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Etoposide is a substrate for P-glycoprotein, CYP3A4, CYP3A5, and UGT1A1. Glucocorticoids modulate CYP3A and P-glycoprotein in preclinical models, but their effect on clinical etoposide disposition is unknown. We studied the pharmacokinetics of etoposide and its catechol metabolite in children with acute lymphoblastic leukemia, along with polymorphisms in CYP3A4, CYP3A5, MDR1, GSTP1, UGT1A1, and VDR. Plasma pharmacokinetics were assessed at day 29, after 1 month of prednisone (n = 102), and at week 54, without prednisone (n = 44). On day 29, etoposide clearance was higher (47.4 versus 29.2 mL/min/m2, P < .0001) than at week 54. The day 29 etoposide or catechol area under the curve (AUC) was correlated with neutropenia (P = .027 and P = .0008, respectively). The relationship between genotype and etoposide disposition differed by race and by prednisone use. The MDR1 exon 26 CC genotype predicted higher day 29 etoposide clearance (P = .002) for all patients, and the CYP3A5 AA and GSTP1 AA genotypes predicted lower clearance in blacks (P = .02 and .03, respectively). The UGT1A1 6/6, VDR intron 8 GG, and VDR Fok 1 CC genotypes predicted higher week 54 clearance in blacks (P = .039, .036, and .052, respectively). The UGT1A1 6/6 genotype predicted lower catechol AUC. Prednisone strongly induces etoposide clearance, genetic polymorphisms may predict the constitutive and induced clearance of etoposide, and the relationship between genotype and phenotype differs by race.

Inappropriate expression of the multidrug resistance(MDR1) gene encoding the P-glycoprotein (Pgp) has been frequently implicated in resistance to different chemotherapeutic drugs. We have previously generated chronic myeloid leukemia (CML) cell lines resistant to the tyrosine kinase inhibitor imatinib mesylate (STI571), and one line (LAMA84-r) showed overexpression not only of the Bcr-Abl protein but also of Pgp. In the present study, we investigated this phenomenon in other cell lines overexpressing exclusively Pgp. Thus, cells from the K562/DOX line, described as resistant to doxorubicin due to MDR1 gene overexpression, grew continuously in the presence of 1 μM imatinib, but died in 4 to 5 days if the Pgp pump modulators verapamil or PSC833 were added to the imatinib-treated culture. Analysis of cell proliferation by the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay confirmed the differential sensitivity of K562/DOX to imatinib, which was also reversed by verapamil or PSC833. Flow cytometric analysis of the total phosphotyrosine content by intracytoplasmic staining after a 2-hour incubation with escalating doses of imatinib showed that the inhibitory concentrations of 50% (IC50) for inhibition of cellular protein tyrosine phosphorylation were 15, 10, and 5 μM for K562/DOX, K562/DOX plus verapamil, and K562, respectively. Retroviral-mediated transfection of theBCR-ABL+ AR230 cell line with theMDR1 gene decreased its sensitivity to imatinib, an effect that was also reversed by verapamil. The possible role of MDR overexpression in clinical resistance to imatinib remains to be defined. We therefore confirm that imatinib should be added to the extensive list of drugs that can be affected by the MDR phenomenon.

The novel prodrug of butyric acid, pivaloyloxymethyl butyrate (AN-9), a histone deacetylase inhibitor, shows great promise as an effective and relatively nontoxic anticancer agent for solid malignancies. However, little is known about its effects on hematopoietic malignancies. In this study, we show that 21 primary samples of acute leukemia were sensitive to the antiproliferative effects of AN-9, with a 50% inhibitory concentration (IC50) of 45.8 ± 4.1 μM. In colony-forming assays, primary T-cell acute lymphoblastic leukemia (T-ALL) cells were 3-fold more sensitive to AN-9 than the normal hematopoietic progenitors, erythroid burst-forming units and granulocyte/monocyte colony-forming units. AN-9 induced apoptosis in the T-ALL cell line CEM. A common problem with cancer is chemoresistance, which is often typical of relapsed cancers. Remarkably, a T-ALL sample at diagnosis and an acute myeloid leukemia sample at relapse that were resistant to doxorubicin in vitro were sensitive to AN-9, with an IC50 of 50 μM for both samples. More strikingly, samples from 2 infants with t(4;11) ALL obtained at diagnosis and relapse each were the most sensitive to AN-9, with IC50values of 25 μM and 17 μM, respectively. Furthermore, a doxorubicin-resistant clone of HL60, HL60/ADR, obtained by the transfection of the MDR-1 gene, was equally sensitive to AN-9 cytotoxicity as the parental cells. AN-9 induced the expression of p21 in an infant leukemia sample with 11q23 rearrangement, but not in T- or B-precursor ALL. Collectively, our results suggest that AN-9 is a selective agent for hematopoietic malignancies that can circumvent the mechanisms of chemoresistance limiting most conventional chemotherapy.

Transduction of murine stem cells with a multidrug-resistance 1 gene (MDR1) retrovirus results in dramatic ex vivo and in vivo expansion of repopulating cells accompanied by a myeloproliferative disorder. Given the use ofMDR1-containing vectors in human trials, investigations have been extended to nonhuman primates. Peripheral blood stem cells from 2 rhesus monkeys were collected, CD34-enriched, split into 2 portions, and transduced with eitherMDR1 vectors or neo vectors and continued in culture for a total of 10 days before reinfusion. At engraftment, the copy number in granulocytes was extremely high from bothMDR vectors and neo vectors, but the copy number fell to 0.01 to 0.05 for both. There were no perturbations of the leukocyte count or differential noted. After 3 cycles of stem cell factor/granulocyte colony-stimulating factor, there were no changes in the levels of MDR1 vector– or neovector–containing cells. There was no evidence for expansion ofMDR1 vector–transduced cells. Long-term engraftment with MDR1 vector– and neo vector–transduced cells occurred despite prolonged culture.

To understand the molecular basis for multidrug-resistant (MDR) cancer cells in vivo, this study analyzed molecular changes of the mdr1a gene region in leukemia cells in mice during continuous treatment with vincristine. An inverse insertion of murine leukemia retrovirus (MuLV) into the 5′-flanking region of the mdr1a gene was found. This insertion was concomitantly accompanied by up-regulation of themdr1a gene and the loss of chemosensitivity. Deletion of long-terminal repeat (LTR) sequences dramatically decreased themdr1a promoter-driven reporter activity. The MuLV LTR insertion appears to exert its enhancer activity onmdr1a transcription during the appearance of MDR leukemia cells. Two mechanisms were postulated to explain the mdr1agene activation by retrovirus insertion during in vivo chemotreatment: de novo insertion of MuLV induced by vincristine treatment and selection of a small fraction of pre-existing cells carrying MuLV insertion during vincristine treatment. No rearranged sequence was detected by polymerase chain reaction in parental cells. This result argued for the first mechanism. The randomly altered distribution of MuLV during repetitive chemotreatment might also be consistent with this hypothesis. On the other hand, the retrovirus insertion was detected at the same site of the mdr1a promoter region in 2 independent experiments, which suggests the second mechanism. It should be noted that in vivo chemotreatment using vincristine could generate the mdr1a-overexpressing cells through retrovirus insertion and the enhancer effect of the LTR.

The expression of P-glycoprotein (P-gp), encoded by the MDR1 gene, is an independent adverse prognostic factor for response and survival in de novo acute myeloid leukemia (AML). Little is known about MDR1 expression during the development of disease. The present study investigated whether MDR1 gene- related clonal selection occurs in the development from diagnosis to relapsed AML, using a genetic polymorphism of the MDR1 gene at position 2677. Expression and function of P-gp were studied using monoclonal antibodies MRK16 and UIC2 and the Rhodamine 123 retention assay with or without PSC 833. No difference was found in the levels of P-gp function and expression between diagnosis and relapse in purified paired blast samples from 30 patients with AML. Thirteen patients were homozygous for the genetic polymorphism of MDR1 (n = 7 for guanine, n = 6 for thymidine), whereas 17 patients were heterozygous (GT). In the heterozygous patients, no selective loss of one allele was observed at relapse. Homozygosity for the MDR1 gene (GG or TT) was associated with shorter relapse-free intervals (P =.002) and poor survival rates (P =.02), compared with heterozygous patients. No difference was found in P-gp expression or function in patients with AML with either of the allelic variants of the MDR1 gene. It was concluded that P-gp function or expression is not upregulated at relapse/refractory disease and expression of one of the allelic variants is not associated with altered P-gp expression or function in AML, consistent with the fact that MDR1 gene-related clonal selection does not occur when AML evolves to recurrent disease. (Blood. 2001;97:3605-3611)

P-glycoprotein (P-gp), a transmembrane efflux pump encoded by theMDR1 gene, has been found to be expressed in many normal bone marrow and peripheral blood cells. Among normal leukocytes, CD3−CD16+ or CD3−CD56+ lymphocytes, ie, natural killer (NK) cells, express relatively high levels of P-gp, but little is known about P-gp in abnormally expanded NK cells. In this study, we examined the expression and activity of P-gp on NK cells derived from three normal donors, six patients with indolent NK cell-lineage granular lymphocyte-proliferative disorder (NK-GLPD), three patients with aggressive NK cell tumors (one NK cell leukemia and two nasal NK cell lymphoma), and two NK cell lines. By flow cytometric analysis using the monoclonal antibody (MoAb) MRK16 and rhodamine 123 dye (Rh123), P-gp expression and the efflux of Rh123 were found in all NK samples except one NK cell line. The Rh123 efflux of NK cells was inhibited by cyclosporin A (CsA) and its analogue PSC 833, but the aggressive NK tumor cells were less inhibited than were the other NK cells. The percent inhibition of efflux in the normal NK cells, indolent NK-GLPD cells and aggressive NK cell tumors was 81.8% ± 0.9%, 93.4% ± 3.1% and 36.9% ± 11.7%, respectively, by 1 μmol/L CsA, and 80.2% ± 3.6%, 91.7% ± 2.6% and 32.7% ± 10.1%, respectively, by 1 μmol/L PSC833. In reverse transcription-polymerase chain reaction (RT-PCR) analysis, the low inhibitory effect of P-gp modulators in aggressive NK cell tumors did not correlate to the expression level of MDR1 gene, multidrug resistance-associated protein gene, or human canalicular multispecific organic anion transporter gene. This phenomenon could be related to the presence of other transporters or to unknown cellular or membrane changes. Some patients with NK cell tumors have been reported to show a highly aggressive clinical course and to be refractory to chemotherapy, and this could be related to the expression of P-gp on NK cells. Our results suggest that, although the inhibitors for P-gp have been used in combination with chemotherapy in some hematologic tumors, these inhibitors may be less effective against aggressive NK cell tumors.

Scott syndrome is a rare inherited bleeding disorder in which platelets and other blood cells fail to promote normal assembly of the membrane-stabilized proteases of the plasma coagulation system. The defect in Scott blood cells is known to reflect inability to mobilize phosphatidylserine from inner plasma membrane leaflet to the cell surface in response to an elevation of Ca2+ at the endofacial surface. To gain insight into the molecular basis of this membrane defect, we examined the expression in Scott cells of plasma membrane proteins that have been implicated to participate in the accelerated transbilayer movement of plasma membrane PL. By both reverse transcriptase-polymerase chain reaction (RT-PCR) and functional assay, the level of expression of the multidrug resistance (MDR)1 and MDR3 P-glycoproteins in immortalized B-lymphoblast cell lines from the patient with Scott syndrome were indistinguishable from matched cell lines derived from normal controls. Whereas the plasma membrane of Scott cells are insensitive to activation of the plasma membrane PL scramblase pathway, it had been shown that PL scramblase protein isolated from detergent-solubilized Scott erythrocytes exhibits normal function when incorporated into proteoliposomes (Stout JG, Basse F, Luhm RA, Weiss HJ, Wiedmer T, Sims PJ: J Clin Invest 99:2232, 1997). Consistent with this finding in Scott erythrocytes, we found that Scott lymphoblasts expressed normal levels of PL scramblase mRNA and protein, and that the deduced sequence of PL scramblase in Scott cells is identical to that of normal controls. These data suggest that the defect in Scott syndrome is related either to aberrant posttranslational processing of the PL scramblase polypeptide or to a defect or deficiency in an unknown cofactor that is required for normal expression of plasma membrane PL scramblase function in situ, or alternatively, reflects the presence of a detergent-dissociable inhibitor of this pathway.© 1998 by The American Society of Hematology.

To overcome the problem of multidrug resistance, we investigated the effectiveness of phosphrothioate antisense oligonucleotides (MDR1-AS) in suppressing multidrug resistance gene (mdr1) expression in drug-resistant acute myelogenous leukemia (AML) blast cells and the K562 adriamycin-resistant cell line K562/ADM. The percentage of cells with the mdr1 gene product P-glycoprotein (P-gp) was decreased from 100% to 26% by 20 μmol/L MDR1-AS in the K562/ADM cells, and from 48.1% to 10.2% by 2.5 μmol/L MDR1-AS in the AML blast cells. Western blot analysis also showed a decrease in the amount of P-gp in the MDR1-AS–treated K562/ADM cells. This effect was specific to MDR1-AS, and not observed with sense or random control oligonucleotides. The expression of mdr1 mRNA in K562/ADM and AML blast cells treated with MDR1-AS was decreased compared with the random control. Intracellular rhodamine retention and [3H]daunorubicin also increased after antisense treatment. Chemosensitivity to daunorubicin increased in MDR1-AS–treated blast cells up to 5.9-fold in the K562/ADM cells and 3.0- to 6.4-fold in the AML blast cells. The expression of mdr1mRNA derived from colony cells decreased in the MDR1-AS–treated groups. No inhibitory effect of the oligonucleotides on normal bone marrow progenitors was observed. These findings suggest that MDR1-AS is useful to overcome multidrug resistance in the treatment of leukemia.

Complete remission is achieved in a high proportion of patients with acute promyelocytic leukemia (APL) after all-trans retinoic acid (RA) treatment, but most patients relapse and develop RA-resistant APL. We have previously reported that both RA-resistant HL-60 (HL-60R) and APL cells express P-glycoprotein and MDR1 transcripts; and these cells differentiate to mature granulocytes after culture with RA and P-glycoprotein antagonist. Ribozymes have been shown to be able to intercept a target RNA by catalytic activity. To address the role of MDR1 in overcoming RA-resistance in APL cells, we investigated the biologic effects of ribozymes against the MDR1 transcript in HL-60R cells. These ribozymes efficiently cleaved MDR1 mRNA at a specific site in vitro. The 196 MDR1 ribozyme was cloned into an expression vector, and stably transfected (HL-60R/196Rz) cells were obtained. Expression of MDR1 transcripts was decreased in HL-60R/196Rz cells compared with parental HL-60R and empty vector-transfected (HL-60R/neo) cells. Interestingly, RA inhibited cellular proliferation and induced differentiation of HL-60R/196Rz cells in a dose-dependent manner, suggesting reversal of drug resistance in HL-60R cells by the MDR1 ribozyme. These data are direct evidence that P-glycoprotein/MDR1 is responsible in part for acquired resistance to RA in myeloid leukemic cells. The MDR1 ribozyme may be a useful tool for investigating the biology of retinoid resistance and may have therapeutic potential for patients with RA-resistant APL.

By using RNase protection analysis, residues 2677 and 2995 ofMDR-1 were identified as sites of genetic polymorphism. Through use of oligonucleotide hybridization, the genomic content and expression of individual MDR-1 alleles were examined in normal tissues, unselected and drug selected cell lines, and malignant lymphomas. In normal tissues, unselected cell lines, and untreated malignant lymphoma samples, expression of MDR-1 from both alleles was similar. In contrast, in drug selected cell lines, and in relapsed malignant lymphoma samples, expression of one allele was found in a large percentage of samples. To understand how expression of one allele occurs, two multidrug resistant sublines were isolated by exposing a Burkitt lymphoma cell line to increasing concentrations of vincristine. The resistant sublines expressed only one allele and had a hybrid MDR-1 gene composed of non–MDR-1 sequences proximal to MDR-1. Previous studies showing hybridMDR-1 genes after rearrangements provided a potential explanation for activation and expression of one MDR-1 allele. We conclude that oligonucleotide hybridization can be used as a sensitive tool to examine relative allelic expression of MDR-1,and can identify abnormal expression from a single allele. Acquired drug resistance in vitro and in patients is often associated with expression of a single MDR-1 allele, and this can be a marker of a hybrid MDR-1 gene.

The multidrug resistance 1 (MDR1) gene product P-glycoprotein (P-gp) is frequently implicated in cross-resistance of tumors to chemotherapeutic drugs. In contrast, acute promyelocytic leukemia (APL) cells do not express MDR1 and are highly sensitive to anthracyclines. The combination of ATRA and the novel histone deacetylase inhibitor (HDACI) depsipeptide (FK228) induced P-gp expression and prevented growth inhibition and apoptosis in NB4 APL cells subsequently exposed to doxorubicin (DOX). ATRA/FK228 treatment after exposure to DOX, however, enhanced apoptosis. Both agents, ATRA or FK228, induced MDR1 mRNA. This effect was significantly enhanced by ATRA/FK228 administered in combination, due in part to increased H4 and H3-Lys9 acetylation of the MDR1 promoter and recruitment of the nuclear transcription factor Y alpha (NFYA) transcription activator to the CCAAT box. Cotreatment with specific P-gp inhibitor PSC833 reversed cytoprotective effects of ATRA/FK228. G1 cell-cycle arrest and p21 mRNA induction were also observed in response to ATRA/FK228, which may restrict DOX-induced apoptosis of cells in G2 phase. These results indicate that epigenetic mechanisms involving NF-YA transcription factor recruitment and histone acetylation are activated by ATRA and HDACI, induce MDR1 in APL cells, and point to the critical importance of mechanism-based sequential therapy in future clinical trials that combine HDAC inhibitors, ATRA, and anthracyclines.

Previous studies have demonstrated leukemic complications in mice after high-copy retroviral gene transfer of the multidrug resistance 1 (MDR1) cDNA, encoding a membrane-located efflux pump expressed in hematopoietic stem cells. In contrast, no such complications or MDR1-associated alterations of hematopoiesis were observed in numerous other studies exploring MDR1 gene transfer into cell lines, mice, dogs, nonhuman primates, and human subjects. Here, we show that leukemias associated with retroviral expression of MDR1 depend on high vector dose, and involve the selection of clones with combinatorial insertional mutagenesis of proto-oncogenes or other signaling genes. Compared with insertion patterns in normal long-term repopulating hematopoietic cells, such hits were overrepresented in leukemic clones, pointing to a causal role. A similar constellation of insertion sites was also observed in a leukemia arising after high-copy retroviral gene transfer of a fluorescent protein. Spectral karyotyping demonstrated additional chromosomal translocations in a subset of cases, indicative of secondary genetic instability. We also show that insertional mutants can be amplified in vitro prior to transplantation. On the basis of these findings, we suggest the use of preclinical dose-escalation studies to define a therapeutic index for retroviral transgene delivery.

In their recent article, Mahon et al[1][1] demonstrate that MDR1 gene overexpression can confer resistance to imatinib mesylate (Gleevec; Novartis, Basel, Switzerland) in leukemia cell lines. Several other cellular mechanisms of resistance to imatinib have also been identified,[2][2]-[4][3] but the

P-glycoprotein (pgp), which is the product of the MDR1 (multidrug resistance-1) gene, has an established role as a mediator of cytotoxic drug resistance in acute myeloid leukemia (AML). To study the role of pgp in mediating apoptosis resistance in AML cells deprived of serum and growth factors, apoptosis was quantified by flow cytometry using uptake of the dye 7-amino-actinomycin D (7-AAD) alongside low forward scatter. In pgp+ve primary AML samples, there was a significant increase in apoptosis in the presence of the pgp-specific antibody UIC2 (mean increase: 58%; range: 11%-95%; P. 05). Likewise, apoptosis in growth factor-deprived TF1 cells cultured for 30 hours increased 2.5-fold in the presence of 25 microg/mL UIC2. The pgp reversal agent PSC-833 (1 micromol/L) augmented in vitro apoptosis by a median of 52% in pgp+ve patient samples and to a comparable degree in 6 pgp-ve samples. To determine whether the sphingomyelin-ceramide (SM-ceramide) pathway of apoptosis occurs in AML blasts in response to cytotoxic drugs, cells were incubated with daunorubicin at the patient-specific IC(30) (the concentration of daunorubicin that caused apoptotic cell death in 30% of cells) in the presence of the ceramide synthase inhibitor fumonisin B1, which inhibited apoptosis by 18%-81% (median: 40%). Exogenous SM failed to augment apoptosis induced by growth factor withdrawal in pgp+ve TF1 cells and was significantly more effective at augmenting apoptosis in pgp-ve patient blasts (median increase in cell death: 33%; range: 19%-88%) than in pgp+ve samples (median: 7%; range: 0%-27%; P =.028). Cellular accumulation of exogenous SM was associated with apoptosis and also occurred in nonapoptotic patient cells treated with PSC-833. However, this effect was not seen following treatment with the UIC2 antibody. These results indicate that pgp is able to exert a protective effect on AML cell viability and that this is associated with a reduced effect of exogenous SM on apoptosis. The pgp reversal agent PSC-833 acts, at least in part, by a pgp independent mechanism to alter SM distribution and to augment apoptosis induced in AML cells by serum and growth factor withdrawal. (Blood. 2000;95:2897-2904)

Mobilized peripheral blood progenitor cells (PBPC) are a potential target for the retrovirus-mediated transfer of cytostatic drug-resistance genes. We analyzed nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse-repopulating CD34+ PBPC from patients with cancer after retroviral transduction in various cytokine combinations with the hybrid vector SF-MDR, which is based on the Friend mink cell focus-forming/murine embryonic stem-cell virus and carries the human multidrug resistance 1 (MDR1) gene. Five to 13 weeks after transplantation of CD34+ PBPC into NOD/SCID mice (n = 84), a cell dose-dependent multilineage engraftment of human leukocytes up to an average of 33% was observed. The SF-MDR provirus was detected in the bone marrow (BM) and in its granulocyte fractions in 96% and 72%, respectively, of chimeric NOD/SCID mice. SF-MDR provirus integration assessed by quantitative real-time polymerase chain reaction (PCR) was optimal in the presence of Flt-3 ligand/thrombopoietin/stem-cell factor, resulting in a 6-fold (24% +/- 5% [mean +/- SE]) higher average proportion of gene-marked human cells in NOD/SCID mice than that achieved with IL-3 alone (P.01). A population of clearly rhodamine-123(dull) human myeloid progeny cells could be isolated from BM samples from chimeric NOD/SCID mice. On the basis of PCR and rhodamine-123 efflux data, up to 18% +/- 4% of transduced cells were calculated to express the transgene. Our data suggest that the NOD/SCID model provides a valid assay for estimating the gene-transfer efficiency to repopulating human PBPC that may be achievable in clinical autologous transplantation. P-glycoprotein expression sufficient to prevent marrow aplasia in vivo may be obtained with this SF-MDR vector and an optimized transduction protocol. (Blood. 2000;95:1237-1248)

A major problem with treating patients with cancer by traditional chemotherapeutic regimes is that their tumors often develop a multidrug resistant (MDR) phenotype and subsequently become insensitive to a range of different chemotoxic drugs. One cause of MDR is overexpression of the drug-effluxing protein, P-glycoprotein. It is now apparent that P-glycoprotein may also possess a more generic antiapoptotic function that protects P-glycoprotein-expressing cancer cells and normal cells from cell death. Herein we show that cells induced to express P-glycoprotein either by drug selection or by retroviral gene transduction with MDR1 cDNA are resistant to cell death induced by a wide range of death stimuli, such as FasL, tumor necrosis factor (TNF), and ultraviolet (UV) irradiation, that activate the caspase apoptotic cascade.However, P-glycoprotein-expressing cells were not resistant to caspase-independent cell death mediated by pore-forming proteins and granzyme B.MDR P-glycoprotein-expressing cells were made sensitive to caspase-dependent apoptosis by the addition of anti-P-glycoprotein antibodies or verapamil, a pharmacological inhibitor of P-glycoprotein function. Clonogenic assays showed that P-glycoprotein confers long-term resistance to caspase-dependent apoptotic stimuli but not to caspase-independent cell death stimuli. This study has confirmed a potential novel physiological function for P-glycoprotein and it now remains to dissect the molecular mechanisms involved in the inhibition of capsase-dependent cell death by P-glycoprotein.

Attempts to expand repopulating hematopoietic cells ex vivo have yielded only modest amplification in stem cell numbers. We now report that expression of an exogenous human multi-drug resistance 1 (MDR1) gene enables dramatic ex vivo stem cell expansion in the presence of early acting hematopoietic cytokines. Bone marrow cells were transduced with retroviral vectors expressing either the MDR1 gene or a variant of human dihydrofolate reductase (DHFR), and then expanded for 12 days in the presence of interleukin-3 (IL-3), IL-6, and stem cell factor. When these cells were injected into nonirradiated mice, high levels of long-term engraftment were only seen with MDR1-transduced grafts. To verify that expansion of MDR1-transduced repopulating cells had occurred, competitive repopulation assays were performed using MDR1 expanded grafts. These experiments showed progressive expansion of MDR1-transduced repopulating cells over the expansion period, with a 13-fold overall increase in stem cells after 12 days. In all of the experiments, mice transplanted with expanded MDR1-transduced stem cells developed a myeloproliferative disorder characterized by high peripheral white blood cell counts and splenomegaly. These results show that MDR1-transduced stem cells can be expanded in vitro using hematopoietic cytokines without any drug selection, but enforced stem cell self-renewal divisions can have adverse consequences.

Overexpression of P-glycoprotein (P-gp), the protein product of the multidrug resistance gene (MDR1), confers a drug resistant phenotype on cells. This phenotype is reminiscent of human T-cell leukemia virus (HTLV)-transformed leukemic cells, for which no consistently effective chemotherapeutic regime has been found. The presence of an active multiple drug resistance (MDR) phenotype in freshly isolated peripheral blood mononuclear cells (PBMC) from HTLV-I-infected subjects was investigated. Significant P-gp-mediated efflux activity and enhanced MDR1 mRNA expression was observed in nine of 10 HTLV-infected subjects. The development of MDR phenotypes was found to be independent of disease type or status with significant MDR activities being observed in adult T-cell leukemia (ATL), HTLV-associated myelopathy (HAM)/tropical spastic paraparesis (TSP), and asymptomatic HTLV-infected individuals. P-gp-mediated drug efflux was also found to be restricted to CD3+ T-cell populations. Furthermore, we show the novel finding that the MDR1 gene promoter is transcriptionally activated by the HTLV-I tax protein, suggesting a molecular basis for the development of drug resistance in HTLV-I infections. These observations open up the possibility of new chemotherapeutic approaches to HTLV-associated diseases through the use of P-gp inhibitors.

The 110-kD lung resistance protein (LRP) is overexpressed in P-glycoprotein–negative multidrug-resistant cell lines and most likely involved in the multidrug resistance (MDR) of these cell lines. To determine the clinical significance of LRP, we have studied LRP expression of leukemic blasts and its association with clinical outcome in patients with de novo acute myeloid leukemia (AML). LRP expression of leukemic blasts obtained from peripheral blood or bone marrow of previously untreated patients (n = 86) was determined by immunocytochemistry by means of monoclonal antibody LRP-56. LRP expression at diagnosis was detected in 31 (36%) patients. LRP expression was independent of age and sex of the patients, French-American-British subtype, cytogenetic abnormalities, and lactate dehydrogenase levels, but correlated with white blood cell count (P = .01). Eighty-two patients received standard induction chemotherapy that included cytarabine and MDR drugs (daunorubicin in most patients, additional etoposide in the majority of patients). The complete remission rate of induction chemotherapy was 72% (95% confidence interval [CI] = 61% to 82%) for the total study population. The complete remission rate was 81% (95% CI = 67% to 91%) for patients without LRP expression but only 55% (95% CI = 36% to 74%) for patients with LRP expression (P = .01). Overall survival and disease-free survival were estimated according to Kaplan-Meier in 82 and 59 patients, respectively. Overall survival was significantly longer in patients without LRP expression than in patients with LRP expression. At a median follow-up of 16 months, median overall survival was 17 months (95% CI = 12 to 38 months) for LRP-negative patients but only 8 months (95% CI = 4 to 12 months) for -positive patients (P = .006). Disease-free survival was 9 months (95% CI = 7 to 11 months) for LRP-negative patients and 6 months (95% CI = 5 to 8 months) for -positive patients (P = .078). Outcome was best in patients lacking both LRP and P-glycoprotein expression. In conclusion, LRP predicts for poor outcome and thus theLRP gene appears to be another clinically relevant drug resistance gene in AML.

Retroviral transfer of the multidrug-resistance 1 (mdr1) cDNA into primary human hematopoietic progenitor cells (HPC) of cancer patients undergoing high-dose chemotherapy has been proposed to protect the bone marrow from the dose-limiting cytotoxicity of cytostatic agents. Preclinical studies performed with vectors derived from the Moloney murine leukemia virus (MoMuLV) or the related Harvey murine sarcoma virus have established that chemoprotection of HPC is feasible. The efficacy of vector-mediated multidrug-resistance under high doses of cytostatic agents, however, remained unclear. We report here that this goal can only be achieved with improved vector design. Novel vectors termed SF-MDR and MP-MDR, which are based on the spleen focus-forming virus or the myeloproliferative sarcoma virus for the enhancer and the murine embryonic stem cell virus for the leader, significantly elevate survival of transduced primary human HPC under moderate doses of colchicine and paclitaxel in vitro when compared with a conventional MoMuLV-based vector. Importantly, SF-MDR and also MP-MDR confer an absolute advantage at high doses of paclitaxel in vitro corresponding to peak plasma levels achieved in patients during chemotherapy. This observation has important consequences for a variety of ongoing and planned gene therapy trials.

The prognostic significance of multidrug resistance (MDR) gene expression is controversial. We investigated whether multidrug resistance gene 1 (MDR1), multidrug resistance-related protein (MRP) and lung resistance protein (LRP) mRNA expression are associated with outcomes in acute leukemia patients. At diagnosis we examined MDR1, MRP and LRP mRNA expression in bone marrow samples from 71 acute leukemia patients (AML 39, ALL 32) using nested reverse transcription polymerase chain reaction (RT-PCR). The expression of each of these genes was then expressed as a ratio in relation to ß-actin gene expression, and the three genes were categorized as being either not expressed (0), weakly expressed (1+), moderately expressed (2+) or strongly expressed (3+). MDR1, MRP and LRP mRNA expression was detected in 23.9%, 83.1% and 45.1%, respectively, of acute leukemia patients. LRP mRNA expression was significantly associated with resistance to induction chemotherapy in acute leukemia patients, and in the AML proportion of these patients, compared to LRP-negative patients (p=0.02 and p=0.03, respectively). MRP and high MDR1 mRNA expression was associated with poorer 2-year survival (P=0.049 and p=0.04, respectively) compared to MRP-negative and non-high MDR1 mRNA-expressing patients, respectively. Patients expressing both MRP and LRP mRNA had poorer outcomes in response to induction chemotherapy and had worse 2-year survival compared to patients not expressing both genes. The present data suggest that MDR gene expression affects CR and survival rates in acute leukemia patients. Thus, determination of MDR gene expression at diagnosis appears likely to provide useful prognostic information for acute leukemia patients.