ABCG2 Gene Expression Research Articles

Glucocorticoid Regulation of Placental Breast Cancer Resistance Protein (Bcrp1) in the Mouse

Placental breast cancer resistance protein (Bcrp1; encoded by the Abcg2 gene) limits maternal-fetal transplacental transfer of numerous endogenous and exogenous substrates; however, the regulation of placental Abcg2 and Bcrp1 and is not well understood. Placental Abcg2 messenger RNA (mRNA) levels decrease with advancing gestation in the mouse, and this corresponds to increasing levels of maternal and fetal plasma glucocorticoid. Glucocorticoids, including dexamethasone (DEX), downregulate Bcrp1 expression and function in both breast cancer cell lines and the blood-brain barrier in vitro; whether this occurs in the placenta is not known. The potential regulatory role of synthetic glucocorticoids on placental Bcrp1 is of interest, given that approximately 10% of pregnant women are treated with synthetic glucocorticoid for threatened preterm labor. We hypothesized that (1) exposure of pregnant mice to DEX will downregulate placental Abcg2 mRNA and Bcrp1 protein, and (2) results in increased fetal accumulation of [(3)H]mitoxantrone. Pregnant mice were treated with DEX (low-dose: 0.1 mg/kg or high-dose: 1 mg/kg) or vehicle (saline) from embryonic day (E) E9.5 to E15.5 or E12.5 to E18.5. In placentae derived from female fetuses, high-dose DEX significantly downregulated Abcg2 mRNA expression on E15.5 (P < .05) and significantly inhibited Bcrp1 function (P < .05). Similarly, high-dose DEX significantly inhibited Bcrp1 function in the placentae derived from male fetuses (P < .05). In conclusion, there is a dose-dependent regulatory effect of synthetic glucocorticoid on placental Abcg2 mRNA and Bcrp1 function in vivo. Further, it appears that, at the level of Abcg2 gene expression, the female-derived placentae are more susceptible to the effects of DEX than male placentae.

Abstract 1746: Modulation of ABCG2 mediated multidrug resistance by xanthine derivatives

Abstract ABCG2 is an ATP-binding-cassette (ABC) transporter that confers multidrug resistance (MDR) in tumor cells by extruding a broad variety of chemotherapeutic agents, ultimately leading to failure of cancer therapy. Accordingly, the downregulation of ABCG2 expression and/or function has been proposed as part of a regimen to improve cancer therapeutic efficacy. Adenosine receptors (ARs), belong to the G-protein coupled receptor superfamily, and play important roles in stimulating neuronal cells, regulating cell proliferation and controlling immune responses. Adenosine receptor antagonists, a large collection of small molecules mainly composed of methylxanthines and other xanthine derivatives, have been used or implicated in many therapeutic applications, such as treatment of asthma, chronic bronchitis, emphysema and neural degenerative diseases. Previously, we have shown that the non-selective AR antagonist 1,3,7-methyxanthine (caffeine) can dramatically downregulate ABCG2 expression levels in cancer cell lines, increasing the intracellular retention of the ABCG2 specific substrate Mitoxantrone (MX) and markedly sensitizing cells to this chemotherapeutic agent. In order to find more potent and specific modulators of ABCG2 expression, we recently tested other ARs antagonists. Among the compounds we have examined, the non-selective antagonists theophylline, theobromine, paraxanthine, and the A2R selective antagonists 8-(3-chlorostyryl), caffeine (CSC) and 3,7-dimethyl-1-propargylxanthine (DMPX), were capable of downregulating ABCG2 protein levels to varying degrees. An A1R selective antagonist, 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX), currently in clinical trials, also decreased the expression of ABCG2 significantly at physiologically- relevant concentrations. In addition, the natural ligand of the ARs, adenosine, completely blocked the downregulation of ABCG2 by caffeine, suggesting that adenosine receptors and their downstream signaling pathways are involved in the regulation of ABCG2 gene expression. These findings suggest that xanthine adenosine receptor antagonists could be developed as combination therapy to improve the efficacy of drugs that are ABCG2 substrates. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1746. doi:10.1158/1538-7445.AM2011-1746

Irinotecan Synergistically Enhances the Antiproliferative and Proapoptotic Effects of Axitinib In Vitro and Improves Its Anticancer Activity In Vivo

To demonstrate the synergistic antiproliferative and proapoptotic activity of irinotecan and axitinib in vitro and the improvement of the in vivo effects on angiogenesis and pancreatic cancer. Proliferation and apoptotic assays were performed on human dermal microvascular endothelial cells and pancreas cancer (MIAPaCa-2, Capan-1) cell lines exposed to SN-38, the active metabolite of irinotecan, axitinib, or their simultaneous combination for 72 hours. ERK1/2 and Akt phosphorylation, the vascular endothelial growth factor (VEGF), VEGF receptor-2, and thrombospondin-1 (TSP-1) concentration were measured by ELISAs. ATP7A and ABCG2 gene expression was performed with real-time polymerase chain reaction and SN-38 intracellular concentrations were measured by high-performance liquid chromatography. Capan-1 xenografts in nude mice were treated with irinotecan and axitinib alone or in simultaneous combination. A strong synergistic effect on antiproliferative and proapoptotic activity was found with the axitinib/SN-38 combination on endothelial and cancer cells. ERK1/2 and Akt phosphorylation were significantly inhibited by lower concentrations of the combined drugs in all the cell lines. Axitinib and SN-38 combined treatment greatly inhibited the expression of the ATP7A and ABCG2 genes in endothelial and cancer cells, increasing the SN-38 intracellular concentration. Moreover, TSP-1 secretion was increased in cells treated with both drugs, whereas VEGFR-2 levels significantly decreased. In vivo administration of the simultaneous combination determined an almost complete regression of tumors and tumor neovascularization. In vitro results show the highly synergistic properties of simultaneous combination of irinotecan and axitinib on endothelial and pancreas cancer cells, suggesting a possible translation of this schedule into the clinics.

Correlation BetweenPXRandABCG2Patterns of mRNA Expression in a MCF7 Breast Carcinoma Cell Derivative upon Induction by Proinflammatory Cytokines

In this study, we aimed to evaluate the relationship between PXR and ABCG2 gene expression patterns induced by proinflammatory cytokines in MCF7 and MCF7/MX breast carcinoma cell lines. The effects of proinflammatory cytokines on ABCG2 and PXR mRNA expression were studied using the real-time polymerase chain reaction method. Significant inductions in the ABCG2 and PXR mRNA levels were observed in MCF7 cells, upon treatment with interleukin-1β and tumor necrosis factor-α, whereas MCF7/MX cells did not significantly respond to the treatment. The results also show that in comparison to MCF7 cell line the basal mRNA expression level of PXR was higher in the MCF7/MX cell line. In conclusion, interleukin-1β and tumor necrosis factor-α induced ABCG2 and PXR mRNAs in the MCF7 breast cancer cell line; no significant changes on expression of the same genes in MCF7/MX cells were observed. This differential effect of the cytokines on two different cell lines seems to be influenced by basal levels of the mRNAs, which are very high in MCF7/MX cells. Similar patterns of induction in PXR and ABCG2 genes suggest a probable relationship between these two factors.

A novel xenobiotic responsive element regulated by aryl hydrocarbon receptor is involved in the induction of BCRP/ABCG2 in LS174T cells

Induction of the breast cancer resistance protein (BCRP/ABCG2) expression has been found in various tissues and cell-types after exposure to chemicals including 17β-estradiol, rosiglitazone, imatinib, as well as aryl hydrocarbon receptor (AhR) activators such as 2,3,7,8-tetrachlorodibenzodioxin, 3-methylcholanthrene (3MC), and omeprazole. However, the mechanism(s) underlying AhR-related induction of ABCG2 is largely unknown. Here, we demonstrate the AhR-dependent induction of ABCG2 expression in human colon adenocarcinoma LS174T cells. Importantly, a novel distal AhR-responsive element (AhRE5) located −2357/−2333 bp upstream of the ABCG2 transcriptional start site has been identified and characterized as a functional unit pivotal to 3MC-mediated induction of ABCG2. Cell-based reporter assays revealed that deletion of AhRE5 and 4 dramatically attenuated 3MC-induced activation of ABCG2 reporter activity, while further deletion of the proximal AhRE3 and 2 only moderately changed the luciferase activities. Notably, site-directed mutation of the AhRE5 in the BCRP-3.8 kb reporter construct alone resulted in approximately 80% decrease in 3MC activation of the ABCG2 promoter; additional mutation of the AhRE4 site had negligible effect on the ABCG2 promoter activity. Moreover, chromatin immunoprecipitation assays demonstrated that treatment with 3MC significantly enhanced the recruitment of AhR to the AhRE5 occupied region, and mutation of the AhRE5 site clearly dissociated AhR protein from this promoter region. Together, these data show that the novel distal AhRE5 is critical for AhR-mediated transcriptional activation of ABCG2 gene expression in LS174T cells, and it may offer new strategies for early identification of ABCG2 inducers, which would be of benefit for preventing transporter-associated drug–drug interactions.

Chemotherapeutic Drug-Induced ABCG2 Promoter Demethylation as a Novel Mechanism of Acquired Multidrug Resistance

ABCG2 is an efflux transporter conferring multidrug resistance (MDR) on cancer cells. However, the initial molecular events leading to its up-regulation in MDR tumor cells are poorly understood. Herein, we explored the impact of drug treatment on the methylation status of the ABCG2 promoter and consequent reactivation of ABCG2 gene expression in parental tumor cell lines and their MDR sublines. We demonstrate that ABCG2 promoter methylation is common in T-cell acute lymphoblastic leukemia (T-ALL) lines, also present in primary T-ALL lymphoblast specimens. Furthermore, drug selection with sulfasalazine and topotecan induced a complete demethylation of the ABCG2 promoter in the T-ALL and ovarian carcinoma model cell lines CCRF-CEM and IGROV1, respectively. This resulted in a dramatic induction of ABCG2 messenger RNA levels (235- and 743-fold, respectively) and consequent acquisition of an ABCG2-dependent MDR phenotype. Quantitative genomic polymerase chain reaction and ABCG2 promoter-luciferase reporter assay did not reveal ABCG2 gene amplification or differential transcriptional trans-activation, which could account for ABCG2 up-regulation in these MDR cells. Remarkably, mimicking cytotoxic bolus drug treatment through 12- to 24-hour pulse exposure of ABCG2-silenced leukemia cells, to clinically relevant concentrations of the chemotherapeutic agents daunorubicin and mitoxantrone, resulted in a marked transcriptional up-regulation of ABCG2. Our findings establish that antitumor drug-induced epigenetic reactivation of ABCG2 gene expression in cancer cells is an early molecular event leading to MDR. These findings have important implications for the emergence, clonal selection, and expansion of malignant cells with the MDR phenotype during chemotherapy.

Prognostic Significances of ABCG-2 and hOCT-1 Gene Expression in Chronic Phase Chronic Myeloid Leukemia Patients Treated with Imatinib Mesylate.

Abstract 3299Poster Board III-187 BackgroundImatinib mesylate is a substrate for both drug efflux transporter ATP-binding cassette transporters (ABCG-2; BCRP) and influx transporter human Organic Cation Transporter 1 (hOCT-1; SLCA22). The correlation between the expression of these two transporters of imatinib and the clinical outcome of imatinib in CML patients remain clarified. MethodsABCG-2 and hOCT-1 mRNA levels were determined by performing real-time polymerase chain reaction assays on seventy eight BM samples obtained at initial diagnosis from the patients with chronic phase CML treated with imatinib (treatment started at 400mg/day). ResultsMedian age of the patients was 51 years (16-83 years) and median follow up time from the start of imatinib treatment was 50.8 months (1-95.4 months). Patients with low ABCG-2 mRNA expression (<13.53 a.u.) at diagnosis could achieve higher rates of MCyR by 6 months (74.3% vs. 51.2%, P=0.039), CCyR by 12 months (79.4% vs. 59.0%, P=0.049) and MMR by 18 months (68.8% vs. 26.3%, P=0.000) than those with high ABCG-2 expression (≥13.53 a.u.). Along with these data, patients with high ABCG-2 showed higher rate of suboptimal responses (fail to achieve CHR by 3 months, MCyR by 6 months, CCyR by 12 months and MMR by 18 months) than those with low ABCG-2 expression (69.3% vs. 22.0%, P=0.000). Furthermore, patients with high ABCG-2 also showed higher rate of primary resistance (fail to achieve CHR by 3 months, PCyR by 6 months and MCyR by 12 months) to imatinib than those with low ABCG-2 expression (19% vs. 2.8%, P=0.025). Patients with low ABGC-2 expression are also demonstrated higher rate of MMR achievement during the whole period of treatment (68.3% vs. 46.2%, P=0.043) and the time to MMR achievement was significantly shorter than those with high ABCG-2 expression (10.5 months vs. 21.8 months, P=0.001). However, there was no statistical significance in response and resistance rates to imatinib according to the hOCT-1 expression levels. Although time to achieving MCyR was significantly shorter in patients with high hOCT-1 expression (≥34.53 a.u.) than those with low hOCT-1 expression (<34.53 a.u.)(6.1 months vs. 6.4 months, P=0.029) and they showed a trend of shorter time to achieving CCR or MMR, there was no statistical significance. Fifteen patients were tested their imatinib plasma concentration at 18 months of treatment. There was no significant correlation between imatinib plasma concentration and ABCG-2 or hOCT-1 expression levels. Although patients with primary resistance to imatinib demonstrated lower imatinib plasma concentration than other patients, there was no statistical significance (958 ng/ml vs. 1476.9 ng/ml, P=0.425). ConclusionsThis study demonstrates that pretreatment assay of ABCG-2 gene expression may help us to identify the treatment outcome in the chronic phase CML patients treated with imatinib. DisclosuresNo relevant conflicts of interest to declare.

Resistance to Danusertib (formerly PHA-739358) in BCR-ABL-Positive Cells Is Mediated by Upregulation of the Drug Transporter Abcg2 and Can Be Suppressed in Vitro by Combination Treatment with Imatinib.

Abstract 1724Poster Board I-750The success of imatinib (IM, formerly STI571, Gleevec®) in the treatment of chronic myeloid leukemia (CML) is compromised by the development of primary or acquired IM-resistance, particularly in advanced phase disease as well as by a limited IM-effect on immature hematopoietic stem cells, emphasizing the need for novel therapeutic strategies. The small molecule inhibitor Danusertib (formerly PHA-739358) potently inhibits Aurora and ABL kinases. Here, the individual contribution of each pathway to the effect of Danusertib was investigated. Starting at very low concentration, a dose-dependent reduction of BCR-ABL activity was observed, whereas inhibition of Aurora kinase activity, assessed by phosphorylation of histone H3-Ser10, required substantially higher concentrations. In primary CD34+ CML cells, including initially quiescent leukemic stem cells, combination therapy with IM and Danusertib revealed a synergistic anti-proliferative activity, which also affected immature CD34+38- cells. Neither mono- nor combination therapy led to substantial induction of apoptosis in quiescent stem cells. Interestingly, under treatment with Danusertib, the emergence of resistant clones in a well-established murine Ba/F3-p210 cell model was considerably less frequent than with IM. Surprisingly, Danusertib-resistant cells did not have mutations in BCR-ABL or Aurora kinase domains and remained IM-sensitive. Analysis of resistance mechanisms using DNA-microarray suggests an overexpression of Abcg2 efflux transporter to be causative for the resistance arising under Danusertib treatment. In support of this finding, stable retroviral overexpression of Abcg2 in parental Ba/F3-p210 cells induced a resistant phenotype against Danusertib. Furthermore, the Abcg2 inhibitor Fumitremorgin C (FTC) could restore the sensitivity of resistant cells to Danusertib. Finally, significant re-expression of Abcg2 in parental Ba/F3-p210 cells upon treatment with the demethylating agent 5-Azacytidine suggests that an epigenetic mechanism might play a role in the regulation of Abcg2 gene expression in resistant clones. Detailed analyses of the methylation patterns of the Abcg2 promoter region are currently being performed.In conclusion, simultaneous in vitro exposure of Ba/F3-p210 cells to Danusertib and IM significantly reduced the emergence of drug resistance, raising hope that both epigenetic modulation of drug transporters involved in development of resistance as well as hypothesis-driven combinations of kinase inhibitors may eventually achieve durable disease control even in 2nd and 3rd line treatment of CML. DisclosuresBrummendorf:Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Moll:Nerviano MS: Employment. Jost:MSD: Research Funding. Bokemeyer:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees. Holyoake:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Myers Squib: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Balabanov:Novartis : Research Funding; Bristol Myers Squibb: Research Funding.

Abcb1a and Abcb1b expression in senescence-accelerated mouse (SAM)

It was recently reported that some strains of senescence-accelerated mouse (SAM) including SAMR1 had a spontaneous retroviral insertional mutation in the ATP-binding cassette, sub-family B, member 1A ( Abcb1a) gene, while other strains including SAMP8 had not. The Abcb1 gene product, P-glycoprotein, is a representative efflux transporter of cerebral vessels. In this study, using brain samples of SAMR1, Abcb1a gene-mutant mice, and of SAMP8 without that mutation, we examined the gene expression of some representative ATP-binding cassettes, such as Abcb1a, Abcb1b, Abcc, and Abcg2, and the protein expression of P-glycoprotein by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting, and immunohistochemical techniques. The gene expression of Abcb1a was decreased in the brain samples of SAMR1 compared with those of SAMP8, while that of Abcb1b was increased in the samples of SAMR1 compared with those of SAMP8. There were no differences in the gene expression of Abcc and Abcg2 between the samples of SAMR1 and SAMP8. The protein expression of P-glycoprotein was decreased in the brain samples of SAMR1 compared with those of SAMP8. Immunosignals of P-glycoprotein were seen in vessels walls, mainly CD34-positive endothelial cells and partially astrocytic cells, in both mice. These findings indicate that SAMR1, Abcb1a-mutant mice, showed decreased expression of Abcb1a gene and P-glycoprotein and increased gene expression of Abcb1b, compared with those of SAMP8 without that mutation, suggesting no clear effect of increased gene expression of Abcb1b on decreased expression of P-glycoprotein. The combination of SAMR1 and SAMP8 may be a good tool to investigate which transporter, Abcb1a or Abcb1b, can be used in drug delivery into the brain.

Interaction of Imatinib with Human Organic Ion Carriers

The activity of imatinib in leukemia has recently been linked with expression of the organic cation transporter 1 (OCT1) gene SLC22A1. Here, we characterized the contribution of solute carriers to imatinib transport in an effort to further understand mechanisms involved in the intracellular uptake and retention (IUR) of the drug. IUR of [3H]imatinib was studied in Xenopus laevis oocytes and HEK293 cells expressing OATP1A2, OATP1B1, OATP1B3, OCT1-3, OCTN1-2, or OAT1-3. Gene expression was determined in nine leukemia cell lines using the Affymetrix U133 array. Imatinib was not found to be a substrate for OCT1 in oocytes (P = 0.21), whereas in HEK293 cells IUR was increased by only 1.20-fold relative to control cells (P = 0.002). Furthermore, in 74 cancer patients, the oral clearance of imatinib was not significantly altered in individuals carrying reduced-function variants in SLC22A1 (P = 0.99). Microarray analysis indicated that SLC22A1 was interrelated with gene expression of various transporters, including ABCB1, ABCC4, ABCG2 (negative), and OATP1A2 (positive). Imatinib was confirmed to be a substrate for the three efflux transporters (P < 0.05) as well as for OATP1A2 (P = 0.0001). This study suggests that SLC22A1 expression is a composite surrogate for expression of various transporters relevant to imatinib IUR. This observation provides a mechanistic explanation for previous studies that have linked SLC22A1 with the antitumor activity of imatinib. Because of its high expression in the intestine, ciliary body, gliomas, and leukemia cells, OATP1A2 may play a key role in imatinib pharmacokinetics-pharmacodynamics.

Single-step doxorubicin-selected cancer cells overexpress the ABCG2 drug transporter through epigenetic changes

Understanding the mechanisms of multidrug resistance (MDR) could improve clinical drug efficacy. Multidrug resistance is associated with ATP binding cassette (ABC) transporters, but the factors that regulate their expression at clinically relevant drug concentrations are poorly understood. We report that a single-step selection with low doses of anti-cancer agents, similar to concentrations reported in vivo, induces MDR that is mediated exclusively by ABCG2. We selected breast, ovarian and colon cancer cells (MCF-7, IGROV-1 and S-1) after exposure to 14 or 21 nM doxorubicin for only 10 days. We found that these cells overexpress ABCG2 at the mRNA and protein levels. RNA interference analysis confirmed that ABCG2 confers drug resistance. Furthermore, ABCG2 upregulation was facilitated by histone hyperacetylation due to weaker histone deacetylase 1-promoter association, indicating that these epigenetic changes elicit changes in ABCG2 gene expression. These studies indicate that the MDR phenotype arises following low-dose, single-step exposure to doxorubicin, and further suggest that ABCG2 may mediate early stages of MDR development. This is the first report to our knowledge of single-step, low-dose selection leading to overexpression of ABCG2 by epigenetic changes in multiple cancer cell lines.

ABCG2 Expression and Side Population Abundance Regulated by a Transforming Growth Factor β–Directed Epithelial-Mesenchymal Transition

We describe here the regulation of ABCG2 expression and side population (SP) abundance in MCF7 human breast cancer cells. The level of ABCG2 mRNA and protein were increased in purified MCF7 SP relative to non-SP cells, and incubation with an ABCG2-specific inhibitor or ABCG2 short interfering RNA eliminated the MCF7 SP. The purified MCF7 SP could generate a heterogeneous population containing both SP and non-SP cells in culture. In vivo tumorigenicity experiments showed that the purified MCF7 SP has an increased ability to colonize the mouse mammary gland. Importantly, the MCF7 SP was depleted by a transforming growth factor-beta (TGFbeta)-directed epithelial-mesenchymal transition (EMT), and this effect was associated with a strong down-regulation of ABCG2 gene expression, and an increased sensitivity to mitoxantrone. ABCG2 expression and SP abundance were restored upon the removal of transforming growth factor-beta and reversion of the cells to an epithelial phenotype. Knock-down of E-cadherin also reduced SP abundance, but this effect was not accompanied by the loss of ABCG2 mRNA or protein. We conclude that ABCG2 expression in MCF7 cells is regulated during an EMT, and that the EMT effect reflects posttranslational regulation of ABCG2 function by E-cadherin as well as transcriptional repression of the ABCG2 gene.

Isolation of side population cells from gallbladder carcinoma of human being and the expression of ABCG2 gene

To investigate whether the side population cells (SP cells) exist in human gallbladder carcinoma cell line and the differences of drug resistance gene ABCG2 expression in SP cells, non-SP cells and GBC-SD cell lines. Fluorescence activated cell sorter (FACS) was used to sort the SP and non-SP cells from GBC-SD cell line of gallbladder carcinoma of human being. Then, the sorting cells were cultured and detected the expression of ABCG2 gene among the SP cells, non-SP cells and GBC-SD cell lines by using reverse transcription polymerase chain reaction (RT-PCR), immunofluorescence chemistry, western blot and flow cytometry techniques. A very small fraction cells (0.64 ± 0.08%) were isolated through FACS analysis which had the potency of stem cells and highly expressed ABCG2 gene (89.56 ± 3.86%). On the contrary, there were nearly no expression in non-SP cells (1.32 ± 0.49%) and lower expression in GBC-SD cell line (12.37 ± 1.61%). The side population cells that had the potency of stem cells existed in human gallbladder carcinoma cell line and over-expressed the drug resistance gene ABCG2. They may be play an important role in drug resistance of tumor.

Evaluation of the MDR1, ABCG2, Topoisomerases IIα and GSTπ gene expression in patients affected by aggressive mantle cell lymphoma treated by the R-Hyper-CVAD regimen

The genomic profile of mantle cell lymphoma (MCL) has been reported to be significantly different from that of other indolent lymphoproliferative disorders, Topoisomerase IIα, glutathione-s-transferaseπ (GSTπ) and ABCG2 (BCRP) chemoresistance genes being over-expressed in MCL. In our study, expression levels of the above mentioned genes plus MDR1 were tested on bone marrow samples from 20 patients treated with Rituximab plus hyper-CVAD regimen, in order to evaluate a possible impact of the chemoresistance phenomenon on this promising treatment regimen. All patients expressed ABCG2 and MDR1 genes; 85% of cases expressed GSTπ and topoisomerase IIα. Only ABCG2 were over-expressed in comparison both with marrow from healthy donors and tonsilar CD5+/CD20+ lymphocytes (adopted as normal counterpart of the neoplastic population). The overall response rate of the entire series was 87.5%, with 44% of complete responses. Fifty-seven percent of patients achieved the clearance of minimal residual disease. Levels of tested genes did not condition either quality of clinical response or PFS (76% at 24 months). Nevertheless, an ABCG2 higher expression appeared associated with a worse PFS and levels of this gene paralleled the status of minimal residual disease. A further evaluation of ABCG2 expression in larger series of MCL patients would be suitable.

Clinical and Functional Evidence That Drug Transporter Expression Dictates the Response to Imatinib in Chronic Myeloid Leukaemia.

We have previously shown that imatinib is transported into cells by hOCT1. More recently, we and others have presented data that hOCT1 expression may affect the clinical response to imatinib. Here we extend these observations in two main ways: 1) We have assessed the expression of hOCT1, MDR1 (ABCB1), MRP1 and ABCG2 (BCRP) in a cohort of 70 CML patients, of which 36 achieved complete cytogenetic response (CCR), 4 partial response and 30 no cytogenetic response (NCR). Peripheral blood was taken at baseline and after 4 weeks and 3 months of imatinib treatment. hOCT1, MDR1, MRP1 and ABCG2 gene expression levels were assessed by using real-time quantitative RT-PCR on a LightCycler. Patients were allocated to high-expression and low-expression groups using the median expression as the cut-off level. With a median follow-up of 22 months (from the start of imatinib treatment), patients with high-expression of hOCT1 at baseline had better progression free and overall survival (both p=0.005, log rank test) and a higher probability of achieving CCR (p=0.001, log rank test). MDR1, MRP1 and ABCG2 expression levels at the start of treatment had no correlation with either survival or the achievement of CCR. In addition, amongst responding patients there was a trend for patients with higher MDR1 expression to have a higher incidence of later relapse, typically with the emergence of mutations in the BCR-ABL kinase domain. In 3 of 4 patients who have lost their initial CCR, the MDR1 expression levels were greater than 10% at 3 months and all 4 cases developed BCR-ABL kinase mutation. In contrast, only 4 of 28 patients who had sustained their CCR had greater than 10% of MDR1 expression and none of them had BCR-ABL kinase mutation. 2) In order to further explore the pivotal role of hOCT1 in imatinib uptake into CML cells, we have established stably transfected KCL22 CML cells with high hOCT1 expression. In comparison to untransfected and mock transfected controls, high hOCT1 expressing cells show increased uptake of the fluorescent cation 4-(4-(dimethyl-amino)styryl)-N-methylpyridinium iodide (ASP + ), 14C-TEA and 14C-radiolabelled imatinib (p=0.0002). This enhanced uptake was blocked by the hOCT1 inhibitors amantidine (p=0.0003) and verapamil (p=0.0001). Taken together, the data demonstrate that the baseline expression of hOCT1 (but not MRP1 or ABCG2) is important in dictating the clinical response to imatinib, by means of increased cellular imatinib delivery. In addition, patients with high MDR1 expression may have a higher probability of loss of imatinib response and of kinase domain mutations, and may benefit from closer surveillance of BCR-ABL transcripts and mutations.