Derivation and preliminary characterisation of adriamycin resistant lines of human lung cancer cells

Abstract

Summary We have produced adriamycin (ADM)-resistant variants of the human lung cancer cell lines NCI-H69 (small cell), MOR (adenocarcinoma) and COR-L23 (large cell) but have failed to produce resistant variants of two other small cell lines. In each case, the derivation protocol took 7-9 months and included a period of drug-free growth. All three resistant lines show reduced cellular content of ADM after 1 h exposure when compared with their controls. During prolonged incubation of control and resistant NCI-H69 cells in 0.4 jgml -ADM, the ADM content of resistant cells was 6-7 times lower than that of control cells. The ratio of ADM doses to suppress growth of the two lines, however, was in the range 40-200X. The ADMresistant variant of NCI-H69 was also resistant to vincristine, colchicine, VP16, mitozantrone, 4'epiadriamycin and 4'deoxyadriamycin, somewhat resistant to melphalan but not resistant to aclacinomycin A, bleomycin or CCNU. The resistance to ADM could be partially overcome by the use of verapamil, an inhibitor of calcium transport.There are numerous reports in the literature of the development of cell lines which are resistant to the anthracycline drugs daunorubicin and doxorubicin (adriamycin, ADM). Most of these lines are either leukaemias or ascites tumours of rodent origin (Dano, 1972;Johnson et al., 1976;Nishimura et al., 1978;Belli & Harris, 1979). Recently, however, ADM-resistant sublines have been obtained from a murine fibrosarcoma (Giavazzi et al., 1983) and from human ovarian cancer lines (Hamilton et al., 1983). A number of mechanisms of resistance have been identified including defective drug accumulation due to increased active efflux (Dano, 1973), increased intracellular glutathione (Babson et al., 1981) and decreased amounts of cellular enzymes (Mungikar et al., 1981). Adriamycin resistance is closely associated with the phenomenon of pleiotropic or multi-drug resistance (Bech-Hansen et al., 1976) where collateral resistance to vincristine and colchicine is observed. This form of resistance is thought to be associated with the presence in the cell membrane of a glycoprotein of 170 K molecular weight (the pglycoprotein) (Ling, 1982) and a gene amplification (Roninson et al., 1984). 530 P.R. TWENTYMAN et al. ADM in which cell numbers would increase. After allowing them to grow in this concentration for a period of 2-4 weeks we then doubled the concentration. If cell multiplication continued we doubled the concentration again after a further 2-4 weeks and so on. When a concentration was reached at which cell multiplication was inhibited, the cells were maintained at this concentration for an indefinite period of time in order to allow a resistant subline to emerge.2nd Series In the second series of experiments, when a concentration of ADM was reached at which no significant growth was occurring, the cells were allowed to remain at this concentration for a period of 4 weeks and the ADM was then removed from the medium. If and when cell numbers began to increase, ADM was re-added to the...

Retention of activity by selected anthracyclines in a multidrug resistant human large cell lung carcinoma line without P-glycoprotein hyperexpression

“…Cell lines and culture conditions The human large cell lung cancer parental cell line COR-L23/P was derived in this laboratory (Baille-Johnson et al, 1985) and doxorubicin-resistant variant COR-L23/R was developed from it by continuous step-wise in vitro incubation with increasing concentrations of doxorubicin (Twentyman et al, 1986b). COR-L23/R was maintained in culture in the presence of 0.2 jig ml-' doxorubicin.…”

Differential recognition of mdr1a and mdr1b gene products in multidrug resistant mouse tumour cell lines by different monoclonal antibodies

“…It has been shown previously that expression of human mdrl RNA is present in the resistant small cell lung lines (Reeve et al, 1989a). These resistant lines were derived as described previously (Twentyman et al, 1986) and were maintained in RPMI 1640 medium supplemented with 10% foetal calf serum, penicillin and streptomycin (all Gibco Europe Ltd). The two mouse cell lines investigated were EMT6 originating from a mouse alveolar tumour nodule transplanted into a mammary fat pad (Rockwell et al, 1972) and RIF-1 obtained from a fibrosarcoma (Twentyman et al, 1980).…”

Modification of cytotoxic drug resistance by non-immuno-suppressive cyclosporins

“…This resulted in a suspension consisting of single cells and small aggregates of up to 10 cells. From this mechanicallydisaggregated suspension an aliquot was taken and formally disaggregated using trypsin and versene (Twentyman et al, 1986b which was actually used for setting up experiments. This procedure was considered necessary as enzymatic reduction of H69 cultures to a single cell suspension results in a prolonged 'lag' in subsequent growth whereas mechanical disaggregation appears to be much less growth inhibitory.…”

“…This resulted in a suspension consisting of single cells and small aggregates of up to 10 cells. From this mechanicallydisaggregated suspension an aliquot was taken and formally disaggregated using trypsin and versene (Twentyman et al, 1986b). A haemocytometer count was then carried out and the figure obtained was used as an estimate of the cell concentration in the mechanically-disaggregated suspension…”