Biomedical subjects

S Hegewisch-Becker

Publications and source records attributed to S Hegewisch-Becker.

16 recordsLinked to original sources

PubMed1996

High-dose multidrug resistance in primary human hematopoietic progenitor cells transduced with optimized retroviral vectors.

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.

Animals↗

PubMed1996

The MDR phenotype in hematologic malignancies: prognostic relevance and future perspectives.

The multidrug resistant (MDR) phenotype has been suspected as a major cause of treatment failure in hematologic malignancies. Numerous studies have investigated the expression of the MDR1 gene product, P-glycoprotein, in leukemia, lymphoma and myeloma. Studies in myelogenous leukemia and myeloma have so far provided best evidence for a significant correlation between P-glycoprotein expression and response to chemotherapy, although large discrepancies in the proportion of positive cells limit any definite conclusion. Differences in P-glycoprotein detection techniques and methodology may account for the divergent results thus emphasizing the necessity for standardized methods of detection. Despite this, encouraging clinical results have been obtained using MDR modulators in combination with conventional chemotherapy to inhibit the activity of the P-glycoprotein pump. The paper summarizes currently available clinical data and provides guidelines for future trials aimed to reverse the MDR phenotype. The potential of idarubicin to overcome the MDR phenotype is also discussed.

Drug Resistance, Multiple↗

PubMed1996

Improved retroviral vectors for hematopoietic stem cell protection and in vivo selection.

Therapeutic gene transfer into hematopoietic cells is critically dependent on the evolution of methods that allow ex vivo expansion, high-frequency transduction, and selection of gene-modified long-term repopulating cells. Progress in this area needs elaboration of defined culture and transduction conditions for long-term repopulating cells and improvement of gene transfer systems. We have optimized retroviral vector constructions based on murine leukemia viruses (MuLV) to overcome the transcriptional repression encountered with the use of conventional Moloney MuLV (MoMuLV) vectors in early hematopoietic progenitor cells (HPC). Novel retroviral vectors, termed FMEV (for Friend-MCF/MESV hybrid vectors), were cloned that mediate greatly improved gene expression in the myeloerythroid compartment. Transfer of the selectable marker multidrug resistance 1 (mdr1), FMEV, in contrast to conventional MoMuLV-related vectors currently in use for clinical protocols, mediated background-free selectability of transduced human HPC in the presence of myeloablative doses of the cytostatic agent paclitaxel in vitro. Furthermore, FMEV also greatly improved chemo-protection of hematopoietic progenitor cells in a murine model system in vivo. Finally, when a second gene was transferred along with mdr1 in an FMEV-backbone, close to 100% coexpression was observed in multidrug-resistant colonies. These observations have significant consequences for a number of ongoing and planned gene therapy trials, for example, stem cell protection to reduce the myelotoxic side effects of anticancer chemotherapy, correction of inherited disorders involving hematopoietic cells, and antagonism of HIV infection.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1996

MDR1 reversal: criteria for clinical trials designed to overcome the multidrug resistance phenotype.

Multidrug resistance (MDR) is a widely studied mechanism of cellular resistance to structurally unrelated cytotoxic agents. The MDR phenotype is related to the overexpression of the MDR1 gene product, P-glycoprotein (P-gp), a transmembrane drug efflux pump. The capacity to compete with the cytotoxic drug for the active outward transport process, thus inhibiting the activity of the P-gp pump, has been demonstrated for numerous non-cytotoxic compounds, termed MDR modulators. The possibility of modulating the activity of the P-gp pump has initiated numerous clinical trials, using a wide range of chemosensitizers. This article reviews these substances, discusses problems that may arise in connection with the concurrent administration of P-gp modulators and chemotherapeutic agents and provides guidelines for the design of future clinical trials. Furthermore, now data are presented concerning the potential of idarubicin to overcome the MDR phenotype.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1995

Transduction of MDR1 into human and mouse haemopoietic progenitor cells: use of rhodamine (Rh123) to determine transduction frequency and in vivo selection.

The MDR1 gene product P-glycoprotein (P-gp) extrudes several anticancer drugs including taxol and fluorescent dyes such as rhodamine (Rh123). Modulation of the level of P-gp expression has the potential of overcoming multidrug resistance. One possible approach is the retroviral transfer of the human MDR1 gene into murine and human bone marrow (BM) progenitor cells. The rationale for this approach is increased chemoprotection, which allows chemotherapy of a greater level of intensity to be delivered. In this study, flow cytometric measurement of Rh123 extrusion was used to test P-gp function in human and mouse haemopoietic progenitor cells, which had been transduced with a virus containing the human MDR1 transcription unit. Human CD34+ selected cells were analysed immediately following transduction. In two successive experiments MDR1 cDNA transduction resulted in a 7% and 11% increase of P-gp expressing Rh123 dull cells. To monitor transduction efficiency over time as well as the possibility of in vivo selection of drug-resistant BM cells in mice treated with increasing numbers of taxol cycles, the assay was also successfully applied to peripheral blood lymphocytes of mice transplanted with MDR1 transduced BM cells, demonstrating increased Rh123 efflux in transduced cells. Analysis of another fluorescence assay using fluorescein di-beta galactopyranoside as a substrate for beta-galactosidase in cells transduced with a MDR1: beta-gal activity. We conclude that the Rh123 efflux assay is a sensitive method to monitor P-gp function in MDR1 cDNA transduced cells, and may be used to enrich transduced cells via flow cytometric cell sorting for Rh123 dull cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1995

Novel retroviral vectors for efficient expression of the multidrug resistance (mdr-1) gene in early hematopoietic cells.

We present data that retroviral gene expression in early hematopoietic cells is subjected to transcriptional controls similar to those previously described for embryonic stem cells. Transient transfection experiments revealed that both the viral enhancer region in the U3 region of the long terminal repeat as well as a repressor element coincident with the primer binding site of Moloney leukemia viruses are limiting for expression in hematopoietic cells in a differentiation-dependent manner. Within the group of Moloney leukemia virus-related viruses, only the myeloproliferative sarcoma virus showed high enhancer activity in myeloid (including erythroid) cells. In contrast, enhancer regions related to the Friend mink cell focus-forming viruses mediate much higher gene expression levels in both multipotent and lineage-committed myeloid cells. In addition, transcriptional repression related to sequences in the primer binding site of Moloney leukemia virus-derived vectors is also found in early hematopoietic cells and can be overcome by using the corresponding sequences of the murine embryonic stem cell virus. On the basis of these results, two types of novel retroviral hybrid vectors were developed; they combine the U3 regions of either the Friend mink cell focus-forming virus family or the myeloproliferative sarcoma virus with the primer binding site of the murine embryonic stem cell virus. When used to express the human multiple drug resistance gene, these vectors substantially improve protection to cytostatic drugs in transduced hematopoietic cell lines FDC-Pmix, TF-1, and K-562 in comparison with Moloney leukemia virus-derived vectors presently used for the stem cell protection approach in somatic gene therapy.

3T3 Cells↗

PubMed1995

Chemotherapy resistance to taxol in clonogenic progenitor cells following transduction of CD34 selected marrow and peripheral blood cells with a retrovirus that contains the MDR-1 chemotherapy resistance gene.

A retrovirus containing the multiple drug resistance (MDR-1) cDNA, was used to transduce cultures of CD34 selected human marrow cells, on stromal monolayers in the presence of hematopoietic growth factors IL-3 and IL-6, following collection from patients recently recovered from chemotherapy-induced myelosuppression. In one experiment, these CD34 selected cells were grown in Dexter cultures for 35 days or more following MDR-1 transduction, and then plated in methylcellulose. Polymerase chain reaction (PCR) analysis of colonies picked after 10-14 days of methylcellulose culture, using a set of primers that are specific for the endogenous or the retrovirally transduced MDR-1, showed that the long-term culture initiating cells (LTCICs) were transduced by the MDR-1 virus. Analysis of the colonies from the CD34 selected MDR-1 transduced cells, with a reverse transcription (RT) PCR assay that could distinguish viral MDR-1 mRNA from endogenous MDR-1 mRNA, showed that the viral MDR-1 mRNA levels were much higher than that of the MDR-1 mRNA from the endogenous MDR-1 gene in the transduced CD34 selected cells. Fluorescence activated cell sorting (FACS) analysis of the CD34 selected transduced marrow cells within 48 h after the transduction, using the C219 and UIC2 monoclonal antibodies for p-glycoprotein, showed that the transduction frequency under these conditions varied from 7 to 20%. Rhodamine efflux studies showed that this additional p-glycoprotein was functional and that the frequency of cells with high p-glycoprotein levels was higher in the transduced cells than in the non-transduced cells. The resistance to taxol of the CD34 selected transduced cells, as judged by the plating efficiency of clonogenic progenitor cells derived from these cells by growth in methylcellulose supplemented with taxol was much higher in the transduced cells than in untransduced cells. In order to test the reproducibility of the transduction frequency of the retroviral supernatants from PA317 MDR-1 viral producer cells on CD34 selected cells, the virus produced from 12 different lots of supernatants from the PA317 MDR-1 producer cell line was used to transduce CD34 selected marrow cells from four different patients, and to transduce the peripheral blood cells of two additional patients collected following chemotherapy-induced myelosuppression. The supernatant lots used for these transduction experiments were tested by Microbiological Associates (Rockville, MD, USA), by the Mus dunni co-cultivation and amplification tests in the S+L-assay and found to be negative for replication-competent retrovirus, and later approved for human use by the Food and Drug Administration.(ABSTRACT TRUNCATED AT 400 WORDS)

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1995

Infusion of tumor-contaminated bone marrow for autologous rescue after high-dose therapy leading to long-term remission in a patient with relapsed Philadelphia chromosome-positive acute lymphoblastic leukemia.

We report a female patient with relapsed Philadelphia chromosome-positive acute lymphoblastic leukemia remaining in long-term second remission after high-dose radiochemotherapy followed by autologous stem cell rescue with bone marrow showing evidence of residual disease. The Philadelphia chromosome and a positive signal for the BCR/ABL p185 translocation by the polymerase chain reaction were detected in the harvested marrow. After mafosfamide-purged marrow failed to engraft, her unpurged 'back-up' bone marrow was also infused. Control marrow examinations after recovery were repeatedly positive for the BCR/ABL translocation on PCR analysis turning negative 30 months after high-dose therapy. She remains in unsustained complete clinical remission for 48+ months showing no evidence of leukemia by cytological and cytogenetic analyses.

Adult↗

PubMed1995

Bone marrow transplantation for Philadelphia-chromosome-positive acute lymphoblastic leukemia.

The outcome of 14 bone marrow transplants (BMT) (autologous 4; allogeneic 10) for Philadelphia-chromosome (Ph1) positive acute lymphoblastic leukemia (ALL) was analyzed. Preparative regimens consisted of etoposide (VP16) (30 or 45 mg/kg BW) (n = 14), cyclophosphamide (CY)(120 mg/kg BW) (n = 14), and total body irradiation (TBI)(12 Gy) (n = 13) or busulfan (Bu)(16 mg/kg) (n = 1). All patients receiving autologous marrow were in complete remission (CR) (three patients in 1.CR, one patient in 2.CR) at the time of BMT. For allogeneic BMT (nine related, one unrelated donor), seven patients were in first CR, two patients in first refractory relapse, and one patient in second relapse. With a median follow-up of 503 days (range 93-1522 days), eight out of 14 patients are alive in remission (six out of 10 patients receiving allogeneic, and two out of four patients receiving autologous BMT). Disease-free survival for all patients is 46%. Causes of death were relapse (n = 3) and transplant-related toxicity (n = 3). All patients tested for the bcr/abl rearrangement by reverse transcriptase-polymerase chain reaction (RT-PCR) were negative 4 weeks post-BMT. Two of the three patients who subsequently relapsed were repeatedly RT-PCR positive prior to relapse (test not done in the third). Considering the negligible cure rate of Ph1-positive ALL with conventional chemotherapy regimens, our data support the concept of early (> or = 1 CR) BMT (allogeneic > autologous (purged) following triple therapy with TBI, VP16, and CY.

Adult↗

PubMed1994

Flow cytometric determination of the multidrug-resistant phenotype in acute leukemia.

Expression of the multidrug-resistant (MDR) phenotype was investigated in acute leukemia using a monoclonal antibody (HYB-241) directed against a cell surface epitope of the 180 kd P-glycoprotein (gp180) by flow cytometric analysis of clinical samples. Samples from sixty-four patients were tested (37 with acute myelocytic leukemia, 20 with acute lymphocytic leukemia, and 7 with blastic chronic myelocytic leukemia). A D value (derived from Kolmogorov-Smirnov test) greater than 0.15 was considered positive (+). Eight of 32 newly diagnosed patients were positive for gp180 compared with 22 of 32 relapsed/refractory (R/R) patients (P < 0.001). Of the new patients, vinca/anthracycline-based induction therapy failed in 3/6 gp180(+) and 5/18 gp180(-) patients. In the R/R group, 15/16 gp180(+) and 3/6 gp180(-) patients failed to achieve complete remission (P < 0.05). In vitro drug accumulation studies performed with verapamil failed to show a correlation with clinical response. However, in a subset of patients, a striking correlation (r = .97, P = .001) was noted between the presence of gp180 as determined by the D value and the functional activity of the P-glycoprotein as expressed by increased daunorubicin accumulation in the presence of verapamil. The results suggest that 1) newly diagnosed patients can express gp180, 2) P-glycoprotein is expressed in 69% of R/R patients, 3) response in R/R patients is effected by the presence of gp180, and 4) expression of gp180 is highly correlated with its function as a drug-efflux pump in a subset of the patients studied. The complexity of clinical drug resistance is underscored by the finding that the MDR model is not applicable to all cases. In such instances, other mechanisms may play a predominant role.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1994

An optimized electroporation protocol applicable to a wide range of cell lines.

A number of transfection methods for mammalian cells are available; however, many cell lines may appear resistant to efficient transfection, or at best, necessitate lengthy optimization procedures in recommended protocols. We describe here an electroporation protocol that yields highly efficient gene transfer (20%-100% of surviving cells) in all 19 cell lines tested so far, including lymphoid, myeloid, glial, fibroblast and COS cells. Unlike earlier electroporation protocols, adaptation of this protocol to a cell line of interest only requires the optimization of a single parameter, i.e., the voltage, and can be performed within a day. Furthermore, it is also superior to transfection protocols for other methods (calcium phosphate precipitation, lipofection, DEAE-dextran transfection and transferrinfection) in terms of reproducibility, economy and average transfection efficiency for a wide variety of cell lines.

Animals↗

PubMed1993

GCSF gene is expressed but not rearranged in a patient with isochromosome 17q positive acute nonlymphocytic leukemia.

We describe a patient with acute nonlymphocytic leukemia (ANLL) and isochromosome 17q as the sole cytogenetic abnormality. ANNL with i(17q) may represent a distinct entity with certain clinical features, such as male sex, hepatosplenomegaly, and characteristic findings in bone marrow (BM) cytology, including hypercellularity, marked basophilia and eosinophilia, and massive increase in abnormal megakaryocytes. Molecular studies of peripheral blood (PB) cells of our patient, by polymerase chain reaction (PCR) analysis, showed expression of the GCSF gene, which is located on 17q. Southern blots hybridized with a GCSF probe showed no rearrangement of this gene as has been described in some patients with i(17q) positive chronic myeloid leukemia (CML).

Blotting, Southern↗

PubMed1993

P-glycoprotein expression in normal and reactive bone marrows.

The expression of mdr1 gene product P-glycoprotein (P-gp) was investigated in 53 normal and reactive bone marrows by means of immunocytochemistry, using the monoclonal antibody (mAb) C219 and the alkaline phosphatase anti-alkaline phosphatase method. In a limited number of patients, data were confirmed by using the mAb MRK16 or a polymerase chain reaction assay for mdr1 gene expression. There was no history of prior chemotherapy or any malignancy in this group. Bone marrow aspirates were obtained as part of a routine diagnostic programme in bone marrow donors or in patients presenting with a variety of diagnoses such as unexplained gammopathy, fever, anaemia, other changes in peripheral blood smear, rheumatoid arthritis, vasculitis, or urticaria pigmentosa. Morphologically the bone marrow was normal in 23 patients, a megaloblastic erythropoiesis was seen in two patients and unspecific changes were seen in 28 patients. Twenty-seven of 53 samples were found to be positive for P-gp expression with the percentage of positive cells ranging from 2%-80% (mean = 24%). With a cutoff point of 10%, five of 23 normal (22%) and 13 of 28 reactive bone marrows (46%) were considered positive for P-gp expression. There was no obvious correlation between diagnosis or age and P-gp expression. Additional staining for the early surface marker CD-34 was performed in 12 samples, with none of them revealing more than 1% positivity. Since P-gp expression has so far been described only in CD-34 positive bone marrow cells, data suggest that P-gp expression may be reinduced in CD-34 negative cells under conditions which remain to be determined.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1993

Exposure to lipophilic industrial solvents leads to increased P-glycoprotein expression in peripheral blood cells.

The MDR1 gene product P-glycoprotein (P-gp), a transmembrane efflux pump, is expressed in many normal tissues including subpopulations of normal peripheral blood. To test the potential impact of cigarette smoke and/or a variety of lipophilic industrial solvents present in paint and varnish, P-gp expression in unsorted peripheral blood cells of 51 healthy volunteers was measured by means of immunocytochemistry and daunorubicin uptake studies. 10 of these volunteers were heavy smokers and 10 were working as painters or sprayers, and consequently in regular and intense contact with industrial solvents. Cigarette smoke had no impact with regard to increased P-gp expression and function. However, extensive contact with industrial solvents of lipophilic origin was shown to cause a significant increase in the level of P-gp expression (P < 0.001), thus providing new evidence for the broad substrate specificity of the P-gp pump.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1990

Colony-stimulating factors (rhG-CSF, rhGM-CSF, rhIL-3, and BCGF) recruit myeloblastic and lymphoblastic leukemic cells and enhance the cytotoxic effects of cytosine-arabinoside.

Prognostic models for acute myeloid and lymphoid leukemias are presented that demonstrate that cell kinetic quiescence in acute leukemia is associated with poor response to chemotherapy, short remission duration, and survival. Recruitment of cells into the cell cycle should therefore enhance cytotoxic effects of cell cycle - specific chemotherapeutic agents. We previously demonstrated recruitment of myeloid leukemic cells by cytokines. We have now investigated whether recruitment can be used to increase cell killing by cytosine arabinoside (Ara-C). Blast cells from 16 acute leukemias were stimulated with cytokines as follows: 13 acute myeloid leukemias (AML) and 3 chronic myeloid leukemia (CML) in blastic phase (1 lymphoid, 2 myeloid) were treated with recombinant human granulocyte colony stimulating factor (rhG-CSF), recombinant human granulocyte-macrophage colony stimulating factor (rhG-CSF, AMGEN, 500 U/ml each), and recombinant human interleukin-3 (rhIL-3, IMMUNEX, 20 ng/ml), alone and in combination. After 48 h, at the time of maximal DNA synthesis, Ara-C (10(-3) M) was added and cell counts, cytokinetics (DNA/RNA, DNA/bromodeoxyuridine and DNA/Ki67 flow cytometry), and cell viability/clonogenicity (fluorescein diacetate/propidium iodide exclusion flow cytometry) were investigated. In all 13 cases of AML recruitment was found; in 6 of these cases over a three fold increase in S phase (P = 0.008) and a significant (P = 0.004) depletion of G0 was demonstrated. In 9 of 13 patients with AML, the effect of Ara-C was investigated, and in 3 of 5 patients with over three fold increase in S phase, Ara-C toxicity was enhanced. None of the patients with less than a three fold increase in S phase and no demonstrable recruitment from G0 had increased Ara-C cytotoxicity. Ara-C cytoreduction was paralled by reduction in clonogenicity as demonstrated by fluorescein diacetate/propidium iodide (FDA/PI) flow cytometry. Four samples of acute lymphoblastic leukemia (ALL) were treated with low molecular weight B-cell growth factor (15 kDa) and recruitment of aneuploid cells from G0 to G1 was found in all patients (from 19.3% to 84.9%). These results indicate that recruitment of leukemic cells is inducible by cytokines and that the cytotoxicity of cell cycle-specific drugs such as Ara-C can be increased. This concept is presently being tested in vivo.

Adult↗

PubMedDate not supplied

Prolongation of medium exchange is associated with a decrease in function but not expression of the P-glycoprotein pump in leukaemic cells.

Daunorubucin (DNR) accumulation studies as functional tests of the multidrug resistance (MDR1) gene product P-glycoprotein have produced diverging results when correlated to response to chemotherapy in acute leukaemia. To investigate possible reasons for this diversity a starvation experiment, based upon prolongation of medium exchange, was set up in the multidrug resistant cell line CEM/VBL100. DNR accumulation (1 microgram/ml) was measured flow cytometrically in the presence or absence of Verapamil (10 micromol/l). In cells permanently kept under ideal growth conditions, addition of Verapamil resulted in an average 90% increase in DNR enhancement in five successive experiments. In contrast, DNR accumulation increased by only 26% when the medium exchange was prolonged by 30 h to 42 h. This effect was not accompanied by changes in the MDR1 gene expression at the RNA or protein level. Consequently, 53 leukaemic blast samples of 30 newly diagnosed and 18 relapsed or refractory patients with acute leukaemia (ALL-18, AML-37) were processed without any delay and under the most stringent conditions possible. Evidence of the classical MDR phenotype was arbitrarily defined by a greater than 20% enhancement in DNR accumulation in response to Verapamil (10 micromol/l) or Cyclosporin A (3 micromol/l). Using this cutoff point for analysis of newly diagnosed leukaemia we found DNR uptake better correlated to response to treatment (p = 0.002) than P-gp detection by means of immunocytochemistry, using a panel of monoclonal antibodies (p = 0.03). We conclude that DNR accumulation studies are a sensitive method for predicting therapy outcome in acute leukaemia when performed with necessary precautions.

ATP Binding Cassette Transporter, Subfamily B, Mem↗