Peter R. Twentyman

Chemosensitisation and drug accumulation effects of cyclosporin A, PSC-833 and verapamil in human MDR large cell lung cancer cells expressing a 190k membrane protein distinct from P-glycoprotein

The doxorubicin-selected multidrug resistant (MDR) human large cell lung cancer line COR-L23/R, lacks P-glycoprotein but shows a drug accumulation deficit. It does however overexpress a 190k membrane protein which shares an epitope with, but is otherwise distinct from, P-glycoprotein. The resistant cells show only a small sensitisation to vincristine and daunorubicin on treatment with cyclosporin A and its more potent analogue, PSC-833 despite an increase in drug accumulation. Verapamil, another effective resistance modifier in P-glycoprotein MDR cells, is slightly more effective. Fluorescent daunorubicin distributes in the cytoplasm and nucleus of sensitive parent COR-L23 cells but is confined to cytoplasmic perinuclear vesicles in resistant cells. Addition of cyclosporin A or PSC-833 slightly increases cytoplasmic fluorescence whereas verapamil also increases nuclear fluorescence. Resistance in this non-P-glycoprotein MDR line, COR-L23/R where these resistance modifiers have little effect may be associated with expression of the 190k protein.

Intermittent exposure to doxorubicin in vitro selects for multifactorial non‐P‐glycoprotein‐associated multidrug resistance in RPMI 8226 human myeloma cells

The purpose of the present study was to evaluate whether intermittent exposure to a constant dose of doxorubicin selects for multidrug resistance (MDR) in RPMI 8226 human myeloma cells and, if so, to determine the molecular mechanism. In an attempt to approximate clinical doxorubicin treatment in vitro, cells were exposed to a fixed dose of doxorubicin for 4u2003d alternating with growth in drug‐free medium for 17u2003d. An MDR subline emerged, termed 8226/DOXint5, which was 3–4‐fold resistant to doxorubicin, etoposide and m‐AMSA, and 1.6‐fold resistant to vincristine. Sensitivity to docetaxel, melphalan and cisplatin was normal. Verapamil normalized vincristine sensitivity but had little effect on resistance to the other agents. Cellular uptake and retention of daunorubicin and vincristine were reduced by approximately 10%. The 8226/DOXint5 cells showed diminished DNA topoisomerase IIα expression and increased expression of the multidrug resistance protein MRP. Expression of MDR1/P‐glycoprotein was not detected. Immunostaining showed 70% of the cells to over‐express the lung‐resistance protein LRP. This new MDR myeloma cell line may prove to be a useful model for the development of strategies to overcome low‐level, multifactorial MDR, which might be a common phenomenon in clinical myeloma treated with doxorubicin.

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.

Hypotonicity-induced anion fluxes in cells expressing the multidrug-resistance-associated protein, MRP.

Anion transport in human multidrug-resis-tant large cell lung tumour cells (COR-L23/R) which overexpress the multidrug-resistance-associated protein (MRP) has been compared with that in cells of the parent line (COR-L23/P). Whole-cell patch-clamp recordings reveal variability between individual cells in basal anion conductance and in anion conductance increases following exposure to hypotonic media. The increase of stimulated over basal conductance is significantly larger for resistant cells than for parent cells. The chloride channel blocker, diisothiocyanatostilbene-2-2′-disulphonic acid (DIDS), rapidly and reversibly inhibits the increase in outward but not inward conductance when applied externally at 10−4 M during recording, but it is without effect when introduced into the cells via the patch pipette. Preincubation with DIDS greatly reduces both inward and outward conductance. 125I− efflux has been used to measure anion movement in cell populations. Basal efflux is similar in the two cell lines, but following a hypotonie challenge, the increase in rate constant for efflux from COR-L23/R cells is at least double that from COR-L23/P cells. This increase in efflux is greatly reduced by incubation with DIDS at 10−4 M. Replacement of external chloride by glu-conate does not affect efflux, thus excluding the possible involvement of DIDS-sensitive chloride exchange. Results from both techniques suggest that DIDS-sensitive, hypotonicity-induced anion channel activity is augmented in COR-L23/R multidrug-resistant variant cells which overexpress MRP. This augmentation may be caused by MRP itself or by other genes coexpressed with MRP.

Modifiers of multidrug resistance.

Over the next few years, hundreds of patients around the world with haematological malignancies will be treated with agents intended to overcome multidrug resistance. It has taken a quarter of a century for this stage to be reached. As in many developments in science and medicine, much of the story is based around a relatively small number of seminal papers. The first observation that induction of resistance in cultured cells to one of a well-defined group of cytotoxic agents could lead to cross-resistance to various other agents was made by Biedler & Riehm (1 9 70). It was then shown that such classic ‘multidrug resistance’ (MDR) was associated with decreased cellular drug accumulation (Dano, 1973) and with over-expression of a membrane glycoprotein subsequently named ‘P-glycoprotein (Pgp)’ (Juliano & Ling. 1976). Tsuruo P t a1 (1981) demonstrated that certain membrane-active agents, including the calcium transport blocker verapamil, were able selectively to restore drug sensitivity to MDR cells. When Pgp was first described it was widely perceived lo be a protein expressed only on drugresistant cells and hence potentially that elusive goal of chemotherapy, the ‘tumour-specific’ target. When, however, antibodies to Pgp became available. it became clear that the protein was widely expressed in normal tissues (Thiebaut et ul, 1987) and hence the ever-present dilemma of cancer chemotherapy the narrow therapeutic ratio was still present. And still, in 1995, there are a great many questions waiting to be resolved. We do not know the constitutive role of Pgp in many of the normal tissues in which it is expressed. We do not know how the transcriptional control of MDRl gene expression works. On a more practical level, we really do not know how to measure Pgp expression in a sufficiently precise way to determine its clinical significance, and it is still quite unclear as to whether resistance circumvention strategies based on competitive inhibition by agents such as verapamil and cyclosporin A are going to prove of major clinical benefit. Two major international meetings were held in 1994 at which these two practical issues were exhaustively addressed. The first of these meetings was the MDR Methods Workshop held in Memphis, Tennessee, in May. Sixteen groups had each carried out determinations of MDRl jPgp status in a variety of leukaemia/solid tumour specimens which had been centrally distributed. The detailed results of these inter-laboratory comparisons have been written up and will be published during 1995. It would be premature to

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.