The nature of inactivating lesions produced by platinum (II) complexes in phage λ DNA

Chem.-Biol. Interactions, 32 (1980) 321--330 © Elsevier/North-Holland Scientific Publishers Ltd.

321

THE N A T U R E O F INACTIVATING LESIONS P R O D U C E D BY PLATINUM(H) COMPLEXES IN PHAGE k DNA

J. FILIPSKI, K.W. K O H N and W.M. B O N N E R

Laboratory of Molecular Pharmacology, Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute, National Institutes of Health, Bethesda, MD 20205 (U.S.A.) (Received April 9th, 1980) (Accepted July 12th, 1980)

SUMMARY

L a m b d a DNA loses transfectivity and acquires interstrand cross-links after treatment with either trans-Pt(II) or cis-Pt(II). With trans-Pt(II) there is close to an equivalence between the fraction of ~, DNA cross-linked and the fraction inactivated. In contrast, with cis-Pt(II) there are approx. 5 inactivating lesions for each ~, DNA interstrand cross-link. These results suggested that trans-Pt(II) does n o t introduce intrastrand inactivating lesions into k DNA while cis.Pt(II) does so. To verify this conclusion, the crosslinked and uncross-linked fractions of ~, DNA treated with trans-Pt(II) or cis-Pt(II) were separated on alkaline sucrose gradients. After trans-Pt(II) treatment, the uncross-linked fraction of ~ DNA was transfective when renaturated. However after cis-Pt(II) treatment the uncross-linked fraction of k DNA was not transfective when renatured. Thiourea treatment restored transfectivity to all inactivated fractions, s h o w i n g that these lesions are reversible. We conclude that trans-Pt(II) inactivates ~ DNA primarily by introducing interstrand cross-links b u t that c/s-Pt(II), although it also introduces interstrand cross-links, inactivates ~ DNA primarily b y introducing intrastrand lesions.

INTRODUCTION

Several types of compounds used in cancer chemotherapy are k n o w n to form DNA interstrand cross-links, and this feature may be directly related to their antitumor activity [1]. The diamminedichloroplatinum(II) isomers are especially convenient for the study of the molecular mechanisms of bifunctional cross-linking compounds, since both the c/s and trans isomers are known to form DNA interstrand cross-links [ 2 ] , but only the cis isomer is a useful chemotherapeutic

322 agent. Previously, we had shown that the cross-linking and inactivation of k DNA by c/s and trans Pt(II) could be completely reversed b y incubation with thiourea [3]. These results indicated that a more detailed study o f the interaction of c/s and trans Pt(II) with k DNA m a y furnish some further insight into their mechanism o f action. In addition we felt that the interaction o f Pt(II) with k DNA might be a useful model system for the interaction of Pt(II) with nuclear DNA in living cells. In this paper we show that trans-Pt(II) inactivates k DNA primarily by introducing interstrand cross-links, while cis-Pt(II), although also introducing interstrand cross-links, inactivates ~, DNA mainly by introducing intrastrand lesions. MATERIALS AND METHODS

Complex formation. Cis-Pt(II) (cis~lichlorodiammine-Pt(II)) and transPt(II) (trans-dichlorodiammine-Pt(II)) were dissolved in water at 1 mM and then kept at 37°C for at least 3 days. 20 /11 of k phage DNA (Bethesda Research Laboratories, Rockville, MD), dissolved in 10 mM Tris--HC1 (pH 7.6), 1 mM Na2 EDTA, 50 mM NaC1 (TES buffer) was mixed with 3 pl of cis- or trans-Pt(II) solution o f appropriate concentration. After incubation at 37°C, the reaction was terminated b y dialyzing the mixtures at 0--2°C against TES buffer. Complex reversal by thiourea was performed by transferring the complex to a vial in which the appropriate a m o u n t of 1 M thiourea in methanol had been dried. Transfectivity assays were performed as described earlier [3]. Analytical CsCl density gradient centrifugation. 0.1 ml of solution conraining 3--5 pg of DNA in 5 mM Tris--HC1, 2 mM EDTA (pH 7.5) was mixed with 0.2 ml of 85 mM NaOH, 1 mM EDTA. After 2 min incubation at r o o m temperature, 0.2 ml of 0.1 N citric acid, 0.03 M Tris base, 0.02% sarcosyl were added (final pH = 8) followed b y 10 #1 of 55 pg/ml Clostridium perfringens DNA ( b u o y a n t density, p = 1.709 g/cm 3) and 0.85 g CsC1 (Harshaw Chemical Co. optical grade), and the refractive index was adjusted to 1.400 with water. Centrifugation was performed at approx. 23°C for 24 h at 44 700 rev./min using an AN-G titanium analytical rotor. Scans were performed at 260 nm. The relative amounts of native and denatured DNA (cross-linked and non-cross-linked) were determined from the areas of the corresponding peaks. Preparative alkaline sucrose gradients. 50 pl samples containing 20 #g of DNA, previously treated with Pt(II) and dialysed against 0.1 M Tris--HC1 (pH 7.2) were layered on 4--20% w/v sucrose gradients containing 0.04 M Na2EDTA, 0.02% sarcosyl, 0.03 M NaOH. Centrifugation was performed in an SW65 rotor of a Spinco L2-65B centrifuge at 45 000 rev./min for 2 h at 20°C. Gradients were fractionated into 23 fractions using an LKB Uvicord and fraction collector. Transmission at 257 nm was recorded. Fractions showing absorbance were dialysed against 0.1 M Tris--HC1 (pH 7.2), adjusted to 120 pl with dialysate, and divided into 3 aliquots for

323 subsequent processing. Aliquot 1: Transfections were performed by diluting a 40/~l aliquot with buffer A (0.1 M Tris--HC1 (pH 7.2), 50/~g/ml thymidine) to 1 ml; 0.1 ml portions were then mixed with 0.2 ml of bacterial suspension in CGT buffer (50 mM CaCI2, 50/Jg/ml thymidine, 10% glycerol). After 1 h in ice, the mixture was mixed with 2 ml of hot (45°C) 0.6% plain DIFCO agar and poured on TB plates. Aliquot 2 : 4 0 / l l aliquots were mixed with 40/~1 of formamide and kept at room temperature overnight. After dialysis against 0.1 M Tris--HCI (pH 7), fractions were diluted to 1 ml with buffer A to perform the transfection assay. Aliquot 3 (trans-Pt(II) experiments): 40/~l aliquots were mixed with 10/~l of 1 M thiourea in H20, kept overnight at 3°C, dialysed against buffer A, and diluted to 1 ml with buffer A for the transfection assay. Aliquot 3 (cis-Pt(II) experiments): 40 /ll aliquots were mixed with 40 /J1 of formamide at room temperature overnight, dialysed against 0.1 M Tris--HC1 (pH 7), diluted to 1 ml with 1 M thiourea, 5 M NaC1, kept overnight at 37°C, and dialysed against buffer A for the transfection assay. RESULTS Preliminary attempts to compare the reaction of c/s- and trans-Pt(II) with DNA revealed two factors that required attention. The first factor is the activity of the solution of the Pt(II), especially trans.Pt(II). Freshly dissolved cis-and trans-Pt(II) did not significantly inactivate ~ DNA or cross-link it (data not presented}, presumably because it is the aquated species which reacts with DNA [4]. Cis-Pt(II) becomes aquated in a few hours [5]. For aquation times of 3 days or more, cis-Pt(II) gave reproducible k DNA inactivation curves such as shown in Fig. 1. Trans-Pt(II) is known to become aquated very much more slowly than cis.Pt(II) [6], a fact which is reflected in Fig. 1. Trans-Pt{II) aquated for 3 days inactivates ~ DNA much less efficiently than cis.Pt(II) aquated for 3 days. Trans-Pt(II) requires approximately one month's incubation at 37°C to become fully activated. Figure 1B shows that trans-Pt(II) aquated for 5 months inactivates ~, DNA as well as does cis-Pt(II). Although the age of the trans-Pt(II) solution affects its efficiency of inactivation and cross-linking, we found that it does not affect the ratio of these two activities. The second factor was the termination of the reaction between c/s- or trans-Pt(II) and k DNA. In the cross-linking assay, the reaction is terminated due to strand separation when the DNA is denatured and centrifuged in alkaline sucrose or CsC1. In the transfection assay, however, the DNA remains double-stranded throughout the assay, during which time cross-link formation may continue to occur. In effect, then, unless the reaction is terminated, monofunctionally bound Pt(II) molecules could continue to inactivate initially active k DNA molecules as the transfection test is being performed. Two methods were tested for terminating the reaction of c/s-Pt{II) and ~, DNA. The first was to treat the k DNA for 15 rain with cis-Pt(II), dialyse at 4°C for 1 h, then reincubate at 37°C until the cross-linking assay and the

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transfection assay reached constant values. Figure 2 shows t h a t this h a p p e n e d after 14 h. Th e second m e t h o d was to t r eat the reaction m i xt ure briefly with 0.1 M thiourea. These conditions were designed to prevent f u r t h e r cross-linking and inactivation, b u t at the same time to cause very little reversal o f crosslinks or o f inactivating lesions. Figure 2 (triangles) shows t h a t t he results of the transfection and cross-linking assays were similar at 1 h or 20 h after th io u r ea t r e a t m e n t , indicating t h a t no f u r t h e r cross-linking or inactivation t o o k place after thiourea t r e a t m e n t . With trans-Pt(II) the reaction was allowed t o proceed to completion. Figure 2 (diamonds) shows t h a t with transPt(II) there was no f u r t h e r inactivation after 20 h. With trans-Pt(II), even mild thiourea t r e a t m e n t caused some reactivation and cross-link removal (data n o t shown). As will he discussed later, a small p r o p o r t i o n of t he trans-Pt(II) cross-links were reversed even during the t r a nf ect i on assay. T h e data f or cis-Pt(II) in Fig. 2 shows t h a t cis-Pt(II) caused m uch more inactivation o f k DNA than interstrand cross-linking. Figure 3 shows a

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direct comparison of these two parameters for trans-Pt(II) and cis-Pt(II). In the case of trans-Pt(II), the inactivation and cross-linking curves lie close together while for cis-Pt(II) these two curves are not close. In other words, for trans-Pt(II) there is close to a one-to-one correlation between inactivation and interstrand cross-linking, while for cis-Pt(II) there is considerably more inactivation then interstrand cross-linking. These findings suggest the following hypothesis. Both trans-Pt(II) and cisPt(II) produce interstrand cross-links which are inactivating lesions in X DNA. In addition cis-Pt(II), but not trans-Pt(II), produces other inactivating lesions, possibly intrastrand cross-links, which are responsible for most of the inactivation.

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This hypothesis predicts that if ~, DNA were treated with trans- and cisPt(II) so that approximately half the DNA molecules would carry one interstrand cross-link, then the fraction without an interstrand cross-link would be active in the case of trans-Pt(II) treatment b u t inactive in the case of cis-Pt(II). This prediction was tested by separating k DNA molecules carrying one or more interstrand cross-links from non-cross-linked molecules on alkaline sucrose, then assaying the various fractions for transfectivity. The concept of t h e experiment is outlined in Fig. 4; the expected transfection activity of each class of DNA molecules is indicated in parentheses. It is hypothesized that molecules bearing inter- or intrastrand cross-links would be inactive. Denatured single-strands also would be inactive. The experiment is presented in Fig. 5. Following reaction with drug, the molecules having interstrand cross-links were separated in alkaline sucrose on the basis of their increased sedimentation velocity due to the double size of the molecules. Upon subsequent neutralization, the interstrand crosslinked molecules reform into double helices, whereas the non-cross-linked molecules or molecules containing only intrastrand cross-links, remain single-stranded. As expected, little or no activity was observed, because the

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Fig. 5. Inactivation and reactivation o f k DNA. Left: trans-Pt-(II) aged 3 months, was incubated w i t h k D N A (r = 7.5 × 10 -5) for 20 h at 37°C. A f t e r separation of crosslinked (k) and non-cross-linked (k/2) D N A on alkaline sucrose, each D N A containing f r a c t i o n was neutralized, dialysed and split to 3 aliquots: (. • .o. • .) u n t r e a t e d ; ( - - - - A - - - - ) reannealed in f o r m a m i d e and dialysed; (-----o----) treated with t h i o u r e a at 4°C and dialysed. The transfection activity o f each aliquot was t h e n d e t e r m i n e d . Right: cis-Pt(II) aged 3 m o n t h s was i n c u b a t e d w i t h ~, D N A (r = 1.6 × 10 -4 ) for 20 h at 37°C. A f t e r f r a e t i o n a t i o n as above, each D N A - c o n t a i n i n g fraction was neutralized, dialyzed and split into 3 aliquots. ( ' ' . o . . . ) u n t r e a t e d ; ( - - - - ~ - - - - ) reannealed in f o r m a m i d e ; ( - - - - n - - - - ) reannealed in f o r m a m i d e , dialyzed, t h e n treated w i t h thiourea. The transfection activity o f each a l i q u o t was d e t e r m i n e d . The inserts show the D N A profile of the alkaline sucrose gradients. F o r details see Materials and Methods.

double-stranded molecules had interstrand cross-links, and single-strands are inactive. (Actually, a small degree of activity was observed in the DNA bearing interstrand cross-links due to trans-Pt(II), (Fig. 5, solid circles), perhaps due to some reversal of these cross-links during the transfection process. This interpretation was supported by the finding that dialysis o f the trans-Pt(II)DNA complex against L-broth for 30 min at 37°C reduced the cross-linking observed in CsC1 gradient centrifugation by 50%. This cross-link reversal, to which trans-Pt(II) is more sensitive than cis-Pt(II), could be caused by sulfurcontaining substances in the medium.) Part of the activity in the region of the gradient between cross-linked and non-cross-linked fraction of DNA (Fig. 5, closed circles, fraction 6 and adjacent) could be due to the presence of the small a m o u n t of non-cross-linked circular DNA molecules [7]. This is supported by the finding that even non-treated X DNA subjected to alkaline sucrose gradient showed some transfecting activity shifted to higher sucrose concentration as compared to bulk DNA.

329 The double-strand and single-strand fractions were then annealed in formamide (FA). Double-strands would n o t be expected to be affected by this treatment, and no change in activity was observed. Single-strands however reassociate to form double-helices which, in the case of trans-Pt(II), recovered their activity (Fig. 5, triangles). Contrariwise, when the trans-Pt(II) treated DNA fractions were treated with thiourea (TU), which removes the Pt adducts, the double-stranded fraction recovered its activity, but the single-strand fraction did not (Fig. 5, open circles) in accord with the scheme in Fig. 4. In the case of the cis-Pt(II) treated DNA fractions, annealing in formamide failed to restore activity to the single,strand fraction, indicating the presence of inactivating lesions other than interstrand cross-links. Both DNA fractions recovered activity if subsequently treated with thiourea (Fig. 5, squares), so that the absence of activity noted above is attributable to Pt adducts. DISCUSSION This comparison of the biological and physical properties o f ~ DNA reacted with cis and trans-Pt(II) has led us to the following conclusions. Trans-Pt(II) forms interstrand cross-links in ), DNA in sufficient n u m b e r to account for the loss of transfectivity. The finding that the non-cross-linked fraction o f trans-Pt(II) treated DNA recovers its transfectivity a~ter renaturation while the cross-linked fraction remains inactive, indicates that the interstrand cross-link is the lesion which destroys transfectivity. C/s-Pt(II) also forms interstrand cross-links in ~ DNA, but these are n o t in sufficient number to account for the loss of transfectivity. There were approximately five lethal lesions for each interstrand cross-link. The results show that the non-cross-linked ~ DNA is not transfective after renaturation; lethal lesions are still present. These intrastrand lesions continue to form after the removal of free cis-Pt(II), indicating that a two step mechanism is involved in the formation of the lethal lesion, as would be the case for intrastrand cross-links. Our results indicate that in the first step c/s-Pt(II) is bound to the DNA in a non-lethal lesion; in the second step the bound c/s-Pt(II) forms the lethal lesion, perhaps by forming an intrastrand crosslink. Cis-Pt(II) but n o t trans-Pt(II) can prevent the unstacking o f the dinucleotide CpA under denaturing condition [8] indicating that cis-Pt(II) may be able to link adjacent nucleotides together, but that trans-Pt(II) is unable to to this. The intrastrand lethal lesion, like the interstrand cross-link, can be removed by thiourea treatment, indicating that the lesion is reversible. Two other differences between cis- and trans-Pt(II) are relevant to this discussion. The first is that the trans-Pt(II) DNA interstrand cross-link seems to be more easily reversed than the cis-Pt(II) DNA interstrand cross-link. We could not find conditions of thiourea treatment that would stop the transPt(II) cros~linking without significant reversal. Incubation of the trans-Pt(II)

330 t r e a t e d k D N A w i t h t h e L B r o t h o f t h e t r a n s f e c t i o n assay led to some reversal. This was lessened b u t n o t e l i m i n a t e d b y s u b s t i t u t i n g 0.6% plain D I F C O agar f o r LB agar. This is p r o b a b l y w h y t h e ), D N A t r e a t e d with transPt(II) shows slightly m o r e cross-links t h a n lethal lesions in Fig. 3 a n d shows s o m e a c t i v i t y in Fig. 5. In c o n t r a s t , t h e cis-Pt(II) i n t e r s t r a n d cross-link seems to be c o n s i d e r a b l y m o r e stable a n d r a t h e r difficult t o reverse w i t h t h i o u r e a . T h e s e c o n d d i f f e r e n c e is t h e rate o f a q u a t i o n o f trans- and cis-Pt(II) b y the f o l l o w i n g e q u a t i o n Pt(NH3)2 C12 + 2 H 2 0 -% Pt(NH3)2(H20)2 ÷ + 2C1-. ( T h e s t e r e o c h e m i s t r y is p r e s e r v e d in e a c h case.) With cis-Pt(II) the r e a c t i o n is relatively fast c o m p a r e d t o trans-Pt(II) [ 6 ] . T h e a q u a t e d f o r m s o f cis- and trans-Pt(II) d e s t r o y t r a n s f e c t i v i t y w i t h similar efficiency. H o w e v e r w h e n freshly dissolved Pt(II) is used, t h e trans i s o m e r is m u c h less p o t e n t t h a n t h e cis isomer, because o f t h e slower aquat i o n rate. This m a y in p a r t explain t h e l o w e r c y t o t o x i c i t y o f t h e trans i s o m e r in cell culture [ 9 ] . T h e s e results suggest t h a t the i n e f f i c i e n c y o f trans-Pt(II) in cell killing m a y result f r o m a c o m b i n a t i o n o f its slow rate o f a q u a t i o n , its m o r e r e s t r i c t e d t y p e s o f lethal lesions, a n d the easy reversibility o f these lesions. REFERENCES

1 K.W. Kohn, Drug induced macromolecular damage of nuclear DNA, in: H. Busch et al. (Eds.), Effects of Drugs on the Cell Nucleus, Academic Press, 1979, p. 207. 2 J.J. Roberts and J.M. Pascoe, Cross-linking of complementary strands of DNA in mammalian cells by antitumor platinum compounds, Nature, 235 (1972) 282. 3 J. Filipski, K.W. Kohn, R. Prather and W.M. Bonner, Thiourea reverses cross-links and restores biological activity in DNA treated with dichlorodiammineplatinum(II), Science, 204 (1979) 181. 4 P. Horacek and J. Drobnik, Interaction of cis-dichlorodiammineplatinum(II)with DNA, Biochim. Biophys. Acta, 254 (1971) 341. 5 J.W. Reishus and D.S. Martin, Jr., Cis-dichlorodiammineplatinum(II)-acid hydrolysis and isotopic exchange of the chloride ligands, J. Am. Chem. Soc., 83 (1961) 2457. 6 V.D. Panasyuk and N.F. Malashok, Kinetic studies of the acid hydrolysis of various platinum (II) complexes in aqueous solution, Russ. J. Inorg. Chem. (Engl.), 13 (1968) 1405. 7 L.W. Enquist and A. Skalka, Replication of bacteriophage }, DNA dependent on the function of host and viral genes. I. Interaction of red, gam and rec, J. Mol. Biol., 75 (1973) 185. 8 LA.G. Roos, A.J. Thomson and S. Mansy, Interaction of platinum compounds with dinucleotides, J. Am. Chem. Soc., 96 (1974) 6484.