In vitro drug sensitivity of normal peripheral blood lymphocytes and childhood leukaemic cells from bone marrow and peripheral blood

Abstract

Summary In vitro drug sensitivity of leukaemic cells might be influenced by the contamination of such a sample with non-malignant cells and the sample source. To study this, sensitivity of normal peripheral blood (PB) lymphocytes to a number of cytostatic drugs was assessed with the MTT assay. We compared this sensitivity with the drug sensitivity of leukaemic cells of 38 children with acute lymphoblastic leukaemia. We also studied a possible differential sensitivity of leukaemic cells from bone marrow (BM) and PB. The following drugs were used: Prednisolone, dexamethasone, 6-mercaptopurine, 6-thioguanine, cytosine arabinoside, vincristine, vindesine, daunorubicin, doxorubicin, mafosfamide (Maf), 4-hydroperoxy-ifosfamide, teniposide, mitoxantrone, L-asparaginase, methotrexate and mustine.Normal PB lymphocytes were significantly more resistant to all drugs tested, except to Maf. Leukaemic BM and PB cells from 38 patients (unpaired samples) showed no significant differences in sensitivity to any of the drugs. Moreover, in 11 of 12 children with acute leukaemia of whom we investigated simultaneously obtained BM and PB (paired samples), their leukaemic BM and PB cells showed comparable drug sensitivity profiles. In one patient the BM cells were more sensitive to most drugs than those from the PB, but the actual differences in sensitivity were small.We conclude that the contamination of a leukaemic sample with normal PB lymphocytes will influence the results of the MTT assay. The source of the leukaemic sample, BM or PB, does not significantly influence the assay results.

Cell proliferation is related to in vitro drug resistance in childhood acute leukaemia

“…In correspondence with our previous data, no differences were found in resistance to any of the drugs between bone marrow and peripheral blood samples (Kaspers et al, 1991;Klumper et al, 1995a). For this reason, the drug resistance data of bone marrow and peripheral blood samples were analysed all together.…”

“…The MTT assay was performed as described previously (Kaspers et al, 1991). In short, isolated leukaemia cells were exposed to six different concentrations of the selected drugs.…”

“…After 4 days of incubation in 5% CO 2 humidified air at 371C, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide (Sigma, St Louis MO, USA) was added and cells were incubated for another 6 h. Subsequently, formazan crystals formed were dissolved in acidified isopropanol. The optical density, which appeared linearly related to the number of viable cells (Kaspers et al, 1991), was measured at 562 nm (Bio-Kinetics Reader, Bio-Tek Instruments, Winooski VT, USA). Samples with less than 70% of leukaemia cells in control wells after 4 days of culture and/or an optical density below 0.050 arbitrary units (adjusted for blank values) were considered not valuable and were excluded from the study.…”

True

“…6,7,19 Briefly, cells were incubated in microtiter plates for 4 days with six DNR concentrations (0.002-2 g/ml) in duplicate wells. Results are expressed as DNR LC 50 values: the DNR concentration lethal to 50% of the cells as compared to cells from the same sample cultured in the same plate without DNR.…”

“…The authors concluded that a low proliferative activity (S phase fraction) as well as low 'spontaneous cell death' (percentage apoptotic cells as measured with propidium iodide staining) correlated better with DNR cytotoxicity than Pgp-related parameters (modulation of DNR accumulation by cyclosporin A). As a further contribution to the development of predictive assays for clinical efficacy of anthracycline-based chemotherapy we have used an MTT assay according to the same methodology 7,19 in AML to determine DNR cytotoxicity with or without the Pgp modulator PSC 833. This test was compared with a rhodamine-based Pgp functional assay, as well as a DNR accumulation assays.…”