Karen A. Wright

Radiation response of human lung cancer cells with inherent and acquired resistance to cisplatin

We have derived sublines of three human lung cancer cell lines with acquired resistance to cisplatin. The cisplatin resistant sublines of NCI-H69 (small cell), COR-L23 (large cell), and MOR (adenocarcinoma) show 5.3 fold, 3.1 fold, and 3.8 fold resistance, respectively, determined in a 6-day MTT assay. Although the parent lines show a wide range of glutathione content per cell, the sublines each show similar values to their corresponding parent line. Radiation response curves have been obtained using a soft agar clonogenic assay. Values obtained for the parent lines (95% CL in parentheses) were: NCI-H69: Do = 0.99 Gy (0.87-1.16), n = 2.9 (1.6-5.2), GSH = 14 ng/10(4) cells; COR-L23: Do = 1.23 Gy (1.05-1.49), n = 1.3 (0.7-2.2), GSH = 47 ng/10(4) cells; MOR: Do = 1.66 Gy (1.48-1.88), n = 3.0 (1.9-4.8), GSH = 86 ng/10(4) cells. The cisplatin resistant variants of NCI-H69 and COR-L23 showed 31% and 63% increases, respectively, in Do compared to their parent lines, whereas no change in radiation response was seen in MOR. In this panel of lines, therefore, although there is a correlation between glutathione content and radiosensitivity of the parent cell lines, acquired resistance to cisplatin is not accompanied by increased glutathione content. However, two of the three cisplatin resistant lines do show a significantly reduced radiosensitivity.

Identification of a novel calcium-binding protein (CP22) in multidrug-resistant murine and hamster cells

Analysis of cytoplasmic extracts of multidrug‐resistant murine and hamster cells by SDS gel and 2D gel electrophoresis showed that they expressed an abundant 22 kDa protein which was absent from the drugsensitive parent lines. SDS gel electrophoresis in the presence of EGTA and direct binding tests with 45Ca2+ showed that the resistance‐associated protein is a specific calcium‐binding protein. Thus the development of multidrug resistance in both colchicine‐selected hamster cells and adriamycin‐selected murine cells is associated with a major change in calcium metabolism. These observations provide the first molecular basis for the hypothesis that Ca2+ plays a central role in the development of the multidrug resistance phenomenon.

CB 1954 revisited

SummaryAlthough it has been the subject of considerable interest for 15 years, originally as a cytotoxic agent and more recently as a radiosensitizer, there is very little pharmacokinetic information on CB 1954 (2,4-dinitro-5-aziridinylbenzamide). We have developed a rapid high-performance liquid chromatography assay for the drug and its metabolites and applied it to detailed examination of the pharmacokinetics of CB 1954 in mice and dogs. With IV administration a dose of 50 mg/kg gave peak blood concentrations of 100 μg/ml in mice, while 25 mg/kg gave peak palsma concentrations of 27 μg/ml in dogs. Peak concentrations were 3 to 5-fold lower for the IP route in mice and the oral route in dogs, and the bioavailabilities were 85% and 40%, respectively. Elimination t1/2 values were 1.4–2 h in mice and 2.5–4 h in dogs and were independent of route of administration. Plasma protein binding was 57% but tissue penetration in mice was generally good. Tumour: plasma ratios were 50%–90%, while brain: plasma ratios were lower, at 37%–50%. The parent drug and several metabolites were identified and quantified in mouse urine, the total recovery being 24%–29%, of which 16%–25% was parent drug. The metabolites were also found in the circulation and in tissues. No changes in pharmacokinetics were seen with repeated dosing in mice or with administration of the protective agent phenyl AIC. Phenobarbitone pretreatment produced a small reduction in elimination t1/2, mainly by accelerating aziridine ring removal. Allopurinol increased the blood levels of the 5-amino nitroreduction product. These studies provide a pharmacokinetic basis for interpreting the antitumour activity and toxicity of CB 1954, as well as for the development of new ‘mixed-function’ sensitizers.

Localisation of the multidrug resistance-associated protein, MRP, in resistant large-cell lung tumour cells

The drug transport protein, P-glycoprotein, confers multidrug resistance (MDR) by expelling drugs across the cell surface. The structurally similar multidrug resistance-associated protein, or MRP, is also involved with drug efflux. In MDR variants of the human lung tumour cell line COR-L23 that overexpress MRP, there are also changes in intracellular drug distribution. To ascertain whether MRP could be involved in either process, experiments were performed to identify where MRP was located in these cells. Following separation of membranes by sucrose gradient centrifugation, MRP was found predominantly in the lighter membrane fractions containing plasma membrane enzyme activity. Immunofluorescent staining with a monoclonal antibody raised against MRP confirmed that MRP is present at the cell surface of these MDR lung tumour cells.

Differential properties of cisplatin and tetraplatin with respect to cytotoxicity and perturbation of cellular glutathione levels

SummaryWe compared the cytotoxicity and the effects on the levels of cellular glutathione (GSH) of cisplatin and tetraplatin in the mouse mammary tumour cell line EMT6/P and a cisplatin-resistant variant, EMT6/CPR. EMT6/CPR showed a 2.5-fold resistance to cisplatin but no cross-resistance to tetraplatin. Basal cellular GSH levels in the resistant line were 1.7-fold those in the parent cell line. On exposure of cells to cisplatin, cellular GSH levels initially dropped in both cell lines but subsequently rose. At between 1 and 4 h after the onset of drug exposure, GSH levels were up to 2-fold the baseline levels. The cisplatin-resistant subline displayed higher GSH levels than the parent cell line at all time points. Tetraplatin failed to influence cellular GSH levels in either of the cell lines.

Rapid recovery of a functional MDR phenotype caused by MRP after a transient exposure to MDR drugs in a revertant human lung cancer cell line.

Prior studies have shown that, in some human tumour cells, increased expression of the multidrug resistance gene MDR1 can be induced in response to certain stress conditions such as a transient exposure to cytotoxic agents. Little is known about the possibility of increasing the expression of the recently cloned multidrug resistance-associated protein (MRP) in response to a transient exposure to cytotoxic drugs. In order to examine this possibility, we have used sensitive assays (RT-PCR, flow cytometry) and the sensitive large cell lung cancer cell line, COR-L23/P, and the revertant line (COR-L23/Rev), generated by growing the doxorubicin-selected, MRP-overexpressing resistant variant COR-L23/R without drug exposure for 24-28 weeks. COR-L23/Rev overexpresses MRP, but to a lesser extent than COR-L23/R. COR-L23/Rev rapidly recovered similar levels of MRP mRNA, protein expression, resistance and drug accumulation deficit as COR-L23/R after a 48-72 h exposure to cytotoxic concentrations of doxorubicin or vincristine but not cisplatin. The increase in MRP mRNA could only be detected 3 to 4 days after the transient exposure to drugs. However, when the parental line, COR-L23/P, was exposed to equitoxic doses of doxorubicin, vincristine or cisplatin, no increase in the levels of MRP mRNA could be observed at higher doses (5- to 10-fold the IC50) of doxorubicin or vincristine (but not of cisplatin), we detected a transient increase in the levels of MDR1 mRNA immediately after short-term exposure. In conclusion, we have shown that a human revertant lung cancer cell line (COR-L23/Rev) has the ability to recover quickly, similar levels of MRP expression and resistance as COR-L23/R after a transient exposure to the MDR-drugs doxorubicin and vincristine.

Derivation and characterisation of a mouse tumour cell line with acquired resistance to cyclosporin A

Cyclosporin A (CsA) is an effective modifier of multidrug resistance. We have studied (a) the possibility that cells grown in increasing concentrations of CsA acquire cellular resistance to the agent and, (b) whether such cells have a multidrug resistant phenotype. Sublines of the EMT6 mouse tumour cell line were developed which were able to grow in 75 and 200 micrograms/ml of CsA, respectively. The resistant sublines grew slowly in the presence of CsA but reverted to control growth rates, whilst maintaining resistance, when the drug was removed. P-glycoprotein (Pgp) was not detectable in the resistant sublines by immunocytochemistry. The CsA-resistant cells were not cross-resistant to doxorubicin or vincristine but showed a clear degree of cross-resistance to the calcium transport blocker, verapamil. Cellular accumulation of both [3H]CsA and [3H]daunorubicin was significantly increased in the EMT6/CsA200R subline compared with the parent line. In the EMT6 parent line, which expresses very low levels of Pgp, 10-30-fold sensitisation to doxorubicin may be achieved using 0.1-5 microgram/ml of CsA. Similar sensitisation by CsA was also seen in the CsA-resistant sublines.

Chemosensitisation of a drug-sensitive parental cell line by low-dose cyclosporin A.

SummaryWe investigated the chemosensitisation of the parental EMT6 mouse mammary tumour cell line by low doses of cyclosporin A (CsA). This cell line has not previously been exposed to cytotoxic drugs but expresses low levels of P-glycoprotein. We produced >2-fold sensitisation to doxorubicin, colchicine and vincristine using 0.084 μM (0.1 μg/ml) CsA. Cellular accumulation of doxorubicin and daunorubicin was also increased by this dose. In the MDR subline EMT6/AR1.0, much higher doses of CsA were required to effect optimal restoration of doxorubicin or daunorubicin accumulation. The effects of CsA on the parent line could not be increased by extended preincubation of cells with the sensitiser. These, effects of CsA in the EMT6 parent cell line occur at a dose that is 1 order of magnitude lower than those previously reported to produce significant chemosensitisation.