Biomedical subjects

R J Scheper

Publications and source records attributed to R J Scheper.

At least 163 records · Page 9Linked to original sources

PubMed1995

Regulation by glutathione of drug transport in multidrug-resistant human lung tumour cell lines overexpressing multidrug resistance-associated protein.

Previous studies have shown that multidrug resistance (MDR) in the doxorubicin-selected lung tumour cell lines COR-L23/R, GLC4/ADR and MOR/R is associated with overexpression of the MRP gene. In this study we report that resistance to daunorubicin, vincristine and rhodamine 123 can be partially reversed in these cell lines by exposing the cells to buthionine sulphoximine (BSO), an inhibitor of glutathione (GSH) synthesis. This effect of BSO on drug resistance was associated with an increased intracellular accumulation of daunorubicin and rhodamine 123, owing to inhibition of the enhanced drug efflux. In contrast, the accumulation of daunorubicin was not increased by BSO treatment in a P-glycoprotein (P-gp)-mediated MDR cell line. BSO treatment (25 microM, 20 h) of the cell lines resulted in 60-80% depletion of cellular GSH levels. The effects of BSO on daunorubicin accumulation in the COR-L23/R and GLC4/ADR cells were associated with cellular GSH depletion. In addition, increase of cellular GSH levels in BSO-treated COR-L23/R and GLC4/ADR cells as a result of incubation with 5 mM GSH ethyl ester restored the accumulation deficit of daunorubicin. However, the transport of daunorubicin did not increase the GSH release in any of the cell lines. These results demonstrate that drug transport in MRP- but not in P-gp-overexpressing MDR tumour cell lines can be regulated by intracellular GSH levels.

Biological Transport↗

PubMed1995

Altered MRP is associated with multidrug resistance and reduced drug accumulation in human SW-1573 cells.

We have analysed the contribution of several parameters, e.g. drug accumulation, MDR1 P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP) and topoisomerase (topo) II, to drug resistance in a large set of drug-resistant variants of the human non-small-cell lung cancer cell line SW-1573 derived by selection with low concentrations of doxorubicin or vincristine. Selection with either drug nearly always resulted in MDR clones. The resistance of these clones could be explained by reduced drug accumulation and was associated with a decrease rather than an increase in the low MDR1 mRNA level. To test whether a decrease in MDR1 mRNA indirectly affected resistance in these cells, we introduced a MDR1-specific hammerhead ribozyme into wild-type SW-1573 cells. Although this led to a substantial reduction in MDR1 mRNA, it did not result in resistance. In all resistant clones we found an altered form of the multidrug resistance-associated protein (MRP), migrating slightly slower during SDS-polyacrylamide gel electrophoresis than MRP in parental cells. This altered MRP was also present in non-P-gp MDR somatic cell hybrids of the SW-1573 cells, demonstrating a clear linkage with the MDR phenotype. Treatment of crude cellular membrane fractions with N-glycanase, endoglycosidase H or neuraminidase showed that the altered migration of MRP on SDS-PAGE is due to a post-translational modification. There was no detectable difference in sialic acid content. In most but not all doxorubicin-selected clones, this MDR phenotype was accompanied by a reduction in topo II alpha mRNA level. No reduction was found in the clones selected with vincristine. We conclude from these results that selection of the SW-1573 cell line for low levels of doxorubicin or vincristine resistance, predominantly results in MDR with reduced drug accumulation associated with the presence of an altered MRP protein. This mechanism can be accompanied by other resistance mechanisms, such as reduced topo II alpha mRNA in case of doxorubicin selection.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1995

Functional detection of MDR1/P170 and MRP/P190-mediated multidrug resistance in tumour cells by flow cytometry.

Multidrug resistance (MDR) in tumour cells is often caused by the overexpression of the plasma membrane drug transporter P-glycoprotein (P-gp) or the recently discovered multidrug resistance-associated protein (MRP). In this study we investigated the specificity and sensitivity of the fluorescent probes rhodamine 123 (R123), daunorubicin (DNR) and calcein acetoxymethyl ester (calcein-AM) in order to detect the function of the drug transporters P-gp and MRP, using flow cytometry. The effects of modulators on the accumulation and retention of these probes were compared in several pairs of sensitive and P-gp- as well as MRP-overexpressing cell lines. R123, in combination with the modulator PSC833, provided the most sensitive test for detecting P-gp-mediated resistance. Moreover, in a 60 min drug accumulation assay R123 can be regarded as a P-gp-specific probe, since R123 is not very efficiently effluxed by MRP. In contrast to R123, a 60 min DNR or calcein-AM accumulation test could be used to detect MRP-mediated resistance. The MRP-specific modulator genistein could be used in combination with DNR, but not with calcein-AM. Vincristine (VCR) can be used to increase the cellular uptake of calcein-AM in MDR cells, but is not specific for MRP. Thus, although the combination of DNR with genistein appeared to be as sensitive as the combination of calcein-AM with VCR, the former may be used to probe specific MRP activity whereas the latter provides a combined (P-gp + MRP) functional MDR parameter. With these functional assays the role and relative importance of P-gp and MRP can be studied in, for example, haematological malignancies.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1995

The drug resistance-related protein LRP is the human major vault protein.

Multidrug-resistant cancer cells frequently overexpress the 110-kD LRP protein (originally named Lung Resistance-related Protein). LRP overexpression has been found to predict a poor response to chemotherapy in acute myeloid leukaemia and ovarian carcinoma. We describe the cloning and chromosome localization of the gene coding for this novel protein. The deduced LRP amino acid sequence shows 87.7% identity with the 104-kD rat major vault protein. Vaults are multi-subunit structures that may be involved in nucleo-cytoplasmic transport. The LRP gene is located on chromosome 16, close to the genes coding for multidrug resistance-associated protein and protein kinase C-beta, and may mediate drug resistance, perhaps via a transport process.

Amino Acid Sequence↗

PubMed1995

A monoclonal antibody against human beta-glucuronidase for application in antibody-directed enzyme prodrug therapy.

The selectivity of anticancer agents may be improved by antibody-directed enzyme prodrug therapy (ADEPT). The immunogenicity of antibody-enzyme conjugates and the low tumor to normal tissue ratio calls for the use of a human enzyme and the development of a monoclonal antibody (MAb) against that enzyme for rapid clearance of the conjugate from the circulation. We isolated beta-glucuronidase from human liver. BALB/c mice were immunized with the roughly purified human liver beta-glucuronidase and we obtained an MAb designated 105. Immunoblotting showed reactivity with native tetrameric human beta-glucuronidase. MAb 105 neither bound to enzyme from bovine liver, rat liver, or mouse liver nor reacted with other human lysosomal enzymes. The antibody appeared to be useful to further purify human beta-glucuronidase from human liver or human placenta to homogeneity by affinity chromatography. MAb 105 did not inhibit the activity of human beta-glucuronidase. When human beta-glucuronidase was injected i.v. into BALB/c mice, the newly generated MAb 105 could indeed accelerate the clearance of the enzyme with a 50% drop in its activity within 5 min.

Animals↗

PubMed1995

Cross-reactivity of human nickel-reactive T-lymphocyte clones with copper and palladium.

Twenty Ni-reactive T-lymphocyte clones were obtained from eight different donors and analyzed for their ability to cross-react with other metals. All Ni-reactive T-lymphocyte clones were CD4+CD8- and recognized Ni in association with either HLA-DR or -DQ molecules. Based on the periodic table of the elements, the metals Cr, Fe, Co, Cu, and Zn from the same horizontal row as Ni, and Pd and Pt from the same vertical row, were selected to study T-lymphocyte clone cross-reactivity. Distinct cross-reactivity patterns were found that could be divided into three major groups: Ni-reactive T-lymphocyte clones i) cross-reacting with Cu, ii) cross-reacting with Pd, or iii) without cross-reactivity. Major histocompatibility complex class II-restriction patterns of Cu- and Pd-induced proliferative responses did not differ from those for the Ni-induced responses. In vitro cross-reactivities with Cu and Pd may be favored by their bivalency and location next to Ni in the periodic table, and the similarity of these metals to Ni in binding to histidine residues of peptides in the pocket of major histocompatibility complex class II molecules. The present findings suggest that Cu and Pd hypersensitivities, which are occasionally observed in Ni-allergic patients, may be due to cross-reactivities at the T-cell clonal level rather than to concomitant sensitization.

Cells, Cultured↗

PubMed1995

Differences in the intrinsic capacity of peripheral blood mononuclear cells to produce tumor necrosis factor alpha and beta in patients with inflammatory bowel disease and healthy controls.

BACKGROUND: Tumor necrosis factor alpha (TNF alpha) and beta (TNF beta) appear to play an important role in the regulation of the inflammatory response. The aim of the present study was to investigate the intrinsic capacity of peripheral blood mononuclear cells (PBMC) to produce these cytokines. METHODS: PBMC from 41 patients with Crohn's disease (CD), with ulcerative colitis (UC), and 23 healthy controls (HC) were cultured for 48 h in the presence of the T-cell activators anti-CD3 and anti-CD28. Biologically active total TNF (TNF alpha and beta), TNF alpha, and TNF beta production were measured using a bioassay for biologically active TNF and specific TNF alpha and TNF beta enzyme-linked immunosorbent assays. RESULTS: Large interindividual differences in TNF production were observed. Production of biologically active TNF after T-cell stimulation was significantly decreased in UC patients as compared with HC and CD patients (median, 337 U/ml, 800 U/ml, and 1050 U/ml, respectively). Stimulated TNF alpha production in UC patients (median, 432 U/ml) and in CD patients (median, 537 U/ml) did not differ statistically significantly from HC (median, 730 U/ml) as compared with HC and UC patients(median, 800 U/ml and 837 U/ml, respectively). CONCLUSIONS: These findings support the concept that UC and CD are homogeneous, clearly distinguishable disease entities but rather a heterogeneous group of diseases. Studies directed to assess the immunogenetic background of these different disease manifestations in IBD are underway.

Adult↗

PubMed1995

Expression of the multidrug resistance-associated protein (MRP) gene in human cancers.

We determined the expression of a newly recognized drug resistance gene, the multidrug resistance-associated protein (MRP) gene, [Cole et al., Science (Washington DC), 258: 1650-1654, 1992], in normal human tissues and in >370 human tumor biopsies using a quantitative RNase protection assay and immunohistochemistry. MRP mRNA appeared to be ubiquitously expressed at low levels in all normal tissues, including peripheral blood, the endocrine glands (adrenal and thyroid), striated muscle, the lymphoreticular system (spleen and tonsil), the digestive tract (salivary gland, esophagus, liver, gall bladder, pancreas, and colon), the respiratory tract (lung), and the urogenital tract (kidney, bladder, testis, and ovary). The human cancers analyzed could be divided into three groups with regard to MRP expression. Group 1 consists of tumors that often exhibit high to very high MRP mRNA levels (e.g., chronic lymphocytic leukemia). Group 2 comprises the tumors that often exhibit low, but occasionally exhibit high MRP mRNA expression (e.g., esophagus squamous cell carcinoma, non-small cell lung cancer, and acute myelocytic leukemia). Group 3 comprises the tumors with predominantly low levels of MRP mRNA, comparable to the levels found in normal tissues (e.g., other hematological malignancies, soft tissue sarcomas, melanoma, and cancers of the prostate, breast, kidney, bladder, testis, ovary, and colon). Using the MRP-specific mAbs MRPr1 and MRPm6, we confirmed the elevated MRP mRNA levels in tumor tissues by immunohistochemistry. We conclude that hyperexpression of MRP is observed in several human cancers, and that additional studies are needed to assess the clinical relevance of MRP.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1994

Overexpression of the gene encoding the multidrug resistance-associated protein results in increased ATP-dependent glutathione S-conjugate transport.

The multidrug resistance-associated protein (MRP) is a 180- to 195-kDa glycoprotein associated with multidrug resistance of human tumor cells. MRP is mainly located in the plasma membrane and it confers resistance by exporting natural product drugs out of the cell. Here we demonstrate that overexpression of the MRP gene in human cancer cells increases the ATP-dependent glutathione S-conjugate carrier activity in plasma membrane vesicles isolated from these cells. The glutathione S-conjugate export carrier is known to mediate excretion of bivalent anionic conjugates from mammalian cells and is thought to play a role in the elimination of conjugated xenobiotics. Our results suggest that MRP can cause multidrug resistance by promoting the export of drug modification products from cells and they shed light on the reported link between drug resistance and cellular glutathione and glutathione S-transferase levels.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1994

PEG-IL-2 therapy of advanced cancer in the guinea pig. Impact of the primary tumor and beneficial effect of cyclophosphamide.

The efficacy of tumor therapy using polyethylene-glycol-modified interleukin-2 (PEG-IL-2), alone or in combination with cyclophosphamide, was studied in advanced metastatic disease in the guinea pig. Line 10 (L10) tumor cells appeared in the axillary lymph node only 7 days after intradermal tumor-cell inoculation, and lymph-node leukocytes were almost completely replaced by tumor cells on day 28. Local treatment of the intradermally growing L10 hepatocarcinoma in the guinea pig with a relatively low dose of PEG-IL-2 resulted in regression of the primary tumor and prevention of lymph-node metastases. Therapy was completely curative (4 out of 5 animals) when started on day 7 or 14 after tumor-cell inoculation. When started on day 21, therapy was effective in only some (2 out of 5 cured) of the treated animals. Anti-tumor effects against the primary tumor and against lymph-node metastases were observed only after intratumoral (i.t.) administration of PEG-IL-2. Injection of the agent into or near lymph-node metastases in the absence of the primary tumor had no curative effect. In PBS/BSA-treated control animals the primary tumor and metastases grew progressively. In the treatment of far advanced metastatic disease, the combination of i.t. administration of PEG-IL-2 and i.p. injection of cyclophosphamide (Cy) resulted in improved anti-tumoral effects (5/5 guinea pigs were cured) when compared with monotherapy using either agent (one and none out of 5 animals cured, respectively). PBS/BSA heated controls showed progressive tumor-growth. We conclude that large primary tumors and lymph-node metastases can be treated effectively with PEG-IL-2. The i.t. route of administration is of major importance in the treatment of metastases, since administration of PEG-IL-2 near or into the lymph node had no therapeutic effect. Combination of PEG-IL-2 therapy with systemic injections of Cy significantly improved the curative effects of the treatment of advanced metastatic cancer.

Animals↗

PubMed1994

The human multidrug resistance-associated protein MRP is a plasma membrane drug-efflux pump.

The multidrug-resistance associated protein MRP is a 180- to 195-kDa membrane protein associated with resistance of human tumor cells to cytotoxic drugs. We have investigated how MRP confers drug resistance in SW-1573 human lung carcinoma cells by generating a subline stably transfected with an expression vector containing MRP cDNA. MRP-overexpressing SW-1573 cells are resistant to doxorubicin, daunorubicin, vincristine, VP-16, colchicine, and rhodamine 123, but not to 4'-(9-acridinylamino)methanesulfon-m-anisidide or taxol. The intracellular accumulation of drug (daunorubicin, vincristine, and VP-16) is decreased and the efflux of drug (daunorubicin) is increased in the transfectant. The decreased accumulation of daunorubicin is abolished by permeabilization of the plasma membrane with digitonin, showing that MRP can lower the intracellular daunorubicin level against a concentration gradient. Anti-MRP antisera predominantly stain the plasma membrane of MRP-overexpressing cells. We conclude that MRP is a plasma membrane drug-efflux pump.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1994

Immunochemical detection of the multidrug resistance-associated protein MRP in human multidrug-resistant tumor cells by monoclonal antibodies.

We have generated rat and murine monoclonal antibodies against multidrug resistance-associated protein (MRP), a M(r) 180,000-195,000 membrane glycoprotein involved in a non-P-glycoprotein multidrug resistance of human tumor cells. The antibodies were raised against two different segments of MRP and found to be suitable for protein blot analyses, immunohistochemical and cytochemical studies, as well as flow cytometry of permeabilized cells. The antibodies do not cross-react with the human P-glycoproteins. Immunocytochemistry using MRP-overexpressing tumor cells of different histogenetic origins showed that MRP is predominantly located in the plasma membrane. Immunoelectron microscopy confirmed the plasma membrane location of MRP. The MRP antibodies provide a sensitive and specific tool for studies on MRP-mediated multidrug resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1994

Immunoregulation of T cell-mediated skin hypersensitivity.

The recent extensive research on the different functions of T cells differing in cytokine production profiles has opened promising venues for further research on mechanisms and therapeutic options. Clearly, the routing hypothesis as described above still leaves many questions unanswered, such as the question why some chemicals may elicit strong Th2 responses and IgE antibody production even when applied to the skin, without apparent reduction of delayed allergic reactivity (Dearman et al., 1991). The preliminary understanding of regulatory mechanisms in allergic contact dermatitis has not yet led to further therapeutic progress. So far, no methods of permanent desensitization have been devised. Nevertheless, major cell types and mediators involved in allergic contact dermatitis have been identified. How T cells specifically recognize distinct allergens, and how these and other inflammatory cells interact to generate inflammation has begun to be understood. Moreover, the recently defined cellular interaction molecules and mediators provide promising targets for anti-inflammatory drugs. Obviously, drugs found to be effective in preventing severe T cell-mediated conditions, e.g. rejection of a vital organ graft, should be very safe before their use in allergic contact dermatitis would seem appropriate. To date, prudence favours any measure to prevent allergic contact dermatitis, be it through legal actions to outlaw the use of certain materials, or through avoiding personal contacts with these materials. In the meantime, for difficult-to-avoid allergens, further studies on the potential value of tolerogenic treatments should be intensified.

Animals↗

PubMed1994

Persistent augmentation of natural-killer- and T-cell-mediated cytotoxicity in peripheral blood mononuclear cells pulsed in vitro with high-dose recombinant interleukin-2 prior to culturing with a low maintenance dose.

The toxicity of high-dose recombinant interleukin-2 (rIL-2) treatment limits its use in tumour therapies. This paper describes in vitro studies of whether a single, peak rIL-2 dose, followed by low maintenance doses, could enhance the cytotoxic potential of peripheral blood mononuclear cells (PBMC) without inducing a significant sustained release of secondary cytokines, known to contribute to undesirable side-effects of therapy. Pre-pulsing of PBMC with high-dose rIL-2 (16,000 IU/ml for 30 min), followed by low-dose (5 IU/ml) maintenance culturing, was found to induce persistent augmentation of cytotoxic activity towards natural-killer(NK)-sensitive and -insensitive tumour targets, as well as increased T-cell-mediated target cell killing. Under these conditions the level of killing was as high as that achieved by higher maintenance doses (20-100 IU/ml). Although not reflected by overexpression of cell surface markers, enhanced activation of cytotoxic capacities by high-dose pre-pulsing remained clearly apparent for at least 12 days of culture. Increased secondary cytokine production (tumour necrosis factor, interleukin-6 and interferon gamma) was only evident during the first 24-72 h after pulsing, and not at later stages of culturing at the low maintenance dose of 5 IU rIL-2/ml. These results may warrant a human phase-1 B study to investigate the in vivo effect of high-dose prepulsing, followed by low-dose maintenance.

Animals↗

PubMed1994

Reduced expression of distinct T-cell CD molecules by collagenase/DNase treatment.

DNase/collagenase treatments are widely used to obtain single-cell suspensions of tumour cells and tumour-infiltrating T lymphocytes (TIL) from solid tumours. Since the functional integrity of such cells has been questioned, we have studied whether treatments with commonly used preparations of these enzymes could affect the expression of lymphocyte surface molecules and lymphocyte proliferative responsiveness. With peripheral-blood-derived T cells as a model, flow-cytometric analysis revealed strongly reduced expression of distinct CD molecules for each enzyme, notably CD2, CD4, CD8 and CD44 for DNase, and CD4, CD14, CD16, and CD56 for collagenase. The effects were found to be due to protease contaminations present in all but the purest enzyme preparations tested. Addition of serum or trypsin inhibitor abolished the effects. Since serum-free media are widely used to expand tumour-infiltrating T cells for clinical therapeutic use, data from early phenotypic analyses can be strongly misleading. Even after an 18-h rest period following the enzyme treatments, re-expression of the affected membrane markers was still far from complete. On the other hand, despite strongly reduced expression of CD2 molecules on the lymphocyte membrane, anti-CD2-induced proliferation was not affected, showing the redundancy of this signal molecule. Since other important T cell activation molecules (TCR, CD3, CD28) were not affected by enzymatic treatment, the use of expensive, highly purified collagenase/DNase preparations does not seem to be mandatory in clinical studies with expanded TIL.

Antigens, CD↗

PubMed1994

The development of anti-interleukin-2 (IL-2) antibodies in cancer patients treated with recombinant IL-2.

Serum samples from 217 cancer patients participating in phase I/II clinical trials were analysed for the development of anti-interleukin-2 (IL-2) antibodies. Patients received recombinant human IL-2 (rIL-2) by continuous intravenous infusion (c.i.v.; n = 86) or by subcutaneous (s.c.) injections (n = 131). Both patient groups developed anti-rIL-2 antibodies as detected by ELISA with similar frequencies and titres: 52% (median titre, 23) and 47% (median titre, 24), respectively. Using an IL-2-dependent T-cell proliferation assay, sera from 5 c.i.v.-treated patients (6%) and 13 s.c.-treated patients (10%) exhibited neutralising activity. Immunoabsorption studies with rIL-2-coated beads, demonstrated that in 8 of 15 patients with neutralising sera, the neutralising activity was correlated with specific anti-rIL-2 immunoglobulin. All 8 patients had received at least two cycles of rIL-2 by s.c. injections. Specific IL-2 neutralising activity affected both recombinant and natural IL-2 in all 8 patients. Development of anti-rIL-2 antibodies, irrespective of whether these exhibited neutralising activity or not, did not affect the frequency or duration of clinical responses.

Antibodies, Neoplasm↗

PubMed1994

Non-sensitizing epicutaneous skin tests prevent subsequent induction of immune tolerance.

Oral administration of nickel or chromium to naive guinea pigs results in immune unresponsiveness to subsequent induction of allergic contact hypersensitivity. Such "oral tolerance" depends on the oral dose, is antigen specific, T-suppressor-cell mediated, and very persistent. In contrast, oral antigen administration to sensitized animals results at best in transient desensitization. Here we report that even non-sensitizing epicutaneous skin contacts prevented the subsequent induction of oral tolerance. These data support the view that primed T cells are less sensitive to suppressor T-cell function than naive T cells.

Administration, Oral↗

PubMed1994

Augmentation of antigen-specific lymphoproliferative responses in vitro by biological response modifiers.

The detection of antigen-specific T cell responsiveness, particularly of resting memory lymphocytes, in cultures of peripheral blood mononuclear cells (PBMC) may be hampered by a less than optimal antigen presentation in vitro. Augmented sensitivity of the test system may be achieved by the addition of reagents with a beneficial effect on lymphocyte and antigen-presenting cell (APC) functions. In this study the effect of several biological response modifiers on antigen-specific T cell proliferation was determined, using nickel sulphate and tetanus toxoid as test antigens. IL-1 alpha (100 U/ml), interferon-gamma (IFN-gamma) (10 U/ml), and indomethacin (2 microM) were found to significantly enhance nickel-induced proliferation in PBMC cultures from nickel-hypersensitive donors (n = 6). Tetanus-induced proliferation (n = 5) was similarly enhanced, both by the above supplements and by the addition of polyethylene glycol (PEG) or a neuraminidase treatment of the PBMC before culture. The addition to PBMC cultures of a combination of IL-1 alpha (30 U/ml), IFN-gamma (10 U/ml), and indomethacin (2 microM) is recommended to specifically enhance antigen-induced lymphoproliferative signals.

Antigen Presentation↗