DNA Interactions of a Novel Platinum Drug, cis-[PtCl(NH₃)₂(N7-Acyclovir)]⁺

Results and Discussion

Stability of cisPt-ACV and its DNA binding.

We found conditions for RP-HPLC analysis that allowed quantitative detection of ACV in the presence of cisPt-ACV (Fig.2A). The lower limit of this determination was 0.5% ACV in the presence of cisPt-ACV. cisPt-ACV was dissolved at concentrations of 1–5 × 10^-4m in water, 0.01 m and 1.0 mNaClO₄ or NaCl and incubated in the dark at 37° for 1 week. No peak in the RP-HPLC profile corresponding to free ACV was observed, indicating that no ACV spontaneously dissociated from cisPt-ACV in any of the solutions. Solutions of calf thymus DNA at a concentration of 0.32 mg/ml were incubated with cisPt-ACV at r_i values of 0.01–0.1 in 10 mmNaClO₄ at 37° (r_i is defined as the molar ratio of free platinum complex to DNA nucleotide phosphates at the onset of incubation with DNA). At various time intervals, an aliquot of the reaction mixture was withdrawn and precipitated by ethanol, and the supernatant was assayed by RP-HPLC for free cisPt-ACV (not bound to DNA). In this way, we also were able to check directly whether cisPt-ACV on its binding to double-helical DNA is decomposed so its ACV moiety is released into the solution. The HPLC peak corresponding to the free (unbound) cisPt-ACV complex decreased with time of its incubation with DNA and completely disappeared after 24 hr [half-time of this reaction (t_1/2) was∼1.7 hr] (shown for r_i = 0.1 in Fig. 2B). This result indicates that cisPt-ACV binds to DNA with a similar rate as cisplatin (Kim et al., 1990) and that all molecules of cisPt-ACV are bound to DNA within 24 hr. No peak appeared in the HPLC profile corresponding to the released ACV after 24 hr of the incubation. It was verified that a small quantity of free ACV (corresponding to 1% of the total amount of cisPt-ACV present in the reaction with DNA) added to this DNA sample after incubation lasting 24 hr yielded a well defined peak in the HPLC profile appearing at the same retention time as free ACV (Fig. 2A). The samples of DNA, to which all molecules of Pt-ACV present in the reaction mixture were bound after incubation in 10 mm NaClO₄ for 24 hr, were further incubated for additional 48 hr at 37° in 0.01, 0.05, and 1 m NaClO₄ or in 0.05 and 1m NaCl. These subsequent incubations resulted in only the occurrence of a very small peak coeluting with ACV, indicating that only a negligible amount of ACV was released; the amount of ACV released did not exceed ∼3%.

In addition, we investigated labilization of ammonia in free cisPt-ACV or in adducts formed by cisPt-ACV on double-helical DNA by means of an assay using Nessler reagent. No ammonia was found in the solutions of 5 × 10^-4m cisPt-ACV dissolved in water or 0.01, 0.05, and 1 mNaClO₄ or in 0.05 and 1.0 m NaCl. In addition, no ammonia was liberated in the presence of excess of double-helical DNA (0.032 mg/ml, r_i = 0.1 in 10 mm NaClO₄ at 37°) within 24 hr or even after prolonged incubations (additional 5 days at 37° in 0.01–1m NaClO₄ or NaCl); it was verified that the assay allowed determining amounts of ammonia as small as 2% under the conditions used. The same binding rate was noticed if DNA was incubated with transPt-ACV and free platinum assayed by differential pulse polarography (Kim et al., 1990).

Solutions of calf thymus DNA were incubated under identical conditions with cisPt-ACV or transPt-ACV using fresh stock solutions (added to DNA samples immediately after dissolving the platinum complex). No changes in DNA binding were observed compared with the experiments in which the same stock solutions stored ≥1week were used. In addition, pH of the reaction mixture containing DNA and cisPt-ACV or transPt-ACV was measured within 24 hr after mixing DNA with the platinum complex, and no changes in pH were noticed.

Sequence specificity of cisPt-ACV binding to DNA.

Transcription mapping studies were performed to determine the preferential binding of cisPt-ACV to specific sites or regions in DNA. Recent work has shown that the in vitro RNA synthesis by RNA polymerases on DNA templates containing several types of bidentate adducts of platinum complexes can be prematurely terminated at the level or in the proximity of adducts (Lemaire et al., 1991;Brabec and Leng, 1993; Brabec et al., 1994;Nováková et al., 1995). Importantly, monofunctional DNA adducts of several platinum(II) complexes are unable to terminate RNA synthesis (Lemaire et al., 1991; Brabec and Leng, 1993; Brabec et al., 1994).

Cutting of pSP73 DNA by NdeI and HpaI restriction endonucleases yielded a 221-bp fragment containing T7 RNA polymerase promotor in the upper strand close to the 3′-end of the fragment (Lemaire et al., 1991; Brabec and Leng, 1993). The experiments were carried out using this linear DNA fragment, modified by cisplatin, dienPt, or cisPt-ACV at r_b = 0.01, for RNA synthesis by T7 RNA polymerase (Fig.3A) (r_b is defined as the number of platinum atoms fixed per nucleotide residue). RNA synthesis on the template modified by cisplatin yielded fragments of defined sizes, which indicates that RNA synthesis on DNA modified by these platinum complexes was prematurely terminated. The major stop sites observed for cisplatin (Fig. 3A, cisDDP) were at d(GG) or d(AG) sites. In contrast, RNA synthesis on the template modified by monofunctional dienPt yielded no fragments that would correspond to premature termination of the RNA synthesis (Fig. 3A,dienPt). The results obtained with the fragments modified by cisplatin or dienPt were in agreement with previous reports (Lemaireet al., 1991; Brabec and Leng, 1993; Brabec et al., 1994). Importantly, RNA synthesis on the template globally modified by cisPt-ACV (Fig. 3A, cisPt-ACV) was clearly terminated although somewhat less efficiently than the synthesis on the template modified by cisplatin. The major stop sites observed for cisPt-ACV were distributed less regularly [i.e., not only at single dG sites in some d(GG) or 5′-d(AG)-3′ sequences, which are preferential DNA binding sites of cisplatin (Fichtinger-Schepmanet al., 1985; Eastman, 1987), but also at single dG sites in various other sequences] (Fig. 3B). Importantly, RNA synthesis on the unplatinated template in the presence of ACV at r_i = 0.01 and 0.05 yielded no termination sites (not shown).

The most striking feature of these transcription experiments is that the adducts in DNA globally modified by cisPt-ACV prematurely terminate RNA synthesis. This is in remarkable contrast with RNA synthesis on the template modified by monofunctional platinum complexes, such as dienPt or [Pt(NH₃)₃Cl]Cl or monofunctional adducts of cisplatin or transplatin (Lemaire et al., 1991; Brabec and Leng, 1993; Brabec et al., 1994). In addition, it is generally accepted that monofunctional DNA adducts of cisplatin are not relevant to the cytostatic effects of this metal complex and that monofunctional dienPt or [Pt(NH₃)₃Cl]Cl exhibits no anticancer activity. Thus, the results of the transcription mapping experiments (Fig. 3) are consistent with the hypothesis that cisPt-ACV forms some type of monofunctional DNA adduct that modifies DNA in a manner fundamentally different from that of monofunctional dienPt or [Pt(NH₃)₃Cl]Cl.

Transcription mapping has revealed (Fig. 3) that d(GG) sequence is among those at which cisPt-ACV is bound. The d(GG) site in double-helical DNA is a preferential binding site of antitumor cisplatin, at which this bifunctional drug forms the 1,2-intrastrand cross-link. To further corroborate the conclusion that the DNA binding mode of cisPt-ACV is different from that of cisplatin, chemical studies of a synthetic oligodeoxyribonucleotide duplex containing a single adduct of cisPt-ACV at the d(GG) site were conducted.

The duplex d(TGGT)/d(ACCA) (19 base pairs) was designed (see Experimental Procedures); it contains in the top pyrimidine-rich strand a central d(GG) sequence. The single-stranded d(TGGT) [the top, pyrimidine-rich strand of the duplex d(TGGT)/d(ACCA)] was modified so it contained a single adduct of cisPt-ACV (see Experimental Procedures). The platinated sites in this oligonucleotide then were identified using enzymatic digestion analysis. The platinated or nonmodified oligonucleotides d(TGGT) were treated with DNase I, P1 nucleases, and finally with alkaline phosphatase to yield corresponding deoxyribonucleosides. An RP-HPLC analysis of the products of this enzymatic digestion procedure is shown in Fig.4. Peaks C, G, and T are nonmodified deoxyribocytidine, deoxyriboguanosine, and thymidine, respectively, assigned by coinjection with samples of pure deoxyribonucleosides. Thecis-[Pt(NH₃)₂(N7-ACV)(dG)]²⁺cation, prepared by reaction ofcis-[Pt(NH₃)₂(N7-ACV)(Cl)]Cl with dG, was used to assign peak X by coinjection. Because this is the only new product observed in the enzymatic digestion analysis, it can be concluded that product X is a monofunctional adduct of cisPt-ACV at a guanine residue. This conclusion is further supported by the observation (Fig. 4) that the area under the peak G yielded by the platinated oligonucleotide was exactly half of that found for the nonmodified, control sample. Nevertheless, the HPLC enzymatic digestion analysis could not determine which of the two guanine residues in the TGGT sequence was a preferential binding site for the monofunctional coordination of cisPt-ACV to DNA.

Further insight into the nature of the single cisPt-ACV adduct in single-stranded oligonucleotide d(TGGT) was provided by DMS footprinting of platinum coordinated to DNA. This assay is based on the fact that DMS methylates the N7 position of guanine residues in DNA, producing alkali-labile sites (Lemaire et al., 1991; Brabec and Leng, 1993). However, if N7 is covalently bound to platinum, it cannot be methylated. The platinated and nonmodified d(TGGT) strands were 5′-end labeled with ³²P and subsequently reacted with DMS. The oligonucleotides then were treated with NaCN and hot piperidine and analyzed by denaturing polyacrylamide gel electrophoresis (Fig. 5, ss noPt and ss Pt), and the autoradiograms were evaluated by densitometry. For the nonmodified oligonucleotide, shortened fragments due to the cleavage of the strand at the two methylated guanine residues were observed in the gel (Fig. 5, ss noPt). For the oligonucleotide containing the single adduct of cisPt-ACV, shortened fragments due to the cleavage of the strand at the two methylated guanine residues also were observed, but their intensity was exactly half of those of the nonmodified oligonucleotide (Fig. 5, ss Pt). The same result was obtained if the platinated oligomer was incubated before reaction with DMS for 48 hr in 0.05 and 1.0m NaClO₄ or NaCl. The only reasonable explanation of this result is that cisPt-ACV forms stable monofunctional adducts without any preference at either guanine residue in the central sequence TGGT of the oligomer 20 nucleotides long.

The same studies based on enzymatic digestion analysis and DMS footprinting were performed with the oligomer d(TGGT) (containing the single adduct of cisPt-ACV and characterized as described in the preceding paragraphs), which was further hybridized with its complementary strand d(ACCA). The goal of these studies was to show whether the single monofunctional adducts formed by cisPt-ACV at either guanine residues of the single-stranded d(TGGT) oligonucleotide also are stable in double-helical DNA. The bottom complementary strand was added to either the nonmodified or platinated d(TGGT), and the mixture of the two oligonucleotides in the medium of 50 mmNaClO₄ was incubated to form the duplex (see Experimental Procedures). Both platinated and nonmodified oligonucleotide duplexes d(TGGT)/d(ACCA) were treated with the enzymes to yield corresponding deoxyribonucleosides in the same way as single-stranded d(TGGT). The peaks C, G, T, and A in the HPLC profiles (Fig. 4B) are nonmodified deoxyribocytidine, deoxyriboguanosine, thymidine, and deoxyriboadenosine, respectively, assigned by coinjection with samples of pure deoxyribonucleosides. The profile obtained for the platinated duplex only contained one new peak X, identified ascis-[Pt(NH₃)₂(N7-ACV)(dG)]²⁺by coinjection with authentic material. Thus, these results suggest that the monofunctional adduct of cisPt-ACV is not converted to a bifunctional lesion in double-helical DNA. This conclusion is further supported by the observation (Fig. 4B) that the area under the peak G yielded by the platinated duplex was exactly eight-ninths of that found for the nonmodified, control duplex (containing nine guanine residues).

In further experiments, the platinated and nonmodified d(TGGT) single strands 5′-end labeled with ³²P were hybridized with the complementary strand d(ACCA) (which was not radioactively labeled). These duplexes were reacted with DMS and further analyzed by gel electrophoresis (Fig. 5, ds noPt and ds Pt) in the same way as single-stranded oligonucleotides (vide supra). The intensities of the bands yielded by the two guanine residues contained in the top strand of the platinated duplex were half of those of the nonmodified duplex. It was verified (not shown) that the intensities of these bands were lowered in the same extent if the duplexes were reacted with a lower amount of DMS, which methylated only 10% molecules of the unplatinated duplex (in contrast to 20% in the experiment demonstrated in Fig. 5). The same result was obtained when the platinated duplex was incubated before reaction with DMS for 48 hr in 1.0 m NaClO₄ or 0.05 and 1.0m NaCl. This result is consistent with the view that the monofunctional adducts of cisPt-ACV formed at guanine residues are stable in double-helical DNA. Taken together, the results of the current study clearly demonstrate that monofunctional coordination of cisPt-ACV to single-stranded or double-helical DNA results in no elimination of ACV or NH₃ ligands, which could allow closure of these monofunctional adducts to bifunctional lesions.

Unwinding induced in DNA by cisPt-ACV binding.

Electrophoresis in native agarose gel was used to determine the unwinding induced in pSP73 plasmid by cisPt-ACV through monitoring of the degree of supercoiling (Keck and Lippard, 1992) (Fig.6). A compound that unwinds the DNA duplex reduces the number of supercoils. This decrease in supercoiling (on binding of unwinding agents) causes a decrease in the rate of migration through agarose gel, which makes it possible for the unwinding to be observed and quantified. Fig. 6 shows electrophoresis gels in which increasing amounts of cisPt-ACV have been bound to a mixture of relaxed and supercoiled pSP73 DNA. The unwinding angle is given by F = 18 s/r_b(c), where s is the superhelical density and r_b(c) is the value of r_b at which the supercoiled and relaxed forms comigrate (Keck and Lippard, 1992). Under the current experimental conditions, s was calculated to be −0.063 on the basis of the data of cisplatin for which the r_b(c) was determined in this study and F was taken 13° (Keck and Lippard, 1992). Unwinding angles for cisPt-ACV calculated in this way were 6°. The unwinding angles for cisplatin and dienPt taken from the literature (Bellonet al., 1991; Keck and Lippard, 1992) are 13° and 6°, respectively.

Although the molecular details of the interactions of cisPt-ACV with DNA are not fully understood, the information obtained from the unwinding experiments is nevertheless valuable. The values of unwinding angles are affected by the nature of the ligands in the coordination sphere of platinum and the stereochemistry at the platinum center. A previous systematic work by others (Keck and Lippard, 1992) revealed that the platinum(II) compounds fall into different classes according to their DNA binding modes. It has been shown that platinum(II) compounds with the smallest unwinding angles (6°) are those that can bind DNA only monofunctionally [dienPt or [Pt(NH₃)₃Cl]Cl]. The observation that cisPt-ACV, which unwound DNA by 6°, can be grouped with other monofunctional platinum(II) compounds is readily understood in terms of an adduct structure in which cisPt-ACV is preferentially coordinated to DNA in a monodentate manner. In this configuration, the ACV ligand in cisPt-ACV either has only little or no opportunity to interact with the double helix or interacts in such a way that it does not contribute to the unwinding induced by this complex.

DNA ICL by cisPt-ACV.

On electrophoresis in agarose gel under denaturing conditions, 3′-end labeled strands of linearized pSP73 plasmid containing no ICLs migrate as a 2464-base single strand, whereas the ICL strands migrate more slowly as a species of higher molecular mass (Lemaire et al., 1991; Brabec and Leng, 1993). The weak bands corresponding to more slowly migrating ICL fragments were only noticed if cisPt-ACV complex was used to modify DNA in linearized form at r_b of 0.002 (not shown). The intensity of the more slowly migrating band increased with the growing level of the modification. The radioactivity associated with the individual bands at each r_b was measured to obtain estimates of the fraction of ICL molecules. These estimates gave the frequency of ICLs formed by cisPt-ACV in linear DNA (amount of ICLs/molecule of cisPt-ACV complex bound to DNA) of only ∼0.2% almost independently of r_b. This result indicates that the ICL efficiency of cisPt-ACV in linear DNA is negligible [cisplatin forms under identical conditions ∼6% ICLs (Brabec and Leng, 1993; Vrána et al., 1995)].

Melting of DNA modified by cisPt-ACV.

Calf thymus DNA was modified by cisPt-ACV, cisplatin, or dienPt to various r_b (0–0.2) (at 37° for 48 hr so all molecules of the platinum complexes were coordinated to DNA). The effect of these modifications on DNA melting temperature,t_m, was measured (Fig.7). The results indicate that cisPt-ACV has a similar effect on DNA melting as cisplatin (i.e., both complexes reduce thermal stability of DNA). In contrast, clinically ineffective monofunctional dienPt or bidentate transplatin enhances thermal stability of DNA under identical conditions. The observation that thet_m values of DNA modified by cisPt-ACV were decreased is consistent with an occurrence of conformational alterations induced in DNA by cisPt-ACV that destabilize the duplex (Z̆aludová et al., 1996).

Fluorescence of complexes of terbium ion with DNA modified by cisPt-ACV.

Terbium ion (Tb³⁺) fluorescence is used to investigate local perturbations induced in conformation of double-helical DNA by various physical or chemical agents, including cisplatin pimprivate ENRf8 (Arquilla et al., 1983;Balcarová and Brabec, 1989). This assay is based on the observation that Tb³⁺ fluorescence is strongly enhanced when the ion is bound to the N7 atoms of guanine residues in distorted DNA regions pimprivate ENRf8 (Topal and Fresco, 1980). The modification of double-helical DNA by cisplatin has been shown to result in substantial increase of the fluorescence of this lanthanide cation (Arquilla et al., 1983; Balcarová and Brabec, 1989). This enhancement takes place due to its binding to unplatinated guanine residues in distorted regions around the platination site (Balcarová and Brabec, 1989). In contrast, the coordination of ineffective complexes, such as transplatin or monofunctional dienPt to double-helical DNA results in no distortions, which would increase the accessibility of base residues for their reaction with terbium leading to enhancement of its fluorescence (Arquilla et al., 1983;Balcarová and Brabec, 1989).

Calf thymus DNA was modified by cisplatin, dienPt, and cisPt-ACV at r_b values in the range of 0–0.1, the resulting samples were treated with TbCl₃, and the fluorescence was measured as described in Experimental Procedures (Fig.8). In accordance with previous results (Arquilla et al., 1983; Balcarová and Brabec, 1989), the modification by cisplatin resulted in a marked enhancement of terbium fluorescence, whereas the modification by dienPt had only a negligible effect. The modification by cisPt-ACV also resulted in a significant enhancement of the fluorescence, but the fluorescence intensity was considerably lower than that seen in the case of DNA modified by cisplatin. These results can be interpreted to mean that cisPt-ACV induces in double-helical DNA local conformational alterations that are, at least in some features, similar to those induced by cisplatin. The extent of the conformational changes induced by cisPt-ACV (as detected by terbium fluorescence) is, however, considerably lower than that induced by cisplatin. On the other hand, this observation is in contrast to the modification of DNA by monofunctional dienPt, which does not induce in DNA this type of distortions.

Immunochemical analysis of DNA modified by cisPt-ACV.

The antibodies raised against DNA modified by cisplatin have been found useful for probing the structures of DNA adducts formed by a number of different platinum complexes (e.g., Sundquist et al., 1987;Hollis et al., 1991; Vrána et al., 1992;Nováková et al., 1995; Vrána et al., 1995; Z̆aludová et al., 1997). We prepared antibodies that bind specifically to DNA modified by cisplatin and its analogues (Ab_cis). Ab_cis were found to exhibit strict requirements for two neighboring purine residues in one strand of DNA coordinated incis geometry with the platinum atom ofcis-[Pt(NH₃)₂]²⁺unit; moreover, this antibody is insensitive to the chemical nature of the inert cis platinum amine ligand. Importantly, Ab_cis do not recognize DNA adducts of transplatin and monofunctional adducts of several platinum(II) complexes (Sundquistet al., 1987; Vrána et al., 1992).

Several results indicating that cisPt-ACV does not form bifunctional adducts on DNA through loss of an amine ligand have been presented (vide supra). We intended to support this conclusion by using Ab_cis. Using competitive ELISA, the inhibition of the binding of Ab_cis to their immunogens (calf thymus DNA modified by cisplatin at r_b = 0.08 for 48 hr, respectively) by DNA modified by cisPt-ACV to various r_b values in the range of 0.005–0.1 was measured (Fig. 9). The results (Fig. 9) demonstrate that Ab_cisrecognized DNA modified by cisPt-ACV in a broad range of r_b values (0.01–0.1) (shown in Fig. 9 for r_b = 0.05) although somewhat less efficiently than DNA modified by cisplatin.

To explain this observation, we have taken into consideration the fact that cisPt-ACV preferentially forms on double-helical DNA stable monofunctional adducts at guanine residues. If this type of DNA binding occurs, then the platinum atom in cisPt-ACV still coordinates two guanine residues in cis position: the guanine residue contained in the ACV and the guanine residue in DNA to which cisPt-ACV is monodentately coordinated. Apparently, the structural motif recognized by Ab_cis could also occur in double-helical DNA due to the monofunctional binding of the cisPt-ACV. Consistent with this view is the observation that Ab_cis do not recognize DNA modified bycis-[PtCl(NH₃)₂(Am)]⁺, where Am is an amine ligand derived from pyrimidine derivatives (Holliset al., 1991). Thus, it seems reasonable to suggest that DNA adducts of cisPt-ACV mimic to some extent the structural motif produced by the major DNA adduct of cisplatin.

Conclusions.

A new platinum(II) antiviral and antitumor agent,cis-[Pt(NH₃)₂(Am)Cl]⁺, was synthesized, in which Am is the known antiviral agent ACV (Colucciaet al., 1995). The current results suggest that this new compound can coordinate to DNA and preferentially forms in double-helical DNA stable monofunctional adducts at guanine residues. The results also demonstrate that cisPt-ACV, when monodentately coordinated at d(GG) sites in single-stranded or double-helical DNA, is bound with equal preference to either guanine residue. This is in contrast to DNA binding mode of the similar monofunctional complexcis-[Pt(NH₃)₂(4-methylpyridine)Cl]⁺, which also forms monofunctional lesions at the d(GG) sites but with preference to their 5′ residues (Lempers et al., 1990).

The monofunctional adducts of cisPt-ACV inhibit DNA synthesis (Colucciaet al., 1995) and block specifically RNA polymerase at platinated guanine residues in vitro (Fig. 3). This observation is particularly interesting because the monofunctional lesions of several platinum(II) complexes are unable to terminate RNA synthesis by the RNA polymerase in vitro (Lemaire et al., 1991; Brabec and Leng, 1993; Brabec et al., 1994), whereas bifunctional adducts of all platinum compounds so far tested inhibit RNA synthesis at the level of these adducts (Lemaire et al., 1991; Anin et al., 1992; Brabec and Leng, 1993;Brabec et al., 1994). A question remains concerning the mechanism by which RNA synthesis is inhibited (i.e., in particular, what structural features of a monodentate fashion of DNA binding, previously expected not to result in inhibition of RNA synthesis, are required for blocking the RNA polymerase activity).

The results of immunochemical analysis (Fig. 9) demonstrate that some monofunctional lesions formed by cisPt-ACV at guanine residues in double-helical DNA inhibit the antibodies that specifically recognize and bind to intrastrand cross-links formed by cisplatin between neighboring purine residues. The latter observation is consistent with an idea that DNA adducts of cisPt-ACV have some structural motif similar to that produced by the major adduct of cisplatin (vide supra). We speculate that the ACV ligand itself interacts with DNA in a noncovalent manner, producing certain structural feature similar to the major adduct of cisplatin. These suggestions are corroborated by the observation that cisPt-ACV adducts destabilize DNA (Fig. 7) and affect conformation of DNA (Fig. 8) in ways that are qualitatively similar to the effects of bifunctional cisplatin and distinct from the effects of monofunctional, clinically ineffective dienPt. Thus, DNA adducts of cisPt-ACV could be recognized and further processed by some components of tumor cells (e.g., by some DNA or RNA polymerases) by a fundamentally different way than other monofunctional platinum adducts, including those of dienPt.

Unique properties of DNA adducts of cisPt-ACV have been revealed in the current work. However, the fact that there are some similarities with the effects of antitumor cisplatin (terbium fluorescence, melting behavior, recognition by specific antibodies, termination of RNA synthesis) might be of a fundamental importance in explaining some aspects of anticancer activity of this new platinum(II)-triamine complex. On the other hand, the antitumor activity of cisPt-ACV is not identical with that exhibited by cisplatin and its bifunctional analogues (Coluccia et al., 1995). Thus, the results of this work support the view that the platinum drugs, which bind to DNA in a manner different from that of cisplatin and related analogues, can differ in activity profile. Further studies are in progress in our laboratories to reveal other details of the modification of DNA by cisPt-ACV most likely relevant to its cytostatic properties.

The current work has yielded new information on DNA interactions of the platinum(II)-triamine compounds. However, it also shows that cisPt-ACV modifies DNA in a way that is in some aspects similar to that of anothercis-[Pt(NH₃)₂(Am)Cl]⁺complex (Hollis et al., 1991). It implies that the effect of the ACV ligand in the platinum-triamine complexes of this type on its DNA binding mode need not be strictly specific. The current work describes coordination of cisPt-ACV to DNA, consequences of this reaction for DNA conformation, and its processing by proteins (DNA or RNA polymerases and antibodies). However, this coordination of the drug to DNA must be involved, for obvious reasons, in the mechanism of antiviral activity of unplatinated nucleosides and their analogues such as unplatinated ACV. On the other hand, cisPt-ACV exhibits antiviral activity in vitro (Coluccia et al., 1995), so it cannot be excluded that at least some consequences of DNA interactions of cisPt-ACV revealed in the current work may play a role in the molecular mechanisms of antiviral activity ofcis-[Pt(NH₃)₂(Am)Cl]⁺complexes, as they do in their antitumor effect. Further studies are therefore warranted to reveal the importance of DNA binding modes of the novel platinum(II) compounds containing, as a part of the coordination sphere, certain selected nucleosides for their antiviral efficiency.