Development and validation of an immunoperoxidase antigen detection test for improved diagnosis of rabies in Indonesia.

Rabies continues to pose a significant threat to human and animal health in regions of Indonesia. Indonesia has an extensive network of veterinary diagnostic laboratories and the 8 National laboratories are equipped to undertake diagnostic testing for rabies using the commercially-procured direct fluorescent antibody test (FAT), which is considered the reference (gold standard) test. However, many of the Indonesian Provincial diagnostic laboratories do not have a fluorescence microscope required to undertake the FAT. Instead, certain Provincial laboratories continue to screen samples using a chemical stain-based test (Seller's stain test, SST). This test has low diagnostic sensitivity, with negative SST-tested samples being forwarded to the nearest National laboratory resulting in significant delays for completion of testing and considerable additional costs. This study sought to develop a cost-effective and diagnostically-accurate immunoperoxidase antigen detection (RIAD) test for rabies that can be readily and quickly performed by the resource-constrained Provincial laboratories. This would reduce the burden on the National laboratories and allow more rapid diagnoses and implementation of post-exposure prophylaxis. The RIAD test was evaluated using brain smears fixed with acetone or formalin and its performance was validated by comparison with established rabies diagnostic tests used in Indonesia, including the SST and FAT. A proficiency testing panel was distributed between Provincial laboratories to assess the reproducibility of the test. The performance of the RIAD test was improved by using acetone fixation of brain smears rather than formalin fixation such that it was of equivalent accuracy to that of the World Organisation for Animal Health (OIE)-recommended FAT, with both tests returning median diagnostic sensitivity and specificity values of 0.989 and 0.993, respectively. The RIAD test and FAT had higher diagnostic sensitivity than the SST (median = 0.562). Proficiency testing using a panel of 6 coded samples distributed to 16 laboratories showed that the RIAD test had good reproducibility with an overall agreement of 97%. This study describes the successful development, characterisation and use of a novel RIAD test and its fitness for purpose as a screening test for use in provincial Indonesian veterinary laboratories.

A rare variant in human fibroblast activation protein associated with ER stress, loss of enzymatic function and loss of cell surface localisation.

Fibroblast activation protein (FAP) is a focus of interest as a potential cancer therapy target. This membrane bound protease possesses the unique catalytic activity of hydrolysis of the post-proline bond two or more residues from the N-terminus of substrates. FAP is highly expressed in activated fibroblastic cells in tumours, arthritis and fibrosis. A rare, novel, human polymorphism, C1088T, encoding Ser363 to Leu, occurring in the sixth blade of the ß propeller domain, was identified in a family. Both in primary human fibroblasts and in Ser363LeuFAP transfected cells, we showed that this single substitution ablates FAP dimerisation and causes loss of enzyme activity. Ser363LeuFAP was detectable only in endoplasmic reticulum (ER), in contrast to the distribution of wild-type FAP on the cell surface. The variant FAP showed decreased conformational antibody binding, consistent with an altered tertiary structure. Ser363LeuFAP expression was associated with upregulation of the ER chaperone BiP/GRP78, ER stress sensor ATF6, and the ER stress response target phospho-eIF2α, all indicators of ER stress. Proteasomal inhibition resulted in accumulation of Ser363LeuFAP, indicating the involvement of ER associated degradation (ERAD). Neither CHOP expression nor apoptosis was elevated, so ERAD is probably important for protecting Ser363LeuFAP expressing cells. These data on the first loss of function human FAP gene variant indicates that although the protein is vulnerable to an amino acid substitution in the ß-propeller domain, inactive, unfolded FAP can be tolerated by cells.

Targeting the intrinsic apoptosis pathway as a strategy for melanoma therapy.

Melanoma drug resistance is often attributed to abrogation of the intrinsic apoptosis pathway. Targeting regulators of apoptosis is thus considered a promising approach to sensitizing melanomas to treatment. The development of small-molecule inhibitors that mimic natural antagonists of either antiapoptotic members of the BCL-2 family or the inhibitor of apoptosis proteins (IAPs), known as BH3- or SMAC-mimetics, respectively, are helping us to understand the mechanisms behind apoptotic resistance. Studies using BH3-mimetics indicate that the antiapoptotic BCL-2 protein MCL-1 and its antagonist NOXA are particularly important regulators of BCL-2 family signaling, while SMAC-mimetic studies show that both XIAP and the cIAPs must be targeted to effectively induce apoptosis of cancer cells. Although most solid tumors, including melanoma, are insensitive to these mimetic drugs as single agents, combinations with other therapeutics have yielded promising results, and tests combining them with BRAF-inhibitors, which have already revolutionized melanoma treatment, are a clear priority.

CB2 and TRPV1 receptors mediate cannabinoid actions on MDR1 expression in multidrug resistant cells.

BACKGROUND: Cannabis is the most widely used illicit drug in the world that is often used by cancer patients in combination with conventional anticancer drugs. Multidrug resistance (MDR) is a major obstacle in the treatment of cancer. An extensively characterized mechanism of MDR involves overexpression of P-glycoprotein (P-gp), which reduces the cellular accumulation of cytotoxic drugs in tumor cells. METHODS: Here we examined the role of cannabinoid receptors and transient receptor potential vanilloid type 1 (TRPV(1)) receptors in the effects of plant-derived cannabinoids on MDR1 mRNA expression in MDR CEM/VLB(100) cells which overexpress P-gp due to MDR1 gene amplification. RESULTS: We showed that both cannabidiol (CBD) and Δ(9)-tetrahydrocannabinol (Δ(9)-THC) (10 µM) transiently induced the MDR1 transcript in P-gp overexpressing cells at 4 but not 8 or 48 h incubation durations. CBD and THC also concomitantly increased P-gp activity as measured by reduced accumulation of the P-gp substrate Rhodamine 123 in these cells with a maximal inhibitory effect observed at 4 h that slowly diminished by 48 h. CEM/VLB(100) cell lines were shown to express CB(2) and TRPV(1) receptors. Δ(9)-THC effects on MDR1 expression were mediated by CB(2) receptors. The effects of CBD were not mediated by either CB(2) or TRPV(1) receptors alone, however, required activation of both these receptors to modulate MDR1 mRNA expression. CONCLUSION: This is the first evidence that CB(2) and TRPV(1) receptors cooperate to modulate MDR1 expression.

Synchronized defibrillation for ventricular fibrillation.

OBJECTIVE: Optimization of defibrillation success is important to improve efficacy and minimize post-shock sequelae. Previous work has suggested an improvement in shock success when an intracardiac shock is delivered synchronized to the upslope of a VF wave. We investigated the efficacy of transthoracic defibrillation success using a novel external biphasic defibrillator which delivers shocks synchronized to the upslope of the surface ECG. METHODS: A prospective, controlled, randomized study in a research institute laboratory of male and female pigs (54.2±1.8 kg). Ventricular fibrillation (VF) was induced in 10 anaesthetized and ventilated pigs. Shocks were delivered randomly from a biphasic defibrillator in synchronized or non-synchronized mode via self-adhesive electrode pads following 30 s of VF. Energy settings at 50, 70, 80, and 100J were randomly tested. VF amplitude, impedance, and shock outcome were recorded and analysed digitally. RESULTS: A total of 300 shocks were delivered. Synchronized shocks were delivered on the upslope of the VF wave in 99% of cases. There was no significant difference in shock success between shocks delivered in synchronized or non-synchronized modes (p=0.695). There was no significant difference in the amplitude of VF between successful and unsuccessful shocks (p=0.163). Furthermore, there was no association between shock success and transthoracic impedance. CONCLUSION: The novel defibrillator used in this study was able to consistently deliver shocks on the upslope portion of the VF wave but did not show an improvement in shock success.

Effect of vitamin D3 supplementation on the pharmacokinetics of digoxin--a pilot study.

Emerging evidence from preclinical, clinical and epidemiological studies suggests that vitamin D3 plays vital roles in several diseases in addition to bone disorders. According to new medical evidence, it is being recommended that vitamin D3 intake to be increased for maximal benefits in human health. However, it is necessary to consider potential side effects of increased intake of vitamin D3. Vitamin D3 exerts its actions through the vitamin D receptor, which is known to be an important regulator of P-glycoprotein (P-gp). As P-gp plays a significant role in limiting drug bioavailability, we undertook a study to compare single-dose digoxin (a P-gp substrate) pharmacokinetics in eight healthy male subjects before and after vitamin D3 supplementation (1000 IU per day). The geometric mean ratios for AUC(0-3h), AUC(0-48h) and C(max) were 1.06 (90% CI 0.92, 1.21) and 1.02 (90% CI 0.97, 1.08) and 1.03 (95% CI 0.86, 1.24), respectively. The median for digoxin T(max) was 0.75 h before and after vitamin D3 ingestion. The mean plasma 25-hydroxyvitamin D3 (25(OH)D3) levels remained constant after the intake of vitamin D3 (15.4 ± 3.7 and 14.4 ± 3.6 ng/mL, respectively), while there was a modest but statistically significant increase in plasma calcium levels, from 9.32-9.68 mg/dL (P = 0.0277). These results suggest that vitamin D3 supplementation (1000 IU per day) in human volunteers does not produce a P-gp-mediated drug interaction with orally administered digoxin.

Modulation of NOXA and MCL-1 as a strategy for sensitizing melanoma cells to the BH3-mimetic ABT-737.

PURPOSE: Drug resistance in melanoma is commonly attributed to ineffective apoptotic pathways. Inhibiting antiapoptotic BCL-2 and its relatives is an attractive strategy for sensitizing lymphoid malignancies to drugs but it has been largely unsuccessful for melanoma and other solid tumors. ABT-737, a small-molecule BH3-mimetic, selectively inhibits BCL-2, BCL-XL, and BCL-w and shows promise for treating leukemia, lymphoma, and small-cell lung cancer. Melanoma cells are insensitive to ABT-737, but MCL-1 inhibition reportedly increases the sensitivity of other tumors to the compound. EXPERIMENTAL DESIGN: The efficacy of MCL-1 and BFL-1 inhibition for sensitizing melanoma cells to ABT-737 was investigated by short hairpin RNA-mediated knockdown or overexpression of their antagonist NOXA in two-dimensional cell culture, a three-dimensional organotypic spheroid model, and an in vivo model. RESULTS: MCL-1 downregulation or NOXA overexpression strongly sensitized melanoma cells to ABT-737 in vitro. NOXA-inducing cytotoxic drugs also strongly sensitized melanomas to ABT-737 but, surprisingly, not vice versa. The drugs most suitable are not necessarily those normally used to treat melanoma. Resistance to ABT-737 occurred quickly in three-dimensional melanoma spheroids through reduced NOXA expression, although experiments with both xenografts and three-dimensional spheroids suggest that penetration of ABT-737 into tumor masses may be the principal limitation, which may be obviated through use of more diffusible BH3-mimetics. CONCLUSION: Sensitization of tumors to BH3-mimetics by cytotoxic drugs that induce NOXA is a therapeutic strategy worth exploring for the treatment of melanoma and other solid cancers.

Response of myeloma to the proteasome inhibitor bortezomib is correlated with the unfolded protein response regulator XBP-1.

BACKGROUND: Multiple myeloma, a malignancy of the antibody-secreting plasma cells, remains incurable by current therapy. However, the proteasome inhibitor bortezomib and other new drugs are revolutionizing its treatment. It remains unclear why myelomas are peculiarly sensitive to bortezomib, or what causes primary or acquired resistance. The 'unfolded protein response' is necessary for folding and assembly of immunoglobulin chains in both normal and malignant plasma cells, as well as for the disposal of incorrectly folded or unpaired chains via the ubiquitin-proteasome pathway. We tested the hypothesis that levels of transcription factor XBP-1, a major regulator of the unfolded protein response, predict response to bortezomib. DESIGN AND METHODS: Expression of XBP-1 and other regulators of the unfolded protein response were measured in myeloma and other cancer cell lines and two cohorts of patients with refractory myeloma and correlated with sensitivity/response to bortezomib. Bortezomib-resistant myeloma cell lines were derived and the effects on expression of unfolded protein response regulators, immunoglobulin secretion, proteasome activity and cross-resistance to cytotoxic drugs and tunicamycin determined. The consequences of manipulation of XBP-1 levels for sensitivity to bortezomib were tested. RESULTS: Low XBP-1 levels predicted poor response to bortezomib, both in vitro and in myeloma patients. Moreover, myeloma cell lines selected for resistance to bortezomib had down-regulated XBP-1 and immunoglobulin secretion. Expression of ATF6, another regulator of the unfolded protein response, also correlated with bortezomib sensitivity. Direct manipulation of XBP-1 levels had only modest effects on sensitivity to bortezomib, suggesting it is a surrogate marker of response to bortezomib rather than a target itself. CONCLUSIONS: The unfolded protein response may be a relevant target pathway for proteasome inhibitors in the treatment of myeloma and its regulator XBP-1 is a potential response marker. (The BIR study was registered with Australian Clinical Trial Registry Number 12605000770662).

Evaluation of cell cycle arrest in estrogen responsive MCF-7 breast cancer cells: pitfalls of the MTS assay.

Endocrine resistance is a major problem with anti-estrogen treatments and how to overcome resistance is a major concern in the clinic. Reliable measurement of cell viability, proliferation, growth inhibition and death is important in screening for drug treatment efficacy in vitro. This report describes and compares commonly used proliferation assays for induced estrogen-responsive MCF-7 breast cancer cell cycle arrest including: determination of cell number by direct counting of viable cells; or fluorescence SYBR®Green (SYBR) DNA labeling; determination of mitochondrial metabolic activity by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay; assessment of newly synthesized DNA using 5-ethynyl-2'-deoxyuridine (EdU) nucleoside analog binding and Alexa Fluor® azide visualization by fluorescence microscopy; cell-cycle phase measurement by flow cytometry. Treatment of MCF-7 cells with ICI 182780 (Faslodex), FTY720, serum deprivation or induction of the tumor suppressor p14ARF showed inhibition of cell proliferation determined by the Trypan Blue exclusion assay and SYBR DNA labeling assay. In contrast, the effects of treatment with ICI 182780 or p14ARF-induction were not confirmed using the MTS assay. Cell cycle inhibition by ICI 182780 and p14ARF-induction was further confirmed by flow cytometric analysis and EdU-DNA incorporation. To explore this discrepancy further, we showed that ICI 182780 and p14ARF-induction increased MCF-7 cell mitochondrial activity by MTS assay in individual cells compared to control cells thereby providing a misleading proliferation readout. Interrogation of p14ARF-induction on MCF-7 metabolic activity using TMRE assays and high content image analysis showed that increased mitochondrial activity was concomitant with increased mitochondrial biomass with no loss of mitochondrial membrane potential, or cell death. We conclude that, whilst p14ARF and ICI 182780 stop cell cycle progression, the cells are still viable and potential treatments utilizing these pathways may contribute to drug resistant cells. These experiments demonstrate how the combined measurement of metabolic activity and DNA labeling provides a more reliable interpretation of cancer cell response to treatment regimens.

The effect of vitamin D3 and ketoconazole combination on VDR-mediated P-gp expression and function in human colon adenocarcinoma cells: implications in drug disposition and resistance.

The vitamin D3 metabolite 1,25-dihydroxycholecalciferol (DHC) and analogues derived from it are being investigated as potential agents for the treatment of cancer. Combining ketoconazole (KTZ) with DHC has been recommended to enhance the anticancer activity of DHC. DHC exerts its biological activities through the vitamin D receptor (VDR). VDR is recognized to be a regulator of P-glycoprotein (P-gp), a member of the ABC transporter family well known for its role in multidrug resistance in cancer chemotherapy. We have investigated the effect of DHC and adding KTZ together with DHC on P-gp and VDR expression and the functional consequences of P-gp induction in intestinal human colonic adenocarcinoma cells LS174T cells. DHC increased P-gp expression by two times, and the addition of KTZ further increased the expression to four times. The combination of DHC + KTZ also significantly increased VDR expression, consistent with the enhanced increase in P-gp expression by this combination. The increase in P-gp expression was accompanied by increased P-gp function, as measured by decreased Rh123 accumulation in the LS174T cells. In addition, DHC significantly decreased colchicine cytotoxicity in a dose-sensitive manner, and the addition of KTZ further decreased the colchicine cytotoxicity, indicating the chemo-protective effect of DHC is enhanced by KTZ, consistent with the enhanced expression of P-gp. The results of this study raise the possibility that DHC and the addition of KTZ to DHC treatment may decrease the effectiveness of cancer chemotherapy by promoting P-gp-mediated drug resistance.

Optimization of the antitumor efficacy of a synthetic mitochondrial toxin by increasing the residence time in the cytosol.

Plasma membrane drug efflux pumps of the multidrug resistance associated protein (MRP) family blunt the effectiveness of anticancer drugs and are often associated with drug resistance. GSAO, a tripeptide trivalent arsenical that targets a key mitochondrial transporter in angiogenic endothelial cells, is an example of a compound whose efficacy is limited by tumor cell expression of MRP isoforms 1 and 2. A cysteine mimetic analogue of GSAO was made, PENAO, which accumulates in cells 85 times faster than GSAO due to increased rate of entry and decreased rate of export via MRP1/2. The faster rate of accumulation of PENAO corresponds to a 44-fold increase in antiproliferative activity in vitro and approximately 20-fold better antitumor efficacy in vivo. This information could be used to improve the efficacy of other small molecule cancer therapeutics.

Changes in the frequency spectrum, the P-P interval, and the bispectral index during ventricular fibrillation are physiologic indicators of ventricular fibrillation duration.

The 3-phase time-sensitive model by Weisfeldt and Becker in 2002 has resulted in a redirection of efforts toward developing treatment algorithms specific to each phase of cardiac arrest. In this study, a number of physiologic indicators of ventricular fibrillation (VF) duration were investigated. The bispectral index was recorded at 15-second intervals over 12 minutes and recordings of the atrial electrocardiogram and lead II electrocardiogram were acquired simultaneously using Notocord data acquisition software during sinus rhythm, ventricular tachycardia, and VF, and analyzed using a total of 30 porcine models. A number of frequency markers (fast Fourier transform and density and amplitude of peaks [DA]) were derived. There was a direct relationship between VF duration and bispectral index with a Pearson correlation coefficient (mean) of r = -0.91. The P-P interval recorded in the atria during VF, demonstrated similar findings (r = 0.97) when measured against VF duration. It was interesting to note that P waves were still apparent during VF despite the on-going chaotic activity in the ventricles. The DA was calculated for each episode of prolonged VF and an exponential relationship with VF duration was observed. The dominant frequency during VF, DA, the P-P interval, and the BIS index are all potential physiologic indicators of VF duration.

Interaction of plant cannabinoids with the multidrug transporter ABCC1 (MRP1).

The ATP-binding cassette (ABC) transporter ABCC1, or multidrug resistance-related protein 1 (MRP1) is implicated in Phase II metabolism and multidrug resistance as it effluxes substrate anticancer drugs. As cannabinoids inhibit two related ABC transporters, P-glycoprotein and ABCG2, here we examined whether they also inhibit ABCC1. Indeed, the cannabinoids enhanced the intracellular accumulation of two ABCC1 substrates, Fluo3 and vincristine, in ovarian carcinoma cells over-expressing ABCC1 (2008/MRP1) with a rank order of potency: cannabidiol>cannabinol>Delta(9)-tetrahydrocannabinol. Cannabinoid inhibition of ABCC1 was confirmed using insect cell membrane MRP1 ATPase assays. These results demonstrate that cannabinoids inhibit ABCC1.

Androgen regulation of multidrug resistance-associated protein 4 (MRP4/ABCC4) in prostate cancer.

BACKGROUND: MRP4/ABCC4 is an ATP-binding cassette transporter expressed in normal prostate. This study aimed to define the pattern of MRP4/ABCC4 expression in normal and malignant prostate tissue and the relationship of MRP4/ABCC4 expression and function in response to androgen signaling. METHODS: Eighty-four radical prostatectomy specimens from patients with localized prostate cancer (PC) (22 neoadjuvant androgen ablation, AA, 62 no AA), 42 non-cancer and 16 advanced PCs were assessed for MRP4/ABCC4 mRNA/protein expression. The effect of DHT and bicalutamide on LNCaP cells was assessed by immunoblotting. HEK293 cells (+/-MRP4/ABCC4) were assessed for the ability to efflux androgens and anti-androgens. RESULTS: MRP4/ABCC4 mRNA/protein levels were higher in localized PC compared to non-cancer (P = 0.006). MRP4/ABCC4 levels were significantly decreased in PCs treated with AA compared to cancers exposed to normal testosterone levels (P < 0.0001). MRP4/ABCC4 expression in normal human tissues was limited to the prostate and the renal tubules. MRP4/ABCC4 protein levels increased in LNCaP cells after DHT which was partially blocked by bicalutamide. However, DHT did not alter the activation of the MRP4/ABCC4 promotor in luciferase reporter assays and testosterone, DHT, flutamide and hydroxy-flutamide were not substrates for MRP4/ABCC4. DISCUSSION: Elevated MRP4/ABCC4 expression is found in malignant compared to benign prostate tissue while lower MRP4/ABCC4 expression is seen after AA. Furthermore, MRP4/ABCC4 is upregulated by androgen and downregulated by anti-androgen treatment in vitro potentially through an indirect mode of action. These data strongly suggest that MRP4/ABCC4 is an androgen-regulated gene important in the progression to PC and may be a potential drug target.

Mechanism of selectivity of an angiogenesis inhibitor from screening a genome-wide set of Saccharomyces cerevisiae deletion strains.

BACKGROUND: The synthetic tripeptide arsenical 4-(N-(S-glutathionylacetyl)amino) phenylarsenoxide (GSAO) is an angiogenesis inhibitor that targets the mitochondria of actively dividing but not quiescent endothelial cells, arresting their proliferation and causing apoptosis. Normal endothelial cells are much more sensitive to GSAO than tumor cells. To elucidate the mechanism of tumor cell resistance, we identified yeast genes that are necessary for resistance to GSAO. METHODS: We screened a genome-wide set of 4546 Saccharomyces cerevisiae deletion strains to identify GSAO-sensitive strains. We then examined GSAO accumulation in and proliferation activity of endothelial cells (BAECs) and tumor cells treated with GSAO and modulators of pathways and proteins identified in the yeast screen. We also examined GSAO effects on proliferation of mammalian cells transfected with transporter protein constructs. RESULTS: Eighty-eight deletion strains were sensitive to GSAO. The most sensitive strains had deletions of genes whose products are involved in vacuolar function (corresponding to drug transport in mammalian cells) and glutathione synthesis. BAECs were more sensitive to GSAO than tumor cells, and cell sensitivity to GSAO was approximately proportional to cellular glutathione levels. Treatment of BAECs and tumor cells with MK-571, an inhibitor of multidrug resistance-associated protein (MRP), or with buthionine sulfoximine, an inhibitor of glutathione synthesis, increased their sensitivity to GSAO. Mammalian cells transfected with MRP1 or MRP2 were resistant to GSAO, whereas cells transfected with MRP3, MRP4, MRP5, P-glypoprotein, or breast cancer resistance protein were not. CONCLUSIONS: Differences in MRP activity and cellular glutathione levels contribute to the selectivity of GSAO for endothelial versus tumor cells. MRP1 and/or MRP2 may transport GSAO from resistant cells, with glutathione acting as a cotransporter. Genetic screening in yeast is a powerful tool for understanding drug action in mammalian cells.

Expression of multidrug transporter MRP4/ABCC4 is a marker of poor prognosis in neuroblastoma and confers resistance to irinotecan in vitro.

Members of the multidrug resistance-associated protein (MRP) family of transporters are believed to contribute to cytotoxic drug resistance and chemotherapy failure. We observed frequent MRP4 overexpression in aggressive primary neuroblastoma, a disease for which we have previously shown MRP1 to be a prognostic indicator. High MRP4 expression correlated with MYCN oncogene amplification and was significantly associated with poor clinical outcome. Although MRP4 is known to transport some nucleoside analogues, it has not previously been associated with resistance to drugs used to treat solid tumors. We now show that it mediates substantial resistance in vitro to the topoisomerase I poison irinotecan/CPT-11 and its active metabolite SN-38. These results suggest that MRP4 will be a useful prognostic marker for neuroblastoma and that clinical trials of irinotecan as a neuroblastoma treatment should monitor MRP4 expression. The same may be true for other tumor types expressing high levels of the transporter.

Glucuronidation as a mechanism of intrinsic drug resistance in colon cancer cells: contribution of drug transport proteins.

We have recently shown that drug conjugation catalysed by UDP-glucuronosyltransferases (UGTs) functions as an intrinsic mechanism of resistance to the topoisomerase I inhibitors 7-ethyl-10-hydroxycamptothecin and NU/ICRF 505 in human colon cancer cells and now report on the role of drug transport in this mechanism. The ability of transport proteins to recognise NU/ICRF 505 as a substrate was evaluated in model systems either transfected with breast cancer-resistance protein 1 (Bcrp1), multidrug-resistance protein 2 (Mrp2) or Mrp3, or overexpressing MRP1 or P-170 glycoprotein. Results from chemosensitivity assays suggested that NU/ICRF 505 was not a substrate for any of the above proteins. In drug accumulation studies in human colon cancer cell lines NU/ICRF 505 was taken up avidly and retained in cells lacking UGTs (HCT116), whereas, following equally rapid uptake, it was cleared rapidly from cells displaying UGT activity (HT29) as glucuronide metabolites. HT29 cells were shown to express MRP1 and 3, but not P-170 glycoprotein, MRP2 or breast cancer-resistance protein. The major glucuronide of NU/ICRF 505 inhibited ATP-dependent transport of estradiol 17-beta-glucuronide in Sf9 insect cell membrane vesicles containing MRP1 or MRP3, while co-incubation of HT29 cells with the MRP antagonist, MK571, significantly restored intracellular concentrations of NU/ICRF 505. These data lead us to conclude that the presence of a glucuronide transporter is essential for glucuronidation to represent a major de novo resistance mechanism and that UGTs will contribute more as a primary resistance mechanism when the parent drug (e.g. NU/ICRF 505) is not itself recognised by transport proteins.

Mouse breast cancer resistance protein (Bcrp1/Abcg2) mediates etoposide resistance and transport, but etoposide oral availability is limited primarily by P-glycoprotein.

The breast cancer resistance protein [BCRP (BCRP/ABCG2)] has not previously been directly identified as a source of resistance to epipodophyllotoxins.However, when P-glycoprotein (P-gp)- and Mrp1-deficient mouse fibroblast and kidney cell lines were selected for resistance to etoposide, amplification and overexpression of Bcrp1 emerged as the dominant resistance mechanism in five of five cases. Resistance was accompanied by reduced intracellular etoposide accumulation. Bcrp1 sequence in all of the resistant lines was wild-type in the region spanning the R482 mutation hot spot known to alter the substrate specificity of mouse Bcrp1 (mouse cognate of BCRP) and human BCRP. Transduced wild-type Bcrp1 cDNA mediated resistance to etoposide and teniposide in fibroblast lines and trans-epithelial etoposide transport in polarized Madin-Darby canine kidney II cells. Bcrp1-mediated etoposide resistance was reversed by two structurally different BCRP/Bcrp1 inhibitors, GF120918 and Ko143. BCRP/Bcrp1 (inhibition) might thus impact on the antitumor activity and pharmacokinetics of epipodophyllotoxins. However, treatment of P-gp-deficient mice with GF120918 did not improve etoposide oral uptake, suggesting that Bcrp1 activity is not a major limiting factor in this process. In contrast, use of GF120918 to inhibit P-gp in wild-type mice increased the plasma levels of etoposide after oral administration 4-5-fold. It may thus be worthwhile to test inhibition of P-gp in humans to improve the oral availability of etoposide.

Function of the ABC transporters, P-glycoprotein, multidrug resistance protein and breast cancer resistance protein, in minimal residual disease in acute myeloid leukemia.

BACKGROUND AND OBJECTIVES: Relapse is common in acute myeloid leukemia (AML) because of persistence of minimal residual disease (MRD). ABC-transporters P-glycoprotein (Pgp) and multidrug resistance protein (MRP), are thought to contribute to treatment failure, while it is unknown whether breast cancer resistance protein (BCRP) does so. However, whether up-regulation of pump activity or selection of subpopulations with higher pump activity occurs during chemotherapy is unclear. The aim of this study was to elucidate whether ABC-transporter function changes during the course of disease. DESIGN AND METHODS: MRD cells were identified using leukemia-associated phenotypes combined with a fluorescent probe assay with substrate/modulator: Syto16/ PSC833 (Pgp), calcein-AM/probenecid (MRP) and BODIPY-prazosin/Ko143 (BCRP); efflux profiles were directly compared with blasts at diagnosis and relapse from the same patient. RESULTS: At diagnosis BCRP activity was undetectable in AML blasts from 23/26 cases, while Pgp activity was present in 36/45 and MRP activity in 26/44 of the cases. Furthermore, no subpopulations of blasts with considerably higher drug efflux capacities were found. Overall, no consistent changes were observed at follow-up [during chemotherapy (n=20), MRD (n=37), relapse (n=26))] in forty-five patients, the mean activities (as percentages of values at diagnosis) were 97% (Pgp), 103% (MRP) and 102% (BCRP). INTERPRETATION AND CONCLUSIONS: Emergence of MRD is thus not accompanied by either upregulation of ABC-transporter function during or after chemotherapy or by selection of pre-existing highly resistant subpopulations. The prognostic value of Pgp and MRP is, therefore, likely related to drug efflux capacity homogeneously distributed in the whole blast population, while BCRP probably has a limited function in drug efflux-related resistance in AML.