Chromosomal Characterizations of Human Nasal and Nasopharyngeal Cells Immortalized by Human Papillomavirus Type 16 DNA Maria Debiec-Rychter, Kim Zukowski, Ching Yung Wang, and Wu-Nan Wen
A B S T R A C T : ('~11 lines N W - I (m(t N I } ( ; - N n'er~ ~sl(lblisht~d M' trm~slt,cli,ln (~t h l l m . n ntis(t1 . m l tl.s~)l}hIJrv'n~{~(Jl etlitht~lital c~lls, r(:slmCti~ clv. w i t h h t m l . n pt~l}illt)m{n'MIs lypP l fi [11t}V - l fil I ) N A . / \ ( I ( l i th}n(d tr(!lllm{!nt t11 N/}C-N {:{'lls n'itll n)i/{}myt:h] C .(4(l'cl~rise l{} the (:ell litm NP('=N-,\I. {31rtm.}s{}m(ll (m(~l'csis >;hc,w{!(l lh(~l 40 47% 111N'I.V-I. Nt'(;-N. Im(l Nt}C-N-M (:(dis hml (1 n(!m' tctrlll}l{}id 11[111111{!r Of C]ll'Ol]11)N()lllt)N(111(I S(?V(!I"(I] Utll"{}lll()H()lll{ll (11111(11"111111ilij!s. "l'hes{r ,:ells s l ] . n ~ . l m'i(t{m(:P (if e.cnt! (inllllili(:l~lil}n in the l(n'm {}l (l(}ttllh! nlil}tlh!s ((tminsl. 111 sil~ hrbriltizl~li(}n willl lll)V lfi I}N/\ I}r()t)t~ (hml(}nslr(111xt th(~t tim rh'uN ,.\tin inl(~,.,r(ltc{t (~t tim 2(121 (:hr(ml{}s(ml{~l r~5,~i(}n hi NI}C-N 1:~11:.;. Mtlllil)hr intcpr(lli(m s/los n'ithin Ill3& 1(t31 q/J2, 21J2,3. 1111i[ 21121 r{'[AJ()lD; r.l~rll (}t}sm'x'(r(l hi NI~.' 1 (:ells. Allh{m,4h ~tll ()l lt].st~ h]]n)(}rtJH (:{~lls t:(,.ht ~4r.tv i . n m l l n l~ clhtl{}~{'. none (}t thrum (l{ml(}nstr(tt(~(t ltltll()t'i[41!ni{:ily m nH(lo tni(:t).
INTRODUCTION
t | u m a n p a p i l l o m a v i r u s t y p e 16 { H P V - I 6 ) D N A is f r e q u e n t l y d e t e c t e d in b i o p s i e s from c e r v i c a l , v u l v a r , a n d p e n i l e c a n c e r s [1 31. O t : c a s i t ) n a l l y . it is fotm(t in n n n l [4[, oral [5], n a s a l [6[, t o n g u e 17l, l a r y n g e a l [8], a n d pharyn~4eal 191 c a r c i n ( ) m a s . T r a n s f e c t i o n s of H P V - 1 6 or H P V - 1 8 D N A se{tuen(:es ilah} l m m a n f i b r o b l a s t s I I 0 l , ker~flint}t:yles 111 13]. a n d exo(:ervi(:al epilheli~d (:ells 114. 151 hav(; restllte(t in i n t e g r a l i o n a n d e x l } r e s s i o n (}f v i r a l s e q u e n ( : e s , a n d h n m ( ) r t a l i z a t i o n (}f t r e a t e d {:ells. N(}ne {}1 t h e s e i m m { } r t a l i z e d h u m a n {;ell l i n e s is t u m o r i g e n i { : in t h e n u d e m o u s e I1(} 14]. h{}wever. suggestin(q t h a t o t h e r a g e n t s , s u c h as h ( } r m o n e s {}r {:hemi{:al {:()ml}(}t(n{ls I161 m i g h t he r e q u i r e d f(}r full [rallSf{}rlllati{}ll (}| t h e s e (:ells. I m m ( ) r t a l i z a t i { ) n {}f n ( ) r m a l h i , m a n (:(dis I}y v i r a l D N A is a s s o c i a t e d u s u a l l y w i t h aneul}h}idy' a n d stru(:ttH'al r{!;Irrailgelll{;ll|S {}] (:hr{}nl(}s(}nles [ 1 1 - 1 3 , 1 7 - 2 1 ]. H{)wevei', {}wiNg l{} t]]{,~ r t ; l a t i v e l y h}w iltlllll}ef {}f {:ell l i n e s a n a l y z e d , specifi(; {:hr{}m{}sonw, a l t e r a t i o n s ass(}ciate{t w i t h vir~d transf(}rmnti(}n a r e still n o t {-lear. I]e(_:ause viral [ } N A m a y a(:t as a n inserti(}nal at:livat()r (}t n e a r b y (:elluh(r g e n e s , t h e integrati{}n s i t e {}f v h ' a l se(itlen{;(,'s {}f t h e s e (:(dis m a y b e imt)()rhlnt, b u t n{} {;()(nm(}n i n t e g r a t i o n site ()f ItI}V-16 D N A h a s vet b e e n (h,qe(;le(t. T(} o u r k n o w l e d g e , t h e i m m ( } r t a l i z a t i o n o( n a s a l a n d n a s t } t ) h a r y n g e a l e p i t h e l i a l (:ells by ttl}V-l{i
F'rom th[~ I}epartn]ent o[ {]henli{:al (:ar(:ino~cneMs. ~li(:hi!4an {]nn{:cr I"{}unih]liim (,",,I. D.-I'~.. K. Z.. { : Y. ~,V.I
I}etr(}it, MI. aml Inslitute of Bi{}{:hemistr\. C{dJe~4e.t Mmlidn.. Nati{.~al Taiwan [!ni~ ersily (W. N. W.I 'I'.i pei I {11)01, Tai'¢val/.
A d d r e s s rcprinl rcqtl(!sts tt}: C h i n o Y. VV(m~, l)(ef)m'ltnent t}f Chmnit:{d (;(~rcino[4cnesis, ,\li{:hi g(m (;c;nccr F o t m t l a l i ( m , I l t} F. V~/arren A r c . . l}elroit. ,\/1 48201. Hec{~ivcd June 12. 1{)9o; (t(:{;cplcd ]tin{! 27. 1!:190.
51 c, 1991 Elsevier S{:ielme Pulllishing Co.. Ira:. 655 Avt!nue id tlu~ Allll!rit:as, N{!w York. NY l(}{)ll}
Cam:er C(~nl~t('.ylogml{q 32:51 61 (1!1911 {}lti3 -ttil}Sff}l SHfL3(I
52
M. Debiec-Rvchter et al.
DNA has not been rel)orte(I I)reviously. We (tes(:ribe (:hromosomal changes and integration sites of normal nasal and nasopharyngeal (:ells transfe(:ted with ltPV-1[~ DNA in vitro.
MATERIALS AND METHODS Materials MCDB-151 basal an(t l';agle's m i n i m u m essential medium (EMEM), epidermal growth factor (EGF), bovine pituitary extract {BPE), insuline, cholera toxin {CT). transferrin (TR), hydrocortisone, quina(:rine mustard, RNase, and (:ah:ium (:hh)ride were (ll)tained from Sigma Chemical, St. Louis, M(). Vitrogen 100 was purchased from (k)llagen (;orporation (Palo Alto, (;/\). N(h'-nu nude mi(e were ol)tained fr(lm National Can(:er Institute. Belhesda, MI).
Cell Cultures Nasal polyps were obtained from a 27-year-old male t)atienl un(terg(ling an ()tmrati(m for nasal obstruction. A l)as(lpharyngeal biopsy was tlerl(lrlne(t in a 45-vein-old male t)atient undergoing an operation for removal (if a (:an(:er(ms mass of the n,s()pharyn,:. A histologi(:ally n(irma] I)ortiol) of the lliopsy was used for (:ell (:ulture eXll(:rimenls. Epithelial (:ells were isolated tr(,n the tissue samples by pro(ease digestion 1221. The (:ell ilellets were resust)el)de,(t in me(It(urn MCDB-15I (:()nhfiniuIM ().l mM t:al(:ium chloride, 15 ng/ml BPE, 2 ng/ml (71', 1()nM/ml E(;I:, O.l /zM/ml hy(h'o(:ortisone, 5t~,ml insulin, 10/,,e,/ml TR, 50 [b'ml penicillin, and 50 #g,'ml slrel)hmly(:in and were see(led in Vitrogen (:ollagel>coat(~d (:ulture dishes whi(:h wen'c then incubated at 37'C in , 5% C(), humi(tified atl)lOSllhere. [J1)(leF lhese (:ultur(! (:on(liti(ms, the epithelial (:ells ()btailmd (:oul(t lie tlassage(I only eight times, with apl)roximatt',ly 25 ~:umulalive cell l)opulati(ln (t()ublings. At the t'illal stage of (:ulture, the (:ells ~ither (lillereJ)tiah~d or became senescent. In situ hybridization did nol shovx either HPV-I(i ()r t!',t)stein-l{arr virus DNA sequen(:es in the (:ultured (:ells at (hal time (W-N. Wen, unpublishe(I observations). Secondary cultures ot nasal and naSOldmryngeal ~qJithelial ~:~lls lhat had u n d e r g o n e 5 10cell doublings w e r e used tortransfe(:tion.
Transfection and Selection of Transfected Cells The ret:omlfinant HPV-III I)NA, pMflPl~16d, was constructed .s described II0]: i.e., tile conaplete HPV-IIi genomi(: DNA was subcloned into tile BamHI site ()f plasmid pdMMTneo as a head-to-hill I IPV-16 DNA dimer. Both nasal and nasol)haryngeal epithelial cells were transfected by a calcium I)h()spllate-DNA pre(:ipitatiotl method [10] modified as follows: approximately 4 × 1(1r'h'¢+d ¢:hr¢+it]¢+scm]~+nhav~+ %l'~tiJIs at lh~ S¢11111)
sit(,.
in c e l l u l a r D N A [191. V i r a l i n t e g r a t i o n h a s b e e n s h o w n to oc¢:ur ill d i f f e r e n t ¢ : h r o m o s()mes a n d at d i f f e r e n t s i t e s in c e r v i c a l (:an(:er (lerive(l litles I2~ 31] a m t h u m a n e l ) i t h e l i a l (:ells i m m o r t a l i z e d w i t h H P V - 1 6 D N A [ 1 1 - 1 4 , 19, 20]. (~ertain g e n o t n i ( : s i t e s are p r e f e r e n t i a l t a r g e t s for v i r a l i n t e g r a t i o t l , h o w e v e r , a n d it u s t m l l v o¢:(:urs at ¢ : h r o m o s o m a l f r a g i l e s i t e s or n e a r t h e l o ( : a t i o n of ( : e l h l l a r t ) r o t o o t l ( : o g e n e s or b o t h [17, +19, 20I. Of t h e i n t e g r a t i o n s i t e s o b s e r v e ( | in t h e t)reset~t stu(ly, 2(t21 r e g i o u is uot a site for a n y k n o w t l o n ( : o g e n e s , b u t is a ( : o m m c m , a p h i ( l i c o l i n - t y l ) e , f r a g i l e site [321.
M. I)elfi(.x:-Ry(:hter et al.
60
1 p 3 6 a n d l q 3 1 r e g i o n s a r e a l s o a ( : ( i m m o n fragile site. In a d d i t i o n , in t h e 1t)36 regi(m, b o t h ( ; a r d e t m r - R a s h e e d f e l i n e s a r t : o m a viral o n ( : o g e n e h o m o l o g u e (F(;R) a n d a ( ] e n o v i r u s - 1 2 ( : h r o m o s ( ) m e mo(lifi(:ation site 1A ( A 1 2 M 2 ) h a v e b e e n lo(:ate(] [33]. T h e s e i n t e g r a t i o n mites for [ ] P V - 1 6 h a v e n o t b e e n d e s c r i b e d t ) r e v i o u s l y . In o u r stu(ly, viral i n t e g r ~ d i o n w a s n o t f o u n d o n a l ) n o r m a l ( : h r o m o s o m e s as has b e e n s h o w n in l+mman f o r e s k i n k e r a t i n o ( : y t e - ( l e r i v e ( l (:ell l i n e s 120]. Be(:ause tim H P V - 1 6 I)NA w a s i n t e g r a t e d i n t o a u n i q u e site it+ t h e q21 r e g i o n of tlor(nal ( : h r o m o s o m e 2 i n / ) o t h (:ell l i n e s , a sear(:h for o t h e r g e n e s in tiffs r e g i o n for p o s s i b l e i n v o l v e m e n t in ( : e l h d a r i m m o r t a l i z a t i o t t m a y be w a r r a n t e d . All i t n t n o r t a l n a s o l ) h a r y n g e a l l i n e s (h~,st:ril)(,'(] in t h i s s t u d y lack tutnorigetfi(: i)otential. T h i s is ( : o n s i s t e n t w i t h t h e o t ) s e r w t t i ( m that humat~ (:ells are g e n e r a l l y m o r e r e s i s t a n t h) t h e t r a n s f o r m i n ~ effe(:t (if v i r a l a.'4ents or (:hemi(:al (:ar(:inogens t h a n are ro(let~t (:ells.
This slutlv Irt)nl the A. Alfred Taubnmn l"auilitv of Michigan (lancer l"otmtlation wits supl)(]rt(!(] hy it.S, Publiu Health Servi(:~+ (Mint N(), (]A49783 and an instilulional u,rattt from tlm tlnited W a y of (;rearer Detroit. W.-N. W. w~ts supp(.'ted hy the N~ttimlal St:htnt:e Council m M file Acadetnh: Sinh:a. Taiwan.
REFERENCES
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,";2:5t"'.3 5fiB. 4. Beck(harm AM. I)aling IR. Slterlllalt lq. l'vl.den (L Milh." BA, (:o.les P.I, Ki~.,iut NIL Mvtu's.n I), Wuiss N,'S, Hish~p '1'(;, Ihta,~i'i~ M. Md)uugall IK (l.qmfl: tlunum imlfilloln~l virus i.i.t:tio. and at'~aI cam:er, ltlt J (]anl:er 43:1042 1049. 5. Syrjatmn SM, Syrj.nen K], l,amh~rg MA (l!W;ti): I)etl~(:lioll ()t htlma]l papilhmun;irus I)NA in (]r.I mm:(]sal ll~sit)tt,'-; rising in silt] I)N,'\ hvbritlizali()n apl)lit~t] on I)araftin sl~(:tilms. C)ral l'alh()l ii2:1i(~0-61i7. G, Syrjdllen ~N], I lil])pOllml RP, Vil'olltitl(!II I~], ,Siiv(]nim I,, ,';yl'i(ltlttlt KJ (1987): Deter:riot1 (fl htllllHll ]}it[li[ltHll4X. il'tlS(Hi)\,I) strt]i:lurld mdi!4etts ~lnd I)NA typlts in iiv,,'itI'h!dI)alfillmnas and sqtHmOtlS (:~tllcan:ires.is ()I"the nasal cavilhts a~td pitranasa] siHuses. Acla ()hd.rvHgol 104:334 341. 7. de VilIiurs EM. Wifidm.w H. ()IIo H. zur Hat~slm II[ I~,W;,S):I'~qfilhmutvh'us I)NA in htunall t()ngtm um'uinom.s, lnI I ('sneer 311:575 B78.
8. I~I'itlt(lslltll It,. Shfind)eru, BM. Al)r.mson A I . 'Winkler I) (19~m): lh'estm(:e oI humml I).lfil]o tllil'++'Jl'tlS t\l)tt lfi rlthdi!iI Bi!(ltlitlti:its ill \Itl'l'tl(:()tlS (;41"(:ill()lllO ()]' lhlt lilt'VII\. (',iltt(:t+r I{(~ 4(+:2185 21 BY,. ,9. I'h'undstnlt It,, ,'\l)ramson ,'\1+ ( 19891: Associiflion ifl' Ilapilltm.r, irus with t:itlt( ei's ot the httil(I .rid t.+(:k. Arch ()t()lilt'ytlgo[ Ileud Nuuk ~III'U 115:(121 1i2.3.
10. Pirisi L. Yasunloto ~. Feller M, l)oni+4er J, I)iP.(flo JA I19fi7): 'l+ransh>rmatiol~ M hutnatt tibrohlusts itnd kerlttin.u','tes with ht.tt.+t imtfilhm.r,.'irus Ivpe tii I)NA. l Viral 61:1061 1()liIi. 11. Durst M. l)zarliex, a l'eh'uslwskll P.T. l;t)ukaml) P, t"us.niy, NI",, (;issmm~n 1, t 1!)~;7): Nhfleuul.r .,.n (:~+,rtt)giwlelit: analysis . t imrn(trtaliz(~d human l)rimurv ktwalino(:vtes ( ] t ) t . i n d att~u" trans fet:li(]n with hu]nun lmj)ilhin(uvirus tyl:,t 16 I)NA. ()m:.u,et.~ 1:251 251L 12. I)iPaM~) IA, Woo(lwl)rlh (',I),I%I)eSuu N(',. N()I.ri(i V. I)uni~er I { 19.+m): I.dt.:ti.n .I h u m . n (:i!l'vi(:a]S(itlilill()iis(3!]] ciIrciIIOItIH [)}/se(luuntial II'mt.shti:li()ttwith h u m + m I~UIfiIhmm',,irt~s lfi I)NA m M vh'al II,rx.w r.s. ()nco,~e.e 4:395 :i.ml.
1'3. Pirisi I,. (;ret~k KI"+, I)t)ni~4er J. l)il).oh) IA (l!)figl: (:ontitH~(lus uell litu~s v,:ilh allm'l!d ~rl)v,'th
Human Epiflmlial (:ells llnm(}rtalize{t t}v HPV-16 DNA
61
and d i f f ( ; r t m t i a l i o n i)rol}erties (}ri~il~ate alter tr~mstc(:tion of hum~u] k(~r.linocvt{~s w i t h huram}. F.al}illomavirtL'-; l y p e 16 I)N/\. (]ar(:ino~{mesis !t:157:{ 157'.L 14. W o o d w o r l h CI). []{);v(l(m PI:. l)onig(.' I. Pirisi 1.. I}arll(!s VV. I,an(:8,'.;lcr WI}, [}il}mflo IA { 1'~)88B ( ] h a r a ( : t e r i z a l i o n of u{irma] h u m a i ] i!xol:(!rvil:M ~fl}iflwliM (:{ills imml)rt;lliz(xl in vilr{} b \ i)al}illomavirus l y p c s 16 ~tlld 18 I)NA. (]an{:(+r Res 48:41i211 4628, 15. Mt:(]all(:e I)1, Ko[}~lll R. l:ut:hs E. IJ~iilllillS I,A 11988): Htllll~lll pal}illclminVil'US Iyllc 16 alters h m n a u (qlith(~iiaI {:.lI (liff{u'(mtiati(m in vitro. Pro(: N~(II A(:m] ,
A B S T R A C T : ('~11 lines N W - I (m(t N I } ( ; - N n'er~ ~sl(lblisht~d M' trm~slt,cli,ln (~t h l l m . n ntis(t1 . m l tl.s~)l}hIJrv'n~{~(Jl etlitht~lital c~lls, r(:slmCti~ clv. w i t h h t m l . n pt~l}illt)m{n'MIs lypP l fi [11t}V - l fil I ) N A . / \ ( I ( l i th}n(d tr(!lllm{!nt t11 N/}C-N {:{'lls n'itll n)i/{}myt:h] C .(4(l'cl~rise l{} the (:ell litm NP('=N-,\I. {31rtm.}s{}m(ll (m(~l'csis >;hc,w{!(l lh(~l 40 47% 111N'I.V-I. Nt'(;-N. Im(l Nt}C-N-M (:(dis hml (1 n(!m' tctrlll}l{}id 11[111111{!r Of C]ll'Ol]11)N()lllt)N(111(I S(?V(!I"(I] Utll"{}lll()H()lll{ll (11111(11"111111ilij!s. "l'hes{r ,:ells s l ] . n ~ . l m'i(t{m(:P (if e.cnt! (inllllili(:l~lil}n in the l(n'm {}l (l(}ttllh! nlil}tlh!s ((tminsl. 111 sil~ hrbriltizl~li(}n willl lll)V lfi I}N/\ I}r()t)t~ (hml(}nslr(111xt th(~t tim rh'uN ,.\tin inl(~,.,r(ltc{t (~t tim 2(121 (:hr(ml{}s(ml{~l r~5,~i(}n hi NI}C-N 1:~11:.;. Mtlllil)hr intcpr(lli(m s/los n'ithin Ill3& 1(t31 q/J2, 21J2,3. 1111i[ 21121 r{'[AJ()lD; r.l~rll (}t}sm'x'(r(l hi NI~.' 1 (:ells. Allh{m,4h ~tll ()l lt].st~ h]]n)(}rtJH (:{~lls t:(,.ht ~4r.tv i . n m l l n l~ clhtl{}~{'. none (}t thrum (l{ml(}nstr(tt(~(t ltltll()t'i[41!ni{:ily m nH(lo tni(:t).
INTRODUCTION
t | u m a n p a p i l l o m a v i r u s t y p e 16 { H P V - I 6 ) D N A is f r e q u e n t l y d e t e c t e d in b i o p s i e s from c e r v i c a l , v u l v a r , a n d p e n i l e c a n c e r s [1 31. O t : c a s i t ) n a l l y . it is fotm(t in n n n l [4[, oral [5], n a s a l [6[, t o n g u e 17l, l a r y n g e a l [8], a n d pharyn~4eal 191 c a r c i n ( ) m a s . T r a n s f e c t i o n s of H P V - 1 6 or H P V - 1 8 D N A se{tuen(:es ilah} l m m a n f i b r o b l a s t s I I 0 l , ker~flint}t:yles 111 13]. a n d exo(:ervi(:al epilheli~d (:ells 114. 151 hav(; restllte(t in i n t e g r a l i o n a n d e x l } r e s s i o n (}f v i r a l s e q u e n ( : e s , a n d h n m ( ) r t a l i z a t i o n (}f t r e a t e d {:ells. N(}ne {}1 t h e s e i m m { } r t a l i z e d h u m a n {;ell l i n e s is t u m o r i g e n i { : in t h e n u d e m o u s e I1(} 14]. h{}wever. suggestin(q t h a t o t h e r a g e n t s , s u c h as h ( } r m o n e s {}r {:hemi{:al {:()ml}(}t(n{ls I161 m i g h t he r e q u i r e d f(}r full [rallSf{}rlllati{}ll (}| t h e s e (:ells. I m m ( ) r t a l i z a t i { ) n {}f n ( ) r m a l h i , m a n (:(dis I}y v i r a l D N A is a s s o c i a t e d u s u a l l y w i t h aneul}h}idy' a n d stru(:ttH'al r{!;Irrailgelll{;ll|S {}] (:hr{}nl(}s(}nles [ 1 1 - 1 3 , 1 7 - 2 1 ]. H{)wevei', {}wiNg l{} t]]{,~ r t ; l a t i v e l y h}w iltlllll}ef {}f {:ell l i n e s a n a l y z e d , specifi(; {:hr{}m{}sonw, a l t e r a t i o n s ass(}ciate{t w i t h vir~d transf(}rmnti(}n a r e still n o t {-lear. I]e(_:ause viral [ } N A m a y a(:t as a n inserti(}nal at:livat()r (}t n e a r b y (:elluh(r g e n e s , t h e integrati{}n s i t e {}f v h ' a l se(itlen{;(,'s {}f t h e s e (:(dis m a y b e imt)()rhlnt, b u t n{} {;()(nm(}n i n t e g r a t i o n site ()f ItI}V-16 D N A h a s vet b e e n (h,qe(;le(t. T(} o u r k n o w l e d g e , t h e i m m ( } r t a l i z a t i o n o( n a s a l a n d n a s t } t ) h a r y n g e a l e p i t h e l i a l (:ells by ttl}V-l{i
F'rom th[~ I}epartn]ent o[ {]henli{:al (:ar(:ino~cneMs. ~li(:hi!4an {]nn{:cr I"{}unih]liim (,",,I. D.-I'~.. K. Z.. { : Y. ~,V.I
I}etr(}it, MI. aml Inslitute of Bi{}{:hemistr\. C{dJe~4e.t Mmlidn.. Nati{.~al Taiwan [!ni~ ersily (W. N. W.I 'I'.i pei I {11)01, Tai'¢val/.
A d d r e s s rcprinl rcqtl(!sts tt}: C h i n o Y. VV(m~, l)(ef)m'ltnent t}f Chmnit:{d (;(~rcino[4cnesis, ,\li{:hi g(m (;c;nccr F o t m t l a l i ( m , I l t} F. V~/arren A r c . . l}elroit. ,\/1 48201. Hec{~ivcd June 12. 1{)9o; (t(:{;cplcd ]tin{! 27. 1!:190.
51 c, 1991 Elsevier S{:ielme Pulllishing Co.. Ira:. 655 Avt!nue id tlu~ Allll!rit:as, N{!w York. NY l(}{)ll}
Cam:er C(~nl~t('.ylogml{q 32:51 61 (1!1911 {}lti3 -ttil}Sff}l SHfL3(I
52
M. Debiec-Rvchter et al.
DNA has not been rel)orte(I I)reviously. We (tes(:ribe (:hromosomal changes and integration sites of normal nasal and nasopharyngeal (:ells transfe(:ted with ltPV-1[~ DNA in vitro.
MATERIALS AND METHODS Materials MCDB-151 basal an(t l';agle's m i n i m u m essential medium (EMEM), epidermal growth factor (EGF), bovine pituitary extract {BPE), insuline, cholera toxin {CT). transferrin (TR), hydrocortisone, quina(:rine mustard, RNase, and (:ah:ium (:hh)ride were (ll)tained from Sigma Chemical, St. Louis, M(). Vitrogen 100 was purchased from (k)llagen (;orporation (Palo Alto, (;/\). N(h'-nu nude mi(e were ol)tained fr(lm National Can(:er Institute. Belhesda, MI).
Cell Cultures Nasal polyps were obtained from a 27-year-old male t)atienl un(terg(ling an ()tmrati(m for nasal obstruction. A l)as(lpharyngeal biopsy was tlerl(lrlne(t in a 45-vein-old male t)atient undergoing an operation for removal (if a (:an(:er(ms mass of the n,s()pharyn,:. A histologi(:ally n(irma] I)ortiol) of the lliopsy was used for (:ell (:ulture eXll(:rimenls. Epithelial (:ells were isolated tr(,n the tissue samples by pro(ease digestion 1221. The (:ell ilellets were resust)el)de,(t in me(It(urn MCDB-15I (:()nhfiniuIM ().l mM t:al(:ium chloride, 15 ng/ml BPE, 2 ng/ml (71', 1()nM/ml E(;I:, O.l /zM/ml hy(h'o(:ortisone, 5t~,ml insulin, 10/,,e,/ml TR, 50 [b'ml penicillin, and 50 #g,'ml slrel)hmly(:in and were see(led in Vitrogen (:ollagel>coat(~d (:ulture dishes whi(:h wen'c then incubated at 37'C in , 5% C(), humi(tified atl)lOSllhere. [J1)(leF lhese (:ultur(! (:on(liti(ms, the epithelial (:ells ()btailmd (:oul(t lie tlassage(I only eight times, with apl)roximatt',ly 25 ~:umulalive cell l)opulati(ln (t()ublings. At the t'illal stage of (:ulture, the (:ells ~ither (lillereJ)tiah~d or became senescent. In situ hybridization did nol shovx either HPV-I(i ()r t!',t)stein-l{arr virus DNA sequen(:es in the (:ultured (:ells at (hal time (W-N. Wen, unpublishe(I observations). Secondary cultures ot nasal and naSOldmryngeal ~qJithelial ~:~lls lhat had u n d e r g o n e 5 10cell doublings w e r e used tortransfe(:tion.
Transfection and Selection of Transfected Cells The ret:omlfinant HPV-III I)NA, pMflPl~16d, was constructed .s described II0]: i.e., tile conaplete HPV-IIi genomi(: DNA was subcloned into tile BamHI site ()f plasmid pdMMTneo as a head-to-hill I IPV-16 DNA dimer. Both nasal and nasol)haryngeal epithelial cells were transfected by a calcium I)h()spllate-DNA pre(:ipitatiotl method [10] modified as follows: approximately 4 × 1(1r'h'¢+d ¢:hr¢+it]¢+scm]~+nhav~+ %l'~tiJIs at lh~ S¢11111)
sit(,.
in c e l l u l a r D N A [191. V i r a l i n t e g r a t i o n h a s b e e n s h o w n to oc¢:ur ill d i f f e r e n t ¢ : h r o m o s()mes a n d at d i f f e r e n t s i t e s in c e r v i c a l (:an(:er (lerive(l litles I2~ 31] a m t h u m a n e l ) i t h e l i a l (:ells i m m o r t a l i z e d w i t h H P V - 1 6 D N A [ 1 1 - 1 4 , 19, 20]. (~ertain g e n o t n i ( : s i t e s are p r e f e r e n t i a l t a r g e t s for v i r a l i n t e g r a t i o t l , h o w e v e r , a n d it u s t m l l v o¢:(:urs at ¢ : h r o m o s o m a l f r a g i l e s i t e s or n e a r t h e l o ( : a t i o n of ( : e l h l l a r t ) r o t o o t l ( : o g e n e s or b o t h [17, +19, 20I. Of t h e i n t e g r a t i o n s i t e s o b s e r v e ( | in t h e t)reset~t stu(ly, 2(t21 r e g i o u is uot a site for a n y k n o w t l o n ( : o g e n e s , b u t is a ( : o m m c m , a p h i ( l i c o l i n - t y l ) e , f r a g i l e site [321.
M. I)elfi(.x:-Ry(:hter et al.
60
1 p 3 6 a n d l q 3 1 r e g i o n s a r e a l s o a ( : ( i m m o n fragile site. In a d d i t i o n , in t h e 1t)36 regi(m, b o t h ( ; a r d e t m r - R a s h e e d f e l i n e s a r t : o m a viral o n ( : o g e n e h o m o l o g u e (F(;R) a n d a ( ] e n o v i r u s - 1 2 ( : h r o m o s ( ) m e mo(lifi(:ation site 1A ( A 1 2 M 2 ) h a v e b e e n lo(:ate(] [33]. T h e s e i n t e g r a t i o n mites for [ ] P V - 1 6 h a v e n o t b e e n d e s c r i b e d t ) r e v i o u s l y . In o u r stu(ly, viral i n t e g r ~ d i o n w a s n o t f o u n d o n a l ) n o r m a l ( : h r o m o s o m e s as has b e e n s h o w n in l+mman f o r e s k i n k e r a t i n o ( : y t e - ( l e r i v e ( l (:ell l i n e s 120]. Be(:ause tim H P V - 1 6 I)NA w a s i n t e g r a t e d i n t o a u n i q u e site it+ t h e q21 r e g i o n of tlor(nal ( : h r o m o s o m e 2 i n / ) o t h (:ell l i n e s , a sear(:h for o t h e r g e n e s in tiffs r e g i o n for p o s s i b l e i n v o l v e m e n t in ( : e l h d a r i m m o r t a l i z a t i o t t m a y be w a r r a n t e d . All i t n t n o r t a l n a s o l ) h a r y n g e a l l i n e s (h~,st:ril)(,'(] in t h i s s t u d y lack tutnorigetfi(: i)otential. T h i s is ( : o n s i s t e n t w i t h t h e o t ) s e r w t t i ( m that humat~ (:ells are g e n e r a l l y m o r e r e s i s t a n t h) t h e t r a n s f o r m i n ~ effe(:t (if v i r a l a.'4ents or (:hemi(:al (:ar(:inogens t h a n are ro(let~t (:ells.
This slutlv Irt)nl the A. Alfred Taubnmn l"auilitv of Michigan (lancer l"otmtlation wits supl)(]rt(!(] hy it.S, Publiu Health Servi(:~+ (Mint N(), (]A49783 and an instilulional u,rattt from tlm tlnited W a y of (;rearer Detroit. W.-N. W. w~ts supp(.'ted hy the N~ttimlal St:htnt:e Council m M file Acadetnh: Sinh:a. Taiwan.
REFERENCES
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,";2:5t"'.3 5fiB. 4. Beck(harm AM. I)aling IR. Slterlllalt lq. l'vl.den (L Milh." BA, (:o.les P.I, Ki~.,iut NIL Mvtu's.n I), Wuiss N,'S, Hish~p '1'(;, Ihta,~i'i~ M. Md)uugall IK (l.qmfl: tlunum imlfilloln~l virus i.i.t:tio. and at'~aI cam:er, ltlt J (]anl:er 43:1042 1049. 5. Syrjatmn SM, Syrj.nen K], l,amh~rg MA (l!W;ti): I)etl~(:lioll ()t htlma]l papilhmun;irus I)NA in (]r.I mm:(]sal ll~sit)tt,'-; rising in silt] I)N,'\ hvbritlizali()n apl)lit~t] on I)araftin sl~(:tilms. C)ral l'alh()l ii2:1i(~0-61i7. G, Syrjdllen ~N], I lil])pOllml RP, Vil'olltitl(!II I~], ,Siiv(]nim I,, ,';yl'i(ltlttlt KJ (1987): Deter:riot1 (fl htllllHll ]}it[li[ltHll4X. il'tlS(Hi)\,I) strt]i:lurld mdi!4etts ~lnd I)NA typlts in iiv,,'itI'h!dI)alfillmnas and sqtHmOtlS (:~tllcan:ires.is ()I"the nasal cavilhts a~td pitranasa] siHuses. Acla ()hd.rvHgol 104:334 341. 7. de VilIiurs EM. Wifidm.w H. ()IIo H. zur Hat~slm II[ I~,W;,S):I'~qfilhmutvh'us I)NA in htunall t()ngtm um'uinom.s, lnI I ('sneer 311:575 B78.
8. I~I'itlt(lslltll It,. Shfind)eru, BM. Al)r.mson A I . 'Winkler I) (19~m): lh'estm(:e oI humml I).lfil]o tllil'++'Jl'tlS t\l)tt lfi rlthdi!iI Bi!(ltlitlti:its ill \Itl'l'tl(:()tlS (;41"(:ill()lllO ()]' lhlt lilt'VII\. (',iltt(:t+r I{(~ 4(+:2185 21 BY,. ,9. I'h'undstnlt It,, ,'\l)ramson ,'\1+ ( 19891: Associiflion ifl' Ilapilltm.r, irus with t:itlt( ei's ot the httil(I .rid t.+(:k. Arch ()t()lilt'ytlgo[ Ileud Nuuk ~III'U 115:(121 1i2.3.
10. Pirisi L. Yasunloto ~. Feller M, l)oni+4er J, I)iP.(flo JA I19fi7): 'l+ransh>rmatiol~ M hutnatt tibrohlusts itnd kerlttin.u','tes with ht.tt.+t imtfilhm.r,.'irus Ivpe tii I)NA. l Viral 61:1061 1()liIi. 11. Durst M. l)zarliex, a l'eh'uslwskll P.T. l;t)ukaml) P, t"us.niy, NI",, (;issmm~n 1, t 1!)~;7): Nhfleuul.r .,.n (:~+,rtt)giwlelit: analysis . t imrn(trtaliz(~d human l)rimurv ktwalino(:vtes ( ] t ) t . i n d att~u" trans fet:li(]n with hu]nun lmj)ilhin(uvirus tyl:,t 16 I)NA. ()m:.u,et.~ 1:251 251L 12. I)iPaM~) IA, Woo(lwl)rlh (',I),I%I)eSuu N(',. N()I.ri(i V. I)uni~er I { 19.+m): I.dt.:ti.n .I h u m . n (:i!l'vi(:a]S(itlilill()iis(3!]] ciIrciIIOItIH [)}/se(luuntial II'mt.shti:li()ttwith h u m + m I~UIfiIhmm',,irt~s lfi I)NA m M vh'al II,rx.w r.s. ()nco,~e.e 4:395 :i.ml.
1'3. Pirisi I,. (;ret~k KI"+, I)t)ni~4er J. l)il).oh) IA (l!)figl: (:ontitH~(lus uell litu~s v,:ilh allm'l!d ~rl)v,'th
Human Epiflmlial (:ells llnm(}rtalize{t t}v HPV-16 DNA
61
and d i f f ( ; r t m t i a l i o n i)rol}erties (}ri~il~ate alter tr~mstc(:tion of hum~u] k(~r.linocvt{~s w i t h huram}. F.al}illomavirtL'-; l y p e 16 I)N/\. (]ar(:ino~{mesis !t:157:{ 157'.L 14. W o o d w o r l h CI). []{);v(l(m PI:. l)onig(.' I. Pirisi 1.. I}arll(!s VV. I,an(:8,'.;lcr WI}, [}il}mflo IA { 1'~)88B ( ] h a r a ( : t e r i z a l i o n of u{irma] h u m a i ] i!xol:(!rvil:M ~fl}iflwliM (:{ills imml)rt;lliz(xl in vilr{} b \ i)al}illomavirus l y p c s 16 ~tlld 18 I)NA. (]an{:(+r Res 48:41i211 4628, 15. Mt:(]all(:e I)1, Ko[}~lll R. l:ut:hs E. IJ~iilllillS I,A 11988): Htllll~lll pal}illclminVil'US Iyllc 16 alters h m n a u (qlith(~iiaI {:.lI (liff{u'(mtiati(m in vitro. Pro(: N~(II A(:m] ,
