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Background: The expression of side-population (SP) cells and their relation to tumour-initiating cells (T-ICs) have been insufficiently studied in breast cancer (BC). We therefore evaluated primary cell cultures derived from patients and a panel of human BC cell lines with luminal- or basal-molecular signatures for the presence of SP and BC stem cell markers., Methods: The SPs from luminal-type BC were analysed for BC T-IC characteristics, including human epidermal growth factor receptor 2 (HER2), ERalpha, IGFBP7 expression and their ability to initiate tumours in non-obese diabetic severe combined immunodeficiency (NOD/SCID) mice. Pharmacological modulators were used to assess the effects of HER2 signalling and breast cancer-resistance protein (BCRP) expression on SPs., Results: The SP was more prevalent in the luminal subtype of BC compared with the basal subtype. HER2 expression was significantly correlated with the occurrence of an SP (r(2)=0.75, P=0.0003). Disappearance of SP in the presence of Ko143, a specific inhibitor of the ATP-binding cassette transporter BCRP, suggests that BCRP is the predominant transporter expressed in this population. The SP also decreased in the presence of HER2 signalling inhibitors AG825 or trastuzumab, strengthening the notion that HER2 contributed to the SP phenotype, likely through downstream AKT signalling. The SP cells from luminal-type MCF-7 cells with enforced expression of HER2, and primary cells with luminal-like properties from a BC patient, displayed enrichment in cells capable of repopulating tumours in NOD/SCID mice. Engraftment of SP cells was inhibited by pretreatment with AG825 or by in vivo treatment with trastuzumab., Interpretation: Our findings indicate an important role of HER2 in regulating SP and hence T-ICs in BC, which may account for the poor responsiveness of HER2-positive BCs to chemotherapy, as well as their aggressiveness.

Multidrug resistance (MDR) remains a major obstacle in the treatment of human cancers. The recently discovered breast cancer resistance protein (BCRP/ABCG2) has been found to be an important mediator of chemotherapeutic MDR. Fumitremorgin C (FTC) is a selective and potent inhibitor of BCRP that completely inhibits and reverses BCRP-mediated resistance at micromolar concentrations. We report a study of the pharmacokinetics and tissue distribution of FTC when administered intravenously (IV) at a dose of 25 mg/kg to female SCID mice bearing the BCRP-overexpressing human ovarian xenograft Igrov1/T8 tumors. Plasma pharmacokinetics and tissue distribution of FTC in various organs and tissues were studied. In addition, the effect of FTC administration on the expression of BCRP in T8 tumors was also assessed by RT-PCR. Administration of a single FTC IV dose did not appear to cause any major toxicities. The resulting pharmacokinetic data were fit to a two-compartment model using NONMEM and the FTC clearance was determined to be 0.55 ml/min (25.0 ml/min/kg) with a 56% inter-animal variability. Area under the plasma concentration time curve was determined by Bailer's method and was calculated to be 1128+/-111 microg min/ml. FTC was widely distributed in all tissues assayed with highest concentrations found in lungs, liver and kidney in decreasing order, respectively. FTC did not appear to have any effect on the expression of BCRP in T8 tumors. Less than 2% of the administered dose was recovered in the urine and feces after 24 h, suggesting hepatic metabolism as a primary mechanism of elimination. The current study can be used as a basis for future animal or in vivo studies with FTC designed to further understand the impact of BCRP on drug resistance.

Data on breast cancer resistance protein (BCRP, MXR, ABCG2) expression in acute myeloid leukemia (AML) have been inconsistent, possibly due to use of different assays in different studies. BCRP mRNA was studied by the reverse-transcription polymerase chain reaction and BCRP protein expression (BXP-21, BXP-34 or anti-ABCG2 antibody, with anti-CD34 and anti-CD33) and function (fumitremorgin C modulation of mitoxantrone retention) by flow cytometry in eight cell lines and in pretreatment blasts from 31 AML patients. BCRP mRNA levels, antibody staining and function correlated strongly in cell lines (Pearson r values, 0.73-0.97), but not in AML samples. AML sample BCRP mRNA levels were between those in parental 8226 and 35-fold mitoxantrone-resistant 8226/MR20 cells in all but one case, and BCRP mRNA had the wild-type sequence at codon 482 in all. In AML, unlike in cell lines, BCRP protein expression or function, when present, was only detected in small subpopulations. BCRP mRNA and protein expression did not correlate, nor did staining with different BCRP antibodies, and function did not correlate with mRNA nor protein expression. Presence of BCRP only in subpopulations and discordance among BCRP measurements suggest complex biology of BCRP in AML and incomplete modeling by cell lines.

The breast cancer resistance protein (BCRP) gene, formally known as ATP-binding cassette transporter G2 (ABCG2) gene, encodes an ABC half transporter that causes resistance to certain cancer chemotherapeutic drugs when transfected and expressed in drug sensitive cancer cells. Here we report the organization of the BCRP gene, and the initial characterization of the BCRP promoter. We identified the genomic sequence of BCRP and its promoter by screening a human genomic lambda phage library, as well as a BAC library, and by searching the human genome database. The BCRP gene spans over 66 kb and consists of 16 exons and 15 introns. The exons range in size from 60 to 532 bp. The translational start site is found in the second exon. The first exon contains the majority of the 5' UTR. Promoter activity was characterized by a luciferase reporter assay using transient transfection of the human breast cancer cell line MCF7, and the human choriocarcinoma cell lines JAR, BeWo and JEG-3, which we find to have high endogenous expression of BCRP. The BCRP gene is transcribed by a TATA-less promoter with several putative Sp1 sites, which are downstream from a putative CpG island. The sequence 312 bp directly upstream from the BCRP transcriptional start site conferred basal promoter activity. The 5' region upstream of the basal promoter is characterized by both positive and negative regulatory domains.

Breast cancer resistance protein (BCRP), an ABC half-transporter, is overexpressed in cancer cell lines selected with doxorubicin/verapamil, topotecan, or mitoxantrone. BCRP-overexpressing cells show cross-resistance to camptothecin derivatives such as irinotecan, SN-38 (the active metabolite of irinotecan), and topotecan. To test whether BCRP confers SN-38 resistance, we selected two SN-38 resistant sublines from PC-6 human small-cell lung cancer cells by SN-38, and then characterized these cells. Compared to PC-6 cells, the resistant sublines PC-6/SN2-5 and PC-6/SN2-5H were approximately 18- and 34-fold resistant, respectively. The intracellular SN-38 accumulation was reduced in the sublines, and BCRP mRNA was overexpressed in proportion to the degree of SN-38 resistance. These findings suggest that BCRP confers SN-38 resistance in the sublines. To confirm this hypothesis, PC-6/SN2-5 cells were transfected with antisense oligonucleotides complementary to portions of BCRP mRNA. The antisense oligonucleotides significantly suppressed BCRP mRNA expression, and enhanced SN-38 sensitivity in the subline. These data indicate that BCRP is directly involved with SN-38 resistance, by efflux transport of SN-38.

Because the activities of HER family members are elevated and/or aberrant in a variety of human neoplasms, these cell surface receptors are receiving increasing attention as potential therapeutic targets. In the present study, we examined the effect of combining the HER family tyrosine kinase inhibitor CI1033 (PD 183805) with the topoisomerase (topo) I poison 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of irinotecan, in a number of different cell lines. Colony-forming assays revealed that the antiproliferative effects of simultaneous treatment with CI1033 and SN-38 were synergistic in T98G glioblastoma cells and HCT8 colorectal carcinoma cells, whereas sequential treatments were additive at best. In additional studies examining the mechanistic basis for these findings in T98G cells, immunoblotting revealed that the inhibitory effects of CI1033 on epidermal growth factor receptor autophosphorylation were unaffected by SN-38. Likewise, CI1033 had no effect on topo I polypeptide levels, localization, or activity. Nonetheless, CI1033 markedly enhanced the number of covalent topo I-DNA complexes stabilized by SN-38 or the related agent topotecan (TPT). Analysis of intracellular SN-38 levels by high-performance liquid chromatography and intracellular TPT levels by flow microfluorometry revealed that CI1033 increased the steady-state accumulation of SN-38 and TPT by 9.4 +/- 1.9- and 1.8 +/- 0.2-fold, respectively. Further evaluation revealed that the initial rate of TPT uptake was unaffected by CI1033, whereas the rate of efflux was markedly diminished. Additional studies demonstrated that T98G and HCT8 cells express the breast cancer resistance protein (BCRP), a recently cloned ATP binding cassette transporter. Moreover, CI1033 enhanced the uptake and cytotoxicity of SN-38 and TPT in cells transfected with BCRP but not empty vector. Conversely, CI1033 accumulation was diminished in cells expressing BCRP, suggesting that CI1033 is a substrate for this efflux pump. These results indicate that CI1033 can modulate the accumulation and subsequent cytotoxicity of two widely used topo I poisons in cells that have no history of previous exposure to these agents.

We sought to characterize the interactions of flavopiridol with members of the ATP-binding cassette (ABC) transporter family. Cells overexpressing multidrug resistance-1 (MDR-1) and multidrug resistance-associated protein (MRP) did not exhibit appreciable flavopiridol resistance, whereas cell lines overexpressing the ABC half-transporter, ABCG2 (MXR/BCRP/ABCP1), were found to be resistant to flavopiridol. Flavopiridol at a concentration of 10 microM was able to prevent MRP-mediated calcein efflux, whereas Pgp-mediated transport of rhodamine 123 was unaffected at flavopiridol concentrations of up to 100 microM. To determine putative mechanisms of resistance to flavopiridol, we exposed the human breast cancer cell line MCF-7 to incrementally increasing concentrations of flavopiridol. The resulting resistant subline, MCF-7 FLV1000, is maintained in 1,000 nM flavopiridol and was found to be 24-fold resistant to flavopiridol, as well as highly cross-resistant to mitoxantrone (675-fold), topotecan (423-fold), and SN-38 (950-fold), the active metabolite of irinotecan. Because this cross-resistance pattern is consistent with that reported for ABCG2-overexpressing cells, cytotoxicity studies were repeated in the presence of 5 microM of the ABCG2 inhibitor fumitremorgin C (FTC), and sensitivity of MCF-7 FLV1000 cells to flavopiridol, mitoxantrone, SN-38, and topotecan was restored. Mitoxantrone efflux studies were performed, and high levels of FTC-reversible mitoxantrone efflux were found. Northern blot and PCR analysis revealed overexpression of the ABCG2 gene. Western blot confirmed overexpression of ABCG2; neither P-glycoprotein nor MRP overexpression was detected. These results suggest that ABCG2 plays a role in resistance to flavopiridol.

Cellular resistance to the antifolate methotrexate (MTX) is often caused by target amplification, uptake defects, or alterations in polyglutamylation. Here we have examined MTX cross-resistance in a human breast carcinoma cell line (MCF7/MX) selected in the presence of mitoxantrone, an anticancer agent associated with the multidrug resistance (MDR) phenotype. Examination of protein expression and enzyme activities showed that MCF7/MX cells displayed none of the classical mechanisms of MTX resistance. They did, however, exhibit an ATP-sensitive accumulation defect accompanied by reduced polyglutamylation. Although the kinetics of drug uptake was similar between parental and resistant cells, the resistant cells exhibited increased energy-dependent drug efflux. This suggested the involvement of an ATP-binding cassette (ABC) transporter. However, cells transfected with the breast cancer resistance protein (BCRP)-the ABC transporter known to be highly overexpressed in MCF7/MX cells and to confer mitoxantrone resistance (D. D. Ross et al., J. Natl. Cancer Inst. 91: 429-433, 1999)-were not MTX resistant, which suggested that this transporter is not involved in MTX cross-resistance. Moreover, members of the MRP protein family of transport proteins, which had previously been implicated in MTX resistance, were not found to be overexpressed in the MCF7/MX cells. Thus, our data suggest that a novel MTX-specific efflux pump may be involved in this unusual cross-resistance phenotype.

Breast cancer resistance protein (BCRP) is a novel member of the adenosine triphosphate-binding cassette superfamily of transport proteins. Transfection and enforced expression of BCRP in drug-sensitive cells confer resistance to mitoxantrone, doxorubicin, daunorubicin, and topotecan. We studied blast cells from 21 acute leukemia patients (20 acute myeloid leukemia, 1 acute lymphocytic leukemia) for the expression of BCRP mRNA using a quantitative reverse-transcription polymerase chain reaction assay. BCRP mRNA expression varied more than 1000-fold among the samples tested, with low or barely detectable expression in half of the samples. Seven samples (33%) had relatively high expression of BCRP mRNA. High expression of BCRP did not correlate strongly with high expression of P-glycoprotein, suggesting that BCRP may cause resistance to certain antileukemic drugs in P-glycoprotein-negative cases. High expression of BCRP mRNA is sufficiently frequent in AML to warrant more extensive investigations to determine the relation of disease subtype and treatment outcome to BCRP expression and function.

Mechanisms of drug resistance other than P-glycoprotein are of increasing interest as the list of newly identified members of the ABC transport family has grown. We sought to characterize the phenotype of the newly discovered ABC transporter encoded by the mitoxantrone resistance gene, MXR, also known as ABCP1 or BCRP. The pharmacodynamics of mitoxantrone and 12 other fluorescent drugs were evaluated by confocal microscopy in four multidrug-resistant human colon (S1) and breast (MCF-7) cancer cell lines. We utilized two sublines, MCF-7 AdVp3000 and S1-M1-80, and detected overexpression of MXR by PCR, immunoblot assay and immunohistochemistry. These MXR overexpressing sublines were compared to cell lines with P-glycoprotein- and MRP-mediated resistance. High levels of cross-resistance were observed for mitoxantrone, the anthracyclines, bisantrene and topotecan. Reduced levels of mitoxantrone, daunorubicin, bisantrene, topotecan, rhodamine 123 and prazosin were observed in the two sublines with high MXR expression. Neither the P-glycoprotein substrates vinblastine, paclitaxel, verapamil and calcein-AM, nor the MRP substrate calcein, were extruded from MCF-7 AdVp3000 and S1-M1-80 cells. Thus, the multidrug-resistant phenotype due to MXR expression is overlapping with, but distinct from, that due to P-glycoprotein. Further, cells that overexpress the MXR protein seem to be more resistant to mitoxantrone and topotecan than cells with P-glycoprotein-mediated multidrug resistance. Our studies suggest that the ABC half-transporter, MXR, is a potent, new mechanism for conferring multiple drug resistance. Definition of its mechanism of transport and its role in clinical oncology is required.

Tumor cells may display a multidrug resistant phenotype by overexpression of ATP-binding cassette transporters such as multidrug resistance (MDRI) P-glycoprotein, multidrug resistance protein 1 (MRP1), and breast cancer resistance protein (BCRP). The presence of BCRP has thus far been reported solely using mRNA data. In this study, we describe a BCRP-specific monoclonal antibody, BXP-34, obtained from mice, immunized with mitoxantrone-resistant, BCRP mRNA-positive MCF-7 MR human breast cancer cells. BCRP was detected in BCRP-transfected cells and in several mitoxantrone- and topotecan-selected tumor cell sublines. Pronounced staining of the cell membranes showed that the transporter is mainly present at the plasma membrane. In a panel of human tumors, including primary tumors as well as drug-treated breast cancer and acute myeloid leukemia samples, BCRP was low or undetectable. Extended studies will be required to analyze the possible contribution of BCRP to clinical multidrug resistance.

Fumitremorgin C (FTC) is a potent and specific chemosensitizing agent in cell lines selected for resistance to mitoxantrone that do not overexpress P-glycoprotein or multidrug resistance protein. The gene encoding a novel transporter, the breast cancer resistance protein (BCRP), was recently found to be overexpressed in a mitoxantrone-selected human colon cell line, S1-M1-3.2, which was used to identify FTC. Because the drug-selected cell line may contain multiple alterations contributing to the multidrug resistance phenotype, we examined the effect of FTC on MCF-7 cells transfected with the BCRP gene. We report that FTC almost completely reverses resistance mediated by BCRP in vitro and is a pharmacological probe for the expression and molecular action of this transporter.

Clinical studies are currently in progress to evaluate functional modifiers of P-glycoprotein (Pgp), an efflux pump associated with resistance to cancer chemotherapy. However, the effects of these modifiers on a more recently discovered efflux pump, the multidrug resistance associated protein (MRP), have not yet been fully characterized. MRP is expressed in most human tissues and is overexpressed in several tumor types. For these reasons, we have investigated the effects of three prototype Pgp modifiers, which act by different modes on the function of Pgp, on the function of MRP in two MRP-overexpressing cell lines: UMCC/VP lung and MCF-7/VP breast cancer cells. Clinically optimal plasma levels of verapamil, cremophor, and PSC833 have been shown to completely block the function of Pgp in Pgp-over expressing cells. However, in the two MRP-over expressing cell lines, these modifiers only partially blocked the function of MRP and combinations of these optimal concentrations acted antagonistically. Similar antagonistic effects were seen with combinations of suboptimal concentration levels of these blockers, while these combinations resulted in synergistic effects in Pgp overexpressing cells. For two biophysical parameters measured at the plasma membrane, membrane fluidity and membrane potential, the effects of these modifiers were essentially similar in Pgp and MRP expressing cells. We suggest that the 170 kD Pgp and the 190 kD MRP glycoproteins, imbedded in the plasma membranes, respond differently to simultaneous effects of the investigated prototype resistance modifiers. These results also suggest that the identification of the specific mechanism of drug resistance is important for the selection of chemotherapeutic strategies to block the efflux pump on the cancer cell.

MCF-7/AdrVp is a multidrug-resistant human breast cancer subline that displays an ATP-dependent reduction in the intracellular accumulation of anthracycline anticancer drugs in the absence of overexpression of known multidrug resistance transporters such as P glycoprotein or the multidrug resistance protein. RNA fingerprinting led to the identification of a 2.4-kb mRNA that is overexpressed in MCF-7/AdrVp cells relative to parental MCF-7 cells. The mRNA encodes a 655-aa [corrected] member of the ATP-binding cassette superfamily of transporters that we term breast cancer resistance protein (BCRP). Enforced expression of the full-length BCRP cDNA in MCF-7 breast cancer cells confers resistance to mitoxantrone, doxorubicin, and daunorubicin, reduces daunorubicin accumulation and retention, and causes an ATP-dependent enhancement of the efflux of rhodamine 123 in the cloned transfected cells. BCRP is a xenobiotic transporter that appears to play a major role in the multidrug resistance phenotype of MCF-7/AdrVp human breast cancer cells.

This review paper will focus on the molecular biological, biochemical and biophysical aspects of the following three efflux pumps: P-glycoprotein (Pgp), Multidrug Resistance Protein (MRP) and Lung cancer Resistance-related Protein. Since suppression of the function of these pumps has clinical implications, novel approaches developed in our laboratory for blocking the function of Pgp and MRP are also discussed. The second part of this review will summarize the clinical significance and the results of clinical trials designed to suppress the effects of these efflux pumps.

A specific and quantitative reverse-transcription polymerase chain reaction (RT-PCR) assay was developed for measuring the mRNA of the multidrug resistance-associated protein (MRP). A region corresponding to bp 3897-4471 of MRP cDNA is amplified, which encompasses approximately half of the second nucleotide-binding domain (NBD2). In two multidrug resistant (MDR) sublines of the HL-60 human acute myeloid leukemia (AML) cell line which overexpress MRP but not P-glycoprotein, the assay detects elevated levels of MRP mRNA (4- to 8-fold) relative to the drug-sensitive parental cells (designated HL-60/W). Blast cells from 24 patients with AML were also studied for MRP expression using this RT-PCR method. Expression of MRP was normalized for that of beta-actin in the blast cells, which was also determined by RT-PCR. All of these blast cell samples had MRP expression that was detectable after 35 PCR cycles. Eighteen of these patients samples had levels of expression of MRP mRNA equal to or less than that expressed by HL-60/W cells. In six patient blast cell specimens, the expression of MRP mRNA was up to 1.7-fold higher than that of HL-60/W cells. In 21 specimens, the steady-state intracellular accumulation of daunorubicin (1 microgram/ml, 3h) was also determined. The blast cells with MRP mRNA expression higher than HL-60/W had a lower median accumulation of daunorubicin compared to those whose MRP expression was less than HL-60/W, suggesting a functional defect in drug transport in the cells with higher MRP expression; a similar trend toward lower daunorubicin accumulation was also noted in the one-third of samples that displayed the highest expression of MDR1 mRNA (also determined by RT-PCR). These studies illustrate the range of expression of MRP in AML blast cell specimens. The identification of MRP overexpression in MDR AML cell lines and in some AML patient blast cells with low intracellular daunorubicin accumulation warrants further study of MRP as a component of clinical drug resistance in AML.

We have characterised an etoposide-resistant subline of the small-cell lung cancer cell line, UMCC-1, derived at our centre. Subline UMCC-1/VP was developed by culturing the parent line in increasing concentrations of etoposide over 16 months. UMCC-1/VP is 20-fold resistant to etoposide by MTT assays, relative to the parent line, and is cross-resistant to doxorubicin, vincristine and actinomycin D, but not to taxol, cisplatin, melphalan, thiotepa or idarubicin. Topoisomerase II immunoblotting demonstrates a 50% reduction of the protein in the resistant subline. The UMCC-1/VP subline demonstrates a marked decrease in the accumulation of [3H]etoposide relative to the parent line, as well as a modest reduction in the accumulation of daunorubicin. Reverse transcription-polymerase chain reaction assays demonstrate no detectable mdr1 expression but marked expression of the multidrug resistance-associated protein (MRP) gene in the resistant subline. Northern blotting with an MRP cDNA probe confirms marked overexpression of the MRP gene only in the UMCC-1/VP subline. Western blotting with antisera against MRP peptide confirms a 195 kDa protein band in the UMCC-1/VP subline. Southern blotting experiments demonstrate a 10-fold amplification of the MRP gene in the resistant subline. Depletion of glutathione with buthionine sulphoximine sensitised UMCC-1/VP cells to daunorubicin and etoposide. Our studies indicate that MRP gene expression may be induced by etoposide and may lead to reduced accumulation of the drug.