Pt-DNA Adducts Research Articles

Competitive reactions of interstrand and intrastrand DNA-Pt adducts: A dinuclear-platinum complex preferentially forms a 1,4-interstrand cross-link rather than a 1,2 intrastrand cross-link on binding to a GG 14-mer duplex.

A study of the kinetics and mechanism of the reaction between the dinuclear Pt complex [(trans-PtCl(NH(3))(2))(2)(mu-NH(2)(CH(2))(6)NH(2))](2+) (1) and the 14-mer duplex 5'-d(ATACATG(7)G(8)TACATA)-3'.5'-d(TATG(25)TACCATG(18)TAT)-3' is reported. [(1)H,(15)N]-HSQC NMR was used to follow the reaction at 298 K, pH 5.4. The product is primarily the 5'-5' 1,4-interstrand cross-link between G(8) and G(18) bases and exists in two conformational forms. No evidence for the possible 1,2-intrastrand G(7)G(8) adduct was seen, confirming the preferential formation of interstrand cross-links by these dinuclear complexes. An initial electrostatic association of (15)N-1 with the duplex is indicated by changes in its (1)H/(15)N chemical shifts, followed by aquation of 1 to form the monoaqua monochloro species 2, with a rate constant of 4.00+/-0.03x10(-5) s(-1). Monofunctional binding to the duplex occurs primarily at G(8), the 3' base of the nucleophilic GG grouping, with a rate constant of 1.5+/-0.7 M(-1) s(-1). Changes in the (1)H/(15)N shifts indicate there is an electrostatic interaction between the unbound (PtN(3)Cl) group of the monofunctional adduct and the duplex. No peaks for a transient aquated monofunctional species are seen and closure of 3 to form the 1,4-G(8)G(18) interstrand cross-link (5) was treated as direct, with a rate constant of 4.47+/-0.06x10(-5) s(-1). The G(8)G(18) cross-link was confirmed from analysis of the NOESY NMR spectrum of the final product. Structural perturbations for the 1,4-interstrand cross-link extend over approximately four base-pairs and are similar to those found for a 1,4-interstrand cross-link with a shorter 8-mer -GTAC- sequence. A major distortion was evident for the 5'T (T(17)) adjacent to the platinated G(18), consistent with the findings from the use of chemical probes to investigate the conformation of 1,4-interstrand cross-links.

Translesion synthesis past platinum DNA adducts by human DNA polymerase mu.

DNA polymerase mu (pol mu) is a member of the pol X family of DNA polymerases, and it shares a number of characteristics of both DNA polymerase beta (pol beta) and terminal deoxynucleotidyl transferase (TdT). Because pol beta has been shown to perform translesion DNA synthesis past cisplatin (CP)- and oxaliplatin (OX)-GG adducts, we determined the ability of pol mu to bypass these lesions. Pol mu bypassed CP and OX adducts with an efficiency of 14-35% compared to chain elongation on undamaged DNA, which is second only to pol eta in terms of bypass efficiency. The relative ability of pol mu to bypass CP and OX adducts was dependent on both template structure and sequence context. Since pol mu has been shown to be more efficient on gapped DNA templates than on primed single-stranded DNA templates, we determined the ability of pol mu to bypass Pt-DNA adducts on both primed single-stranded and gapped templates. The bypass of Pt-DNA adducts by pol mu was highly error-prone on all templates, resulting in 2, 3, and 4 nt deletions. We postulate that bypass of Pt-DNA adducts by pol mu may involve looping out the Pt-GG adduct to allow chain elongation downstream of the adduct. This reaction appears to be facilitated by the presence of a downstream "acceptor" and a gap large enough to provide undamaged template DNA for elongation past the adduct, although gapped DNA is clearly not required for bypass.

Novel apoptosis-inducing trans-platinum piperidine derivatives: synthesis and biological characterization.

The synthesis, chemical characterization, and interaction with cells of new sterically hindered trans- and cis-diaminedichloroplatinum(II) complexes are described. The amine ligands include monofunctional piperidine (pip) and piperazine (pz). The poor solubility of trans-diaminedichloroplatinum complexes was overcome by introducing the positively charged pz ligand, which allows retaining of the classic platinum coordination sphere. In vitro evaluation in OV-1063 and C-26 tumor cells revealed that replacing one NH3 of the inactive transplatin by an aromatic planar ligand (4-picoline, 4-pic) or by an aliphatic nonplanar heterocyclic ligand (pip) or replacing both NH3 groups with a 4-pic ligand and a pip or pz ligand significantly increases the cytotoxic activity of these complexes. The unsymmetric complexes trans-[PtCl2(4-pic)(pip)] and trans-[PtCl2(4-pic)(pz)]HCl were the most cytotoxic compounds against the cisplatin-sensitive tumor cell line C-26 (IC50 = 4.5 and 5.5 microM, respectively) and the cisplatin-sensitive tumor cell line OV-1063 (IC50 = 6.5 and 7.4 microM, respectively). In contrast, replacing one NH3 of the cis isomer by an aromatic planar ligand (4-pic) or by an aliphatic amine lowered their cytotoxiciy in comparison to cisplatin. Cell penetration and Pt-DNA adduct formation were also evaluated, and it was clearly shown that both trans-[PtCl2(4-pic)(pip)] and trans-[PtCl2(4-pic)(pz)]HCl penetrate efficiently the cellular membrane of the tumor cells and platinate the cellular DNA. When comparing cellular DNA platination, positively charged trans-[PtCl2(4-pic)(pz)]HCl was 7-fold higher than both cisplatin and its neutral analogue trans-[PtCl2(4-pic)(pip)]. Moreover, in contrast to cisplatin, in the cell lines used, cell death caused by both complexes appeared to be apoptotic according to several criteria including early phosphatidylserine exposure, activation of caspases, and characteristic morphological changes. Our results suggest that these novel mixed nonclassical trans-Pt(II) complexes are biologically and mechanistically distinct from known Pt complexes and deserve evaluation of their efficacy in tumor-bearing animals.

JM216-, JM118-, and cisplatin-induced cytotoxicity in relation to platinum-DNA adduct formation, glutathione levels and p53 status in human tumour cell lines with different sensitivities to cisplatin

The aim of this study is to establish anti-tumour potency of the new oral platinum drug JM216 and its metabolite JM118 in relation to the platinum (Pt)-DNA adduct formation, glutathione (GSH)-levels, and p53 status in human cancer cell lines with different sensitivities to cisplatin (CDDP). These parameters were studied in the CDDP sensitive human germ cell cancer cell line Tera and the small-cell lung cancer cell line GLC 4 and their sublines with in vitro acquired CDDP resistance, Tera-CP and GLC 4-CDDP, in a human ovarian cancer cell line transfected with mutant p53 (A2780/mt273) and with an empty vector as control (A2780/cmv), and in the intrinsic CDDP resistant human non-small-cell lung cancer cell line SW1573/S1 and colon carcinoma cell line Caco-2. Cytotoxicity was tested with the microculture tetrazolium (MTT)-assay. Pt-DNA adduct levels were assessed immunocytochemically. Quantitative analysis was performed by double fluorescence video microscopy. Results were correlated with GSH levels and p53 status of the cell lines. This study showed that both JM216 and JM118 can partially circumvent intrinsic and acquired resistance to CDDP. Drug-induced cytotoxicity only correlated negatively with GSH levels for JM216 and CDDP in the tested unselected cell lines. At equimolar basis, JM216 induced lower levels of Pt-DNA adducts in the various cell lines than JM118 and CDDP, whereas the JM118-induced amount and pattern of Pt-DNA adducts was comparable to CDDP. No difference in initial Pt-DNA adducts levels was observed between cell lines sensitive, acquired or intrinsic resistant to CDDP suggesting a Pt-resistance mechanism based on tolerance or increased repair, rather than decreased initial Pt-DNA adduct formation.

In vitro studies on the mechanisms of oxaliplatin resistance.

We have previously reported that elevation of glutathione mediated by gamma-glutamyl transpeptidase is one mechanism of oxaliplatin resistance. This study explored other potential oxaliplatin resistance mechanisms with two aims: (1) to identify the differences between cisplatin and oxaliplatin in terms of drug accumulation, DNA-Pt adduct formation and repair, and (2) to determine whether defects in drug accumulation and enhanced repair of the DNA-Pt adduct contribute to oxaliplatin resistance. The human ovarian carcinoma cell line A2780, an oxaliplatin-resistant variant A2780/C25 and a cisplatin-resistant variant A2780/CP along with an inherently cisplatin-resistant HT-29 colon carcinoma cell line were used in the study. The methods consisted of sulforhodamine-B assays, atomic absorption spectrophotometry and real-time quantitative RT-PCR. Significantly higher drug accumulation and DNA-Pt adduct formation were observed after exposure to cisplatin compared to after oxaliplatin in the parent A2780 cells and the oxaliplatin-resistant A2780/C25 cells. The DNA-Pt adduct formed after treatment with either drug was repaired with equal efficiency by all cell lines except A2780/CP, which repaired the DNA-cisplatin adduct more efficiently than the DNA-oxaliplatin adduct. Relative to the parent line, oxaliplatin-resistant A2780/C25 cells showed reduced Pt accumulation and DNA-Pt adduct levels following exposure to oxaliplatin, but only reduced accumulation after exposure to cisplatin. The cisplatin-resistant A2780/CP cells showed reduced accumulation and DNA-Pt adduct levels after exposure to cisplatin, but only reduced DNA-Pt adduct after exposure to oxaliplatin. In comparison to A2780 cells, the inherently cisplatin-resistant HT-29 cells showed lower accumulation and DNA-Pt adduct levels after exposure to cisplatin, but displayed no difference after exposure to oxaliplatin. An enhanced repair of the DNA-cisplatin adduct was observed only in A2780/CP cells relative to A2780 cells in an 8-h period. The steady-state levels of ERCC-1 mRNA, but not of XPA, were moderately elevated in the resistant cells. Exposure to either one of the drugs resulted in an induction of XPA in all the cell lines and of ERCC-1 in cisplatin-resistant cells. There was no relationship between the level of expression of the repair genes and the DNA-Pt adduct levels or repair. Relative to cisplatin a lower intracellular concentration and fewer DNA-Pt adducts are sufficient for oxaliplatin to exert its cytotoxicity. Resistance to oxaliplatin is mediated by similar mechanisms of reduced drug accumulation and DNA-Pt adduct formation as resistance to cisplatin. There is no clear evidence that enhanced repair is a mechanism of oxaliplatin resistance in the cell line (A2780/C25) studied here. The findings are suggestive of yet unidentified differences between the two drugs with respect to cellular uptake and/or efflux and repair of DNA-Pt adducts.

The Effect of DNA Structure on the Catalytic Efficiency and Fidelity of Human DNA Polymerase β on Templates with Platinum-DNA Adducts

DNA adducts formed by platinum-based anticancer drugs interfere with DNA replication. The carrier ligand of the platinum compound is likely to affect the conformation of the Pt-DNA adducts. In addition, the conformation of the adduct can also change upon binding of damaged DNA to the active site of DNA polymerase. From the crystal structures of pol beta ternary complexes it is evident that undamaged gapped and primed single-stranded (non-gapped) DNA templates exist in very different conformations when bound to pol beta. Therefore, one might expect that the constraints imposed on the damaged templates by binding to the polymerase active site should also affect the conformation of the Pt-DNA adducts and their ability to inhibit DNA replication. In support of this hypothesis we have found that the efficiency, carrier ligand specificity, site of discrimination (3'-G versus 5'-G of the Pt-GG adducts), and fidelity of translesion synthesis past Pt-DNA adducts by pol beta are strongly affected by the structure of the DNA template. Previous studies have suggested that the conformation of Pt-DNA adducts may be affected by the sequence context of the adduct. In support of this hypothesis, our data show that sequence context affects the efficiency, fidelity, and pattern of misincorporation by pol beta.

Oxaliplatin-induced damage of cellular DNA.

Damage to cellular DNA is believed to determine the antiproliferative properties of platinum (Pt) drugs. This study characterized DNA damage by oxaliplatin, a diaminocyclohexane Pt drug with clinical antitumor activity. Compared with cisplatin, oxaliplatin formed significantly fewer Pt-DNA adducts (e.g., 0.86+/-0.04 versus 1.36+/- 0.01 adducts/10(6) base pairs/10 microM drug/1 h, respectively, in CEM cells, P<.01). Oxaliplatin was found to induce potentially lethal bifunctional lesions, such as interstrand DNA cross-links (ISC) and DNA-protein cross-links (DPC) in CEM cells. As with total adducts, however, oxaliplatin produced fewer (P<.05) bifunctional lesions than did cisplatin: 0.7+/-0.2 and 1.8+/-0.3 ISC and 0.8+/-0.1 and 1.5+/-0.3 DPC/10(6) base pairs/10 microM drug, respectively, after a 4-h treatment. Extended postincubation (up to 12 h) did not compensate the lower DPC and ISC levels by oxaliplatin. ISC and DPC determinations in isolated CEM nuclei unequivocally verified that oxaliplatin is inherently less able than cisplatin to form these lesions. Reactivation of drug-treated plasmids, observed in four cell lines, suggests that oxaliplatin adducts are repaired with similar kinetics as cisplatin adducts. Oxaliplatin, however, was more efficient than cisplatin per equal number of DNA adducts in inhibiting DNA chain elongation ( approximately 7-fold in CEM cells). Despite lower DNA reactivity, oxaliplatin exhibited similar or greater cytotoxicity in several other human tumor cell lines (50% growth inhibition in CEM cells at 1.1/1.2 microM, respectively). The results demonstrate that oxaliplatin-induced DNA lesions, including ISC and DPC, are likely to contribute to the drug's biological properties. However, oxaliplatin requires fewer DNA lesions than does cisplatin to achieve cell growth inhibition.

The Efficiency and Fidelity of Translesion Synthesis past Cisplatin and Oxaliplatin GpG Adducts by Human DNA Polymerase β

DNA polymerase beta (pol beta) is the only mammalian DNA polymerase identified to date that can catalyze extensive bypass of platinum-DNA adducts in vitro. Previous studies suggest that DNA synthesis by pol beta is distributive on primed single-stranded DNA and processive on gapped DNA. The data presented in this paper provide an analysis of translesion synthesis past cisplatin- and oxaliplatin-DNA adducts by pol beta functioning in both distributive and processive modes using primer extension and steady-state kinetic experiments. Translesion synthesis past Pt-DNA adducts was greater with gapped DNA templates than with single-stranded DNA templates. In the processive mode pol beta did not discriminate between cisplatin and oxaliplatin adducts, while in the distributive mode it displayed about 2-fold increased ability for translesion synthesis past oxaliplatin compared with cisplatin adducts. The differentiation between cisplatin and oxaliplatin adducts resulted from a K(m)-mediated increase in the efficiency of dCTP incorporation across from the 3'-G of oxaliplatin-GG adducts. Rates of misincorporation across platinated guanines determined by the steady-state kinetic assay were higher in reactions with primed single-stranded templates than with gapped DNA and a slight increase in the misincorporation of dTTP across from the 3'-G was found for oxaliplatin compared with cisplatin adducts.

Efficient translesion replication past oxaliplatin and cisplatin GpG adducts by human DNA polymerase eta.

Platinum anticancer agents form bulky DNA adducts which are thought to exert their cytotoxic effect by blocking DNA replication. Translesion synthesis, one of the pathways of postreplication repair, is thought to account for some resistance to DNA damage and much of the mutagenicity of bulky DNA adducts in dividing cells. Oxaliplatin has been shown to be effective in cisplatin-resistant cell lines and less mutagenic than cisplatin in the Ames assay. We have shown that the eukaryotic DNA polymerases yeast pol zeta, human pol beta, and human pol gamma bypass oxaliplatin-GG adducts more efficiently than cisplatin-GG adducts. Human pol eta, a product of the XPV gene, has been shown to catalyze efficient translesion synthesis past cis, syn-cyclobutane pyrimidine dimers. In the present study we compared translesion synthesis past different Pt-GG adducts by human pol eta. Our data show that, similar to other eukaryotic DNA polymerases, pol eta bypasses oxaliplatin-GG adducts more efficiently than cisplatin-GG adducts. However, pol eta-catalyzed translesion replication past Pt-DNA adducts was more efficient and less accurate than that seen for previously tested polymerases. We show that the efficiency and fidelity of translesion replication past Pt-DNA adducts appear to be determined by both the structure of the adduct and the DNA polymerase active site.

Effect of 6-aminonicotinamide and other protein synthesis inhibitors on formation of platinum-DNA adducts and cisplatin sensitivity.

The present study was undertaken to examine the mechanistic basis for the recent observation that the pyridine nucleotide derivative 6-aminonicotinamide (6AN, NSC 21206) enhances the accumulation and resulting cytotoxicity of cisplatin in a variety of tumor cell lines. When A549 lung cancer cells or K562 leukemia cells were treated with 62.5 microM 6AN for 21 h and then pulse-labeled with [(35)S]methionine for 1 h, increased labeling of five polypeptides, one of which corresponded to a M(r) approximately 78,000 glucose-regulated protein (GRP78), was observed. Two subsequent observations, however, suggested that up-regulation of these polypeptides was unlikely to explain the interaction between 6AN and cisplatin: 1) the concentration of 6AN required to induce GRP78 was 4-fold higher than the dose required to sensitize cells to cisplatin; and 2) simultaneous treatment of cells with 6AN and cycloheximide prevented the increase in GRP78 but not the sensitizing effect of 6AN. On the contrary, treatment with the protein synthesis inhibitors cycloheximide, anisomycin, or puromycin as well as prolonged exposure to the RNA synthesis inhibitor actinomycin D mimicked the biochemical modulating effects of 6AN on cisplatin action. Conversely, 6AN inhibited protein synthesis, whereas 18 6AN analogs that failed to enhance Pt-DNA adducts and cisplatin cytotoxicity failed to inhibit protein synthesis. These observations are consistent with a model in which 6AN and other inhibitors of protein synthesis act as modulating agents by increasing cisplatin accumulation, thereby enhancing the formation of Pt-DNA adducts and subsequent cisplatin-induced cell death.

Increased DNA-binding activity of cis-1,1-cyclobutanedicarboxylatodiammineplatinum(II) (carboplatin) in the presence of nucleophiles and human breast cancer MCF-7 cell cytoplasmic extracts: activation theory revisited

The molecular mechanism of carboplatin [ cis-1,1-cyclobutanedicarboxylatodiammineplatinum(II)] activation is still unresolved. We studied the binding of carboplatin to calf thymus DNA in the presence of thiourea, glutathione, and human breast cancer MCF-7 cell cytoplasmic extracts by measurement of DNA-dependent ethidium bromide fluorescence and atomic absorption spectroscopy. After a 96-hr period of reaction, the decrease in the DNA-dependent fluorescence yield of ethidium bromide due to the formation of platinum (Pt)-DNA adducts increased significantly in the presence of thiourea (6-fold) and glutathione (3- to 4-fold) as compared to the controls in the absence of the nucleophiles. There was also a marked elevation in the levels of platinum incorporated into DNA, measured by atomic absorption spectroscopy (2- to 3-fold and 5- to 7-fold for thiourea and glutathione, respectively). More remarkably, the Pt-DNA adducts formed in the presence of cytoplasmic extracts of MCF-7 human breast cancer cells also showed similar results in a dose-related fashion. Carboplatin, therefore, displayed a characteristic increase in DNA binding/damaging in the presence of the very same S-containing nucleophiles that showed the expected quenching effects in the case of cisplatin [ cis-diamminedichloroplatinum (II)]. We propose a nucleophile-facilitated release of the active species of carboplatin prior to binding with DNA.

Solution structure of a DNA duplex containing a cis-diammineplatinum(II) 1,3-d(GTG) intrastrand cross-link, a major adduct in cells treated with the anticancer drug carboplatin.

The platinum 1,3-d(GXG) intrastrand cross-link is one of the adducts formed in the reaction of the antitumor drug cisplatin with DNA, and in fact the major adduct found in cells treated with the cisplatin analogue carboplatin. To determine the 3D structure of this adduct, the duplex d(CTCTGTGTCTC).d(GAGACACAGAG)], where GTG denotes a platinum 1,3-intrastrand cross-link, was prepared and studied with high-resolution (1)H NMR. The solution structure was determined using the SPEDREF protocol, which includes an iterative NOE-restrained refinement procedure. Calculated and recorded NOE spectra were found to be in good agreement (NMR R factor 22%). The studied duplex is more distorted from B-DNA than previously determined structures of the 1,2-d(GG) intrastrand adducts. The base pairing is lost for the 5'G-C and the central T-A base pair in the GTG lesion, and the central thymine is extruded from the minor groove. To accommodate this lesion, the minor groove is widened, and the 5'-guanine ribose adopts an N-type conformation. The helix is unwound locally and is significantly bent toward the major groove. Significant difference between the structural distortion of the 1, 3-d(GTG) cross-link and other Pt-DNA cross-links sheds new light on the observed differences in protein recognition of these lesions, and thus on the possible differences in mechanisms of action of the various Pt-DNA adducts formed in treatment with platinum anticancer complexes.

Murine pharmacokinetics of 6-aminonicotinamide (NSC 21206), a novel biochemical modulating agent

The pyridine nucleotide 6-aminonicotinamide (6AN) was shown recently to sensitize a number of human tumor cell lines to cisplatin in vitro. The present studies were undertaken to compare the drug concentrations and length of exposure required for this sensitization in vitro with the drug exposure that could be achieved in mice in vivo. Human K562 leukemia cells and A549 lung cancer cells were incubated with 6AN for various lengths of time, exposed to cisplatin for 1–2 hr, and assayed for Pt-DNA adducts as well as the ability to form colonies. K562 cells displayed progressive increases in Pt-DNA adducts and cisplatin sensitivity during the first 10 hr of 6AN exposure. An 18-hr 6AN exposure was likewise more effective than a 6-hr 6AN exposure in sensitizing A549 cells to cisplatin. HPLC analysis of 6AN and its metabolite, 6-amino-NAD +, permitted assessment of exposures achieved in vivo after i.v. administration of 10 mg/kg of 6AN to CD2F 1 mice. 6AN reached peak serum concentrations of 80–90 μM and was cleared rapidly, with T 1/2α and T 1/2β values of 7.4 and 31.3 min, respectively. Bioavailability was 80–100% with identical plasma pharmacokinetics after i.p. administration. At least 25% of the 6AN was excreted unchanged in the urine. The metabolite 6-amino-NAD + was detected in perchloric acid extracts of brain, liver, kidney, and spleen, but not in serum. Efforts to prolong systemic 6AN exposure by administering multiple i.p. doses or using osmotic pumps resulted in lethal toxicity. These results demonstrated that 6AN exposures required to sensitize tumor cells to cisplatin in vitro are difficult to achieve in vivo.

Selective cell kill of the combination of gemcitabine and cisplatin in multilayered postconfluent tumor cell cultures.

Both gemcitabine (2',2'-difluorodeoxycytidine, dFdC) and cisplatin (cis-diammine-dichloroplatinum) have significant anticancer activity against ovarian, head and neck, and non-small cell lung cancer (NSCLC). dFdC can be incorporated into DNA and RNA, and inhibit DNA repair, while cisplatin can form Pt-DNA adducts. We previously observed schedule-dependent synergism of the combination of dFdC and cisplatin in monolayer cell cultures. We now evaluated whether the combination would also enable selective cell kill in multilayered postconfluent cell cultures, since each compound showed variable activity in multilayered cells. The combination was tested in multilayered cultures from cell lines with a different histological origin: the human head and neck squamous cell carcinoma cell line UMSCC-22B (22B), the human NSCLC cell line H322, and ADDP, a cisplatin-resistant variant of the human ovarian cancer cell line A2780. Sensitivity of the multilayered cells was dependent on exposure duration and sequence of the drug combinations, which were added in a constant molar ratio (dFdC:cisplatin 1:100), with a total exposure time of 96 h. The type of interaction was related to the degree of resistance of the cell lines to either dFdC or cisplatin. Thus, the very sensitive 22B cells only showed an additive effect when cells were preincubated for 24 h with dFdC prior to exposure to the combination. In contrast, in the resistant ADDP and H322 cells, synergism was the most common profile (three out of four schedules tested). This is of special relevance when we take into account that these drugs only show cytostatic effects when administered alone, whereas the combination produced cytotoxic cell killing. In conclusion, combining dFdC with cisplatin can be at least additive, but synergistic in multilayered postconfluent cells resistant to dFdC and cisplatin.

Pharmacokinetic schedule finding study of the combination of gemcitabine and cisplatin in patients with solid tumors

To determine possible schedule dependent pharmacokinetic and pharmacodynamic interactions between gemcitabine (2',2'-difluorodeoxycytidine, dFdC) and cisplatin (cis-diammine-dichloroplatinum, CDDP) in patients with advanced stage solid tumors in a phase I trial. A total of 33 patients with advanced stage solid tumors were treated with gemcitabine (30-min infusion, 800 mg/m2) and cisplatin (one-hour infusion, 50 mg/m2). Sixteen patients had a four-hour interval between gemcitabine (days 1, 8, 15) and cisplatin (days 1 and 8), followed by the reverse schedule and seventeen patients had a 24-hour interval between gemcitabine (days 1, 8, 15) and cisplatin (days 2 and 9), followed by the reverse schedule. Gemcitabine and cisplatin pharmacokinetics were measured in plasma and white blood cells (WBC), isolated from blood samples taken at several time points after the start of treatment. A four-hour time interval between both agents did not reveal major differences in plasma pharmacokinetics of gemcitabine, dFdU (deaminated gemcitabine) and platinum (Pt), and of gemcitabine-triphosphate (dFdCTP) accumulation and Pt-DNA adduct formation in WBC between the two different sequences of gemcitabine and cisplatin. In the patients treated with the 24-hour interval, cisplatin before gemcitabine did not significantly change peak gemcitabine levels and the AUC of plasma dFdU, but tended to increase dFdCTP AUC in WBC 1.5-fold (P < 0.06). Gemcitabine before cisplatin decreased the plasma AUC of Pt 2.1-fold (P = 0.03). No significant differences in Pt-DNA adduct levels in WBC were found, although gemcitabine before cisplatin tended to increase the 24-hour retention of Pt-DNA adducts. Creatinine clearance on day 28 was related to the peak plasma levels of total Pt (linear regression coefficient (r) = 0.47, P = 0.02, n = 26). Furthermore, the increase in the Pt-GG to Pt-AG ratio 24 hours after cisplatin treatment was related to the overall response of patients (r = 0.89, P < 0.01, n = 8). Of all schedules the treatment of patients with cisplatin 24 hours before gemcitabine led to the highest dFdCTP accumulation in WBC and total Pt levels in plasma. These characteristics formed the basis for further investigation of this schedule in a phase II clinical study.

Pharmacokinetically guided dose escalation of carboplatin in epithelial ovarian cancer: Effect on drug-plasma AUC and peripheral blood drug-DNA adduct levels

Platinum based drugs are active agents in epithelial ovarian cancer and increased platinum drug dose intensity is thought to lead to improved survival, because of the largely untested assumption that increased dose intensity results in an increased interaction of the platinum drug with its target, DNA. In a previously reported phase I trial (Lind et al., J Clin Oncol 1996; 14: 800-5), carboplatin dose intensity was increased by the use of G-CSF to support the bone marrow and using pharmacokinetically-guided carboplatin dosing. The objectives of this study were to validate the carboplatin dosing formula during high dose intensity therapy and evaluate the relationship between systemic carboplatin exposure and Pt-DNA adduct levels in peripheral blood leucocytes. A total of 17 patients were studied over four levels of dose intensification. The carboplatin dose was calculated using the 'Calvert formula'. Levels of drug-target interaction in peripheral blood leukocytes were measured using an immunoassay based on a monoclonal antibody that recognises DNA-platinum adducts. Pharmacokinetic measurements were carried out using a previously validated single sample method. The area under the curve of concentration of unbound carboplatin in plasma versus time (AUC) for target AUC values of 5, 7 and 9 mg/ml x min were: 5.6 +/- 1.0, 7.3 +/- 0.7 and 9.8 +/- 0.5 mg/ml x min (mean +/- S.D.). There was a good correlation between target and achieved dose intensities (r2 = 0.899) and the slope of the linear regression line was 0.95 (+/- 0.09 SD) not significantly different to 1.0 (P > 0.6). The levels of immunoreactive DNA adducts were not detectable at a target AUC of 5 mg/ml x min but increased progressively at the higher AUC levels. Accumulation of adducts between courses was not detected. Pharmacokinetically-based carboplatin dosing during high intensity therapy accurately predicted the dose required to achieve a target AUC and resulted in consistent patient exposure to active drug. During the dose escalation study, peripheral blood leucocyte DNA platinum-DNA adduct levels were positively related to drug dose and drug AUC.

Mechanism of Bracken Fern Carcinogenesis: Evidence for H-Ras Activation Via Initial Adenine Alkylation by Ptaquiloside

Bracken fern (Pteridium spp.) causes cancer of the oesophagus and the urinary bladder in cattle and sheep. Ptaquiloside (PT) is believed to be the carcinogenic principle which alkylates DNA when activated to its unstable dienone form (APT) under alkaline conditions. In this report we present evidence for the presence of PT-DNA adducts in the ileum of bracken fem-fed calves using the 32P-postlabelling assay. H-ras mutations were also observed in the ileum using single strand conformation polymorphism (SSCP) technique. Mutations corresponding to adenine to pyrimidine transversions in the codon 61 of H-ras were identified by the cycle sequencing method. In vitro DNA alkylation studies showed that APT alkylated H-ras primarily at the adenines. In addition, the rate of depurination of alkylated adenine was sequence dependent. Investigation of DNA template activity using a plasmid DNA showed that DNA synthesis by T7 DNA polymerase was terminated by the presence of all alkylated bases but certain apurinic sites allowed the DNA synthesis to continue. These results suggest that initial alkylation of adenine by PT in codon 61 followed by depurination and error in DNA synthesis lead to activation of H-ras proto-oncogene.

Studies on the Chromosomes of Murine Dalton's Lymphoma Cells and the Effect of Cisplatin on Chromosomes of These Cells and Bone Marrow Cells in vivo.

Chromosome studies of Dalton's lymphoma (DL) revealed only one rb-marker in the cells and the chromosome numbers varied between 59 and 68 with a peak at 66 chromosomes. Thus, the modal number of chromosomes of this DL is suggested to be 66. Cisplatin treatment of tumor bearing hosts resulted the regression of the tumor and induced the formation of chromosomal aberrations/aberrant metaphases (80-90%) in the DL cells which may be responsible for the anticancer activity of cisplatin. Cisplatin treatment of the hosts caused the significant decrease in GSH levels in DL as well as bone marrow cells. Cisplatin's mutagenic potentials in the host is also supported from the observation of the induction of chromosomal aberrations in the bone marrow cells of the same host. Differential effect of cisplatin on the chromosomes of bone marrow and Dalton's lymphoma cells is noted. Bone marrow cells developed significantly less aberrant metaphases as compared to that of DL cells after cisplatin treatment in vivo. Interestingly, aberrant metaphases in bone marrow cells decrease appreciably with time of treatment (48-96 hr) which may suggest the possibility of an efficient clearance/repair of Pt-DNA adducts in bone marrow cells but in DL cells it remained more or less constant.

Platinum-DNA adduct formation in leucocytes of children in relation to pharmacokinetics after cisplatin and carboplatin therapy.

Platinum (Pt)-DNA adducts were measured in peripheral blood leucocytes (PBLs) from 24 children with solid tumours after standard cisplatin and/or carboplatin treatment. The relationship between Pt-DNA adduct levels and pharmacokinetics of cisplatin and carboplatin was investigated. Adduct measurements were performed by competitive enzyme-linked immunosorbent assay (ELISA) and plasma unbound Pt concentrations were measured by atomic absorption spectrophotometry (AAS). There was considerable interindividual variation in Pt-DNA adduct level that was weakly correlated (r2 = 0.32) with the area under the unbound drug concentration vs time curve (AUC) at 6 h after the start of cisplatin infusion, indicating that the variation in Pt-DNA adduct levels was primarily determined by factors other than AUC. No clear relationship between AUC and adduct levels was seen at 24 and 48 h after cisplatin or at 6, 24 or 48 h after carboplatin. Carboplatin produced lower levels of immunoreactive adducts than did cisplatin (1.3 +/- 0.6 nmol Pt g-1 DNA vs 3.2 +/- 1.7 nmol Pt g-1 DNA), despite a 20-fold higher unbound drug AUC for carboplatin (8.0 +/- 3.5 mg ml-1 min vs 0.4 +/- 0.2 mg ml-1 min). This study demonstrates that, after cisplatin and carboplatin treatment the drug-target interaction is determined by both pharmacokinetic and, predominantly, cellular factors. Intrinsic differences between the two complexes, primarily reactivity, probably explain the lower adduct levels observed after carboplatin treatment.

Improved 32P-postlabelling assay for the quantification of the major platinum-DNA adducts.

For the improvement of chemotherapy with platinum (Pt)-containing drugs a sensitive assay to detect the induced Pt-DNA adducts is needed. Therefore, the 32P-postlabelling assay, described by Blommaert and Saris (Nucleic Acids Res., 1995, 23, 1300-1306), to detect the major adducts Pt-GG and Pt-AG has substantially been improved and compared with ELISA and AAS. For the quantification of the adducts, TpT was added as an internal standard immediately after isolation of the Pt-adducts from digested DNA samples. It was found that 32P-labelling of both GpG and ApG, the dinucleotides obtained after deplatination of the adducts, was equally efficient as that of TpT. To isolate the Pt-adducts on basis of a positive charge, the pH of DNA digests was adjusted to approximately 3 prior to separation by strong cation-exchange chromatography. For the subsequent deplatination a volume of only 12 microl of 0.2 M NaCN was used, which did not interfere with the following labelling step. The quantification of the 32P-labelled dinucleotides was performed by phosphorimaging of spots after separation on TLC as well as by 32P-counting of fractions collected after separation by HPLC. The method was used to determine adduct levels in in vitro cisplatin-treated DNA and in DNA isolated from cisplatin-treated cultured cells, tumor xenografts from cisplatin-treated mice, and from white blood cells and (tumor) tissues from cisplatin-treated patients. The results show a significant correlation with the adduct levels as determined with atomic absorption spectroscopy (high levels) or with specific antibodies (low levels). This assay appears to be useful for the determination of low levels of Pt-adducts in small DNA samples as present in clinical specimens such as blood and tumor tissue, but also in buccal mucosal cells and fine needle aspirates.