Further characterization of acquired-resistance to Cisplatin in a rat ovarian tumor-cell line

The proliferation of a rat ovarian tumor cell line (O-342) was completely inhibited by a 2 h exposure to 20 muM cisplatin (DDP) in vitro up to 120 h after its removal, while in its DDP resistant subline (O-342/DDP), the same treatment only caused a transient growth inhibition within the first 24 h post the exposure, followed by the recovery of proliferation at a similar rate as the control cells. DNA interstrand cross links (ISCL) were maximally formed 12 h post DDP treatment in either O-342 or O-342/DDP cells, with a 2.8-fold increase in the sensitive cells at this time (262 vs. 95 rad eq.). After further 12 h incubation, however, 75% of DNA-ISCL was removed in O-342/DDP cells, while only 22% of them were repaired in O-342 cells. DNA single strand breaks (SSB) were produced to a similar extent in both lines but reached a maximum at 12 and 24 h in the resistant and the sensitive cells, respectively. ADP-ribosyl transferase (ADPRT) activity, a DNA repair-associated enzyme, was 2.6-fold higher in O-342/DDP cells compared to the sensitive subline. Following DDP treatment, the activity was stimulated in the sensitive cells with a maximum of about 1.5-fold at 24 h, whereas the inhibitory effect was observed in the resistant cells, although at 12 h it recovered almost to the control level. Flow cytometric analysis showed that there were at least two sub-populations (2n and 4n) in O-342 cells, while only 2n population was observed in O-342/DDP cells. Following DDP exposure, O-342/DDP cells progressed through the cell cycle with only a small and transient accumulation of cells in S-phase at 12 h and 24 h, while in the sensitive cells, it was impossible to distinguish cell-cycle distribution at 12 h due to severe damage, at 24 h most of the cells became arrested in G2-phases, which persisted until the end of the observation (48 h). Our results suggest that both reduced interaction of cellular DNA with DDP and increased DNA repair are contributing factors for development of DDP resistance, which might be directly or indirectly subsequent to alterations of poly (ADP-ribose) metabolism in the resistant cells.     

Multiple effects of 3-aminobenzamide on DNA damage induced by cisplatin (DDP) in DDP-sensitive and -resistant rat ovarian tumor cell lines.

To investigate the mechanisms by which 3-aminobenzamide (3AB) reverses cisplatin (DDP) resistance in a rat ovarian tumor cell line, the effects of 3AB on DDP-induced DNA damage and repair were kinetically determined over a post-exposure period of 48 h. DNA single strand breaks (SSB) occurred maximally 12 h and 24 h following DDP exposure in DDP-resistant (O-342/DDP) and -sensitive (O-342) rat ovarian tumor cells, respectively. 3AB, present during and after the exposure, significantly increased SSB formation by DDP at 24 h (P < 0.02) and 48 h (P < 0.01) in O-342/DDP cells. To a lesser extent (P > 0.05), a similar tendency was also observed in O-342 cells. Formation of DNA interstrand cross-links (ISCL) by DDP reached a maximum by 12 h in either O-342 or O-342/DDP cells, but in the resistant cells they were both much lower and more rapidly removed. 3AB decreased ISCL in the sensitive cells at 12 h and thereafter with a maximum at 24 h (P < 0.05), while in the resistant cells the same treatment decreased ISCL at 12 h, had no effect at 24 h and increased ISCL at 48 h following DDP treatment. Therefore, it is concluded that 3AB has multiple effects on DNA damage and repair induced by DDP in both cell lines and increase of DNA-ISCL by 3AB at 48 h after the exposure in O-342/DDP cells might be related to its chemosensitizing effect in this line.     

Antitumor activity of thaliblastine (NSC-68075) in experimental ovarian tumor cell lines sensitive and resistant to cisplatin.

Cytotoxicity of thaliblastine (thalicarpine, TBL; NSC-68075) and/or cisplatin (DDP) in DDP-sensitive (O-342) and-resistant (O-342/DDP) rat ovarian tumor cell lines was comparatively determined using the MTT assay. The 50% inhibitory dose (ID50) of DDP was found to be 6.2 microM in O-342 cells and 23.4 microM in O-342/DDP cells, while, vice versa, the ID50 of TBL was 39.3 micrograms/ml in the sensitive line and 27.3 micrograms/ml in the resistant line. Furthermore, simultaneous exposure of cells to DDP and TBL showed a significant superiority over DDP alone in O-342 cells, as evaluated with variance analysis (P less than 0.001). This enhancing effect of TBL on DDP cytotoxicity, however, was not observed in the resistant cells.     

Chemoresistance in rat ovarian tumours

In a cisplatin resistant subline (O-342/DPP) of an intraperitoneally growing transplantable rat ovarian tumour (O-342), intracellular glutathione (GSH) was approximately doubled (mean [S.E.] 1.5 [0.26] vs. 0.8 [0.2] nmol/10⁶ cells). GSH reductase activity was higher (30.64 [4.07] vs. 20 [0.92] nmol/min per mg protein), although no difference was found for GSH-S-transferase. 24 h after exposure to cisplatin, formation of DNA interstrand cross-links was at a maximum in both lines and significantly higher in O-342 (162 [23] vs. 88 [22] rad eq). Combination treatment of O-342/DDP with buthionine sulphoximine plus cisplatin resulted in a marginal increase in survival compared with cisplatin treatment; treatment of this line with 3-aminobenzamide plus cisplatin was also superior to cisplatin alone. In the sensitive line both combinations were likewise superior to cisplatin alone. In vitro, at equimolar concentration, a new platinum complex (CTDP) was at least as active as cisplatin in both lines, which suggests a superior therapeutic index because its LD₅₀ in mice is threefold higher than that of cisplatin. A ruthenium complex (ICR) had a higher activity in the resistant line. A titanium complex (budotitane) was not active.     

Augmentation of cisplatin (DDP) cytotoxicity in vivo by DL-buthionine sulfoximine (BSO) in DDP-sensitive and-resistant rat ovarian tumors and its relation to DNA interstrand cross links.

DDP treatment (1.2 mg/kg x 5) prolonged the mean survival time (MST) of rats bearing an experimental ovarian tumor (0-342) from 16.4 to 51.1 days, with one of ten rats surviving more than 90 days. Administration of D,L - buthionine sulfoximine (BSO) (24 and 2 h prior to DDP, respectively) before the last two doses of DDP had no significant effect on DDP therapeutic activity, while daily combination of DDP with BSO (BSO 2 h prior to DDP) throughout the treatment significantly increased MST to 69 days (p less than 0.05, vs. DDP alone), with three of ten rats surviving more than 90 days. In the DDP resistant counterpart (0-342/DDP), on the other hand, DDP alone showed only a slight increase of MST (11.6 days in DDP group vs. 10.7 days in control group), addition of BSO to DDP treatment further prolonged MST to 13.3 days (p less than 0.01 vs. DDP alone). The formation of DNA interstrand cross links (DNA-ISCL) was found to be higher in 0-342 than in 0-342/DDP cells in vitro with a maximum at 24 h following 1 h exposure to DDP. BSO depleted the intracellular GSH level in a dose - and time - dependent manner in the two cell lines. Pretreatment with BSO resulted in a 7.4% increase in DNA-ISCL by DDP in 0-342 cells but a 39% increase in 0-342/DDP cells, which may partially account for chemosensitization of BSO to DDP in vivo. Our result that the chemosensitizing effect of BSO, through depletion of cellular GSH, is more significant in the DDP sensitive O-342 tumor than in its DDP resistant subline in vivo underlines that BSO should be used as a chemosensitizer in combination with DDP at the beginning of chemotherapy for clinical trial.     

Synergistic potentiation of Cisplatin cytotoxicity by caffeine in a Cisplatin-resistant rat ovarian tumor-cell line

The combined cytotoxicity of cisplatin (DDP) and caffeine (CA) against DDP-sensitive (O-342) and -resistant (O-342/DDP) rat ovarian tumor cell lines in vitro was investigated. 0-342/DDP cells showed a similar sensitivity to CA as O-342 cells did. Simultaneous administration of DDP and CA resulted in infra-additive to additive cytotoxicity in O-342 cells, whereas in O-342/DDP cells, combination of DDP with CA produced effects from infra-additivity to synergism. The strength of this enhancement of DDP cytotoxicity by CA in both cell lines varied in a CA-dose dependent manner but inversely with DDP concentrations. ADP-ribosyl transferase (ADPRT) activity was 2.6-fold in O-342/DDP cells compared to that in O-342 cells. CA (2.5 mM) caused 53.4% and 28.9% inhibition of ADPRT activity in control and DDP-treated O-342/DDP cells, respectively. This inhibitory effect, however, was not observed in 0-342 cells. Our results suggest that CA may have some potential in combination with DDP for treatment of DDP-resistant malignancies in the clinic. One of the possible mechanisms involved in this process might be that CA inhibits ADPRT-associated DNA repair in the resistant cells.     

Reversal of acquired cisplatin resistance by nicotinamide in vitro and in vivo

At a concentration of 2.5 mM, nicotinamide (NA), an inhibitor of poly(ADP-ribose) polymerase (PARP), significantly potentiated the cytotoxicity of cisplatin (DDP) in a DDP-resistant rat ovarian tumor cell line (O-342/DDP) in vitro, whereas the same treatment had no substantial effect on DDP's cytotoxic activity against the DDP-sensitive parental line (O-342). Furthermore, in a nude mouse model where the O-342/DDP tumor grew intraperitoneally, whereas DDP given alone at 1 mg/kg ×3 exhibited no antitumor activity as compared with control values due to the resistance, NA given at a nontoxic dose (5 mmol/kg ×3) significantly increased the mean survival time (MST) of the tumor-bearing NMRI nude mice from 20.7 days in the DDP-treated group to 29.0 days in the combination group. Mechanism studies showed that endogenous PARP activity (incorporation of tritiated nicotinamide adenine dinucleotide, [³H]-NAD) was 2.6 times higher in O-342/DDP than in O-342 cells and that the presence of 2.5 mM NA during the incubation with the isotope resulted in 73.3% inhibition of the enzyme activity in O-342/DDP cells but in only about 30% inhibition in the sensitive line. However, treatment with NA during and after DDP exposure failed to produce any significant effect on the formation of DNA single-strand breaks (SSB) but decreased the induction of DNA interstrand cross-links (ISCL) by DDP in the sensitive and resistant cell lines. These results suggest that NA might have some clinical potential in reversing DDP resistance, and further studies are therefore warranted to confirm the resistance-reversing effect of NA in other DDP-resistant cell lines.Abbreviations DDP cisplatin,cis-diamminedichloroplatinum(II) - ID₅₀ 50% inhibitory dose - ISCL DNA interstrand cross-links; MST, mean survival time - NAD nicotinamide adenine dinucleotide - PARP poly(ADP-ribose) polymerase (formerly ADPRT; ADP-ribosyl transferase) - SSB DNA single-strand breaks - TCA trichloroacetic acid     

Enhancement of cisplatin (DDP) antitumor activity by 3-aminobenzamide in rat ovarian tumors sensitive and resistant to DDP in vivo

A cisplatin (DDP)-resistant rat ovarian tumor cell line (O-342/DDP) and its parental sensitive counterpart (O-342) were used to investigate the combination effect of DDP plus 3-aminobenzamide (3-AB), an inhibitor of adenosine diphosphate ribose transferase (ADPRT). Treatment with six doses of DDP in NMRI nude mice bearing O-342/DDP produced an increase in mean survival of only 1 day over that of controls (P less than 0.05). The addition of nontoxic doses of 3-AB (5 mM/kg x 6) increased the mean survival to 6.4 days compared with that obtained with DDP treatment alone (P less than 0.001). In the sensitive ovarian tumor line (O-342), the combination effect of DDP plus 3-AB was even more impressive: simultaneous treatment of NMRI nude mice bearing O-342 with three doses of DDP plus 3-AB increased the mean survival by 2 weeks and the median survival by 3 weeks over that achieved with DDP treatment alone. Possible mechanisms involved in the potentiation of DDP activity and the possible clinical potential of this combination are discussed.     

The relationships between glutathione, glutathione-S-transferase and cytotoxicity of platinum drugs and melphalan in eight human ovarian carcinoma cell lines

The role of glutathione (GSH) and GSH-S-transferase (GST) activity in modulating the cytotoxicity of four platinum drugs and melphalan was evaluated in eight human ovarian carcinoma cell lines. The cell lines were established from solid and ascitic tumours from pretreated and untreated patients, and showed a wide spectrum of sensitivity to several platinum II and platinum IV drugs; cisplatin, carboplatin, CHIP and tetraplatin. Intracellular glutathione concentration measured in the cell lines showed a significant (P = 0.05) correlation with IC50 values for cisplatin (r = 0.91), carboplatin (r = 0.87) and CHIP (r = 0.88). The correlation between GSH levels and IC50 values for melphalan (r = 0.76) or tetraplatin (r = 0.60) was not as significant. GST activity showed no correlation with IC50 values, for the four platinum drugs. To determine the significance of the elevated GSH concentration in the refractory cell lines, the effect of D,L-buthionine-S, R-sulfoximine (BSO) mediated GSH depletion on platinum drug cytotoxicity was examined in one of the most sensitive (CH1) and two of the least sensitive (relatively resistant; SKOV-3, HX/62) cell lines. Comparison was made with the effect of GSH depletion on melphalan cytotoxicity in these three lines. These lines were differentially sensitive to BSO, with the two most platinum drug resistant lines being more tolerant to BSO than the sensitive CH1 line. Depletion of cellular GSH, ranging between 61 and 88%, had a differential effect on the sensitivity to PtII vs PtIV drugs in the three cell lines: cytotoxicity of the PtIV drugs, tetraplatin and CHIP, was substantially enhanced in both the resistant and sensitive cell lines; in contrast, the cytotoxicity of the PtII drugs, cisplatin and carboplatin, was only significantly increased in one of the two relatively resistant lines (SKOV-3) and in the sensitive (CH1) line after GSH depletion. Moreover the dose modification factor (DMF) for the PtII agents were lower than those for PtIV agents in the three cell lines. The dose modification factor for tetraplatin after BSO treatment was similar to that observed for melphalan in all three cell lines. In the SKOV-3 cell line extending the BSO pretreatment period to 48 h from 24 h marginally reduced the cytotoxicity of cisplatin, whereas the cytotoxicity of the other three drugs remained similar to that observed after 24 h BSO pretreatment. In contrast, extending the BSO treatment to 24 h after drug exposure potentiated the cytotoxicity of cisplatin, CHIP and tetraplatin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Multifactorial resistance to adriamycin: relationship of DNA repair, glutathione transferase activity, drug efflux, and P-glycoprotein in cloned cell lines of adriamycin-sensitive and -resistant P388 leukemia

Cloned lines of Adriamycin (ADR)-sensitive and -resistant P388 leukemia have been established, including P388/ADR/3 and P388/ADR/7 that are 5- and 10-fold more resistant than the cloned sensitive cell line P388/4 (Cancer Res., 46: 2978, 1986). A time course of ADR-induced DNA double-strand breaks revealed that in sensitive P388/4 cells, evidence of DNA repair was noted 4 h after removal of drug, whereas in resistant clone 3 and 7 cells repair was observed 1 h after drug removal. The earlier onset of DNA repair was statistically significant (p = 0.0154 for clone 3 cells, and p = 0.0009 for clone 7 cells). By contrast, once the repair process was initiated, the rate of repair was similar for all three cell lines. The level of glutathione transferase activity was determined in whole cell extracts. Enzyme activity (mean +/- SE) in sensitive cells was 9.49 +/- 1.00 nmol/min/mg protein, that in resistant clone 3 cells was 13.36 +/- 1.03 nmol/min/mg, and that in clone 7 cells was 13.96 +/- 1.44 nmol/min/mg; the 1.44-fold increase in enzyme activity in resistant cells was statistically significant (p = 0.01). Further evidence of induction of glutathione transferase was provided by Northern blot analysis using a 32P-labeled cDNA for an anionic glutathione transferase, which demonstrated approximately a twofold increase in mRNA in resistant clone 7 cells. Western blot analysis with a polyvalent antibody against anionic glutathione transferase also revealed a proportionate increase in gene product in resistant cells. Dose-survival studies showed that ADR-resistant cells were cross-resistant to actinomycin D, daunorubicin, mitoxantrone, colchicine, and etoposide, but not to the alkylating agent melphalan; this finding provided evidence that these cells are multidrug resistant. Using a cDNA probe for P-glycoprotein, a phenotypic marker for multidrug resistance, Northern blot analysis showed an increase in the steady state level of mRNA of approximately twofold in resistant clone 3 and 7 cells. Southern analysis with the same cDNA probe showed no evidence of gene amplification or rearrangement. Western blot analysis with monoclonal C219 antibody demonstrated a distinct increase in P-glycoprotein in resistant cells. Efflux of Adriamycin as measured by the efflux rate constant was identical in all three cell lines. Furthermore, the metabolic inhibitors azide and dinitrophenol did not augment drug uptake in either sensitive or resistant cells. These findings suggest that despite the increase in P-glycoprotein, an active extrusion pump was not operational in these cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

In vitro evaluation of platinum,titanium and ruthenium metal complexes in cisplatin-sensitive and-resistant rat ovarian tumors

Summary The antitumor activity of eight new metal complexes (three platinum, one titanium, four ruthenium derivatives) was investigated in a cisplatin (DDP)-sensitive (O-342) and a DDP-resistant (O-342/DDP) ovarian tumor line using the bilayer soft-agar assay. A continuous exposure set up at logarithmically spaced concentrations was used to test the drugs; to uncover possible pharmacokinetic features, a short-term exposure was additionally included for selected compounds. DDP served as the reference drug. The following compounds were investigated: 18-crown-6-tetracarboxybis-diammineplatinum(II) (CTDP),cis-aminotrismethylenephosphonato-diammineplatinum(II) (ADP),cis-diamminecyclohexano-aminotrismethylenephosphonato-platinum(II) (DAP), diethoxybis(1-phenylbutane-1,3-dionato)titanium(IV) (DBT, budotitane),trans-imidazolium-bisimidazoletetrachlororuthenate(III) (ICR),trans-indazolium-tetrachlorobisindazoleruthenate(III) (IndCR),cis-triazolium-tetrachlorobistriazoleruthenate(III) (TCR) andtrans-pyrazolium-tetrachlorobispyrazoleruthenate(III) (PCR). Of the new metal complexes, CTDP was the most active compound in O-342, resulting in a percentage of control plating efficiency (±SE) of 1±1, 12±8 and 40±21 following continuous exposure to 10, 1 and 0.1 m, respectively, and was thus comparable to DDP at equimolar concentrations. In the resistant line, 10 m CTDP reduced colony growth to 18%±8%, whereas an equimolar concentration of DDP effected a reduction to 26%±9%. During short-term exposure, CTDP was inferior to DDP, which may be ascribed to the stability of the bis-dicarboxylate platinum ring system. The titanium compound DBT, in contrast, showed promising effects at its highest concentration (100 m) during short-term exposure in both lines; at this concentration the activity in O-342/DDP was higher than that in O-342 (7%±7% vs 34%±17% of control plating efficiency at 100 m). All ruthenium complexes showed higher activity in the resistant line O-342/DDP than in the sensitive counterpart. ICR was the most active compound. Following continuous exposure of O-342/DDP cells to 10 m ICR, colony growth was reduced to 18%±4% that of controls. Further studies should concentrate on CTDP and ICR for the following reasons: the activity of CTDP was equal to that of DDP at equimolar concentrations during continuous exposure; considering that the in vivo toxicity of DDP was 3-fold that of CTDP, an increase in the therapeutic index of CTDP would be expected. ICR showed the best effect of all ruthenium complexes; it was superior to DDP in the resistant line.     

Glutathione metabolism during Yoshida ascites sarcoma growth

Glutathione (GSH) metabolism and protein synthesis were observed over a period of about two weeks in Yoshida ascites sarcoma and intracellular concentration relative to days 7, 10 and 13 assumed as 'markers' of different stages of tumor development. During this period the decrease in rate of cell proliferation was followed by decrease in protein synthesis, GSH, oxidized glutathione (GSSG), adenosine triphosphate (ATP), glutathione-S-transferase (GSH-S-transferase) and gamma-glutamyl-cysteine synthatase (gamma-GCS); by increase in glutathione-peroxidase (GSH-peroxidase); while glutathione-reductase (GSH-reductase) and glucose-6-phosphate-dehydrogenase (G-6-PD) remained unchanged. In relation to growth curve of the tumor, GSH concentration was very high up to day 7 (logaritmic phase), decreased till quantity similar to that of corresponding normal cells up to day 10 (plateau phase) and on day 13 was significantly smaller. Correlation between high concentration of GSH in tumor cells and their survival and proliferation after intraperitoneal implantation is discussed.     

Inhibition of O6-alkylguanine-DNA alkyltransferase in animal and human ovarian tumor cell lines by O6-benzylguanine and sensitization to BCNU

O⁶-Alkylguanine-DNA alkyltransferase (O⁶-AGT) activity in rat ovarian tumor lines O-342 and O-342/DDP was 103.4±18.4 and 240.9±40.2 fmol/mg protein, respectively; thus, cisplatin (DDP) resistance was paralleled by an increase in O⁶-AGT activity by a factor of approximately 2.3. The DDP-resistant line expressed a collateral resistance to BCNU. Both lines could be sensitized to BCNU by O⁶-BG, with sensitization factors of 6.0 and 2.1, respectively. In neither line did depletion of O⁶-AGT have any sensitizing effect towards DDP. In the human ovarian cancer lines SK-OV-3 and OAW 42, O⁶-AGT activity was 337.6±18.2 and 180.0±39.9 fmol/mg protein, respectively; in these lines depletion of O⁶-AGT activity by O⁶-BG treatment resulted in sensitization factors of 3.0 and 4.1, respectively. The increase in sensitivity of ovarian tumor cell lines against a chloroethylating agent by O⁶-AGT depletion and possible pharmacological advantages of regional (i.p.) administration of this combination might be beneficial in advanced ovarian cancer.Abbreviations BCNU (1,3-bis(2-chloroethyl)-1-nitrosourea) - CENU 2-chloroethylnitrosourea - DDP cisplatin ED₅₀, the effective dose required to inhibit colony formation or cell proliferation by 50% - O⁶-AGT O⁶-alkylguanine-DNA alkyltransferase - O⁶-BG O⁶-benzylguanine SF sensitization factor     

Characterization of Acquired Resistance to cis-Diamminedichloroplatinum(II) in Mouse Leukemia Cell Lines

We have established in vivo cisplatin-resistant mouse leukemia cell lines, L-1210/DDP and P388/ DDP, in order to elucidate the mechanism of acquired resistance to cisplatin. Resistance indices were 22 and 14, respectively, when the cells were exposed to cisplatin for 48 h. Uptake of cisplatin by both resistant lines was significantly reduced, compared with values for the respective parent lines (17% for L-1210/DDP and 27% for P388/DDP, at 100 μM for 1 h). While glutathione contents in the resistant cells were 1.7-1.9 times higher than those in the sensitive ones, their reduction by preincubation with buthionine sulfoximine did not influence the sensitivity of the cells to cisplatin. In addition, the resistant lines dill not show lower sensitivity to CdCl₂, than the respective sensitive ones, suggesting that intracellular SH groups might contribute little to the mechanism of cisplatin resistance in these cells. Postincubation with DNA repair inhibitors, caffeine and aphidicolon, did not selectively enhance the sensitivity of the resistant cells to cisplatin. These results suggested that reduced drug uptake would be a primary mechanism of cisplatin resistance in L-1210/DDP and P388/DDP. Cross-resistance patterns to platinum complexes were quite different between L-1210/DDP and P388/DDP. Colon 26/DDP, another cisplatin-resistant mouse tumor showed a different pattern from those observed with L-1210/DDP and P388/DDP. In the development of new platinum complexes we should use plural resistant lines for examining cross-resistance patterns to candidate platinum complexes.     

Influence of glutathione S-transferases on cellular glutathione determination by flow cytometry using monochlorobimane

Intracellular glutathione (GSH) levels for seven mammalian cell lines (four human tumors, two rodent, one monkey) were determined by flow cytometry following staining with monochlorobimane (MBCl), and the results were compared with GSH levels measured by the Tietze assay. The mean fluorescence intensity for all but the two rodent lines did not correlate with GSH levels determined biochemically. Good agreement between the two assays was observed for the rodent lines following depletion of GSH by buthionine sulfoximine, but the level of GSH depletion achieved in the human and monkey lines was always underestimated by MBCl/flow cytometry. These discrepancies were not resolved by increasing stain concentration or staining time. Total glutathione S-transferase (GST) activity and GST isozyme profiles were determined for each of the cell lines. Western analysis with antibodies raised against rat Ya, Yb1, and Yc and human pi isozymes revealed that the rodent cell lines expressed abundant alpha (Ya, Yc subunits) and mu (Yb1 subunits) class isozymes. In contrast, GST-pi was the predominant isozyme detected in the human tumor cell lines and Cos-7 monkey cells. Michaelis-Menten analysis with purified GSTs from rat liver as well as purified human placental (pi) GST revealed that the conjugation of MBCl and GSH catalyzed by the alpha (1-1 and 2-2) and mu (3-3 and 3-4) class GST isozymes was approximately 10 and 80 times more efficient than was conjugation by the GST pi form, respectively. These data indicate that the GST-catalyzed conjugation of GSH and MBCl is isozyme dependent and that MBCl is a relatively poor substrate for the pi isozyme. As a consequence of this isozyme rate differential, the MBCl/flow cytometry technique for GSH quantitation must be applied cautiously, particularly with human tumor cells, many of which have been shown to have high GST-pi activity. Application to other cell types should also be made after careful characterization of GSH levels and GST isozyme composition and only after comparison with other independent assays of GSH concentration.     

Cellular glutathione and thiol measurements from surgically resected human lung tumor and normal lung tissue

Cellular glutathione (GSH) levels were measured from 27 human lung tumor biopsies, enzymatically disaggregated, and compared with cells isolated from normal lung of the same patients. GSH levels from normal lung were similar among patients with a mean value of 11.20 +/- 0.58 (SEM) nmol GSH/mg protein (24 patients) with a range from 6.1 to 17.5 nmol GSH/mg protein. GSH levels varied considerably within and across histological tumor types with the following values: adenocarcinomas, 8.83 +/- 0.96 nmol/mg protein (8 patients); large cell carcinomas, 8.25 +/- 2.51 nmol/mg protein (3 patients); and squamous cell carcinomas, 23.25 +/- 5.99 nmol/mg protein (8 patients). The cyclic GSH reductase assay gave only average GSH values and could not distinguish possible GSH variation among subpopulations of cells isolated. Cell volume measurements and microscopic evaluation of cells isolated from both tumors and normal lung revealed heterogeneity with respect to cell types present. To determine the extent of thiol variation among tumor cell subpopulations, tumor cell suspensions were stained with the thiol-specific stain, monochlorobimane (MCB). The accuracy of MCB staining was tested by flow cytometric analysis of 12 in vitro human tumor cell lines and 3 rodent cell lines. A linear relationship was found between the bimane cellular fluorescence and the cyclic GSH reductase assay for cell lines having less than 80 nmol GSH/mg protein (R2 = 0.82). Above 80 nmol GSH/mg protein the rate of change of the bimane fluorescence intensity with respect to increasing GSH concentrations was much reduced. However, by labeling cells with MCB it was possible to distinguish between cell lines with low versus high GSH content. MCB staining of tumor samples revealed multiple populations of cells with respect to thiol levels. In particular, 2 of 8 squamous cell carcinomas had a proportion of cells with elevated fluorescence intensities (from 10 to 35% of the population) suggesting the presence of cells with greatly elevated thiol levels. These findings underscore the complexity of quantitating intracellular GSH levels from tumor biopsies. The combined use of MCB with flow cytometry and conventional GSH assays may help to delineate subpopulations of cells within tumors with different thiol levels.     

Hepsulfam sensitivity in human breast cancer cell lines: the role of glutathione and glutathione S-transferase in resistance.

Hepsulfam (NSC 329680, 1,7-heptanediol disulfamate) is an alkylating agent that showed excellent activity against mouse and human mammary carcinoma in preclinical studies. We therefore studied the cytotoxicity of this drug in six human breast cancer cell lines (AdrRMCF7, WTMCF7, Hs578T, MDA-MB-231, T47D, and MDA-MB-468). Clonogenic assays of these cell lines showed a range of sensitivity with the 90% inhibitory concentration ranging from 3.1 microM hepsulfam (MDA-MB-468) to 32.3 microM hepsulfam (AdrRMCF7) after 24-h exposure to the drug. To evaluate possible mechanisms responsible for this observed variation in sensitivity to hepsulfam, we have studied glutathione S-transferase (GST) activity and glutathione (GSH) in these cell lines. Total cytoplasmic GST activity correlated with sensitivity; the most sensitive cell lines had the lowest GST activity, while the two most resistant cell lines, AdrRMCF7 and Hs578T, had the highest GST levels of the six cell lines. Western blot analysis showed that the only detectable isoenzyme was GST-pi. The amount of GST-pi isoform correlated with hepsulfam sensitivity in the three most resistant cell lines and was undetectable in the three most sensitive cell lines. Cellular concentrations of GSH did not correlate with hepsulfam sensitivity. However, GSH depletion with buthionine sulfoximine increased sensitivity to hepsulfam in a dose-dependent fashion in all six cell lines. Evaluation by mass spectrometry revealed that glutathione can form conjugates with hepsulfam. We conclude that the GST/GSH detoxication system plays a role in the sensitivity of these breast cancer cell lines to hepsulfam.     

Role of cystine transport in intracellular glutathione level and cisplatin resistance in human ovarian cancer cell lines

Transport system x(c)(-) is a member of plasma membrane heterodimeric amino-acid transporters and consists of two protein components, xCT and 4F2hc. This system mediates cystine entry coupled with the exodus of intracellular glutamate and regulates the intracellular glutathione (GSH) levels in most mammalian cultured cells. We studied the activity of system x(c)(-) and GSH content in human ovarian cancer cell line (A2780) and its cisplatin (CDDP)-resistant variant (A2780DDP). The rate of cystine uptake was approximately 4.5-fold higher in A2780DDP cells than in A2780 cells and the cystine uptake in A2780DDP cells was mediated by system x(c)(-). Intracellular GSH content was much higher in A2780DDP cells but it fell drastically in the presence of excess glutamate, which inhibited the cystine uptake competitively. xCT and 4F2hc mRNAs were definitely expressed in A2780DDP cells, but far less in A2780 cells. Expression of system x(c)(-) activity by transfection with cDNAs for xCT and 4F2hc made A2780 cells more resistant to CDDP. Similar results on the cystine uptake were obtained in human colonic cancer cell lines. These findings suggest that the system x(c)(-) plays an important role in maintaining the higher levels of GSH and consequently in CDDP resistance in cancer cell lines.     

Increased removal of DNA-bound platinum in a human ovarian cancer cell line resistant to cis-diamminedichloroplatinum(II)

A human ovarian cancer cell line resistant to cis-diamminedichloroplatinum(II) (DDP) (2780CP) was compared with its DDP-sensitive parental cell line (A2780) to determine whether differences in the removal rate of DNA-bound platinum were related to resistance. Both cell lines were treated in vitro with various doses of DDP for 2 h and subsequently incubated in arginine-deficient Eagle's minimum essential medium with 2.5% dialyzed fetal bovine serum in the presence or absence of aphidicolin. After 0, 12, and 24 h, DNA was isolated from the cells and DNA-bound platinum was determined by flameless atomic absorption spectrophotometry. Binding of platinum to DNA of either cell line was a linear function of concentration ranging from 20 to 80 microM DDP. Platinum binding was almost equal at each dose in both cell lines. 2780CP cells that were 3-fold resistant to DDP lost 30.5 to 40.1% of their total DNA-bound platinum, compared with a 1.3 to 16.1% loss for A2780 cells, 12 to 24 h after a 2-h exposure to 40 microM DDP, respectively. Aphidicolin (3.0 micrograms/ml) increased the cytotoxicity in 2780CP cells by about 2-fold and caused a significant delay in the time required for platinum removal in the resistant cells (14.6 and 18.9% at 12 and 24 h). These studies indicate that the mechanism of DDP resistance in the 2780CP cell line is related to an increased ability to remove platinum-DNA adducts, and not to a difference in initial DDP binding to DNA.     

Glutathione, glutathione S-transferases, and related redox enzymes in Adriamycin-resistant cell lines with a multidrug resistant phenotype

Friend erythroleukemia cells (FLC) selected by exposure to Adriamycin (doxorubicin) express an approximate 2.5-fold (ARN1) or 13-fold (ARN2) resistance to the drug with various degrees of cross-resistance to other anthracyclines, vinca alkaloids, and epipodophyllotoxins. Because the redox cycling of the quinone moiety of Adriamycin is known to produce oxidative stress, however, an analysis of glutathione (GSH) and related enzyme systems was undertaken in the wild-type and selected resistant cells. In ARN1 and ARN2, superoxide dismutase (SOD) and catalase activities were slightly decreased, intracellular GSH and GSH reductase were essentially unchanged, and total GSH peroxidase, glutathione S-transferase (GST), and DT-diaphorase activities were slightly elevated. In each case there was no stoichiometric relationship between degree of resistance and level of activity. GST isozymes were purified from each cell line by HPLC GSH affinity column chromatography. Two-dimensional gel electrophoresis and western blot immunoreactivity against a battery of GST isozyme polyclonal antibodies determined that both the resistant and sensitive cells expressed isozymes of the alpha, pi, and mu classes (alternative murine nomenclature: M1, M2, M3). Of significance, both ARN1 and ARN2 cell lines expressed a unique alpha subunit which was absent from the parent FLC cell line. This isozyme presumably accounted for the increased GSH peroxidase activity (cumene hydroperoxide as substrate) found in ARN1 and ARN2 and may play a role in the small incremental resistance to melphalan found for both resistant lines. Expression of the isozyme was not stoichiometric with respect to degree of resistance. The presence of this isozyme may contribute to the resistant phenotype or may be the consequence of a more general cellular response to oxidative stress.