HMG-domain proteins specifically inhibit the repair of the major DNA adduct of the anticancer drug cisplatin by human excision nuclease.

Abstract

The most frequent DNA adduct made by the anticancer drug ciplatn, the 1,2-tarand d(GpG) crosslink, as well as the minor 1,3-intrasrand d(GpTpG) adduct, were both repaired by an us vitro human excision repair system.Fragments of 27-29 nt containg the platinum damage were excised. The high mobility group (HMG)-domain proteins HMG1 and human mitochondrial rnscription factor specifIcaly inhibited repair of the 1,2-intrd cross-link by the human excision nuclease. These results suggest that the types and levels of E1MG-domain proteins in a given tumor may nfluence the responsiveness of that cancer to cisplatin chemotherapy and they provide a rational basis for the synthesis of new platinum anticancer drug candidates.Cisplatin [cis-diamminedichloroplatinum(II)] is one of the most commonly used anticancer drugs, being particularly effective against testicular tumors (1). Although cisplatin reacts with many cellular components (2), DNA adducts are thought to be the main cytotoxic lesions, and cells deficient in excision repair are extremely sensitive to the drug (3, 4). Recently, it was discovered that proteins with high mobility group (HMG)-domain motifs (5, 6) bind specifically to the major cisplatin-DNA adducts, 1,2-intrastrand d(GpG) and d(ApG) cross-links, which comprise =90o ofthe lesions, but not to the rare 1,3-intrastrand d(GpNpG) cross-links (7,8). Initially, it was thought that the binding of HMG-domain proteins might aid in damage recognition and thus promote repair. When a yeast HMG-domain protein, Ixrl, was deleted, however, the cells became less sensitive to the drug (9). This result demonstrated that HMG-domain proteins can indeed play a role in mediating the cytotoxicity of cisplatin but suggested that repair was inhibited rather than promoted. To evaluate this possibility further, we used two HMGdomain proteins, HMG1 (10) and the human mitochondrial transcription factor (h-mtTFA) (11-13), and DNA fragments containing site-specific platinum adducts to investigate directly the effects of HMG-domain proteins on the excision repair of cisplatin-damaged DNA.Cisplatin adducts as well as other bulky lesions are removed from DNA by an ATP-dependent multisubunit enzyme system called the excision nuclease (excinuclease) (14,15). In humans the excinuclease removes cyclobutane pyrimidine dimers in 27-to 29-nt oligomers by hydrolyzing the 5th phosphodiester bond 3' and the 22nd-24th phosphodiester bonds 5' to the modified base (16,17 (see refs. 8, 20, and 21). Each oligomer had a single cis-diammineplatinum(II) moiety coordinated to N7 of guanine at the sites designated by an asterisk. The oligomer (0.5 ug) was labeled in 50 j4 with [y-32P]ATP (7000 Ci/mmol; 1 Ci = 37 GBq) and mixed with 1.0 tig each of the other seven oligomers, which were phosphorylated (in 100 t4) with nonradioactive ATP.The mixture was precipitated with ethanol and the oligomers were resuspended in 40 1A of annealing buffer, which contained 20 mM Tris HCl (pH 7.4), 50 mM NaCl, and 2 mM MgCl2. The mixture was heated at 700C for 2 min an...

Cisplatin: mode of cytotoxic action and molecular basis of resistance

“…The HMG1 protein, for instance, has been implicated in promoting cytotoxicity by first interacting with the DNA adduct and then shielding it from repair (Huang et al, 1994). This action of HMG1 is supported by the finding that overexpression of this recognition protein by preexposure to estrogen sensitizes breast tumor cells to cisplatin (He et al, 2000).…”

Loss of DNA mismatch repair facilitates reactivation of a reporter plasmid damaged by cisplatin

“…Thus, steric hindrance by the MMR proteins would be expected to slow repair by the nucleotide excision repair system and reduce generation of luciferase activity. Such a mechanism has been proposed to explain the ability of another group of cisplatin adduct-binding proteins, the HMG proteins, to interfere with adduct repair (Huang et al, 1994). However, in a recent study, (Mu et al, 1997) reported that addition of the hMSH2/hMSH6 heterodimer to a cell-free excision repair system did not impair the ability of the nucleotide excision repair system to remove cisplatin adducts from DNA.…”

Solution Structures of a DNA Dodecamer Duplex with and without a Cisplatin 1,2-d(GG) Intrastrand Cross-Link: Comparison with the Same DNA Duplex Containing an Oxaliplatin 1,2-d(GG) Intrastrand Cross-Link,

“…Similarly, a number of damage recognition proteins and transcription factors, especially those with HMG domains, have been shown to discriminate between CP-and OX-GG adducts (14,15). The mechanism(s) by which the binding of these proteins to Pt-DNA adducts influences the cytotoxic response is not known but has been postulated to involve shielding of the adducts from DNA repair and tolerance mechanisms (16)(17)(18)(19), activation of signaling pathways leading to cell cycle arrest or apoptosis, and/or hijacking of transcription factors needed for DNA replication or cell division (15,20). The binding specificity has been determined for only a few of these proteins, but where it has been studied, these proteins bind to CP-GG adducts with higher affinity than to OX-GG adducts (14,15,21).…”