Apoptin gene therapy in head and neck cancer Schoop, R.A.L.
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Hele tekst
(2) 2 Bcl-xL inhibits p53- but not apoptin-induced apoptosis in head and neck squamous cell carcinoma cell line Remilio A. L. Schoop Klaas Kooistra Robert J. Baatenburg de Jong . . .
(3) . . !.
(4) Abstract
(5) ! "# $%"& "!' (!)*% %$+$ disease and poor prognosis of patients suffering head and neck squamous ,-
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(8) /!$+$ 5//!(3 $%" "% ! "# 1" "+$) %"%! and induced the release of mitochondrial cytochrome c. Up-regulation (!)* 5/!( % $$ 4 " "!$ $ " " %%34%&'+&$"# !$ $ (!)* %'7& $%$ "# !$ $ & , %3 apoptin-triggered one. Our data demonstrate that apoptin induces apoptosis $"$ (!)*$"# 3$,& % " " ' ,
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(10) Introduction. $$
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(12) /0 % , : ,'%1. Despite recent treatment advances with surgery, , "&$$
(13) /%%, ', %$$% world2 and rates of local recurrence and distant metastases are high . Impairment of apoptosis, the physiological process in which cells undergo self-destruction, plays an important role in the development of + %%% %
(14) /4. Tumor cells overwhelmingly contain genetic lesions in the apoptotic decision pathway, such as mutated tumor-suppressor " "# 5 +!)"%% " " " % (! $ (!)*6. In " 3(!)* +!)"%% % '& % $+ ",
(15) /7 (!)* % " " %% + "+ & , release from mitochondria into the cytosol, which is a crucial event during apoptosis for this step activates the downstream effector caspases resulting +%) " " " %%6,8..
(16) /%% $' " " %%%% %'" " signal is offered to them8. Unfortunately, many conventional therapies fail %&$ " " %%+"# 9 and/or are inhibited by antiapoptotic " % % % (!)*10 M , 3 % % 4 %"& (!)*% $4%% $ "&11 and chemotherapeutic '%% % ,&3%"3 " %$3+%3$$ ) 12. +3 +!)"%% (!)* "&% "+ $ %%$"$ "# % %12$% +!)"%% %% metastatic activity of cancer cells . N 3 +%'% "%'
(17) /$$ to bypass the resistance for undergoing apoptosis due to high levels of Bcl)*6. Previous in-vitro studies with the chicken anemia virus-derived protein apoptin demonstrated that this 121 amino acid protein can induce apoptosis $"$ "# 14 and is even stimulated by the antiapoptotic Bcl-215. M , 3 " " $ % " ',,$ $ ,, Q$. 25.
(18) and transformed cells, but not in normal, diploid cells16. Apoptin-induced " " %% $ % 9 "%, %"%% + %"%! and possibly other downstream caspases are essential for a rapid apoptininduced apoptosis17. Therefore, apoptin is a potential antitumor agent. In "% % $&3 4 )" $ " "V% " & " %% ,
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(20) /!$+$ % "% ' +% (!)* $$"$ "# . Materials and Methods Cell culture. The earlier described human head and neck squamous carcinoma cell line UMSCC-14B was a generous gift of Dr TE Carey, University of Michigan Medical Center, MI, USA. The cell line was established from a poorly $$%9 , % , [ , $% , $"# 18 N4% &,$\ V%, $7$ ]'V% ,$ , -\]0 ' : % ,3 " $ %" ,& -* N '%3 ^ 8+3 $ 0 % 4 $ _oC and passed twice a week by trypsinization. Eight hours after been "%%$`!,!$,$%% `!4"%3!: [ % of UMSCC-14B cells were transiently transfected with plasmid DNA. Plasmids and transfection. N "%,$ "x!x{ $' " " $ "%,$ "x!"# $' "# + $%$ 19 N "%,$ "\
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(22) $%0 $% '+ " " %% *| 4 & $ %!' $ !, %3 ' $ & x " , N "%,$ "x!(!)* 4% % $ & ' ," (!)* %9 ] ^2. 26.
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(25) 1 -+ '0$ 4%, N_$x promoters203$4%8$&" +$$&\/~ %,&3 +$$ / 3 ( % 3 13 5/10 N % 4 %$ 4 M ' ` according the protocols of the manufacturer Roche Molecular Biochemicals, Almere, The Netherlands. Two μg DNA was transfected per dish. Subcellular fractionation and Western Blot Analysis. Subcellular fractionation was essentially performed as described by Juin et al.21. After transfection, the UMSSC-14B-cell monolayer was washed 44! $" %"! $%-{(/0 N, &-! `%2,,$%04%%$44! ${(/3%"$$ '$' #, N%4% %"$$) - ,% %3, "%" _ 3#,~3#,]}N13 # , '3 , $ 3 , "&,&% & [ $3 $ & % (03 ,3 $ , 'Q$ & ,% + )' &%% 5 8 % $ 4 "$ & ' # , ' +& ,,% 4 , +$ from the resulting supernatant by centrifugation for 5 min at 14000 g. The resulting supernatant is the crude cytosolic fraction. All samples were frozen in liquid nitrogen and stored at -80° C until analysis by sodium dodecyl % 7$!" &&,$ ' " %% -/\/!{1}]0 ]9 , % & % " ) 4 $ $ ' %," # ,3 $$ % $ ,$ $&% 7$!#:" &&,$-/\/!{110 '3 $ $ ,, !{ ,,% -" 3 ($ $3 135/10 N,,%4 8$ +'{(/N! -, N%! 3 " _ 3 # ,
(26) 3 #: N4!0 ' #: $$ ,8 ( % 4 $ 4 ",& $& (!)* 4% $$ 4 , $& !` -$ / Q ( '&3 / Q3 13 5/10 $ & , 4 , $& _ -$ {,'3 / \' 3 13 5/10 Positive signals were visualized by enhanced chemiluminescence according. 27.
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(29) At different time points after transfection, UMSCC-14B cells were 7)$ 4 : , % $ ,, [ % assays were performed as described by Danen-van Oorschot et al.17. Prior ' % 4 $%3 % 4 $ , %4{(/ ' #:N4!$#: ,' % , 1" " 4% $$ 4 , % , $& x!1x! -% "3$ 0143,&!''$*|4!,& $& ] -$ 0223"# 4 $&\!-$ 03$(!)*4 , $&1(`-$ 0-, 0 1 " & $& -$ 0 4% %$ $ + %"%! -{,'3 / \' 3 13 5/10 / $& $% 4 [ % % &!$ ' !, % $% $,!$ ' !, % $% -8% ,, ^% * %3 W% } +3 {13 5/10 N , 4% %$ 4 3 $,$ !!"&$ -\1{0 " " %3 , % $%$3 %% ',$3$7&\1{%'% % due to fragmentation of the DNA22 N%4&Q$&[ % , % "& -&," %3 | 4 $3 N
(30) $%0 )"%% %$ " 3 , " '& %"%! +&3 indicating the apoptotic state of the cell. At least 100 cells per independent time point were counted to determine the percentage of apoptotic cells.. 28.
(31) Results Apoptin induces aoptosis in UMSCC-14B cells, which express nonfunctional p53. N ),4" "$4$!&""# $ " " %%
(32) / % ' ! "# 3 ,
(33) / line UMSCC-14B was transiently transfected with plasmids encoding " "3 4$!&" "# % " %+ 3 *| % '+ The cells were screened for production of the various transgenes by $ ,, [ % 1" " %% 4% &Q$ & %' 4 DAPI, which is known to stain intact nuclei strongly but apoptotic ones ' & $ 48& N4 $&% % 3 "" ),& : "# ! $ " "!" %+ % , " " 3 4 %$ $&% "" ),& : "# !$" "! " %+ % -M' 0 N 5//!( % )"%%' !" " *| $4 " #: $3 4 % , % 8& $ used transfection method15. These data indicate that apoptin can induce " " %%%9 , % ,!$+$%8' "# Figure 1. 1" "$ %" " %%
(34) /!$+$5//!(%8' "# N %4%&%$4"x!x{ $'" "-8%03"x!"# -'&%0 "x!*|- "%0 %47)$$&%-"0 $&%-' "0 % $ &Q$ & $ ,, [ % N "' of cells that stained abnormally with DAPI is given as a relative measure for apoptosis. ^% % ,% % $"$ )",% )", % % 4 ),$ 4 " %+ %$ %' " $ . 29.
(35) Apoptin induces cytochrome c release and caspase-3 activity.
(36) )3 4 +%'$ 4 & , % %$ , mitochondria during apoptin-induced apoptosis. To that end, UMSCC-14B %4%$4"%,$% $'" "3"# *| N $&%3& % )%4""$&% $ &Q$&W% ' %%$4" " "# 3 levels of cytochrome c were increased compared to cells transfected with '+ *| -M' 03 4 $% & , % released from mitochondria during apoptin-induced apoptosis. Figure 2 *|. "# . apoptin Cytochrome c. Cytochrome c release in apoptin-induced apoptosis. UMSCC-14B cells were transfected with "%,$% $'*|-'+ 03" " "# -" %+ 0 N$&% 3& % )%4""$$&Q$&W% ' & ,+%. "% $%34),$" %%+ %"%! & " " ,"% "# $ *| 5//!( % 4 %$ 4 "%,$% $' " "3 "# *|3 7)$ $&% $%$4 $&%"7 %'$4 $&%"7 +%"%! , & "# !%4% " "!)"%%' %3 + %"%! $ $$3 & 4*!|!" %+5/%-M' 0 N, & " "!" %+ " " 5/!(%""$ ++%"%! 3$ &% , !" " " "!" %+ % %$ " %+ + %"%! Our results indicate that upon apoptin induction of apoptosis in UMSCC( % %$% & , %3 % %"%! ,% +$. 30.
(37) Figure 3 1" "-0. DNA. 1+%"%! . A. 1" "- 0. B. "# . C. *|. D. 1+%"%! %"%5/!(% $' '" "!$ $" " %% % 4%$4"%,$% $'*|-'+ 03" "3 4"x"# -" %+ 0 1+ %," %% 3%47)$$%$4 $% 'Q'" "3"# 3*| +%"%! 3%$4\1{$&Q$ &[ %, % "& N% 4%4%$4" "$7)$4 $&% -0 $&%- 0% 3 %$4"# 4*|$7)$ $&% { '"%48 "%+%,'7 ). 31.
(38) Bcl-xL interferes with p53-induced apoptosis, but not with apoptin-induced apoptosis. M&3 4 ),$ +!)"%% (!)* " "!$"# !$ $" " %%5/!(% M%35/!( %4%&%$4"%,$ $'(!)* *| ),(!)*!%"7%&%%$% Q W%! &%% +$ & (!)*!%$ 5/!( % '+% (!)*" 4)"$W 8\-M' 10 1 $ ,, [ % %%& 4 $& %"7& $$'%(!)*% 4$%$5/!(% $ $(!)*" & "%,-M' (0$%$% resembled that of mitochondria, as reported previously . Figure 4 *|. (!)*. A. *|. (!)*. B. 5/!( % +!" $ ' " " (!)* " % 4 %& %$ 4 "%,$ "x!(!)* 4 "x!*| W% -" 10 $ ,, [ % -" (0 5/!( % %$ 4 "x!(!)* -0 "x!*|-!0$%$ (!)*" 4(!)*!%"7 $% !`.
(39) )3 4 % $$ 4 (!)* " " $2 "# ! induced apoptosis. To that end, UMSCC-14B cells were transfected with "%,$% $'" " 4$!&""# !%$4(! )*$" " 44$!&""# M $&%% 3%4 7)$$%$4 $% 'Q'" "3(!)*$2 "# $ &Q$&[ %, % "& $ " " %%4%% $&. 32.
(40) &%% , " '&&\1{%' M $&%" %!% 3 !)"%% (!)*$$ " "+&34%"# !$ $ " " %%4%%'7&$ $$ !)"%% (!)*-M' #10 Figure 5 A. (!)* $ % " "!$ $ " " %% % 4 %$ 4 "%,$% $' " " -8 03 "# - " 03 !%$ 4 (!)* $ " " -%"$ 0 4 "# -$ $ 0 % 4 7)$ $ &Q$ & $ ,, [ % N"' %%$ ,&4\1{4% %$% ,% " " %% ^% %'+, $"$)",% )",%%4),$4" %+ %$%' product.. N%% %$ '(!)*%' "# ! induced apoptosis, it clearly does not interfere with the apoptin-induced " " %% " %%% N 3 ) %" 4% $, 4 & , 4% % %$ " "!)"%%' % % ' " " %% ,"$ % )"%%' 4$!&" "# 5/!( % 4 %$ 4 "%,$% $' 4$!&" "# " " 3 !%$ 4 "%,$% $' (!)* $ " " (!)*$4$!&""# N$&%% 3& % % 4'$%$%$$ $%% W%! &%%&% 4$(!)*$ %% & , 5/!( % !)"%%' 4$!&" "# $ (!)*3 $ no effect on the cytochrome c release when cells co-produced apoptin $ (!)* -M' #(0 N 3 (!)* %,% '+& 4 "# !$ $ " " %% & "+ & , . 33.
(41) release, which is not the case upon induction of apoptosis by apoptin. Figure 5 B 1" " (!)* *| (!)*. "# (!)*. Cytochrome c (!)* $ % " "!$ $ & , % 5/!( % 4 !%$ 4 "%,$% $' (!)* $ *|3 " " "# N $&% 3 & % )% 4 ""$3 $ &Q$ & W% ' & , +% /, % % 4 $ $"$ )",%. Discussion " % 4% " "!$ $ " " %% "# ! ! functional UMSCC-14B cells is accompanied by release of cytochrome c $2 + %"%! ]+ , ," % %+ (!)*$$ " "!$ $" " %%%
(42) /% The observation that apoptin induces apoptosis in UMSCC( % 8' "# % ," ' /,Q al.24 " $ $% " % "# !' $ " " %% " %%% could lead to the rapid emergence of cellular drug resistance. Cells )"%%' % , ,% , "# % 4 $ , ' potential with increased resistance to chemotherapy and radiation25. Noutomi et al.10 generated squamous cell carcinoma cell lines 4"# , % ,$4'+% (!)* N % 4 "# , '$ ,$ %% ' "3 4% % 4 "# , $ ' +% (!)* 4 '& %% , "& $ $ W % 4$ 5/! ( % !)"%%' (!)* $ "# $ %'7 4 " " %% 4 ,"$ )"%% 4$!&" "# 3 4 correlates with the inhibition of cytochrome c. Therefore, it is surprising !)"%% (!)* $ " " $ %, " " %% +. 34.
(43) ,"% 4 " " 4% )"%%$ N% % %,% 8$ & (!)* %,% ," " "!$ $ release of cytochrome c. These features indicate that apoptin can induce " " %%"% '+% (!)*34%8 4 "+ induction by a large variety of conventional anti-cancer therapies26-28. The. majority. of. apoptin-positive. morphologically. apoptotic. 5/!( % ""$ + + %"%! 3 4% % , morphologically non-apoptotic apoptin-positive cells stained positive + %"%! 3 $' %"%! + "$% " " ) "% \!+ % $ ' %17 " $ & " "!)"%%' , " '& " " , % % , / %! % $ + %"%! N& $$ %"%! % +$ $ ' " "!$ $ $3 % + %,% %' N$%"%! + % )"$ & / %! % %"%! + results in immediate morphological cellular changes, whereas UMSCC( % % 4 $&$ ) " " %% %"% + 1 '(!)*$(! ' " " ,,% same protein family, they act in a different way on apoptin-induced apoptosis. Previously, it has been shown that Bcl-2 stimulates15,22 the effect of apoptin$ $" " %% , , %34% %+$ (!)* /, $ % 4 % % " ( $ *!/ $ 4 8 3 " ," $ " 7$ , ' 7% . " ( $ *!/ % +!)"%%' (! $ 8 $ $ $3 4 $ $ (!)*29. Nevertheless, +!)"%% (!$(!)*% $% & , $% %9 + $ 4%,%"%! " " "!$ $ " " %% 4+3 & 4 ,%, " " $ % of cytochrome c in the presence of the antiapoptotic proteins Bcl-217 and (!)*,% +$ N" "$ %" " %% , !%"74&22 in cases where therapeutic agents are known to fail. 35.
(44) % % +!)"%% (!)*3 ,8% " ! , ' In conclusion, we showed that apoptin induces apoptosis independent "#
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(47) References . . . . . . . }^N3 ! , (3 &N3N %%%3 1# #! ` (Q\31%]3M%/^3/^*3^%,W3 %x3} ' /*3 '1N3{ %Q*^ &" $$ 4 4 concurrent chemotherapy for locally advanced head and neck cancer. N ]'$ _ ! /{3*&$W N, $$8 1 ## #!` }%,(^ 1" " %%$%," $
(48) 8 ! 25. # x %$~ 3* *+ $ V%%" % "#
(49) ^+ 2002; 2: 594-604. ` ^$ %,% " " %% 1,{ #_ #! _ {3N ,"% (3^W3x 8%]]3^ $ (!)*$(! )"%% %9 , % , $$8 85: 164-70. 8. Reed JC. Apoptosis-based therapies. Nat Rev Drug Discov 2002; 1: 111-21. %~33N8%13~3% , 3%N N% , "# '$"%%%!&! \\{!$ $" " %% , %9 , % , $$8 1" " %%_ `_!_
(50) ,N3 3 3N & 3Q ' (!)-*0 %, ! drug resistance in several squamous cell carcinoma cell lines. Oral Oncol ! | 3 1, 3 ~3 ( % * 3 & (3 N % }3 V {3 M 13 N" " %%!%% $',,!&%" % (*!-*0 $"$% ,"# ' ` _! 13 ^ $ 3 ( % * 3 N ,"% ( ])"%% !)* , $ '%%" &" ( $ # ` ! M$Q 3 ]%" *3 % /3 M 13 / 1 (!)* " , % metastasis of breast cancer cells by induction of cytokines resistance. Cell \\_ #! | ' /3 /+% 13 + , $ 3 ,% 1}3 + $ ] 13
(51) 1" "3 " $+$ , 8 , + %3 $ % "# !$"$ " " %% , % % , % Res 1995; 55: 486-9.. 37.
(52) . . . . . . . . . . 38. # \!+ % 113$ $13N8&,/3^$3+$ ]13
(53) (1}!%"# !$ $ " "!$ $ " " %% 1" " %% _ #! ` \!+ % 113M%\M3},'3~(3| '/3 M8 ' 3 (8$ 3 ) { 3 + $ ] 13
(54) Apoptin induces apoptosis in human transformed and malignant cells but ,% {
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(56) N 8 , virus-derived protein apoptin requires activation of caspases for induction of apoptosis in human tumor cells. J Virol 2000; 74: 7072-8. / /3 {3 *3 \4% 3 / (3 *," WW3 &, ^13 $^13/ $ ~3 'W~3* ^ $7 " ! selective retinoids that are potent inhibitors of the growth of human head $8%9 , % ,% ^%` #` !_
(57) 3 N $$ \3 x% 13 $ } 4 W3 W*3 Veldkamp S, Douglas AJ, McNulty MS, van der Eb AJ, Koch G. A single 8,+ %" $ %" " %% x ` `!# | 3 $ 3 %" + ~3 8 3 W8%, }3 ~ %,& / ( $ , (1\ % 9 $ $,Q 4 (*!* $ " ! apoptotic activity. J Biol Chem 1997; 272: 24101-4. {3 13*4 $ N3]+} !&!$ $ %%Q " " %% % ,$$ ' & , % }% \+ `_! \!+ % 111 1 + " $ % , !%"7 apoptosis. Thesis. 2001, Leiden University, Leiden, The Netherlands. |3 $ 3']3 83~ %,&/ /" %" & $' %(1\%" % % ++ % %$' ! ! (*!-*0 ` _ ` ! /,Q 3 * 3 * 3 N3 W3 N
(58) 3 } 8 13 ] Translational regulation as a novel mechanism for the development of $ '%% %%%^+ ! # ~% \}3 ~% ( N , !% ""%% "# ," % , $+ ",$" ' %% ` # !` ` N%8\~3W }N3($ $^3M%/}3\+&~3 % 3/' N3 W ])"%% (! ,& " % $+$ &' squamous cell carcinoma: correlation with response to chemotherapy and '"%+ *&' % " ` !.
(59) . . _ \'+ 13 * ' +%8 & 13 $ 3 & (3 W' }3 % N3 $7 %,!, % 4 ( $ ,$(!)*
(60) ( _ ! 28. Kroemer G, Reed JC. Mitochondrial control of cell death. Nat Med 2000; 6: # ! '/]3 %N3N%N 3 % /* \ 4!, $ (!*3% of cytochrome c and sequential activation of caspases during honokiol$ $ " " %% , %9 , % ' _ % ( , {, ` `!#. 39.
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