BIOCHIMIE, 1978, 60, 859-867. Mbcanismes d'action

Platinum complex-DNA interactions and anticancer activity.

Barnett ROSEN BE~RG. Depar tment of B iophys ics , Michigan Slate Universi ty , East Lansing, Michigan ~882/t, USA.

Metal coordina t ion complexes, par t icular ly those of p la t inum, have now been proven to be active an t icancer agents in animals and man. The most wide ly investigated complex is cisplat in (the generic name for cis -d ichlorodiammine- plat inum(II)) . This drug has now completed pha- ses I, II and ]II of the cl inical trials. In combina- t ion chemotherapy it appears to be of meri t in the

s tand the b iochemical mechan i sm of action of this drug. One of the most powerful tools for stu- dying elusive mechanisms of act ion is the corre- lat ion of b iochemical and molecular biology phe- nomenon wi th the activity of the drugs as anti- cancer agents. I shall use the s t ructure-act ivi ty relat ion to develop a hypothesis of the par t icu la r mechan i sm of act ion of cisplat in at the cellular

Active Anti- Tumor Compounds

gr

/ P,(]z)/c=o NH3/ /Cl N~ pt(]I)/ NH3/ "'0

NH3//Pt(]I)/C' NH 3 ` 'Br NH~ / ~/-0 "C=O cis-dichloro- ci.js- dibromo- OxQIotodi(]mmine- diemminepletinum diommineplatinum platinum(rl)

(~) (TT) CI CI

,NHz/--.----. / Cl N H3r--J(--/C I ,NH2/--'~/C t H2C / mc H2C / ~"*"/

Dichloroethylene- cI cI diomineplotinum(]I) cis-tetrachloro- Tetrachloroethylene-

diommineplotinum(]~Z') diomineplotinum(IE)

FIG. 1. - - Molecular structures of the f irst group of anticancer active platinum coordination complexes.

t reatment of the fol lowing human cancers : testi- cular cancers, ovar ian cancers, head and neck cancers, b ladder cancers, prostate cancers, and lung cancers. Some of the cl inical results are des- cr ibed in the papers inc luded in this symposium.

I should like to present here some basic studies wi th cisplat in, under taken in an attempt to under-

level ; to present the evidence leading to this hy- pothesis ; and finally, to describe a pred ic t ion of the hypothesis which can lead to an exper imental test.

The structures of the coord ina t ion complexes of p la t inum that are active an t icancer agents are shown in figure 1. The first such complex, cispla-

860 B. R o s e n b e r g .

tin, was chosen some yea r s ago by the Nat ional Cancer Ins t i tu te to be en te red onto c l in ica l t r ials . Therefore , most of the expe r imen ta l w o r k has, of necessi ty, been .done wi th this p a r t i c u l a r complex. Some newer complexes are p r e sen t l y in pr.elimi- na ry p h a r m a c o l o g y and tox ico logy studies, or in Phase I c l in ica l t r ia ls . I t is by no means cer ta in that the chemis t ry or the b iochemis t ry of these n e w e r complexes is necessar i ly iden t i ca l to, or even re la ted to, the chemis t ry of c ispla t in . W i t h th is caveat in mind , I shal l desc r ibe some of the k n o w n chemis t ry of c ispla t in .

C i sp la t in is in jec ted in t r avenous ly into pat ients . The h igh ch lo r ide concen t r a t i on of the ex t ra cellu- la r f luid limit.s or p revents the exchange of the ch lo r ide l igands, and the in t eg r i ty of the molecu- lar s t ruc ture of the drug is ma in ta ined . In animals most of the drug is exc re t ed in the u r ine w i t h i n 1 1/2 hours af ter a pulse in jec t ion . By th in layer chromatography tests, the p l a t i n u m excre ted in the u r ine is at least 90 pe rcen t in the form of the unchanged c isp la t in . A p p r o x i m a t e l y 5 pe rcen t ap- pea r s to be p ro t e in bound. The ,drug is pass ive ly t r a n s p o r t e d th rough the cell membranes to the in te r io r s of all cells of the body (with the sole except ion of the bra in) . The in t r ace l lu l a r f luid is .lower in c h l o r i d e concen t r a t i on than the extra- ce l lu la r f luid by a fac tor of 30 and u n d e r these c i r cums tances the ,chloride l igands of c i sp la t in are exchanged for w a t e r or h y d r o x y l igands and the o r ig ina l ly neu t ra l molecule is now charged. The aquat ion reac t ion has been s tud ied extensi- vely in vitro. At 37°C. the ha l f t ime for the ex- change process is a p p r o x i m a t e l y 1.7 hours (de- t e r m i n e d by con,ductometry) ; the ac t iva t ion en- t ha lpy for th is aqua t ion reac t ion is a p p r o x i m a t e l y 18 k i loca lo r i e s pe r mole. The aquated species m a y have three di f ferent combina t ions of exchanged l igands. At pH 's above 7.37 the molecule is pre- dominan t ly in the d i h y d r o x y form ; wh i l e at pH ' s be low 5.51 i t is p r e d o m i n a n t l y in the d iaquo form. In the reg ion be tween the~.e two pH's the d rug is p re sen t as the m o n o a q u o - m o n o h y d r o x y species. Therefore , in the i n t r ace l lu l a r fluid, at roughly neu t ra l pH, i t is this last species tha t is p r e sen t and, therefore , poss ib ly the reac t ive in ter - media te w i t h the cel l rece ivers . These s t ruc tures a re shown in ~gure 2.

The rmture of the cel l r ece ive r s has been exten- s ive ly s tud ied by m a n y l abora to r i es over these pas t five years . Two p a r t i c u l a r l ines of s tudy im- p l i ca te in te rac t ions w i t h the ce l lu lar DNA~ as be ing the essent ia l lesion l ead ing to cy to tox ic i ty a r i d /o r an t i cance r act ivi ty . H a r d e r ~nd Rosen- berg [1] showed tha t ceils t r ea ted in vitro wi th a

concen t ra t ion of c i sp la t in equivalent to a thera- peu t ic in an imals exh ib i ted select ive and pers is - tent i nh ib i t i on of new DN.A synthesis . The effects

A /OH A A ' \ K., \ /0H - -.--~ Pt( I I ) ~ Pt( ] ] )

\ , \

H / A / "Hp A / \oH

A = -NH 3 pKa, pKaz

5,51 7.37

Fro. 2. - - The aquated species of cisplatin.

on new RNA and new p ro t e in synthes is were mi- n imal at this dose level, but became s ignif icant at dose levels a p p r o a c h i n g the equivalent LD~o in animals , at w h i c h po in t the re was a large amount of cell death. Howte and Gale [2] i n d e p e n d e n t l y and s imul taneous ly a r r i ve d at the same conclu- sions, using both in oivo and in vitro techniques . It was also shown that the exposu re to c i sp la t in d id not i nh ib i t the fo rma t ion of essent ia l p recur - sors to D,NA, or the i r p e r m e a b i l i t y th rough cell membranes , or the ab i l i ty of the necessa ry enzy- mes to po ly lner ize the p recu r so r s into macromole - cu lar nucle ic acids.

The second t echn ique that imp l i ca t ed DNA as the ce l lu lar target is tha t of in:direct i nduc t ion of lys is in lysogenic bac te r ia , as measu red by Res- lova-Vasi lukova [3]. In this expe r imen t a non- lysogenic s t r a in of E. coli K12 was exposed to luw concen t ra t ions of c i sp la t in as the aquated species. These .cells, w h i c h con ta ined the F* sexual t r a n sduc t i on factor , were then sepa ra t ed f rom the c isp la t in , and then incuba ted wi th an F- s t r a in of E. colt K12 con ta in ing the l a m b d a bacte- r iophage genome. Upon sexual conjugat ion the p l a t i num t rea ted plasmi,ds (DNA) of the 1 ~ s t r a in we re t r a n s f e r r e d to the F- s t ra in . The F- s t ra in p r o m p t l y exh ib i t ed lysis . This s t rongly stcggests that n,ucl, eic ac ids c a r r y i n g p l a t i num les ions we re sufficient to induce lysis. This expe r imen t takes on a d d e d s igni f icance f rom tl~e fact that the re is an excel len t c o r r e l a t i o n be tween the class of co- o rd ina t i on complexes exh ib i t ing an t i cance r act i- v i ty anLd the class of coo rd ina t ion complexes ca- pab le of i nduc ing lysis in lysogenic bac te r ia .

The~e exper iments , and a number of o thers w h i c h s t rong ly suggest that DNA is the ce l lu lar ta rge t for the p l a t i n u m complex , have r ecen t ly

B[OCHIMIE, 1'9'78, 60, n ° 9 .

P l a t i n u m c o m p l e x - D N A in t e rac t i ons , a n t l c a n c e r a c t i v i t y . 861

been reviewed by Roberts and Thomson [4]. At this time, no other cellular target has been impli- cated i,n the antican.cer activity, but any such oth,ers may not be excluded wi th cer ta inty on the basis of present evidence.

The chemist ry of the equat ion react ion has tu rned out to be somewhat nmre complicated than expected. Indeed, if one prepared the diaquo spe- cies by react ing cisplat in wi th silver nitrate, (which replaces the chloride by ni trate l igands which then exchange very rap id ly for water), the drug propert ies, w h e n injected into mice, change drastically. The diaquo species produces an im- mediat, e toxicity, wh ich kills the mice at much lower doses than the LDs0 of c isplat in (13 mill i- grams per Mlogram). Cisplat in, even at high over- doses, requires some three to four days to ki l l the animal. Also, even at much lower doses of the diaquo species, wh ich is well tolerated by the

to be the activated form of the molecule in the ceil showed little or no activity as an an t icancer agent but only exhibi ted severe toxicity. While

rv-

.,"-'~ N(2) . ' .: 0(I)

Fie. 4. - - X-ray crystallographic structure of the cen- trosymmetrie dimer [(NHa)~ Pt(ll) (OH)~ Pt(II) (NH~),] .2* of the nitrate salt.

extracel lular react ions of the diaquo species could be invoked to expla in this difference in behavior, we have no evidence that this is the case.

0(4) N(2)

0(5) ~ "~

' 5 )

o,j%o., FIO. 3. - - X-ray crystallographic structure of the mo-

nomer cis-Pt(ll) (NHs)~ (NOs)t.

{('-'~' N q)

d..~, N(5) / ' ~ ,r~-:'.,

,1 ; a(5) 'I . ' : 'N(6) - ' . . j Z

l a

[b

~:~'~N(5) "~l~: ,' iN(I)

\-,p,(2) N~)

Fro. 5. - - X-ray crystallographic structure of the trimer cation [(NH~)e Pt(II) (OH] ~' of the nitrate salt.

mice, no significant an t i tumor activity was de- teCtable. These results lead to the somewhat pa- radoxical s i tuat ion i,n that what was cons idered

BIOCHIMIE, t978, 60, n ° 9.

Lippert, in our laboratory, then discovered that the aquat ion react ion, depending upon pH, may yield at least three and possibly four ol igomeric

5 9

862 B. Rosenberg.

forms of the p la t inum complex. The chemical analysis, physical chemistry m,easurements, and finally, and most conclusively, the molecular con- figurations by X-ray crysta l lography 'was done by Lippert in cooperat ion with Lock and his group at McMasters Univers i ty [5, 6, 7, 81. We have found thai at very low and very high pH's the aquat ion product is a monomer 'whose s tructure is shown in figure 3. At in termedia te pH's, a hy- d'roxy bridged, ce,ntrosy,mmeiric .dimer can be crystall ized whose .structure is shown in figure 4. This s tructure had been predic ted by Martin Eg~ after having found the equivalent s t ructure for pal ladium. The p la t inum dimer s tructure was si- mul taneous ly and independen t ly discovered by Hoeschele and Stanko [10]. In addit ion, Lippert, Lock and co~workers foun~d the tr imers, shown in figure 5, as 'wel, l as a te t ramer consis t ing of a di-

Chi.kuma [11], in our laboratory, has found that each of th.ese oligomeric species can be dist in- guished from the others by the use of 195PtNMR. The broad signals we obtained show extensive high field shifts from the s tandard Na2PtC16. Pre- para t ion of the monomer , dimer, and t r imer mo- lecules with 15N in the ammonia groups p.roduce a dis t inct sha rpen ing of lhe spectra. One such spect rum is sho'wn in figure 6. Ini t ial ly, the mono- mer solution, at ..(~5 Molar concentra t ion , was pla- ced in the Bruker WH-180 NMR and the pH was adjusted wi th a suitable buffer to a value a round 6. The signal p ropor t iona l to the monomer con- centrat ion, decreased with time, wi th a ,corres- pond ing bui ldup of the signal due to the dimer con.centration, and finally of the t r imer concen- t ra t ion signal. The kinet ics of th.ese processes are shown, as p r e l imina ry results, in figure 7. The

DIMER 195pT - FT - NMR

SPECTRA OF MOilOMER, DIIIER AND TRIMER OF cIs- PT(II)((NH3)2(OH)H20) ' ~03

J -I155

INSTRUMENT

SOLVENT

TEMP,

15N ENRICHED

STANDARD

- BRUKER - WH 180

- 100% D20

- 24oc

- NA?PTCL6

PD = 6,4

TRIMER

MOIIOFiER

CHEMICAL SHIFT (PPM)

HIGH FIELD

FIG. 6.

-1510 -1586

mer of dimers wi th p la t inum-p la t inum interac- tions. These hydroxy bridged o,ligomeric species are apparent ly more stable than we would have anticipated.

BIOCHIMIE, 1978, 60, n ° 9.

half t ime for the d isappearance of the monomer band was de te rmined as a funct ion of pH. The re- sults are shown in figure 8. The oligomerization react ion is most rapid in the neutral pH region

P l a t i n u m c o m p l e x - D N A in terac t ions , a n t i c a n c e r ac t iv i ty . 863

where the monoaquo-monohydroxy species is pre- domina.nt. For compar ison purposes, a plot of the concent ra t ion of this species as a funct ion of pH is inc luded in the figure.

cisplat in in high concent ra t ions may form a si- gnificant quant i ty of the d imer as a compet ing react ion to that of the monomer wi th DNA. This may occur in the kidneys, thymus, and spleen of

I-- z

o n,, ILl O.

5 0

, l I I I I i l f l

I 00 140 180 2 2 0

&

~ ° f o

o "

2 0 6 0

,oo

I I I I I 5.0 6.0 7.0 8.0 9.0

Ti me, min.

FIG. 7. - - 195Pt(II)-FT-NMR determined kinetics of the oligomerization reaction of the monomer, cis-Pt(ll) [(NHo),(OH) (H~O)]* in PIPES Buffer at pH 6.60 and in water. Monomer : ©, • ; Dimer : £x, • : Trimer : [5, • (solide marks are without buffer).

What is perhaps most in t r igu ing about this pro- cess of ol igomerizat ion is that the biological acti- vities are quite different for the monomer, dinaer, and t r imer species. While all three, when injected into animals are more toxic than cisplatin, the monomer requires 3 days to produce death (as does cisplat in) whi le wi th the dimer and tr imer, p rompt mortal i ty ocours in the animals. At lower concent ra t ions the monomer exhibits high anti- cancer activity, whi le the dimer and t r imer exhi- bit little or no such activity. Fur ther studies on all animal effects, and on the in teract ions of these oligomers wi th DNA are under way in our labo- ratory at this time.

These results not only p ro : id~ a reasonable explanat ion for the unexpected toxicity of the diaquo species prepared by silver nitrate, but may also provide a rat ionale for the toxicities caused by cisplat in . While the kinet ics of the oligomeri- zation react ions are still incom.pletely worked out, our evidence suggests that the dimer formation rate is p ropor t iona l to the square of the lnonomer concentra t ion. Since the dimer is the more toxic of the two, then those tissues which accumulate

BIOCHIMIE, 19'78, 60, n ° 9.

Z 6(3

5C c; 0

IM o._

zD _.3 < 2c > I

h_ I(3

"i-

-,- o D O \ . , - / ' I -

Q_

20 z / \

I.-- 6 0 z

t.~

9 o w o_

I00

pH

Fro. 8. - - Half-value period (minutes) [or the disap- pearance of the monomer, cis-Pt(II) [(NH~) (OH) (HtO)] ÷, I'3~Pt(II)-FT-NMR signal, and the percent concentration of th is form of the aquated species as functions of the pH.

the animals since these are the major depots for the cisplat in. This suggests that lower concent ra- t ions of the monomer may decrease the toxici ty and, therefore, explain why divided doses a n d / o r slow infus ion processes produce less toxicity to the pat ient than the same dose given as a rapid IV push. This needs to be s tudied in more detail.

Studies in our laboratory and others, par t icu- larly those of Tobe and Connors, have clearly shown that those p la t inum coordina t ion com- plexes which are an t i tumor active are invar iab ly in the cis configuration. The cor responding trans isomers are without any significant activity. The best quant i ta t ive evidence, taken from the work of Connors et al. El2] is shown in figure 9 for a variety of subst i tuted amine l igands in the plati- num(II) dichlor ide complex. The residual amount of activity in the trans complexes of the cyclopen- tyl- and cyclohexylamine structures co,uld be at- t r ibuted to a small con taminan t of the correspon- ding cis complexes. Because of their extremely high activity, only a small amount of contamina- t ion is suffici'ent to account for the results. The ini t ia l evidence had been publ ished earl ier by Cleare and Hoeschele [131. This stereospecificity provides us with a powerful tool in the studies of the significant lesion, assumed to occur on DNA,

864 B. R o s e n b e r g .

leading to an t icancer activity. Generally, the che- mis t ry of the cis and trans complexes are not suf- ficiently diss imilar to account for the marked dif- ferences in biologic activity. Also, the react ions wi th nucleic acids should not be too diss imilar because of the plast ici ty of the DNA structrure. This urges us to look for a specific react ion site wi th a r igid molecule as the site -which distin- guishes the cis from the trans complexes. Pre- vious work had indica ted that cisplat in interac- t ions wi th DNA occur p r edominan t ly in regions

Therapeutic am Isomer LDs0 IDgo index (LI)~/

(mg/kg) (mg/kg) IDgo)

et$ trans

NH3

~NH 2 ~"NH 2 I~NH2

NH

H 3 c ' ~ H 2

ClS

lrans

c l s

lrans

CIS lrans

ClS

trans

ClS lrans

ClS

trans

CI$ lrans

13 1.6 8.1 27 > 27 - -

56.5 2.6 21.7 18.0 > 18.0 --

240 17.5 13.7 72 > 72

57 2.3 25 27 > 27 - -

90 2.9 31 250 > 11o - -

480 2.4 200 180 72 2.5

< 3200 12 > 267 680 260 2.6

990 1180 - - 360 > 800 --

FZG. 9. -- Anticaneer activitg of cis and trans isomers of Pt(II) (amine)~ CI~ complexes. Tumor system : ADJ/ PC6A in C- mice. (after Tube, Connors et at., ref. 12).

r ich in G-C content . This has been most clearly shown by Stone and his col laborators [14]. In vi- tro studies lead us to the conc lus ion that all pos- sible attack sites on D~NA react at l~east to some extent. Thus, in te r s t rand crossl inking, in t r a s t r and crossl inking, and react ions wi th the ind iv idua l bases have all been looked for and found. The most l ikely in te rac t ion now appears to be one bet- ween the mono-hydroxo mono-aquo p l a t inum spe- cies wi th guanine. The nucleophi l ic sites of the DNA structure are : the phosphate groups ; the C8, N7, 06, N3 and 2 NH 2 of guanine ; the C8, N7, N3, N1 and 6 NH 2 of adenine ; the N3 and 4 NH.., of cytosine ; and the N3 and 04 of thymine. These

sites differ in nucleophi l ic i ty for attack by elec- t rophi l ic reactants such as the alkylat ing agents and the aquated c ispla t in species. It is k n o w n that this p la t inum species does not in teract wi th DNA by in tercala t ion. B a u e t al. [15] have analyzed a s t ructure showing a react ion of a p la t inum com- plex wi th a phosphate group, but this is not yet considered to be a signif icant in terac t ion in DNA.

The quest ion of the par t icu la r react ion which is stereospecific for cis compared to trans com- plexes has attracted much at tention. In recent years Theophanides et aI. [16], Goodgame, [17] and Jordanov and Dehand [18] have all suggested the possibi l i ty of forming a closed r ing chelate between the p l a t inum species in the cis canfigu-

0 OPO-

~ / o . . . . .

. . . . . \ . / ,, \1 I/I I I

F / o OPO- H 3 N ~ t~.~NH3 O

CI- L CI

O OPO-

...... cC / ~ N ~ ~, j O - ~ [

...... H-N~, ~--~--\I I/

2+ r _//--'~h~ I - - ! ' I o " - \ / ooo - i ~ " / O

,c,- /

FIG. 10. - - Postulated structures of the reaction pro- ducts of cis-Pt(lI) (NHj)jCls and trans-Pt(II) (NHs)2Clt with guanine showing the possible closed ring chelate complex of the cis configuration with the N7 and 06 sites.

ra t ion and N7 and O6 sites of guanine. The trans configurat ion cannot form such a closed r ing chelate. Such configurat ions are shown in figure 10 (one modification, related to our discus-

BIOCHIMIE, 1978, 60, n ° 9.

Pla t i num complex-DNA interactions, anticancer activity. 865

sions fu r the r on, w o u l d be remova l of the p ro ton f rom the N1 pos i t ion of guanine in fo rming the chelate complex invo lv ing 06. This enables the guanine base to m i spa i r w i th t hymine dur ing cell r ep l i ca t ion ins tead of w i t h cytosine) .

Suff icient ly h a r d chemica l ev idence for th is N7-O6 guanine chelate complex does not exist. I t is now obvious that, if we reac t guanine wi th the aquated p l a t i n u m species, a number of different p roduc t s should occur . Reac t ion at the N7 site, s ince it is the most uuc leoph i l i c , w o u l d be expec- ted to be p redominan t . This may occur via a mo- nodenta te a t tack or via a p l a t i num coupl ing bet- ween two guanines at the N7 sites. Therefore , s im- ple chemis t ry tells us that of the mul t ip l i c i t y of poss ib le p roduc t s those w i th N7 a t tachments ~vill be dominant . This s i tua t ion is ve ry r emin i scen t of the c i r cums tances involv ing ca rc inogenes i s by a lky la t ing agents. Here too, in the ear ly days, the N7 reac t ion was co,nsidered to be the s ignif icant lesion l ead ing to the fu r the r s teps in cell t rans- fo rmat ion to ma l ignancy . ,However, Schoenta l in 1958 [19] first suggested tha t a lky la t ion of the oxygen of guanine m a y be impor t an t for ca rc ino- genesis. Loveless in 196.9 [20] p r o v i d e d the first expe r imen ta l ev idence suppor t i ng this sugges t i on In recen t years many repor ts have ten ted to sup- po r t this idea [21]. Whi l e the cu r ren t s tudies are not unambiguous , the bu lk of the expe r imen ta l evidence suggests that N7 a lky la t ion undergoes decompos i t ion r a p i d l y in the ce l lu lar DNA, wi- thout the necessa ry in te rven t ion of a r epa i r en- zyme. In those organs of the an imal in w h i c h a cancer ar ises af ter in jec t ion of an a lky la t ing agent, the a lky la t ion at the 06 pos i t ion is the lon- gest l ived lesion. Repa i r of this les ion requi res an enzyme, as has been shown in p r o k a r y o t i c sys- tems, and more recen t ly by Pegg [22], in eukaryo- t ic sys tems as vcell.

The invoca t ion of the 06 guanine site is very in t r igu ing in that , if the DNA rep l ica tes p r i o r to r epa i r of the lesion, the 06 h y d r o g e n bond wi l l not form, and a m i spa i r i ng w i th t hymine should occur. In vitro tests by Lud lum et al. [23] have verif ied this. They have fo rmed me thy la t ed 06 on po ly (G, C) and incuba ted i t ~vith RNA polyme- ruse i~n the p resence of a va r ie ty of nuc leos ide t r iphospha tes . They showed tha t the a lky la t ed 06 guanine exh ib i t ed a s t rong p re fe rence to m i s p a i r wi th UMP, even in the p resence of h igh concen- t ra t ions of CTP. They aIso showed that for the N7 ine thyl guanine there was no s ignif icant mi- s i nco rpo ra t i on of UM'P. The s tudies w i th a lkyla- t ing agents show tha t the b io log ica l ly s ignif icant lesion (for carc inogenes i s ) is a m i n o r componen t

of the poss ib le p roduc t s of the reac t ions of a lkyla- r ing agents wi th guanine ; that it is the ce l lu la r r epa i r system, a n d / o r spontaneous decomposi - t ion, w h i c h p icks out the 06 lesion as the longest

HF'LC nF SrlL',AT':D clS-"LATI 'I-%UA'~I'I~ PEAC[Ir,q pPqDIICT~

INJECT

SCA,~ I - WITH 2 C4UIVALENT? OF NAOH Irl REAGION ~IXTURE

SCAN 2 WITN i E'%UIVALENT OF NAOH IN ~EACTION MIXTURE

ELUENI ].01 ~ NH4H2PO 4 ; PH : 4

INJECT

GUANINE

Fro. 11. - - HPLC spectra of the reaction products of aquated, monomeric cisplatin with guanine. The diffe- rence in the reaction conditions for the two spectra is described in the text.

k_

l ived one ; that th is leads to m i s p a i r i n g of the bases af ter DNA rep l ica t ion . This then p roduces a base subs t i tu t ion muta t ion after a second repl i - ca t ion and, therefore , fixes the lesion more or less p e r m a n e n t l y in the DNA..

We are now act ively s tudy ing the va r ie ty of complexes c rea ted by r eac t ing guanine wi th the aqua ted p l a t i n u m complexes . Dr. Pol lock, in our l abora to ry , is ana lyz ing the reac t ion p roduc t s wi th high pressure l iquid c h r o m a t o g r a p h y (both ana ly t ic and p repa ra t ive ) . The first a t tempt to de- t e rmine the mu l t i p l i c i t y of p roduc t s is shown in figure 11 taken by Dr. Pol lock. Two di f ferent scans of the HPJLC c h r o m a t o g r a m are shown for two s l ight ly different r eac t ion condi t ions . In the first, most of the guanine has reacted, and one dominan t peak occ,urs a c c o m p a n i e d by a number of m i n o r peaks. The iden t i f ica t ion of these peaks is p r e sen t l y u n d e r w a y . In the second reac t ion , most of the guanine has not reacted, but again a mu l t i p l i c i t y of reac t ion p roduc t s are seen. This would appea r to be a p a r t i c u l a r l y potent tool to invest igate the cent ra l p rob lem, and a l r eady the p r e l i m i n a r y resul ts do indeed suggest tha t the r eac t ion be tween these two c o m p o u n d s y ie lds a

BIOCHIMIE, 1978, 60, n ° 9.

866 B. R o s e n b e r g .

mul t ip l i c i t y of p roduc t s at least as r i ch as that occur ing wi th a lky la ted guanine.

REACTIONS LEADING TO BASE SUBSTITUTION MUTATIONS

I~js.-Pt(n)((NH3) z (OH) HzO). NO3+ Guanine--~ (Guanine)"

G* -- C

DNA ~ REPLICATION

G'--T

ONA ,[, REPLICATION

A - - T

Fro. 12. - - Postu la ted reaction of the aquated, mono- mer of c isplat in w i th guanine leading to the f i x ing of the long lived lesion into a base subs t i tu t ion mu ta t i on in DNA. (After Tbeophanides et al., ref. 16).

i I I I I I I I I I I TA I00

I000 0

0

o~ coo

g o

g

2 0

I I I I I I I I I 2 4 6 8 ]0

P io tmum compound per pla'te (ug)

Fro. 1 3 . - Revers ion of Sahnonella typhimurium s train TAIO0 to h i s t id ine prototrophg induced by expo- sure of bacteria to p l a t i n u m compounds in Ames plate incorporation mutagenes is assays. Data represented are averages of 9 plates. Dose-response curves after subtraction of spontaneous revertants are (@) trans- platinum(II)-diamminodichloride, (A) oxalatodlammine platinum(II), ([:3) malonatodiammine platinum(II), ( I ) platinum(II)-ethylenediaminedichloride and (©) cis- platinum(II)-diamminodichloride. (After Beck et al., ref. 27).

I,f then the p l a t i num in te rac t ion wi th guanine leads upon DNA rep l i ca t ion to the same type of mi spa i r i ng d iscussed before, then, a second rep l i - cat ion, where the t hymine now cor rec t ly pa i r s

wi th adenine , nvould p r o d u c e a base subs t i tu t ion muta t ion as shown in figure 12. If the t rans com- p lex cannot form such a complex wi th the 06, it may not be a base subs t i tu t ion mutagen.

The mutagen ic i ty of the cis complex has been s tud ied in four di f ferent l abora to r ies us ing the Ames TA 100 tes ter s t ra in of the S a l m o n e l l a Tg- p h i m u r i u m [24, ~i, 26, 27]. In all cases i t has been sh(~wn tha t the c is complex is an act ive mutagen in this s t ra in w h i c h is sensi t ive to base subst i tu- t ion mutagens . No mutagen ic i ty occurs in those s t ra ins ~¢hich are sensi t ive to f rame shif t muta- gens.

Beck et al. [27] have u n d e r t a k e n the test of our p red ic t ion . The i r resul ts are shown in figure 13. T h e c is c o m p l e x is an act ive mutagen in this TA 100 s t ra in whi le the t rans complex is not. They also showed tha t u n d e r these cond i t ions and doses, the cis complex is much more cyto- toxic than is the t rans complex. ~nterest ingly, i t is also appa ren t in th is figure tha t o ther an t i tumor complexes w h i c h r equ i re h igher doses for thera- peu t i c ac t iv i ty in an imals are a p p a r e n t l y also less mutagen ic than c i sp la t in , thus showing a rough co r re l a t ion be tween the mutagen ic i ty and ant i tu- m o r act ivi ty . This rough co r re l a t ion ve ry ob- v ious ly needs to be s tud ied more in tens ive ly . Re- cen t ly Z~celling et al. [28] have r e p o r t e d that the c is complex is also mutagen ic in the V79 mamma- l ian cell l ine, ~¢hile the t rans is not.

This en t i r e l ine of a rgumen t leads to an inte- res t ing hypo thes i s to account for the thus far u n e x p l a i n e d select ive des t ruc t ion of t umor cells by the p l a t i n u m drug at dose levels tha t p r o d u c e only minima} s ide effects. I f w e make the assump- t ion tha t at /east some cancers are in i t i a t ed by ca rc inogens and have c loned f rom cells w h i c h are deficient, comp~ared to no rma l t issue, in the i r abi.lity to r e p a i r 06 lesions of guanine ; and if we assume that the p l a t i n u m complexes p r o d u c e le- s ions at the 06 si te of guanine ; then i t is poss ib le tha t DNA damage due to the p l a t i num drug is re- p a i r e d in all no rma l cells p r i o r to cell r ep l i ca t ion , but is not r e p a i r e d in the tumor cells before cell r ep l i ca t ion . Therefore , the p Ia t inum lesions get f ixed as base subs t i tu t ion muta t ions in the cancer ceils, Jeading to t he i r eventua l des t ruc t ion . This hypo thes i s is not a unique one, in that one could conce ive of o ther les ions on the DNA lead ing to the base subs t i tu t ion muta t ion , but at the p re sen t t ime it is the most p laus ib le one. I t is easi ly tes- table, and such tests are cu r r en t ly u n d e r w a y in our l abora to ry .

BIOCHIMIE, 1978, 60, n ° 9.

Platinum complex-DNA interactions, anticancer actil~ily. 867

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21. For a review see Rajewsky, M. F. et al., Nervous- System-Specific Carcinogenesis by E thy ln i t ro - sourea in the Rat : ~o l eeu l a r and Cellular Aspects, In << Origins of H u m a n Cancer >> (edited by Hiatt , Watson , Wins ten) Cold Spring Harbor Laboratory , 700-726 (1977).

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23. Gerchman, L. L. it Ludlum, D. B. (1973)The Proper- t ies of O6-Methylguanine in Templates for RNA Polymerase . Bioehimiea et Biophysica Acta, 308, 310-316.

24. Monti-Bragadin, C., Tamaro, M. ~ Banff, E. (1975) Mutagenic Activity of P l a t i n u m and R u t h e n i u m Complexes. Chem. Biol. Interactions, 11, 469-472.

25. Leeointe, P., Macquet, J.-P., Butour , J.-L. a Paolet t i , C. (1977) Relat ive efficiencies of a Series of Squa re -P lana r P l a t i n u m (II) Compounds on Sal- monel la Mutagenesis. Mutat ion Research, 48, 139-144.

26. Benedict, W. F., Baker, M. S., Haroun, L., Choi, E. ,~ Ames, B. N. (1977) M~tagenici ty of Cancer Che- mo the rapeu t i c Agents in the Salmonel la /Micro- some Test. Cancer Research, 37, 2209-2213.

27. Beck, D. J. it Fisch, J. E., Mutagenic i ty of P l a t i n u m Coordinat ion Complexes in Salmonel la Typhi - mur ium, Submi t ted for Pub l ica t ion (1978).

28. Zwelling, L. A., Kohn, K. ~V. ~ Anderson, T., DNA Crosslinking, Cytotoxici ty and Mutagenici ty in Cells Treated wi th Cis- and Trans-Pla t inum (II) Diammine Dichloride (PDD), In Abstract 930, Proceedings, S ix ty -n in th Annual Meeting of the American Associat ion for Cancer Research April, 1978.

BIOCHIMIE, 1978, 60, n ° 9.