Ross A. Davey

P-Glycoprotein and transporter MRP1 reduce HIV protease inhibitor uptake in CD4 cells: potential for accelerated viral drug resistance?

BackgroundThe multidrug transporters P-glycoprotein (P-gp) and MRP1 are functionally expressed in several subclasses of lymphocytes. HIV-1 protease inhibitors interact with both; consequently the transporters could reduce the local concentration of HIV-1 protease inhibitors and, thus, influence the selection of viral mutants. ObjectivesTo study the effect of the expression of P-gp and MRP1 on the transport and accumulation of HIV-1 protease inhibitors in human lymphocytes and to study the effects of specific P-gp and MRP1 inhibitors. MethodsThe initial rate and the steady-state intracellular accumulation of radiolabelled ritonavir, indinavir, saquinavir and nelfinavir was measured in three human lymphocyte cell lines: control CEM cells, CEM-MDR cells, which express 30-fold more P-gp than CEM cells, and CEM-MRP cells, which express fivefold more MRP1 protein than CEM cells. The effect of specific inhibitors of P-gp (GF 120918) and MRP1 (MK 571) was also examined. ResultsCompared with CEM cells, the initial rates of uptake and the steady-state intracellular concentrations of all protease inhibitors are significantly reduced in CEM-MDR cells. The intracellular concentrations of the protease inhibitors are increased upon co-administration with GF 120918, in some cases to levels approaching those in CEM cells. The intracellular concentrations of the protease inhibitors are also significantly reduced in CEM-MRP cells. Co-administration with MK -571 can partially overcome these effects. ConclusionsThe overexpression of multidrug transporters significantly reduces the accumulation of protease inhibitors at this major site of virus replication, which, potentially, could accelerate the acquisition of viral resistance. Targeted inhibition of P-gp may represent an important strategy by which this problem can be overcome.

Differences in the intracellular accumulation of HIV protease inhibitors in vitro and the effect of active transport.

ObjectivesTo investigate the intracellular accumulation of HIV protease inhibitors (PI) and to assess the effect of active transport on this accumulation. MethodsCEM cells were incubated with a PI for 18 h and the intracellular concentration determined using cell number and radioactivity. The effect of active transport was investigated using cells expressing P-glycoprotein (CEMVBL) and cells expressing multidrug resistance-associated protein 1 (MRP1; CEME1000). Incubations were also carried out at 4°C and in the presence of 2-deoxyglucose plus rotenone to examine the effect of inhibiting active transport. ResultsNelfinavir (NFV) accumulated to the greatest extent (> 80-fold) followed by saquinavir (SQV; ∼ 30-fold), ritonavir (RTV; 3–7-fold) and finally indinavir (IDV; extracellular equivalent to intracellular). In CEMVBL cells there was a significant reduction in the intracellular accumulation of NFV, SQV and RTV and in CEME1000 cells there was reduced accumulation of SQV and RTV. Inhibition of active transport processes caused a reduction in SQV and RTV accumulation but had no effect on IDV accumulation in all cell types. NFV accumulation was increased in CEMVBLcells as a result of inhibition of active transport. ConclusionsMarked differences can be detected in the intracellular accumulation of HIV PI drugs in vitro. Both P-glycoprotein and MRP1 may play a role in limiting the intracellular concentration of the PI and active influx mechanisms may contribute to drug accumulation.

The MTT cell viability assay for cytotoxicity testing in multidrug-resistant human leukemic cells

The MTT cell viability assay is widely used in determining drug sensitivity profiles for patients with hematological malignancies and in primary screening of potential chemotherapeutic drugs. Because the multidrug resistance (MDR) phenotype is associated with these malignancies, and since many vital dyes are effluxed from MDR expressing cells, we have investigated whether the MDR phenotype interferes with the MTT assay. In CCRF-CEM and K562 human leukemic cell lines and drug-resistant sub-lines developed from them, comparison of the MTT assay with other cell viability assays showed significant variation in IC50 concentrations, although the resistance relative to the sensitive parent cell was correlated. Inclusion of verapamil, an inhibitor of drug efflux activity, had no effect on the MTT assay.

Activated protein C prevents inflammation yet stimulates angiogenesis to promote cutaneous wound healing

Activated protein C (APC) is a serine protease that plays a central role in physiological anticoagulation, and has more recently been shown to be a potent anti‐inflammatory mediator. Using cultured human cells, we show here that APC up‐regulates the angiogenic promoters matrix metalloproteinase‐2 in skin fibroblasts and umbilical vein endothelial cells, vascular endothelial growth factor in keratinocytes and fibroblasts, and monocyte chemoattractant protein‐1 in fibroblasts. In the chick embryo chorioallantoic membrane assay, APC promoted the granulation/remodeling phases of wound healing by markedly stimulating angiogenesis as well as promoting reepithelialization. In a full‐thickness rat skin‐healing model, a single topical application of APC enhanced wound healing compared to saline control. APC‐treated wounds had markedly more blood vessels on day 7 and a significantly lower infiltration of neutrophils at days 4 and 7. The broad spectrum matrix metallo‐proteinas, GM6001, prevented the ability of APC to promote wound healing. In summary, our results show that APC promotes cutaneous wound healing via a complex mechanism involving stimulation of angiogenesis and inhibition of inflammation. These unique properties of APC make it an attractive therapeutic agent to promote the healing of chronic wounds.

DRUG RESISTANCE MECHANISMS AND MRP EXPRESSION IN RESPONSE TO EPIRUBICIN TREATMENT IN A HUMAN LEUKAEMIA CELL LINE

A drug resistant series of sublines were developed by treating the human leukaemia CCRF-CEM cell line with 16-1000 ng/ml of the anthracycline, epirubicin. The sublines developed resistance in two stages, neither involving detectable levels of P-glycoprotein. Treatment with up to 50 ng/ml epirubicin produced sublines with cross resistance limited to the anthracyclines and etoposide. Treatment with 100-1000 ng/ml epirubicin produced sublines with increased expression of the mrp gene, increased resistance to the anthracyclines and etoposide, additional cross resistance to vincristine and colchicine, decreased drug accumulation and reversal of resistance by verapamil and by buthionine sulphoximine (BSO; an inhibitor of glutathione synthesis). Our results indicate an interaction between MRP and glutathione metabolism as a mechanism for multidrug resistance.

Fractionated irradiation of H69 small-cell lung cancer cells causes stable radiation and drug resistance with increased MRP1, MRP2, and topoisomerase IIα expression

PURPOSE After standard treatment with chemotherapy and radiotherapy, small-cell lung cancer (SCLC) often develops resistance to both treatments. Our aims were to establish if fractionated radiation treatment alone would induce radiation and drug resistance in the H69 SCLC cell line, and to determine the mechanisms of resistance. METHODS AND MATERIALS H69 SCLC cells were treated with fractionated X-rays to an accumulated dose of 37.5 Gy over 8 months to produce the H69/R38 subline. Drug and radiation resistance was determined using the MTT (3,-4,5 dimethylthiazol-2,5 diphenyltetrazolium bromide) cell viability assay. Protein expression was analyzed by Western blot. RESULTS The H69/R38 subline was resistant to radiation (2.0 +/- 0.2-fold, p < 0.0001), cisplatin (14 +/- 7-fold, p < 0.001), daunorubicin (6 +/- 3-fold, p < 0.05), and navelbine (1.7 +/- 0.15-fold, p < 0.02). This was associated with increased expression of the multidrug resistance-associated proteins, MRP1 and MRP2, and topoisomerase IIalpha and decreased expression of glutathione-S-transferase pi (GSTpi) and bcl-2 and decreased cisplatin accumulation. Treatment with 4 Gy of X-rays produced a 66% decrease in MRP2 in the H69 cells with no change in the H69/R38 cells. This treatment also caused a 5-fold increase in topoisomerase IIalpha in the H69/R38 cells compared with a 1.5-fold increase in the H69 cells. CONCLUSIONS Fractionated radiation alone can lead to the development of stable radiation and drug resistance and an altered response to radiation in SCLC cells.

A systematic review of genes involved in the inverse resistance relationship between cisplatin and paclitaxel chemotherapy: role of BRCA1.

A systematic review of cell models of acquired drug resistance not involving genetic manipulation showed that 80% of cell models had an inverse resistance relationship between cisplatin and paclitaxel. Here we systematically review genetically modified cell lines in which the inverse cisplatin/paclitaxel resistance phenotype has resulted. This will form a short list of genes which may play a role in the mechanism of the inverse resistance relationship as well as act as potential markers for monitoring the development of resistance in the clinical treatment of cancer. The literature search revealed 91 genetically modified cell lines which report toxicity or viability/apoptosis data for cisplatin and paclitaxel relative to their parental cell lines. This resulted in 26 genes being associated with the inverse cisplatin/paclitaxel phenotype. The gene with the highest number of genetically modified cell lines associated with the inverse resistance relationship was BRCA1 and this gene is discussed in detail with reference to chemotherapy response in cell lines and in the clinical treatment of breast, ovarian and lung cancer. Other genes associated with the inverse resistance phenotype included dihydrodiol dehydrogenase (DDH) and P-glycoprotein. Genes which caused cross resistance or cross sensitivity between cisplatin and paclitaxel were also examined, the majority of these genes were apoptosis associated genes which may be useful for predicting cross resistance. We propose that BRCA1 should be the first of a panel of cellular markers to predict the inverse cisplatin/paclitaxel resistance phenotype.

Comparison of drug accumulation in P-glycoprotein-expressing and MRP-expressing human leukaemia cells

P-glycoprotein- and multidrug resistance-associated protein (MRP)-mediated multidrug resistance is associated with decreased drug accumulation. The P-glycoprotein-expressing CCRF-CEM/VLB100 subline and the MRP-expressing CCRF-CEM/E1000 subline are both 50-fold resistant to daunorubicin. However, accumulation of daunorubicin and rhodamine 123 was > 85% reduced in the P-glycoprotein-expressing subline compared to 40-50% in the MRP-expressing subline. Further, the CCRF-CEM/E1000 cells were 30-fold resistant to idarubicin, without reduced accumulation. Verapamil and SDZ PSC 833 restored daunorubicin and rhodamine 123 accumulation, while buthionine sulphoximine affected only the CCRF-CEM/ E1000 subline. We conclude that the verapamil associated change in rhodamine 123 accumulation provides a sensitive functional assay for both P-glycoprotein- and MRP-mediated MDR.

Verapamil-stimulated glutathione transport by the multidrug resistance-associated protein (MRP1) in leukaemia cells.

Multidrug resistance mediated by the multidrug resistance-associated protein MRP1 is associated with decreased drug accumulation, which is in turn dependent on cellular glutathione. We have reported that verapamil, an inhibitor of drug transport, caused a decrease in cellular glutathione in CCRF-CEM/E1000 MRP1-overexpressing leukaemia cells (Biochem Pharmacol 55;1283--9, 1998). We now demonstrate that other inhibitors of MRP1-mediated drug transport (e.g. MK571, indomethacin, genistein, and nifedipine) deplete cellular glutathione in these leukaemia cells (>30% decrease; P < 0.01) while having no effect on the parental CCRF-CEM cells. However, treatment with etoposide or vincristine (at similar molar concentrations) caused a 20% decrease in glutathione. Verapamil-stimulated glutathione transport correlated with MRP1 expression in a series of drug-resistant cells, and glutathione was quantitatively recovered in the extracellular media. Further, verapamil-stimulated glutathione transport was rapid (50% decrease in 10 min), dose-dependent, and inhibited by vanadate, an inhibitor of ATPase activity, but not by sulphobromophthalein (BSP) or methionine, inhibitors of hepatic glutathione transporters. Incubation of CCRF-CEM/E1000 cells in 25 mM glutathione not only showed that verapamil-mediated efflux occurred against the concentration gradient, but also demonstrated the MRP1-mediated uptake of glutathione (P < 0.01 compared to the parental CCRF-CEM cells), which was not inhibited by vanadate. These results demonstrate that while MRP1 transports glutathione in the presence of inhibitors of drug transport, there is no convincing evidence for co-transport of glutathione with drug. They further demonstrate that MRP1 mediates the facilitated transport of glutathione into the MRP1-overexpressing CEM/E1000 cells, suggesting that MRP1 may play a major role in cellular glutathione homeostasis.

Identification of a multidrug resistance associated antigen (P-glycoprotein) in normal human tissues

The multidrug resistance (NDR) phenotype describes a pattern of cross-resistance to unrelated compounds observed in mammalian cell lines selected in vitro for resistance to a single agent. Overexpression of a 170,000 dalton cell membrane glycoprotein (P-glycoprotein) is associated consistently with this phenotype in these cell lines. Recently, several human tumours have been shown to contain P-glycoprotein and expression was greatest in tumours exhibiting clinical drug resistance. To explore further the significance of P-glycoprotein, we examined normal human tissues obtained at autopsy by polyacrylamide gel electrophoresis and immunoblotting using a monoclonal antibody directed against P-glycoprotein. We showed expression of P-glycoprotein in normal liver and small bowel mucosa but not in other organs examined. This suggests there may be significant expression of P-glycoprotein in certain normal human tissues and any plan to exploit P-glycoprotein clinically must take these findings into account.