Augmentation of adriamycin, melphalan, and cisplatin cytotoxicity in drug-resistant and -sensitive human ovarian carcinoma cell lines by buthionine sulfoximine mediated glutathione depletion

Hamilton, Thomas C.; Winker, Margaret A.; Louie, Karen G.; Batist, Gerald; Behrens, Brent C.; Tsuruo, Takashi; Grotzinger, Karen R.; McKoy, Wilma M.; Young, Robert C.; Ozols, Robert F.

doi:10.1016/0006-2952(85)90551-9

Cited by 494 publications

(183 citation statements)

References 5 publications

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“…Thus, A2780R cells were approximately 7 times more resistant to L-PAM than the parenthal cell line A2780'. Such difference in sensitivity is close to the 9 x degree of resistance described by Hamilton et al (10).…”

Section: Sensitivity Differences Between Parenthal Andsupporting

confidence: 87%

Detection of cells resistant to alkylating agents by flow cytometric analysis of DNA damage

Frankfurt¹,

Seckinger²,

Sugarbaker³

1990

Cytometry

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DNA damage was measured by flow cytometric analysis of cells sensitive and resistant to alkylating agents. Human ovarian carcinoma cell line A2780 and a subline which is 7 times more resistant to L-phenylalanine mustard (L-PAM) were treated with the drug, fixed, and stained with monoclonal antibody (MOAB) F7-26 which detects single-stranded regions in alkylated DNA. Mean fluorescent intensity was measured on a flow cytometer. Cells were heated before staining to amplify single-strandedness in alkylated DNA. Significantly larger amount of MOAB was bound to DNA in sensitive than in resistant cells. We conclude that staining with MOAB F7-26 can be used as an indicator of cell sensitivity or resistance to alkylating agents. Detection of minor subsets of resistant cells in heterogeneous populations by FCM analysis may be useful for monitoring emerging drug resistance.Key terms: Monoclonal antibodies, flow cytometry, human ovarian carcinoma cell line A2780 Emergence of tumor cell populations which have resistance to anticancer drugs is a major problem in the successful application of cancer chemotherapy. Detection of resistant cells in heterogeneous populations may, therefore, have a critical role for selection and monitoring of tumor treatment.DNA damage is considered to be a target for antitumor and cytotoxic actions of alkylating agents. Biochemical methods were applied extensively for the analysis of DNA damage induced by these drugs (2,5,11,12,15,19). However, no methods for the measurement of DNA damage a t a cellular level applicable to heterogeneous cell populations were developed.In previous reports, the detection of DNA damage in individual cells using anti-DNA monoclonal antibody (MOAB) was described (7,s). The binding of MOAB F7-26 to DNA, in fixed EDTA-treated cells, correlated well with the decrease in cell viability induced by alkylating agents. However, the fluorescence intensity was not sufficient for complete separation between populations of control and drug-treated cells in fluorescence histograms or for the detection of cells with clinically relevant levels of drug resistance.In the current work, a modified highly sensitive procedure for FCM analysis of DNA damage was applied. The new procedure is based on the amplification of single-strandedness in alkylated DNA by heating in the presence of Mgzi. The effects of drug treatment on the binding of MOAB F7-26 to DNA in the cells sensitive and resistant to alkylating agents were compared in order to evaluate the ability of staining procedure to predict drug resistance. Since fluorescence difference in sensitive and resistant cells was sufficient to distinguish between two cell types, the method was then applied to heterogeneous populations of tumor cells. Mixed populations of sensitive and resistant cells were used as a model of tumors containing minor subsets of resistant cells.

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Section: Sensitivity Differences Between Parenthal Andsupporting

confidence: 87%

Detection of cells resistant to alkylating agents by flow cytometric analysis of DNA damage

Frankfurt¹,

Seckinger²,

Sugarbaker³

1990

Cytometry

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“…GSH, the main nonprotein thiol present in cells, has been shown to be an important determinant of the sensitivity of cells to a wide variety of drugs including cytostatic agents (Arrick & Nathan, 1984). Elevated levels of GSH have been associated with drug-resistant phenotypes developed in cells exposed to a number of electrophilic drugs (Hamilton et al, 1985;Green et al, 1984;Tan et al, 1987;Evans et al, 1987). Moreover, cells with elevated levels of GSH also appeared to be resistant to CDDP (Richon et al, 1987;Waud, 1987;Hamilton et al, 1985;Hromas et al, 1987;Behrens et al, 1987).…”

Section: Discussionmentioning

confidence: 99%

“…Several mechanisms responsible for this resistance to CDDP including reduced membrane permeability, enhanced drug detoxification, changes at the level of DNA adduct formation and increased efficiency of DNA repair have been previously reported (Teicher et al, 1987;Richon et al, 1987;Waud, 1987;Kraker & Moore, 1988;Andrews et al, 1988;Hamilton et al, 1985;Hromas et al, 1987;Behrens et al, 1987;Eastman & Schulte, 1988).…”

mentioning

confidence: 92%

The role of glutathione in resistance to cisplatin in a human small cell lung cancer cell line

Mulder²,

et al. 1990

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Summary The role of glutathione (GSH) in resistance to cisplatin (CDDP) was studied in a human small cell lung carcinoma cell line (GLC4) and a CDDP-resistant subline (GLC4-CDDP). In addition to studying the steady state of GSH, the kinetics of this defence system were also studied via the monitoring of the GSH status of the cells under continuous pressure of CDDP. GLC4-CDDP maintained its elevated GSH level whereas GLC4 (under pressure of CDDP) quickly synthesised GSH to about twice its intitial level, corresponding with 80% of the GSH level of GLC4-CDDP. D,L-buthionine-S,R-sulphoximine (BSO) was used to analyse the role of GSH in resistance to CDDP. Pretreatment with BSO (48 h, 50 tAM, GSH not detectable) increased the CDDP-induced cytotoxicity 2.8-fold in GLC4-CDDP and 1.7-fold in GLC4. In GLC4 no changes in the amount of platinum (Pt) bound to DNA could be observed after GSH depletion. Changes in formation of interstrand cross-links or the main Pt-containing intrastrand cross-link in digested DNA, the Pt-GG adduct, were also not observed. In GSH depleted GLC4-CDDP cells, an increase in the amount of Pt bound to DNA and in the Pt-GG adduct was observed. Pretreatment with BSO substantially reduced the repair of Pt bound to DNA in both cell lines. We conclude that an increased GSH level and GSH synthesis capacity were demonstrated in CDDP resistant cells. The observations after BSO treatment suggest two roles for GSH in CDDP resistance, namely that of a cytosolic elimination resulting in less DNA platination and a nuclear effect on the formation and repair of DNA platinum adducts.

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“…Recently it has been proposed that tumour GSH is an important factor influencing the effectiveness of a variety of cancer chemotherapeutic agents (Arrick & Nathan, 1984;Russo & Mitchell, 1985;Hamilton et al, 1985): these authors state that tumours with a low GSH content will be more susceptible to the action of these agents, whereas tumours with a high GSH level will be protected and therefore resistant. However, in normal tissues a high GSH content is desirable to protect them from the toxicity of these anticancer agents.…”

Section: Discussionmentioning

confidence: 99%

Glutathione localisation in benign and malignant human breast lesions

Murray¹,

Burke²,

Ewen³

1987

Br J Cancer

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Summary Reduced glutathione (GSH) has been demonstrated in benign and malignant human breast lesions using a newly.developed histofluorescence technique. GSH was present in every lesion and in each case was localised to the epithelium. A semi-quantitative assessment revealed a moderate amount of GSH in normal epithelium and fibroadenoma and a high level in apocrine metaplasia, epitheliosis and intraduct carcinoma. Invasive ductal carcinoma contained a variable amount of GSH. Correlation between fluorescence intensity and histological grade of ductal carcinomas was almost statistically significant but a relationship to oestrogen receptor status was not detected. The rapid assessment of GSH in breast cancer may aid in the selection of optimum chemotherapeutic regimens.Reduced glutathione (GSH) is a thiol containing tripeptide which is involved in a variety of cellular functions. GSH has a role in the detoxification of drugs and carcinogens, protection of cells from free radical damage and reactive oxygen compounds, participates in protein and DNA synthesis and in the regulation of enzyme activity (Meister & Anderson, 1983;Orrenius & Moldeus, 1984;Chasseaud, 1979). Altered levels of GSH are frequently observed in experimental animal neoplasia (Roomi et al., 1985, Fiala et al., 1976 but GSH levels in human neoplasia have been reported only for carcinomas of the stomach and colon (Siegers et al., 1983;Siegers et al., 1984). GSH in human breast tissue has not been investigated previously, although GSH-associated enzymes have been studied both biochemically (Di Ilio et al., 1985) and histochemically (Levine et al., 1983;Bard et al., 1986).Tumour GSH appears to be an important factor in determining the effectiveness of a variety of anti-cancer chemotherapeutic drugs, in general a low level of GSH being associated with increased chemotherapeutic efficiency (Arrick & Nathan, 1984;Romine and Kessel, 1986;Crook et al., 1986).We have studied the localisation and amount of GSH in normal and pathological human breast tissue using our recently developed specific histofluorescence method for GSH (Murray et al., 1986 (dichroic mirror) for epi-illumination (set at position 1, 50% transmission at 400 nm), a UGI excitation filter (band pass filter, peak transmission 365nm) and a K430 high pass emission filter (50% transmission at 430 nm).The distribution of fluorescence in each section was recorded and the intensity of GSH fluorescence assessed in semi-quantitatively on a 4-point scale (0=no fluorescence, + = slight fluorescence, + + =moderate fluorescence, + + + = bright fluorescence).Relationship between GSH concentration and histofluorescence Five known concentrations of authentic GSH (2, 4, 6, 8, 10mM) in 0.1 M formic acid were each mixed with 1% polyvinylpyrrolidone (PVP: 2 ml GSH solution plus 1 ml PVP), droplets applied to glass slides and then dried in air. The slides were then reacted with o-phthalaldehyde as described above, examined with fluorescence microscopy and the fluorescence intensity assessed semi-quantitati...

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Augmentation of adriamycin, melphalan, and cisplatin cytotoxicity in drug-resistant and -sensitive human ovarian carcinoma cell lines by buthionine sulfoximine mediated glutathione depletion

Cited by 494 publications

References 5 publications

Detection of cells resistant to alkylating agents by flow cytometric analysis of DNA damage

Detection of cells resistant to alkylating agents by flow cytometric analysis of DNA damage

The role of glutathione in resistance to cisplatin in a human small cell lung cancer cell line

Glutathione localisation in benign and malignant human breast lesions

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