Platinum Adducts Research Articles

Platinum Interference with siRNA Non-seed Regions Fine-Tunes Silencing Capacity

Knowledge concerning the molecular mechanisms governing the influence of non-coding RNAs on protein production has emerged rapidly during the past decade. Today, two main research areas can be identified, one oriented toward the use of artificially introduced siRNAs for manipulation of gene expression, and the other one focused on the function of endogenous miRNAs. In both cases, the active molecule consists of a ∼20-nucleotide-long RNA duplex. In the siRNA case, improved systemic stability is of central interest for its further development toward clinical applications. With respect to miRNA processing and function, understanding its influence on mRNA targeting and the silencing ability of individual miRNAs, e.g., under pathological conditions, remains a scientific challenge. In the present study, a model system is presented where the influence of the two clinically used anticancer drugs, cisplatin and oxaliplatin, on siRNA's silencing capacity has been evaluated. More specifically, siRNAs targeting the 3' UTR region of Wnt-5a mRNA (NM_003352) were constructed, and the biologically active antisense RNA strand was pre-platinated. Platinum adducts were detected and characterized by a combination of gel electrophoresis and MALDI-MS techniques, and the silencing capacity was evaluated in cellular luciferase-expressing systems using HB2 cells. Data show that platination of the antisense strand of the siRNAs results in adducts with protection against hydrolytic cleavage in the proximity of the platination sites, i.e., with altered degradation patterns compared to native RNAs. The MALDI-MS method was successfully used to further identify and characterize platinated RNA, with the naturally occurring platinum isotopic patterns serving as sensitive fingerprints for metalated sites. Expression assays all confirm biological activity of antisense-platinated siRNAs, here with platination sites located outside of the seed region. A significant reduction of silencing capacity was observed as a general trend, however. Of the two complexes studied, oxaliplatin exhibits the larger influence, thus indicating subtle differences between the abilities of cis- and oxaliplatin to interfere with si- and miRNA processing.

Determination of the binding sites for oxaliplatin on insulin using mass spectrometry-based approaches

Using insulin as a model protein for binding of oxaliplatin to proteins, various mass spectrometric approaches and techniques were compared. Several different platinum adducts were observed, e.g. addition of one or two diaminocyclohexane platinum(II) (Pt(dach)) molecules. By top-down analysis and fragmentation of the intact insulin-oxaliplatin adduct using nano-electrospray ionisation quadrupole time-of-flight mass spectrometry (nESI-Q-ToF-MS), the major binding site was assigned to histidine5 on the insulin B chain. In order to simplify the interpretation of the mass spectrum, the disulphide bridges were reduced. This led to the additional identification of cysteine6 on the A chain as a binding site along with histidine5 on the B chain. Digestion of insulin-oxaliplatin with endoproteinase Glu-C (GluC) followed by reduction led to the formation of five peptides with Pt(dach) attached. Identification of several of the binding sites was obtained using matrix-assisted laser desorption/ionization (MALDI)-ToF-ToF-MS and liquid chromatography-nESI-Q-ToF-MS. Upon comparing the top-down and bottom-up approaches, the suitability of the bottom-up approach for determining binding sites was questioned, as the release and possible re-association of Pt(dach) were demonstrated upon enzymatic digestion. The associated advantages and disadvantages of ESI and MALDI were also pointed out.

Sequence specific effects on DNA and cell damage with the PARP inhibitor olaparib (AZD2281) and carboplatin.

5025 Background: We have found clinical activity of carboplatin (C) with the PARP inhibitor olaparib (O) in BRCA1/2mut or BRCA-like breast and ovarian cancers. Current clinical trials are testing the hypothesis that PARP inhibition will sensitize tumors to platinum agents. We have examined sequence specificity of C and O combinations in cell lines, including two BRCA1mut (HCC1937, UWB1.289) and two BRCA-WT lines (OVCAR8, HeyA8), on the development of DNA damage and cell injury. Methods: Cell injury was measured with XTT assays. DNA damage was examined using Comet assay with tail quantification and γH2AX foci by immunofluorescence. Platinum DNA-adduct formation was measured using atomic absorption spectrometry and normalized to DNA input. Results: Exposure to O prior to C for 24 hours (O>C) reduced the efficiency of double stranded DNA damage compared to C alone or O+C (p<0.05 for O>C vs. C or O+C) measured by γH2AX foci/nucleus in BRCA1mut cell lines, UWB1.289: 11.0 (O>C), 40.9 (C), 46.4 (O+C); HCC1937: 14.7 (O>C), 53.2 (C), 60.6 (O+C). This trend was not observed in BRCA-WT cell lines. Comet assay demonstrated decreased DNA damage as a reduced percentage of DNA in the comet tail in BRCA1mut cells treated with O>C compared to C (p<0.05) but not O+C (p=not significant), HCC1937: 11.3% (C), 3.4% (O>C), 5.1% (O+C); UWB1.289: 7.3% (C), 2.4% (O>C), 5.1% (O+C). Cell cytotoxicity by XTT assay indicated O>C had decreased cytotoxic effects compared to C or O+C (p<0.05 for O>C vs. C or O+C) in BRCA1mut cell lines, UWB1.289: 69% (O>C), 44% (C), 40% (O+C); HCC1937: 88% (O>C), 68% (C), 63% (O+C). Platinum adduct formation was not affected by treatment with O in either BRCA1mut cell lines or BRCA-WT cell lines. Conclusions: The sequencing of O with C yields changes in cytotoxicity and the quantity of DNA damage observed in both comet analysis and γH2AX focus formation while platinum adducts were not altered by O exposure. This suggests pretreatment of BRCA-deficient cell lines with PARP inhibition may up-regulate other DNA repair pathways and lead to paradoxically less DNA damage than seen with C alone or C+O concurrently. Further studies are ongoing to evaluate this question.

Abstract 5494: CX-4945, an inhibitor of protein kinase CK2, potentiates the antitumor activity of platinum chemotherapy in models of ovarian cancer by preventing phosphorylation of XRCC1 and MDC1 and disrupting DNA damage repair

Abstract Platinum-based chemotherapeutics are commonly used to treat solid tumors but may be restricted in their application by dose limiting toxicity and inherent or acquired resistance. Because efficient DNA damage repair mechanisms contribute to resistance, targeting components of the repair machinery has emerged as a strategy to increase the effectiveness of these and other DNA-damaging anti-cancer drugs. Protein kinase CK2 is a serine/threonine kinase that has emerged as an attractive molecular target due to its overexpression in cancer and regulatory role in key cellular processes including the cell cycle, apoptosis and DNA damage repair. Multiple lines of evidence suggest an increasingly important role for CK2 in the DNA damage response, including the phosphorylation and activation of the mediator/adaptor proteins XRCC1 and MDC1. XRCC1 is an essential component for nucleotide excision repair which is the major repair pathway responsible for removing platinum adducts. MDC1 is the predominant γ-H2AX recognition factor in mammalian cells and is essential for homologous recombination repair. CX-4945 is a first-in-class, selective, oral inhibitor of CK2 currently in Phase 1 clinical trials. We sought to determine if inhibiting CK2 activity with CX-4945 would prevent phosphorylation of XRCC1 and MDC1 and potentiate the activity of platinum-based drugs by preventing DNA damage repair. Treatment of the ovarian cancer cell lines A2780 and SKOV3 with CX-4945 led to decreased phosphorylation of XRCC1 at the CK2 specific phosphorylation sites and reduced total XRCC1 protein levels. Likewise, immunoprecipitation of MDC1 from SKOV3 cells treated with CX-4945 revealed significant reductions in phosphorylation at the CK2 specific sites, while in A2780 cells MDC1 protein levels were decreased. The reduction of MDC1 protein levels was reproduced by CK2 siRNA, confirming that CK2 activity supports MDC1 expression levels. Combined treatment of A2780 cells with CX-4945 and cycloheximide revealed a faster rate of MDC1 degradation than with cycloheximide alone but did not affect MDC1 mRNA levels, indicating that CK2 regulates MDC1 protein stability. When combined with cisplatin, CX-4945 enhanced the activation of CHK2 and increased levels of γ-H2AX and cleaved PARP. In antiproliferative experiments, CX-4945 exhibited synergy with cisplatin in A2780 and SKOV3 cell lines. The combination of CX-4945 with cisplatin or carboplatin significantly enhanced antitumor activity in ovarian xenograft models and was well tolerated. Thus, the inhibition of CK2 by CX-4945 enhanced the antitumor activity of platinum agents by preventing DNA damage repair and inducing apoptosis. These data provide compelling preclinical support for pursuing CX-4945 in combination with platinum chemotherapy in the clinic. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5494. doi:10.1158/1538-7445.AM2011-5494

Late Activation of Stress-activated Protein Kinases/c-Jun N-terminal Kinases Triggered by Cisplatin-induced DNA Damage in Repair-defective Cells

Although stress-activated protein kinases/c-Jun N-terminal kinases (SAPK/JNK) are rapidly activated by genotoxins, the role of DNA damage in this response is not well defined. Here we show that the SEK1/MKK4-mediated dual phosphorylation of SAPK/JNK (Thr-183/Tyr-185) correlates with the level of cisplatin-DNA adducts at late times (16-24 h) after drug treatment in both human and mouse cells. Transfection of platinated plasmid DNA also caused SAPK/JNK activation. A defect in transcription-coupled nucleotide excision repair resting on a mutation in Cockayne syndrome group B protein promoted the late SAPK/JNK activation following cisplatin exposure. Signaling to SAPK/JNK was accompanied by activation of Ataxia telangiectasia mutated- and Rad3-related kinase, replication protein A, and checkpoint kinases as well as by the formation of DNA double strand breaks (DSBs). Ionizing radiation-induced DSBs did not provoke SAPK/JNK activation, and inhibition of transcription also failed to provoke this response. Late activation of SAPK/JNK stimulated by cisplatin-induced DNA lesions was reduced in the absence of specific DNA repair proteins, such as xeroderma pigmentosum protein C, pointing to an essential function of individual repair factors in DNA damage signaling to SAPK/JNK. Collectively, the data indicate that late SAPK/JNK activation is triggered by non-repaired cisplatin adducts in transcribed genes and involves replication-associated events, DSBs, tyrosine kinases, Rho GTPases, and specific repair factors.

Overcoming biochemical pharmacologic mechanisms of platinum resistance with a texaphyrin-platinum conjugate.

In our effort to investigate further texaphyrin conjugation as a means of increasing delivery and accumulation of known anticancer platinum agents in cancer cells, we have continued our studies on the mode of action of a texaphyrin-platinum conjugate, particularly in cisplatin-resistant tumor cells that are characterized by several mechanisms of resistance, including reduced drug accumulation. Our results provide support for the proposal that intracellular platinum and Pt-DNA adduct levels were significantly increased using our conjugate relative to corresponding Pt controls. Moreover, no differences were found in cellular accumulation and Pt-DNA adduct formation between Pt sensitive and Pt resistant ovarian cells. As a result, resistance to the conjugate was lower than cisplatin in resistant cells. Based on these results we conclude that texaphyrin conjugation provides a promising strategy for overcoming biochemical pharmacologic mechanisms of resistance.

Cisplatin induced Mitochondrial DNA damage in dorsal root ganglion neurons

Cisplatin is a platinum-based chemotherapeutic agent that induces peripheral neuropathy in 30% of patients. Peripheral neuropathy is the dose limiting side effect, which has no preventative therapy. We have previously shown that cisplatin induces apoptosis in dorsal root ganglion (DRG) sensory neurons by covalently binding to nuclear DNA (nDNA), resulting in DNA damage, subsequent p53 activation and Bax-mediated apoptosis via the mitochondria. We now demonstrate that cisplatin also directly binds to mitochondrial DNA (mtDNA) with the same binding affinity as nDNA. Cisplatin binds 1 platinum molecule per 2166 mtDNA base pairs and 1 platinum molecule per 3800 nDNA base pairs. Furthermore, cisplatin treatment inhibits mtDNA replication as detected by 5-bromo-2′-deoxy-uridine (BrdU) incorporation and inhibits transcription of mitochondrial genes. The relative reduction in mtDNA transcription is directly related to the distance the gene is located from the transcription initiation point, which implies that randomly formed platinum adducts block transcription. Cisplatin treated DRG neurons exhibit mitochondrial vacuolization and degradation in vitro and in vivo. Taken together, this data suggests that direct mtDNA damage may provide a novel, distinct mechanism for cisplatin-induced neurotoxicity separate from the established nDNA damage pathway.

Consequences of Cisplatin Binding on Nucleosome Structure and Dynamics

The effects of cisplatin binding to DNA were explored at the nucleosome level to incorporate key features of the eukaryotic nuclear environment. An X-ray crystal structure of a site-specifically platinated nucleosome carrying a 1,3-cis-{Pt(NH₃)₂}²+-d(GpTpG) intrastrand cross-link reveals the details of how this adduct dictates the rotational positioning of DNA in the nucleosome. Results from in vitro nucleosome mobility assays indicate that a single platinum adduct interferes with ATP-independent sliding of DNA around the octamer core. Data from in vitro transcription experiments suggest that RNA polymerases can successfully navigate along cisplatin-damaged DNA templates that contain nucleosomes, but stall when the transcription elongation complex physically contacts a platinum cross-link located on the template strand. These results provide information about the effects of cisplatin binding to nuclear DNA and enhance our understanding of the mechanism of transcription inhibition by platinum anticancer compounds.

ChemInform Abstract: Effects of Asymmetry and Bulky Substituents in Amine Ligands of cis-Di( amine)dichloroplatinum Complexes on the Structure of Adducts Formed with Adjacent Guanines in Nucleic Acids.

Platinum adducts obtained from the reaction of 3′-O,5′-O-phosphodiguanosine, r(GpG), with the asymmetric complexes cis-[PtCl2L(L′)](L = NH3; L′= alkylamine such as NH2Me, NH2Et, NHMe2and NHEt2; LL′=N,N-dimethylethylenediamine) have been structurally characterized by 1H NMR techniques. Two different adducts, in which platinum co-ordinates to the N7 sites of both guanines, were obtained as geometrical isomers, one with the NH3group cis to the 5′ base and the other with the alkylamine cis to it. Apparently, the structures of these platinum adducts, cis-[PtL(L′)(GpG-N7,N7)]+, are similar to that of the known cis-[Pt(NH3)2(GpG-N7,N7)]+, i.e. with an anti-anti configuration and with the N conformer of the sugar as the 5′ residue. However, the asymmetric character of the non-leaving group induces a slight but significant deviation in the relative orientation of the two guanines which increases with increasing bulkiness of the alkylamine group.

Fever-range whole body thermotherapy combined with oxaliplatin: A curative regimen in a pre-clinical breast cancer model

Purpose: Studies were conducted to test whether fever-range whole body thermal therapy would boost the efficacy of oxaliplatin chemotherapy without substantial toxicity.Materials and methods: The effect of mild heat (40°C) on oxaliplatin cytotoxicity, cellular uptake, and platinum-DNA adduct formation was studied in vitro using the MTLn3 tumour cell line. In vivo oxaliplatin was administered at various doses and times before, during and after fever-range thermal therapy (6 h at 40°C) to rats bearing an MTLn3 mammary adenocarcinoma. Tumour growth, survival, and toxicity were measured to determine treatment outcome.Results: Heating halved the oxaliplatin IC-50 dose for MTLn3 cells. Cellular uptake of platinum and platinum adducts increased by 34% and 36%, respectively, with heat. In vivo, 50% of all rats given 10 mg/kg oxaliplatin 24 h before thermal therapy were completely immunologically cured, while a further 11% regressed their primary tumour but ultimately succumbed to metastases, and 17% experienced a limited response with increased survival. The curative response occurred only in a narrow range of doses, with most cures at 10 mg/kg. Thermochemotherapy-treated, but uncured, animals had delayed incidence and slowed growth of metastases. Anti-tumour efficacy was greatest, and toxicity was least, when oxaliplatin was administered 12 or 24 h before fever-range whole body thermal therapy.Conclusions: When properly dosed and scheduled, oxaliplatin thermochemotherapy achieved permanent eradication of all primary and metastatic tumours in 50% of animals, seemingly through an immune response. Successful clinical translation of this protocol would yield hitherto unseen cures and substantial improvement in quality of life.

Genotoxic effects of irinotecan combined with the novel platinum(II) complexes in human cancer cells

Combination chemotherapy has become increasingly important as synergistic drugs enable to achieve therapeutic effects at substantially lower doses and limited spectrum of side effects. Irinotecan as a one of the camptothecin analogues has shown a broad spectrum of antitumor activity against various malignancies. It is commonly used in treatment of gastrointestinal and pulmonary cancer. In this study were measured the effect of the novel platinum(II) complexes: cis-[PtCl 2(4-pmOpe) 2] and trans-[PtCl 2(4-pmOpe) 2], with diethyl (pyridine-4-ylmethyl)phosphates (4-pmOpe) as non-leaving ligands, on genotoxicity of irinotecan in human cancer cells. Irinotecan showed genotoxic activity in combination with the new platinum(II) derivatives in cancer cells. Combination of irinotecan with cis-[PtCl 2(4-pmOpe) 2] or trans-[PtCl 2(4-pmOpe) 2] resulted in significant increase in DNA damage in A549 and HT29 cells when compared to effects of irinotecan or platinum(II) complexes used separately. The highest degree of DNA migration in the comet tails was found after the cancer cells were treated with irinotecan and trans-[PtCl 2(4-pmOpe) 2]. In addition, the analysis of DNA repair revealed that irinotecan in combination with trans-[PtCl 2(4-pmOpe) 2] induced unrepairable DNA damage suggesting the poor recognition of the damage by HMG proteins and other repair mechanisms. Thus, genotoxicity of irinotecan was modulated by the structurally different DNA–platinum adducts formed by novel platinum(II) complexes. These data suggest that the use of irinotecan with novel platinum(II) complexes may represent a new strategy for pharmacotherapy in cancer.

Platinum(II) Complexes with Bioactive Carrier Ligands Having High Affinity for the Translocator Protein

Peripheral benzodiazepine receptors (PBRs, also named TSPO) are overexpressed in many tumor types, with the grade of TSPO overexpression correlating with the malignancy of the tumor. For this reason, TSPO-binding ligands have been widely explored as carriers for receptor-mediated drug delivery. In this paper we have selected a ligand with nanomolar affinity for TSPO, [2-(4-chlorophenyl)-8-aminoimidazo[1,2-a]pyridin-3-yl]-N,N-di-n-propylacetamide (3), for preparing platinum adducts that are structural analogues to picoplatin, cis-[PtCl(2)(NH(3))(2-picoline)] (AMD0473, 6), a platinum analogue currently in advanced clinical investigation. In vitro studies assessing receptor binding and cytotoxicity against human and rat glioma cells have shown that the new compounds cis-[PtX(2)(NH(3)){[2-(4-chlorophenyl)-8-aminoimidazo[1,2-a]pyridin-3-yl]-N,N-di-n-propylacetamide}] (X = I, 4; X = Cl, 5) keep high affinity and selectivity for TSPO (nanomolar concentration) and are as cytotoxic as cisplatin. Moreover, they appear to be equally active against sensitive and cisplatin-resistant A2780 cells. Similar to cisplatin, these compounds induce apoptosis but show a favorable 10- to 100-fold enhanced accumulation in the glioma cells.

Platination of telomeric DNA by cisplatin disrupts recognition by TRF2 and TRF1

Telomeres, the nucleoprotein complexes located at the ends of chromosomes, are involved in chromosome protection and genome stability. Telomeric repeat binding factor 1 (TRF1) and telomeric repeat binding factor 2 (TRF2) are the two telomeric proteins that bind to duplex telomeric DNA through interactions between their C-terminal domain and several guanines of the telomeric tract. Since the antitumour drug cisplatin binds preferentially to two adjacent guanines, we have investigated whether cisplatin adducts could affect the binding of TRF1 and TRF2 to telomeric DNA and the property of TRF2 to stimulate telomeric invasion, a process that is thought to participate in the formation of the t-loop. We show that the binding of TRF1 and TRF2 to telomeric sequences selectively modified by one GG chelate of cisplatin is markedly affected by cisplatin but that the effect is more drastic for TRF2 than for TRF1 (3-5-fold more sensitivity for TRF2 than for TRF1). We also report that platinum adducts cause a decrease in TRF2-dependent stimulation of telomeric invasion in vitro. Finally, in accordance with in vitro data, analysis of telomeric composition after cisplatin treatment reveals that 60% of TRF2 dissociate from telomeres.

Enzymatic Processing of Platinated RNAs

The broadly prescribed antitumor drug cisplatin coordinates to DNA, altering the activity of cellular proteins whose functions rely upon sensing DNA structure. Cisplatin is also known to coordinate to RNA, but the effects of RNA-Pt adducts on the large number of proteins that process the transcriptome are currently unknown. In an effort to address how platination of an RNA alters the function of RNA processing enzymes, we have determined the influence of [Pt(NH(3))(2)](2+)-RNA adducts on the activities of 3'-->5' and 5'-->3' phosphodiesterases, a purine-specific endoribonuclease, and a reverse transcriptase. Single Pt(II) adducts on RNA oligonucleotides of the form (5'-U(6)-XY-U(5)-3': XY = GG, GA, AG, GU) are found to block exonucleolytic digestion. Similar disruption of endonucleolytic cleavage is observed, except for the platinated XY = GA RNA where RNase U2 uniquely tolerates platinum modification. Platinum adducts formed with a more complex RNA prevent reverse transcription, providing evidence that platination is capable of interfering with RNA's role in relaying sequence information. The observed disruptions in enzymatic activity point to the possibility that cellular RNA processing may be similarly affected, which could contribute to the cell-wide effects of platinum antitumor drugs. Additionally, we show that thiourea reverses cisplatin-RNA adducts, providing a chemical tool for use in future studies regarding cisplatin targeting of cellular RNAs.

Polymerization by DNA polymerase is blocked by cis-diamminedichloroplatinum(II) 1,3-d(GpTpG) cross-link: implications for cytotoxic effects in nucleotide excision repair-negative tumor cells

cis-Diamminedichloroplatinum(II) (cisplatin) forms DNA adducts that interfere with replication and transcription. The most common adducts formed in vivo are 1,2-intrastrand d(GpG) cross-links (Pt-GG) and d(ApG) cross-links (Pt-AG), with minor amounts of 1,3-d(GpNpG) cross-links (Pt-GNG), interstrand cross-links and monoadducts. Although the relative contribution of these different adducts to toxicity is not known, literature implicates that Pt-GG and Pt-AG adducts block replication. Thus, nucleotide excision repair (NER), by which platinum adducts are excised, and translesion DNA synthesis (TLS), which permits adduct bypass, are thought to be associated with cisplatin resistance. Recent studies have reported that the clinical benefit from platinum-based chemotherapy is high if tumor cells express low levels of NER factors. To investigate the role of platinum-DNA adducts in mediating tumor cell survival by TLS, we examined whether 1,3-intrastrand d(GpTpG) platinum cross-links (Pt-GTG), which probably exist in NER-negative tumor cells but not in NER-positive tumor cells, are bypassed by the translesion DNA polymerase eta (pol eta), which is known to bypass Pt-GG. We show that pol eta can incorporate the correct deoxycytidine triphosphate opposite the first 3'-cross-linked G of Pt-GTG but cannot insert any nucleotides opposite the second intact T or the third 5'-cross-linked G of the adducts, thereby suggesting that TLS does not facilitate replication past Pt-GTG adducts. Thus, our findings implicate Pt-GNG adducts as mediating the cytotoxicity of platinum-DNA adducts in NER-negative tumors in vivo.

Abstract A125: Profound impact of the nucleotide excision repair proteins XPC and CSB, but not ERCC1, on the resistance to an antitumor alkylator destabilizing local DNA duplex structure

Abstract Backgound: Nucleotide excision repair (NER) is one of our most versatile DNA repair pathways. NER plays an important role in the repair of platinum adducts, and expression levels of the ERCC1 endonuclease, the last step in the NER process, is widely considered as a useful clinical marker for NER activity. S23906 is an antitumor alkaloid forming bulky monofunctional adducts with the capacity to destabilize local base pairing, which is a unique feature among anticancer agents. The purpose of the present study was to determine the influence of NER on the sensitivity to this unusual agent. Experimental: A panel of primary and transformed cell lines derived from patients with xeroderma pigmentosum or Cokayne syndrome was used to determine the role of each step in the NER pathway on the response to S23906. KB carcinoma cells with acquired resistance to S23906 were used for further characterization. Results: We here show that cells deficient for either global genome NER or transcription-coupled NER are profoundly sensitive to S23906, which for cells deficient in XPC, CSB and XPA far exceed the sensitivity observed for cisplatin and UV, two classical NER substrates. In contrast, cells deficient in ERCC1 and XPF, the last step in the NER pathway, showed modest sensitivity to S23906, compared to wt cells. Acquired resistance to S23906 was accompanied by cross-resistance to cisplatin and UV irradiation as well as by an important increase in NER activity as determined by unscheduled DNA synthesis and transcriptional recovery. Accordingly, resistant cells show up to 5-fold upregulation of XPC, CSB and XPA proteins. In contrast, no detectable alterations were observed for ERCC1. Conclusions: These results show that upregulation of NER activity in cells with acquired resistance to S23906 is associated with upregulation of XPC and CSB, the two proteins needed for the initial steps of the NER pathway, but without detectable changes in ERCC1 expression, which is needed for the last step of the pathway. Our results suggest that ERCC1 is neither rate-limiting nor representative of NER activity, and is unlikely to be of value as a predictive marker for bulky monofunctional compounds. We further suggest that the value of ERCC1 as a predictive marker for platinum compounds and other cross-linking agents may be based on its functions in recombination repair, cross link repair and telomere maintenance, rather than its role in NER. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A125.

Abstract C93: Novel monosubstituted oxaliplatin analogs with improved characteristics

Abstract Background: Oxaliplatin has unique activity against colon cancer cells distinctly different from cisplatin. Aim of this study was to determine the anticancer properties of new mono-substituted oxaliplatin analogs, namely KP1537 and KP1691, to define the underlying cellular and molecular mechanisms. Methods: Cytotoxic/antiproliferative effects were tested against selected human cancer cell lines by MTT analysis. The impact of the DNA damage response pathway on the drug activities were determined using HCT-116 human colon carcinoma cells together with p53 or p21/WAF1 deleted sublines. Furthermore, DNA damage, cell cycle distribution and induction of apoptosis were analysed by Western Blotting, FACS analysis and immunofluorescence. Platinum accumulation was determined by ICP-MS. For in vivo toxicity experiments, mice were treated with KP1691 and oxaliplatin. Results: Both oxaliplatin analogues had IC50 values in the low µM range, with those for KP1691 being significantly lower in comparison to oxaliplatin. Additionally, KP1691 was less affected by the p53 status of the cells as compared to oxaliplatin. The new compounds were more rapidly taken up by the tumor cells and accumulated over time to higher intracellular concentrations as compared to oxaliplatin. In HCT-116 cells all platinum drugs inhibited the DNA synthesis in a p53-dependent manner, associated with a distinct induction of p53 and p21 in the HCT-116 p53 cell model. This suggests that inhibition of DNA replication due to platinum adducts leads to a p53-dependent cell cycle arrest. KP1537 and KP1691 induced potent G1/S and G2/M arrests in parental HCT-116. While at wild-type p53 conditions the platinum drugs resulted in almost complete loss of S-phase, this fraction was widely unchanged in the p53- deleted subline. Accordingly, expression of cellcycle related genes including cyclin A, cyclin B1, and CDK1 were down-regulated in a p53-dependent manner. Platinum drug-induced DNA damage was confirmed by the phosphorylation of H2AX. First in vivo toxicity experiments in mice indicated significantly less side effects (weight loss) after KP1691 than after oxaliplatin treatment. Conclusion: Taken together, these findings suggest that KP1537 and KP1691 have promising anticancer properties in vitro. Several aspects, including the lower IC50 values, higher accumulation rates and less side effects of KP1537 and KP1691 as compared to the parental compound oxaliplatin, suggest them for further (pre-) clinical development. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C93.

Cisplatin Adducts on a GGG Sequence within a DNA Duplex Studied by NMR Spectroscopy and Molecular Dynamics Simulations

The antitumor drug cisplatin(cis-[PtCl2(NH3)2]) reacts with cellular DNA to form GG intrastrand adducts between adjacent guanines as predominant lesions. GGG sites have been shown to be hotspots of platination. To study the structural perturbation induced by binding of cisplatin to two adjacent guanines of a GGG trinucleotide,we examined here the decanucleotide duplex d[(G1C2C3G*4 G*5 G6T7-C8G9C10).d(G11C12G13A14C15C16C17G18-G19C20)] (dsCG*G*G) intrastrand cross-linked at the G* guanines by cis-{Pt(NH3)2}2+ using NMR spectroscopy and molecular dynamics (MD) simulations.The NMR spectra of dsCG*G*G were found to be similar to those of previously characterized DNA duplexes cross-linked by cisplatin at apyG*G*X site (py=pyrimidine; X=C,T, A). This similarity of NMR spectra indicates that the base at the 3'-side of the G*G*-Pt cross-link does not affect the structure to a large extent. An unprecedented reversible isomerization between the duplex dsCG*G*G (bearing a G*4 G*5 -Pt chelate) and duplex dsGG*G*T (bearing a G*5 G*6 -Pt chelate)was observed, which yielded a 40:60 equilibrium between the two intrastrand GG-Pt cross-links. No formation of interstrand cross-links was observed.NMR spectroscopic data of dsCG*G*G indicated that the deoxyribose of the 5'-G* adopts an N-type conformation, and the cytidines C3, C15,and C16 have average phase angles intermediate between S and N. The NMR spectroscopic chemical shifts of dsGG*G*T showed some fundamental differences to those of pyG*G*-platinum adducts but were in agreement with the NMR spectra reported previously for the DNA duplexes crosslinked at an AG*G*C sequence by cisplatin or oxaliplatin. The presence of apurine instead of a pyrimidine at the 5'-side of the G*G* cross-link seems therefore to affect the structure of the XG* step significantly.

Photoaffinity isolation and identification of proteins in cancer cell extracts that bind to platinum-modified DNA.

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.

Expanding the chemical nature of siRNAs: Oxaliplatin as metalation reagent

Short interfering RNAs (siRNAs) can down-regulate protein production by site specific degradation of mRNA. We here report the in vitro efficiency of three siRNAs metalated with oxaliplatin. All siRNAs were selectively platinated in the sense strand, and designed to target the AU-rich 3′ UTR region of Wnt-5a mRNA cloned into a luciferase reporter plasmid. Two of the studied siRNAs reveal luciferase protein suppression levels well above 90% when used in nano-molar concentrations for both metalated and the corresponding native siRNA. The platinated siRNAs were also characterized with respect to thermal melting properties, and the number of platinum adducts on the different sense strands were determined by MALDI-ToF MS. In all cases, platination was accompanied by a decrease in melting temperature. Further, the dominating oxaliplatin metalation site was the r(GpG)-adduct. The study indicates that metalation can be used as a general strategy to further expand the chemical nature of siRNAs.