Pharmacological modification of multi‐drug resistance (mdr) in vitro detected by a novel fluorometric microculture cytotoxicity assay. Reversal of resistance and selective cytotoxic actions of cyclosporin a and verapamil on mdr leukemia t‐cells

Abstract

A novel fluorometric microculture cytotoxicity assay (FMCA), based on measurements of fluorescein diacetate (FDA) hydrolysis and DNA staining by Hoechst 33342, was used for drug sensitivity testing and detection of resistance reversal in acute lymphoblastic leukemia (ALL) cell lines. The 72-hr assay was found to be sensitive, reproducible and linearly related to the number of viable cells within a broad range of cell concentrations. At clinically achievable drug concentrations, the calcium channel blocker Verapamil (ver) and the immunosuppressant Cyclosporin A (csA) were found to partly reverse acquired Vincristine (vcr) resistance in multi-drug resistant (MDR) T-ALL L100 cells with little or no effect on the drug-sensitive parental L0 cell line. By combining the fluorometric indices, we found that low concentrations of csA were growth-inhibitory, whereas higher concentrations (greater than 10 micrograms/ml) were progressively cytotoxic for drug-sensitive L0 cells. In MDR L100 cells, on the other hand, csA produced significant cell kill even at low drug concentrations. Ver had no effects on sensitive L0 cells but showed considerable cytotoxic action towards MDR L100 cells. There was no apparent relationship between drug reversal of vcr resistance and the cytotoxic actions of the drug per se since the calcium channel blocker diltiazem (dil) significantly potentiated the actions of vcr on MDR L100 cells without being more toxic to these cells (compared to vcr-sensitive L0 cells).

In vitro testing of chemotherapeutic drug combinations in acute myelocytic leukaemia using the fluorometric microculture cytotoxicity assay (FMCA)

“…Empirically derived cut-off concentrations (EDCC) were selected according to principles described previously and are in most cases in the range achievable in plasma (Larsson et al, 1992a). EDCC concentrations of each drug were used in the combinations (Table II) Table I. FMCA procedure The principal steps of the assay procedure have been described previously (Larsson et al, 1992a;Larsson & Nygren, 1990). Day 1 180 pl of the leukaemia preparation at 2.5-5 x 10' cells ml-' in culture medium were seeded into the wells of V-shaped 96 well experimental microtiter plates (Nunc, Roskilde, Denmark) prepared as described above.…”

“…We have previously described the fluorometric microculture cytotoxicity assay (FMCA) for drug sensitivity testing of cell lines and tumour cell samples from patients with acute myelocytic leukaemia (AML; Larsson et al, 1992a;Larsson & Nygren, 1990). In the present study we employed this method for the study of drug interactions in AML.…”

Effects of Interferons and Tumour Necrosis Factor-α on Human Lung Cancer Cell Lines and the Development of an Interferon-Resistant Lung Cancer Cell Line

“…It has therefore been suggested that in vitro testing may be a valuable tool in the preclinical evaluation of new anticancer agents (23,24). In the present study we have employed a fluorometric assay based on detection of esterase activity in cells with intact plasma membranes (16). Indeed, this assay system has been found to give promising individual clinical correlations ( 18).…”

“…The histocytic lymphoma cell line U-937 (15) was used as a TNF-(r and IFN-y sensitive control. Cell survival after culture in the presence and absence of cytokines was determined by using the fluorometric microculture cytotoxicity assay (FMCA) as described elsewhere (16). The assay is based on hydrolysis of non-fluorescent fluorescein diacetate (FDA; Sigma Chem.…”

“…The assay is based on hydrolysis of non-fluorescent fluorescein diacetate (FDA; Sigma Chem. Co., St Louis, MO, USA) to a strongly fluorescent product by viable cells with intact plasma membranes (16). Briefly, after 72 h of microtitre plate culture, the plates were centrifuged (200 g, 5 min), the medium removed by flicking the plate, and the wells washed once with 2 0 0~1 PBS.…”