Protein interactions with platinum–DNA adducts: from structure to function

Abstract

Because of the efficacy of cisplatin and carboplatin in a wide variety of chemotherapeutic regimens, hundreds of platinum(II) and platinum(IV) complexes have been synthesized and evaluated as anticancer agents over the past 30 years. Of the many third generation platinum compounds evaluated to date, only oxaliplatin has been approved for clinical usage in the United States. Thus, it is important to understand the mechanistic basis for the differences in efficacy, mutagenicity and tumor range between cisplatin and oxaliplatin. Cisplatin and oxaliplain form the same types of adducts at the same sites on DNA. The most abundant adduct for both compounds is the Pt-GG intrastrand diadduct. Cisplatin-GG adducts are preferentially recognized by mismatch repair proteins and some damage-recognition proteins, and this differential recognition of cisplatin- and oxaliplatin-GG adducts is thought to contribute to the differences in cytotoxicity and tumor range of cisplatin and oxaliplatin. A detailed kinetic analysis of the insertion and extension steps of dNTP incorporation in the vicinity of the adduct shows that both pol β and pol η catalyze translesion synthesis past oxaliplatin-GG adducts with greater efficiency than past cisplatin-GG adducts. In the case of pol η, the efficiency and fidelity of translesion synthesis in vitro is very similar to that previously observed with cyclobutane TT dimers, suggesting that pol η is likely to be involved in error-free bypass of Pt adducts in vivo. This has been confirmed for cisplatin by comparing the cisplatin-induced mutation frequency in human fibroblast cell lines with and without pol η. Thus, the greater efficiency of bypass of oxaliplatin-GG adducts by pol η is likely to explain the lower mutagenicity of oxaliplatin compared to cisplatin. The ability of these cellular proteins to discriminate between cisplatin and oxaliplatin adducts suggest that there exist significant conformational differences between the adducts, yet the crystal structures of the cisplatin- and oxaliplatin-GG adducts were very similar. We have recently solved the solution structure of the oxaliplatin-GG adduct and have shown that it is significantly different from the previously published solution structures of the cisplatin-GG adducts. Furthermore, the observed differences in conformation provide a logical explanation for the differential recognition of cisplatin and oxaliplatin adducts by mismatch repair and damage-recognition proteins. Molecular modeling studies are currently underway to analyze the mechanistic basis for the differential bypass of cisplatin and oxaliplatin adducts by DNA polymerases.

Introduction

Cis-diamminedichloroplatinum(II) (cisplatin) and cis-diammine-1,1-cyclobutane dicarboxylate (carboplatin) are widely used in chemotherapy, and are particularly effective in the treatment of testicular, ovarian, head, neck and non-small cell lung cancer. However, cisplatin and carboplatin have significant toxicity and are mutagenic in cell culture and animal model systems [1], [2]. Resistance is also a major limitation of cisplatin and carboplatin chemotherapy, with many tumors displaying intrinsic resistance or acquiring resistance during the course of treatment [3], [4], [5]. Cisplatin and carboplatin share the cis-diammine carrier ligands (Fig. 1). They form the same Pt–DNA dducts in vivo and are generally not effective in cell lines or tumors that have developed resistance to either agent.

(Trans-R,R)1,2-diaminocyclohexaneoxalatoplatinum(II) (oxaliplatin) is a second generation platinum complex that is often effective in cisplatin-resistant cell lines and tumors [6], [7] and appears to be less mutagenic than cisplatin [8], [9]. Oxaliplatin has recently been approved for the treatment of colon cancer in the US. Although oxaliplatin has been studied for over 30 years, it is still not clear why it is so much more effective than the hundreds of platinum analogs that have been evaluated over that time span. The elucidation of the molecular basis for the efficacy of oxaliplatin in cisplatin resistant tumors would represent a major advance that might allow the design of even more effective platinum drugs and/or the development of prognostic indicators capable of identifying those tumors most likely to respond to oxaliplatin-based therapy. Oxaliplatin and the related compound ormaplatin form Pt–DNA adducts with the (trans-R,R)1,2-diaminocyclohexane carrier ligand (Fig. 1). For simplicity the cis-diammine-Pt adducts will be referred to as cisplatin adducts even though they can be formed by both cisplatin and carboplatin, and the (trans-R,R)1,2-diaminocyclohexane-Pt adducts will be referred to as oxaliplatin adducts even though they can be formed by both oxaliplatin and ormaplatin.

The cytotoxicity of platinum compounds is thought to result primarily from the formation of Pt–DNA adducts. The effectiveness of oxaliplatin in cisplatin-resistant cell lines is thought to be due to repair or damage-recognition processes that discriminate between cisplatin and oxaliplatin adducts. This has been best established for mismatch repair. For example, the binding of the mismatch repair complex appears to increase the cytotoxicity of Pt–DNA adducts [10], [11], [12], [13], either by activating downstream signaling pathways that lead to apoptosis [14], [15] or by causing “futile cycling” during translesion synthesis past Pt–DNA adducts [16]. These effects appear to be specific for cisplatin adducts. Thus, defects in mismatch repair increase net translesion synthesis past cisplatin DNA adducts [16], decrease cisplain-induced expression of damage-response genes [14], and increase cellular resistance to cisplatin adducts [10], [11], [12], [17], but have no effect on oxaliplatin adducts [16]. As one might predict from these biological differences, hMSH2 [11] and MutS [18] bind with greater affinity to cisplatin adducts than to oxaliplatin adducts.

Some damage-recognition proteins, especially those of the HMG-domain family, also bind more tightly to cisplatin adducts than to oxaliplatin adducts [19], [20]. The biological consequences of these effects are less clear, but the binding of damage-recognition proteins to cisplatin–DNA adducts has been postulated to inhibit nucleotide excision repair [21], [22] and/or translesion DNA synthesis [23], [24]. Finally, since many of the damage-recognition proteins are low abundance transcription factors, some of the cytotoxicity of cisplatin–DNA adducts has been proposed to be due to their ability to “hijack” the transcription factors away from their natural promoters [20], [25]. To the extent that these damage-recognition proteins bind with different affinities to cisplatin and oxaliplatin adducts, they could obviously influence the differential cytotoxicity of cisplatin and oxaliplatin adducts. For example, HMG1 has been shown to inhibit translesion synthesis past cisplatin adducts to a greater extent than past oxaliplatin adducts [24].

Finally, both pol η and pol β appear to discriminate between cisplatin and oxaliplatin adducts based on the kinetics of dCTP incorporation opposite the Pt-GG adduct [24], [26], [27]. However, the previous studies may have been incomplete because they only measured dNTP incorporation opposite the adducts, and recent studies have shown that extension from the adduct is also strongly inhibited [28]. The ability of translesion polymerases to discriminate between cisplatin and oxaliplatin adducts could help explain the greater mutagenicity of cisplatin compared to oxaliplatin. However, the significance of these observations with respect to translesion synthesis in vivo has not been previously described.

We [29] and others [30], [31] have found that cisplatin and oxaliplatin form the same types of adducts at the same sites on the DNA. Both cisplatin and oxaliplatin form approximately 60–65% intrastrand GG, 25–30% intrastrand AG, 5–10% intrastrand GNG, and 1–3% interstrand GG diadducts [32]. X-ray crystallographic structures have been reported for both the cisplatin-GG [33] and oxaliplatin-GG [34] adducts in the same dodecamer DNA sequence. The two structures were virtually identical [34]. However, all of the mismatch repair proteins, DNA polymerases, and damage-recognition proteins that discriminate between cisplatin and oxaliplatin adducts and for which structural information is available appear to bind DNA primarily in the minor groove and bend DNA in the direction of the major groove [35], [36], [37], [38]. Thus, the basis for the differential recognition of cisplatin and oxaliplain adducts by these proteins was not clear from the existing crystal structures of the cisplatin and oxaliplatin adducts.