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1.  Platinum(II)-Acyclovir Complexes: Synthesis, Antiviral and Antitumour Activity 
Metal-Based Drugs  1995;2(5):249-256.
A platinum(II) complex with the antiviral drug acyclovir was synthesized and its antiviral and anticancer properties were investigated in comparison to those of acyclovir and cisplatin. The platinum-acyclovir complex maintained the antiviral activity of the parent drug acyclovir, though showing a minor efficacy on a molar basis (ID50  =   7.85 and 1.02 μΜ for platinum-acyclovir and cisplatin, respectively). As anticancer agent, the platinum-acyclovir complex was markedly less potent than cisplatin on a mole-equivalent basis, but it was as effective as cisplatin when equitoxic dosages were administered in vivo to P388 leukaemia-bearing mice (%T/C = 209 and 211 for platinum-acyclovir and cisplatin, respectively). The platinum-acyclovir complex was also active against a cisplatin-resistant subline of the P388 leukaemia (%T/C = 140), thus suggesting a different mechanism of action. The DNA interaction properties (sequence specificity and interstrand cross-linking ability) of platinum-acyclovir were also investigated in comparison to those of cisplatin and [Pt(dien)Cl]+, an antitumour-inactive platinum-triamine compound. The results of this study point to a potential new drug endowed, at the same time, with antiviral and anticancer activity and characterized by DNA interaction properties different from those of cisplatin.
PMCID: PMC2364982  PMID: 18472776
2.  Revealing Three Stages of DNA-Cisplatin Reaction by a Solid-State Nanopore 
Scientific Reports  2015;5:11868.
The dynamic structural behavior in DNA due to interaction with cisplatin is essential for the functionality of platinum-based anti-cancer drugs. Here we report a novel method to monitor the interaction progress in DNA-cisplatin reaction in real time with a solid-state nanopore. The interaction processes are found to be well elucidated by the evolution of the capture rate of DNA-cisplatin complex, which is defined as the number of their translocation events through the nanopore in unit time. In the first stage, the capture rate decreases rapidly due to DNA discharging as the positive-charged hydrated cisplatin molecules initially bond to the negative-charged DNA and form mono-adducts. In the second stage, by forming di-adducts, the capture rate increases as DNA molecules are softened, appears as the reduced persistence length of the DNA-cisplatin adducts. In the third stage, the capture rate decreases again as a result of DNA aggregation. Our study demonstrates a new single-molecule tool in exploring dynamic behaviors during drug-DNA reactions and may have future application in fast drug screening.
PMCID: PMC4493569  PMID: 26148968
3.  Identification of genes associated with cisplatin resistance in human oral squamous cell carcinoma cell line 
BMC Cancer  2006;6:224.
Cisplatin is widely used for chemotherapy of head and neck squamous cell carcinoma. However, details of the molecular mechanism responsible for cisplatin resistance are still unclear. The aim of this study was to identify the expression of genes related to cisplatin resistance in oral squamous cell carcinoma cells.
A cisplatin-resistant cell line, Tca/cisplatin, was established from a cisplatin-sensitive cell line, Tca8113, which was derived from moderately-differentiated tongue squamous cell carcinoma. Global gene expression in this resistant cell line and its sensitive parent cell line was analyzed using Affymetrix HG-U95Av2 microarrays. Candidate genes involved in DNA repair, the MAP pathway and cell cycle regulation were chosen to validate the microarray analysis results. Cell cycle distribution and apoptosis following cisplatin exposure were also investigated.
Cisplatin resistance in Tca/cisplatin cells was stable for two years in cisplatin-free culture medium. The IC50 for cisplatin in Tca/cisplatin was 6.5-fold higher than that in Tca8113. Microarray analysis identified 38 genes that were up-regulated and 25 that were down-regulated in this cell line. Some were novel candidates, while others are involved in well-characterized mechanisms that could be relevant to cisplatin resistance, such as RECQL for DNA repair and MAP2K6 in the MAP pathway; all the genes were further validated by Real-time PCR. The cell cycle-regulated genes CCND1 and CCND3 were involved in cisplatin resistance; 24-hour exposure to 10 μM cisplatin induced a marked S phase block in Tca/cisplatin cells but not in Tca8113 cells.
The Tca8113 cell line and its stable drug-resistant variant Tca/cisplatin provided a useful model for identifying candidate genes responsible for the mechanism of cisplatin resistance in oral squamous cell carcinoma. Our data provide a useful basis for screening candidate targets for early diagnosis and further intervention in cisplatin resistance.
PMCID: PMC1586025  PMID: 16978399
4.  DNA-Bound Platinum Is the Major Determinant of Cisplatin Sensitivity in Head and Neck Squamous Carcinoma Cells 
PLoS ONE  2013;8(4):e61555.
The combination of systemic cisplatin with local and regional radiotherapy as primary treatment of head and neck squamous cell carcinoma (HNSCC) leads to cure in approximately half of the patients. The addition of cisplatin has significant effects on outcome, but despite extensive research the mechanism underlying cisplatin response is still not well understood.
We examined 19 HNSCC cell lines with variable cisplatin sensitivity. We determined the TP53 mutational status of each cell line and investigated the expression levels of 11 potentially relevant genes by quantitative real-time PCR. In addition, we measured cisplatin accumulation and retention, as well as the level of platinum-DNA adducts.
We found that the IC50 value was significantly correlated with the platinum-DNA adduct levels that accumulated during four hours of cisplatin incubation (p = 0.002). We could not find a significant correlation between cisplatin sensitivity and any of the other parameters tested, including the expression levels of established cisplatin influx and efflux transporters. Furthermore, adduct accumulation did not correlate with mRNA expression of the investigated influx pumps (CTR1 and OCT3) nor with that of the examined DNA repair genes (ATR, ATM, BRCA1, BRCA2 and ERCC1).
Our findings suggest that the cisplatin-DNA adduct level is the most important determinant of cisplatin sensitivity in HNSCC cells. Imaging with radio-labeled cisplatin might have major associations with outcome.
PMCID: PMC3629194  PMID: 23613873
5.  Rapid Cross-Linking of an RNA Internal Loop by the Anticancer Drug Cisplatin 
Cisplatin is the most prominent member of a series of platinum(II) antitumor drugs that demonstrate activity based on binding to adjacent purines on genomic DNA. The interactions between cisplatin and alternate biomolecules, including chemically similar RNA, are less understood than are those for DNA. In order to investigate potential implications of platinum(II) drug binding to a structurally complex RNA, we have characterized the reaction between cisplatin and the internal loop of a 41-nucleotide subdomain derived from the U2:U6 spliceosomal RNAs. This “BBD” RNA subdomain consists of a hairpin structure containing a purine-rich asymmetric internal loop. Aquated cisplatin is found to cross-link G nucleobases on opposing sides of the internal loop, forming an intramolecular internal loop cross-link in BBD and an analogous intermolecular cross-link in a two-piece construct containing the same internal loop sequence. The two opposing guanine residues involved in the cross-link were identified via limited alkaline hydrolysis. The kinetics of aquated cisplatin binding to the BBD RNA, a related RNA hairpin, and its DNA hairpin analogue were investigated in an ionic background of 0.1 M NaNO3 and 1 mM Mg(NO3)2. Both BBD and the RNA hairpin react 5–6-fold faster than the DNA hairpin, with calculated second-order rate constants of 2.0(2), 1.7(3), and 0.33(3) M−1 s−1, respectively, at 37 °C, pH 7.8. MALDI-MS data corroborate the biochemical studies and support a model in which kinetically preferred platinum binding sites compete with less reactive sites in these oligonucleotides. Taken together, these data indicate that cisplatin treatment has potential to create internal loop and other unusual cross-links in structurally complex RNAs, on a time scale that is relevant for RNA-dependent biological processes.
PMCID: PMC2822432  PMID: 19566097
6.  Use of Top-down and Bottom-up Fourier Transform Ion Cyclotron Resonance Mass Spectrometry for Mapping Calmodulin Sites modified by Platinum Anticancer Drugs 
Analytical chemistry  2011;83(24):9507-9515.
Calmodulin (CaM) is a highly conserved, ubiquitous, calcium-binding protein; it binds to and regulates many different protein targets, thereby functioning as a calcium sensor and signal transducer. CaM contains 9 methionine (Met), 1 histidine (His), 17 aspartic acid (Asp), and 23 glutamine acid (Glu) residues, all of which can potentially react with platinum compounds; thus, one third of the CaM sequence is a possible binding target of platinum anticancer drugs, which represents a major challenge for identification of specific platinum modification sites. Here, top-down electron capture dissociation (ECD) was used to elucidate the transition metal-platinum(II) modification sites. By using a combination of top-down and bottom-up mass spectrometric (MS) approaches, ten specific binding sites for mononuclear complexes, cisplatin and [Pt(dien)Cl]Cl, dinuclear complex [{cis-PtCl2(NH3)}2(μ-NH2(CH2)4NH2)] on CaM were identified. High resolution MS of cisplatin-modified CaM revealed that cisplatin mainly targets Met residues in solution at low molar ratios of cisplatin-CaM (2:1), by cross-linking Met residues. At a high molar ratio of cisplatin:CaM (8:1), up to 10 platinum(II) bind to Met, Asp, and Glu residues. [{cis-PtCl2(NH3)}2(μ-NH2(CH2)4NH2)] forms mononuclear adducts with CaM. The alkanediamine linker between the two platinum centres dissociates due to a trans-labilization effect. [Pt(dien)Cl]Cl forms {Pt(dien)}2+ adducts with CaM, and the preferential binding sites were identified as Met51, Met71, Met72, His107, Met109, Met124, Met144, Met145, Glu45 or Glu47, and Asp122 or Glu123. The binding of these complexes to CaM, particularly when binding involves loss of all four original ligands, is largely irreversible which could result in their failure to reach the target DNA or be responsible for unwanted side-effects during chemotherapy. Additionally, the cross-linking of cisplatin to CaM might lead to the loss of the biological function of CaM or CaM-Ca2+ due to limiting the flexibility of the CaM or CaM-Ca2+ complex to recognize target proteins or blocking the binding region of target proteins to CaM.
PMCID: PMC3237854  PMID: 22032417
7.  Systems biology of cisplatin resistance: past, present and future 
Cell Death & Disease  2014;5(5):e1257-.
The platinum derivative cis-diamminedichloroplatinum(II), best known as cisplatin, is currently employed for the clinical management of patients affected by testicular, ovarian, head and neck, colorectal, bladder and lung cancers. For a long time, the antineoplastic effects of cisplatin have been fully ascribed to its ability to generate unrepairable DNA lesions, hence inducing either a permanent proliferative arrest known as cellular senescence or the mitochondrial pathway of apoptosis. Accumulating evidence now suggests that the cytostatic and cytotoxic activity of cisplatin involves both a nuclear and a cytoplasmic component. Despite the unresolved issues regarding its mechanism of action, the administration of cisplatin is generally associated with high rates of clinical responses. However, in the vast majority of cases, malignant cells exposed to cisplatin activate a multipronged adaptive response that renders them less susceptible to the antiproliferative and cytotoxic effects of the drug, and eventually resume proliferation. Thus, a large fraction of cisplatin-treated patients is destined to experience therapeutic failure and tumor recurrence. Throughout the last four decades great efforts have been devoted to the characterization of the molecular mechanisms whereby neoplastic cells progressively lose their sensitivity to cisplatin. The advent of high-content and high-throughput screening technologies has accelerated the discovery of cell-intrinsic and cell-extrinsic pathways that may be targeted to prevent or reverse cisplatin resistance in cancer patients. Still, the multifactorial and redundant nature of this phenomenon poses a significant barrier against the identification of effective chemosensitization strategies. Here, we discuss recent systems biology studies aimed at deconvoluting the complex circuitries that underpin cisplatin resistance, and how their findings might drive the development of rational approaches to tackle this clinically relevant problem.
PMCID: PMC4047912  PMID: 24874729
BCL-2; carboplatin; CTR1; DNA damage response; oxaliplatin; p53
8.  A polymerase chain reaction-based method to detect cisplatin adducts in specific genes. 
Nucleic Acids Research  1991;19(22):6209-6214.
Every bulky lesion in DNA can potentially inhibit the Taq DNA polymerase and thereby decrease the amplification produced in the polymerase chain reaction. We investigated the feasibility of using this inhibition to quantify DNA lesions produced by the anticancer drug cisplatin. Products were detected by electrophoresis followed by ethidium bromide staining. Quantitation was obtained by including [32P]dCTP in the amplification reaction and subsequently assessing the incorporated radioactivity. Hamster genomic DNA was platinated in vitro to defined levels and amplified with primers that produce either a 150, 750 or 2,000 base pair fragment. The degree of inhibition of PCR agreed with the predicted level of DNA platination in each size of fragment, suggesting that the polymerase was inhibited by every cisplatin-induced lesion. This method was used to detect cisplatin-induced lesions in the adenine phosphoribosyltransferase gene of CHO cells. Cells were incubated with 0-125 microM cisplatin for 2 h, the DNA was purified and subjected to PCR. A significant decrease in amplification of the 2 kbp fragment was observed in DNA from cells incubated with cisplatin at 75 microM. The degree of inhibition agreed closely with the amount of DNA damage in the overall genome as measured by atomic absorption. No change was detected in amplification of the 150 base fragment which can therefore be used to normalize data for any variations between DNA samples. This assay has the same sensitivity as other methods currently used for the analysis of gene-specific damage. The advantage of this assay is that it obviates the need for specific endonuclease complexes to recognize and cleave DNA adducts as previously required when analyzing damage in specific genomic sequences.
PMCID: PMC329126  PMID: 1956780
9.  Carboplatin- and cisplatin-induced potentiation of moderate-dose radiation cytotoxicity in human lung cancer cell lines. 
British Journal of Cancer  1995;72(6):1406-1411.
The interaction between moderate-dose radiation and cisplatin or carboplatin was studied in a cisplatin-sensitive (GLC4) and -resistant (GLC4-CDDP) human small-cell lung cancer cell line. Cellular toxicity was analysed under oxic conditions with the microculture tetrazolium assay. For the platinum and radiation toxicity with the clinically relevant dose ranges applied, this assay was used to obtain information on cell survival after the treatments. Apart from effects on cell survival effects on DNA were also investigated. Configurational DNA changes could be induced by platinum drugs and thereby these drugs might change the frequency of DNA double-strand breaks (dsbs). DNA fragmentation assayed with the clamped homogeneous electric field (CHEF) technique was used as a measure for dsbs in DNA. The radiosensitising effect of the platinum drugs was expressed as enhancement ratio (ER) calculated directly from survival levels of the initial slope of the curve. The highest ER for cisplatin in GLC4 was 1.39 and in GLC4-CDDP 1.38. These were all at 75% cell survival. Carboplatin showed increased enhancement with prolonged incubation up to 1.21 in GLC4 and was equally effective as cisplatin in GLC4-CDDP. According to isobologram analysis, prolonged incubation with both platinum drugs showed at least additivity with radiation for both cell lines at clinically achievable doses. GLC4-CDDP showed cross-resistance to radiation. The radiosensitising capacity of both lung cancer cell lines was not dependent on their platinum sensitivity. The formation of dsbs in DNA directly after radiation was not influenced by pretreatment of either drug in the sensitive or in the resistant cell line. Drug treatment resulted in decreased DNA extractability in control as well as in irradiated cells. Modest enhancement ratio for radiosensitisation by platinum drugs cannot be explained on the level of dsb formation in DNA in both cell lines. Interaction of radiation with the clinically less toxic carboplatin can be improved by prolonged low-dose carboplatin exposure before irradiation and is as potent as cisplatin in the resistant lung cancer cell line. This suggests an advantage in combining radiation and carboplatin in lung cancer patients.
PMCID: PMC2034078  PMID: 8519652
10.  Evidence for different mechanisms of ‘unhooking’ for melphalan and cisplatin-induced DNA interstrand cross-links in vitro and in clinical acquired resistant tumour samples 
BMC Cancer  2012;12:436.
DNA interstrand cross-links (ICLs) are critical lesions produced by several cancer chemotherapy agents including platinum drugs and nitrogen mustards. We have previously shown in haematological (multiple myeloma) and solid tumours (ovarian cancer) that clinical sensitivity to such agents can result from a defect in DNA ICL processing leading to their persistence. Conversely, enhanced repair can result in clinical acquired resistance following chemotherapy. The repair of ICLs is complex but it is assumed that the ‘unhooking’ step is common to all ICLs.
Using a modification of the single cell gel electrophoresis (Comet) assay we measured the formation and unhooking of melphalan and cisplatin-induced ICLs in cell lines and clinical samples. DNA damage response in the form of γ-H2AX foci formation and the formation of RAD51 foci as a marker of homologous recombination were also determined. Real-time PCR of 84 genes involved in DNA damage signalling pathways was also examined pre- and post-treatment.
Plasma cells from multiple myeloma patients known to be clinically resistant to melphalan showed significant unhooking of melphalan-induced ICLs at 48 hours, but did not unhook cisplatin-induced ICLs. In ovarian cancer cells obtained from patients following platinum-based chemotherapy, unhooking of cisplatin-induced ICLs was observed at 48 hours, but no unhooking of melphalan-induced ICLs. In vitro, A549 cells were proficient at unhooking both melphalan and cisplatin-induced ICLs. γ-H2AX foci formation closely followed the formation of ICLs for both drugs, and rapidly declined following the peak of formation. RPMI8226 cells unhooked melphalan, but not cisplatin-induced ICLs. In these cells, although cross-links form with cisplatin, the γ-H2AX response is weak. In A549 cells, addition of 3nM gemcitabine resulted in complete inhibition of cisplatin-induced ICL unhooking but no effect on repair of melphalan ICLs. The RAD51 foci response was both drug and cell line specific. Real time PCR studies highlighted differences in the damage response to melphalan and cisplatin following equi-ICL forming doses.
These data suggest that the mechanisms by which melphalan and cisplatin-induced ICLs are ‘unhooked’ in vitro are distinct, and the mechanisms of clinical acquired resistance involving repair of ICLs, are drug specific.
PMCID: PMC3522549  PMID: 23020514
DNA interstrand cross-linking; Acquired drug resistance; DNA repair; DNA cross-linking agent; Melphalan; Cisplatin; Multiple myeloma; Ovarian cancer; DNA damage response
11.  β-Elemene enhances susceptibility to cisplatin in resistant ovarian carcinoma cells via downregulation of ERCC-1 and XIAP and inactivation of JNK 
International Journal of Oncology  2013;43(3):721-728.
β-Elemene is a promising new plant-derived drug with broad-spectrum anticancer activity. It also increases cisplatin cytotoxicity and enhances cisplatin sensitivity in resistant human carcinoma cells. However, little is known about the mechanism of its action. To explore the potential therapeutic application of β-elemene as a drug-resistance modulator, this study investigated the underlying mechanism of β-elemene activity in cisplatin-resistant ovarian cancer cells. β-Elemene enhanced cisplatin sensitivity to a much greater extent in chemoresistant A2780/CP70 and MCAS human ovarian carcinoma cells compared to the chemosensitive parental cell line A2780. The dose-modifying factors for cisplatin were between 35 and 60 for A2780/CP70 cells and between 1.6 and 2.5 for A2780 cells. In the cisplatin-resistant ovarian carcinoma cells, β-elemene abrogated cisplatin-induced expression of excision repair cross-complementation group-1 (ERCC-1), a marker gene in the nucleotide excision repair pathway that repairs cisplatin-caused DNA damage. In addition, β-elemene not only reduced the level of X-linked inhibitor of apoptosis protein (XIAP), but also downregulated cisplatin-mediated XIAP expression in chemoresistant cells. Furthermore, β-elemene blocked the cisplatin-stimulated increase in the level of phosphorylated c-Jun NH2-terminal kinase (JNK) in these cells. These novel findings suggest that the β-elemene enhancement of cisplatin sensitivity in human chemoresistant ovarian cancer cells is mediated at least in part through the impairment of DNA repair activity and the activation of apoptotic signaling pathways, thereby making resistant ovarian cancer cells susceptible to cisplatin-induced cell death.
PMCID: PMC3787889  PMID: 23817665
apoptosis; cisplatin resistance; β-elemene; Chinese medicine; ovarian cancer; excision repair cross-complementation group-1; X-linked inhibitor of apoptosis protein; c-Jun NH2-terminal kinase
12.  Characterization of RNA-Pt Adducts Formed from Cisplatin Treatment of Saccharomyces cerevisiae 
ACS Chemical Biology  2011;7(1):218-225.
The numerous regulatory roles of cellular RNAs suggest novel potential drug targets, but establishing intracellular drug-RNA interactions is challenging. Cisplatin (cis-diamminedichloridoplatinum(II)) is a leading anticancer drug that forms exchange-inert complexes with nucleic acids, allowing its distribution on cellular RNAs to be followed ex vivo. Although Pt adduct formation on DNA is well-known, a complete characterization of cellular RNA-Pt adducts has not been performed. In this study, the action of cisplatin on S. cerevisiae in minimal media was established with growth curves, clonogenic assays, and tests for apoptotic markers. Despite high toxicity, cisplatin-induced apoptosis in S. cerevisiae was not observed under these conditions. In-cell Pt concentrations and Pt accumulation on poly(A)-mRNA, rRNA, total RNA, and DNA quantified via ICP-MS indicate ~4–20 fold more Pt accumulation in total cellular RNA than in DNA. Interestingly, similar Pt accumulation is observed on rRNA and total RNA, corresponding to one Pt per (14,600 ± 1,500) and (5760 ± 580) nucleotides on total RNA following 100 and 200 µM cisplatin treatments, respectively. Specific Pt adducts mapped by primer extension analysis of a solvent-accessible 18S rRNA helix occur at terminal and internal loop regions and appear as soon as 1 hr post-treatment. Pt per nucleotide accumulation on poly(A)-mRNA is 4–6-fold lower than on rRNA, but could have consequences for low copy-number or highly regulated transcripts. Taken together, these data demonstrate significant accumulation of Pt adducts on cellular RNA species following in cellulo cisplatin treatment.
These and other small molecule-RNA interactions could disrupt processes regulated by RNA.
PMCID: PMC3262962  PMID: 22004017
13.  Phosphodiester-Mediated Reaction of Cisplatin with Guanine in Oligodeoxyribonucleotides 
Biochemistry  2008;47(48):12931-12938.
The cancer chemotherapeutic agent cis-diamminedichloroplatinum(II) or cisplatin reacts primarily with guanines in DNA to form 1,2-Pt-GG and 1,3-Pt-GNG intrastrand cross-links, and to a lesser extent, G-G interstrand cross-links. Recent NMR evidence has suggested that cisplatin can also form a coordination complex with the phosphodiester internucleotide linkage of DNA. We have examined the effects of the phosphodiester backbone on the reactions of cisplatin with oligodeoxyribonucleotides that lack or contain a -GTG- sequence. Cisplatin forms a stable adduct with TpT that can be isolated by reversed phase HPLC. The cis-Pt-TpT adduct contains a single Pt, as determined by atomic absorption spectroscopy (AAS) and by electrospray ionization mass spectrometry (ESI-MS), and is resistant to digestion by snake venom phosphodiesterase. Treatment of the adduct with sodium cyanide regenerates TpT. Similar adduct formation was observed when T(pT)8 was treated with cisplatin, but not when the phosphodiester linkages of T(pT)8 were replaced with methylphosphonate groups. These results suggest that the platinum may be coordinated with the oxygens of the thymine and possibly with those of the phosphodiester group. As expected reaction of a 9-mer containing a -GTG- sequence with cisplatin yielded an adduct that contained a 1,3-Pt-GTG intrastrand cross-link. However, we found that the number and placement of phosphodiesters surrounding a -GTG- sequence significantly affected intrastrand cross-link formation. Increasing the number of negatively charged phosphodiesters in the oligonucleotide, increased the amount of -GTG- platination. Surrounding the -GTG- sequence with non-ionic methylphosphonate linkages reduced or eliminated cross-link formation. These observations suggest that interactions between cisplatin and the negatively charged phosphodiester backbone may play an important role in facilitating platination of guanine nucleotides in DNA.
PMCID: PMC2646366  PMID: 18991396
14.  Overexpression of DDB2 enhances the sensitivity of human ovarian cancer cells to cisplatin by augmenting cellular apoptosis 
Cisplatin is one of the most widely used anticancer agents, displaying activity against a wide variety of tumors. However, development of drug resistance presents a challenging barrier to successful cancer treatment by cisplatin. To understand the mechanism of cisplatin resistance, we investigated the role of damaged DNA binding protein complex subunit 2 (DDB2) in cisplatin-induced cytotoxicity and apoptosis. We show that DDB2 is not required for the repair of cisplatin-induced DNA damage, but can be induced by cisplatin treatment. DDB2-deficient noncancer cells exhibit enhanced resistance to cell growth inhibition and apoptosis induced by cisplatin than cells with fully restored DDB2 function. Moreover, DDB2 expression in cisplatin-resistant ovarian cancer cell line CP70 and MCP2 was lower than their cisplatin-sensitive parental A2780 cells. Overexpression of DDB2 sensitized CP70 cells to cisplatin-induced cytotoxicity and apoptosis via activation of the caspase pathway and downregulation of antiapoptotic Bcl-2 protein. Further analysis indicates that the overexpression of DDB2 in CP70 cells downregulates Bcl-2 expression through decreasing Bcl-2 mRNA level. These results suggest that ovarian cancer cells containing high level of DDB2 become susceptible to cisplatin by undergoing enhanced apoptosis.
PMCID: PMC4180185  PMID: 20013802
cisplatin; DDB2; apoptosis; Bcl-2; cisplatin resistance
15.  New Insights into the Mechanism Underlying the Synergistic Action of Ionizing Radiation with Platinum Chemotherapeutic Drugs: The Role of Low-Energy Electrons 
International journal of radiation oncology, biology, physics  2013;87(4):10.1016/j.ijrobp.2013.06.2037.
To investigate the efficiencies of platinum chemotherapeutic drugs (Pt-drugs) in the sensitization of DNA to the direct effects of ionizing radiation and to determine the role of low-energy electrons (LEEs) in this process.
Methods and Materials
Complexes of supercoiled plasmid DNA covalently bound to either cisplatin, carboplatin or oxaliplatin were prepared in different molar ratios. Solid films of DNA and DNA modified by Pt-drugs were irradiated with either 10-KeV or 10-eV electrons. DNA damages were quantified by gel electrophoresis, and the yields for damage formation were obtained from exposure-response curves.
The presence of an average of two Pt-adducts in 3199-bp plasmid DNA increases the probability of a double-strand break by factors of 3.1, 2.5 and 2.4 for carboplatin, cisplatin and oxaliplatin, respectively. Electrons with energies of 10-eV and 10-KeV interact with Pt-adducts to preferentially enhance the formation of cluster lesions. The maximum increase in radiosensitivity per Pt-adduct is found at ratios up to 3.1 × 10−4 Pt-adducts per nucleotide which is equivalent to an average of two adducts per plasmid. Carboplatin and oxaliplatin show higher efficiencies than cisplatin in the radiosensitization of DNA. Since carboplatin and cisplatin give rise to identical reactive species which attach to DNA, carboplatin must be considered as a better radiosensitizers for equal number of Pt-adducts.
Pt-drugs preferentially enhance the formation of cluster damage to DNA induced by the direct effect of ionizing radiation and LEEs are the main species responsible for such an enhancement via the formation of electron resonances.
PMCID: PMC3817081  PMID: 23910707 CAMSID: cams3606
16.  Bifunctional Binding of Cisplatin to DNA: Why Does Cisplatin Form 1,2-Intrastrand Cross-links with AG, But Not with GA? 
The bifunctional binding of the anticancer drug cisplatin to two adjacent nucleobases in DNA is modeled using density functional theory. Previous experimental studies revealed that cisplatin binding to adjacent guanine and adenine is sensitive to nucleobase sequence. Whereas AG 1,2-intrastrand cross-links are commonly observed, the analogous GA adducts are not known. This study focuses on understanding this directional preference by constructing a full reaction profile using quantum chemical simulation methods. Monofunctional and bifunctional cisplatin adducts were generated and the transition states that connect them were located for the dinucleotides d(pApG) and d(pGpA), assuming that initial platination takes place at the guanine site. Our computer simulations reveal a significant kinetic preference for formation of the AG over the GA adduct. The activation free energies of ~23 kcal/mol for AG and ~32 kcal/mol for GA suggest that bifunctional closure is ~6 orders of magnitude faster for AG than for GA. Responsible for the stabilization of the transition state that affords the AG adduct is a strong hydrogen bond between one of the ammine ligands of cisplatin and the 5′ phosphate group of the DNA backbone. This interaction is absent in the transition state that leads to the GA adduct, because the right-handed helix of the DNA backbone places the phosphate out of reach for the ammine ligand. We found only an insignificant thermodynamic difference between AG and GA adducts and conclude that the preference of AG over GA binding is largely under kinetic control. The puckering of the deoxyribose ring plays an important role in determining the energetics of the bifunctional platination products. Whereas the 3′-nucleoside remains in the native C2′-endo/C3′-exo form of B-DNA, the deoxyribose of the 5′-nucleoside always adopts the C2′-exo/C3′-endo puckering in our simulations. A detailed analysis of the energies and structures of the bifunctional adducts revealed that the observed sugar puckering patterns are necessary for platinum to bind in a relaxed coordination geometry.
PMCID: PMC2495024  PMID: 17402732
17.  Comparison of cisplatin sensitivity and the 18F fluoro-2-deoxy 2 glucose uptake with proliferation parameters and gene expression in squamous cell carcinoma cell lines of the head and neck 
The survival of patients with locally advanced head and neck cancer is still poor, with 5-year survival rates of 24–35%. The identification of prognostic and predictive markers at the molecular and cellular level could make it possible to find new therapeutic targets and provide "taylor made" treatments. Established cell lines of human squamous cell carcinoma (HNSCC) are valuable models for identifying such markers.
The aim of this study was to establish and characterize a series of cell lines and to compare the cisplatin sensitivity and 18F fluoro-2 deoxy 2 glucose (18F-FDG) uptake of these cell lines with other cellular characteristics, such as proliferation parameters and TP53 and CCND1 status.
Explant cultures of fresh tumour tissue were cultivated, and six new permanent cell lines were established from 18 HNSCC cases. Successfully grown cell lines were analysed regarding clinical parameters, histological grade, karyotype, DNA ploidy, and index and S-phase fraction (Spf). The cell lines were further characterized with regard to their uptake of 18F-FDG, their sensitivity to cisplatin, as measured by a viability test (crystal violet), and their TP53 and CCND1 status, by fluorescence in situ hybridization (FISH), polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) with DNA sequencing and, for cyclin D1, by immunohistochemistry.
Patients with tumours that could be cultured in vitro had shorter disease-free periods and overall survival time than those whose tumours did not grow in vitro, when analysed with the Kaplan-Meier method and the log-rank test. Their tumours also showed more complex karyotypes than tumours from which cell lines could not be established. No correlation was found between TP53 or CCND1 status and 18F-FDG uptake or cisplatin sensitivity. However, there was an inverse correlation between tumour cell doubling time and 18F-FDG uptake.
In vitro growth of HNSCC cells seem to be an independent prognostic factor, with cell lines being more readily established from aggressive tumours, a phenomenon more dependent on the molecular genetic characteristics of the tumour cells than on tumour location or TNM status.
PMCID: PMC2654548  PMID: 19216775
18.  Triptolide potentiates lung cancer cells to cisplatin-induced apoptosis by selectively inhibiting the NER activity 
Biomarker Research  2015;3:17.
Cisplatin and many other platinum-based compounds are important anticancer drugs that are used in treating many cancer types. The development of cisplatin-resistant cancer cells, however, quickly diminishes the effectiveness of these drugs and causes treatment failure. New strategies that reverse cancer cell drug resistance phenotype or sensitize cancer cells to these drugs, therefore, need to be explored in order to improve platinum drug-based cancer treatment. Triptolide is a bioactive ingredient isolated from Tripterygium wilfordii, a Chinese herbal medicine. Triptolide binds to the TFIIH basal transcription factor and is required for both transcription and nucleotide excision repair (NER), a DNA repair pathway involved in repairing DNA damage generated by the platinum-based anticancer drugs.
Caspase-3 activation and cell growth inhibition assays were used to determine the effect of triptolide on cisplatin-induced apoptosis and cell growth in lung cancer cells. Real time PCR, immunoblotting, and expression of reef coral red protein were used to determine a mechanism through which the presence of triptolide increased cisplatin-induced apoptosis of the lung cancer cells.
Our caspase-3 activation studies demonstrated that the presence of low-levels of triptolide greatly increased the cisplatin-induced apoptosis of HTB182, A549, CRL5810, and CRL5922 lung cancer cells. The results of our cell growth inhibition studies revealed that the presence of low-levels triptolide itself had little effect on cell growth but greatly enhanced cisplatin-induced cell growth inhibition in both A549 and HTB182 cells. The results of our reef coral-red protein reporter expression studies indicated that the presence of low-levels triptolide did not affect expression of the reef coral-red protein from pDsRed2-C1 plasmid but greatly inhibited expression of the reef coral-red protein from cisplatin-damaged pDsRed2-C1 plasmid DNA in A549 cells. In addition, the results of our protein phosphorylation studies indicated that the presence of low-levels triptolide caused a decrease for cisplatin-induced CHK1 phosphorylation at Ser317/345 but an increase for cisplatin-induced ATM phosphorylation at Ser1981 in both HTB182 and A549 cells.
The results of our studies suggest that the presence of low-levels of triptolide potentiates lung cancer cells to cisplatin treatment by selectively inhibiting NER activity, resulting in an increase in apoptosis of the lung cancer cells.
PMCID: PMC4496860  PMID: 26161259
Cisplatin; Triptolide; Lung cancer cells; Apoptosis; NER; Inhibition of NER; Potentiation of lung cancer cells
19.  Cisplatin anti-tumour potentiation by tirapazamine results from a hypoxia-dependent cellular sensitization to cisplatin 
British Journal of Cancer  1999;80(8):1245-1251.
Tirapazamine (TPZ) is a new anticancer drug that is activated specifically at the low oxygen level typically found in solid tumours. It exhibits preferential cytotoxicity towards hypoxic cells and has been shown in preclinical studies with transplanted tumours and in phase II and III clinical trials to potentiate the anti-tumour efficacy of cisplatin without increasing its systemic toxicity. At present, the mechanism for this potentiation is unknown. Here we show that there is a schedule-dependent enhancement of cisplatin cytotoxicity by TPZ for cells in vitro that is similar to that seen with transplanted murine tumours. This cisplatin potentiation depends on the TPZ exposure being at oxygen concentrations below 1%, which are typical of many cells in tumours but not in normal tissues. Also, the interaction between TPZ and cisplatin does not occur in cells mutant in ERCC4, a protein essential for repair of DNA interstrand cross-links. Incubation of the cells with TPZ under hypoxia prior to cisplatin treatment increases cisplatin-induced DNA interstrand cross-links with kinetics suggesting that TPZ inhibits or delays repair of the DNA cross-links. In conclusion, we show that the tumour-specific potentiation of cisplatin cytotoxicity is likely the result of an interaction between TPZ and cisplatin at the cellular level that requires the low oxygen levels typical of those in solid tumours. The mechanism of the interaction appears to be through a potentiation of cisplatin-induced DNA interstrand cross-links, possibly as a result of a diminished or delayed repair of these lesions © 1999 Cancer Research Campaign
PMCID: PMC2362366  PMID: 10376978
tirapazamine; cisplatin; hypoxia; DNA interstrand cross-links; nucleotide excision repair
20.  Organometallic Palladium Complexes with a Water-Soluble Iminophosphorane Ligand as Potential Anticancer Agents 
Organometallics  2012;31(16):5772-5781.
The synthesis and characterization of a new water-soluble iminophosphorane ligand TPA=N-C(O)-2BrC6H4 (C,N-IM; TPA = 1,3,5-triaza-7-phosphaadamantane) 1 is reported. Oxidative addition of 1 to Pd2(dba)3 affords the orthopalladated dimer [Pd(μ-Br){C6H4(C(O)N=TPA-kC,N)-2}]2 (2) as a mixture of cis and trans isomers (1:1 molar ratio) where the iminophosphorane moeity behaves as a C,N-pincer ligand. By addition of different neutral or monoanionic ligands to 2, the bridging bromide can be cleaved and a variety of hydrophilic or water-soluble mononuclear organometallic palladium(II) complexes of the type [Pd{C6H4(C(O)N=TPA-kC,N)-2}(L-L)] (L-L = acac (3); S2CNMe2 (4); 4,7-Diphenyl-1,10-phenanthrolinedisulfonic acid disodium salt C12H6N2(C6H4SO3Na)2 (5)); [Pd{C6H4(C(O)N=TPA-kC,N)-2}(L)Br] (L = P(mC6H4SO3Na)3 (6); P(3-Pyridyl)3 (7)) and, [Pd(C6H4(C(O)N=TPA)-2}(TPA)2Br] (8) are obtained as single isomers. All new complexes were tested as potential anticancer agents and their cytotoxicity properties were evaluated in vitro against human Jurkat-T acute lymphoblastic leukemia cells, normal T-lymphocytes (PBMC) and DU-145 human prostate cancer cells. Compounds [Pd(μ-Br){C6H4(C(O)N=TPA-kC,N)-2}]2 (2) and [Pd{C6H4(C(O)N=TPA-kC,N)-2}(acac)] 3 (which has been crystallographically characterized) display the higher cytotoxicity against the above mentioned cancer cell lines while being less toxic to normal T-lymphocytes (peripheral blood mononuclear cells: PBMC). In addition, 3 is very toxic to cisplatin resistant Jurkat shBak indicating a cell death pathway that may be different to that of cisplatin. The interaction of 2 and 3 with plasmid (pBR322) DNA is much weaker than that of cisplatin pointing to an alternative biomolecular target for these cytotoxic compounds. All the compounds show an interaction with human serum albumin (HSA) faster than that of cisplatin.
PMCID: PMC3466594  PMID: 23066172
Palladium; water-soluble; iminophosphorane ligands; cytotoxic; leukemia; prostate cancer
21.  Mechanism of action of an orally administered platinum complex [ammine bis butyrato cyclohexylamine dichloroplatinum (IV) (JM221)] in intrinsically cisplatin-resistant human ovarian carcinoma in vitro. 
British Journal of Cancer  1994;69(1):1-7.
Intrinsic resistance to existing clinical platinum drugs is a major cause of treatment failure; moreover, these agents have the drawbacks of cross-resistance and intravenous administration. The mechanism of intrinsic cisplatin resistance and the mechanism of circumvention of intrinsic resistance by a member (JM221) of the ammine/amine platinum (IV) dicarboxylate class of platinum complex was studied in intrinsically resistant (SKOV-3) and sensitive (41M) human ovarian carcinoma cell lines. JM221 reduced the cisplatin resistance factor nine- to 2.7-fold, was more potent than cisplatin and showed marked time-dependent cytotoxicity. Cellular platinum accumulation was 20- to 40-fold greater (P < 0.001), and DNA platination was fourfold greater (P < 0.02), immediately following 2 h equimolar exposure to JM221, compared with cisplatin. DNA platinum levels decreased following cisplatin exposure with a half-life approximating 48 h in both lines, while no net removal of DNA-bound platinum was recorded following JM221 exposure. JM221 caused DNA interstrand cross-linking, but this was 10-20% less frequent with JM221 than with cisplatin when expressed as a proportion of total DNA platinum lesions. Cisplatin DNA interstrand cross-linking was twofold greater in the intrinsically sensitive line (41M) than in the resistant line (SKOV-3) over a range of concentrations and time-points. Neither cellular platinum accumulation, levels of DNA platination nor the rate of removal of DNA-bound platinum in the two cell lines related to their ninefold difference in cisplatin sensitivity. Intrinsic cisplatin resistance appears to be attributable to the inhibition of formation of bifunctional DNA lesions, while the circumvention of intrinsic resistance by JM221 seems to be the result of both improved transport properties and circumvention of DNA repair mechanisms.
PMCID: PMC1968778  PMID: 8286188
22.  Cisplatin-Induced Toxicity Is Associated with Platinum Deposition in Mouse Kidney Mitochondria in vivo and with Selective Inactivation of α-Ketoglutarate Dehydrogenase Complex in LLC-PK1 Cells† 
Biochemistry  2006;45(29):8959-8971.
The anti-cancer drug cisplatin is nephrotoxic and neurotoxic. Previous data support the hypothesis that cisplatin is bioactivated to a nephrotoxicant. The final step in the proposed bioactivation is the formation of a platinum-cysteine S-conjugate followed by a pyridoxal 5'-phosphate (PLP)-dependent cysteine S-conjugate β-lyase reaction. This reaction would generate pyruvate, ammonium and a highly reactive platinum (Pt)-thiol compound in vivo that would bind to proteins. In the present work, the cellular location and identity of the PLP-dependent cysteine S-conjugate β-lyase was investigated. Pt was shown to bind to proteins in kidneys of cisplatin-treated mice. The concentration of Pt-bound proteins was higher in the mitochondrial fraction than in the cytosolic fraction. Treatment of the mice with aminooxyacetic acid (AOAA, a PLP-enzyme inhibitor), which had previously been shown to block the nephrotoxicity of cisplatin, decreased the binding of Pt to mitochondrial proteins, but had no effect on the amount of Pt bound to proteins in the cytosolic fraction. These data indicate that a mitochondrial enzyme catalyzes the PLP-dependent cysteine S-conjugate β-lyase reaction. PLP-dependent mitochondrial aspartate aminotransferase (mitAspAT) is a mitochondrial enzyme that catalyzes β-elimination reactions with cysteine S-conjugates of halogenated alkenes. We reasoned that the enzyme might also catalyze a β-lyase reaction with the cisplatin-cysteine S-conjugate. In the present study mitAspAT was stably overexpressed in LLC-PK1 cells. Cisplatin was significantly more toxic in confluent monolayers of LLC-PK1 cells that overexpressed mitAspAT when compared to control cells containing vector alone. AOAA completely blocked the cisplatin toxicity in confluent mitAspAT-transfected cells. The Pt-thiol compound could rapidly bind proteins and inactivate enzymes in close proximity to the PLP-dependent cysteine S-conjugate β-lyase. Treatment with 50- or 100 µM cisplatin for 3 h, followed by removal of cisplatin from the medium for 24 h, resulted in a pronounced loss of α-ketoglutarate dehydrogenase complex (KGDHC) activity in both mitAspAT transfected cells and control cells. Exposure to 100 µM cisplatin resulted in a significantly greater loss of KGDHC activity in the cells overexpressing mitAspAT compared to control cells. Aconitase activity was diminished in both cell types, but only at the higher level of exposure to cisplatin. AspAT activity was also significantly decreased by cisplatin treatment. By contrast, several other enzymes (both cytosolic and mitochondrial) involved in energy/amino acid metabolism were not significantly affected by cisplatin treatment in the LLC-PK1 cells, whether or not mitAspAT was overexpressed. The susceptibility of KGDHC and aconitase to inactivation in kidney cells exposed to cisplatin metabolites may be due to the close proximity of mitAspAT to KGDHC/aconitase in mitochondria. The present findings support the hypothesis that a mitochondrial cysteine S-conjugate β-lyase converts the cisplatin-cysteine S-conjugate to a toxicant and the data are consistent with the hypothesis that mitAspAT plays a role in the bioactivation of cisplatin.
PMCID: PMC4133109  PMID: 16846239
23.  Micro-RNA expression in cisplatin resistant germ cell tumor cell lines 
Molecular Cancer  2011;10:52.
We compared microRNA expression patterns in three cisplatin resistant sublines derived from paternal cisplatin sensitive germ cell tumor cell lines in order to improve our understanding of the mechanisms of cisplatin resistance.
Three cisplatin resistant sublines (NTERA-2-R, NCCIT-R, 2102EP-R) showing 2.7-11.3-fold increase in drug resistance after intermittent exposure to increasing doses of cisplatin were compared to their parental counterparts, three well established relatively cisplatin sensitive germ cell tumor cell lines (NTERA-2, NCCIT, 2102EP). Cells were cultured and total RNA was isolated from all 6 cell lines in three independent experiments. RNA was converted into cDNA and quantitative RT-PCR was run using 384 well low density arrays covering almost all (738) known microRNA species of human origin.
Altogether 72 of 738 (9.8%) microRNAs appeared differentially expressed between sensitive and resistant cell line pairs (NTERA-2R/NTERA-2 = 43, NCCIT-R/NCCIT = 53, 2102EP-R/2102EP = 15) of which 46.7-95.3% were up-regulated (NTERA-2R/NTERA-2 = 95.3%, NCCIT-R/NCCIT = 62.3%, 2102EP-R/2102EP = 46.7%). The number of genes showing differential expression in more than one of the cell line pairs was 34 between NTERA-2R/NTERA-2 (79%) and NCCIT-R/NCCIT (64%), and 3 and 4, respectively, between these two cell lines and 2102EP-R/2102EP (about 27%). Only the has-miR-10b involved in breast cancer invasion and metastasis and has-miR-512-3p appeared to be up-regulated (2-3-fold) in all three cell lines. The hsa-miR-371-373 cluster (counteracting cellular senescence and linked with differentiation potency), as well as hsa-miR-520c/-520h (inhibiting the tumor suppressor p21) were 3.9-16.3 fold up-regulated in two of the three cisplatin resistant cell lines. Several new micro-RNA species missing an annotation towards cisplatin resistance could be identified. These were hsa-miR-512-3p/-515/-517/-518/-525 (up to 8.1-fold up-regulated) and hsa-miR-99a/-100/-145 (up to 10-fold down-regulated).
Examining almost all known human micro-RNA species confirmed the miR-371-373 cluster as a promising target for explaining cisplatin resistance, potentially by counteracting wild-type P53 induced senescence or linking it with the potency to differentiate. Moreover, we describe for the first time an association of the up-regulation of micro-RNA species such as hsa-miR-512-3p/-515/-517/-518/-525 and down-regulation of hsa-miR-99a/-100/-145 with a cisplatin resistant phenotype in human germ cell tumors. Further functional analyses are warranted to gain insight into their role in drug resistance.
PMCID: PMC3120796  PMID: 21575166
germ cell tumor cell line; cisplatin resistance; microRNA; gene expression
24.  Interaction of 2-aminopyrimidine with dichloro-[1-alkyl-2-(naphthylazo) imidazole]palladium(II) complexes : Kinetic and mechanistic studies 
The anticancer properties of cisplatin and palladium(II) complexes stem from the ability of the cis-MCl2 fragment to bind to DNA bases. However, cisplatin also interacts with non-cancer cells, mainly through bonding molecules containing -SH groups, resulting in nephrotoxicity. This has aroused interest in the design of palladium(II) complexes of improved activity and lower toxicity. The reaction of DNA bases with palladium(II) complexes with chelating N,N/donors of the cis-MCl2 configuration constitutes a model system that may help explore the mechanism of cisplatin's anticancer activity. Heterocyclic compounds are found widely in nature and are essential to many biochemical processes. Amongst these naturally occurring compounds, the most thoroughly studied is that of pyrimidine. This was one of the factors that encouraged this study into the kinetics and mechanism of the interaction of 2-aminopyrimidine (2-NH2-Pym) with dichloro-{1-alkyl-2-(α-naphthylazo)imidazole}palladium(II) [Pd(α-NaiR)Cl2, 1] and dichloro-{1-alkyl-2-(β-naphthylazo)imidazole}palladium(II) [Pd(β-NaiR)Cl2, 2] complexes where the alkyl R = Me (a), Et (b), or Bz (c).
2-NH2-Pym reacts with 1a, 1b, and 1c to yield [{1-alkyl-2-(α-naphthylazo)imidazole}bis(2-aminopyrimidine)]palladium(II) (3a, 3b, 3c) dichloride and with 2a, 2b, and 2c to yield [{1-alkyl-2-(β-naphthylazo)imidazole}bis(2-aminopyrimidine)]palladium(II) (4a, 4b, 4c) dichloride in an acetonitrile (MeCN) medium. The products were characterized using spectroscopic techniques (FT-IR, UV-Vis, NMR). The ligand substitution reactions follow second order kinetics – first order dependence on the concentration of the Pd(II) complex and 2-NH2-Pym. Addition of LiCl to the reaction does not influence its rate. The thermodynamic parameters (standard enthalpy of activation, Δ‡H° and standard entropy of activation, Δ‡S°) were determined from variable temperature kinetic studies. The magnitude of the second order rate constant, k2, at 298 K, was shown to increase thus: b
The kinetics of the reaction between Pd(II) complexes (1 and 2) and 2-NH2-Pym were examined spectrophotometrically at 530 nm in MeCN under pseudo-first-order conditions. The reaction rate is largely influenced by the π-acidity of the chelating ligand, with substitution in the naphthyl azoimidazole backbone influencing the rate of the substitution process. The activation parameters, Δ‡H° and Δ‡S°, were determined and support the kinetic rate data.
PMCID: PMC2194761  PMID: 17939858
Molecular and Cellular Biology  2006;26(24):9377-9386.
RNA interference technology allows the systematic genetic analysis of the molecular alterations in cancer cells and how these alterations affect response to therapies. Here we used small interfering RNA (siRNA) screens to identify genes that enhance the cytotoxicity (enhancers) of established anticancer chemotherapeutics. Hits identified in drug enhancer screens of cisplatin, gemcitabine, and paclitaxel were largely unique to the drug being tested and could be linked to the drug's mechanism of action. Hits identified by screening of a genome-scale siRNA library for cisplatin enhancers in TP53-deficient HeLa cells were significantly enriched for genes with annotated functions in DNA damage repair as well as poorly characterized genes likely having novel functions in this process. We followed up on a subset of the hits from the cisplatin enhancer screen and validated a number of enhancers whose products interact with BRCA1 and/or BRCA2. TP53+/− matched-pair cell lines were used to determine if knockdown of BRCA1, BRCA2, or validated hits that associate with BRCA1 and BRCA2 selectively enhances cisplatin cytotoxicity in TP53-deficient cells. Silencing of BRCA1, BRCA2, or BRCA1/2-associated genes enhanced cisplatin cytotoxicity ∼4- to 7-fold more in TP53-deficient cells than in matched TP53 wild-type cells. Thus, tumor cells having disruptions in BRCA1/2 network genes and TP53 together are more sensitive to cisplatin than cells with either disruption alone.
PMCID: PMC1698535  PMID: 17000754

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