Membrane glycoprotein changes associated with anthracycline resistance in HL-60 cells

• Published in: Cancer chemotherapy and pharmacology 1991-01, Vol.28 (2), p.93-101
• Main Authors: GERVASONI, J. E, TAUB, R. N, HINDENBURG, A. A, ROSADO, M, KRISHNA, S, STEWART, V. J, KNOWLES, D. M, BHALLA, K, ROSS, D. D, BAKER, M. A, LUZKY, J
• Format: Article
• Language: English
• Subjects: Antibiotics, Antineoplastic - pharmacology; Antineoplastic agents; Autoradiography; Biological and medical sciences; Cell Line; Culture Techniques; Drug Resistance; Electrophoresis, Polyacrylamide Gel; Epitopes - isolation & purification; General aspects; Glycosylation - drug effects; Humans; Leukemia, Myeloid - metabolism; Medical sciences; Membrane Glycoproteins - drug effects; Membrane Glycoproteins - metabolism; Pharmacology. Drug treatments; Phenotype; Tetradecanoylphorbol Acetate - pharmacology; Tretinoin - pharmacology; Tunicamycin - pharmacology
• ISSN: 0344-5704; 1432-0843
• DOI: 10.1007/BF00689695

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. Furthe