Recognition and repair of DNA-cisplatin adducts

Anticancer activity of cisplatin (cis-diamminedichloroplatinum) is believed to result from its interaction with DNA. The drug reacts with nucleophilic sites in DNA forming monoadducts as well as intraand interstrand crosslinks. DNA–cisplatin adducts are specifically recognized by several proteins. They can be divided into two classes. One constitutes proteins which recognize DNA damage as an initial step of the nucleotide excision and mismatch repair pathways. The other class contains proteins stabilizing cellular DNA–protein and protein–protein complexes, including non-histone proteins from the HMG (high-mobility-group) family. They specifically recognize 1,2-interstrand d(GpG) and d(ApG) crosslinks of DNA–cisplatin adducts and inhibit their repair. Many HMG-domain proteins can function as transcription factors, e.g. UBF, an RNA polymerase I transcription factor, the mammalian testis-determining factor SRY and the human mitochondrial transcription factor mtTFA. Moreover, it seems that some proteins, which probably recognize DNA–cisplatin adducts non-specifically, e.g. actin and other nuclear matrix proteins, can disturb the structural and functional organization of the nucleus and whole cell. The formation of complexes between DNA and proteins in the presence of cisplatin and the changes in the cell architecture may account for the drug cytotoxicity.


STRUCTURAL ALTERATIONS OF DNA EVOKED BY CISPLATIN
The N7 atoms of guanine and adenine are the main binding sites for platinum complexes in double-stranded DNA.The interac-tion between cisplatin and DNA can result in mono-and bifunctional adducts, as well as DNA-protein crosslinks.The bifunctional adducts, which can take the form of intra-or interstrand crosslinks, may cause major local distortions of DNA structure, involving both bending and unwinding of the double helix (Table 1).The intrastrand crosslinks cis-Pt(NH 3 ) 2 -d(GpG) (60-65% of the total) and cis-Pt(NH 3 ) 2 -d(ApG) (22-30%) are the most abundant products of the interaction (Fichtinger-Schepman et al., 1985).These crosslinks, which comprise 90% of the DNA-cisplatin adducts, bend the helix by 34°towards its major groove and unwind it by 13° (Bellon et al., 1991).Interstrand crosslinks represent a small amount of the total cisplatin lesions but several studies have suggested that they could also be responsible for the cytotoxicity of the drug.The distortions induced by these crosslinks exhibit unprecedented features such as location of the platinum residue in the minor groove, extrusion of the cytosines of the crosslinked d(GpC) d(GpC) site, bending of the helix axis towards the minor groove and large DNA unwinding (Table 1) (Malinge et al., 1999).

INCREASED EFFICACY OF DNA REPAIR MAY UNDERLIE CELLULAR RESISTANCE TO CISPLATIN
A major clinical limitation of the use of cisplatin is the intrinsic or acquired resistance to the drug.Postulated mechanisms of resistance to cisplatin include reduced drug uptake or decreased drug accumulation, increased levels of intracellular thiols and metallothioneins and increased level of repair of DNA-cisplatin adducts (Parker et al., 1991;Chu, 1994;Chen et al., 1995).More recently, additional pathways have been characterized indicating that altered expression of oncogenes that subsequently limit the formation of DNA-cisplatin adducts and activate antiapoptotic pathways may also contribute to the resistance phenotype.It is suggested that expression of ras oncogenes can confer resistance to cisplatin by reducing drug uptake and increasing DNA repair (Dempke et al., 2000;Niedner et al., 2001).We have recently shown that oncogenic fusion tyrosine kinases (FTKs)  (Bellon et al., 1991) 26 b (Tahakara et al., 1996) -GpA 13 a 35 a (Bellon et al., 1991) -GpTpG 23 a 33 a (Bellon et al., 1991) Interstrand such as BCR/ABL, TEL/ABL, TEL/JAK2, TEL/PDGFbR, TEL/TRKC(L) and NPM/ ALK may stimulate the repair of cisplatin damaged DNA by a mechanism dependent on the RAD51 protein and it this way contribute to the cellular resistance to the drug (Slupianek et al., 2002).The repair of cisplatin adducts occurs primarily by the nucleotide excision repair pathway (NER) (Zamble et al., 1996).Cell lines from patients with XP (xeroderma pigmentosum) are highly sensitive to cisplatin, even 4-fold more sensitive than normal cells (Fraval et al., 1978;Dijt et al., 1988).NER in human cells involves recognition of damage by XPA in complex with the RPA protein, incision by the specific endonucleases XPG at the 3¢ side of the lesion and ERCC1/XPF at the 5¢ side and repair DNA synthesis mediated by a PCNA-dependent DNA polymerase (Aboussekhra & Wood, 1994;Wood, 1996).The more distorting 1,3-intrastrand d(GpTpG)-cisplatin crosslink is repaired about 15-20-fold more effectively by human whole cell extract than the less distorting 1,2-intrastrand d(GpG)cisplatin crosslink (Moggs et al., 1997).The poorer repair of 1,2 crosslinks in comparison with the 1,3 crosslinks is a consequence of different structural alterations of the DNA helix and a repair-shielding mechanism mediated by HMG (high-mobility-group)-domain proteins.
Several earlier studies showed that cisplatin-resistant cell lines exhibited overproduction of nuclear proteins preferentially binding cisplatin-modified DNA (Chao et al., 1991;Nishio et al., 1994).Initially, it was suggested that the binding of these proteins might assist in damage recognition and thus promote repair.It is now accepted that specific recognition of DNA-cisplatin adducts by nuclear proteins, especially from the HMG-domain family, inhibits nucleotide excision repair and may enhance cisplatin sensitivity (Huang et al., 1994;Zamble et al., 1996;Trimmer et al., 1998;Arioka et al., 1999;Trimmer & Essigmannn, 1999;Jordan & Carmo-Fonseca, 2000).It was reported that a yeast strain with an inactivated IXR1 gene was twice as resistant to cisplatin as its parental strain (Brown et al., 1993).This result strongly indicates that Ixr1 -a protein containing two HMG domains -can inhibit the repair of DNA-cisplatin adducts and may confer the drug sensitivity.An increased expression of the human NER repair genes XPA and ERCC1 was reported in tumour tissue of some patients with malignant ovarian cancer who did not respond to platinum-based chemotherapy (Dabholkar et al., 1994;Yu et al., 1996;Li et al., 2000).It was also shown that ERCC1 mRNA level was correlated with clinical resistance to cisplatinbased chemotherapy in human gastric cancer (Metzger et al., 1998).While transfection of an XPA-expressing vector into XPA-deficient cell lines could restore NER activity, overexpression of the same plasmid in repair-proficient cell lines did not appear to have any substantial effect on the activity levels (Cleaver et al., 1995).In addition, transfection of ERCC1 restored repair of interstrand crosslinks in cell lines that were deficient in this protein, while overexpression of ERCC1 appeared to inhibit repair of these crosslinks (Larminat & Bohr, 1994;Chaney & Sancar, 1996).Therefore, there is no clear evidence of overexpression of the NER genes in resistant cell lines or in resistant tumours.Further studies are needed to elucidate the molecular basis of repair-dependent resistance to cisplatin.
It was found that human 3-methyladenine-DNA glycosylase (AAG), a protein involved in the first step of base excision repair, can bind tightly to cisplatin adducts (Kartalou et al., 2000).It is proposed that analogous to "transcription factor hijacking" cisplatin adducts could titrate human AAG away from lesions such as 3-methyladenine and 3-methylguanine, leading to enhanced toxicity because of the inhibition of repair of these lesions by AAG.On the other hand, it is also possible that AAG binds to cisplatin adducts and, be-cause it cannot excise them, it conveys the adducts to the nucleotide excision repair pathway through the interaction of AAG with the hHR23 proteins (Miao et al., 2000;Kartalou & Essigmann, 2001).Thus the interaction between AAG and DNA-cisplatin adducts can increase the efficiency of DNA repair and, in the case of overexpression of this protein, could lead to cisplatin resistance.
In normal human cells, mispaired bases or short insertions/deletions in DNA are recognized by complex proteins of the mismatch repair system (MMR): hMutSa (heterodimer of hMSH2 and hMSH6) or hMutSb (heterodimer of hMSH2 and hMSH3).The hMutS complexes are thought to recruit the hMutLa heterodimer consisting of the hMLH1 and PMS2 proteins.The hMutSa complex and hMSH2 can recognize DNA-cisplatin adducts (Fink et al., 1996;Duckett et al., 1996;Modrich, 1997).A direct comparison among several mismatched platinated DNA molecules showed that the hMutSa complex recognized duplex DNA containing a 1,2-intrastrand d(GpG)-cisplatin crosslink with a much higher affinity when the non-damaged DNA strand contained a mispaired thymine opposite the 3¢ platinated guanine (Yamada et al., 1997).This crosslink was also repaired more effectively by nucleotide excision repair when placed opposite one or two non-complementary thymine residues (Moggs et al., 1997).
Loss of MMR was reported to be associated with low-level resistance to cisplatin (Anthoney et al., 1996;Aebi et al., 1996;Vaisman et al., 1998).These observations indicated defects in hMLH1 or hMSH6, but not in the hMSH3 MMR protein (Vaisman et al., 1998).It was postulated that deficiency in the hMutSa and hMutLa complexes affected cisplatin resistance by facilitating replicative bypass of cisplatin adducts.In cisplatin sensitive cells DNA-cisplatin adducts are bypassed during replication, then the adducts are bound by the hMutSa/hMutLa complexthis would most likely result in removal of the newly synthesized strand of DNA and retention of the parental strand containing the platinum adducts, because mismatch repair is selective for nascent DNA (Vaisman et al., 1998).Therefore, the damage remains unrepaired.Continuous operation of these futile cycles of resynthesis and mismatch repair is likely to generate persistent gaps or strand breaks, which may lead to cell death.In cells with defective mismatch repair, lack of one of the components of the hMutSa/hMutLa complex may allow completion of DNA replication and evoke some tolerance to the drug.Therefore, cells with an inactive mismatch repair system can better tolerate DNA adducts in the short term, but they display increased rates of other DNA damages leading to mutations (Hoffmann et al., 1996;Vaisman et al., 1998;Lin et al., 1999).

RECOGNITION OF DNA-CISPLATIN ADDUCTS BY HMG-DOMAIN PROTEINS
The HMG-domain proteins constitute the largest and most extensively characterized group of non-histone chromosomal proteins (Bustin & Reeves, 1996).They can bind to specific structures in DNA or in chromatin with little or no specificity for the target DNA sequence.
HMG-domains are basic domains of 80 amino acids containing three a-helical regions and well-conserved hydrophobic amino acids within their sequences (Grosschedl et al., 1994).Two families of HMG proteins have been reported.The first consists of proteins containing two or more HMG domains, it includes the HMG1 and HMG2 proteins, the nucleolar RNA polymerase I transcription factor UBF and the mitochondrial transcription factor mtTF.In the second family there are proteins containing single HMG domain, such as tissue-specific transcription factors, e.g. the mammalian testis-determining factor SRY and the lymphoid enhancer-binding factor LEF-1.
To explain the observed effects it was suggested that DNA-cisplatin adducts might act as molecular decoys in cells, titrating away HMG-domain proteins from their natural binding sites and thereby disrupting their function.It was also suggested that after the drug exposure, HMG1 and HMG2 proteins migrated from the cytosol into the nucleus, where they bound to DNA-cisplatin adducts (Chao et al., 1996).It was also observed in this study that UBF distribution changed from its normal, well-defined location, associated with the nucleoli, to a more dispersed pattern of nuclear staining.UBF, being an RNA polymerase I transcription factor, plays an important role in the efficient transcription of rRNA genes and binds to rRNA promoter sequences (Jantzen et al., 1990).It could preferentially bind to DNA-cisplatin adducts in rRNA genes due to its natural close proximity to this region.Another hypothesis supposes that DNA-cisplatin adducts selectively compete with the rRNA promoter for UBF binding resulting in "cisplatin-mediated transcription factor hijacking" which reduces rRNA synthe-sis in growing cells (Treiber et al., 1994).It was shown that cisplatin blocks synthesis of rRNA (Jordan & Carmo-Fonseca, 1998).Besides the redistribution of UBF in the nucleoli of human cells, redistribution of the major components of the rRNA transcription machinery, TBP, TAF I s and RNA polymerase I, was also observed (Chao et al., 1996).
The HMG-domain protein-mediated cytotoxicity of cisplatin may be explained as a result of the recognition of DNA-cisplatin adducts by tissue-specific HMG proteins.Cisplatin is very effective in the treatment of testicular cancer, with over 70% of patients cured (Loehrer & Einhorn, 1984;Brown et al., 1994).Several HMG-domain proteins are specifically expressed in the testis and could potentially contribute to the cisplatin sensitivity of testicular tumours.One of these proteins is human testis-determining factor hSRY.The ability of hSRY to interact with bent DNA structures and to bend linear DNA has suggested that the protein may modulate transcription by acting architecturally in the assembly of a nucleoprotein complex (Pontiggia et al., 1994).It was shown that full length hSRY bound to the major 1,2-d(GpG) cisplatin adduct with a K d(app) of 120 ± 10 nM and exhibited a 20-fold specificity over unmodified DNA (Trimmer et al., 1998).It was also demonstrated in that study that the HMG-domain of hSRY was sufficient for this interaction.The hSRY-HMG domain recognized the 1,2-d(GpG) intrastrand crosslink with higher affinity but with lower specificity than the full-length protein.The affinities of full-length hSRY and the hSRY-HMG domain for a single DNA adduct were comparable to those for the putative target sequence AACAAAG.Other results suggest that DNA-cisplatin adducts may compete with specific DNA sequences in vivo for the binding of hSRY (Trimmer et al., 1998).Recognition of 1,2-d(GpG) intrastrand crosslink of cisplatin by the murine testis-specific HMG-domain protein (tsHMG) was also described (Ohndorf et al., 1997).These results show that the specificity of tsHMG for platinated DNA is much higher than that observed for other HMG-domain proteins, including Ixr1 and hUBF (Treiber et al., 1994;McA'Nulty et al., 1996).
The competition between DNA-cisplatin adducts and structure-and/or sequence-specific sites in DNA in the recognition by testis-specific HMG-domain proteins can, at least in part, explain the high sensitivity of testicular tumours to cisplatin.This competition can reduce transcription of genes important for testis cell viability and finally cause the cell death.

HMG PROTEINS AND EXCISION REPAIR OF DNA-CISPLATIN ADDUCTS
It was suggested that DNA-HMG-domain protein complexes can protect cisplatin ad-ducts against repair.Therefore, HMG proteins can sensitize cells to cisplatin by a repair-shielding mechanism (Huang et al., 1994;Zamble et al., 1996;Trimmer et al., 1998;Arioka et al., 1999).It was shown that HMG proteins, HMG1 and h-mtTFA, specifically inhibited repair of the 1,2-intrastrand d(GpG) crosslink, but the removal of the 1,3-d(Gp-TpG) crosslink was not inhibited (Huang et al., 1994).Similar experiments checking the ability of another HMG protein, to inhibit the repair of DNA-cisplatin adducts were performed with tsHMG.This protein bound specifically to the 1,2-d(GpG) intrastrand adduct, but not to the 1,3-d(GpTpG) crosslink (Zamble et al., 1996).Therefore, it could inhibit repair of the 1,2-intrastrand, but not the 1,3-intrastrand crosslink.Furthermore, human SRY selectively inhibited repair of the 1,2-d(GpG) adduct in an in vitro excision repair assay (Trimmer et al., 1998).Many studies are directed to understand why cisplatin is more effective in treating testicular tumours than tumours of other tissues.Several HMG-domain proteins are specifically expressed in the testis and they can participate in the cisplatin reactivity toward testicular tumours.Testis-specific HMG-domain proteins bind to the most abundant cisplatin adducts and protect then from nucleotide excision repair.

NUCLEAR ACTIN CAN BIND TO DNA-CISPLATIN ADDUCTS
The nuclear matrix contains sequence-specific DNA binding activities of several transcription factors (van Wijnen et al., 1993;Nardozza et al., 1996).DNA loop anchorage sites are localised in the enhancer regions or intronic sequences of several genes within the nuclear matrix.These sequences have been termed matrix-associated regions (MARs) or scaffold associated regions (SARs) and usually are approximately 200 base pairs in length, are A-T-rich and contain topoiso-merase cleavage sites, polyadenylation signals and telomeric DNA sequences (Roberge & Gasser, 1992;de Lange, 1992).
Actin was found to be the major protein crosslinked to the DNA of Chinese hamster ovary cells treated with cisplatin (Miller et al., 1991).Its aggregates colocalize with transcription sites (Clubb & Locke, 1998;Nguyen et al., 1998).More than 90% of mRNA precursor molecules remain tightly bound to the nuclear matrix during the preparation of matrix from leukemia cells.This interaction relies on actin, since mRNA precursors can be extracted under conditions promoting filamentous actin depolymerization (Nakayasu & Ueda, 1985).Small nuclear ribonucleoproteins (snRNP) have also been shown to be associated with actin in the nuclear matrix (Sahlas et al., 1993).The involvement of actin in DNA-protein crosslinks induced by cisplatin can disturb the nuclear metabolism and the spatial organization of chromatin.On the other hand cisplatin, at low concentration, can affect the polymerization of G-actin and induce the depolymerization and crosslinking of the actin polymer at a higher concentration (Zeng et al., 1996).Cisplatin can alter the natural interactions between DNA and actin or actin and other proteins leading finally to the cell death.
The proteins participating in DNA-protein crosslinks by cisplatin can be found among nuclear matrix proteins (Oliñski et al., 1987;Miller et al., 1991;Ferraro et al., 1992;WoŸniak & Walter, 2002).The presence of nuclear proteins of 50 and 53 kDa, in addition to actin, crosslinked to DNA following exposure to cisplatin was shown (Miller et al., 1991).These proteins are abundant in the nuclear matrix fraction.A comparison of the various electrophoretic patterns of proteins crosslinked to DNA allowed the detection of several similarities between various animal species (Ferraro et al., 1992).It was also shown that nuclear proteins of 28, 30, 34.5, 45 and 120 kDa were crosslinked with DNA in human lymphocytes after incubation with cisplatin.The polypeptide of 34.5 kDa crosslinked to DNA after 2-h incubation with cisplatin (WoŸniak & Walter, 2002).When Novikoff hepatoma-bearing rats were given injections of a therapeutic dose of cisplatin, DNA-protein crosslinks could be detected by using antisera to dehistonized chromatin, nuclear matrix and cytoskeletal preparation.The principal proteins crosslinked to DNA by cis-and transplatin were Novikoff hepatoma cytokeratins (Oliñski et al., 1987).
The human telomere sequences consisting of several thousand nucleotides of the repeating sequence (TTAGGG) n are attached to the nuclear matrix (de Lange, 1992).The G-rich sequence of the telomeres is potentially a good target for cisplatin binding.It was demonstrated that treatment of HeLa cells with cisplatin at low doses resulted in degradation and shortening of telomeres (Ishibashi & Lippard, 1998).Several studies indicated that cisplatin could also interfere with telomerase activity (Burger et al., 1997;Asai et al., 1998).Therefore, cisplatin can affect both telomeres and the telomerase enzyme.It is possible that the drug can damage DNA at specific regions, e.g.within the telomeres.Because telomeres are not transcribed, they will not be repaired as effectively as the transcriptionally active part of chromosomes and this will make even low levels of telomere damage sufficient to cause senescence and cell death (Cohen & Lippard, 2001).But we have recently shown that a conjugate of cisplatin with selenium could inhibit telomerase activity independently of its DNA-damaging effect (B³asiak et al., 2002).We hypothesized that the conjugate, like cisplatin itself, could form intrastrand adducts with guanine residues, G-Pt-G, in the DNA/RNA regions essential for the expression or maintenance of telomerase activity.

H1 HISTONE CAN PREFERENTIALLY BIND TO CISPLATIN-MODIFIED DNA
The linker histone H1 binds much more strongly to cisplatinated DNA than to transplatinated or unmodified DNA (Yaneva et al., 1997).H1 binds to cisplatin-damaged DNA with high affinity, about 20-fold higher than HMG1.The data about binding of histone H1 to cisplatin-modified DNA are related to the earlier report on the preferential formation of the intrastrand crosslinks by cisplatin within the linker region of chromatin (Hayes & Scovell, 1991).The DNase I cleavage pattern observed on cisplatin-modified chromatin and core particles indicates that there is very little distortion of the native helical twist of the modified DNA.
The higher affinity of cisplatin-modified DNA for the linker histone as compared with the proteins of the HMG1/2 group, led to the suggestion that this histone can be the most likely occupant of sites of DNA-cisplatin adducts in the cell.Interestingly, the linker, lysine-rich histones H1 and H5 share with the HMG1/2 proteins many DNA binding properties.Both classes of proteins bind preferentially to the linker DNA between adjacent nucleosomes in the bulk chromatin (van Holde, 1988).The HMG1 protein can also compete effectively with H1 for binding to Holliday junctions in vitro (Varga-Weisz et al., 1994).This observation, combined with the 10-fold higher abundance of the linker histone in the cell nucleus led to the conclusion that linker histone binding to cisplatin-damaged DNA sites might play a major role in the cytotoxicity of the drug (Zlatanova et al., 1998;van Holde, 1988).

CONCLUDING REMARKS
The anticancer activity of cisplatin is believed to arise from its binding to DNA, resulting in DNA-adducts.These adducts can be recognized by nuclear proteins, which may be part of the cellular repair systems or they may be DNA-binding proteins that recognize DNA sequences containing specific structural elements.Overexpression of some genes involved in nucleotide excision repair and de-fects in mismatch repair are associated with cisplatin resistance.The recognition of the major DNA-cisplatin adducts by HMG proteins may block their repair and contribute to the therapeutic action of cisplatin.
The clinical success of cisplatin for the treatment of cancer is clear but severe side effects and intrinsic or acquired resistance limit its application.In order to reduce the side effects and improve the effectiveness of cisplatin, a number of cisplatin analogues have been synthesized (Guo & Sadler, 1999).The molecular mechanisms of action and protective strategies of the new platinum compounds are intensively investigated at present (B³asiak et al., 1999;B³asiak et al., 2000;B³asiak & Kowalik, 2001).The results regarding the interaction of platinum compounds with telomeres and telomerase may open a new area in the investigation of platinum-based drugs.

Figure 1 .
Figure 1.Structure of cisplatin and its analogues.

Figure 3 .
Figure 3. Structure of the complex between the non-sequence-specific domain A of HMG1 and cisplatin-modified DNA.The protein backbone is shown in yellow, the intercalating Phe37 residue as van der Waals spheres and DNA in red and blue with the cis-[Pt(NH 3 ) 2 {d(GpG)-N7-(G 8 )-N7(G 9 )}] intrastrand adduct in green.Numbers indicate the first (N terminus) and last (C terminus) ordered residues in the crystal structure (reprinted with permission from Ohndorf U-M, Rould MA, He Q, Pabo CO, Lippard SJ. (1999) Nature (London) 399: 708-12).