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s-Thalidomide has proven efficacy in multiple myeloma. Although it has both antiangiogenic and pro-apoptotic effects, its primary mode of therapeutic action remains unclear. We have investigated the changes to the expression of genes involved with these cellular processes following culture with s-thalidomide in the U266 MM cell line. Cells were cultured with s-thalidomide (0-1000 microM), and cell parameters, including apoptosis, were assessed on day 3. RNA was extracted from cells cultured for 24 h at the IC(50) concentration of s-thalidomide, and changes to gene expression were investigated by microarray methodologies. A reduction in cell viability was observed in U266 cells cultured with s-thalidomide (IC(50): 362 microM), which were mirrored by significant increases in apoptosis (for example, 200 microM on day 3: 40.3+/-3.1% vs. 3.2+/-0.4% on day 0; P

Etoposide is a commonly used anticancer agent that is highly schedule-dependent. The in vitro activity of etoposide (0-10 microM) was investigated in a panel of leukaemic cell lines.Cells were cultured with etoposide in drug schedules of equal exposure duration (ED, durationxconcentration), and the effects of drug exposure on cell parameters, including cell cycle distribution, were assessed over an 8-day period.Proliferation assays indicated a concentration- and duration-dependent induction of cell death by etoposide in CEM and HL60 cells, and flow cytometric analysis indicated that this cell kill was by apoptosis. Efficacy was also dependent upon schedule, with more cell kill seen in schedules of longer duration. As an example, accumulative percent cell kill resulting from a continuous exposure to 0.05 microM etoposide was significantly greater than that in cultures involving either a 4-day exposure to 0.1 microM or a single-day exposure to 0.4 microM etoposide (193.4+/-15.9% vs 125.2+/-5.4% vs 42.3+/-5.9%, respectively; P0.001 in all cases; equi-ED 0.4 microM.days). Efficacy was also dependent upon the ED of the schedule. At very low concentrations, the initial enhancement of cytotoxicity mediated by increasing duration would gradually and paradoxically be lost in the more protracted schedules (e.g. accumulative percent cell kill 66.4+/-7.4%, 158.3+/-12.0% and 40.1+/-6.0% with 100 n M for 2 days, 33 n M for 6 days and 25 n M for 8 days, respectively; P0.001 in all cases; equi-ED 0.2 microM x days).Our results confirm the schedule-dependency of etoposide in vitro, highlighting the importance of total duration of drug exposure in determining cytotoxicity, and emphasizing the requirement to achieve a cytotoxic concentration in longer exposures. It is therefore crucial to ensure that etoposide regimens used clinically involve doses that are effectively cytotoxic.

Prior to work on the influence of dosing and scheduling of the drug etoposide in bone marrow cells, the DNA-damaging effects of three haemopoietic growth factors, either alone or in combination with etoposide, were investigated. Sister chromatid exchange (SCE) frequencies in phytohaemagglutinin-stimulated mononuclear cells of six normal volunteers were used as an indicator of DNA damage. The effects of three growth factors on SCEs were investigated at concentrations ranging between 0 and 100 ng/ml and those of etoposide alone, at concentrations varying between 0 and 2 microM. The effect of combinations of growth factor (GF) and etoposide were assessed at a 40-ng/ml concentration of each cytokine and at 0.4 microM etoposide. Results showed not only a dose-dependent rise in SCE frequency in cells treated with etoposide but also a cytokine effect. Stem-cell factor did not cause a significant change in SCE numbers. However, cytokines with activity at the progenitor cell level induced small but significant increases in SCE numbers at concentrations of 50 and 100 ng/ml (P0.001). Results of combination studies indicated a significant 60% increase in SCE numbers in cells treated with GF and etoposide as compared with etoposide alone (P0.00001). This finding suggests a sensitivity of peripheral blood mononuclear cells to SCE induction by GFs given either as single agents or in combination with etoposide.