MDR1 gene-specific monoclonal antibody C494 cross-reacts with pyruvate carboxylase.
Overexpression of P-glycoprotein, the plasma membrane protein product of the MDR1 gene, is a major determinant in the development of resistance to a large number of cancer chemotherapeutic agents. A battery of antibodies, including the MDR1 gene-specific monoclonal antibody (mAb) C494, is used to evaluate human tissues in clinical multidrug resistance surveillance and modulation trials. In rat liver fractions, we report that mAb C494 strongly cross-reacted with a nonmembranous M(r) approximately 130,000 protein, comigrating with core-glycosylated human MDR1 on 7% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By immunoblotting and microsequence analysis, this protein was identified as pyruvate carboxylase (PC), an abundant mitochondrial enzyme. A search of the National Center for Biotechnology Information data base, using the epitope-specific sequence of mAb C494, revealed that PC (mouse) contains four of the five most reactive amino acids (TLEG), located near the COOH-terminal end of PC at positions 1167-1170. mAb C494 specifically reacted with PC purified from bovine liver; immunoreactivity was completely abolished by preincubating mAb C494 in the presence of excess synthetic C494 epitope-specific peptide. Furthermore, in cryosections of human skeletal muscle, a tissue known not to express P-glycoprotein, peptide-displaceable immunohistochemical staining with mAb C494 showed a distinct mitochondrial pattern specific to type 1 fibers. Variable immunostaining results were obtained with formaldehyde-fixed, paraffin-embedded muscle and isolated liver mitochondrial preparations. In summary, mAb C494 cross-reacted strongly with rat, bovine, and human PC. Caution is warranted in interpretation of immunoblots and immunohistochemical sections with this putative MDR1 gene-specific mAb.
Rao
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Department of Radiology, Harvard Medical School, Brigham and Women's Hospital, Boston, Massachusetts 02115.
Anthony
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Piwnica-Worms
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D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000276:immunology*; D000918:Antibody Specificity; D002352:Carrier Proteins / Q000032:analysis / Q000276:immunology; D002417:Cattle; D002462:Cell Membrane / Q000737:chemistry / Q000201:enzymology; D003429:Cross Reactions; D004351:Drug Resistance / Q000235:genetics; D000939:Epitopes / Q000276:immunology; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D008099:Liver / Q000201:enzymology; D008562:Membrane Glycoproteins / Q000032:analysis / Q000276:immunology; D008930:Mitochondria, Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D009132:Muscles / Q000737:chemistry; D009954:Ornithine Carbamoyltransferase / Q000276:immunology; D011766:Pyruvate Carboxylase / Q000032:analysis / Q000276:immunology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D000254:Sodium-Potassium-Exchanging ATPase / Q000276:immunology; D013347:Subcellular Fractions / Q000737:chemistry / Q000201:enzymology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D000939:Epitopes; D008562:Membrane Glycoproteins; D009954:Ornithine Carbamoyltransferase; D011766:Pyruvate Carboxylase; D000254:Sodium-Potassium-Exchanging ATPase