Cancer Res 1987 Behrens 414 8

7/29/2019 Cancer Res 1987 Behrens 414 8

1/6

1987;47:414-418.Cancer ResBrent C. Behrens, Thomas C. Hamilton, Hidetaka Masuda, et al.Cell Line and Its Use in Evaluation of Platinum Analogues-Diamminedichloroplatinum(II)-resistant Human Ovarian Cancer

cisCharacterization of a

Updated Versionhttp://cancerres.aacrjournals.org/content/47/2/414

Access the most recent version of this article at:

Citing Articleshttp://cancerres.aacrjournals.org/content/47/2/414#related-urls

This article has been cited by 37 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

SubscriptionsReprints and

[email protected] atTo order reprints of this article or to subscribe to the journal, contact the AACR Publications

[email protected] at

To request permission to re-use all or part of this article, contact the AACR Publications

American Association for Cancer ResearchCopyright 1987on February 21, 2013cancerres.aacrjournals.orgDownloaded from
http://cancerres.aacrjournals.org/content/47/2/414http://cancerres.aacrjournals.org/content/47/2/414http://cancerres.aacrjournals.org/content/47/2/414#related-urlshttp://cancerres.aacrjournals.org/content/47/2/414#related-urlshttp://cancerres.aacrjournals.org/content/47/2/414#related-urlshttp://cancerres.aacrjournals.org/cgi/alertshttp://cancerres.aacrjournals.org/cgi/alertsmailto:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]://www.aacr.org/http://www.aacr.org/http://www.aacr.org/http://cancerres.aacrjournals.org/http://www.aacr.org/http://www.aacr.org/http://cancerres.aacrjournals.org/mailto:[email protected]:[email protected]://cancerres.aacrjournals.org/cgi/alertshttp://cancerres.aacrjournals.org/content/47/2/414#related-urlshttp://cancerres.aacrjournals.org/content/47/2/414


2/6

[C AN CE R R ES EA RCH 47,414-4 18, January 15, 1987]Characterizatio n of a c/s-D iam rn in edichloro platin um (II)-resistant Human OvarianCancer Cell Line and Its Use in Evaluation of Platinum Analogues1B re nt C . B ohrens, ' T homas C . H am ilton , H idetaka M asuda, K aren R . G rotz inger, Jacqueline Whang-P eng ,K aren G . Louie, Turid K nutsen, W ilm a M . M cK oy, R obert C . Y oung, and R obert F. O zols3M e di ci ne B ra nc h, D iv is io n o f C a nc er T re at me nt , N at io n al C a nc er I ns ti tu te , N IH , B et he sd a, M a ry la nd 2 08 92

ABSTRACTHuman o va ri an c an ce r c el l l in es w it h s ta bl e c is pla tin re si sta nc e h av eb een d ev elo ped b y ch ro nic e xpo su re o f th e pa ren t c isp latin -se nsitiveA27X0 li ne t o i nc re as in g con cent ra ti on s o f c is pl at in . 2 780e CP8 r ef er st o t hi s c el l l in e' s g row th i n medium con ta in in g 8 MMc is pl at in ) h as s ev er al

c lo na l c yt og en et ic a bn orma lit ie s b ut l ac ks h omog en eo us ly s ta in in g r eg io ns o r d ou ble -m in ut e c hr omos omes . I t h as a s ig nif ic an tl y g re at er m on -o la ye r g rowt h r at e, c lo ni ng e ff ic ie nc y in a ga ro se , a nd t ota l g lu ta th io necontent com pared to the A 2780 line, but sim ilar activities of severalg lu ta th io ne -d ep endent e nz yme s. Th e 2780" '* s ub li ne i s 7 3- fo ld r es is ta ntto cisplatin com pared to the A 2780 line, as w ell as cross-resistant toirrad iatio n a nd m elp halan . It is n ot c ros s-resistan t to A driam yc in , b utth is d ev el op s w ith in cr ea se d c is pla ti n r es is ta nc e ( 14 -f ol d) o bta in ed b yf ur th er c is pl at in e xp os ur e o f 2 78 0" '* . O f t he c is pl at in a na lo gu es t es te dwhi ch a re o f c ur re nt c li nic al in te re st, c ar bo pl at in , i pr op la tin , a nd te tr a-p latin , on ly th e latter is m ore c yto to xic th an c isp latin in th e A 27 80 a nd2780e l in es . Th e 2780e sub li ne i s a ls o c ro ss -r es is ta nt t o t he se a nal og ue s in th e r el at iv e o rd er c ar bo pl at in > i pr op la ti n > te tr ap la ti n (mo stt o l ea st c ro ss -r es is ta nt ). T r ea tment o f a h ig hl y c is pl at in r es is ta nt c el l l in e(2780""") w ith either m elphalan or cisplatin w as associated w ith asig nific an t in cre ase in [3 H)th ym id in e in co rp ora tio n in to DNA in th ep re se nc e o f 1 0 m \i h yd ro xy ur ea c omp ar ed w ith t he p ar en t s en si ti ve c el lline w hich show ed essentially no capacity to repair D NA dam age bythese drugs. A 2780 and its cisplatin-resistant cell lines m ay thus beu se fu l in s tu dy in g d ru g r es is ta nc e m ec ha ni sm s, in s cr ee nin g n ew d ru gsf or a ct iv it y ( es pe ci al ly a ga in st d ru g r es is ta nt t umo rs ), a nd i n f ormul at in gi nd uc ti on a nd s al va ge t he ra pi es f or o va ri an c an ce r.INTRODUCTIONCispl at in ICM-l ianmi i ned ich lo rop la t i nu m(11) |. a c li ni ca ll y important antineoplastic agent w ith activity against a w ide spectrum of tumors (2, 3), has been particularly useful for thetreatm ent of testicular and ovarian cancer. The cure rate forp atien ts w ith ad van ced testicu la r can cer has in cre ased from 30 -40% achievable w ith non cisplatin based therapy to 70-80%w ith cisp latin-b ased c om bin ation reg im en s (2, 4 ). In p atien tsw ith ad van ced epith elial ov arian canc er cisp latin-b ased regim ens have produced increased com plete response rates, and insom e studies prolonged overall survival com pared to single-agent therapy (5, 6). However, the m ajority of patients w ithbulky ovarian cancer are not cured with standard cisplatinregim ens prim arily due to the developm ent of acquired drug

re sista nc e (7 ). In a dd itio n, w hile th e d os e o f c is pla tin is a c ritic alfactor in achieving optim al results in ovarian cancer patients(8), the toxicity of cisplatin has prevented adm inistration ofdoses greater than 200 m g/m 2 per cycle even w hen cisplatin isRec ei ve d 3 /2 7/ 86 ; r ev is ed 7 /2 2/ 86 , 1 0/ 9/8 6; a cc ep te d 1 0/1 5/ 86 .The c osts of publication of this artic le w ere defra yed in pan by the pa ym ento f p ag e c ha rg es. T his a rtic le m ust th ere fo re b e h er eb y m ark ed a dv ertis em en t ina cc ord an ce w ith 1 8 U .S .C . S ec tio n 1 73 4 so le ly to in dic ate th is fa ct.1These data were presented, in part, at the 76th Annual M eeting of theA merican A ssociation for C ancer R esearch, H ouston, TX (1), and the 77thA nn ua l Me et in g, L os A ng el es , CA .2 P re se nt a dd re ss: D iv isio n o f H em ato lo gy /O nc olo gy , D ep ar tm en t o f M ed ic in e, T he O hio S ta te U niv er sity , C olu mb us, OH 4 32 10 .3 To w hom re quests for reprints should be addressed, at M edicine B ranch,N ational C ancer Institute , B uilding 10, R oom 12N 226, 9 000 R ockville P ike,B eth es da , MD 2 08 92 .

a dm in is te re d in h yp erto nic sa lin e w ith v ig oro us c hlo ru re sis (9 ).In an effort to im prove the therapeutic index for cisplatin,sev era l h und red an alo gues o f cisp latin h av e u nd erg on e in itialp re clin ic al e va lu atio n (1 0). A lte ra tio ns in th e c hlo rid e le av in ggroups have led to analogues w hich have the sam e spectrum ofactivity as cisplatin but which may be less toxic and conseq ue ntly p erm it fu rth er e sc ala tio ns o f d ose (1 1). O th er a na lo gu esh av e b ee n d ev elo pe d b y a lte rin g th e n atu re o f th e n on ex ch an ge -ab le am ino lig an ds in an effo rt to d eve lo p an alo gu es n on-cro ss-re sista nt w ith c isp la tin fo r u se in p atie nts w ith c isp la tin -re sis tant tum ors (3, 12).Experimental studies using relevant in vitro and in vivom odels of drug-resistant and drug-sensitive hum an ovariancancer may lead to the improved use of cisplatin and its analogues (13). The in vitro patterns of cross-resistance in suchm atched ovarian cancer cell lines m ay help identify those anal ogue s o f pote nti al u tilit y in th e t re atment o f c is pl atin -r es is ta nttum ors. In addition, the nature of the dose-response relationship for cisplatin and its analogues is im portant for the designo f h ig h d os e c hemo th era py stu die s.C isp la tin -s en sitiv e a nd -re sis ta nt c ell lin es m ay a ls o b e u se fu lin u nra ve lin g th e mole cu la r m ec ha nism (s) o f c isp la tin c yto to x-ic ity . Wh ile th e p rim ary le sio n re sp on sib le fo r th e c yto to xic ityof cisplatin is likely a result of its reaction w ith D NA (14, 15),th e ex act n atu re of th e c ritical cy to to xic lesio n(s) h as no t b eenestablished. Interstrand D NA -DNA and DNA -protein as w ella s in tra stra nd c ro ss -lin ks h av e b ee n id en tifie d (1 6) a lth ou gh th einterstran d cro ss-lin ks (d etected by alk alin e e lutio n) acco un tfor only 1% of the total platinum bound to DNA and thus maybe insufficient to fully account for all of the antitum or effectsof cisplatin. A major adduci of cisplatin is the intrastrandN 7d(G pG )-diammine platinum adduct w hich com prises 40-60% of the platinum bound to D NA (17). The X -ray structureof suc h an in trastra nd bide ntate add uct o n adjacen t de ox yg ua-no sin es ha s recen tly b een ch aracterized (1 8). M easu rem en t o fthe intrastrand N 7d(G pG )-diammine platinum adduct using asp ec ific e nz ym e-lin ke d immunoso rb en t a ss ay a s w ell a s o f in te rstrand cross-links w ith alkaline elution assays in cisplatin-s en si tiv e a nd - re sis ta nt c el l lin es may p rovid e in sig hts r eg ar di ngth e im po rtanc e of th ese lesion s in the cy to to xicity o f p latin umdrugs (19). In addition, an improved understanding of them echanism s responsible for cisplatin resistance m ay lead tob io ch em ic al te ch niq ue s o r p ha rm ac olo gic al s tra te gie s b y whic hth is resistanc e m ay be d im in ish ed o r circumv en ted as w ell as toth e d esig n o f sp ec ific a na lo gu es le ss c ro ss -re sis ta nt w ith c isp latin.In this report w e describe the characteristics of a cisplatin-re sistan t h um an o va rian can cer cell lin e w hich w as d ev elo pe dfrom a c isp la tin -s en sitiv e p are nt c ell lin e. T he p atte rn s o f c ro ss -resistance to other antineoplastic drugs and to irradiation indicate that the cell line m ay be of potential use in studying them ech anism s o f p rim ary cisp latin resistan ce as w ell as o f cross-re sistan ce to o th er d ru gs. W e h ave ex am ined th ese cell lin es fo rth eir cap acity to resp on d to cisp latin an d m elp ha lan d am ag e b yinitiation of unscheduled DNA synthesis as an indicator of

41 4American Association for Cancer ResearchCopyright 1987

on February 21, 2013cancerres.aacrjournals.orgDownloaded from
http://cancerres.aacrjournals.org/http://www.aacr.org/http://www.aacr.org/http://www.aacr.org/http://cancerres.aacrjournals.org/http://www.aacr.org/http://www.aacr.org/http://cancerres.aacrjournals.org/


3/6

C ISP LAT IN-SE NSIT IVE AND -R ESIST ANT O VAR IAN C ANC ER C EL L L INE Srepair capacity . In addition, w e hav e used this m atched pair ofd ru g-s en sitiv e an d d ru g-re sistan t c ell lin es to e valu ate th e re lativ e ac tiv ity an d cross-resistance o f cisp latin an d sev eral o f itsan alo gu es wh ic h are c urre ntly o f c lin ic al in te re st.M AT ER IAL S AND M ETH ODSS upp lie s. C ry stalline U -1 00 in su lin w as ob tain ed f ro m E li L illy an dC o., Indianapolis, IN . G lutam ine-f ree concentrated R PM I 1640 m edium and all other com ponents of com plete tissue culture m edium( te rm e d "c omp le te m e di um " h ere af te r) w e re f rom Gran d Is lan d B i olo g

ical C o., G ran d Islan d, N Y , as w as E ag le's m in im al esse ntial m e diu m .C i sp lat in analogue s NSC-241240 ( carbop lat in ), NSC-256927 ( ip ropla-t in ), an d NSC -3 63 81 2D ( te trap lati n), an d me lp halan we re p ro vi de d b ythe Inv estig atio nal D ru g B ran ch, D iv is io n o f C an cer T re atm e nt, N atio nal C an ce r In stitu te , B e th esd a, MD . [3H ]d Thd 4 (5 0-80 C i/mm o l)and A quassure w ere from N ew England N uclear, B oston, M A . A llo th er c hem ical s and b io ch em ical s we re purchas ed f rom S igma Chem icalC o., S t. L ouis, M O .C ell L in es an d T iss ue C ultu re. T he A 2 78 0 hum an o varian canc er ce lllin e, e stab lish ed f ro m tissu e o btain ed f ro m an u ntre ated patie nt, w ask in dly p ro vide d b y D r. S . A a ron so n of th e N atio nal C an ce r Ins titu te.S ubl in es wh i ch surv i v e i nt erm i tt en t e x po su re t o 8 ,2 0, o r 70 MMc ispl at ini n monolay e r cul tu re ( de si gnat ed as 2780 ,7 80CP2 ,n d 2780CP7 ,res pe ctiv ely ) w e re o btain ed by e xpo su re o f th e A 2 78 0 lin e to step w is e-i nc re as in g c is plati n c on ce ntrati on s. I nit ial c is plati n e xp os ure was at ac onc en tratio n o f 3 DM ,w ith th e ce ll line ex pos ed th ree tim e s f or 3 -d ayp eri od s d uri ng a 3 -6 -w e ek p erio d al low in g f or g row th re co ve ry b etw e ency cles. A f ter the com pletion of three cy cles of drug, the dose w asdoubled and the procedure repeated until the noted drug lev els w ereachiev ed. A ll cell lines w ere m aintained as m onolay ers in drug-f reecom plete m edium [R PM I 1640 m edium supplem ented w ith 10% (v /v )f e tal b ov i ne s erum , 2 pM L -glut am in e, i ns ul in ( 0. 25 uni ts /m l ), p en ic il li n( 10 0 u nit s/m l), an d s tre ptom y ci n ( 10 0 ig /m l )].The cisplatin-resistantcell lines w ere used in the ex perim ents described below 1-6 m o af terc on ti nu ou s g row th i n m e dia w i th ou t c is plati n.C y to to x ic ity A s s ay s. C lo no ge nic c ell s urv iv al f oll ow i ng t re atm en tw ith platinum com pounds or irradiation w as assessed in a tw o-lay ers of t agaro se s y st em , as p re v io us ly d es cri bed (20 , 2 1) . Br ie f ly , s in gl e c el ls uspens io ns we re f irs t p re pared f rom exponent ial ly g row ing monolay e rc ultu re s (4 d ay s af ter s ub cu ltu re ) b y brief e xp osu re to 0 .05% try psin /0.02% EDT A (both w /v in phosphate buf fered saline) follow ed byg entle p as sag e th ro ug h 2 2-g aug e n ee dles . S usp en sio ns w e re th en adju ste d to s tan dard c ell c on ce ntrati on s u si ng an e le ct ro ni c p arti cl e c ou nter (m odel Z B I; C oulter E lectronics, H ialeah, FI.). C ells w ere thenm ix ed w ith agaro se in co m plete m e diu m (0 .3% , w /v , f in al co nce ntratio n) an d 1 -m l aliq uo ts (co ntain in g 1 0,00 0 ce lls) ov erlaid o n ch illed 1 -m l base lay ers of 0.6% agarose in com plete m edium contained in 35-m n i diam e ter p las tic d ish es (30 35 tissu e cu ltu re d is he s; C ostar, C amb ri dg e, MA ) . T o p ag aro se l ay e rs als o c on tai ne d f re sh ly p re pare d d ru gso r an e qu al v olum e o f d ilu ent (n orm al salin e) in stud ies o f co ntin uou sdrug exposure .Ka ryo ty p ic Ana ly s is . Ch romo some p reparat io ns we re e xam in ed af t erb oth co nv en tio nal G iem s a stain in g and try psin -G ie m sa b an ding an dre su lts w e re re po rt ed u sin g s tan dard n onmen cl at ure , as d es crib ed p rev ious ly (22) .O the r A s say s. T he A 2 780 an d th e e isp lal in -res istan t ce ll lin es w ereplated in m ultiple f lask s at a density of 1.5 x IO 6 cells/75-cm 2 tissueculture f lask . T issue culture m edia w as changed on day s 1, 4, and 7af te r p lat in g. C el l s us pe ns io ns w e re t he n p re pare d, as d es crib ed ab ov e,at v arious tim es af ter plating. A portion of each suspension w as usedto m easure total G SH (G SH + G SH disulf ide) content using a m odif ication of the m ethod of S uz uk ak e et ai (23) in w hich the rate (ratherthan the total ex tent) of 2-nitro-5-thiobenz oic acid f orm ation w asmo nit ore d s pe ctro ph ot ome tri call y. T h e i nit ial v el oc it y o f th is re ac ti onis lin early re late d to total G SH co nte nt o ver sam p le co nce ntration s o f2-80 nm ol/m l (data not show n).

4 T h e a b br e via tio ns u se d a r e: d T h d, t hy m id in e; CR I , c r oss- r esist an ce in d ex ;G SH , g lu ta th io ne ; K M , d r u g c on ce nt r at io n c au sin g 5 0% r e du ct io n in su r viv in gf r a ct io n ; UDS, u n sc h ed u le d ONA s yn t h es is .

S tat is ti cal Ana ly s is . D i ff e re nc es b etween mean s we re t es te d u si ng t heu np ai re d two -t ai le d S tu de nt 's / -te st , an d l in ear re gre ss io n an aly si s wasp erf orm e d b y th e le as t- sq uare s m e th od ( 24 ).M e as urem en t o f UDS . T h e c el l l in es w e re s cre en ed f or th eir c ap ac it yt o p erf orm UDS w i th e xp os ure t o m e lp hal an o r c i sp lati n. I n t he ab se nc eo f s em i co ns erv ati ve DNA s yn th es is [3H]d T hd in co rp orat io n i nto ac id -p rec ip itable DNA is tak en as a m e asu re o f re pair o f ind uce d d am ag e toDNA (2 5). A s th es e carcin om a ce ll lin es sh ow little p ro pe nsity to w ardcontac t-assoc ia ted inh ib i tion o f ce ll g rowth and |"HJc lThd incorporat ioni nto DNA d oe s n ot d is crim i nate b etw ee n DNA re pli cati on an d u ns ch edu led DNA sy nthe sis (26 ), s pe cial care w as n ec essary to lim it s em ic on -serv ativ e sy nthesis during the assay of these cells. G row th of cells toc on flu en ce in 2 5-c m 2 c ultu re d is he s u sin g E ag le 's m in im al es sen tialm e diu m co ntainin g 1 0% (v /v ) f etal b ov in e se ru m f ollo w ed b y m ain tenance of cultures f or 3 day s in E agle's m inim al essential m ediumd ef ic ie nt in arg in in e b ut w i th L -g lu tam i ne an d 2 .5% ( v/ v) d ialy z ed f et albov ine serum inhibited f urther grow th of A 2780 and the resistantv ariants b y >9 9% . A t th is p oin t, to f urth er in hib it sem ic ons erv ativ eD NA sy nthesis, hy drox yurea (10 H IM ) w as added to each dish, andaf te r 1 h , d ru g (m e lp halan o r c is platin at a ran ge o f co nce ntratio ns )and 5 /iC i/m l of [3H ]dT hd (50-80 C i/m m ol; N ew E ngland N uclear)w e re ad ded . A f ter a 3-h in cu batio n at c ultu re co nd itio ns, dish es w erewas he d f ou r t im e s w i th ic e- co ld p ho sp hate b uf f ere d s al in e f ol lo w ed b ytw o w ashes w ith cold trichloroacetic acid (10% w /v ). D N A w as thene xtracted as d esc rib ed b y S ch m id t an d T han nh au se r (2 7) an d q uanti-tated b y the m e tho d o f B u rto n (2 8). | 'I I jdT lu l in co rp oratio n in to DNAwas d ete rm in ed b y co un tin g an am o un t o f th e e xtract so lub iliz ed w ithA q uassure in a B eck m an L S 2800 liquid scintillation counter w ith a3 H co un tin g e ff icie nc y o f 4 0-50%. T he UDS ac tiv ity in the resp ectiv ecell lines w as then ex pressed as the ratio of dpm /M g of D NA in drugt re ate d c ell s to in co rp orat io n o f [3H]d T hd i n DNA o f u nt re at ed c on tro lcells.

RESULTSCharac te ri stic s o f C is platin Re sis tan t Ce ll L i ne s. Pe rio dic ICSOd eterm in ation s h av e sh ow n th e d eg ree of cisp latin resistan ce inthe 2780CP8 subline to be stable for at least 9 m o duringsub cu ltu re in d rug -f ree m e diu m (d ata no t sh ow n ). T he 2 78 0CP8

sub line h as a sig nif ican tly sh orte r d oub ling tim e in m o no lay erc ultu re an d h ig he r c lo nin g e ff ic ie nc y (p erc en tag e o f c ells p late dgiv ing rise to colonies) in sof t agarose than the parent line(T able 1). B oth cell lines hav e a m odal chrom osom e num ber of4 6 as w e ll as s ev eral c ommon an d d is tin ctiv e c lo nal c yto ge ne ticabnorm alities (T able 1). D uplications of a specif ic portion ofthe q arm of chrom osom e 1 and the sam e break ing point atchrom osom e 6 are shared by both lines. A b norm alities notedo nly in the 2 78 0CP8 lin e in clu de d eletion s o f sp ecif ic p ortio nsof the X chrom osom e and of chrom osom es 6 and 20, anaddition to the short arm of chrom osom e 13(13p+), and atranslocation inv olv ing the q arm s of chrom osom es 4 and 7.

T ab le 1 C harac te ri sti cs o f t he 4 27 80 an d 2 78 0e r* h um an o vari ancancer ce ll l inesCharacteristicDoublingtimeh)Cloningefficiency%)Relativecellolume'Modal

chromosomeumberChromosomalmarkers'A278025.3

.0"27.5 .9 1.0046du p(l)(q23- l44)t(l;6X q23;q21),13pc'2780e 0.7 *36.4 2.8 '1.0846del(XKp21),

dup(l)(q23-Kj24),del(6)(q21),t(

" M e an SE .* P < 0 .0 5 co m pared to v alu e f or th e A 2 78 0 lin e.' D et erm i n ed w it h a n e le ct r on ic p a n ic le c ou n t er e q ui pp e d w it h a c h an n eli ze r .' N om e nc la tu r e a s p er P a r is C on fe r en ce o f 1 97 1.* pe . p rom inen t c en trome re .415

American Association for Cancer ResearchCopyright 1987on February 21, 2013cancerres.aacrjournals.orgDownloaded from
http://www.aacr.org/http://www.aacr.org/http://www.aacr.org/http://cancerres.aacrjournals.org/http://www.aacr.org/http://www.aacr.org/http://cancerres.aacrjournals.org/


4/6

C ISP LAT IN-SE NSIT IVE AND -R ESIST ANT O VAR IAN C ANC ER C EL L L INE SHomo gen eo usly stain in g reg io ns an d d ou ble-m in ute chro m os ome s , k ary o ty p ic abno rma lit ie s as so ciat ed w i th d rug re sis tan ceacquired in v itro by other cell lines (29), w ere absent in thec ispl at in -re si st an t cel l l ine .Cellular total GS H (G SH + G SH disulf ide) content of the27 80 CP8 lin e is sig nif ican tly h ig her than th at o f th e A 2 78 0 lineat all tim es during grow th in m onolay er culture (Fig. 1). A f ter4 days of culture the total GS H content (nm ol/106 cells) w as10.7 0.3 in 2780C P com pared to 4.46 0.4 in A 2780. T hisdif f erence in GSH content is not due to alterations in cellv olum e betw een these lines (T able 1). T he greatest dif ferencein total G SH content betw een these lines occurs at the earliesttim e af ter p lating , day 2, b ut th is is n ot a result o f a dif feren tialresponse of the tw o lines to try psiniz ation or other aspects ofs ub cu ltu re . T o c on firm th at th es e mark ed e arly d if fe re nc es af te rp latin g w e re n ot d ue to d if feren tial try psin ef fects b etw e en th ec ell lin es, to tal GSH le ve ls in s in gle c ell p re paratio ns sim i larlyprepared but not plated w ere determ ined and found to be onlym inim ally elev ated at 3-6 h after try psiniz ation (1.3- and 1.2-f old in the A 2 78 0 an d 2 78 0CP8 l in es, respe ctiv ely ), an d w ithin18 -2 1 h retu rne d to th e base-lin e le vels f or cells in m o no lay er.R e sis tan ce an d C ro ss-re sistan ce S tu die s. C ro ss -re sis tan ce toirradiation and other drugs comm only used in ov arian cancertherapy w as assessed by concurrent com parisons of the sensitiv ity of a resistant subline to that of the parent line. B ycom parison of U\


5/6

C ISPLATIN -SEN SITIV E AN D -R ESISTAN T OV ARIAN CA NC ER C ELL LIN ES

S 1 0M E LPH AL AN I

20 40 80

10 20 40 80C B -P LA T IN ( uM )Fig. 2. Effect of m elphalan (a) and cisplatin (*) on induction of UDS inh um an o va ria n c an ce r c ell lin es A 27 80 (O ) a nd 2 78 0C P7A ). V alu es s ho wn a reratios of hydroxy urea refractory [3H ]dT hd incorporation in the presence ofin cre asin g c on ce ntr atio ns o f d ru g to in co rp or atio n in th e a bse nc e o f d ru g; e rr orbars, 1 SD. The base- line va lue (no trea tment) for A2780 was 67.2 4 .4 dpm/Mgo f DNA a nd fo r 2 78 0C P t w as 7 3.7 1 .3 d pm /V g o f DNA .

1 .00

0 .7 5 -

0.50 -

0 .5 0 -

0 .2 5 -

0 . 10DO S EF ig . 3 . D os e- re sp on se c ur ve s f or c is pla tin a nd c is pl ati n a na lo gu es in t he A 27 80( top) and 2780"" (bottom) human ovarian cancer cel l l ines. .NSC-363812 D;O , c is pl ati n; ,i pr op la ti n (NSC- 25 69 27 ); a nd O , c ar bo pla tin (NSC -2 41 24 0)(mean SE) .

a nalog ou s to th e m ech an ism s fo r clin ical cisp latin resistan ce,the relativ e cy to to xicities an d th e n atu re o f th e d ose-resp on serelationships for cisplatin and its analogues in 2780C P8 haveproven useful in the design of "high dose" chem otherapy regi

m ens in ovarian cancer and in the preclinical evalution of non-c ros s- re si st an t c isp la ti n anal ogues .Com pared to the sensitive cell line A2780, the cisplatin-resistan t sub line 2 78 0C P8 h as a 2 -fo ld in crease in G SH co ntent(31). Reduction of G SH by exposure of the cells to buthionines ulf ox im in e, a s pe ci fi c i nh ib ito r o f -y -g lu tamylc ys te in e s yn th e-tase, an enzyme required for GSH synthesis, decreases theresistan ce to c isp latin (3 1) an d rev erses th e cro ss-resistan ce tom elphalan (31) and irradiation (21). T he exact m echanism bywhich GSH modulates cisplatin cytotoxicity is unknown.Amon g the p ossibilitie s u nd er in vestigatio n in o ur lab orato ryare: (a) G SH m ay alter the transport of cisplatin; (b) G SH m ayin activa te cisp latin b y form in g an in activ e G SH -cisp latin c onjugate in the cytosol of resistant cells, although w e have previo usly s hown th at th ere is n o sig nific an t d iffe re nc e in th e s pe cificactivities of GSH S-transferase, GSH peroxidase, or GSHred uc ase(31 ); (c) G SH m ay p ro tect critical sites o n DNA an ddecrease form ation of the intrust rand A ^-bidentate adduct onad jacen t deo xy guan osines; (d) G SH m ay fun ctio n in th e re pairof D NA dam age.The finding that induced resistance to cisplatin is accompanied by cross-resistance to other agents which cause DNAdam age, i.e., m elphalan and irradiation, suggests that a comm on repair m echanism m ay be responsible at least in part forprim ary resistance to cisplatin and for the cross-resistance tom elp halan an d irrad iation see n in 2 78 0"'. Ind irect ev id en ce ofa ro le fo r G SH in th e rep air o f irrad iatio n cy to to xicity has bee np rov id ed b y stu dies w hich ha ve d em on strated th at glu ta th io neester admin istered to lym ph oid cells after irrad iatio n w as ab leto m arkedly decrease the lethality of the irradiation (32). Toevaluate DNA repair as a possible mechanism of cisplatinresistance, we developed a variant of A2780 with m arked resistance to the drug, 2780C P7 ,n the hope that supraclinicalle ve ls o f re sista nc e m ig ht in cre ase th e p ro ba bility o f d ete ctin gd iffe re nc es in o ve ra ll re pa ir a ctiv ity u sin g c la ss ic al re pa ir e va luation m ethodology w ith its lim ited sensitivity. Indeed, thep re lim in ary sc re en in g e xp erim en ts re po rte d h ere in dic ate d th atcisplatin and m elphalan dose-dependent increases in U DS area ss oc ia te d w ith induced r es is ta nc e t o c is pl atin in 2780CP7h ilesignificant UDS is not observed in the parental cell line. Exp erim en ts usin g th e m ore comp lex d en sity sh ift m eth od olog y(33) and studies on the effect of repair inhibition on drugc yto to xic ity a re c urre ntly in p ro gre ss . T he se s tu die s sh ou ld h elpclarify w hether the U DS m easured is indicative of D NA repairand if so the overall im portance of D NA repair as a m echanismo f antic an ce r d rug r es is ta nc e.T he dose-response curves for cisplatin and its analogues inthe sensitive and resistant cell lines provided inform ation ofp ote ntia l c lin ic al re le va nc e fo r th e tre atm en t o f o va ria n c an ce r.First, both the A 2780 and 2780C P8 cell lines have a steep dose-response curve w ith cisplatin exposure, Fig. 3, w ith the curvefo r th e re sista nt su blin e s im ply d is pla ce d towa rd s h ig he r c is platin c on cen tration s. T hu s, a chievement o f tumo r cisp latin co nc en tra tio ns e xc ee din g th ose o bta in ed b y c on ve ntio na l c isp la tina dm in istra tio n m ay d ela y eme rg en ce o f c lin ic al d ru g re sis ta nc eor produce responses in patients refractory to standard doses.U sin g H yp erto nie sa lin e a nd v ig oro us c hlo ru re sis to c irc umve ntth e d ose-lim itin g n eph ro to xicity o f cisplatin (9 ), w e h av e d emo nstrated th at h ig h-d ose cisp latin (2 00 m g/m 2) p ro du ces a 3 2%resp on se rate in ov arian can cer p atien ts w ho w ere refra cto ry tostandard dose cisplatin regim ens (8). In addition, it has alsob een d em onstrated th at h ig her dru g lev els asso cia ted w ith i.p.adm inistration o f cisp latin p ro duce re sp onses in patien ts w ho




6/6

CISPLATIN-SENSITIVE AND -RESISTANT OVARIAN CANCER CELL LINEShave been refractory to the i.v. administration of standardc is pla tin r eg imens ( 34 ).T he d ose-resp on se relatio nsh ip s of th e p latin um an alo gue sm ay also b e u seful in ide ntifying th ose an alo gues w hich m ay beparticularly useful in the treatm ent of cisplatin-resistant tum ors. Structure-activity relationships in drug-sensitive and-resistant m urine tum ors have dem onstrated that resistance isa function of the ligands attached to the am ine groups and nota function of the leaving groups (3,10,12). T hus, a high degreeof cross-resistance w ould be expected betw een cisplatin andcarb op latin in asm uch as th e aq uated interm ediate o f cisplatin(the active m etabolite) is likely the sam e as the aquated form ofcarboplatin. In agreem ent w ith this w as the finding that in thecisp latin-resistant cell lin e 2 78 0C P8 , th ere w as m ark ed cross-resistance w ith carboplatin (T able 3). It has recently been rep orted th at p atients refracto ry to fu ll-d ose therap y w ith cisp latin are u nlik ely to re sp ond to th erap y w ith carb oplatin (3 5) th usp ro viding sup po rt fo r th e u se of the cisp la tin -resistan t h um ano varian can cer cell lin e 2 780CP 8 to sc ree n p latin um a nalog uesw hich m ay b e o f u se in ov arian can cer p atien ts. It is o f p articu lari nt ere st th at t etr ap la tin h ad th e lowes t d eg re e o f c ro ss -r es is ta nc ew ith th e c isp la tin -re sista nt c ell lin e. T etra pla tin w as e va lu ate din this system due to initial observations that it had activityagainst th e su blin e o f L 121 0 leu kem ia w ith a cqu ired resistan ceto c is pla tin . In c on tra st to o th er d iamin oc yc lo he xa ne p la tin umcom plexes, tetraplatin has a defined structure and sufficientp urity , s olu bility , a nd sta bility fo r fo rmula tio n d ev elo pm en t.5T etraplatin is currently undergoing toxicity testing at the N ational Cancer Institute, and its m arked activity in cisplatin-resistant hum an ovarian cancer cell lines suggests that thiscom pound m ay be of use in those ovarian cancer patients whobecome r es is ta nt t o c is pla ti n.REFERENCES1 . B eh re ns, B . < .. G ro tz in ge r, K . R ., H am ilto n, T . C ., W ha ng -P en g, J ., B atist,

G., Louie, K . G., Knutsen, T., Tsunio, T., M cKoy, W . M ., Young, R . C.,a nd O zo ls , R . F . C yt oto xi cit y o f 3 c is pl ati n (CP ) a na lo gu es i n a d ru g- se ns it iv eand a new C P resistant hum an ovarian cancer (OC ) cell line. Proc. Am .A ss oc . C an ce r R es ., 2 6: 2 62 , 1 98 5.2. Loe hrer, P . J., and Einhorn, L . H . C isplatin. A nn. Intern. M ed., 100: 704-7 13 , 1 98 4.3 . R os en be rg , B . F un damen ta l s tu di es w ith c is pla tin . C an ce r ( Ph ila .) , 5 5: 23 03 -2 31 6, 1 98 5.4 . E in ho rn , L . H ., a nd D on oh ue , J . P . C u- diamm in ed ic hlo ro pla tin um , v in bla s-ti ne , a nd b le omyc in c ombi na ti on c hemoth er ap y i n d is sem in ate d t es tic ul arc an ce r. A nn . In te rn . M ed ., 8 7: 2 93 -2 98 , 1 97 7.5. O zols, R . F., and Y oung, R . C . Chem otherapy of ovarian cancer. Sem in.O nc ol., //: 2 51 -2 63 , 1 98 4.6. T higpe n, T ., and B lessins, J. A . C urrent therapy of ovarian carc inom a: ano ve rv iew. S em in . O nc ol. , 1 2 ( Su pp l. 4 ): 4 7- 52 , 1 98 5.7. O zols, R . F . P harm acologie reversal of dru g resistance in ovarian cance r.S em in . O nc ol., 1 2 (S up pl. 4 ): 7 -1 1, 1 98 5.8. Ozols, R. F., Ostchega, Y ., Myers, C. E., and Young, R. C. High dosec is pla tin i n h yp er to nic s al in e in r ef ra cto ry o va ria n c an ce r. S em in . O nc ol. , 1 3:1 246-1250, 1 985.9 . O zo ls , R . F ., a nd C ord en , B . J . H ig h d ose c is pla tin in h yp er to nic s alin e. A nn .In te rn . M ed ., 1 00 : 1 9-2 4, 1 98 4.1 0. L ee , F . II., C an etta , R ., I sse ll, B . F ., a nd L en az , L . N ew p la tin um c om ple xe sin c lin ic al t ria l. C an ce r T re at . R ev ., 1 0:3 9- 51 , 1 98 3.11. Ozols, R. F., Behrens, B. C., Ostchega, Y ., and Young, R. C. High dosecispla tin and high do se carboplatin in refra ctory o varian cancer. C ancerT re at. R ev ., 1 2 (S up pl. A ): 5 9-6 6, 1 98 5.12. B urc henal, J. II.. K ala her, K ., D ew , K ., L okys, L ., and G ale, G . S tudies of5M. Wo lp er t-De F il li pe s, Na ti on al Cancer I ns ti tu te , p er so n al communi ca ti on .

c ro ss r es is tance, s yn ergi st ic comb in at io ns , an d b lo ck in g act iv it y o f p la ti numder iv at iv es . B io ch emi st ry , 6 0 :9 61 -965 , 1 978.1 3. H am ilto n, T . C , Y ou ng , R . C ., a nd O zo ls, R . F . E xp erim en ta l m od el s yste mso f o va ria n c an ce r: a pp lic ati on to t he d es ig n a nd e va lu ati on o f n ew t re atme nta pp ro ac he s. S em in . O nc ol ., 1 1: 2 85 -2 98 , 1 98 4.14. R oberts, J. J., and P era, M . F . D NA as a target for anticanc er co ordina tionc omp ou nd s. I n: S . J . L ip pa rd ( ed .) , P la tin um s, G old , a nd O th er Chemo th er -a pe utic A ge nts, p p. 3 -2 5. W ash in gto n, D C: Am eric an C hemic al S oc ie ty ,1983.15. P looy, A . C . M ., v an D iik, M ., a nd 1 oilm an. P . H . W . Induction and rep airof D NA -cross links in C hinese ham ster ovary ce lls tre ated w ith variousp la tin um c oo rd in atio n c om po un ds in re la tio n to p la tin um b in din g to DNA ,c yt oto xic ity , mu ta ge nic ity , a nd a nt it umor a cti vit y. C an ce r R es ., 4 4: 2 04 3-2051, 1 984 .16. Zw elling, L . A ., A nderson, T . A ., and K ohn, K . W . D NA -protein and D NAin terst rand cros s- li nking by r /' .v -and f ran .v -pl a limim (II ) d iammined ich lo r idein 1 ,1 21 0 m ou se le uk em ia a nd its r ela tio n to c yto to xic ity . C an ce r R es, .IV365-369,1979.1 7. P oirie r, M . C , L ip pa rd , S . J., Z we llin g, L . A ., a nd U sh ay , H . M . A ntib od ie se li ci te d aga in st c - di ammin edi ch lo ro pl at in um( II )-DNA adduc ts f ormed i nvi vo a nd in vitro . P ro c. Na ti. Ac ad. Sc i. U SA, 7 9:6 44 3-6 44 7, 1 98 2.18. Sherman, S. E., Gibson, D., Wang, A. H. J., and Lippard, S. J. X-raystruc ture of the m ajor adduci of the anticance r drug cisplatin w ith D NA .S cie nc e (W as h. D C), 2 30 :4 12 -4 17 , 1 98 5.19. R ee d, E ., B ehrens, B . C ., Y uspa, S . H ., P oirie r, M . C ., H am ilton, T . C ., andO zo ls , R . F . D if f re nc esn c is pl ati n-DNA add uc i f orma tio n i n s en si ti ve a ndr esista nt s ub lin es o f h um an o va ria n c an ce r c ells . P ro c. Am. A ss oc . C an ce rR es., 2 7: 2 85 , 1 98 6.2 0. O zo ls, R . F ., W ills on , J. K . V ., G ro tz in ge r, K . R ., a nd Y ou ng , R . C . C lo nin gof hum an ov aria n can cer cells in soft aga r from m alignant effusions andp er it on ea l wash in gs . Cance r Re s. , 4 0: 2 743-2747 ,1 980.2 1. L ou ie , K . G ., B eh re ns, B . C ., K in se lla , J . J., H am ilto n, T . C ., G ro tz in ge r, K .R ., M cK oy, W . M ., W inker, M . A ., and O zols, R. F. R adiation survivalp arame te rs o f a nlin eo pla stic d ru g-se ns itiv e a nd r esista nt h um an o va ria nc an ce r c ell lin es a nd th eir m od ific atio n o f b uth io nin e s ulf ox im in e. C an ce rRe s. , .- 2110 -2115, 1 985.2 2. W ha ng -P en g, J., K nu tse n, T ., D ou gla ss , E . C , C hu , E ., O zo ls, R . F ., H og an ,W . M ., and Young, R. C . C ytogenetic sludies in ovarian cancer. CancerG en et. C yto ge ne t., //: 9 1-1 06 , 1 98 4.23. Suzukake, K., Petro, B. J., and V istica, D . T. R eduction in glutathionec on le nt o f L -PAM r es is ta nt L I 2 10 c el ls c on fe rs d ru g s en si tiv ily . B io ch em .P ha rmac ol. , 3 1: 12 1- 12 4, 1 98 2.2 4. A rm ita ge . P . S ta tistic al M eth od s in M ed ic al R ese ar ch . O xfo rd : B la ck we llSc ien ti fi c Pub li cat ions. 1971 .2 5. M asu da , H ., a nd E nd o., H . C ap ac itie s o f m ou se emb ry o c ells fo r in du clio no f uns ch edu led DNA syn lh es is b y4 -ni lr oqui no li ne 1 -o xi de i n p rimary cul tu resy ste ms. J. N ati. C an ce r In st., 6 7: 1 06 3- 10 70 , 1 98 1.26. T rosk o, J. E ., and Y ager, J. D . A sensilive m ethod to m easure physical andchemical c ar ci nogen- in du ced "un schedul ed DNA syn th es is " i n r ap id ly d iv idin g e uk ar yo tic c el ls . E xp . C eil R es ., S S: 4 7- 55 , 1 97 4.27. Schm idt, G ., and Thannhauser, S. J. A m ethod for the determ ination ofd eo xy ri bo nu cl eic a ci d, r ib on uc le ic a ci d, a nd p ho sp ho pr ote in s i n a nima l ti ssu es. J. B io l. C hem., 1 61 :8 3- 89 , 1 94 5.2 8. B urto n, K . A stu dy o f Ih e c on dilio ns a nd m ec ha nism o f I he d ip he ny lamin er ea d io n f or I he c ol or im et ri e e sti ma tio n o f d eo xy rib on uc le ic a cid . B io ch em .J., 6 2: 3 15 -3 23 , 1 95 6.2 9. C urt, G . A ., C le nd en nin , N . J., a nd C ha bn er , B . A . D ru g r esista nc e in c an ce r.C an ce r T re al. R ep ., 6 8: 8 7- 99 , 1 98 4.3 0. Z we llin g, L . A ., M ic ha els, S ., S ch wa rtz , H ., D pb so n, P . P ., a nd K oh n, K . W .DNA c ro ss- lin kin g a s a n in dic ato r o f se ns itiv ity a nd re sista nc e o f m ou seL I2 10 le uk em ia to c u- diammi ne di ch lo ro pla lin um (I I) a nd L -p he ny la ia ni nem usta rd . C an ce r R es., 4 1: 6 40 -6 49 , 1 98 1.31. H am ilton, T. C ., W inker, M . A ., Louie, K . G ., B atist, G . Behrens, B. C .,T suruo, T., G rotz inger, K . R ., M cK oy, W . M ., Y oung, R . C , and O zols, R .F . A ugment ati on o f a dr iamyc in , m e lp ha la n a nd c is pla tin c yt oto xi ci ty in d ru gr es is ta nl a nd s en si ti ve h uman o va ria n c an ce r c el l l in es b y b uth io nin e s ulf oxim in e medi at ed g lu la th io ne d ep le ti on . B i ochem . Ph armacol ., 3 4: 2583 -2586,1985.32. W ellner, V . P ., A nderson, M . E., P uri, R . N ., Jense n, G . L ., and M eister, A .R ad io pr ote ctio n b y g lu la th io ne e ste r: tra nsp on o f g lu ta th io ne in to h um anlymph oid c el ls a nd f ib ro bl as ts . P ro c. N ai l. A ca d. S ci . U SA , 8 1: 4 73 2- 47 35 ,1984.33. Sm ilh, C . A., C ooper, P. K ., and Hanawalt, P. C . M easurem ent of repairreplication by equilibriu m sedim entation. In: E . C . F riedbe rg and P . C .H an aw al t ( ed s. ), DNA Rep ai r A L ab or ato ry Manu al o fR e se ar ch P ro ce du re s,V ol. 1, P art B , p p. 289 -305, N ew Y ork: M arcel D ekker, 1981.34. O zols, R . F . Intraperitoneal chem oth erapy in th e m anage ment of ova rianc an ce r. S em in . O nc ol., 1 2 (S up pl. 4 ): 7 -1 1, 1 98 5.3 5. O zo ls , R . F ., O stc he ga , Y ., C urt, G ., a nd Y ou ng , R . C . H ig h d ose c arb op la tinin o va ria n c an ce r: p ote nti al f or r ep la cin g c is pl ati n in t he c ombi na tio n c hemo th era py o f o va ria n c an ce r. P ro c. Am . S oc . C lin . O nc ol., 5 :1 18 , 1 98 6.



Cancer Res 1987 Behrens 414 8

Documents

Transcript of Cancer Res 1987 Behrens 414 8