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Overexpression of the breast cancer resistance protein (BCRP) efflux pump in human cancer cell lines results in resistance to a variety of cytostatic agents. The aim of this study was to analyze BCRP protein expression and activity in acute myeloid leukemia (AML) samples and to determine whether it is up-regulated due to clonal selection at relapse/refractory disease. BCRP protein expression was measured flow cytometrically with the monoclonal antibodies BXP-34 and BXP-21 in 20 paired samples of de novo and relapsed/refractory AML. BXP-34/immunoglobulin G1 ratios were observed of 1.6 +/- 0.5 (mean +/- SD,range 0.8-2.7) and BXP-21/immunoglobulin G2a ratios of 4.9 +/- 3.0 (range 1.1-14.5) in the patient samples versus 9.8 +/- 6.8 and 6.5 +/- 2.4,respectively,in the MCF-7 cell line. BCRP activity was determined flow cytometrically by measuring mitoxantrone accumulation in absence and presence of the inhibitor fumitremorgin C. Mitoxantrone accumulation,expressed as mean fluorescence intensity (MFI),varied between 44 and 761 MFI (227 +/- 146 MFI) and correlated inversely with BCRP expression (r = -0.58,P textless.001). Addition of fumitremorgin C showed a small increase in mitoxantrone accumulation (11 +/- 29 MFI,n = 40) apart from the effect of PSC833 and MK-571. No consistent up-regulation of BCRP expression or activity was observed at relapse/refractory disease; some cases showed an increase and other cases a decrease at relapse. Relatively high BCRP expression correlated with immature immunophenotype,as determined by expression of the surface marker CD34 (r = 0.54,P =.001). In conclusion,this study shows that BCRP protein is expressed at low but variable levels in AML,especially in immature CD34(+) cells. BCRP was not consistently up-regulated in relapsed/refractory AML. View Publication

The human ATP-binding cassette superfamily G (White) member 2 (ABCG2) gene and its murine homologue breast cancer resistance protein 1 (Bcrp1) are recently described ATP-binding cassette transporters associated with drug resistance in tumor cell lines,including the MCF-7 cell line,selected for its resistance to mitoxantrone (MCF-7/MitoR). Infection of MCF-7 cells with the retroviral vector containing ABCG2 cDNA (G1-ABCG2) resulted in cells (MCF-7/ABCG2) that were resistant to mitoxantrone at levels similar to those observed in MCF-7/MitoR cells. Previous studies have shown that pluripotent hematopoietic stem cells overexpress the multidrug-resistant transport (MDR1) gene and efflux rhodamine,a substrate for the MDR1 transporter. Other studies have identified a primitive hematopoietic stem cell population,or side population (SP) cells,which are identified by their efflux of the fluorescent dye,Hoechst 33342. In an attempt to identify the transport genes responsible for this phenotype,we examined the uptake of Hoechst 33342 into MCF-7,MCF-7/MitoR,and MCF-7 cells infected with a retroviral vector expressing the ABCG2 gene (MCF-7/ABCG2). MCF-7/MitoR cells as well as MCF-7/ABCG2 cells demonstrated lower levels of Hoechst 33342 uptake compared with the parental MCF-7 cells. We also examined the level of the mouse Bcrp1 RNA in SP cells and non-SP cells isolated from mouse hematopoietic cells. Mouse SP cells expressed relatively high levels of Bcrp1 mRNA relative to non-SP cells. These results suggest that Hoechst 33342 is a substrate for the ABCG2 transporter and that ABCG2/Bcrp1 expression may serve as a marker for hematopoietic stem cells in hematopoietic cells. View Publication

Characterization of molecules with tightly controlled expression patterns during differentiation represents an approach to understanding regulation of hematopoietic stem cell commitment. The multidrug resistance-1 (MDR1) gene product,P-glycoprotein,and the breast cancer resistance protein (BCRP) are expressed differentially during hematopoiesis,with the highest levels in primitive bone marrow stem cell populations that are CD34(low) and CD34(-),respectively. Roles for ATP-binding cassette (ABC) transporter superfamily members in conferring drug resistance have been extensively described. However,recent hematopoietic overexpression studies have begun to reveal previously unknown roles for ABC transporter function in normal and malignant hematopoiesis. Expression of MDR1 and BCRP transporters in the myeloid lineage has been reported in blasts from acute myeloid leukemia,but very low to undetectable in normal myelomonocytic cells. Retroviral-mediated dysregulated expression of the MDR1 transporter resulted in increased hematopoietic repopulating activity and myeloproliferative disease in mice. A distinct functional role for the BCRP transporter as a negative regulator of hematopoietic repopulating activity has recently been demonstrated using the same approach. Additionally,the presence of BCRP expression specifically on hematopoietic side-population stem cells and neural stem/progenitors,makes BCRP an attractive candidate marker for isolation of stem cells with the ability to respond to diverse environmental cues. Regulation of stem cell biology by ABC transporters has emerged as an important new field of investigation. In light of these findings,it will be critical to further characterize this family of proteins in hematopoietic lineage-restricted stem cells and in pluripotent stem cells capable of crossing lineage barriers. View Publication

A promising and increasingly exploited property of hematopoietic stem cells is their ability to efflux the fluorescent dye Hoechst 33342. The Hoechst-negative cells are isolated by fluorescence-activated cell sorting as a so-called side population" (SP) of bone marrow. This SP from bone marrow� View Publication

This report describes an international prevalidation study conducted to optimise the Standard Operating Procedure (SOP) for detecting myelosuppressive agents by CFU-GM assay and to study a model for predicting (by means of this in vitro hematopoietic assay) the acute xenobiotic exposure levels that cause maximum tolerated decreases in absolute neutrophil counts (ANC). In the first phase of the study (Protocol Refinement),two SOPs were assessed,by using two cell culture media (Test A,containing GM-CSF; and Test B,containing G-CSF,GM-CSF,IL-3,IL-6 and SCF),and the two tests were applied to cells from both human (bone marrow and umbilical cord blood) and mouse (bone marrow) CFU-GM. In the second phase (Protocol Transfer),the SOPs were transferred to four laboratories to verify the linearity of the assay response and its interlaboratory reproducibility. After a further phase (Protocol Performance),dedicated to a training set of six anticancer drugs (adriamycin,flavopindol,morpholino-doxorubicin,pyrazoloacridine,taxol and topotecan),a model for predicting neutropenia was verified. Results showed that the assay is linear under SOP conditions,and that the in vitro endpoints used by the clinical prediction model of neutropenia are highly reproducible within and between laboratories. Valid tests represented 95% of all tests attempted. The 90% inhibitory concentration values (IC(90)) from Test A and Test B accurately predicted the human maximum tolerated dose (MTD) for five of six and for four of six myelosuppressive anticancer drugs,respectively,that were selected as prototype xenobiotics. As expected,both tests failed to accurately predict the human MTD of a drug that is a likely protoxicant. It is concluded that Test A offers significant cost advantages compared to Test B,without any loss of performance or predictive accuracy. On the basis of these results,we proposed a formal Phase II validation study using the Test A SOP for 16-18 additional xenobiotics that represent the spectrum of haematotoxic potential. View Publication

Use of oncoretroviral vectors in gene therapy for hemoglobinopathies has been impeded by low titer vectors,genetic instability,and poor expression. Fifteen self- inactivating (SIN) lentiviral vectors using 4 erythroid promoters in combination with 4 erythroid enhancers with or without the woodchuck hepatitis virus postregulatory element (WPRE) were generated using the enhanced green fluorescent protein as a reporter gene. Vectors with high erythroid-specific expression in cell lines were tested in primary human CD34(+) cells and in vivo in the murine bone marrow (BM) transplantation model. Vectors containing the ankyrin-1 promoter showed high-level expression and stable proviral transmission. Two vectors containing the ankyrin-1 promoter and 2 erythroid enhancers (HS-40 plus GATA-1 or HS-40 plus 5-aminolevulinate synthase intron 8 [I8] enhancers) and WPRE expressed at levels higher than the HS2/beta-promoter vector in bulk unilineage erythroid cultures and individual erythroid blast-forming units derived from human BM CD34(+) cells. Sca1(+)/lineage(-) Ly5.1 mouse hematopoietic cells,transduced with these 2 ankyrin-1 promoter vectors,were injected into lethally irradiated Ly5.2 recipients. Eleven weeks after transplantation,high-level expression was seen from both vectors in blood (63%-89% of red blood cells) and erythroid cells in BM (70%-86% engraftment),compared with negligible expression in myeloid and lymphoid lineages in blood,BM,spleen,and thymus (0%-4%). The I8/HS-40-containing vector encoding a hybrid human beta/gamma-globin gene led to 43% to 113% human gamma-globin expression/copy of the mouse alpha-globin gene. Thus,modular use of erythroid-specific enhancers/promoters and WPRE in SIN-lentiviral vectors led to identification of high-titer,stably transmitted vectors with high-level erythroid-specific expression for gene therapy of red cell diseases. View Publication

Stem cells from bone marrow,skeletal muscle and possibly other tissues can be identified by the 'side-population' (SP) phenotype. Although it has been assumed that expression of ABC transporters is responsible for this phenotype,the specific molecules involved have not been defined. Here we show that expression of the Bcrp1 (also known as Abcg2 murine/ABCG2 human) gene is a conserved feature of stem cells from a wide variety of sources. Bcrp1 mRNA was expressed at high levels in primitive murine hematopoietic stem cells,and was sharply downregulated with differentiation. Enforced expression of the ABCG2 cDNA directly conferred the SP phenotype to bone-marrow cells and caused a reduction in maturing progeny both in vitro and in transplantation-based assays. These results show that expression of the Bcrp1/ABCG2 gene is an important determinant of the SP phenotype,and that it might serve as a marker for stem cells from various sources. View Publication

BACKGROUND: Circulation of mature fetal blood cells in the maternal blood for a certain postpartum period has been verified,but detailed study of the fetal HPCs has not been reported. The objective of this study was to evaluate the frequency and clearance of these cells in the peripheral blood of puerperal women. STUDY DESIGN AND METHODS: PBMNCs from 15 puerperal women who gave birth to male infants were cultured in semi-solid medium containing hematopoietic stimulating factors. Colonies formed in the medium were individually characterized,collected,and subjected to PCR amplification of the SRY gene on Y chromosome to confirm fetal origin. RESULTS: The mean numbers of fetal progenitor cell colonies isolated per mL of maternal blood were 1.63,2.48,0.56,0.12,and 0 on the day of delivery,at 4 days,1 month,6 months,and 1 year after delivery,respectively. There was no difference in the ratio of fetal versus maternal colonies between erythroid and granulocyte/macrophage lineages. CONCLUSION: The present study demonstrated that a significant number of fetal HPCs circulate in the maternal blood for a duration of at least 6 months after delivery. View Publication

High expression of the Breast Cancer Resistance Protein (BCRP) gene has been shown to be involved in resistance to chemotherapeutic drugs. Knowledge of the localization of BCRP protein in normal tissues may help unravel the normal function of this protein. Therefore,we characterized the tissue distribution and cellular localization of BCRP in frozen sections of normal human tissues. For this purpose,we used the recently described monoclonal antibody BXP-34 and another independently developed monoclonal antibody directed against BCRP,BXP-21. Both monoclonal antibodies show specific BCRP plasma membrane staining on cytospins obtained from topotecan- or mitoxantrone-selected cell lines,as well as from BCRP-transfected cell lines. Immunoprecipitation experiments using either BXP-21 or BXP-34 yielded a clear M(r) 72,000 BCRP band from BCRP-overexpressing tumor cells. In the topotecan-selected T8 and mitoxantrone-selected MX3 tumor cell lines,BCRP turned out to be differentially glycosylated. In contrast to BXP-34,BXP-21 is able to detect the M(r) 72,000 BCRP protein on immunoblots and is reactive with BCRP in formalin-fixed,paraffin-embedded tissues. Using BXP-21 and BXP-34,prominent staining of BCRP was observed in placental syncytiotrophoblasts,in the epithelium of the small intestine and colon,in the liver canalicular membrane,and in ducts and lobules of the breast. Furthermore,BCRP was present in veinous and capillary endothelium,but not in arterial endothelium in all of the tissues investigated. In the tissues studied,the mRNA levels of BCRP were assessed using reverse transcription-PCR,and these corresponded with the levels of BCRP protein estimated from immunohistochemical staining. The presence of BCRP at the placental syncytiotrophoblasts is consistent with the hypothesis of a protective role of BCRP for the fetus. The apical localization in the epithelium of the small intestine and colon indicates a possible role of BCRP in the regulation of the uptake of p.o. administered BCRP substrates by back-transport of substrate drugs entering from the gut lumen. Therefore,it may be useful to attempt to modulate the uptake of p.o. delivered BCRP substrates,e.g.,topotecan or irinotecan,by using a BCRP inhibitor. Clinical trials testing this hypothesis have been initiated in our institute. View Publication

A careful prognostic evaluation of patients referred for high-dose therapy (HDT) is warranted to identify those who maximally benefit from HDT as well as those who clearly fail current HDT and are candidates for more innovative treatments. In a series of 110 patients with myeloma who received HDT as first-line therapy,times to event (disease progression and death) were studied through proportional hazard models,in relation to different prognostic factors,including a chromosome 13 fluorescence in situ hybridization (FISH) analysis using a D13S319 probe. Delta13 was detected in 42 patients (38%). Follow-up time among surviving patients and survival time were 48 +/- 3 and 51 +/- 7 months,respectively (median +/- SE). In the univariate analysis,Delta13 was the most powerful adverse prognostic factor for all times to event,especially for the survival time (P textless.0001) and was followed by beta2-microglobulin (beta2m) levels 2.5 mg/L or higher (P =.0001). The comparison of survival prognostic models including beta2m 2.5 mg/L or greater and another factor favored the Delta13/beta2m combination. In 22 patients (20%) with no unfavorable factor,the median survival time was not reached at 111 months. In contrast,among 55 patients (50%) with one unfavorable factor and 33 patients (30%) with 2 unfavorable factors,median survival times were 47.3 +/- 4.6 months and 25.3 +/- 3.2 months,respectively (P textless.0001). We conclude that delta13,adequately detected by FISH analysis,is a very strong factor related to poor survival,especially when associated with a beta2m level of 2.5 mg/L or higher. Routine FISH Delta13 assessment is strongly recommended for patients considered for HDT. View Publication

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. View Publication

The zinc finger protein GATA-1 functions in a concentration-dependent fashion to activate the transcription of erythroid and megakaryocytic genes. Less is understood,however,regarding factors that regulate the GATA-1 gene. Presently elements within intron 1 are shown to markedly affect its erythroid-restricted transcription. Within a full-length 6. 8-kilobase GATA-1 gene construct (G6.8-Luc) the deletion of a central subdomain of intron 1 inhibited transcription textgreater/=10-fold in transiently transfected erythroid SKT6 cells,and likewise inhibited high-level transcription in erythroid FDCW2ER-GATA1 cells. In parental myeloid FDCER cells,however,low-level transcription was largely unaffected by intron 1 deletions. Within intron 1,repeated GATA and Ap1 consensus elements in a central region are described which when linked directly to reporter cassettes promote transcription in erythroid SKT6 and FDCER-GATA1 cells at high rates. Moreover,GATA-1 activated transcription from this subdomain in 293 cells,and in SKT6 cells this subdomain footprinted in vivo. For stably integrated GFP reporter constructs in erythroid SKT6 cells,corroborating results were obtained. Deletion of intronic GATA and Ap1 motifs abrogated the activity of G6.8-pEGFP; activity was decreased by 43 and 56%,respectively,by the deletion of either motif; and the above 1800-base pair region of intron 1 per se was transcribed at rates uniformly greater than G6.8-pEGFP. Also described is the differential utilization of exons 1a and 1b among primary erythromegakaryocytic and myeloid cells. View Publication