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MDR1 Gene-specific Monoclonal Antibody C494 Cross-Reacts with Pyruvate Carboxylase¹

Laboratory of Molecular Radiopharmacology, Department of Radiology [V. V. R., D. P-W.], and Department of Pathology [D. C. A.], Harvard Medical School, Brigham and Women's Hospital and Children's Hospital, Boston, Massachusetts 02115

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 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, paraffinembedded 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.

¹ This work was supported in part by a grant from the Milton Fund, Harvard Medical School, and by NIH Grant CA 09536.

² To whom requests for reprints should be addressed, at Department of Radiology, Brigham and Women's Hospital, 75 Francis Street, Boston, MA 02115.

³ D. P-W. is an Established Investigator of the American Heart Association.

Received 7/20/93. Accepted 1/17/94.

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