Changes in sensitivity to cytotoxic agents occurring during the life history of monolayer cultures of a mouse tumour cell line

Abstract

Our studies of the cytotoxic drug, bleomycin, have shown Bleehen, 1973, 1975) that in early plateau phase the sensitivity is reduced from that shown by exponentially growing cells. In late plateau phase, however, the sensitivity increases again and becomes greater than that of exponential phase cells. In this paper we report results obtained in exponential, early plateau and late plateau phases for 5 other cVtotoxic drugs. MATERIALS AND METHODSFull details of the cell line and of the drug response assay have been described previously (Twentyman and Bleehen, 1975). Briefly, 105 EMT6 mouse tumour cells were inoculated on Day 0 into Falcon tissue culture flasks containing 5 ml of Eagle's medium supplemented with 20% calf serum. Change of medium was carried out daily from Day 2. Drugs were added directly to the growth medium approximately 24 h after the previous medium change. At the end of the drug exposure period, the medium was removed, the monolayer was rinsed with fresh medium and cells were then trypsinized from the surface. After resuspension, counting and diluting, the cells were plated into plastic Petri dishes and incubated for 10 days. At the end of this period the dishes were fixed and stained and then colonies containing more than 50 cells were counted.The drugs used in the current series of experiments are shown in Table I. All drugs were added in a volume of between 0-04 and 0-2 ml. As a control for the alcohol solvent used for BCNU and CCNU, it was established in a preliminary experiment that the addition of 0-2 ml of absolute methanol to cultures for 1 h had no effect on cell survival.

Cytokinetic factors in drug resistance of Lewis lung carcinoma: comparison of cells freshly isolated from tumours with cells from exponential and plateau-phase cultures

“…The other intercalating agent studied, doxorubicin, also shows higher activity against exponential than against plateau-phase cultures (Figure 4b), as has been demonstrated with EMT6 (Twentyman & Bleehen, 1975;Kwok & Twentyman, 1985), CHO (Barranco & Novak, 1974), L1210 (Bhuyan et al, 1977) and LoVo (Drewinko et al, 1981) cells. Such cycle selectivity of intercalating drugs appears to reflect the activity of topoisomerase II, which declines in noncycling cells (Sullivan et al, 1986).…”

“…Since Bruce and his associates (reviewed by Valeriote & van Putten, 1975;van Putten, 1974) first demonstrated the importance of proliferative state in relation to drug sensitivity in vivo, most studies have utilized in vitro models of non-cycling cells such as plateau-phase cultures (Barranco & Novak, 1974;Drewinko et al, 1981;Hahn & Little, 1972;Twentyman & Bleehen, 1975) and multicellular spheroids (Sutherland & Durand, 1984;Nederman & Twentyman, 1984;Kwok & Twentyman, 1985). The question remains as to whether these provide adequate models for non-cycling cells in tumours (Valeriote & van Putten, 1975;Twentyman & Bleehen, 1975).…”

“…The question remains as to whether these provide adequate models for non-cycling cells in tumours (Valeriote & van Putten, 1975;Twentyman & Bleehen, 1975). In the work described here a novel and complementary approach has been adopted by investigating changes in drug sensitivity in short-term primary cultures derived from an advanced murine solid tumour.…”

“…Important differences in drug sensitivity between cycling and non-cycling cells have been demonstrated with antimetabolites, alkylating agents and DNA intercalating drugs (Barranco & Novak, 1974;Drewinko et al, 1981;Kwok & Twentyman, 1985;Twentyman & Bleehen, 1975). The present study focuses on 4-methyl-5-(N-methyl)carboxamideamsacrine (CI-921), a new 9-anilinoacridine derivative which has recently entered phase I clinical trials (Baguley et 1984).…”

The response to cytotoxic drugs of EMT6 cells treated either as intact or disaggregated spheroids

“…Unlike EMT6 cells which are more sensitive to HN2 in exponential than in plateau phase (Twentyman & Bleehen, 1975) In addition to possible differences in initial sensitivity of isolated spheriod cells and cells in intact spheroids to drugs, an additional factor comes into operation when considering the relationship between cellular response and the "overall' response of intact spheroids. "Recovery of potentially lethal damage" by cells in drug-treated EMT6 spheroids was reported by Twentyman (1980).…”