Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

The antitumor drug cisplatin [cis-diamminedichloroplatinum (II)] exerts its effect primarily by interacting with cellular DNA. When cisplatin passes from the blood to the cells, the drug aquates, producing cationic species that bind to nitrogen atoms on the bases of DNA (Zwelling et al., 1979). Although cisplatin can form many types of covalent adducts with DNA, an important lesion is an intrastrand cross-link at two adjacent purine bases, with binding to the sequence guanine-guanine being the most common (Gelasco and Lippard, 1999; Legenre et al., 2000). Cisplatin binding alters the structure of DNA and affects its ability to act as a template in transcription (Bellon et al., 1991). If the rate of DNA platination DNA exceeds the rate at which Pt adducts are removed by repair, cells enter apoptosis and die (Chu, 1994; Demarcq et al., 1994).

The cytotoxic activity of cisplatin correlates with the amount of Pt bound to DNA (Zwelling et al., 1979; Knox et al., 1986; Lindauer and Holler, 1996). Factors controlling DNA platination include the drug uptake, the rate of Pt adduct formation and repair, and the concentration of cellular thiols. The uptake of cisplatin varies among different cells. For example, in cisplatin-sensitive ovarian cancer cells, the uptake occurs by passive diffusion and active transport. By contrast, in the resistant cells, the uptake occurs by passive diffusion (Sharp et al., 1995). In a clinical study, disproportional increments in the number of Pt adducts were found in patients receiving higher doses of cisplatin (Fichtinger-Schepman et al., 1990), confirming the role of adduct removal by repair (O'Neill et al., 1999).

Thiol (sulfhydryl) groups, such as those on glutathione (GSH²) and metallothionein (MT), defend the cell against cisplatin (Kraker et al., 1985; Zhang et al., 1995, 2001; Bose et al., 1997). Since the thiolate anion has a high affinity for Pt⁺², Pt ions entering the cell may preferentially bind to sulfur atoms rather than the bases of DNA (Dedon and Borch, 1987; Lai et al., 1989; Ishikawa and Ali-Osman, 1993). Although it is easy to overwhelm this protective mechanism in first-time patients receiving cisplatin, continued exposure to the drug ultimately produces resistance due to increased sulfhydryl levels (e.g., GSH and MT) (Schilder et al., 1990; Godwin et al., 1992).

Drug thiols are known to modulate cisplatin toxicity, with WR-2721 [amifostine; Ethyol; S-2-(3-aminopropylamino)ethyl phosphorothioic acid; ⁺H₃N-(CH₂)₃-NH₂⁺-(CH₂)₂-S-PO₃H] and mesna (sodium 2-mercaptoethanesulfonate; HS-CH₂-CH₂SO₃Na), being the most commonly used (Brock et al., 1982; Kempf and Ivankovic, 1987; Treskes et al., 1992; Treskes and van der Vijgh, 1993; Reedijk and Teuben, 1999). WR-2721 is a "pro-drug", which (after hydrolysis by alkaline phosphatase) produces the thiol WR-1065 [S-2-(3 aminopropylamino)ethanethiol; ⁺H₃N-(CH₂)₃-NH₂⁺-(CH₂)₂-SH]. The cytoprotective mechanism of WR-1065 and mesna presumably involves formation of Pt-thiolate adducts (Dedon and Borch, 1987; Leeuwenkamp et al., 1991; Reedijk and Teuben, 1999).

The distribution of WR-1065 and mesna differs markedly. WR-1065 distributes equally between the extra- and intracellular compartments, whereas mesna distributes mostly in the extracellular compartment (Souid et al., 2001). Modeling the kinetics of DNA platination provides a framework for thinking about the reaction of cisplatin with DNA and is helpful in designing effective treatment strategies involving cisplatin.

In this study, we measure the amount of Pt bound to DNA in peripheral blood mononuclear cells (PBMC) and ovarian cancer cells as a function of [CDDP], time of incubation, and thiol content. The data are used to construct a kinetic model, which takes into account the passage of cisplatin through the cell membrane, the transit through the nuclear envelope, the reaction of cisplatin with cellular thiols, and the binding of cisplatin to DNA.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Reagents. Cisplatin [mol. wt., ~300; purchased as 1 mg/ml of solution (~3.3 mM); working solution, 0.33 mM; freshly diluted in dH₂O immediately before addition) was purchased from American Pharmaceutical Partners (Los Angeles, CA); mesna [mol. wt., 164.18; purchased as 100 mg/ml of solution (~609 mM)] from Bristol-Myers Squibb Co. (Princeton, NJ); WR-1065.2HCl · (mol. wt., 207.16) from U.S. Bioscience (West Conshohocken, PA); EDTA, GSH, N-ethylmaleimide (NEM; working solution, 0.1 M; made fresh in dH₂O), diamide [DA; azodicaroxylic acid bis(dimethylamide); working solution, 0.1 M; made fresh in dH₂O], phenol (purchased as a saturated solution with 10 mM Tris-Cl, pH 8.0, 1 mM EDTA, and stored at 20°C), proteinase k (working solution, 20 mg/ml; made in dH₂O and stored at 20°C), ribonuclease A [DNase-free from bovine pancreas; working solution, 10 mg/ml (containing ~80 units/mg); made in 10 mM sodium acetate, pH 5.5, and stored at 20°C], ribonuclease T1 (from aspergillus oryzae; working solution, 50 units/µl; diluted in ammonium sulfate and stored at 20°C), SDS (working solution, 10% in dH₂O), and Tris from Sigma (St. Louis, MO); monobromobimane (mBBr) from Molecular Probes (Eugene, OR); methanesulfonic acid from Fluka BioChemika (Ronkonkoma, NY); perchloric acid from Aldrich (Milwaukee, WI); Pt atomic spectroscopy standard (H₂PtCl₆; purchased as 1 mg/ml of solution in 10% HCl) from PerkinElmer (Norwalk, CT); nitric acid Ultrex II Ultrapure reagent from J. T. Baker, (Phillipsburg, NJ); ethyl alcohol (200 proof; USP grade) from Pharmco Products (Brookfield, CT); Ficoll-Paque from Amersham Biosciences AB (Uppsala, Sweden); RPMI 1640 medium (without L-glutamine), pH 7.15, from Mediatech (Herndon, VA); phosphate-buffered saline (PBS; without calcium or magnesium) from Bio Whittaker (Walkersville, MD); and CD45 MicroBeads, OctoMACS magnet, and MiniMACS high-gradient magnetic separation columns (capacity 2 × 10⁸ cells) from Miltenyi Biotec (Auburn, CA).

Tumor Cell Suspension. Ovarian cancer specimens were collected in RPMI medium immediately following tumor resection. Cell suspensions were produced by passing small pieces of tumors through a cell sieve (Thomas Scientific 8323-T41; 0.425-mm screen; Swedesboro, NJ) into 50 ml of RPMI medium. The suspensions were spun at 500g for 15 min, and the cell pellets were washed and respun twice. The final pellets were suspended in 20 ml of RPMI medium, and 0.8 ml was placed in each reaction mix. Each suspension was examined microscopically to confirm a pure tumor cell population.

PBMC Collection. Blood was freshly drawn from healthy volunteers into heparin-containing Vacutainer tubes (BD Biosciences, Franklin Lakes, NJ); ~10 ml of blood (containing ~1-2 × 10⁷ PBMC) was required per condition. PBMC were collected and counted as previously described (Souid et al., 2001). The yield was ~3 × 10⁶ cells/1.0 ml of blood. Cell morphology was evaluated on cytospin smears prepared with the Wright stain. Two-hour incubations with and without additions produced no noticeable morphologic changes.

Thiol Solutions. The WR-1065 and mesna solutions were prepared in dH₂O and stored at 70°C. Their concentrations were determined by 5,5'-dithio-bis(2-nitrobenzoic acid) titration immediately before additions and during the course of incubation, as previously described (Souid et al., 1998).

Incubation with Drugs. In all experiments, ~1 to 2 × 10⁷ cells/condition were incubated in RPMI medium in a final volume of 1.0 ml at 37°C. A control sample with no addition was incubated along with the experimental conditions. Mixing was by frequent, rapid inversions. The volume of each addition was 100 µl. Cells were incubated at 37°C with various concentrations of cisplatin for the indicated time before rapid DNA extraction. Cells were also incubated without or with indicated concentrations of WR-1065 or mesna at 37°C for 5 min. Cisplatin was then added to a final concentration of 5 or 7 µM, and the incubation continued at 37°C for 2 h before rapid DNA extraction.

Modification of Cellular Thiol Groups. In all experiments, ~2.0 × 10⁷ cells/condition were incubated in RPMI in a final volume of 1.0 ml at 37°C. A control sample without any addition was incubated along with the experimental conditions. PBMC were first incubated with 5 mM NEM or 5 mM DA at 37°C for 15 min. Cisplatin was then added at various concentrations. The mixtures were incubated at 37°C for the indicated time before rapid DNA extraction.

Repair of Platinated DNA. The ability of the cells to remove Pt from DNA was measured. PBMC (4.0 × 10⁷/condition in RPMI) were incubated at 37°C for 15 min without or with 5 mM NEM. Cisplatin was then added to a final concentration of 7 µM to normal and thiol-blocked cells, and the incubation continued at 37°C for 2 h (final volume, 1.0 ml). The cells were collected by centrifugation, and the DNA was extracted immediately. In parallel, identical incubations the cells were resuspended in 1.0 ml of RPMI, and incubated at 37°C for additional 2 h to allow for adduct repair before DNA extraction.

GSH Determination. Cellular thiols that were not modified by NEM were alkylated with the fluorescent probe mBBr. The low molecular weight alkylated thiols in the acid-soluble supernatants were separated by high-pressure liquid chromatography (HPLC) and detected by fluorescence (Souid et al., 1999, 2001). Briefly, at the end of the 15-min incubation without or with 5 mM NEM, cells (2.1 × 10⁷/condition; mean ± S.D. cell volume, 190 ± 50 fl) were collected by centrifugation, suspended in 20 mM Tris-methanesulfonic acid, pH 8.0, and 5 mM mBBr (final volume, 0.5 ml). After incubation in the dark at RT for 15 min, the cells were collected by centrifugation, and their acid-soluble supernatants were prepared by the addition of 300 µl of 2.5% perchloric acid/2 M sodium methanesulfonate. After vigorous vortexing, the supernatants were collected by centrifugation, and the amount of GS-bimane adducts was determined by HPLC (Souid et al., 1998).

Cellular Drug Thiol Determination. PBMC were collected from a healthy volunteer and purified with CD45 MicroBeads on a MiniMAC column attached to a magnet exactly as described (Souid et al., 2001). Purified PBMC (~10⁷ cells/condition) were incubated at 37°C for 15 min with 1.0 ml of PBS without or with 2 mM WR-1065 or 2 mM mesna. mBBr was then added to a final concentration of 30 mM, and the derivatization continued in the dark at RT for 20 min. The cells were collected by centrifugation and washed twice with PBS. The concentrations of WR-1065 and mesna in the acid-soluble supernatants were determined by HPLC.

Isolation of DNA. Cells were collected by centrifugation and resuspended in 10 mM Tris-Cl, 10 mM EDTA, pH 8, and 1% SDS (w/v) in the presence of 0.5 mg/ml proteinase k (final volume, 2.0 ml). The solutions were incubated overnight at 37°C and transferred to glass tubes. An equal volume of phenol was added, and the samples were mixed by vigorous vortexing and centrifuged as above. DNA was precipitated with 2 volumes of cold (20°C) absolute ethyl alcohol, collected on glass rods, rinsed with 1.0 ml of cold 80% ethyl alcohol, and air-dried at RT for 15 min. After resuspension (in 0.5 ml of 10 mM Tris-Cl), 10 mM EDTA, pH 8, 40 µg of ribonuclease A, and 25 units of ribonuclease T1 were added, and the solutions were incubated overnight at 37°C. DNA was then precipitated, collected, rinsed, and dried as described above. The final DNA pellets were suspended in 200 µl of dH₂O and incubated overnight at 37°C to rehydrate. DNA concentrations were calculated on a Beckman spectrophotometer (Model DU 640B; Beckman Coulter, Inc., Fullerton, CA) in a 1-cm cell, using the formula of 50 µg/ml double-stranded DNA corresponding to 1.0 absorbance unit at 260 nm (₂₆₀, ~12,000 M¹ · cm¹). The DNA concentration was 606 ± 42 (55) µg/ml [mean ± S.D. (n)], and A₂₆₀/A₂₈₀ was 1.73 ± 0.03.

Pt Analysis. Pt analysis was performed using the graphite furnace of a Shimadzu AAS (Model AA-6800; Kyoto, Japan), with an ist hollow cathode Pt lamp (Imaging and Sensing technology, Horseheads, NY), deuterium arc background correction, and pyrolytically coated graphite tubes. The graphite tubes were changed after 100 ignitions. Argon gas and tap water flowed through the furnace hoses. The instrument operated at a lamp current of 14 mA, wavelength of 266.0 nm, and slit width of 0.5 nm. The Pt standard (H₂PtCl₆) was a 51.3 nM (0.01 mg/liter) solution, freshly prepared by serial dilutions of the Pt atomic spectroscopy standard stock in dH₂O plus 1% HNO₃ (v/v). A calibration curve was generated immediately before each measurement. It was linear from 0 to 1.0 pmol (r > 0.98); the lower limit of detection was ~20 pg of atomic Pt (~0.1 pmol). Each sample was measured in triplicate. The injection volume was 10 µl, containing 4 to 8 µg DNA. The furnace program used sequential drying (70°C for 10 s, 90°C for 10 s, and 120°C for 10 s), charring (250°C for 10 s and 800°C for 25 s), cooling (30°C for 20 s), and atomization (2600°C for 5 s) phases. The control samples (i.e., blood incubated without any addition) gave absorbance values that were the same as those of dH₂O. Calculations were based on 1 pg of Pt/µg of DNA = 5.13 fmol of Pt/µg of DNA (based on the molecular weight of Pt; 195.078), and 1 fmol of Pt/µg of DNA = ~ 0.34 Pt molecules/10⁶ nucleotides (nt) (based on the average molecular weight of the nucleotides; ~343 g mol¹) (McGahan and Tyczkowska, 1987; Reed et al., 1988).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

PBMC Treated with Cisplatin. A plot showing the amount of Pt bound to DNA, after a 2 h exposure, as a function of [CDDP] in the incubation medium is shown in Fig. 1 (left panel). Over the concentration range of the study, the plot is nonlinear. However, it appears to consist of two linear portions. For [CDDP] less than about 5 µM, the slope of the plot is ~1.3 adducts/µM cisplatin. For [CDDP] >~5 µM, the slope is much larger, with ~9.5 adducts/µM cisplatin. For higher concentrations (up to 80 µM; data not shown), the amount of Pt bound to DNA remains linear in [CDDP].

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Normal (left panel) and thiol-blocked (right panel) PBMC treated with cisplatin. In each condition, ~2.5 × 107 cells were incubated at 37°C with 1 to 9 µM cisplatin for 2 h. In the thiol-blocked condition, PBMC were incubated at 37°C for 15 min with 5 mM NEM before the addition of cisplatin. At the end of the incubation period, DNA was extracted, and the number of Pt adducts determined using AAS. The values are mean ± S.D. of three separate measurements.

Thiol-Blocked PBMC Treated with Cisplatin. To study how thiol functions affect Pt binding to DNA, cells were treated with a large excess of NEM or DA; both agents selectively modify thiol groups so that they cannot bind Pt. Analysis using HPLC showed that the amount of unmodified GSH remaining after treatment of cells with NEM or DA was 3% (Table 1). Treatment with NEM produced 8-fold enhancement in the level of Pt adducts and with DA about 4-fold enhancement (Table 1).

Kinetics of Cisplatin Binding to PBMC DNA. The manner in which the amount of Pt adducts changes with the incubation time for normal and thiol-blocked cells (treatment with NEM) is shown in Fig. 2. Although there is much scatter in the points, each plot shows an initial rapid increase followed by a slower increase up to 2 h. At each time, cells having blocked thiols had increased amount of Pt bound to DNA by a factor of 2 or 3. 

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Kinetic curve of normal and thiol-blocked cells. In each condition, ~2.5 × 107 cells were incubated at 37°C for 15 min without (open squares) and with (closed squares) 5 mM NEM. Treatment with NEM blocks thiols so that they cannot bind platinum. At the end of the incubation period, cisplatin was added to a final concentration of 7 µM, and the reactions were continued at 37°C for the indicated time. DNA was then extracted, and the number of Pt adducts determined using AAS. The values are mean ± S.D. of three separate measurements.

DNA-Pt Adduct Repair. Incubating PBMC with platinated DNA in cisplatin-free medium for 2 h resulted in substantial removal of Pt from DNA via repair. The numbers of Pt adducts per 10⁶ nt for normal and thiol-blocked cells before the 2 h repair period were 75 ± 5 and 185 ± 5, respectively, whereas the number of adducts after repair were 5 ± 2 and 40 ± 18, respectively. Control cells to which no cisplatin was added gave 5 ± 2 adducts/10⁶ nt. Thus, in normal cells, 100% of the Pt-bound DNA was repaired in 2 h, and in thiol-blocked cells, ~80% was repaired. Similar results were obtained in cells treated with DA and iodomethane (data not shown).

Effects of WR-1065 and Mesna on DNA-Pt Adducts. The data in Table 2 show the number of Pt adducts in PBMC of healthy volunteers exposed to 5 µM cisplatin and 300 µM WR-1065 or mesna (volunteer 1) or 7 µM cisplatin and 150 µM WR-1065 or mesna (volunteer 2). These levels are routinely achieved in patients receiving cisplatin, WR-1065, and mesna (see Discussion). At these concentrations, neither agent affected DNA platination (Table 2).

Cellular Drug Thiol Concentrations. The concentration of WR-1065 in highly purified PBMC (see Materials and Methods) following incubation (37°C for 15 min) with 2 mM WR-1065 was ~2440 µM (that is, ~112% of that of the incubation medium). In contrast, the concentration of mesna following incubation (37°C for 15 min) with 2 mM mesna was only ~130 µM (that is, ~6% of that of the incubation medium).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The cytotoxicity of cisplatin is related to its ability to bind to DNA (Zwelling et al., 1979; Knox et al., 1986). Competing with DNA for the drug are numerous thiol functions, such as those on GSH and MT (Kraker et al., 1985; Dedon and Borch, 1987; Eastman, 1991; Zhang et al., 1995; Bose et al., 1997; Zhang et al., 2001). If these groups are blocked from binding Pt (by modification with NEM or DA), the amount of cisplatin available to form Pt-DNA adducts is increased. Figures 1 and 2 and Table 1 support this.

The fact that significant cellular GSH depletion does not produce the same level of Pt-DNA adduct enhancement by NEM (Table 1) suggests an important role for other cellular thiol pools in binding cisplatin (e.g., MT and thiol groups on the chromatin) (Zhang et al., 2001).

For normal PBMC, the initial slope of the plot of the number of adducts after a 2-h incubation versus [CDDP] is small (Fig. 1, left panel), with ~1.3 adducts/µM cisplatin (using the first three points). This indicates that much of the cisplatin entering the cell is bound by thiols and unavailable for binding DNA. However, at high [CDDP], the slope of the plot rises sharply (Fig. 1, left panel), with ~9.5 adducts/µM cisplatin (using the last three points). Thus, small increments in cellular cisplatin concentration above 5 µM give substantial enhancements in adduct levels (e.g., increasing [CDDP] from 5 to 9 µM increased the number of bound Pt atoms per 10⁶ nt of DNA from 10 to 47). Such enhancement is expected for cisplatin concentrations high enough to saturate the cellular thiols that compete for Pt binding.

One also expects the slope of such a plot to be large when thiols are blocked, so they cannot bind Pt (as by treatment with NEM or DA). The slope of Fig. 1 (right panel) is in fact about twice the high-[CDDP] slope of Fig. 1 (left panel). However, the latter is calculated from the last three points of Fig. 1 (left panel), whereas the slope is probably still increasing at [CDDP] = 9 µM. It is expected that the plot becomes linear at higher [CDDP]. Once the thiols are blocked, there should be a simple linear relationship between [CDDP] and the number of Pt atoms bound to DNA for all [CDDP]. Indeed, Fig. 1 (right panel) shows that for thiol-blocked cells the relationship is linear.

Efficient Pt removal from DNA occurs for both normal and thiol-blocked cells (see Results). Thus, treatment with the thiol-modifying agents does not compromise cellular repair mechanisms. The enhancement in the number of Pt adducts in cells treated with NEM or DA (Table 1; Fig. 1, right panel) is, therefore, mostly due to an increase in the amount of Pt available for binding to DNA (Fig. 2).

The extent to which thiols added as cytoprotective agents affect the amount of DNA platination by cisplatin in fresh human cells has not been previously reported, especially at therapeutic cisplatin levels. The data show that neither WR-1065 nor mesna, when added at clinically achievable concentrations (i.e., ~300 µM), affected DNA platination (Tables 2-4). In the current Children Oncology Group trial (9970), the peak free plasma Pt levels following a 1-h infusion of 30 mg/m² cisplatin were 4.7 ± 1.6 (15) µM [mean ± S.D. (n)] (A.-K. Souid, unpublished data). Other investigators reported a peak level of ~10 µM following a 1-h infusion of 70 mg/m² cisplatin (Korst et al., 1998). Thus, the concentrations of cisplatin in Figs. 1 and 2 and Tables 1 to 4 are within the therapeutic range.

Furthermore, we previously reported on the WR-1065 plasma and blood cell levels following intravenous infusion of these agents (Souid et al., 1999, 2001). Briefly, immediately following a 15-min infusion of WR-2721 (850 mg/m² or ~3.1 mmol/m²), WR-1065 peaked in the plasma at 76 ± 31 (6) µM and blood cells at 77 ± 25 (6) µM [mean ± S.D. (n)]. These levels are similar to the previously reported peak plasma WR-1065 value (~50 µM) (Korst et al., 1996, 1997). However, following a 15-min infusion of mesna (400 mg/m² or ~2.4 mmol/m²), mesna level peaked in the plasma at ~340 µM and blood cells at ~85 µM (Souid et al., 2001). The plasma levels are similar to those reported by others (Goren et al., 1998). Thus, the clinical studies demonstrate that patients receiving conventional doses of WR-271 and mesna achieve plasma levels in the micromolar range, and the cellular concentrations do not markedly exceed those found in the plasma (see Results under Cellular Drug Thiol Concentrations).

Both WR-1065 and mesna decay rapidly by oxidation, with a t_1/2 of ~16 min (Goren et al., 1998 and references therein; Souid et al., 1999, 2001), which is similar to measured decay rates in the incubation medium (see Materials and Methods under Incubation with Drugs).

The data in Tables 2 to 4 show that the number of Pt adducts in PBMC and ovarian cancer cells is not affected by concentrations ~300 µM WR-1065 or mesna. This finding confirms the insignificant effect on DNA platination when patients received WR-2721 before cisplatin (Korst et al., 1998). In contrast, millimolar concentrations of WR-1065 or mesna significantly diminish DNA platination (Tables 3-4), presumably due to cisplatin binding to the thiolate ions. The discrepancy in the concentrations of these two thiol agents required to abolish DNA platination (~1.25 mM WR-1065 versus ~5 mM mesna; Table 3) reflects the higher uptake of WR-1065 by PBMC (see Results under Cellular Drug Thiol Concentrations). Furthermore, since the reactive moieties are thiolate anions, the pK_a of the thiol groups and the pH of the medium play an important role in determining the efficacy of the drug thiols. The experiments are performed at a pH value of ~7.2. The pK_a value of WR-1065 is ~7.7, GSH ~8.7, and mesna ~9.1 (Shaked et al., 1980; Newton et al., 1992). Thus, WR-1065 is expected to be the most reactive among the three thiols studied (since more of the WR-1065 thiol group is ionized at pH 7.2 and available for nucleophilic attack on Pt).

Similarly, in the two ovarian cancers, WR-1065 and mesna at concentrations ~300 µM produced no effect on DNA platination (Table 4) and at 5 mM produced ~90% inhibition (Table 4; patient 1).

Phenomenological Model. To show the relation between the slopes for normal cells at high or low [CDDP] and thiol-blocked cells (Fig. 1, left and right panels, respectively), we propose a simple model to show the relations between the processes that take place for platination of DNA by cisplatin. For cisplatin to bind to DNA, the following processes must be taken into account: 1) passage of cisplatin into the cytoplasm; 2) passage of cisplatin into the nucleus; 3) reaction of cisplatin with cellular sulfhydryl groups; 4) binding of cisplatin to DNA; and 5) repair (eliminating Pt from DNA). If repair occurs, sites with bound Pt are replaced, but the Pt is not returned to the pool of Pt available for binding to DNA. If drug thiols are present, their passage into the cells must be included in the model.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Diagram of the cell with important steps indicated. The rate constants for cisplatin entering the cell and the nuclear envelope are ka and kc, respectively.

(1)

(2)

(3)

(4)

1

1

1

1

1

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Results of calculations using a simple phenomenological model (for values of parameters, see text). Incubation time is 2 h. Solid- and dashed-line plots show the amount of binding of Pt to DNA after a 2-h incubation with cisplatin as a function of extracellular [CDDP]. Asterisks and dashed lines are for sulfhydryl initially available at a 2 mM concentration, filled circles and the solid line for sulfhydryl blocked and unavailable. Triangles represent available sulfhydryl concentration after 2 h as a function of extracellular [CDDP].

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Results of calculations using a simple phenomenological model (for values of parameters, see text). Extracellular [CDDP] is 7 µM. Solid- and dashed-line plots show the amount of binding of Pt to DNA after incubation with cisplatin as a function of incubation time. Asterisks and the dashed line are for sulfhydryl initially available at a concentration of 2 mM, filled circles and the solid line for sulfhydryl blocked and unavailable. Triangles represent available sulfhydryl concentration as a function of extracellular [CDDP].

1

1

1

1

View larger version (14K): [in this window] [in a new window]   Fig. 6.   Effect of drug thiol concentration on the amount of Pt bound to DNA, according to the kinetic model described in text.