Internal duplication and homology with bacterial transport proteins in the mdr1 (P-glycoprotein) gene from multidrug-resistant human cells

We have characterized the normal human tissue distribution and tumor expression of the human multidrug resistance gene (MDR1) product P-glycoprotein (Pgp) by immunohistochemical staining of frozen tissue sections of human normal and tumor tissues, using three mouse monoclonal antibodies (MAb) which recognize at least two different epitopes of Pgp. Pgp expression on normal human tissues was detected in specialized epithelial cells with secretory/excretory functions, trophoblasts in the placenta, and on endothelial cells of capillary blood vessels at blood-tissue barrier sites. There were significant differences in the staining patterns of these MAb. Mouse MAb HYB-241 and HYB-612 each recognize an extracellular epitope of Pgp, whereas mouse MAb C219 detects a carboxy terminal intracellular epitope and has recently been reported to crossreact with the MDR3 gene product. HYB-241 and HYB-612 strongly stain endothelial cells and trophoblasts, whereas C219 is weakly positive or unreactive on these cells. Likewise, C219 strongly stains the biliary pole of hepatocytes, skeletal and heart muscle fibers, whereas HYB-241 and HYB-612 are unreactive on these cells. Immunopathological studies were performed on a wide variety of human tumors. Pgp expression on human tumors was most commonly detected in colon. renal, and adrenal carcinomas; rarely in lung and gastric carcinomas and certain germ cell tumors; and was undetectable in breast and endometrial carcinomas tested. Few sarcomas and none of the melanomas, neuroblastomas, gliomas, and pheochromocytomas had detectable Pgp expression. Intensity and pattern of staining varied among different cases of a given tumor type; although homogeneous immunoreactivity was observed, heterogeneity of expression in a single histological section was more common. The finding of Pgp expression in a variety of normal tissues with diverse physiological functions suggests that the role of Pgp may not be limited to excretion of xenobiotics. Pgp expression in capillaries of the brain and testis may explain the failure of drugs such as vincristine and actinomycin-D to penetrate into these tissues, allowing them to remain as pharmacological sanctuaries for malignant cells. Although Pgp expression can now be detected in a variety of human tumors, further studies are needed to establish the possible significance of this finding.

Expression of the multidrug resistance gene product (P-glycoprotein) in human normal and tumor tissues.

The plasma membranes of hamster, mouse, and human tumor cell lines that display multiple resistance to drugs were examined by gel electrophoresis and immunoblotting. In every case, increased expression of a 170,000-dalton surface antigen was found to be correlated with multidrug resistance. This membrane component is of identical molecular size and shares some immunogenic homology with the previously characterized P-glycoprotein of colchicine-resistant Chinese hamster ovary cells. This finding may have application to cancer therapy.

https://science.sciencemag.org/content/sci/221/4617/1285.full.pdf

Cell surface P-glycoprotein associated with multidrug resistance in mammalian cell lines.

Overexpression of a novel anionic glutathione transferase in multidrug-resistant human breast cancer cells.

Certain calcium channel blockers bind specifically to multidrug-resistant human KB carcinoma membrane vesicles and inhibit drug binding to P-glycoprotein.

Glycoproteins and gangliosides of spontaneously transformed Chinese hamster lung cells (DC-3F) and of vincristine- and daunorubicin-resistant sublines of DC-3F have been compared. These resistant cells and also actinomycin D-resistant cells have been shown to have reduced permeability to drug and to exhibit some degree of mutual cross-resistance to all three agents. Glycoproteins of tritiated glucosamine-labeled membrane preparations from drug-sensitive and-resistant cells were separated by sodium dodecyl sulfate-polyacrylamide and two-dimensional gel electrophoresis. Analysis of the cylindrical gels revealed that drug-resistant cells synthesize a major glycoprotein species which is much larger (Mr about 150,000) than the major species synthesized by control DC-3F cells (Mr about 100,000). Data obtained with a revertant DC-3F/AD × subline exhibiting a 45-fold decrease in resistance as well as with vincristine-resistant DC-3F/VCRd revertant cells with a 20-fold decrease in resistance to the Vinca compound indicate a concomitant decrease in amount of the Mr 150,000 species and an increase in the Mr 100,000 species characterizing drugsensitive DC-3F cells, thereby substantiating the observed relationship between drug resistance and plasma membrane glycoprotein expression. Examination of the major glycopeptide species characteristic of the drug-resistant cell lines by two-dimensional electrophoresis revealed that the Mr 150,000 glycopeptide appears to be a family of glycopeptides with an isoelectric point range of 6.5 to 7.5. The Mr 100,000 species of control cells also comprises a heterogeneous group of acidic peptides with apparent isoelectric points near 5. Gangliosides labeled with [14C]glucosamine were extracted and separated by thin-layer chromatography. DC-3F cells synthesize a full complement of gangliosides, whereas daunorubicin-resistant cells appear to be blocked at the level of hematosides (GM3), as observed previously for actinomycin D-resistant cells. Cells highly resistant to vincristine synthesize the full complement of gangliosides seen in the parental line but contain relatively more GM3 and GM2. Membrane glycoprotein and ganglioside changes in the drug-resistant cells are concomitants of the development of resistance to vincristine, daunorubicin, or actinomycin D. The changes are also correlated with the previously observed alterations in cell morphology and the suppression of the malignant phenotype occurring in the cells selected for resistance to these drugs.

Alteration of Plasma Membrane Glycopeptides and Gangliosides of Chinese Hamster Cells Accompanying Development of Resistance to Daunorubicin and Vincristine

Development of resistance to actinomycin D, daunomycin, or vincristine in Chinese hamster cells growing in vitro resulted in reversion to or retention of normal phenotypes in comparison to spontaneously transformed drug-sensitive parent populations Sublines resistant or cross-resistant to actinomycin D showed reduced uptake of antibiotic in proportion to degree of resistance The cells with acquired resistance were either weakly tumorigenic or nontumorigenic when tested in the cheek pouches of weanling Syrian hamsters treated with cortisone In contrast, parent cells and several amethopterin (methotrexate)-resistant sublines produced many tumors Antibiotic- and Vinca-alkaloid-resistant cell lines showed oriented growth patterns often associated with the behavior of normal cells in culture; antibiotic-sensitive, tumorigenic lines had morphologic characteristics of malignant cells The altered cell membrane properties that accompanied development of resistance or cross-resistance to actinomycin D appeared to account also for the lower oncogenic potential and greater cell adhesiveness of resistant cells relative to their malignant counterparts

Membrane-Mediated Drug Resistance and Phenotypic Reversion to Normal Growth Behavior of Chinese Hamster Cells

Plasma membrane proteins and glycoproteins have been isolated from Chinese hamster cells of the spontaneously transformed DC-3F parental cell line and the DC-3F/AD X line with a high level of acquired resistance to actinomycin D Plasma membrane preparations from both cell lines band at 116 g/ml after isopycnic centrifugation We present evidence to indicate differences in the leucylpeptide backbones of the antibiotic-sensitive cells and the drug-resistant DC-3F/AD X cells In addition, there are differences in the plasma membrane glycopeptides of the two cell lines as revealed by sodium dodecyl gel electrophoresis Drug-resistant cells synthesize a surface glycopeptide which is much larger than the major one present on the drug-sensitive cells Both of these cell lines are devoid of 5′-nucleotidase and alkaline phosphatase activities The role of plasma membrane protein differences in drug-resistant cells is discussed

Plasma membrane proteins and glycoproteins from Chinese hamster cells sensitive and resistant to actinomycin D.

In developing a chemotherapeutic program for children with disseminated neuroblastoma, we established three human neuroblastoma lines in cell culture to study the effects of dibutyryl cyclic AMP, papaverine, 5-trifluoromethyl-2'-deoxyuridine, and cyclophosphamide on cell growth, biochemical behavior, and morphology. Based upon our studies, a clinical treatment program was designed. We have treated 15 patients with disseminated neuroblastoma and have established the optimum dose range and sequence of these drugs. Early results were promising; plans for continuation of clinical and experimental studies were discussed.

/pdf/a-rationale-for-the-treatment-of-metastatic-neuroblastoma-158jbjao1y.pdf

A Rationale for the Treatment of Metastatic Neuroblastoma

Modulation of IL-1R on human neutrophils was investigated after in vitro treatment of cells with human recombinant (hr) granulocyte (G)-CSF, hrgranulocyte-macrophage (GM)-CSF, hrCSF-1, hrIL-1 alpha, hrIL-2, hrIL-3, hrIL-4, hrIL-5, hrIL-6, hrIL-7, transforming growth factor-beta 1, or hrTNF-alpha. At 4 degrees C, 125I-IL-1 binding was competed by IL-1 but not by other cytokines tested. At 37 degrees C, GM-CSF, TNF-alpha, and IL-1 decreased 125I-IL-1 binding in a dose-dependent manner. Kinetic studies showed that GM-CSF reduced > 45% IL-1 binding within 15 min but later (8 h) produced a > 2-fold increase. In contrast, TNF decreased > 85% IL-1 binding within 15 min with a recovery of > 80% relative to that of control after 24 h. Scatchard analysis revealed that TNF or GM-CSF down-modulation of IL-1 binding was due to a decrease of IL-1R number. Further studies showed that dexamethasone and GM-CSF (or G-CSF) synergistically increased IL-1 binding after 8 h. This synergistic modulation was a cytokine dose- and time-dependent process, and was due to an increase in IL-1R numbers rather than a change in binding affinity. In addition, human bone marrow neutrophils, cord blood neutrophils, and several human hematopoietic cell lines (HL-60, U-937, and AML-193) responded to dexamethasone and GM-CSF (or G-CSF) with a superadditive increase in IL-1 binding. Because mammalian systems respond to bacterial endotoxins with secretion of TNF, IL-1, glucocorticoids, G-CSF and GM-CSF, our results shed additional light on this highly regulated cytokine network and revealed a novel role for GM-CSF.

Robert H. F. Peterson

Papers

Alteration of Plasma Membrane Glycopeptides and Gangliosides of Chinese Hamster Cells Accompanying Development of Resistance to Daunorubicin and Vincristine

Membrane-Mediated Drug Resistance and Phenotypic Reversion to Normal Growth Behavior of Chinese Hamster Cells

Plasma membrane proteins and glycoproteins from Chinese hamster cells sensitive and resistant to actinomycin D.

A Rationale for the Treatment of Metastatic Neuroblastoma

Cytokines and dexamethasone modulation of IL-1 receptors on human neutrophils in vitro.