Experimental and Theoretical Studies on the Pharmacodynamics of Cisplatin in Jurkat Cells

For Jurkat cells in culture exposed to cisplatin (1), we measured the number of platinum adducts on DNA and showed that it is proportional to the AUC, the area under the concentration vs time curve, for cisplatin. The number of platinum-DNA adducts is measured immediately following exposure to drug. The AUC is calculated either as the product of the initial cisplatin concentration and the exposure time or as the integral under the concentration vs time curve for the unreacted dichloro species, which decreases exponentially. We also show that the number of adducts correlates with decreases in respiration, with the amount of DNA fragmentation, and with cell viability, all measured 24 h after exposure to the drug. To study the reactions of cisplatin at concentrations approaching clinical relevance (65 microM), we use two-dimensional [1H15N]HSQC NMR and the 15N-labeled form of the drug, cis-Pt(15NH3)2Cl2, 1. In the absence of cells, 1 reacts with components of the growth medium and also transforms slowly (k(h) = 0.205 h-1 at 37 degrees C) into the chloro-aquo species, cis-[Pt(15NH3)2Cl(H2O)]+ (2), which at the pH of the medium (pH 7.15), is mainly in the deprotonated chloro-hydroxy form, cis-Pt(15NH3)2Cl(OH) (4). The concentration of 2 (4), as measured by HSQC NMR, decreases due to reaction with components of the medium. In the presence of 5 million or more cells, the concentration of 1 decreases with time, but the NMR signal for 2 (4) is not seen because it is rapidly removed from solution by the cells, keeping its concentration very low. These experiments confirm that the species preferentially removed from the medium by cells is 2 (4) and not 1. Our findings are discussed in the context of a kinetic model for platination of nuclear DNA by cisplatin, which includes aquation of cisplatin outside the cell, passage of 2 (4) through the cell membrane, reaction of reactive platinum species (RPS) in the cytosol with thiols, formation of adducts between RPS and accessible sites on genomic DNA, and removal of platinum from DNA by repair. Some of the rate constants involved are measured, but others can only be estimated. Calculations with this model show that little of the platinum reacts with intracellular thiols before reaching the nuclear DNA, indicating that binding to thiols is not important in cisplatin resistance. The model also predicts the circumstances under which the amount of platination of nuclear DNA is proportional to AUC

Kinetic Analysis of the Reactions of 4-Hydroperoxycyclophosphamide and Acrolein with Glutathione, Mesna, and WR-1065

The kinetics of the reactions of glutathione (GSH) with 4-hydroperoxycyclophosphamide (400H-CP) and acrolein, a metabolite of 400H-CP, were investigated in a cell-free medium (pH ∼7.5) and peripheral blood mononuclear cells. The ability of the thiol drugs, sodium 2-mercaptoethane sulfonate (mesna) and S-2-(3-aminopropylamino)ethanethiol (WR-1065), to affect the reactions of cellular GSH with the alkyalting agents was also studied. The amount of unreacted thiols in the various reactions was determined by derivatization with monobromobimane, followed by separation of fluorescent-labeled thioether adducts using high-pressure liquid chromatography. The second-order rate constants (k2) for reactions of GSH, mesna, and WR-1065 with 4OOH-CP in solution were 38 ± 5, 25 ± 5, and 880 ± 50 M-1s-1, respectively. The corresponding k2 for reactions of GSH, mesna, and WR-1065 with acrolein were 490 ± 100, 700 ± 150, and \u3e2000 M-1s-1, respectively. The apparent rate constants for reactions of cellular GSH with acrolein and 4OOH-CP were smaller than those obtained in solution. Assuming that the k2 is the same inside and outside cells, we estimate the first-order rate constant (k1) for transfer of 4OOH-CP and acrolein across the cell membrane as ∼0.01 and ∼0.04 s-1, respectively. WR-1065 was more effective than mesna in blocking depletion of cellular GSH (because it passes into the cell more quickly and has higher reaction rates with the alkylators than the latter compound). When WR-1065 and mesna were used together, the protection against cellular depletion of GSH was additive. Our results are relevant to the administration of thiol drugs with highdose alkylating agents

Kinetics of Cisplatin Binding to Cellular DNA and Modulations by Thiol-Blocking Agents and Thiol Drugs

DNA platination by cisplatin (CDDP) was investigated in peripheral blood mononuclear cells and ovarian cancer cells using atomic absorption spectroscopy. Plots showing the amount of platinum (Pt) bound to DNA versus the molar concentration of cisplatin in the incubation medium ([CDDP]) were nonlinear. For [CDDP] \u3c about 5 μM, the amount of Pt bound to DNA increased slowly with added drug. However, for larger [CDDP], the slope of the plot increased significantly. To study the role of thiols in affecting cisplatin binding to DNA, cells were treated with N-ethylmaleimide, which modifies thiol groups, rendering them incapable of binding cisplatin. Analysis using high-pressure liquid chromatography showed that ∼99% of cellular glutathione was modified by Nethylmaleimide. A plot of the amount of Pt bound to DNA versus [CDDP] for thiol-blocked cells is linear, with a slope similar to that at unblocked cells at high [CDDP]. Neither S-2-(3 aminopropylamino)ethanethiol (WR-1065) nor mesna, when added at clinically achievable concentrations (i.e., \u3c ∼300 μM), affected DNA platination. However, D]NA platination was totally abolished by millimolar concentrations of the drug thiols (∼1.25 mM WR-1065 or ∼5 mM mesna). Thus, the data show that endogenous thiols intercept cellular cisplatin, but this mechanism is less important at high [CDDP]. Moreover, therapeutic concentrations of drug thiols do not significantly affect DNA platination. A simple model that reproduces the experimental results of the amount of cisplatin binding to DNA as a function of [CDDP], time, and thiol content is proposed. The model takes into account passage of cisplatin and thiols through the cell membrane, binding of cisplatin to cellular thiols, and platination of DNA

Modification and Uptake of a Cisplatin Carbonato Complex by Jurkat Cells

The interactions of Jurkat cells with cisplatin, cis-[Pt( 15NH3)2Cl2] (1), are studied using 1H-15N heteronuclear single quantum coherence (HSQC) NMR and inductively coupled plasma mass spectrometry. We show that Jurkat cells in culture rapidly modify the monocarbonato complex cis-[Pt(15NH 3)2(CO3)Cl]- (4), a cisplatin species that forms in culture media and probably also in blood. Analysis of the HSQC NMR peak intensity for 4 in the presence of different numbers of Jurkat cells reveals that each cell is capable of modifying 0.0028 pmol of 4 within ∼0.6 h. The amounts of platinum taken up by the cell, weakly bound to the cell surface, remaining in the culture medium, and bound to genomic DNA were measured as functions of time of exposure to different concentrations of drug. The results show that most of the 4 that has been modified by the cells remains in the culture medium as a substance of molecular mass \u3c3 kDa, which is HSQC NMR silent, and is not taken up by the cell. These results are consistent with a hitherto undocumented extracellular detoxification mechanism in which the cells rapidly modify 4, which is present in the culture medium, so it cannot bind to the cell. Because there is only a slow decrease in the amount of unmodified 4 remaining in the culture medium after 1 h, -1.1 ± 0.4 μM h -1, the cells subsequently lose their ability to modify 4. These observations have important implications for the mechanism of action of cisplatin

Kinetic Analysis of the Reactions of 4-Hydroperoxycyclophosphamide and Acrolein With Glutathione, Mesna, and Wr-1065

The kinetics of the reactions of glutathione (GSH) with 4-hydroperoxycyclophosphamide (400H-CP) and acrolein, a metabolite of 400H-CP, were investigated in a cell-free medium (pH ∼7.5) and peripheral blood mononuclear cells. The ability of the thiol drugs, sodium 2-mercaptoethane sulfonate (mesna) and S-2-(3-aminopropylamino)ethanethiol (WR-1065), to affect the reactions of cellular GSH with the alkyalting agents was also studied. The amount of unreacted thiols in the various reactions was determined by derivatization with monobromobimane, followed by separation of fluorescent-labeled thioether adducts using high-pressure liquid chromatography. The second-order rate constants (k2) for reactions of GSH, mesna, and WR-1065 with 4OOH-CP in solution were 38 ± 5, 25 ± 5, and 880 ± 50 M-1s-1, respectively. The corresponding k2 for reactions of GSH, mesna, and WR-1065 with acrolein were 490 ± 100, 700 ± 150, and \u3e2000 M-1s-1, respectively. The apparent rate constants for reactions of cellular GSH with acrolein and 4OOH-CP were smaller than those obtained in solution. Assuming that the k2 is the same inside and outside cells, we estimate the first-order rate constant (k1) for transfer of 4OOH-CP and acrolein across the cell membrane as ∼0.01 and ∼0.04 s-1, respectively. WR-1065 was more effective than mesna in blocking depletion of cellular GSH (because it passes into the cell more quickly and has higher reaction rates with the alkylators than the latter compound). When WR-1065 and mesna were used together, the protection against cellular depletion of GSH was additive. Our results are relevant to the administration of thiol drugs with highdose alkylating agents