Mary W. Davey

Increased expression of ABC transport proteins is associated with ivermectin resistance in the model nematode Caenorhabditis elegans

Widespread resistance to chemotherapeutic agents is one of the biggest challenges facing human health and the agricultural industry, with resistance to all current anthelmintics now recorded and few new agents or vaccines available. Understanding the development of drug resistance in parasitic nematodes is critical to prolonging the efficacy of current anthelmintics, developing markers for monitoring drug resistance and is beneficial in the design of new chemotherapeutic agents or targets. This study describes the development of ivermectin-resistant strains of the model nematode Caenorhabditis elegans through step-wise exposure to increasing doses of ivermectin commencing with a non-toxic dose of 1 ng/ml. Resistant strains were developed that displayed a multidrug resistance phenotype with cross-resistance to the related drug moxidectin and to other anthelmintics, levamisole and pyrantel, but not albendazole. Resistance was associated with increased expression of the multidrug resistance proteins (MRPs) and P-glycoproteins. Resistance to ivermectin was reversible by the co-administration of MRP, P-glycoprotein and glutathione biosynthesis inhibitors, confirming the involvement of these proteins in resistance. In our model, resistance to low levels of ivermectin (<or=6 ng/ml) was associated with increased expression of mrp-1 and pgp-1 and decreased glutathione, while higher level resistance (10 ng/ml) was primarily associated with the increased expression of P-glycoproteins. Importantly, resistance was stable after 3 months without ivermectin treatment. This clearly demonstrates the involvement of transport proteins in ivermectin resistance and provides a model to understand drug resistance and its reversal.

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.

Understanding cisplatin resistance using cellular models.

Many mechanisms of cisplatin resistance have been proposed from studies of cellular models of resistance including changes in cellular drug accumulation, detoxification of the drug, inhibition of apoptosis and repair of the DNA adducts. A series of resistant models were developed from CCRF‐CEM leukaemia cells with increasing doses of cisplatin from 100 ng/ml. This produced increasing resistance up to 7‐fold with a treatment dose of 1.6 μg/ml. Cisplatin resistance in these cells correlated with increases in the antioxidant glutathione, yet treatment with buthionine sulphoximine, an inhibitor of glutathione synthesis, had no effect on resistance, suggesting that the increase in glutathione was not directly involved in cisplatin resistance. Two models were developed from H69 SCLC cells, H69‐CP and H69CIS200 using 100 ng/ml or 200 ng/ml cisplatin respectively. Both cell models were 2‐4 fold resistant to cisplatin, and have decreased expression of p21 which may increase the cells ability to progress through the cell cycle in the presence of DNA damage. Both the H69‐CP and H69CIS200 cells showed no decrease in cellular cisplatin accumulation. However, the H69‐CP cells have increased levels of cellular glutathione and are cross resistant to radiation whereas the H69CIS200 cells have neither of these changes. This suggests that increases in glutathione may contribute to cross‐resistance to other drugs and radiation, but not directly to cisplatin resistance. There are multiple resistance mechanisms induced by cisplatin treatment, even in the same cell type. How then should cisplatin‐resistant cancers be treated? Cisplatin‐resistant cell lines are often more sensitive to another chemotherapeutic drug paclitaxel (H69CIS200), or are able to be sensitized to cisplatin with paclitaxel pre‐treatment (H69‐CP). The understanding of this sensitization by paclitaxel using cell models of cisplatin resistance will lead to improvements in the clinical treatment of cisplatin resistant tumours. IUBMB Life, 59: 696‐699, 2007

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.

Members of the Chloride Intracellular Ion Channel Protein Family Demonstrate Glutaredoxin-Like Enzymatic Activity

The Chloride Intracellular Ion Channel (CLIC) family consists of six evolutionarily conserved proteins in humans. Members of this family are unusual, existing as both monomeric soluble proteins and as integral membrane proteins where they function as chloride selective ion channels, however no function has previously been assigned to their soluble form. Structural studies have shown that in the soluble form, CLIC proteins adopt a glutathione S-transferase (GST) fold, however, they have an active site with a conserved glutaredoxin monothiol motif, similar to the omega class GSTs. We demonstrate that CLIC proteins have glutaredoxin-like glutathione-dependent oxidoreductase enzymatic activity. CLICs 1, 2 and 4 demonstrate typical glutaredoxin-like activity using 2-hydroxyethyl disulfide as a substrate. Mutagenesis experiments identify cysteine 24 as the catalytic cysteine residue in CLIC1, which is consistent with its structure. CLIC1 was shown to reduce sodium selenite and dehydroascorbate in a glutathione-dependent manner. Previous electrophysiological studies have shown that the drugs IAA-94 and A9C specifically block CLIC channel activity. These same compounds inhibit CLIC1 oxidoreductase activity. This work for the first time assigns a functional activity to the soluble form of the CLIC proteins. Our results demonstrate that the soluble form of the CLIC proteins has an enzymatic activity that is distinct from the channel activity of their integral membrane form. This CLIC enzymatic activity may be important for protecting the intracellular environment against oxidation. It is also likely that this enzymatic activity regulates the CLIC ion channel function.

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.

Demethylation of the human MDR1 5' region accompanies activation of P-glycoprotein expression in a HL60 multidrug resistant subline.

Chemotherapy is frequently limited by the development of multidrug resistance, a major cause of which is activation of the P-glycoprotein-encodingMDR1 gene. We have previously developed a P-glycoprotein-expressing multidrug resistant subline (HL60/E8) from the non-P-glycoprotein-expressing human HL60 promyelocytic leukemia cell line. A possible cause ofMDR1 silencing in HL60 cells is methylation of the promoter proximal region, thus demethylation occurring as a result of drug treatment may be responsible forMDR1 activation in the multidrug resistant subline. Using the bisulphite genomic sequencing technique we demonstrated that HL60 DNA is methylated at multiple sites within two distinct areas, one upstream and one downstream of the transcription start point. Only a single site in each area was methylated in all strands examined, with the remaining adjacent sites showing partial methylation. In contrast, DNA from the multidrug resistant HL60/E8 subline was unmethylated at essentially all sites in both areas. Thus the development of the P-glycoprotein-expressing multidrug resistant subline was associated with demethylation of theMDR1 5′ region.

Development of drug resistance is reduced with idarubicin relative to other anthracyclines.

Multidrug resistance (MDR) is associated with poor prognosis in leukemia, and anthracyclines, which are used in the treatment of leukemia, are associated with the expression of P-glycoprotein and the development of MDR. We report here that idarubicin, a new anthracycline approved for use in the treatment of acute myelogenous leukemia (AML), did not induce P-glycoprotein expression in the K562 human leukemia cell line under conditions where daunorubicin, doxorubicin and epirubicin did induce expression of P-glycoprotein. The P-glycoprotein expressing, multidrug resistant sublines developed to daunorubicin (K/DNR), doxorubicin (K/DOX) and epirubicin (K/EPR) were cross-resistant to the other anthracyclines and to vinblastine, taxol, colchicine and actinomycin D, but were not resistant to idarubicin or etoposide. The idarubicin treated subline, K/IDA, was only resistant to taxol but was 12-fold sensitized to etoposide, suggesting that idarubicin had affected topoisomerase II in this subline.