ABSTRACT
The activity of the anticancer drug cisplatin is a consequence of its ability to bind DNA. Platinum adducts bend and unwind the DNA duplex, creating recognition sites for nuclear proteins. Following DNA damage recognition, the lesions will either be repaired, facilitating cell viability, or if repair is unsuccessful and the Pt adduct interrupts vital cellular functions, apoptosis will follow. With the use of the benzophenone-modified cisplatin analogue Pt-BP6, 25 bp DNA duplexes containing either a 1,2-d(G*pG*) intrastrand or a 1,3-d(G*pTpG*) intrastrand crosslink were synthesized, where the asterisks designate platinated nucleobases. Proteins having affinity for these platinated DNAs were photocrosslinked and identified in cervical, testicular, pancreatic and bone cancer-cell nuclear extracts. Proteins identified in this manner include the DNA repair factors RPA1, Ku70, Ku80, Msh2, DNA ligase III, PARP-1, and DNA-PKcs, as well as HMG-domain proteins HMGB1, HMGB2, HMGB3, and UBF1. The latter strongly associate with the 1,2-d(G*pG*) adduct and weakly or not at all with the 1,3-d(G*pTpG*) adduct. The nucleotide excision repair protein RPA1 was photocrosslinked only by the probe containing a 1,3-d(G*pTpG*) intrastrand crosslink. The affinity of PARP-1 for platinum-modified DNA was established using this type of probe for the first time. To ensure that the proteins were not photocrosslinked because of an affinity for DNA ends, a 90-base dumbbell probe modified with Pt-BP6 was investigated. Photocrosslinking experiments with this longer probe revealed the same proteins, as well as some additional proteins involved in chromatin remodeling, transcription, or repair. These findings reveal a more complete list of proteins involved in the early steps of the mechanism of action of the cisplatin and its close analogue carboplatin than previously was available.
Subject(s)
Cell Extracts/chemistry , Cisplatin/chemistry , DNA/metabolism , Neoplasms/pathology , Nuclear Proteins/analysis , Nuclear Proteins/metabolism , Photoaffinity Labels/analysis , Base Pair Mismatch , Base Sequence , Binding Sites , Biotinylation , Cell Line, Tumor , Cisplatin/analogs & derivatives , Cisplatin/pharmacology , Cross-Linking Reagents/metabolism , DNA/chemistry , DNA Damage/drug effects , DNA Probes/chemical synthesis , DNA Probes/genetics , DNA Probes/metabolism , DNA Repair/drug effects , Gene Expression Regulation, Neoplastic , Humans , Nuclear Proteins/isolation & purification , Photoaffinity Labels/chemistry , Poly (ADP-Ribose) Polymerase-1 , Poly(ADP-ribose) Polymerases/genetics , Protein Binding , RNA Interference , Substrate SpecificityABSTRACT
The affinity of the poly(ADP-ribose) polymerase-1 (PARP-1) for platinum-damaged DNA was first discovered during photo-cross-linking experiments using the photoactive compound Pt-BP6 [J. Am. Chem. Soc.2004, 126, 6536-6537], an analogue of the anticancer drug cis-diamminedichloroplatinum(II), cisplatin. Although PARP inhibitors sensitize cancer cells to cisplatin, there are conflicting reports in the literature about their efficacy. In order to improve our understanding of the mechanism by which PARP inhibition might potentiate the cell-killing ability of cisplatin, and to shed light on the source of the discrepancy among different laboratories, we have in the present study probed the influence of three PARP inhibitors in four types of cancer cells, cervical (HeLa), testicular (NTera2), pancreatic (BxPC3), and osteosarcoma (U2OS), on the results of Pt-BP6 photo-cross-linking experiments and cytotoxicity assays. We find that the activity of PARP proteins following exposure to platinum-modified DNA results in the dissociation of DNA-bound proteins. PARP inhibitors were able to sensitize some, but not all, of the cell lines to cisplatin. This cell line-dependence and the potential consequences of PARP-initiated protein removal from platinum-DNA lesions are discussed. Control experiments revealed that NTera2 cells are especially sensitive to PARP inhibition.
Subject(s)
Antineoplastic Agents/chemistry , Cisplatin/analogs & derivatives , Cisplatin/chemistry , DNA Damage , Nuclear Proteins/metabolism , Poly(ADP-ribose) Polymerase Inhibitors , Poly(ADP-ribose) Polymerases/metabolism , Antineoplastic Agents/toxicity , Cell Extracts/chemistry , Cell Extracts/genetics , Cross-Linking Reagents , DNA/metabolism , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , HeLa Cells , Heterocyclic Compounds, 4 or More Rings/chemical synthesis , Heterocyclic Compounds, 4 or More Rings/chemistry , Heterocyclic Compounds, 4 or More Rings/pharmacology , Humans , Tumor Cells, CulturedABSTRACT
The industrially important compound methacrylamide crystallizes as concomitant conformational polymorphs; the monoclinic form contains only the s-cis conformer, while the orthorhombic form contains only the s-trans conformer.
