Biomedical subjects

E B Mechetner

Publications and source records attributed to E B Mechetner.

At least 19 recordsLinked to original sources

PubMed1999

Altered expression of the MYCN oncogene modulates MRP gene expression and response to cytotoxic drugs in neuroblastoma cells.

We have recently shown a close correlation between expression of the Multidrug Resistance-associated Protein (MRP) gene and the MYCN oncogene and provided evidence that high MRP expression is a powerful independent predictor of poor outcome in neuroblastoma (Norris et al., New Engl. J. Med., 334, 231-238, 1996). The effect of MYCN down-regulation on MRP expression and response to cytotoxic drugs was investigated in NBL-S neuroblastoma cells transfected with MYCN antisense RNA constructs. Concomitant with MYCN down-regulation, the level of MRP expression was decreased in the NBAS-4 and NBAS-5 antisense transfectants. These cells demonstrated significantly increased sensitivity to the high affinity MRP substrates vincristine, doxorubicin, sodium arsenate and potassium antimony tartrate, but not to the poor MRP substrates, taxol or cisplatin. Similarly, transfection of full-length MYCN cDNA into SH-EP neuroblastoma cells resulted in increased MRP expression and significantly increased resistance specifically to MRP substrates. The results provide evidence for the MYCN oncogene influencing cytotoxic drug response via regulation of MRP gene expression. Our data also provide a link between the malignant and chemoresistant phenotypes of this childhood malignancy.

ATP-Binding Cassette Transporters↗

PubMed1998

Activation of the LRP (lung resistance-related protein) gene by short-term exposure of human leukemia cells to phorbol ester and cytarabine.

Treatment-induced secondary drug resistance of tumor cells is a major cause of relapsed disease and therapeutic failure in cancer patients. It has been shown that the expression of the multidrug resistance MDR1/P-glycoprotein gene could be induced by short-term in vitro exposure of cells to protein kinase C (PKC) agonists or different chemotherapeutic drugs. We studied whether other genes involved in drug resistance are regulated by similar signaling pathways. Transient (up to 24 h) treatment of HL-60 or K562 leukemia cells with phorbol 12-myristate 13-acetate (TPA) resulted in increased steady-state level of LRP (lung resistance-related protein) mRNA and protein. Among conventional chemotherapeutic drugs tested, only cytarabine (Ara C) induced the LRP mRNA expression though no increase in LRP protein was detected. LRP gene activation was not detectable in either H9 T-cell leukemia or in solid carcinoma cell lines (BT-20, ZR-75-1, and SW 1573). None of the agents influenced the levels of MRP (multidrug resistance-associated protein) mRNA in any cell line tested. In HL-60 cells, the LRP activation by TPA or Ara C was sustained for at least 23 days after withdrawal of inducing agents. bis-Indolylmaleimide I, a potent PKC inhibitor, attenuated TPA-induced LRP activation. In contrast, the inhibitor had no effect on the LRP induction by Ara C. These data indicate that the LRP gene can be activated by different mechanisms, some of which involve PKC.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1997

P-glycoprotein function involves conformational transitions detectable by differential immunoreactivity.

The MDR1 P-glycoprotein (Pgp), a member of the ATP-binding cassette family of transporters, is a transmembrane ATPase efflux pump for various lipophilic compounds, including many anti-cancer drugs. mAb UIC2, reactive with the extracellular moiety of Pgp, inhibits Pgp-mediated efflux. UIC2 reactivity with Pgp was increased by the addition of several Pgp-transported compounds or ATP-depleting agents, and by mutational inactivation of both nucleotide-binding domains (NBDs) of Pgp. UIC2 binding to Pgp mutated in both NBDs was unaffected in the presence of Pgp transport substrates or in ATP-depleted cells, whereas the reactivities of the wild-type Pgp and Pgps mutated in a single NBD were increased by these treatments to the level of the double mutant. These results indicate the existence of different Pgp conformations associated with different stages of transport-associated ATP hydrolysis and suggest trapping in a transient conformation as a mechanism for antibody-mediated inhibition of Pgp.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1996

The multidrug resistance phenotype confers immunological resistance.

Multidrug resistance (MDR), which is due, in part, to the overexpression of P-glycoprotein, confers resistance to a variety of natural product chemotherapeutic agents such as daunorubicin, vincristine, and colchicine. RV+ cells are a P-glycoprotein overexpressing variant of the HL60 myeloid leukemia cell line. In addition to classic MDR, RV+ cells displayed relative resistance to complement-mediated cytotoxicity with both immunoglobulin G and M antibodies against different cell surface antigens, but not to antibody-dependent cellular cytotoxicity and lymphokine-activated killing. Complement resistance was reversed both by treatment with verapamil and with specific monoclonal antibodies (mAbs) capable of binding to P-glycoprotein and blocking its function. To further confirm that the resistance of RV+ cells was not a consequence of the selection of the cells on vincristine, a second system involving P-glycoprotein infectants was also investigated. K562 cells infected with the MDR1 gene, which were never selected on chemotherapeutic drugs, also displayed relative resistance to complement-mediated cytotoxicity. This MDR1 infection-induced resistance was also reversed by mAbs that bind to P-glycoprotein. Therefore, the MDR phenotype as mediated by P-glycoprotein provides resistance to complement-mediated cytotoxicity. The increased intracellular pH and the decreased membrane potential due to the MDR phenotype may result in abnormal membrane attack complex function. This observation may have implications for the possible mechanisms of action of P-glycoprotein and for a possible physiologic role for P-glycoprotein in protection against complement-mediated autolysis.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1996

Involvement of MDR1 P-glycoprotein in multifactorial resistance to methotrexate.

Cellular resistance to methotrexate (MTX) is believed to be unaffected by expression of MDR1 P-glycoprotein (Pgp), a pleiotropic efflux pump acting on different hydrophobic compounds that enter cells by passive diffusion. A series of human leukemic CCRF-CEM sublines, isolated by multi-step selection for very high resistance to MTX, exhibit multiple mechanisms of MTX resistance, including decreased carrier-mediated uptake of MTX and DHFR gene amplification. These sublines show cross-resistance to drugs of the multi-drug resistance (MDR) family, which is correlated with relative resistance to MTX. The MTX-selected sublines show increased expression and function of the MDR1 gene, based on the measurement of MDR1 mRNA, Pgp and rhodamine 123 accumulation. Sequence analysis of the MDR1 cDNA from MTX-selected CCRF-CEM cells revealed no mutations in the protein coding region. MTX resistance in these cell lines is partially reversible by a Pgp-specific monoclonal antibody (MAb) UIC2 and a monovalent FaB fragment of UIC2. Our results indicate that Pgp can contribute to multifactorial resistance to MTX.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1996

P-glycoprotein confers methotrexate resistance in 3T6 cells with deficient carrier-mediated methotrexate uptake.

P-glycoprotein (Pgp), a transmembrane efflux pump encoded by the MDR1 gene, transports various lipophilic drugs that enter the cell by passive diffusion through the lipid bilayer. Pgp-expressing multidrug-resistant cell lines are not usually cross-resistant to a hydrophilic antifolate methotrexate (MTX). MTX enters cells primarily through a folate carrier, but passive diffusion becomes the primary mode of MTX uptake in carrier-deficient cells. To test if a deficiency in MTX carrier would allow Pgp to confer resistance to MTX, a MTX carrier-deficient cell line (3T6-C26) was infected with a recombinant retrovirus expressing the human MDR1 gene. The infected 3T6-C26 cells showed increased survival in MTX relative to uninfected cells. Multistep selection of the infected cells with vinblastine led to increased Pgp expression and a concomitant increase in resistance to MTX. MTX resistance of Pgp-expressing 3T6-C26 cells was reduced by Pgp inhibitors, including a Pgp-specific monoclonal antibody UTC2. In contrast, the expression and the inhibition of Pgp had no effect on MTX resistance in 3T6 cells with normal carrier-mediated MTX uptake. Thus, a deficiency in the MTX carrier enables Pgp to confer resistance to MTX, suggesting that hydrophilic compounds may become Pgp substrates when such compounds enter cells by passive diffusion.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1996

A new method for a quantitative assessment of P-glycoprotein-related multidrug resistance in tumour cells.

A rapid, functional and quantitative diagnostic method for the estimation of the P-glycoprotein (P-gp)-dependent multidrug resistance is required in the clinical treatment of human tumours, as chemotherapy protocols and resistance-reversing agents could be applied accordingly. In the present work, by using a calcein accumulation method in combination with immunorecognition and drug-resistance studies, a new method is described for the quantitative estimation of the expression and function of the multidrug transporter. MDR1-transfected and drug-selected tumour cell lines with various levels of drug resistance were examined. The expression of P-gp and its cell-surface appearance were assessed by quantitative immunoblotting and by immunofluorescence cytometry. The transport function of the P-gp was assessed by measuring the extrusion of calcein acetoxymethyl ester (AM) with fluorometry and flow cytometry, while in parallel experiments drug resistance was directly examined in cell survival assays. The MDR1 activity factor (MAF), calculated from the calcein AM extrusion assay, is demonstrated to provide a reliable quantitative measure for MDR1 specific activity, reflecting cellular drug resistance. This relatively simple and rapid new functional P-gp assay surpasses the formerly used techniques in both sensitivity and reproducibility.

3T3 Cells↗

PubMed1996

Monoclonal antibodies against P-glycoprotein, an MDR1 gene product, inhibit interleukin-2 release from PHA-activated lymphocytes.

P-glycoprotein (Pgp), a product of the human MDR1 gene, is a member of the ABC superfamily of transporters responsible for the trafficking of biologically active substances across the membrane. In tumors, Pgp is associated with multidrug resistance (MDR), the phenomenon characterized by the ability of cells to efflux structurally diverse lipophilic compounds. It has been demonstrated that Pgp is also expressed on various types of normal human tissues and cells, including hematopoietic stem cells, T, B, and natural killer (NK) cells. The normal physiologic function of Pgp in immune cells is unclear. In this study, we used highly specific and nontoxic monoclonal antibodies (mAbs) against external epitopes of Pgp (mAb UIC2, its monovalent Fab fragments, and mAb MRK16) to inhibit Pgp-mediated efflux and investigate a possible role of Pgp in activated T lymphocytes. We found that the treatment of phytohemagglutinin (PHA)-stimulated peripheral blood leukocytes (PBL) with these mAbs resulted in a significant reduction of interleukin-2 (IL-2) levels in the culture. Early activation events, as measured by intracellular calcium flux, expression of the CD69 early activation marker, and expression of IL-2 mRNA, were not affected by anti-Pgp mAbs. These results suggest that the Pgp efflux pump may be involved in the transport of IL-2 in T lymphocytes.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1994

Expression of erythroblast antigen (AG-EB) in hematopoietic and non-hematopoietic mouse cells during pre- and postnatal ontogenesis. Immunocytochemical study using monoclonal antibody.

It has been shown previously that erythroblast antigen (AG-EB) is an interspecies antigen expressed on the surface of nucleated red cells and reticulocytes. We have recently produced the monoclonal antibody MAE 15 that reacted specifically with murine epitope of AG-EB. Using MAE15 and immunocytochemical techniques on the light and electron microscopic levels the expression of AG-EB was studied in mouse embryonal and adult tissues. In mouse embryos AG-EB was revealed in erythroid cells of the yolk sac, liver and spleen, in epithelial cells of the intestine, salivary and thyroid glands as well as in the skin. The adult pregnant mice displayed AG-EB in cells of the yolk sac, decidua and trophoblast. The normal adult mice definitely expressed AG-EB not only in nucleated erythroid cells of the bone marrow and spleen but also in cells of non-hemopoietic organs and tissues: in epithelium of intestine, some glands and tubules of the kidney and testis; in endothelium of blood brain capillaries; in cells of the glandular layer of the epidermis; in insertion disks of myofibrils. Our findings suggest a possible transport role of AG-EB in different "barrier" cells.

Animals↗

PubMed1993

Binding properties of monoclonal antibodies recognizing external epitopes of the human MDR1 P-glycoprotein.

Monoclonal antibodies (MAbs) recognizing external epitopes of the human MDR1 P-glycoprotein have been used both for the detection of multidrug-resistant cells and as specific inhibitors of P-glycoprotein-mediated multidrug resistance. Using a panel of recently developed transfected or transgenic cell lines containing variants of the human MDR1 and MDR3 P-glycoproteins, we have compared the specificity and binding properties of the previously isolated MAbs MRK16, HYB-241, UIC2 and 4E3, and of the newly isolated MAb 7G4. The removal of 1, 2 or all 3 of the N-glycosylation sites present in the first extracellular loop of MDR1 P-glycoprotein did not significantly affect the binding of these MAbs. In contrast, 20 amino acid deletion in the first extracellular loop of MDR1 P-glycoprotein completely abolished binding of UIC2, whereas the binding of all other MAbs was hardly affected. None of the MAbs tested bound detectably to cell lines containing a high level of the human MDR3 P-glycoprotein. The differences in the binding specificity between UIC2 and the other tested antibodies parallel the reported functional differences in the ability of these antibodies to inhibit P-glycoprotein-mediated drug efflux.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1993

Cytotoxic effect of ricin A-chain conjugates containing monoclonal antibodies against human erythroid cells.

Selective elimination of human erythroblastoid cells by the conjugate of the A-chain of a plant toxin ricin (RA) and monoclonal antibody (MAb) HAE9 (IgM) directed against human erythroblast antigen (Ag-Eb) has been demonstrated. In comparative experiments, MAb HAE3 (IgM) against human glycophorin-A was used. On average, the conjugates obtained contained two A-chain molecules and one antibody molecule. Efficiency of cytotoxic action of native ricin and conjugates was compared both with the amount of binding sites on the surface of K562 cells and the internalization rate of these proteins. The association constants of the proteins proved to be almost the same (ka = 10(8) M-1). The ID50 values were 1.1 x 10(-11), 3.2 x 10(-10) and 3.1 x 10(-9) M for ricin, HAE9/RA and HAE3/RA, respectively. Ammonium chloride at a concentration of 10 mM increases the cytotoxic effect of the HAE9/RA conjugate approximately 10 times and does not change the activity of the HAE3/RA conjugate.

Antibodies, Monoclonal↗

PubMed1993

Comparison of the cytotoxic activity of the immunotoxins with different internalization rate.

In comparative experiments the conjugates of A-chain of a plant toxin ricin (RTA) and monoclonal antibody (MAb) HAE9 (IgM) directed against human erythroblast antigen (Ag-Eb) or Mab HAE3 against human glycophorin-A and immunotoxins (IT) directed to CD5 and CD7 antigens of human T-lymphocytes have been investigated. Proceeding from the number of receptors on a cell, we compared efficiency of the cytotoxic effects of the conjugates with different internalization rate. The efficiency of immunoconjugates against Ags with an extremely high internalization rate was only slightly higher than that of immunoconjugates against Ags with a lower internalization rate. The enhancing effect of ammonium chloride on immunoconjugate cytotoxicity was more pronounced in the case of immunotoxins with a high internalization rate.

Antibodies, Monoclonal↗

PubMed1992

Expression and activity of the multidrug resistance P-glycoprotein in human peripheral blood lymphocytes.

P-glycoprotein (P-gp), the product of the MDR1 (multidrug resistance) gene, is a transmembrane efflux pump for different lipophilic compounds, including many anticancer drugs and fluorescent dyes. We have previously reported that the efflux of fluorescent dyes from lymphoid cells of human bone marrow was directly correlated with the cellular P-gp content. In the present study, we show that human peripheral blood lymphocytes (PBL) also express P-gp, and that P-gp expression correlates with the efflux of fluorescent dyes from PBL. This efflux was suppressed not only by chemical inhibitors of P-gp but also by a P-gp-specific monoclonal antibody UIC2, thus providing direct evidence that it was mediated by P-gp. We have also characterized dye efflux and UIC2 reactivity in specific PBL subsets. P-gp was expressed in the majority of CD56+, CD8+, and CD20+ lymphocytes, but in less than one half of CD4+ cells. P-gp-mediated dye efflux was highly heterogeneous relative to the expression of CD56RA, CD56RO, Leu-8, and HLA-DR antigens. No significant P-gp activity was detectable in CD14+ monocytes. MDR1 expression in normal lymphocytes may be a determinant of multidrug resistance in the corresponding malignancies.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1992

Efficient inhibition of P-glycoprotein-mediated multidrug resistance with a monoclonal antibody.

P-glycoprotein (Pgp), encoded by the MDR1 gene, is an active efflux pump for many structurally diverse lipophilic compounds. Cellular expression of Pgp results in multidrug resistance (MDR) in vitro and is believed to be a clinically relevant mechanism for tumor resistance to chemotherapy. We have developed a mouse monoclonal antibody, UIC2, that recognizes an extracellular epitope of human Pgp. UIC2 inhibited the efflux of Pgp substrates from MDR cells and significantly increased the cytotoxicity of Pgp-transported drugs, under the conditions where no effect was detectable with other anti-Pgp antibodies. Potentiation of cytotoxicity by UIC2 was observed with all the tested drugs associated with MDR (vinblastine, vincristine, colchicine, taxol, doxorubicin, etoposide, actinomycin D, puromycin, and gramicidin D) but not with any of the drugs to which MDR cells are not cross-resistant (methotrexate, 5-fluorouracil, cis-platinum, G418, and gentamicin). The inhibitory effect of UIC2 in vitro was as strong as that of verapamil (a widely used Pgp inhibitor) at its highest clinically achievable concentrations. Our results suggest that UIC2 or its derivatives provide an alternative or supplement to chemical agents for the reversal of MDR in clinical cancer.

3T3 Cells↗

PubMed1992

Monoclonal antibody to alkaline phosphatase from the intestinal mucosa of the harp seal, Phoca groenlandica.

1. Hybridoma secreting a monoclonal antibody APP.1 to the harp seal alkaline phosphatase (A1Ph) was obtained by fusing murine myeloma Sp 2/0 cells with the splenocytes of BALB/c mice immunized with purified isozyme K. 2. The antibody has no effect on the enzyme activity and shows a high affinity for harp seal A1Ph (KD = 8.5 x 10(-10) M). The antibody has similar affinities for the AlPh of harp seal, fur seal, common seal and deer. 3. The antibody APP.1 was coupled to Sepharose and employed in chromatographic purification of the harp seal intestinal AlPh. Alkaline phosphatase isolated on this immunosorbent has a spec. act. of 20,800 units per mg of protein. 4. The antibody-enzyme complex gives an excellent immunocytochemical labeling of tissue sections, cell cultures and smears.

Alkaline Phosphatase↗

PubMed1991

Determination of boron in tissues and cells using direct-current plasma atomic emission spectroscopy.

We have developed a safe, simple, and efficient method for boron determination by means of direct-current plasma atomic emission spectroscopy. Tissues were solubilized by using concentrated sulfuric acid and 70% hydrogen peroxide to digest the samples without the need of high temperatures and pressures. Boron cluster compounds could be measured with sensitivity, precision, and accuracy similar to those of boric acid standards. Results obtained with [(C2H5)3NH]2B12H12, Cs2B12H11SH.H2O, and C15H32B10O6 show that this analytical method is applicable to a variety of compounds with different chemical structures. A sensitivity of 0.1 ppm has been obtained with known standards alone and in a variety of tissue matrices including tumor, blood, liver, skin, and cell suspensions. The measurement of total boron by direct-current plasma atomic emission spectroscopy (DCP-AES) has been achieved with as little as 50 mg of tissue or as few as 5 x 10(7) cells. The procedure is applicable to the analysis of boron in the ppm range with a high degree of precision and accuracy.

Animals↗

PubMed1991

Heterogeneity of peripheral blood reticulocytes: a flow cytometric analysis with monoclonal antibody HAE9 and thiazole orange.

The expression of a human erythroid cell surface antigen recognized by monoclonal antibody (mAB) HAE9 has been studied on peripheral blood reticulocytes by one- and two-color flow cytometry. Total reticulocyte count was determined using Thiazole Orange (TO) and flow cytometry. In normal individuals, 4.56% of reticulocytes were stained by FITC-labeled mAB HAE9. The correlation between reticulocyte percentage by TO and HAE9 staining was 0.828 (P less than 0.0001) in patients with hematocrits less than 0.25. A HAE9-positive reticulocyte percentage of 6-44% was observed when analyzed by two-color flow cytometry with TO and mAB HAE9. These findings, in conjunction with previous studies, suggest that mAB HAE9 recognizes an early, less differentiated population of peripheral blood reticulocytes. Enumeration of immature reticulocytes may be of clinical utility.

Antibodies, Monoclonal↗

PubMed1991

[Monoclonal antibodies to human small-cell lung cancer].

Murine monoclonal antibodies to human small cell lung cancer (SCLC) have been developed and partially characterized. Primary hybridoma clones were screened in the indirect immunofluorescence assay (IFA) on alive H417 cells. Then five clones (IgG1, IgG2a, IgG3 and IgM) non-reactive with normal human bone marrow cells and positively reactive with SCLC tumors were selected. The H417.3 antibody is directed against 47-50kD surface antigens of H417 cells. The antibodies are supposed to be applied for the immunodetection of SCLC metastases to bone marrow and immunotoxin preparations.

Animals↗