A 300-fold adriamycin resistant variant (DLKP-A) of the human lung squamous cell carcinoma line DLKP was established by stepwise selection in increasing concentrations of adriamycin. Different levels of cross-resistance were observed towards VP-16, VM-26, colchicine, vincristine and, somewhat unexpectedly, cis-platin. Resistance was stable for at least 3 months in culture in the absence of drug. P-glycoprotein overexpression was detected by immunofluorescence and Western Blotting, and a direct causal role for P-glycoprotein overexpression in the resistant phenotype was established by transfection with an mdr1 specific antisense oligonucleotide. A modified cryopreservation procedure was necessary for the resistant variant line. The resistant population displays clonal heterogeneity with respect to resistance level. A higher frequency of double minute chromosomes was observed in DLKP-A when compared with the parental cell line.

The correlation between cellular resistance to radiation and to chemotherapeutic drugs has been investigated in a number of solid tumour cell lines, and preliminary results indicate no direct relationship. The acquisition of a multidrug resistance (MDR) profile by adriamycin-selected variants of a human squamous lung carcinoma, an ovarian carcinoma, a cervical carcinoma and by a colchicine-selected variant of a Chinese hamster ovarian carcinoma resulted in alterations to their radiosensitivity. However, the degree of change in the radiosensitivity of the MDR cell lines could not be predicted from their level of resistance to adriamycin. Clonal populations derived from DLKP-A, an adriamycin-selected MDR variant of the human lung carcinoma cell line DLKP, exhibited individual radiosensitivity profiles, which did not correlate with their chemoresistance. Exposure of DLKP to consecutive increasing doses of radiation did not confer cross-resistance to chemotherapeutic drugs.

of a monoclonal antibody rai8.d to the over expre88.d plasm r d r a n e associated glycoprotein of a multidrug resistant cell line. A murine monoclonal antibody against a multidrug resistant (MDR) variant of the CHOKl cell line (which overexpresses the P170-18OkD glycoprotein receptor) was produced by the fu8ion of NSO myeloma cell and splenocytes from a Balb/c mouse immunised with the whole cell preparations and plasma membrane-enriched microsomal fractions of the MDR variant cell line. The fusion technique was a modification of the method described by Kohler and Hiletein [l].All tissue culture media and supplements were obtained from Flow laboratories, Herts, England. Instead of using mouse feeder cells (e.g.macrophages) Briclone was used. This is a conditioned medium containing high levels of human IL-6 and is produced at the National Cell and Tissue Culture Centre.This product increases the efficiency of both fusion and cloning steps in hybridoma formation.Following cloning by limiting dilution (ZX), one monoclonal antibody, designated P3A8/12H, was selected for further study. This antibody was also grown as an ascite tumor in Balb/c mice. The commercially obtained antibody (Centocor Diagnostics USA) was used concurrently with P3A8/12H during all screening procedures.C219 recognises specifically the P-glycoprotein (P-170) receptor on MDR variant cell lines and could be used as a positive control for the Screening systems used. Initial screening of hybridomas by ELISA was preformed by growing the drug-sensitive parental cell line (CHOK1) and the MDR variant cell line on 96 well polyvinyl plates and fixing with 0.05% glutaraldehyde in PBS.Nitrocellulose bottomed 96 well plates (obtained from Amersham, U.K.) were also used for the ELISAs.These were coated with whole cells or plasma membrane enriched microsomal fractions prior to screening, no fixation of cells was required. microsomal fractions from the CHOKl cell line, the multidrug resistant variant and four other multidrug resistant variants (produced in our laboratories) and their drug sensitive counterparts were prepared by two methods.

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Two multi-drug resistant variants of the human carcinoma line Hep-2 have been selected by adaptation to progressively increasing concentrations of adriamycin. In comparison to the wild-type Hep-2 cells, the variant lines both showed approximately 100-fold resistance to adriamycin, 10 to 20-fold resistance to the vinca alkaloids but only 2-3 fold resistance to VP-16 and VM-26. There was essentially no difference between wild-type and variant cells in regard to sensitivity to threosulfan and 5-fluorouracil. The drug-resistant phenotype is stable for at least 3 months in the absence of drug, and is partially reversible by concomitant treatment with Verapamil. Chromosomal abnormalities consistent with gene amplification were observed in one of the variant lines. Sensitivity of variant cells to adriamycin was enhanced following trypsin-EDTA treatment.