title: Analysis of MRP gene expression and function in HL60 cells isolated for resistance to adriamycin.
abstract: In an effort to define clearly the basis of non-P-glycoprotein multidrug resistance in HL60/ADR cells, we have analyzed expression of MRP mRNA levels and the MRP-encoded protein in resistant cells and also in resistant cells that have undergone a reversion to drug sensitivity. The results demonstrate that an MRP cDNA containing 5'-end coding sequences reacts with a 6-kb RNA, which is overexpressed in the resistant isolate. As resistant cells revert to drug sensitivity there is essentially a complete loss of the 6-kb RNA. Southern blot analysis indicates that the MRP gene is amplified compared to the copy number found in sensitive cells. Revertant cells no longer contain amplified MRP sequences. Western blot analysis has been conducted using an antibody prepared against the carboxyl terminus (15 amino acids) of the deduced sequence of the MRP-encoded protein. The antibody is reactive with a 190-kDa protein (P190) and with two closely migrating proteins of 65 and 70 kDa (P70), which are overexpressed in plasma membranes and endoplasmic reticulum of resistant cells. Both proteins are greatly reduced in revertant cells. Growth of cells in the presence of tunicamycin demonstrates that both P190 and P70 are glycosylated, with the deglycosylated forms migrating in polyacrylamide gels as proteins of 165 kDa and 45 kDa, respectively. Additional antisera have also been prepared against sequence domains contained in the C-terminal region of P190. These antisera are reactive with both P190 and P70. Antisera directed against sequences of the amino terminal region of P190 do not react with P70.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Division of Biology, Kansas State University, Manhattan 66506-4901.
authors.forename: N
authors.initials: N
authors.lastname: Krishnamachary
authors.forename: L
authors.initials: L
authors.lastname: Ma
authors.forename: L
authors.initials: L
authors.lastname: Zheng
authors.forename: A R
authors.initials: AR
authors.lastname: Safa
authors.forename: M S
authors.initials: MS
authors.lastname: Center
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000032:analysis / Q000096:biosynthesis*; D000595:Amino Acid Sequence; D000906:Antibodies; D015152:Blotting, Northern; D015139:Blotting, Southern; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D004273:DNA, Neoplasm / Q000032:analysis; D004317:Doxorubicin / Q000494:pharmacology*; D018432:Drug Resistance, Multiple / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D015473:Leukemia, Promyelocytic, Acute; D008969:Molecular Sequence Data; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D012334:RNA, Neoplasm / Q000032:analysis / Q000096:biosynthesis; D014407:Tumor Cells, Cultured
chemical_list: D018528:ATP-Binding Cassette Transporters; D000906:Antibodies; D004273:DNA, Neoplasm; D004317:Doxorubicin; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D010455:Peptides; D012334:RNA, Neoplasm