cisplatin-DNA Adduct Formation Research Articles

Formation of monofunctional cisplatin-DNA adducts in carbonate buffer

Carbonate in its various forms is an important component in blood and the cytosol. Since, under conditions that simulate therapy, carbonate reacts with cisplatin to form carbonato complexes, one of which is taken up and/or modified by the cell [C.R. Centerwall, J. Goodisman, D.J. Kerwood, J. Am. Chem. Soc., 127 (2005) 12768–12769], cisplatin-carbonato complexes may be important in the mechanism of action of cisplatin. In this report we study the binding of cisplatin to pBR322 DNA in two different buffers, using gel electrophoresis. In 23.8 mM HEPES, N-(2-hydroxyethyl)-piperazine- N′-2-ethanesulfonic acid, 5 mM NaCl, pH 7.4 buffer, cisplatin produces aquated species, which react with DNA to unwind supercoiled Form I DNA, increasing its mobility, and reducing the binding of ethidium to DNA. This behavior is consistent with the formation of the well-known intrastrand crosslink on DNA. In 23.8 mM carbonate buffer, 5 mM NaCl, pH 7.4, cisplatin forms carbonato species that produce DNA-adducts which do not significantly change supercoiling but enhance binding of ethidium to DNA. This behavior is consistent with the formation of a monofunctional cisplatin adduct on DNA. These results show that aquated cisplatin and carbonato complexes of cisplatin produce different types of lesions on DNA and they underscore the importance of carrying out binding studies with cisplatin and DNA using conditions that approximate those found in the cell.

Prediction of Outcome by Cisplatin DNA-adduct Formation in Patients with Stage III/IV Head and Neck Squamous Cell Carcinoma, Treated by Concurrent Cisplatin-Chemoradiation (RADPLAT)

Purpose/Objective: Cisplatin-DNA adduct formation in normal tissue has been shown to correlate with outcome in patients with NSCLC treated with chemoradiation and in advanced stage solid tumors treated with chemotherapy. The purpose of the present study was to explore relationships between adduct levels in tumor and normal tissue in patients with head and neck squamous cell carcinoma (HNSCC), treated with cisplatin-based chemoradiation and investigate relationships with treatment outcome.

Molecular basis of cellular response to cisplatin chemotherapy in non-small cell lung cancer (Review)

Cisplatin is one of the most potent anticancer agents, displaying significant clinical activity against a variety of solid tumors. For more than two decades, the most effective systemic chemotherapy for non-small cell lung cancer (NSCLC), the leading cause of cancer morbidity and mortality among men and women in the western world, was cisplatin-based combination treatment. Unfortunately, the outcome of cisplatin therapy on NSCLC seems to have reached a plateau. Therefore, the biological mechanisms of cisplatin action need to be understood in order to overcome the treatment plateau on NSCLC. Moreover, the development of resistance is a hurdle in the use of this drug. The molecular mechanisms that underlie this chemoresistance are largely unknown. Possible mechanisms of acquired resistance to cisplatin include reduced intracellular accumulation of cisplatin, enhanced drug inactivation by metallothionine and glutathione, increased repair activity of DNA damage, and altered expression of oncogenes and regulatory proteins. In addition, it is generally accepted that cytotoxicity of cisplatin is mediated through induction of apoptosis and arrest of cell cycle resulting from its interaction with DNA, such as the formation of cisplatin-DNA adducts, which activates multiple signaling pathways, including those involving p53, Bcl-2 family, caspases, cyclins, CDKs, pRb, PKC, MAPK and PI3K/Akt. Increased expression of anti-apoptotic genes and mutations in the intrinsic apoptotic pathway may contribute to the inability of cells to detect DNA damage or to induce apoptosis. Towards an understanding of the molecular basis of the cellular response to cisplatin-based chemotherapy in NSCLC, in this review we provide some insights into the pathways involved in cisplatin damage from entering the cells to execution of apoptosis or survival of NSCLC cells. We believe that as more and more molecular mechanisms of response to cisplatin-based therapy are unraveled, this knowledge should provide a basis for further studies to improve our understanding of molecular events associated with lung NSCLC as well as to devise novel and effective therapeutic approaches to overcome the treatment plateau or reverse drug resistance in this disease.

The copper influx transporter human copper transport protein 1 regulates the uptake of cisplatin in human ovarian carcinoma cells.

Cells selected for resistance to cisplatin are often cross-resistant to copper and vice versa, and the major copper influx transporter copper transport protein 1 (CTR1) has been shown to regulate the uptake of cisplatin, carboplatin, and oxaliplatin in yeast. To further define the role of hCTR1 in human tumor cells, the ovarian carcinoma cell line A2780 was molecularly engineered to increase expression of hCTR1 by a factor of 20-fold. Enhanced expression of hCTR1 in the A2780/hCTR1 cells was associated with a 6.5-fold increase in basal steady-state copper content and a 13.7-fold increase in initial copper influx, demonstrating that the exogenously expressed hCTR1 was functional in altering copper homeostasis. The A2780/hCTR1 cells accumulated 46% more platinum after a 1-h exposure to 2 microM cisplatin, and 55% more after a 24 h exposure, than the control A2780/empty vector cells. The initial uptake of cisplatin was 81% higher in the A2780/hCTR1 cells when measured at 5 min. Thus, increased expression of hCTR1 had a substantially larger effect on the cellular pharmacology of copper than cisplatin. Interestingly, the increased uptake of copper and cisplatin was accompanied by only a marginal increase in sensitivity to the cytotoxic effect of copper and cisplatin, and there was no increase in the extent of cisplatin-DNA adduct formation. Thus, although increased expression of hCTR1 mediates greater cellular accumulation of copper and cisplatin, hCTR1 delivers these compounds into intracellular compartments from which they do not have ready access to their key cytotoxic targets.

The interaction of cisplatin and analogues with DNA in reconstituted chromatin

The influence of chromatin structure on cis-diamminedichloroplatinum(II) (cisplatin) DNA damage was investigated in a reconstituted nucleosome system. Nucleosomes were reconstituted on the somatic 5S rRNA gene from Xenopus borealis using the octamer transfer method of reconstitution. Footprinting techniques, utilising bleomycin and DNase I as the damaging agents, were employed to establish the precise location of positioned nucleosomes with respect to the DNA sequence. Reconstituted nucleosomal DNA was treated with cisplatin and drug-induced DNA adduct formation was quantitatively analysed with a polymerase stop assay using Taq DNA polymerase. A densitometric comparison of the relative damage band intensities between purified and reconstituted DNA revealed regions of relative protection corresponding to the sites of the positioned nucleosome cores. This indicated that the preferred site of cisplatin DNA binding was in the linker region of the nucleosome. Statistical analysis showed significant protection from cisplatin DNA damage in the core region of the nucleosome. Three cisplatin analogues were also investigated in this reconstituted nucleosome system. These analogues, cis-diammine(1,1-cyclobutanedicarboxylato)platinum(II) (carboplatin), cis-dichlorobis(cyclohexylamine)platinum(II) ( cis-[PtCl 2(C 6H 11NH 2) 2]) and dichloro( N-[3-[(2-aminoethyl)-amino]propyl]acridine-4-carboxamide)platinum(II) (ac-PtenCl 2(n3)), were also found to target the linker region of the nucleosome. The latter DNA-targeted acridine–platinum complex gave rise to the most predominant footprints of all the Pt compounds tested.

Cisplatin resistance and oncogenes--a review.

Cisplatin is among the most widely used broadly active cytotoxic anticancer drugs; however, its clinical efficacy is often limited by primary or the development of secondary resistance. Several mechanisms have been implicated in cisplatin resistance, including reduced drug uptake, increased cellular thiol/folate levels and increased DNA repair. More recently, additional pathways have been characterized indicating that altered expression of oncogenes that subsequently limit the formation of cisplatin-DNA adducts and activate anti-apoptotic pathways may also contribute to the resistance phenotype. Several lines of evidence suggest that expression of ras oncogenes can confer resistance to cisplatin by reducing drug uptake and increasing DNA repair; however, this is not a uniform finding. Tumor cells, in contrast to normal cells, respond to cisplatin exposure with transient gene expression to protect or repair their chromosomes. The c-fos/AP-1 complex, a master switch for turning on other genes in response to DNA-damaging agents, has been shown to play a major role in cisplatin resistance. In addition, AP-2 transcription factors, modulated by protein kinase A, are also implicated in cisplatin resistance by regulating genes encoding for DNA polymerase beta and metallothionines. Furthermore, considerable evidence indicates that mutated p53 plays a significant role in the development of cisplatin resistance since several genes implicated in drug resistance and apoptosis (e.g. mismatch repair, bcl-2, high mobility group proteins, DNA polymerases alpha and beta, PCNA, and insulin-like growth factor) are known to be regulated by the p53 oncoprotein. Improved understanding of molecular factors for the development of cisplatin resistance may allow the prediction of clinical response to cisplatin-based treatment. Furthermore, the identification of oncogenes involved in cisplatin resistance has already led to in vitro approaches which successfully inactivated these genes using ribozymes or antisense oligodeoxynucleotides, thus restoring cisplatin sensitivity. It is conceivable that these strategies, once transferred to a clinical setting, may have the potential to enhance the efficacy of cisplatin against a great variety of malignancies and thus more fully exploit the antineoplastic and curative potential of this drug.

Cisplatin–DNA adduct formation in rat spermatozoa and its effect on fetal development

Exposure of males to some genotoxic chemicals causes DNA damage in spermatozoa resulting in embryotoxicity and developmental defects in their offspring. This study demonstrates that cisplatin–DNA adducts could be measured in spermatozoa following treatment with the antineoplastic drug, cisplatin. The formation of spermatozoa cisplatin–DNA adducts showed dose and time-dependent increases both in vitro, and in vivo up to 168 h (7 days) after dosing. Treatment of rats with 10 mg cisplatin/kg resulted in spermatozoa Pt–GG adduct levels of approximately 1.0 fmol/μg DNA. When cisplatin-treated male rats were bred to untreated females 6–24 h after cisplatin administration, no adverse developmental effects or decreases in body weight were seen in the offspring although there was a trend towards increased early embryo mortality.

Effects of amifostine on cisplatin induced DNA adduct formation and toxicity in malignant glioma and normal tissues in rat.

The chemoprotective effect of amifostine (WR2721) was studied in a BDIX rat model with intracerebral BT4C glioma implants. Twenty-one rats were given cisplatin 5 mg/kg i.p., 21 were given amifostine 200 mg/kg i.p. + cisplatin 5 mg/kg i.p. Ten rats served as untreated controls. An immunohistochemical method for analysis of cisplatin-DNA adducts was used to elucidate the adduct formation in tumor, normal brain and kidney. Tumor volume and serum creatinine level were analysed 10 days after treatment. In animals pretreated with amifostine there was a delayed adduct formation rate in the normal brain, and in the kidney cortex the number of tubular cells with extremely high adduct level was reduced. No difference in adduct formation was seen in tumors. Tumor volume was significantly larger following amifostine + cisplatin (66% of controls) compared to cisplatin alone (38% of controls). Weight loss was, however, severe in rats given cisplatin alone. In the tumor growth study only 3 out of 11 rats treated with cisplatin 5 mg/kg alone survived until time of sacrifice at 10 days, whereas all those pretreated with amifostine survived. Mean serum creatinine was 48 micromol/l (controls), 146 micromol/l (cisplatin) and 59 micromol/l (amifostine + cisplatin). A marked reduction of histopathological renal changes was found when amifostine was added. Amifostine thus significantly reduced general and renal toxicity of cisplatin. The tumor growth retardation was stronger when cisplatin was given alone but this is probably related to general toxicity and malnutrition indirectly supported by the fact that amifostine did not significantly reduce cisplatin-DNA adduct formation in tumors. The results of the present study suggest that amifostine may have a role in increasing the therapeutic ratio of cisplatin, also in the treatment of malignant glioma.

Synergistic cytotoxicity of cisplatin and topotecan or SN-38 in a panel of eight solid-tumor cell lines in vitro.

The cytotoxicity of cisplatin alone and in combination with topotecan (TPT) or SN-38, two novel topoisomerase I (topo I) inhibitors, was determined in a panel of eight well-characterized human solid-tumor cell lines. Interactions between cisplatin and these topo I inhibitors were investigated using three different administration schedules: (1) simultaneous incubation (C + T and C + S), (2) cisplatin followed by TPT or SN-38 (C --> T and C --> S), and (3) TPT or SN-38 followed by cisplatin (T --> C and S --> C). Median-effect analysis revealed synergistic cytotoxicity in seven of the eight cell lines used. In addition, a significant schedule-dependent synergistic cytotoxicity was found in three of the cell lines used, with C --> T (or C --> S) being the most active schedule. The formation and repair of total cisplatin-DNA adducts in the IGROV-1 ovarian cancer cell line and its cisplatin-resistant subline IGROV(CDDP) was not significantly affected by TPT on simultaneous incubation. In contrast, the number of cisplatin-DNA interstrand cross-links detected in the IGROV-1 and IGROV(CDDP) lines at certain time points was significantly lower after coincubation of the cells with TPT. Assessment of the cell-cycle distribution revealed an accumulation of cells in the G2/M phase after exposure to cisplatin. After exposure to TPT a different pattern was observed that was cell-type-specific and dependent upon the TPT concentration. Although up to 4-fold differences in topo I activity were observed in this panel of cell lines, these differences did not appear to be related to the synergy observed between cisplatin and TPT or SN-38. The observed synergy may at least partly be explained by the increased retention of cisplatin-DNA interstrand cross-links in the presence of topo I inhibitors.

Intraperitoneal cisplatin with regional hyperthermia in advanced ovarian cancer: pharmacokinetics and cisplatin–DNA adduct formation in patients and ovarian cancer cell lines

The purpose of this study was to investigate the influence of hyperthermia on cisplatin pharmaco-kinetics and DNA adduct formation. The latter was investigated both in tumour cell lines in vitro and in tumour cells and buccal cells from cancer patients. The patients had advanced ovarian carcinoma and were entered into a phase I study for cytoreductive surgery followed by hyperthermia in combination with intraperitoneal cisplatin administration. The cisplatin–DNA modifications in vivo and in vitro were studied by an immunocytochemical method with the polyclonal antiserum NKI-A59. The patient samples for pharmacokinetic determinations were analysed by flameless atomic absorption spectrometry. In vitro, the combination of hyperthermia and cisplatin enhanced cell killing compared with either treatment alone, such that the cisplatin-resistant ovarian cell line A2780/DDP became almost as sensitive as the parent A2780 cell line (resistance factor reduced from 30 to 2 at the IC50). In addition, increased cisplatin–DNA adducts were observed in the resistant cell line after the combined treatment compared with cisplatin alone. A good correlation was found between nuclear staining density and surviving fraction for all groups, indicating that the DNA adducts generated are an important determinant of toxicity and that the mechanism by which hyperthermia enhances kill is by increasing adduct levels. In the patients, the ratio of drug concentration in the peritoneal perfusate compared with that in plasma was found to be approximately 15, indicating a favourable pharmacokinetic ratio. Cisplatin–DNA adduct formation in tumour cells from patients was higher than in buccal cells, reflecting this higher drug exposure, i.e. local plus systemic versus systemic only. In addition, the tumour cells but not buccal cells were exposed to hyperthermia. The higher number of tumour adducts also suggests that a favourable therapeutic ratio could be achieved. Platinum–DNA adduct formation was found to decrease with distance from the surface of the tumour nodules. However, at a distance of 3–5mm, the nuclear staining density levels were still measurable and higher than in buccal cells. In conclusion, the combined pharmacokinetic and adduct data in patients support the advantages of the intraperitoneal route for drug administration, and the addition of heat.

The formation and repair of cisplatin-DNA adducts in wild-type and cisplatin-resistant L1210 cells: comparison of immunocytochemical determination with detection in isolated DNA

We have studied the formation and repair of cisplatin-DNA adducts in wild-type mouse leukemia L1210/0 cells and in the sublines L1210/2 and L1210/5, which differ in cisplatin sensitivity. In a colony-formation assay these sublines were 9- and 22-fold more resistant compared to L1210/0, respectively. Cisplatin-induced DNA modification was studied at the cellular level by immunocytochemistry with antiserum NKI-A59 raised against cisplatin-treated DNA. Levels of nuclear staining immediately after a 1-h treatment were similar to those seen after a 24-h post-incubation in drug-free medium. Clear differences in DNA platination were found between the cell lines: immediately after exposure, L1210/2 and L1210/5 showed only 32 and 14%, respectively, of the nuclear staining observed in L1210/0, and 48 and 13% after 24 h. In these experiments, adduct-specific nuclear staining was quantified as the area under the adduct versus concentration curves (AUC). The formation and repair in these cell lines of the bifunctional adducts cis-Pt(NH 3) 2d(pGpG) (Pt-GG), cis-Pt(NH 3) 2d(pApG) (Pt-AG) and cis-Pt(NH 3) 2(dGMP) 2 (G-Pt-G) were studied with an enzyme-linked immunosorbent assay (ELISA). No relation between repair and resistance was observed. The results suggest that differences in induced DNA platination levels, rather than in repair, are responsible––at least in part––for the differences in cisplatin resistance. A mechanism such as an increased tolerance of the resistant cells to platinum-DNA damage may also be involved.

Hyperthermia enhances the cytotoxicity and platinum-DNA adduct formation of lobaplatin and oxaliplatin in cultured SW 1573 cells.

The cytotoxicity of cisplatin and cisplatin-DNA adduct formation in vitro and in vivo is clearly enhanced by hyperthermia. We investigated whether cytotoxicity and platinum-DNA adduct formation of two promising new third-generation platinum derivatives, lobaplatin [1,2-diamminomethylcyclobutane platinum(II) lactate] and oxaliplatin [oxalato-1,2-diaminocyclohexane platinum(II)], are also enhanced by hyperthermia. Cisplatin was used for comparison. SW 1573 cells were incubated with cisplatin, lobaplatin or oxaliplatin at different concentrations for 1 h at 37 degrees, 41 degrees and 43 degrees C. The reproductive capacity of cells was determined by cloning experiments. Immunocytochemical detection of platinum-DNA adducts was performed with the rabbit antiserum NKI-A59. At 37 degrees C, cisplatin was the most cytotoxic, followed by oxaliplatin and lobaplatin. Hyperthermia clearly enhanced the cytotoxicity of cisplatin, lobaplatin and oxaliplatin. There was no further increase in cytotoxicity at 43 degrees C compared to 41 degrees C for cisplatin and oxaliplatin. A further increase in cytotoxicity at 43 degrees C was observed for lobaplatin. At 43 degrees C thermal enhancement was higher for lobaplatin than for oxaliplatin, with the reverse pattern at 41 degrees C. For both drugs, thermal enhancement of cytotoxicity was lower than observed for cisplatin. Immunocytochemical detection of platinum-DNA adducts was feasible for all the drugs. Adduct formation was enhanced at 43 degrees C for cisplatin, lobaplatin and oxaliplatin with a relative increase of 410%, 170% and 180%. These results seem to confirm that an increase in platinum-DNA adduct formation is involved in the in vitro thermal enhancement of cytotoxicity. The observed thermal enhancement of cytotoxicity of lobaplatin and oxaliplatin in vitro warrants further in vivo investigations.

Elevated mitochondrial cisplatin-DNA adduct levels in rat tissues after transplacental cisplatin exposure.

Although there is evidence that the toxic effects of cis-diamminedichloroplatinum(II) (cisplatin) include morphologically abnormal mitochondria, direct demonstrations of mitochondrial DNA damage by this chemotherapeutic agent have rarely been reported. Here we show that, in rats exposed to a single dose of cisplatin during gestation, cisplatin-DNA binding levels in both maternal and fetal liver and brain mitochondrial DNA are higher than those observed in genomic DNA. Pregnant F344/NCr rats were injected i.p. with either 5 or 15 mg cisplatin/kg body wt at 18 days of gestation and killed 24 h later. Cisplatin-DNA adducts were determined by dissociation-enhanced lanthanide fluoroimmunoassay using a cisplatin-DNA standard modified in the same range as the biological samples. Values for genomic cisplatin-DNA adducts in multiple maternal and fetal tissues have been presented elsewhere. Here, genomic DNA adduct levels for liver, brain, kidney and placenta are reported again for comparison with mitochondrial DNA adduct levels in the same tissues. In maternal and fetal brain, mitochondrial DNA adduct levels were approximately 7- to 50-fold higher than genomic DNA adduct levels, and in fetal liver they were approximately 2- to 16-fold higher than genomic DNA adduct levels. These studies demonstrate extensive cisplatin-DNA adduct formation in brain and liver mitochondria of fetal rats exposed transplacentally and suggest that mitochondrial DNA in some organs may be a particular target for cisplatin genotoxicity.

Cross-resistance to cis-diamminedichloroplatinum(II) of a multidrug-resistant lymphoma cell line associated with decreased drug accumulation and enhanced DNA repair

HOB1/VCR, a multidrug-resistant subline of the immunoblastic B lymphoma cell line, was established by sequential selection in increasing concentrations of vincristine. The expression of the human mdr1 gene, as analyzed by reverse transcription and polymerase-chain reaction (RT-PCR), revealed a 10–15-fold overexpression in this resistant cell line. A complete inhibition of vincristine resistance by verapamil was observed in the vincristine-resistant HOB1/VCR cells, which suggests that acquired resistance may be mainly due to P-glycoprotein. HOB1/VCR cells also developed a 67-fold cross-resistance to the anticancer drug cis-diamminedichloroplatinum (cisplatin). DNA repair of the resistant and the parental cell lines was investigated by in situ detection with a cisplatin-DNA adduct-specific antibody and by measurement of repair-associated host cell reactivation of damaged plasmid DNA. HOB1/VCR cells exhibited a 2-fold decrease in the level of cisplatin-DNA adducts, compared to the parental cells. The DNA repair rate following peak accumulation of cisplatin-DNA adducts (which took ∼4 h) was also enhanced in the resistant cells. This was supported by the measurement of the cisplatin level remaining in cells by atomic absorption spectrophotometry, which showed a 2.7-fold reduction in the resistant cells. In addition, the acquired resistance and enhanced DNA repair in HOB1/VCR cells were partially reversed by nontoxic aphidicolin, a DNA polymerase-α and DNA repair inhibitor. Inhibition of the intracellular level of glutathione by dl-buthionine-[ S,R]-sulfoximine demonstrated that cell viability was inhibited 4-fold more in the resistant cells than in the parental cells. The results suggest that the reduced formation of cisplatin-DNA adducts and the increased glutathione content of the multidrug-resistant cells play a major role in phenotypic cross-resistance to cisplatin.

Human Ku Autoantigen Binds Cisplatin-damaged DNA but Fails to Stimulate Human DNA-activated Protein Kinase

We have identified a series of proteins based on an affinity for cisplatin-damaged DNA. One protein termed DRP-1 has been purified to homogeneity and was isolated as two distinct complexes. The first complex is a heterodimer of 83- and 68-kDa subunits, while the second complex is a heterotrimer of 350-, 83-, and 68-kDa subunits in a 1:1:1 ratio. The 83- and 68-kDa subunits in each complex are identical. The 83-kDa subunit of DRP-1 was identified as the p80 subunit of Ku autoantigen by N-terminal protein sequence analysis and reactivity with a monoclonal antibody directed against human Ku p80 subunit. The 68-kDa subunit of DRP-1 cross-reacted with monoclonal antisera raised against the Ku autoantigen p70 subunit. The 350-kDa subunit was identified as DNA-PKcs, the catalytic subunit of the human DNA-activated protein kinase, DNA-PK. DRP-1/Ku DNA binding was assessed in mobility shift assays and competition binding assays using cisplatin-damaged DNA. Results indicate that DNA binding was essentially unaffected by cisplatin-DNA adducts in the presence or absence of DNA-PKcs. DNA-PK activity was only stimulated with undamaged DNA, despite the ability of Ku to bind to cisplatin-damaged DNA. The lack of DNA-PK stimulation by cisplatin-damaged DNA correlated with the extent of cisplatin-DNA adduct formation. These results demonstrate that Ku can bind cisplatin-damaged DNA but fails to activate DNA-PK. These results are discussed with respect to the repair of cisplatin-DNA adducts and the role of DNA-PK in coordinating DNA repair processes.

Effect of DNA conformation on cisplatin adduct formation.

The anticancer drug cis-diamminedichloroplatinum(II) (cisplatin) has been shown previously to form adducts preferentially within internucleosomal or linker DNA rather than to DNA within the nucleosome. To determine whether other "open" regions of chromatin have an increased affinity for cisplatin, adduct formation within specific chromatin domains was analyzed. There was a significant increase in cisplatin-DNA adduct formation for DNA associated with the nuclear matrix (NM) compared with other chromatin domains and total unfractionated DNA. In contrast, treatment of the same cells with trans-diamminedichloroplatinum(II) (transplatin) did not result in preferential adduct formation. These findings led to the hypothesis that it might be possible to alter DNA to make it a more favorable target for cisplatin. The effect of arginine butyrate on cisplatin-DNA adduct formation was analyzed in human cancer cells. The combination of arginine butyrate and cisplatin resulted in a concentration-responsive increase in cisplatin-DNA adduct formation in PC-3 cells and an overall increase in cisplatin-DNA adduct formation in three other human cancer cell lines. The same combination also resulted in a significant increase in drug-induced cytotoxicity at a low concentration of cisplatin. These results suggest that chromatin configuration can affect cisplatin adduct formation.

Cisplatin-DNA adduct formation in maternal and fetal rat tissues after transplacental cisplatin exposure

Cis-diamminedichloroplatinum (II) (cisplatin), given to pregnant rats at 5 mg/kg body weight (bw) is a trans placental carcinogen for fetal liver, kidney, nervous system and lung, resulting in tumor incidences of 22.5, 10.5, 6.1 and 7.5% respectively, in offspring grown to adulthood (B.A. Diwan et al., 1995, Toxicol. Appl. Pharm., 132, 115). In this study, the capacity of cisplatin to pass through the placental barrier and bind covalently to DNA in maternal and fetal tissues was evaluated. Pregnant F344/NCr rats were injected i.p. with single doses of 5, 10 or 15 mg cisplatin/kg bw at 18 days of gestation and sacrificed 24 h later. Cisplatin-DNA adducts were determined by dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA) using both High (90 pmol/micrograms DNA) and Low (0.50 pmol/ microgram DNA) Modified cisplatin-DNA standards and atomic absorbance spectrometry (AAS). The adduct quantities determined by the two DELFIAs varied in concert, but the DELFIA with Low Modified standard gave actual values similar to those observed with AAS. In maternal and fetal tissues, with the exception of placenta in one experiment and maternal kidney in another experiment, the extent of cisplatin-DNA adduct formation increased with dose. In maternal kidney, the low adduct levels observed at the 15 mg/kg dose may reflect kidney toxicity. Fetal kidney, liver and lung contained fewer cisplatin-DNA adducts than the corresponding maternal tissues. In contrast, at 5 and 15 mg/kg, fetal brain DNA contained higher adduct levels than maternal brain DNA. This study demonstrates the presence of DNA damage induced by cisplatin in multiple maternal and fetal rat tissues at tumorigenic doses of drug; the results are therefore consistent with the hypothesis that genotoxic mechanisms play an important role in the drug-induced tumor incidence.

Selective intra-arterial infusion of high-dose cisplatin in patients with advanced head and neck cancer results in high tumor platinum concentrations and cisplatin-DNA adduct formation

Selective intra-arterial infusion of high-dose cisplatin in patients with advanced head and neck cancer results in high tumor platinum concentrations and cisplatin-DNA adduct formation

Effects of thiourea and ammonium bicarbonate on the formation and stability of bifunctional cisplatin-DNA adducts: Consequences for the accurate quantification of adducts in (cellular) DNA

Cisplatin reacts with DNA by forming mainly bifunctional adducts via reactive monofunctional intermediates. When freshly platinated DNA was postincubated with thiourea (10 mM, at 23 or 37°C) for periods of up to 24 h, followed by determination of mono- and diadducts, a rapid initial decrease was seen in the fraction of diadducts, followed by a much slower decrease. About 40% diadducts were found after 10-min postincubation at 23°C, which dropped to some 14% after 24 h at 37°C; total platination was hardly affected. Postincubation of “aged” platinated DNA (no reactive monoadducts) only showed the slower decrease. The rapid process is likely to represent monoadduct inactivation, preventing the formation of diadducts, whereas the slow reaction must be interpreted as diadduct-to-monoadduct conversion. Similar reactions, but less efficient than with thiourea, occurred during dialysis against NH 4HCO 3 (0.1–1 M). Pt-containing (di)nucleotides in digested DNA were hardly affected by thiourea. Rapid reduction of the measured level of bifunctional adducts also occurred when cisplatin-treated Chinese hamster ovary cells were postincubated with thiourea, with concomitant increase in survival. It is concluded that quantification of the real levels of mono- and diadducts in freshly platinated DNA requires a posttreatment with thiourea of 30–60 min at 37°C.

Selective intra-arterial infusion of high-dose cisplatin in patients with advanced head and neck cancer results in high tumor platinum concentrations and cisplatin-DNA adduct formation.

A group of 23 patients with advanced head and neck cancer were treated with highly selective intra-arterial (IA) cisplatin 150 mg/m2 delivered rapidly through microcatheters. The systemic effects of cisplatin were neutralized by concurrent administration of sodium thiosulfate. Two-to-threefold higher tumor platinum contents were detected in tumor biopsies after selective IA cisplatin administration compared to historical controls (treated with 100 mg/m2 IA). Cisplatin-induced DNA modification in human tumor biopsies was quantitated using the antiserum NKI-A59. High levels of cisplatin DNA adducts were detected which correlated linearly with the tumor platinum content (r2 = 0.62). The addition of radiotherapy to this high dose intensity cisplatin treatment resulted in a 92% complete response (CR) rate (12 of 13 patients achieved a CR). Since no difference in tumor platinum content was detected between patients receiving or not receiving radiotherapy (13 and 10 patients, respectively), but the response rate was substantially different (12 CR and 1 partial response with radiotherapy versus 6 partial and 4 non-responders without radiotherapy), these data suggest that the high platinum levels achieved by selective IA infusion were sufficient to produce enough interaction with radiotherapy to cause a 92% CR rate. Whether this interaction is additive or synergistic is as yet unclear.