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Originally published In Press as doi:10.1074/jbc.M010205200 on December 4, 2000

J. Biol. Chem., Vol. 276, Issue 13, 9655-9661, March 30, 2001
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Thermal and Thermodynamic Properties of Duplex DNA Containing Site-specific Interstrand Cross-link of Antitumor Cisplatin or Its Clinically Ineffective Trans Isomer*

Ctirad HofrDagger and Viktor Brabec§

From the Institute of Biophysics, Academy of Sciences of the Czech Republic, CZ-61265 Brno, Czech Republic

Received for publication, November 9, 2000


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The effect of the single, site-specific interstrand cross-link formed by cisplatin or transplatin on the thermal stability and energetics of a 20-base pair DNA duplex is reported. The cross-linked or unplatinated 20-base pair duplexes were investigated with the aid of differential scanning calorimetry, temperature-dependent UV absorption, and circular dichroism. The cross-link of both platinum isomers increases the thermal stability of the modified duplexes by changing the molecularity of denaturation. The structural perturbation resulting from the interstrand cross-link of cisplatin increases entropy of the duplex and in this way entropically stabilizes the duplex. This entropic cross-link-induced stabilization of the duplex is partially but not completely compensated by the enthalpic destabilization of the duplex. The net result of these enthalpic and entropic effects is that the structural perturbation resulting from the formation of the interstrand cross-link by cisplatin induces a decrease in duplex thermodynamic stability, with this destabilization being enthalpic in origin. By contrast, the interstrand cross-link of transplatin is enthalpically almost neutral with the cross-link-induced destabilization entirely entropic in origin. These differences are consistent with distinct conformational distortions induced by the interstrand cross-links of the two isomers. Importantly, for the duplex cross-linked by cisplatin relative to that cross-linked by transplatin, the compensating enthalpic and entropic effects almost completely offset the difference in cross-link-induced energetic destabilization. It has been proposed that the results of the present work further support the view that the impact of the interstrand cross-links of cisplatin and transplatin on DNA is different for each and might also be associated with the distinctly different antitumor effects of these platinum compounds.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The thermal and thermodynamic stability of DNA play an important role in many biological processes. In addition, agents of biological significance that modify DNA may also affect its thermal and thermodynamic stability, which may be associated with the mechanism underlying biological activity of such agents. Thus, the studies of thermal and thermodynamic stability of DNA modified by various agents are of great interest.

It is well established that platinum coordination complexes exhibit antitumor effects (1-4). The success of platinum complexes in killing tumor cells results from their ability to form on DNA various types of covalent adducts that are capable of terminating DNA synthesis (5, 6) and the cellular processes triggered by the presence of those adducts on DNA (7). The first platinum complex introduced in the clinic is cis-diamminedichloroplatinum(II) (cisplatin)1 (1). Although the antitumor effects of cisplatin were discovered more than 30 years ago, the mechanism of its antitumor activity has not yet been fully understood. It has been shown (8, 9) that this bifunctional platinum complex mainly forms intrastrand cross-links on DNA between neighboring purine residues (~90%). Other minor adducts are intrastrand cross-links between two purine nucleotides separated by one or more nucleotides; few adducts remain monofunctional. Importantly, cisplatin also forms interstrand cross-links (~6% in cell-free media in linearized plasmid DNA (10, 11)). Transplatin (the trans isomer of cisplatin) is clinically ineffective, so that both isomers have been used frequently in studies of the structure-pharmacological activity relationship of platinum complexes. Transplatin-DNA adducts are also intrastrand cross-links but between nonadjacent nucleotides (12). Transplatin also forms in DNA interstrand cross-links (~12%) (10), and a relatively large portion of the adducts remains monofunctional even after long periods of DNA modification (13).

It has been postulated (6, 14) that the antitumor properties of cisplatin are mediated by damaged DNA-binding proteins (for instance those containing a HMG (high mobility group) domain). It has been also shown (15) that the recognition of adducts formed on DNA by platinum complexes is dependent on the extent of thermal or thermodynamic destabilization imposed on the duplex by the adduct. The increase of the thermodynamic destabilization results in the reduced recognition and binding of HMG domain proteins to platinated DNA. In addition, the thermal stability of DNA modified by various platinum compounds, which differ in their antitumor effects, has been also studied. These studies have revealed (16-20) that the important factors influencing the thermal stability of platinated DNA are also interstrand cross-links, which contribute to the global stabilization of DNA.

Despite great effort devoted to the understanding of how cisplatin modifies DNA and how these modifications are associated with the antitumor effects of cisplatin, the relative efficacy of its intrastrand and interstrand cross-links is unknown. Whereas the thermal and thermodynamic properties of DNA duplexes containing intrastrand cross-link of cisplatin or its analogues have already been studied in detail (15, 21, 22), no attention has been paid to thermal stability and energetics of DNA interstrand cross-links of platinum drugs. It is so despite the fact that DNA interstrand cross-links of platinum complexes could play a very significant role in the biological activity of these compounds because these covalent cross-links preventing separation of the two strands of DNA could block DNA replication markedly more efficiently than intrastrand adducts (23). In addition, it has been also suggested that nucleotide excision repair may reduce antitumor effects of platinum complexes. Thus, the fact that nucleotide excision repair of interstrand cross-links in general is much more difficult than that of intrastrand cross-links (24-26) may also serve to emphasize the importance of interstrand cross-links of platinum complexes for their antitumor effects even when these cross-links are only minor lesions. Here we examine the effect of the single, site-specific interstrand cross-link formed by cisplatin or transplatin on the thermal stability and energetics of a 20-base pair (bp) DNA duplex. The differential scanning calorimetric (DSC), temperature-dependent UV absorption, and circular dichroism (CD) properties of the platinated or unplatinated 20-bp duplex were investigated.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Chemicals-- Cisplatin and transplatin were from Sigma. The synthetic oligodeoxyribonucleotides d(TGCT) and d(AGCA) (Fig. 1) were purchased from IDT, Inc. (Coralville, IA) and purified as described previously (10, 27, 28). Molar extinction coefficients for the single-stranded oligonucleotides were determined by phosphate analysis (21). The following extinction coefficients at 260 nm and 25 °C were obtained: 148,000 for unmodified d(TGCT) and 189,000 for unmodified d(AGCA). Isothermal mixing experiments (21) using unmodified d(TGCT) and d(AGCA) strands revealed 1:1 stoichiometries for both complexes, a ratio consistent with duplex formation. Dimethyl sulfate was from Sigma.

Platinations of Oligonucleotides-- The single-stranded oligonucleotide d(TGCT) (the top strand in Fig. 1) at a concentration of 125 µM was reacted with a monoaquamonochloro derivative of cisplatin or transplatin generated by allowing these complexes to react with 0.9 molar equivalent of AgNO3 at an input platinum to strand molar ratio of 3:1 or 3.9: 1, respectively, in 10 mM NaClO4 (pH 5.2) at 37 °C. The mixture with cisplatin was incubated for 13 min and the mixture with transplatin for 15 min. Then, the NaCl concentration was adjusted to 0.1 M, and the platinated oligonucleotides were again purified by fast protein liquid chromatography (FPLC). Using platinum flameless atomic absorption spectrophotometry (FAAS) and measurements of the optical density, it was verified that the modified oligonucleotide contained one platinum atom. It was also verified using dimethyl sulfate footprinting of platinum on DNA (10) that in the platinated top strands the N7 position of the central G was not accessible for reaction with dimethyl sulfate, which implies that this G residue was platinated. The platinated strands were allowed to anneal with unplatinated complementary strand d(AGCA) in 0.4 M NaCl (pH 7.4) at 25 °C for 24 h, precipitated by ethanol, dissolved in 0.1 M NaClO4 and incubated for 48 h in the dark at 37 °C. The resulting products were still purified by FPLC in an alkaline gradient. Using this denaturing gradient, non-interstrand cross-linked strands were eluted as 20-nucleotide single strands, whereas the interstrand cross-linked strands were eluted later in a single peak as a higher molecular mass species. This single peak was only collected so that the samples of the interstrand cross-linked duplexes contained no single-stranded molecules. Alternatively, the duplexes containing the interstrand cross-links were separated on a 12% polyacrylamide, 8 M urea denaturing gel, and the single bands corresponding to interstrand cross-linked duplexes were cut off from the gel, eluted, precipitated by ethanol, and dissolved in a solution consisting of 10 mM sodium cacodylate (pH 7.2), 100 mM NaCl, 10 mM MgCl2, and 0.1 mM EDTA. Both procedures of the purification of interstrand cross-linked duplexes provided products of which subsequent analysis (see below) gave identical results. The yields of these interstrand cross-linking reactions were ~15 and 30% for cisplatin and transplatin, respectively. The duplexes were still further analyzed for platinum content by FAAS. Additional quantitation of cross-linked duplex by UV absorption spectrophotometry was used to ascertain that 1:1 adducts (one Pt/duplex) had formed. The sites involved in interstrand cross-links were deduced in the same way as described earlier (10, 27-30), i.e. mainly from Maxam-Gilbert footprinting experiments. It was verified in this way that the interstrand cross-link of cisplatin was formed between guanine residues in neighboring base pairs in the 5'-GC·5'-GC central sequence, whereas the interstrand cross-link of transplatin was formed between central guanine residue in the top strand of the d(TGCT)·d(AGCA) duplex and its complementary cytosine residue. FPLC purification and FAAS measurements were carried out on an Amersham Biotech FPLC system with MonoQ HR 5/5 column and a Unicam 939 AA spectrometer equipped with a graphite furnace, respectively. The concentration of the purified and characterized duplexes containing the interstrand cross-link of cisplatin and transplatin was further estimated by determining the platinum concentration by means of FAAS. Other details can be found in previously published papers (10, 28, 29).

Differential Scanning Calorimetry-- Excess heat capacity (Delta Cp) versus temperature profiles for the thermally induced transitions of d(TGCT)·d(AGCA) duplex, unmodified or containing a unique interstrand cross-link of cisplatin or transplatin, were measured using a VP-DSC calorimeter (Microcal, Northampton, MA). In these experiments, the heating rate was 60 °C/h and a maximum temperature was 95 °C. Enthalpies (Delta Hcal) and entropies (Delta Scal) of duplex formation were calculated from the areas under the experimental Delta Cp versus T and the derived Delta Cp/T versus T curves, respectively, using ORIGIN version 5.0 software (Microcal). The free energy of duplex formation at 25 °C (Delta G25) was calculated using the standard thermodynamic relationship given in Equation 1 and the corresponding values of Delta H and Delta S.


&Dgr;G<SUB>25</SUB>=&Dgr;H−(298.15)&Dgr;S (Eq. 1)
The oligonucleotide duplexes at the concentration of 10 µM were dialyzed against the buffer containing 10 mM sodium cacodylate (pH 7.2), 100 mM NaCl, 10 mM MgCl2, and 0.1 mM EDTA. The samples were vacuum-degassed before the measurement. It was also verified in the same way as described in the previous paper (21) that the melting transition of both the platinated and unmodified duplexes were fully reversible.

UV Absorption Spectrophotometry-- UV absorbance measurements were conducted on a Beckman DU-7400 spectrophotometer equipped with a thermoelectrically controlled cell holder and quartz cells with a path length of 1 cm. Absorbance versus temperature profiles were measured at 260 nm. The temperature was raised using linear heating rate of 1.0 °C/min. For each optically detected transition, the melting temperature (Tm) was determined as described previously (16). The DNA solutions ranged from 0.2 to 10 µM in duplex and contained 10 mM sodium cacodylate (pH 7.2), 100 mM NaCl, 10 mM MgCl2, and 0.1 mM EDTA.

Circular Dichroism Spectrophotometry-- CD spectra were recorded using a Jasco J-720 spectropolarimeter equipped with a thermoelectrically controlled cell holder. The cell path length was 1 cm. Isothermal CD spectra were recorded from 220 to 320 nm in 1-nm increments with an averaging time of 5 s. The DNA concentration was 6 µM in duplex, and buffer conditions were 10 mM sodium cacoldylate (pH 7.2), 100 mM NaCl, 10 mM MgCl2, and 0.1 mM EDTA.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Differential scanning calorimetry measurements were conducted to characterize the thermally induced denaturation of the 20-bp duplex with the specific goal of elucidating the thermal and thermodynamic consequences of modifying and constraining DNA via a single, site-specific interstrand cross-link of antitumor cisplatin or its clinically ineffective trans isomer (Fig. 1). The cross-links were formed in the center of these duplexes between the nucleotide residues preferentially involved in these adducts when high molecular mass DNA is globally modified by cisplatin or transplatin, i.e. between guanine residues in neighboring base pairs in the sequence 5'-GC·5'-GC in the case of cisplatin cross-link (30) or between guanine and complementary cytosine of the same duplex in the case of the cross-link formed by transplatin (10). The results of these studies are shown in Fig. 2 with the associated data listed in Table I. Denaturation (heating) and renaturation (cooling) curves for the unmodified and the platinated duplex were superimposable, which is consistent with the reversibility of this melting equilibrium. Thus, meaningful thermodynamic data from our calorimetric and spectrophotometric measurements described below could be obtained. In addition, the pre- and post-base lines coincide for both unmodified and platinated duplexes, which suggests no differential heat capacity change resulting from the presence of the cross-link. Comparing the calorimetrically determined melting temperatures (Tm) for the cross-linked duplexes and for the unconstrained (unplatinated) duplex reveals that formation of either cross-link results in a substantial increase in the thermal stability of the duplex (Delta Tm (defined as the difference between Tm values of the cross-linked and unplatined duplex) was +8.7 or +5.0 °C for the cross-link of cisplatin or transplatin, respectively).


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  		Fig. 1.   Structures of cisplatin and transplatin along with the base sequence of the synthetic oligodeoxyribonucleotide duplex used in the present study with their abbreviations. The top and bottom strands in the pair of oligonucleotides are designated top and bottom, respectively, in the text. The central part of the duplex containing the base pairs interstrand cross-linked by cisplatin or transplatin is framed and also shown separately with the manifestation of the cross-linking. The bold letters in this central part indicate the location of the interstrand cross-link after modification of the oligonucleotide by cisplatin or transplatin as described under "Experimental Procedures."


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  		Fig. 2.   DSC thermograms for the d(TGCT)·d(AGCA) unplatinated duplex (solid line) containing a single, site-specific interstrand cross-link of cisplatin (dot-dash line) or transplatin (dashed line). The duplex concentration was 10.0 µM, and the buffer conditions were 10 mM sodium cacodylate (pH 7.2), 100 mM NaCl, 10 mM MgCl2, and 0.1 mM EDTA.

                              
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Table I
Calorimetrically derived thermodynamic parameters for the formation of the 20-mer duplex d(TGCT)·d(AGCA), unplatinated or containing a single, site-specific interstrand cross-link of cisplatin or transplatin
The Tm, Delta Hcal and Delta Scal values are averages derived from three independent experiments.

The unconstrained (unplatinated) duplex denatures in a bimolecular reaction to form two single strands. As a consequence, melting of the unplatinated duplex was dependent on the overall oligonucleotide concentration. For instance, increasing the duplex concentration from 0.2 to 10.0 µM increased the Tm of the unplatinated duplex from 61.5 to 68.8 °C. In contrast, the duplexes containing the interstrand cross-link of cisplatin or transplatin melted in a concentration-independent manner to a single-stranded state, which is consistent with the expectation that the molecularity had been reduced from bimolecular to monomolecular. Thus, the observed Delta Tm differences could also result from the change in molecularity. To support this assumption, we performed a "correction" for the concentration dependence of the Tm in the bimolecular unplatinated duplex following the general procedure outlined by Marky and Breslauer (31). Using this approach and our calorimetrically determined enthalpy (Table I), we estimated a reduced concentration-independent Tm of our unplatinated duplex as 88.1 °C. This "reduced" Tm value of the unplatinated duplex is significantly different from the Tm values of the cross-linked duplexes. Hence, the overall impact of the single interstrand cross-link of cisplatin or transplatin should not be associated only with the change in molecularity of the duplex system but another mechanism affecting the thermal stability of the duplex also has to be involved (32).

The transition entropy for a bimolecular complex depends on strand concentration. To eliminate the effect of different molecularities of the unplatinated and interstrand cross-linked oligomer systems, we also performed a correction for this concentration dependence again using the general procedure outlined by Marky and Breslauer (31) to calculate a reduced entropy (Delta S*) (Table I) from the observed Delta Scal values. A comparison of the calorimetrically obtained enthalpy (Delta Hobs) and reduced entropy (Delta S*) values for unplatinated duplex with those measured for the duplex containing the interstrand cross-link of cisplatin revealed a significant decrease of the change of enthalpy of duplex formation by 18 kcal/mol and an increase in the reduced duplex entropy (Delta Delta S* = 37 cal/K · mol so that TDelta Delta S* = 11 kcal/mol at 25 °C). In other words, the structural perturbation resulting from the interstrand cross-link of cisplatin increases entropy of the duplex and in this way entropically stabilizes the duplex. Thus, the 18 kcal/mol enthalpic destabilization of the duplex resulting from the cross-link of cisplatin is partially, but not completely, compensated by the entropic cross-link induced stabilization of the duplex of 11 kcal/mol at 25 °C. The net result of these enthalpic and entropic effects is that the structural perturbation resulting from formation of the interstrand cross-link by cisplatin induces a decrease in duplex thermodynamic stability (Delta Delta G25*) of +7.0 kcal/mol with this destabilization being enthalpic in origin.

By contrast, the cross-link formed by transplatin does not considerably alter the enthalpic stability of the duplex, whereas entropically destabilizing the host duplex by 15 cal/K · mol (Delta Delta S* = -15 cal/K · mol so that TDelta Delta S* = -4.5 kcal/mol at 25 °C). In other words, the interstrand cross-link of transplatin is enthalpically neutral with the cross-link-induced destabilization entirely entropic in origin. These results illustrate that the isomerization of diamminedichloroplatinum(II) can modulate the magnitude of the destabilization induced by the interstrand cross-link as well as the relative enthalpic and entropic contributions to this destabilization. The net result of the enthalpic and entropic effects noted above is that formation of the interstrand cross-link by transplatin at 25 °C reduces the thermodynamic stability (Delta G25*) of the duplex by 6.5 kcal/mol. Thus, formation of the interstrand cross-links of cisplatin and transplatin has an almost identical impact on the reduction in thermodynamic stability of the duplex (Delta G25*), although the origins of these destabilizations (Delta H, Delta S*) are different.

From a comparison of the model-dependent van't Hoff and the model-independent calorimetric transition enthalpies, it is possible in principle to conclude whether a transition occurs in a two-state (all-or-none) manner with no significant thermodynamic contribution from intermediate states (33, 34). Table II lists the directly measured, model-independent calorimetric transition enthalpies (Delta Hcal) and the indirectly derived, model-dependent van't Hoff transition enthalpies (Delta HvH). The Delta HvH values were obtained by analyzing the shapes of each calorimetric curve using the approach described earlier (31). A comparison of the Delta HvH and Delta Hcal data listed in Table II reveals that for the unplatinated duplex the van't Hoff value is identical within the experimental uncertainty to the corresponding model-independent calorimetric value. This result is consistent with an all-or-none, two-state melting behavior of the unplatinated duplex. On the other hand, van't Hoff values were considerably smaller than the corresponding model-independent calorimetric data for the interstrand cross-linked duplexes (Table II). This disparity demonstrates that both interstrand cross-links alter the ability of the duplex to propagate those interactions required for cooperative melting and that the efficiency of the cross-links of cisplatin and transplatin to alter this ability is different.

                              
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Table II
Calorimetric (model-independent) and van't Hoff enthalpies for formation of the 20-mer duplexes d(TGGT)·d(ACCA), unplatinated or containing a single, site specific interstrand cross-link of cisplatin or transplatin

The interpretation of our data described below is also based on the assumption that all thermodynamic parameters for formation of the unmodified and platinated duplexes are ascribed to differences in the initial duplex states. This implies that the final single-stranded states should be thermodynamically equivalent at the elevated temperatures at which they are formed. The CD signals of the high-temperature denatured state (recorded at 95 °C) of the unplatined duplex and those cross-linked by either cisplatin or transplatin agree within the noise of the measurement, whereas significant differences exist in the intensity of the CD signals of the native states (Fig. 3). These results are consistent with the local perturbations in the native duplex state in agreement with the structural studies performed with the duplexes containing an interstrand cross-link of cisplatin or transplatin (23, 28, 35-37). The similarity of the CD signals of the denatured states of unplatinated and cross-linked duplexes may reflect a similar degree of base unstacking in these duplexes at the elevated temperatures, although such a conclusion may well exceed the information content of the CD measurement.


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  		Fig. 3.   CD spectra for the d(TGCT)·d(AGCA) duplex unplatinated or containing a single, site-specific interstrand cross-link of cisplatin (A) or transplatin (B) recorded at 25 or 95 °C. The duplex concentration was 6 µM, and the buffer conditions were 10 mM sodium cacodylate (pH 7.2), 100 mM NaCl, 10 mM MgCl2, and 0.1 mM EDTA. Curves: 1 and 2, unplatinated duplex at 25 and 95 °C, respectively; 3 and 4, the cross-linked duplex at 25 and 95 °C, respectively.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The CD spectrum of the duplex containing single, site-specific interstrand cross-link of cisplatin recorded at 25 °C (Fig. 3A) confirms that this lesion considerably alters the global geometry of the parent duplex. It has been shown (23, 35, 36) that cisplatin interstrand cross-link, which is preferentially formed between opposite guanines in the 5'-GC·5'-GC sequence (30), induces several irregularities in the cross-linked base pairs and their immediate adjacent pairs in a base sequence-independent manner (38). The cross-linked deoxyriboguanosine residues are not paired with hydrogen bonds to the complementary deoxyribocytidines, which are located outside the duplex and not stacked with other aromatic rings. All other base residues are paired, but distortion extends over at least four base pairs at the site of the cross-link (38). In addition, the cis-diammineplatinum(II) bridge resides in the minor groove (23, 35, 36) and the double helix is locally reversed to a left-handed, Z-DNA-like form. The change of the helix sense and the extrusion of deoxyribocytidine residues (complementary to the platinated deoxyriboguanosine residues) from the duplex results in the helix unwinding by ~80° relative to B-DNA (35), which is very likely responsible for a marked reduction of the amplitude of the negative CD band at around 240 nm observed at 25 °C (Fig. 3A). The interstrand cross-link of cisplatin also induces the bending of ~40° of the helix axis at the cross-linked site toward the minor groove (23, 35, 36).

The CD spectrum at 25 °C of the duplex d(TGCT)·d(AGCA) is affected by the site-specific interstrand cross-link of transplatin (formed preferentially between complementary guanine and cytosine (10)) much less than with cisplatin (Fig. 3B). Consistent with this CD behavior is the observation that the conformational alterations induced by the interstrand cross-link of transplatin (28, 37) are much less severe than those induced by the cross-link of cisplatin. The platinated deoxyriboguanosine residue in the cross-link of transplatin adopts a syn conformation. In addition, the duplex is slightly distorted on both sides of the cross-link, but all bases are still paired and hydrogen-bonded. The cross-link of transplatin unwinds the double helix by ~12° and induces a slight, flexible bending of ~20° of its axis toward minor groove (28).

The distinctly different structural features of the interstrand cross-links of cisplatin and transplatin are also reflected by their different thermal and thermodynamic properties (Table I). Importantly, the increase of the thermal stability of the d(TGCT)·d(AGCA) duplex resulting from the interstrand cross-link of cisplatin or transplatin is because of the change in the molecularity of the oligomer system. If the change observed in Tm is due entirely to the molecularity of the system, then one might expect to observe changes only in entropy. This is observed in the case of the formation of the interstrand cross-link of transplatin. On the other hand, significant changes in enthalpy are observed in addition to entropy changes as a consequence of the formation of the interstrand cross-link of cisplatin. Thus, whereas the structural perturbation resulting from the formation of the latter cross-link induces a decrease in duplex thermodynamic stability (Delta Delta G25*) of +7.0 kcal/mol, with this destabilization being enthalpic in origin, the structural distortion associated with the formation of transplatin cross-link induces a very similar decrease in duplex thermodynamic stability (Delta Delta G25*) of +6.5 kcal/mol, but this destabilization is entropic in origin. In other words, for the duplex containing the interstrand cross-link of cisplatin relative to the duplex containing the interstrand cross-link of transplatin, the compensating enthalpic and entropic effects almost completely offset the difference in cross-link-induced energetic destabilization. The observation that the transplatin interstrand cross-link is nearly enthalpically neutral in contrast to the same lesion formed by cisplatin is consistent with a relatively small conformational distortion induced by the cross-link of transplatin in comparison with a markedly more severe distortion induced by the cross-link of cisplatin.

We also attempted to rationalize the enthalpic destabilizing effect of the interstrand cross-link of cisplatin (Delta Delta Hcal = 18 kcal/mol (Table I)) in terms of the cross-link-induced structural perturbations in the host duplex. Conformational changes in DNA induced by the single, site-specific interstrand cross-link of cisplatin have been investigated already (see above) by various techniques. The view that the conformational distortion induced by the interstrand cross-link of cisplatin is not only localized to the platinated base pairs is supported by the results of DNase I footprinting of cisplatin-modified DNA (39). The approach, based on using a chemical nuclease such as 1,10-phenantroline-copper (38), even permitted us to determine the approximate number of base pairs, the secondary structure of which had been perturbed by the cross-link. Importantly, this chemical nuclease does not represent measures of base pair disruption, as they can proceed even if the base pairing is distorted in a nondenaturational manner (40). The analysis of the duplex containing the site-specific interstrand cross-link of cisplatin has revealed that the interstrand cross-link of cisplatin results in distortions between ~4 to 5 base pairs at the d(GC)·d(GC) site involved in the cross-link (38). Such a conformational alteration should be enthalpically unfavorable, which is consistent with observations of the present work. A rough estimation of an upper limit for this cross-link-induced perturbation, based on the enthalpic cost resulting from disruption of 4-5 relevant nearest-neighbor stacking interactions, yields Delta Delta H value of 25-32.5 kcal/mol (41). Our experimental calorimetric data (see Table I) reflect a cross-link-induced Delta Delta Hcal value of 18 kcal/mol.

An attempt to account for the differences between the predicted upper limit Delta Delta H value of 25-32.5 kcal/mol and the measured calorimetric value of 18 kcal/mol may invoke at least some energetic consequences of the formation of the interstrand cross-link of cisplatin. An important feature of the structure of this adduct is that cytosine residues complementary to the platinated guanines are no longer hydrogen-bonded and are extrahelical (see above). On the other hand, the unpaired platinated guanine residues are stacked with the adjacent base pairs, which contributes to the stabilization of the duplex. Similarly, it has been proposed that the hydration of the interstrand cross-link (42) and bending induced by the adducts of cisplatin (21) thermodynamically stabilize the duplex. Although the stabilization resulting from hydration cannot be quantified because of the limited thermodynamic database on DNA hydration, at least a very crude estimate can be obtained in the case of helical bending. It was shown that the bending due to the formation of the 1,2-d(GpG) intrastrand cross-link contributed roughly 6.4 kcal/mol toward stabilization of the global duplex structure (21) so that it seems reasonable to assume that the contribution of the bending induced by the interstrand cross-link of cisplatin is at least that derived for its intrastrand cross-link. Another important conformational parameter of the distortion induced by the formation of the interstrand cross-links of cisplatin is the unwinding of the double helix (lowering of the number of base pairs per helical turn). The energetics of the destabilizing effect of DNA unwinding can be estimated crudely using the same approach as used to calculate the free energy required to twist a 12-bp-long DNA fragment containing a single 1,2-d(GpG) intrastrand adduct of cisplatin about its helix axis (43). The free energy of unwinding of only 0.29 kcal/mol was calculated assuming the unwinding angle is 13° (43). The same calculations were performed, assuming unwinding angles of 80° for the interstrand cross-link of cisplatin (see above) and local twisting within the 20-bp-long fragment (the 20-bp-long fragment was taken for these calculations because the energetics of the duplex d(TGCT)·d(AGCA) of this length containing the single, site-specific interstrand cross-link of cisplatin was characterized in the present work (Fig. 2 and Table I)). These approximate calculations give a rough estimate of the free energy of unwinding of 6.7 kcal/mol. If this rough estimate is justified, then it seems reasonable to suggest that the destabilization of the duplex because of its unwinding as a consequence of the formation of the interstrand cross-link of cisplatin compensates at least partially for the stabilizing effect of bending. Furthermore, a local reversal of the double helix to a left-handed form at the site of the cross-link undoubtedly also contributes to the observed energetic impact of the interstrand cross-link of cisplatin. In general, a prediction of the energetic consequences of conformational changes induced by the interstrand cross-link of cisplatin is difficult because of the limited current knowledge on the thermodynamic consequences of distortions and transitions on DNA duplexes. Despite these possible microscopic interpretations of our macroscopic data, the calorimetric results reported here reveal that the formation of the interstrand cross-link of cisplatin induces on one hand a substantial thermal stabilization associated mainly with the change in the molecularity of the system and on the other hand thermodynamic destabilization of the host duplex that is enthalpic in origin. The interstrand cross-link of transplatin also induces thermal stabilization associated mainly with the change in the molecularity of the system, but it is enthalpically neutral so that the thermodynamic destabilization of the duplex induced by this transplatin adduct is entirely entropic in origin.

From the ratio of the model-dependent van't Hoff and the model-independent calorimetric transition enthalpies (Table II), one can define the fraction of a duplex that undergoes transition as a single thermodynamic unity (31, 33, 34). Thus, for the duplex containing an interstrand cross-link of cisplatin this ratio is 0.74. This value indicates that the largest size of the unit in the host duplex, which melts in the all-or-none manner, should involve 74% of the duplex. We speculate that this value reflects the existence of a severe local distortion of the duplex at the central cross-link of cisplatin (23, 35, 36, 38) and that the base pairs in this distorted segment melt cooperatively and more easily than the rest of the duplex. Hence, one intermediate state of the cisplatin-cross-linked duplex during its thermal melting could involve a short, denatured central structure arising from the segment ~4-5 base pairs long consisting of the two base pairs involved in the cross-link and approximately two or three base pairs flanking the cross-linked base pairs. This size of the denatured central structure, which represents 20-25% of the duplex examined in the present work, is deduced from the observation that severe distortion induced by cisplatin cross-link extends over approximately four or five base pairs at the site of the adduct (38). The remaining ~15 or 16 base pairs of the duplex d(TGCT)·d(AGCA) represent 75 or 80% of its size; this value is very close to the 74% found for the largest size of the unit melting cooperatively in a two-state process in the host duplex on the basis of the ratio of the model-dependent van't Hoff and the model-independent calorimetric transition enthalpies (Table II). The two-state melting of the duplex containing two marginal segments of a similar length and GC content, which are separated by the central denatured region (4-5 base pairs long) around the cross-link, deserves additional discussion. The two-state melting was observed even in the case of an immobile DNA junction composed of the four isolated octameric duplex arms in which not all arms had the same melting temperature (44). For the duplex examined in the present work consisting of the two marginal segments separated by the central denatured region, because of two different sets of circumstances the two-state melting can take place in a manner analogous to that proposed for the melting behavior of the DNA junction structure (44). In one case, the two marginal duplex parts, seven or eight base pairs long, separated by the central denatured region around the cross-link may fortuitously possess the same Tm. When this situation prevails, the remainder of the duplex consisting of the two marginal segments will melt in an apparent two-state manner without requiring any cooperative communication across the central denatured part around the cross-link. A second alternative case might truly reflect an all-or-none melting event. When this case prevails, molecular communication should occur between the marginal parts of the duplex across the denatured central part in a manner that results in a cooperative melting event. Our data do not allow us to differentiate between these two alternatives.

On the other hand, the Delta HvH/Delta Hcal ratio found for the duplex containing an interstrand cross-link of transplatin was 0.55. This value is consistent with this duplex containing two units each melting cooperatively, the size of which is close to one-half of the duplex. The conformational distortion in the central part of the duplex around the cross-link of transplatin is much less severe than that induced by the cross-link of cisplatin. As a consequence of this markedly less pronounced distortion, the base pairs in the short segment of the duplex around the cross-link of transplatin would not melt independently as a separated cooperative unit as in the case of the distorted segment around the cross-link of cisplatin. The schematic representation of the melting of the duplexes, which are interstrand cross-linked by cisplatin or transplatin, proposed on the basis of the results of the present work is shown in Fig. 4.


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  		Fig. 4.   Schematic representation of thermal melting of the duplexes containing central, site-specific interstrand cross-link of cisplatin (A) or transplatin (B).

In the aggregate, our results reveal and characterize the profound but different impacts that interstrand cross-linking by antitumor cisplatin and clinically ineffective transplatin can have on DNA stability and melting behavior. Such assessments are important in a range of applications including those aimed at understanding the molecular mechanisms underlying the biological effects of bifunctional agents that modify DNA. Moreover, the results of the present work further support the view that the impact of the interstrand cross-links of cisplatin and transplatin on DNA is different, which might be also associated with distinctly different antitumor effects of these platinum compounds.

    FOOTNOTES

* This research was supported by the Grant Agency of the Czech Republic (Grants 305/99/0695 and 301/00/0556), the Grant Agency of the Academy of Sciences of the Czech Republic (Grant A5004702), and the Internal Grant Agency of the Ministry of Health of the Czech Republic (Grants NL6058-3/2000 and NL6069-3/2000).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Supported by a doctoral fellowship from the Faculty of Sciences, Masaryk University, Brno, Czech Republic.

§ To whom correspondence should be addressed: Institute of Biophysics, Academy of Sciences of the Czech Republic, Kralovopolska 135, CZ-61265 Brno, Czech Republic. Tel.: 420-5-41517148; Fax: 420-5-41240499; E-mail: brabec@ibp.cz.

Published, JBC Papers in Press, December 4, 2000, DOI 10.1074/jbc.M010205200

    ABBREVIATIONS

The abbreviations used are: cisplatin, cis-diamminedichloroplatinum(II); transplatin, the trans isomer of cisplatin; DSC, differential scanning calorimetry; bp, base pair(s); FPLC, fast protein liquid chromatography; FAAS, flameless atomic absorption spectrophotometry.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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C. Hofr, N. Farrell, and V. Brabec
Thermodynamic properties of duplex DNA containing a site-specific d(GpG) intrastrand crosslink formed by an antitumor dinuclear platinum complex
Nucleic Acids Res., May 15, 2001; 29(10): 2034 - 2040.
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