title: Membrane glycoprotein changes associated with anthracycline resistance in HL-60 cells.
abstract: The glycoproteins on the surface of HL-60/S wild-type, drug-sensitive human leukemia cells and HL-60/AR anthracycline-resistant cells which do not overexpress the P-glycoprotein, were characterized by labeling with [35S]-methionine, NaB[3H4], phosphorus 32, or sodium iodide I 125. HL-60/S and HL-60/AR cell lysates and membrane fractions tagged with [35S]-methionine or phosphorus 32 showed no significant differences in their protein patterns as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by autoradiography. HL-60/S cells labeled with NaB[3H4] yielded glycoproteins that were smeared predominantly in the molecular-weight range of 210,000 and 160,000 Da, with pI values ranging between pH 4 and pH 4.4. In contrast, NaB[3H4]-labeled HL-60/AR cells showed 7-8 discrete glycoproteins within a molecular-weight range of 170,000 and 140,000 Da, with pI values also ranging between pH 4 and pH 4.4. In addition, [3H]-glucosamine incorporation into HL-60/S and HL-60/AR cells revealed that the latter showed lower uptake of [3H]-glucosamine than did the former. Following treatment with tunicamycin, [3H]-glucosamine uptake in HL-60/S cells decreased, whereas that in HL-60/AR cells remained unchanged. Surface-membrane radioiodination of HL-60/S and HL-60/AR cells showed two distinct protein electrophoretic patterns, with differences being observed in both the high-(220-95 kDa) and low-molecular-weight ranges (21 kDa). Flow cytometric analysis of HL-60/S and HL-60/AR cells using myeloid and lymphoid antigen-specific antibodies demonstrated no antigenic differences between HL-60/S and HL-60/AR cells. HL-60/S cells incubated in the presence of tunicamycin, an inhibitor of N-linked glycosylation, or the protein kinase C agonist phorbol 12-myristate 13-acetate (PMA) developed a glycoprotein pattern similar to that observed in HL-60/AR cells. In addition, tunicamycin treatment of HL-60/S cells decreased daunorubicin (DNR) retention and altered its intracellular distribution as compared with that in HL-60/AR cells. These data indicate that HL-60/AR cells do not possess either de novo or amplified high-molecular-weight surface-membrane proteins; instead, existing proteins are hypoglycosylated. These results also show that HL-60/AR cells exhibit the multidrug-resistant phenotype in association with altered membrane glycoproteins of both high (220-95 kDa) and low molecular weight (21 kDa), but without overexpression of the P-glycoprotein. Furthermore, in HL-60/S cells, the multidrug-resistant phenotype is partially inducible by inhibition of N-linked glycosylation of cell-surface proteins.
authors.affiliation: Department of Medicine, Columbia University, New York, NY 10032.
authors.forename: J E
authors.initials: JE
authors.lastname: Gervasoni
authors.forename: R N
authors.initials: RN
authors.lastname: Taub
authors.forename: M
authors.initials: M
authors.lastname: Rosado
authors.forename: S
authors.initials: S
authors.lastname: Krishna
authors.forename: V J
authors.initials: VJ
authors.lastname: Stewart
authors.forename: D M
authors.initials: DM
authors.lastname: Knowles
authors.forename: K
authors.initials: K
authors.lastname: Bhalla
authors.forename: D D
authors.initials: DD
authors.lastname: Ross
authors.forename: M A
authors.initials: MA
authors.lastname: Baker
authors.forename: J
authors.initials: J
authors.lastname: Lutzky
mesh_terms: D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D001345:Autoradiography; D002460:Cell Line; D046508:Culture Techniques; D004351:Drug Resistance; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000302:isolation & purification; D006031:Glycosylation / Q000187:drug effects; D006801:Humans; D007951:Leukemia, Myeloid / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000187:drug effects* / Q000378:metabolism; D010641:Phenotype; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014212:Tretinoin / Q000494:pharmacology; D014415:Tunicamycin / Q000494:pharmacology*
chemical_list: D000903:Antibiotics, Antineoplastic; D000939:Epitopes; D008562:Membrane Glycoproteins; D014415:Tunicamycin; D014212:Tretinoin; D013755:Tetradecanoylphorbol Acetate