a t e d t u m o r m o r p h o l o g y f o r m e d b y G F P - c o n t r o l a n d G F P - T M M C F - 7 c e l l l i n e s
2 4
A
B
F i gu r e 10 T u m or vol u m e s i n xe n ogr af t as s ay
T h e w i d t h a n d l e ngt h of t um or w e r e m e a s u r e d , a nd t he vol um e s w e r e c a l c ul a t e d by us i ng
t he f or m ul a ( w i dt h)
2
× l e ngt h/ 2. A . T u m o r v o l u m e f o r e a c h n u d e m o u s e B . A v e r a g e v o l u m e s
f o r m e d b y e a c h M C F - 7 c e l l s t a b l e l i n e s
M o u s e
n u m b e r
T u m o r v o l u m e
( m m 3 )
M o u s e
n u m b e r
T u m o r v o l u m e
( m m 3 )
L e f t s i d e
( C O N -
s h R N A )
R i g h t s i d e
( 2 2 8 -
s h R N A )
L e f t s i d e
( G F P -
c o n t r o l )
R i g h t s i d e
( G F P -
T M )
M o u s e # 1 1 9 9 1 3 1 . 1 M o u s e # 4 1 1 9 . 2 4 2 . 6
M o u s e # 2 1 9 4 8 7 . 8 M o u s e # 5 1 0 8 4 8 . 7
M o u s e # 3 2 5 2 4 5 . 6 M o u s e # 6 1 0 2 . 7 3 2
A v e r a g e
t u m o r
v o l u m e ( m m
3
)
2 1 5 8 8 . 2
A v e r a g e t u m o r
v o l u m e ( m m
3
) 1 1 0 4 1 . 1
2 5
T u m or s w e r e i s ol a t e d a nd ke pt i n c e l l l ys i s buf f e r . R N A of t he t um or s w a s
e xt r a c t e d a nd t he r e l a t i ve T M E M e xpr e s s i on w a s t e s t e d by R e a l - t i m e R T - P C R . T he
di f f e r e nc e i n T M E M 56 e xpr e s s i on be t w e e n C O N - s hR N A a nd T M - s hR N A , a nd G F P -
c ont r ol a nd G F P - T M i n t he t um or s w a s gr e a t e r t ha n t he di f f e r e nc e s e e n i n t he c e l l s
( s how n i n F i gur e 1 1 ) .
2 6
A
B
F i gu r e 1 1 R e l at i ve T M E M 56 e xp r e s s i on i n t u m or s f r om t h e n u d e m i c e i n oc u l at e d
w i t h T M E M 56 k n oc k d ow n or ove r - e xp r e s s e d M C F - 7 s t ab l e c e l l l i n e s
A . R e l a t i v e T M E M 5 6 e x p r e s s i o n i n t u m o r s f o r m e d b y C O N - s h R N A a n d T M - s h R N A M C F - 7 c e l l s
B . R e l a t i v e T M E M 5 6 e x p r e s s i o n i n t u m o r s f o r m e d b y G F P - c o n t r o l a n d G F P - T M M C F - 7 c e l l s
2 7
T um or s t a t ue s w e r e obs e r ve d w he n t um or s w e r e i s ol a t e d f r om nude m i c e .
T u m or s ge ne r a t e d by G F P - c ont r ol a nd C O N - s hR N A M C F - 7 c e l l s w e r e r e d i n c ol or ,
w hi l e t um or s f r om G F P - T M a nd T M - s hR N A w e r e w hi t e ( s how n i n F i gur e 12) . O ne
s pe c ul a t i on w a s t ha t t he r e d w a s f or m e d by t um or va s c ul a r i z a t i on.
A
B
F i gu r e 12 M or p h ol ogy of t u m or s i s ol at e d f r om n u d e m i c e i n j e c t e d w i t h d i f f e r e n t
M C F - 7 c e l l l i n e s
A . T h e t u m o r s g e n e r a t e d b y C O N - s h R N A ( l e f t ) a n d T M - s h R N A ( r i g h t ) c e l l s
B . T h e t u m o r s g e n e r a t e d b y G F P - c o n t r o l ( l e f t ) c e l l s a n d G F P - T M ( r i g h t ) c e l l s
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S i nc e pr e vi ous s t udy ha s s how n t ha t T M E M 56 pr ot e i n w a s c o- l oc a l i z e d w i t h
A T P a s e α a nd β i n m i t oc hondr i a a nd m a ny pa pe r s ha ve r e por t e d t ha t m i t oc hondr i a l
dys f unc t i on c or r e l a t e d w i t h c a nc e r , a s e c t i on of e a c h t um or w a s pr e s e r ve d i n f or m a l i n
a nd w a s us e d f or i m m unohi s t oc he m i s t r y w i t h A T P a s e β a nt i body . I m m unohi s t oc he m i s t r y
i s w i de l y us e d f or de t e c t i ng pr ot e i ns i n t i s s ue s ba s e d on t he pr i nc i pl e of a nt i ge n a nd
a nt i body i nt e r a c t i on. F i gur e 13 s how s A T P a s e β e xpr e s s i on i n t um or s f or m e d by C O N -
s hR N A , T M - s hR N A , G F P - c ont r ol a nd G F P - T M M C F - 7 c e l l s . T he i m a ge s i ndi c a t e d t ha t
t he e xpr e s s i on s of A T P a s e β s ubuni t s i n T M - s hR N A a nd G F P - T M t um or s w e r e
a ppr oxi m a t e l y t he s a m e w i t h t he i r c or r e s pondi ng c ont r ol s . T he s e w e r e a l s o c onf i r m e d by
W e s t e r n B l ot t ha t e xa m i n e d A T P a s e β e xpr e s s i on i n di f f e r e nt t um or s a nd c e l l l i ne s a t t he
pr ot e i n l e ve l ( a s s how n i n F i gur e 14 ) .
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A
B
F i gu r e 13 I m m u n oh i s t oc h e m i s t r y f or A T P as e β i n t u m or s f or m e d b y f ou r M C F - 7
s t ab l e c e l l l i n e s ( 10 × an d 40× )
T h e t i s s u e s e c t i o n s f r o m t h e t u m o r s w e r e f o r m a l i n f i x e d p a r a f f i n e m b e d d e d , t h e n
i m m u n o h i s t o c h e m i s t r y w a s d o n e w i t h A T P a s e β s u b u n i t . T h e b l u e s p o t s s h o w e d w h e r e t h e c e l l s
l o c a t e d , w h i l e t h e b r o w n s p o t s s t o o d f o r A P T a s e β .
A . A T P a s e β i n t h e t u m o r s f o r m e d b y C O N - s h R N A a n d T M - s h R N A
B . A T P a s e β i n t h e t u m o r s f o r m e d b y G F P - c o n t r o l a n d G F P - T M
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F i gu r e 14 A T P as e e xp r e s s i on i n t u m or s an d c e l l l i n e s
A T P a s e e x p r e s s i o n w a s t e s t e d b o t h i n t u m o r s a n d c e l l l i n e s b y w e s t e r n b l o t . P r i m a r y a n t i b o d y w a s
a n t i - A T P a s e β w i t h d i l u t i o n o f 1 : 1 0 0 0 , a n d s e c o n d a r y a n t i b o d y w a s α a n t i - m o u s e w i t h d i l u t i o n o f 1 :
1 0 0 0 0 . T h e e x p r e s s i o n o f b e t a - a c t i n ( 1 : 1 5 0 0 0 ) w a s e x a m i n e d a s a c o n t r o l . A T P a s e β s u b u n i t w a s
a r o u n d 5 5 k D a a n d β - a c t i n w a s a r o u n d 4 2 k D a .
A . A T P a s e β s u b u n i t s e x p r e s s i o n i n c e l l s a n d t u m o r s g e n e r a t e d b y C O N - s h R N A a n d T M - s h R N A c e l l s . T h e
f i r s t f o u r l a n e s w e r e t h e e x p r e s s i o n s f r o m t u m o r s i n M o u s e # 1 a n d M o u s e # 2 . T h e l a s t t w o l a n e s w e r e t h e
e x p r e s s i o n f r o m C O N - s h R N A a n d T M - s h R N A c e l l s .
B . A T P a s e β s u b u n i t s e x p r e s s i o n i n c e l l s a n d i n t u m o r s g e n e r a t e d b y G F P - c o n t r o l a n d G F P - T M c e l l s . T h e
f i r s t s i x l a n e s w e r e t h e e x p r e s s i o n s f r o m t u m o r s i n M o u s e # 4 , M o u s e # 5 a n d M o u s e # 6 . T h e l a s t t w o l a n e s
w e r e t h e e x p r e s s i o n s f r o m G F P - c o n t r o l a n d G F P - T M c e l l s .
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C H A P T E R F O U R
C O N C L U S I O N A N D D I S C U S S I O N
I n t hi s t he s i s , t he a i m w a s t o s t udy i f T M E M 56 pr ot e i n c ha nge s t um or i ge ni c
gr ow t h of M C F - 7 hum a n br e a s t c a nc e r c e l l l i ne . T r a ns m e m br a ne pr ot e i n T M E M 56 i s a
263A A pr ot e i n t ha t w a s f i r s t i de nt i f i e d a s a n I K K - a s s oc i a t e d pr ot e i n by m a s s
s pe c t r om e t r y i n D r . E br a hi m Z a ndi ’ s l a b. T hi s pr ot e i n w a s s how n t o i nt e r a c t w i t h A T P a s e
α a nd β s ubuni t s i n m i t oc hondr i a . I n a ddi t i on, i t ha d a pot e nt i a l t um or s uppr e s s or f unc t i on
i n H E K 293T c e l l s by r e gul a t i ng t um or pr ogr e s s i on. B a s e d on t he s e pr e vi ous r e s ul t s , w e
w e r e c ur i ous a bout t he pr ot e i n ’ s r ol e i n c a nc e r c e l l s s i nc e T M E M 56 m a y r e gul a t e
m i t oc hondr i a , a nd m i t oc hondr i a l dys f unc t i on i s r e l a t e d t o c a nc e r . M C F - 7 hum a n br e a s t
c a nc e r s t a bl e c e l l l i ne s C O N - s hR N A , T M - s hR N A , G F P - c ont r ol a nd G F P - T M w e r e
e s t a bl i s he d by t r a ns f e c t i on t o knoc k dow n a nd ove r e xpr e s s T M E M 56 ge ne w hi c h w e r e
c onf i r m e d by R e a l - t i m e R T - P C R da t a . C e l l c yt ot oxi c i t y a s s a y a nd pr ol i f e r a t i on a s s a y
f ound no s i gni f i c a nt di f f e r e nc e be t w e e n t he t r a ns f e c t e d c e l l l i ne s a nd t he i r c ont r ol s ,
i ndi c a t i ng t ha t c e l l gr ow t h r a t e a nd s e ns i t i vi t y t o pa c l i t a xe l w e r e not a f f e c t e d by t he
c ha nge i n T M E M 56 e xpr e s s i on. T he f our M C F - 7 s t a bl e c e l l l i ne s w e r e t e s t e d i n
xe nogr a f t a s s a y i n i m m une - de f i c i e nt m i c e . T he da t a s how e d t ha t t he t um or f or m e d by
bot h G F P - T M a nd T M - s hR N A M C F - 7 c e l l s w e r e s m a l l e r t ha n t he t um or f r om G F P -
c ont r ol a nd C O N - s hR N A c e l l s , r e s pe c t i ve l y . T hi s s ugge s t e d t ha t a bnor m a l T M E M 56
e xpr e s s i on m a y i nhi bi t t um or pr ogr e s s i on. I m m unohi s t oc he m i s t r y w i t h A T P a s e β
a nt i body i ndi c a t e d t ha t G F P - T M a nd T M - s hR N A ha d a ppr oxi m a t e l y t he s a m e A T P a s e β
l e ve l s c om pa r e d t o G F P - c ont r ol a nd C O N - s hR N A , r e s pe c t i ve l y . T hi s r e s ul t w a s a l s o
c on s i s t e nt w i t h W e s t e r n B l ot da t a i n e xa m i ni ng t he A T P a s e β l e ve l i n t um or s . I n s um m a r y ,
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c ha ngi ng T M E M 56 pr ot e i n e xpr e s s i on m a y ha ve s om e a dve r s e e f f e c t s i n M C F - 7 c e l l s .
E i t he r knoc ki ng dow n or ove r - e xpr e s s i ng t he pr ot e i n w i l l r e s ul t i n t he i nhi bi t i on of t um or
pr ogr e s s i on. A ddi t i ona l l y , a bnor m a l T M E M 56 e xpr e s s i on w oul d not i nf l ue nc e A T P a s e β
e xpr e s s i on i n c e l l s , but i t s r ol e i n r e gul a t i ng A T P a s e β w a s unknow n a nd ne e d s f ur t he r
r e s e a r c h.
A c c or di ng t o R e a l - t i m e R T - P C R r e s ul t s , T M E M 56 ge ne e xpr e s s i on ha s a n
a ppr oxi m a t e l y 60% de c r e a s e i n T M - s hR N A M C F - 7 c e l l s , a nd 32 t i m e s i nc r e a s e i n G F P -
T M M C F - 7 c e l l s . T he i n vi vo t um or i ge ne s i s s how e d t ha t a l l t he t um or s f or m e d by e i t he r
T M - s hR N A or G F P - T M M C F - 7 c e l l s ha d s m a l l e r t um or s t ha n t he c o r r e s pon di ng c o nt r ol s .
T hi s r e s ul t w a s s om e w ha t di f f e r e nt f r om w ha t ha s be e n s e e n f r om t he s i m i l a r e xpe r i m e nt
us i ng H E K 293T c e l l s . O ne e xpl a na t i on c oul d be t ha t M C F - 7 c e l l s ne e d t o m a i nt a i n a
c e r t a i n l e ve l of T M E M 56 pr ot e i n e xpr e s s i on. I f T M E M 56 pr ot e i n l e ve l w a s out s i de of
t hi s r a nge , i t c oul d i nf l ue nc e gr ow t h r a t e of M C F - 7 c e l l s i n nude m i c e . T ha t c a n e xpl a i n
w hy bot h T M - s hR N A a nd G F P - T M M C F - 7 c e l l s f or m e d s m a l l e r t um or s i n nude m i c e .
T he e xpl a na t i on i s a l s o c ons i s t e nt w i t h t he phe nom e non t ha t t he t um or gr ow t h r a t e f or
T M - s hR N A a nd G F P - T M M C F - 7 c e l l s w e r e not a s f a s t a s t he c ont r ol one s .
I n t he i m m unohi s t oc he m i s t r y da t a a nd W e s t e r n bl ot r e s ul t s , t he l e ve l of A T P a s e β
s ubuni t i n bot h T M - s hR N A a nd G F P - T M M C F - 7 t um or s w a s t he s a m e c om pa r e d t o t he i r
c ont r ol s . H ow e ve r , i t i s pos s i bl e t ha t T M E M 56 c a n r e gul a t e A T P a s e β s ubuni t e xpr e s s i on
i n s om e w a ys w i t hout c ha ngi ng i t s a m ount . S e ve r a l pa pe r s r e c e nt l y r e por t e d t he s ur f a c e
l oc a l i z a t i on of F 1 c om pone nt A T P a s e on non- pe r m e a bi l i z e d M D A - M B - 231( br e a s t c a nc e r
c e l l l i ne ) ha s be e n c onf i r m e d a nd t he i nhi bi t i on of A T P a s e F 1 c om pone nt a t c e l l s ur f a c e
w oul d s i gni f i c a nt l y s uppr e s s t he gr ow t h of M D A - M B - 231 c e l l s [ 34] . I nt e r e s t i ngl y , t he
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s ur f a c e A T P a s e ha s be e n i de nt i f i e d a s c e l l - s ur f a c e r e c e pt or s f or a ppa r e nt l y unr e l a t e d
l i ga nds t ha t m a ybe c or r e l a t e w i t h c a nc e r pr ogr e s s i on [ 35] [ 36] . T hi s di s c ove r y w a s f a i r l y
c ont r a di c t or y t o t he i ni t i a l unde r s t a ndi ng t ha t F - A T P a s e w a s s t r i c t l y l oc a t e d i n
m i t oc hondr i a . T hus , t he m e c ha ni s m f or how T M E M 56 r e gul a t e s t he F - A T P a s e l e ve l w i l l
be a ne w di r e c t i on t o r e s e a r c h on. W i l l T M E M 56 e xpr e s s i on i nf l ue nc e s t he A T P a s e l e ve l
a t c e l l s ur f a c e ? O r i s t he r e a ny r e l a t i on s hi p be t w e e n T M E M 56 a nd F - A T P a s e di s t r i b ut i on?
I n t he f ut ur e , A T P a s e l e ve l c a n be e xa m i ne d bot h a t c e l l s ur f a c e a nd i n t he c yt opl a s m i n
T M - s hR N A , C O N - s hR N A , G F P - T M a nd G F P - c ont r ol M C F - 7 c e l l s .
A ddi t i ona l l y , a s s how n i n F i gur e 4 a nd F i gur e 1 1 , t he di f f e r e nc e i n T M E M 56
e xpr e s s i on i n G F P - T M or T M - s hR N A t um or s a nd t he i r c ont r ol s ha ve be c om e l a r ge r ,
e s pe c i a l l y f or G F P - T M , a s t he di f f e r e nc e c ha nge d f r om 30 t i m e s t o 80 t i m e s . T hi s m a y be
due t o a m or e f a vor a bl e gr ow t h e nvi r onm e nt i n vi vo. T hi s m a y a l s o e xpl a i n t he r e a s on f or
w hy t he i n vi t r o c e l l ul a r e xpe r i m e nt s s uc h a s c e l l c yt ot oxi c i t y a s s a y a nd c e l l pr ol i f e r a t i on
a s s a y di d not s how a ny di f f e r e nc e be t w e e n t he di f f e r e nt c e l l l i ne s a nd t he i r c ont r ol s .
F ur t he r m or e , t he t um or s f or m e d by C O N - s hR N A a nd G F P - c ont r ol ha d r e d c ol or
c om pa r e d t o t he w hi t e t um or s i n G F P - T M a nd T M - s hR N A . W e s pe c ul a t e i t a s t um or
va s c ul a r i z a t i o n. T u m or va s c ul a r i z a t i on i s t he gr ow t h of ne w bl ood ve s s e l s a nd i t i s
ne c e s s a r y f or t he gr ow t h a nd s pr e a d of c a nc e r ous t um or s .
B a s e d on t he pr e vi ous r e s ul t s f r om our l a b, T M E M 56 ge ne w a s be l i e ve d t o be a
t um or s uppr e s s or ge ne i n H E K 293T c e l l s . H ow e ve r , i n M C F - 7 c e l l s , t hi s w a s not t he
c a s e . D i f f e r e nt c e l l l i ne s w i l l ha ve di f f e r e nt c ha r a c t e r i s t i c s a nd di f f e r e nt e xpr e s s i on l e ve l
of T M E M 56 w hi c h m e a ns t he f unc t i on of T M E M 56 pr ot e i n w i l l va r y t o s om e e xt e nt .
O n t he ot he r ha nd, H E K 293T c e l l s a r e nor m a l c e l l s t ha t ha ve be e n t r a ns f or m e d by l a r ge
3 4
T a nt i ge n. T he y a r e not pr i m a r y c a nc e r c e l l s l i ke M C F - 7. T hus , t he di f f e r e nc e s i n c e l l
t ype s s e e m t o be a n i m por t a nt de t e r m i na nt f or f unc t i on of T M E M 56.
I n c onc l us i on, t he f unc t i on a nd r ol e of T M E M 56 pr ot e i n i n M C F - 7 c e l l s a r e s t i l l
unknow n f or t he m os t pa r t . T he c e l l t ype i s a de t e r m i na nt f or T M E M 56 f unc t i on. I n
M C F - 7 c e l l s , T M E M 56 pr ot e i n ne e ds t o be m a i nt a i ne d a t a c e r t a i n l e ve l t o ha ve i t s
f unc t i on. A bnor m a l e xpr e s s i on of t hi s pr ot e i n c a n i nhi bi t t um or pr ogr e s s i on t o s om e
e xt e nt w i t hout i nf l ue nc i ng A T P a s e β l e ve l .
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R e f e r e n c e s
[ 1] M a r kus S ä l l m a n A l m é n, K a r l J V N or ds t r öm , R obe r t F r e dr i ks s on a nd H e l gi B S c hi öt h
( 2009) .M a ppi ng t he hum a n m e m br a ne pr ot e om e : a m a j or i t y of t he hum a n m e m br a ne
pr ot e i ns c a n be c l a s s i f i e d a c c or di ng t o f unc t i on a nd e vol ut i ona r y or i gi n. B M C B i ol . 7: 50.
[ 2] S t e ve n R . G oodm a n. M e di c a l c e l l bi ol ogy ( 2008) . A c a de m i c P r e s s . pp. 37- .
[ 3] L odi s h H , B e r k A , Z i pur s ky S L , e t a l ( 2000) . M ol e c ul a r C e l l B i ol ogy . 4t h e di t i on.
N e w Y o r k.W . H . F r e e m a n.
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c a l c i um - a c t i va t e d c hl or i de c ha nne l s . E xpe r i m e nt a l P hys i ol ogy . D O I :
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1 1 4( 705 – 712) .
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c e l l s . E ndoc r i ne - r e l a t e d C a nc e r 13 ( 4) : 1033–67.
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s t udi e s i n 30 c ount r i e s , i nc l udi ng 50302 w om e n w i t h br e a s t c a nc e r a nd 96973 w om e n
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Abstract (if available)
Abstract
Transmembrane protein TMEM 56 was first identified as an IKK‐associated protein by mass spectrometry in Dr. Ebrahim Zandi’s lab. This 263AA protein was found to be localized in mitochondria and interacts with α and β subunit of ATPase. In some lung, head and neck cancer cells, TMEM 56 was shown to have a correlation with mitochondrial abundance. In addition, it could act as a potential tumor suppressor in HEK 293T cells. This thesis was based on the conclusion of previous studies to research the function of TMEM 56 protein on tumorigenic growth of MCF-7 human breast cancer cell line. Stable cell lines (CON-shRNA, TM-shRNA, GFP-control, and GFP-TM MCF-7) were established to knock‐down and over‐express TMEM 56 expression. Xenograft assay in immune‐deficient mice showed that tumors formed by both GFP-TM and TM-shRNA MCF-7 cells were smaller than GFP‐control and CON-shRNA cells, respectively. This indicated that a deviation from the basal expression level of TMEM 56 would inhibit tumor progression in MCF-7 cells. Immunohistochemistry and Western Blot with ATPase β antibody showed that over‐expressing or down‐regulating TMEM 56 did not change ATPase β expression, suggesting that TMEM 56 protein will not influence ATPase β level. In summary, data in this study suggest that the difference in cell type is an important determinant for the function of TMEM 56 protein. Specifically, in MCF-7 cells, the abnormal TMEM 56 protein level might result in the inhibition of tumor progression without influencing ATPase β expression.
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Creator
Hong, Dai
(author)
Core Title
Studies on the role of a novel protein, TMEM 56 in tumorigenic growth for MCF-7 cells
School
Keck School of Medicine
Degree
Master of Science
Degree Program
Biochemistry and Molecular Biology
Publication Date
04/28/2014
Defense Date
03/18/2014
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University of Southern California
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MCF-7 cells,OAI-PMH Harvest,protein,TMEM 56,tumorigenic growth
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English
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Tokes, Zoltan A. (
committee chair
), Markland, Francis S., Jr. (
committee member
), Zandi, Ebrahim (
committee member
)
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uschongd@gmail.com
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https://doi.org/10.25549/usctheses-c3-404283
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The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...
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Tags
MCF-7 cells
protein
TMEM 56
tumorigenic growth