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Little is understood about the mechanism of acquired resistance to the widely used antineoplastic drug, cisplatin. The cisplatin-resistant murine leukemia L1210 cell line L1210/PAM has an elevated cellular glutathione content, as compared to its sensitive parent cell line, L1210/*. Exposure to D,L-buthionine-S,R-sulfoximine reduced L1210/PAM cells' glutathione content to nearly that of L1210/* cells and abrogated the resistance to cisplatin.

Under anaerobic conditions and with proper electron donors, NADPH-cytochrome P-450 reductase (EC 1.6.2.4) and xanthine oxidase (EC 1.2.3.2) similarly reductively metabolized mitomycin C. Reversed phase high performance liquid chromatography was used to separate, detect, and isolate several metabolites. Three metabolites were identified by mass spectrometry and thin layer chromatography as 1,2-cis- and trans-2,7-diamino-1-hydroxymitosene and 2,7-diaminomitosene. Three metabolites were phosphate-dependent, and two of them were identified to be 1,2-cis- and trans-2,7-diaminomitosene 1-phosphate. The amounts of the five identified metabolites generated during the reduction of mitomycin C varied with pH and nucleophile concentration. At pH 6.5, 2,7-diaminomitosene was essentially the only metabolite formed, whereas from pH 6.8 to 8.0, trans- and cis-2,7-diamino-1-hydroxymitosene increased in quantity as 2,7-diaminomitosene decreased. The disappearance of mitomycin C and the production of metabolites were enzyme and mitomycin C concentration-dependent. Substrate saturation was not reached for either enzyme up to 5 mM mitomycin C. Electron paramagnetic resonance studies demonstrated the formation of mitomycin C radical anion as an intermediate during enzymatic activation. Our results indicate that either enzyme catalyzed the initial activation of mitomycin C to a radical anion intermediate. Subsequent spontaneous reactions, including the elimination of methanol and the opening of the aziridine ring, generate one active center at C-1 which facilitates nucleophilic attack. Simultaneous generation of two reactive centers was not observed. All five primary metabolites were metabolized further by either flavoenzyme. The secondary metabolites exhibited similar changes in their absorbance spectra and were unlike the primary metabolites, suggesting that a second alkylating center other than C-1 was generated during secondary activation. We propose that secondary activation of monofunctionally bound mitomycin C is probably a main route for the bifunctional binding of mitomycin C to macromolecules and that the cytotoxic actions of mitomycin C result from multiple metabolic activations and reactions.

We have determined the effect of glutathione (GSH) depletion on the cytotoxicity of three nitrogen mustards, six platinum complexes, and mitomycin C in a human ovarian carcinoma cell line. GSH levels in COLO 316 cells were depleted by exposure of cell monolayers to 0.5 mM D,L-buthionine-S,R-sulfoximine. GSH depletion significantly potentiated the cytotoxicity of L-phenylalanine mustard, chlorambucil, and mechlorethamine as determined by clonogenic assay on plastic plates. The dose modification factors were 2.6, 2.6, and 1.9, respectively. The same level of GSH depletion had a minimal effect on the cytotoxicity of cis-diamminedichloroplatinum(II) (cis-DDP), carboplatin, dichloro(ethylenediamine)platinum(II), 1,2-diaminocyclohexylplatinum(II) malonate, and iproplatin. The dose modification factors of GSH depletion for these drugs were 1.4 or less. trans-Diamminedichloroplatinum(II) was, however, markedly potentiated by GSH depletion with a dose modification factor of 2.7. Mitomycin C was minimally potentiated by GSH depletion. We have also generated cis-DDP-resistant cells from COLO 316 and 2008 human ovarian carcinoma cells by in vitro selection with cis-DDP. These cis-DDP-resistant cells had identical levels of GSH as the parental cells. GSH depletion sensitized these cells only to the same degree as the parental cells and did not reverse the resistant phenotype. Our results indicate that intracellular GSH levels are not an important determinant of the cytotoxicity of cis-platinum(II) or cis-platinum(IV) complexes in COLO 316 and 2008 cells. In addition, altered GSH metabolism does not appear to be a component of the cis-DDP-resistant phenotype in these cells.

cis-Diamminedichloroplatinum(II) (DDP) was measured in plasma ultrafiltrate following derivatization with sodium diethyldithiocarbamate (DDTC) by quantitation against a nickel chloride internal standard. A chloroform extract containing the Pt(DDTC)2 and Ni(DDTC)2 complexes was separated by reversed-phase high-performance liquid chromatography on a C18 radial compression column. The complex was eluted with methanol/water, 4/1, at a flow rate of 1.5 ml/min, and was detected at 254 nm. The limit of sensitivity was 0.1 microgram/ml DDP in the ultrafiltrate. This analytical approach was validated by comparison to graphite furnace atomic absorption spectrophotometric determinations of duplicate samples. There was clearly a component of the ultrafiltrable platinum present that was resistant to derivatization by DDTC. Evidence is presented that this component, presumably Pt(II) complexed with endogenous small molecules, is non cytotoxic and, hence, that this method may be selective for "active Pt(II)." This method offers an advantage over atomic absorption determination of total platinum in ultrafiltrate which does not discriminate between active and inactive forms, and over off-line FAA detection of parent DDP in HPLC eluates which ignores other active forms. Using this technique we have measured the pharmacokinetics of DDTC-reactive Pt(II) in humans after either i.v. infusion or infusion of DDP into the peritoneal cavity of patients with ovarian carcinoma.

The pharmacokinetics of 5-fluorouracil (5-FU) injected into a surgically isolated pelvic circuit during hyperthermic perfusion was studied in five patients with local recurrence of anorectal cancer. 5-FU doses ranged from 11 to 23 mg/kg. The geometric mean ratio of peak plasma 5-FU in the isolated to systemic circuits was 10, the ratio at the end of the 45-minute perfusion was 12.5. The mean half-life of 5-FU in the isolated circuit was 18.5 minutes. Total drug exposure for the isolated circuit was 7.8-fold greater than for the systemic compartment. These results demonstrate a large pharmacologic advantage for the use of the isolation-perfusion technique.

6-thioguanine (6TG) and its metabolites were analyzed in human plasma with a reversed-phase high-performance liquid chromatographic methods. 6TG and related compounds were extracted from plasma with an equal volume of 2 N perchloric acid at a 50-100% recovery efficiency. The neutralized extracts were chromatographed on a muBondapak C18 column by two separate isocratic conditions. 6TG, 6-thiouric acid, 6-thioxanthine, 6-thioguanosine, and 6-methylthiouric acid were analyzed with 0.01 M sodium acetate, pH 3.5-10% methanol as the mobile phase and 340 nm for detection. 6-Methylthioguanine and three unknown metabolites were separated with acetate-25% methanol and 310 nm detection. One of the unknowns was identified as 6-methylthioguanosine. External standard calibration was used for quantitation. The 6TG detection limit was 0.8 nmol/ml in plasma.

Analysis of partial duplication of chromosome 21 suggests that band 21q22 contains determinants for the Down syndrome. We report two cases of free proximal trisomy 21 without manifestations of the Down syndrome. Phenotypic anomalies included marked microcephaly, short stature, hypoplastic nails, and mental retardation/developmental delay. Our cases are consistent with the assignment of band 21q22 as the causal duplicated segment in the Down syndrome.

We administered cisplatin and etoposide by peritoneal dialysis to 39 patients with i.p. malignancies in order to investigate the toxicity, pharmacokinetics, and clinical activity of this 2-drug combination. All patients received i.v. sodium thiosulfate concurrently with the i.p. chemotherapy. Myelosuppression, nausea, vomiting, and malaise were the primary toxicities encountered. The maximum tolerated dose of etoposide was 350 mg/m2, when administered with a fixed dose of cisplatin, 200 mg/m2. Although the total (free and protein-bound) etoposide exposure for the peritoneal cavity was only 1.5-fold greater than that for the plasma, the free (non-protein bound) etoposide peritoneal exposure was 65-fold greater than the plasma. Tumor regressions were noted in patients with ovarian and pancreatic carcinomas. This study is the first demonstration of the large pharmacokinetic advantage that exists for the i.p. administration of highly protein-bound drugs, and it also documents the clinical activity of i.p. cisplatin and etoposide.

The clinical pharmacology of intraperitoneally administered bleomycin was examined in 11 patients with malignancies confined predominantly to the peritoneal cavity. Bleomycin (60 mg/m2) was mixed in 2 L of 0.9% NaCl, and administered rapidly into the peritoneal cavity via a Port-a-Cath (Pharmacia Nu Tech, Inc, Walpole, MA). The cavity was drained following either a four- or eight-hour dwell time. Bleomycin concentration in the peritoneal cavity and plasma was determined by radioimmunoassay. A total of 15 courses was studied. The peak peritoneal: plasma concentration ratio averaged 22, and total exposure for the peritoneal cavity averaged seven-fold greater than that for the plasma. The peritoneal and plasma half-lives for bleomycin were 3.2 and 6.0 hours, respectively. The peritoneal clearance of bleomycin averaged 11.3 mL/min/m2. Abdominal pain occurred on 47% of courses; other toxicities included fever, skin rash, and mucositis. We conclude that there is a substantial pharmacologic advantage to the peritoneal route for tumors confined to the peritoneum, but that the advantage is less, and the local toxicity greater, than that found with the intraperitoneal administration of many other anticancer drugs.

Certain human DNA sequences are much less methylated at CpG sites in sperm than in various adult somatic tissues. The DNA of term placenta displays intermediate levels of methylation at these sequences (Sp-0.3 sequences). We report here that pluripotent embryonal carcinoma (EC) cells derived from testicular germ cell tumors are hypermethylated at the three previously cloned Sp-0.3 sequences and seven newly isolated sequences that exhibit sperm-specific hypomethylation. In contrast to their hypermethylation in EC cells, the Sp-0.3 sequences are hypomethylated in a line of yolk sac carcinoma cells, which like placenta, represent an extraembryonic lineage. These DNA sequences, therefore, appear to be subject to coordinate changes in their methylation during differentiation, probably early in embryogenesis, despite their diversity in copy number (1 to 10(4] and primary structure. Two of these Sp-0.3 sequences are highly homologous to DNA sequences in human chromosomal regions that might be recombination hotspots, namely, a cryptic satellite DNA sequence at a fragile site and the downstream region of the beta-globin gene cluster.

A ring chromosome 12 (p13.3q24.3) was observed in all cells analyzed from skin fibroblasts and the peripheral blood of a 19-year-old man initially referred for developmental delay with expressive language deficiency. Other phenotypic anomalies included growth deficiency, multiple café-au-lait spots, mild pectus excavatum, glandular hypospadias, left esotropia, clinodactyly of the fifth fingers, and hypothyroidism with elevated antithyroid antibodies. The four previously reported cases of r(12) support the theory of a general ring phenotype which is manifested independently of the specific autosome involved and which is characterized by growth failure, moderate mental retardation, and lack of other major phenotypic anomalies. Breakpoints in all cases of r(12) have been assigned to the telomeric regions, suggesting minimal deletion of chromosome material.

We compared the relative susceptibility of HL-60, a human acute progranulocytic leukemia cell line, and the normal human myeloid progenitor cell, the colony-forming unit in culture, to growth inhibition by thymidine. Normal human myeloid colony formation was more sensitive to thymidine than was HL-60 colony formation, the former being inhibited by greater than or equal to 0.5 mM thymidine and the latter by greater than or equal to 5.0 mM. Colony inhibition by thymidine was irreversible in both cases after a seven-day incubation of the colony-forming unit in culture in liquid medium enriched with thymidine and subsequent replating in thymidine-free soft agar. Thymidine-induced inhibition of HL-60 cloning could be partially prevented by deoxycytidine, whereas normal myeloid cloning could not, suggesting that high-concentration thymidine may affect processes necessary for cloning in addition to deoxyribonucleotide synthesis. HL-60 growth in liquid suspension culture could be suppressed transiently by 1.0 mM thymidine, suppressed totally by greater than or equal to 5.0 mM thymidine, and rescued completely by concomitant addition of deoxycytidine. The development of resistance to 1.0 mM thymidine could not be accounted for by reduction in thymidine pool size or by reduction in thymidine kinase activity. Replating of HL-60 cells growing in the presence of 1.0 mM thymidine in liquid medium in thymidine-free soft agar produced significant colony inhibition, also suggesting that thymidine affects more than just deoxyribonucleotide synthesis or that the clonogenic HL-60 cell presents a distinct subpopulation with more sensitive deoxyribonucleotide-synthetic control mechanisms.

The disposition, metabolism, and excretion of adriamycin octanoylhydrazone (OctAdr) were studied after iv administration to BALB/c mice and New Zealand White rabbits and were compared to similar studies of adriamycin (Adr). In mice, concentrations of OctAdr-derived fluorescence were initially greatest in lung with very low concentrations detectable in kidney, liver, and spleen. Little drug fluorescence was found in brain, heart, or skeletal muscle. Adriamycin had the highest drug fluorescence concentration in the kidney, with progressively lower but easily detectable concentrations in liver, heart, lung, spleen, and skeletal muscle. Drug fluorescence was lost much more rapidly from tissues of mice injected with OctAdr than from tissues of mice injected with Adr. At all times after injection, Adr was the major fluorescent drug species recovered from livers and kidneys of mice treated with OctAdr or Adr, although substantial amounts of unaltered OctAdr were recovered from the former group. In rabbits, plasma concentrations of OctAdr-derived fluorescence declined rapidly but then remained relatively constant from 120 to 480 min after injection. No OctAdr-derived fluorescence was observed in urine. During the 8 hr after injection, biliary excretion of OctAdr represented approximately 25% of the administered dose. At all times after injection, OctAdr was the major biliary fluorescent species, with Adr and adriamycinol (Adrol) the second and third most prominent, respectively. Eight hours after OctAdr administration, lung contained the highest concentration of drug-related fluorescence, with progressively lower amounts in liver, kidney, duodenum, and heart. Brain, skeletal muscle, and spleen contained little or no OctAdr-derived fluorescence. Adr and OctAdr were the major fluorescent species in all tissues. Small amounts of Adrol were detected in all tissues, but aglycones were present only in liver.

We conducted studies to determine whether the cisplatin (DDP)-resistant human ovarian carcinoma cell line designated 2008/DDP has membrane changes which could slow the passive diffusion of the drug into cells, thereby accounting for the observed reduction in DDP accumulation in 2008/DDP cells compared to the sensitive parental 2008 cells. To this end, we compared three indicators of membrane lipid composition and fluidity in the resistant and sensitive cells. In a comparison of major membrane lipid classes, the resistant line had a 17% increase in phosphatidyl choline, and an 11% increase in phosphatidyl ethanolamine relative to the sensitive line. There were no differences in the other major phospholipids or in the free cholesterol content of the cell lines. Secondly, results of fluorescence polarization measurements on whole cells of each line using the plasma membrane specific probe trimethylammonium diphenylhexatriene revealed no difference between the cell lines at both 28 degrees C and 37 degrees C. Finally, an examination of the temperature-dependence of DDP accumulation over 1 h produced Arrhenius plots of similar shape in 2008 and 2008/DDP cells. Both plots were linear from 40 degrees C down to a break point between 4 degrees C and 12 degrees C. The Q10's for DDP accumulation from 30 degrees C to 40 degrees C were greater than 2 in each cell line and were not significantly different. Results of these 3 comparisons point to substantial similarity in the bulk membrane lipid composition and fluidity of the two cell lines. We conclude that decreased DDP accumulation in our resistant cells is not due to membrane changes that would be expected to retard passive diffusion of the drug into the cells.

We have determined the effect of extended glutathione (GSH) depletion on cis-diamminedichloroplatinum(II) (DDP) cytotoxicity in parent and DDP-resistant human ovarian carcinoma cells. Cells were exposed to 50 microM buthionine sulfoximine (BSO) for 48 h and exposed to DDP for the last 24 h of this time. This treatment protocol sensitized 2008 cells to DDP. The dose modification factor (DMF) defined as IC50 control cells/IC50 GSH depleted cells was 1.6 +/- 0.5 (N = 9). DDP-resistant cells selected by acute, high dose DDP exposure were also sensitized by this treatment; the DMF in the 3-6-fold resistant 2008/DDP cells was 2.4 +/- 1.2 (N = 9). The sensitization was not significantly greater in the resistant cells than in the parent cells (P greater than 0.05). When the rebound of GSH following BSO exposure was reexamined, the GSH levels were found to rise rapidly following trypsinizing and plating. BSO treatment following DDP exposure had no effect on DDP cytotoxicity in 2008 and 2008/DDP cells. These results indicate that simply depleting GSH prior to DDP exposure is not sufficient for sensitizing these cells to DDP. In contrast to the potentiation of nitrogen mustard cytotoxicity, exposure to GSH depletion must be maintained during DDP treatment for enhancement of DDP cytotoxicity to occur.

Adriamycin and eight metabolites were separated and characterized with an ammonium formate/tetrahydrofuran solvent system on a muBondapak-Phenyl column. Danorubicin and seven daunorubicin metabolites were separated and characterized by the same method. Adriamycin and its major metabolite, adriamycinol, were extracted with 50-70% efficiency from human plasma with chloroform/2-propanol (1:1) and quantified against a daunorubicin internal standard. Urinary concentrations of parent drug, adriamycinol, and the 4-O-sulfate were determined as well. Daunorubicin and daunorubicinol were extracted from human plasma and quantified against an adriamycinol internal standard. The fluorescence detection system has a detection limit of 1.0 pmol, and levels as low as 25 mmol/ml are measurable in plasma.

We have characterized the accumulation of cisplatin (DDP) into parent and cisplatin-resistant 2008 human ovarian carcinoma cells. Accumulation of DDP at 1 h was a linear function of concentration from 0.25 to 100 microM DDP in both cell types. DDP-resistant cells that were only 3.3-fold resistant had approximately 50% less accumulated platinum at all concentrations examined. Accumulation of 1.0 microM DDP was linear for approximately 3 h and then slowed but did not reach equilibrium at up to 24 h in either cell type. The decreased DDP accumulation in resistant cells did not appear to be due to increased efflux; similar percentages of platinum were available for exodus in parent and resistant cells. Intracellular metabolites of DDP appeared to be decreased by similar amounts in the resistant cells. Native DDP was not driven uphill into cells against a concentration gradient, suggesting that DDP uptake does not involve primary active transport. The metabolic inhibitors, dinitrophenol and NaF, did not decrease DDP accumulation; iodoacetate had a stimulatory effect. Dinitrophenol, however, in combination with NaF or iodoacetate decreased DDP accumulation. A 30-min exposure to 0.2 mM ouabain also decreased DDP accumulation in both parent and resistant cells. A component of DDP accumulation thus appears to be energy dependent. These studies have identified decreased DDP accumulation as an important mechanism of resistance that is expressed early in the acquisition of DDP resistance in human ovarian carcinoma cells.

Nephrotoxicity frequently limits the dose of cisplatin to less than 120 mg/m2 per injection. Sodium thiosulfate is a neutralizing agent for cisplatin that protects against renal damage. To determine whether injection of thiosulfate would permit larger doses of cisplatin to be administered, a fixed 9.9-g/m2 dose of thiosulfate was given intravenously over three hours concurrently with escalating doses of cisplatin. Cisplatin was administered over the last two hours of the thiosulfate infusion. Using this technique, it was possible to escalate the cisplatin dose to 225 mg/m2 before dose-limiting toxicities were encountered. Comparison of cisplatin pharmacokinetics in patients treated with 202.5 mg/m2 plus thiosulfate to those in patients treated with 100 mg/m2 without thiosulfate indicated that there were no changes in the elimination rate constant, volume of distribution, or total body clearance of cisplatin. The total drug exposure for the plasma was approximately twofold at the higher cisplatin dose. This study demonstrates that concurrent administration of thiosulfate permits at least a twofold increase in dose and total exposure to cisplatin.

We have determined the ability of two human ovarian carcinoma cells to over-express metallothioneins (MTs) and the subsequent effect this elevation has on DDP cytotoxicity. Cells of 2008 and COLO 316 human ovarian carcinomas that were resistant to CdCl2 were obtained by stepwise selection and chronic culture in CdCl2 and ZnCl2. The 2008/MT cells were 3.2-fold resistant to CdCl2 and 4.1-fold resistant to DDP; they had 23-fold elevated MTs. The COLO/MT cells were 1.2-fold resistant to CdCl2 and 3.3-fold resistant to DDP, and they had 9-fold elevated MTs. Glutathione (GSH) was also elevated in the Cd-resistant sublines. However, four times more intracellular thiols were contributed by the MTs than by the GSH. 2008 and 2008/MT cells were examined in more detail to elucidate the mechanism of DDP resistance. Depletion of GSH with D,L-buthionine-S,R-sulfoximine (BSO) had no effect on the sensitivity of these cells to either CdCl2 or DDP. Uptake of [195m Pt]DDP in 2008 and 2008/MT cells was identical. Fractionation of the cytosol from [195mPt]DDP-exposed cells on Sephadex G-75 revealed that 17% of the total cellular Pt in 2008/MT cells was associated with the MT fraction, as against 4% in the parent 2008 cells. This increase corresponded to a concomitant loss of Pt from the particulate fraction. Fractionation of 2008 cells selected with DDP (2008/DDP cells) indicated that elevated MTs did not contribute to the DDP resistance of these cells. Only 2% of the total cellular Pt was in the MT fraction in 2008/DDP cells. These results showed that elevation of MTs may be one mechanism of DDP resistance in ovarian carcinoma; however, in vitro selection with DDP does not trigger this mechanism.

Eleven metabolites of the anthracycline antineoplastic antibiotic aclacinomycin A (Acm) were isolated and purified from the urine of four patients treated with 100-120 mg/m2 of Acm. In an initial step, pooled urine was extracted and Acm and its metabolites were concentrated in n-butanol with a horizontal flow-through coil planet centrifuge. Individual metabolites were subsequently isolated and purified by thin-layer chromatography (TLC). Purified urinary species were characterized and compared to standards of Acm and its known metabolites by TLC, high performance liquid chromatography (HPLC), acid and enzymatic hydrolysis, and mass spectrometry. Nine of the urinary species were identified as either Acm or metabolites previously described in fermentation broths of Streptomyces galileus, in vitro reactions, animal plasma, or human plasma. Another urinary species corresponded to the major and previously unidentified Acm metabolite observed in human plasma. Sufficient quantitites of this material were isolated to allow its characterization and identification as bisanhydroaklavinic acid. The most polar urinary species isolated was identified as a beta-glucuronide conjugate of bisanhydroaklavinic acid. As such, these studies demonstrate the ability of humans to produce all of the previously proposed metabolites of Acm and establish the identity of the major Acm metabolite observed in human plasma.

The reaction of superoxide with daunorubicin or its aglycones in the aprotic solvents dimethyl sulfoxide and dimethylformamide was studied. This interaction generated the blue anthracycline phenolate anion as monitored by u.v.-visible spectrometry and molecular oxygen as determined by a modified Clark-type oxygen electrode. The visible spectrum of the phenolate anion (gamma max 604, 652 nm) was subject to considerable shifts dependent on the size of the cation present. The phenolate anion could be further oxidized by molecular oxygen to generate the C-6, C-11 (B-ring) semiquinone as detected by a weak electron paramagnetic resonance spectrometry signal. These results raise the possibility that similar reactions of superoxide with anthracyclines in vivo may play a role in the antitumor activity and/or the etiology of the toxic side effects of this class of drugs.

The biochemical changes responsible for acquired resistance to cisplatin (DDP) are not fully understood. We have developed DDP-resistant sublines of COLO 316 human ovarian carcinoma cells in vitro and characterized a number of biochemical features of these cells. Following selection with either continuous 50 nM DDP (COLO/DDP50 cells) or intermittent 1 microM DDP (COLO/B, COLO/C, or COLO/D cells) the onset of resistance was rapid. The resistance of the COLO/B cells gradually fell from 14-fold to 5-fold over 6 months in drug-free media. Both selection procedures produced cells exhibiting broad cross-resistance to other platinum analogs, natural products and alkylating agents. There was no significant change in the growth rate (doubling time = 36 h, cloning efficiency (28%), protein content (0.55 mg/10(6) cells), or morphology of these cells. Cell cycle distributions of log-phase cells were similar (60% G0/G1, 35% S, 5% G2/M) as determined by flow cytometry. Glutathione (GSH) levels, while not elevated in COLO-B cells at low levels of resistance (2-3-fold), were 30% elevated at higher levels of resistance (9-fold). However, GSH levels in COLO/DDP50 cells with 13-fold resistance were 2.3-fold elevated. The resistance of both cell types could be partially reversed by extended depletion of GSH with D,L-buthionine-S,R-sulfoximine. COLO/D cells had a 48% decrease in DDP accumulation at 1 h while COLO/DDP50 cells had no change in DDP accumulation. The cross-resistance profiles, GSH biochemistry and DDP accumulation data indicate that acquired DDP-resistance is a complex, multifactorial response in these cells. The specific combination of mechanisms expressed in these cells appears to depend upon the selection procedure.

We have investigated the role of glutathione in determining the macromolecular binding and cytotoxicity of cisplatin (DDP) and melphalan (LPAM) in human ovarian carcinoma cells and DDP-resistant L1210 mouse leukemia cells. Glutathione reacted avidly with DDP in normal saline with a bimolecular rate constant of 16.2 M-1 hr-1. Glutathione had no effect on the rate of hydrolysis of LPAM, consistent with the SN1-like reaction mechanism of LPAM. Glutathione protected calf thymus DNA and bovine serum albumin from DDP platination and LPAM alkylation. Glutathione also protected nuclei isolated from human ovarian carcinoma cells from DDP platination. The importance of intracellular glutathione in determining the cytotoxicity of DDP and LPAM was assessed by depletion of glutathione with buthionine sulfoximine in three cell types. Exposure to 0.5 mM buthionine sulfoximine for 20-28 hr depleted glutathione to levels that were 10-20% of control levels. COLO 316 and 2008 human ovarian carcinoma cells, and ZCR9 mouse leukemia cells were all sensitized to LPAM cytotoxicity by this level of glutathione depletion. The dose modification factors, defined as the IC50 control cells/IC50 depleted cells, were: 2.6 +/- 0.5 for COLO 316 cells, 1.6 +/- 0.1 for 2008 cells, and 2.1 +/- 1.1 for ZCR9 cells. In contrast, glutathione depletion had a minimal effect on DDP cytotoxicity in these cells with dose modification factors of: 1.2 +/- 0.2 for COLO 316 cells, 0.8 +/- 0.3 for 2008 cells, and 1.1 +/- 0.1 for ZCR9 cells. The differential potentiation of DDP and LPAM cytotoxicity by glutathione depletion in these cells, despite the similar protection that glutathione affords macromolecules from drug binding, suggests that there are fundamental differences in the intracellular interaction of these electrophilic drugs with glutathione.