Abstract
An earlier report (Matherly, L. H., Czajkowski, C. A., and Angeles, S. M. (1991) Cancer Res. 51, 3420-3426) described a K562 human erythroleukemia line (K562.4CF), characterized by an elevated uptake capacity for methotrexate (MTX) and 5-formyltetrahydrofolate, and the identification of a highly glycosylated membrane transporter (GP-MTX) by radioaffinity labeling with N-hydroxysuccinimide [3H] methotrexate. In the present study, radioaffinity-labeled GP-MTX from K562.4CF cells was isolated by Ricinus communis agglutinin I-agarose affinity chromatography, coupled with gel filtration and preparative electrophoresis. Antiserum to the purified, radio-labeled protein was raised in a rabbit and screened by immunoblot analysis of K562.4CF plasma membrane proteins or purified GP-MTX. The antiserum detected a broad GP-MTX band centered at 92 kDa on 7.5% gels. On 4-10% gels, the apparent molecular mass for GP-MTX shifted to 99 kDa. Antiserum specificity was established by quantitatively converting the immunoreactive glycoprotein in plasma membrane homogenates to its N- and O-deglycosylated forms with N- and O-glycanases, respectively. Whereas the methotrexate uptake capacity of K562.4CF cells was elevated 6.1-fold over parental cells, the GP-MTX content on immunoblots was increased approximately 3-fold. For two methotrexate-resistant K562 lines (33- and 70-fold), decreased drug uptake (28 and 18% of parental levels) closely correlated with their reduced GP-MTX contents. A GP-MTX isoform was also detected on immunoblots of membrane proteins from CCRF-CEM human lymphoblastic leukemia cells. With a transport-impaired CCRF-CEM line (13% of wild type uptake), an aberrant electrophoretic migration for GP-MTX was observed, establishing the presence of structural modifications in the transport protein. These structural differences were independent of carrier glycosylation since they were detected following the glycosidase treatments. These findings implicate important roles for distinct carrier-specific alterations in the expression of diminished drug transport in methotrexate-resistant human tumor cells.
Highlights
Beled GP-MTX from K562.4CF cells was isolated by lysine [5]
Labeled protein was raised in a rabbit and screenbeyd In contrast to thme urine protein, with K562 human erythimmunoblot analysis of K562.4CF plasma membrane proteins or purifiedGP-MTX
Proteins were Binding of GP-MTX to Lectin-Agaroses-Because of the electrophoretically transferred to Immobilon P (Millipore Corporation) for 2h using a Bio-Rad transblot cell and a buffer system composed of 10 mM CAPS, pH 11, and 10% methanol [32]
Summary
Sedimented by centrifugation in a microcentrifuge and the supernatants added to one-third volume of 3 X Laemmli “sample buffer” [31] Estimation of the Absolute GP-MTX Contents of K562 and for loading onto thegels. A preliminary step to attempting to isolate GP-MTX, theabsolute carrier content of these cells was established by measuring the level of specific [3H]MTX ing day, the vials werecooled and radioactivity measured with a cell surface binding [23]. Proteins were Binding of GP-MTX to Lectin-Agaroses-Because of the electrophoretically transferred to Immobilon P (Millipore Corporation) for 2h using a Bio-Rad transblot cell and a buffer system composed of 10 mM CAPS, pH 11, and 10% methanol [32].
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