L1210 Cells Overexpressing ABCB1 Drug Transporters Are Resistant to Inhibitors of the N- and O-glycosylation of Proteins
Abstract
:1. Introduction
2. Results
2.1. Characterization of P-gp Positive Variants of L1210 Cells
2.2. Cytotoxic Effect of O- and N-Glycosylation Inhibitors on S, R and T Cells
2.3. Effect of GalNAc-α-O-benzyl and Tunicamycin on P-gp Glycosylation
2.4. Binding of ConA and GNA to Glycoprotein in S, R and T Cells
2.5. Effect of Tunicamycin on Protein Ubiquitination in R and T Cells
3. Discussion
- (a)
- Tunicamycin may be a substrate for P-gp efflux and therefore could effectively be eliminated from the intracellular volume of P-gp-positive cells. While this idea has previously been discussed [32], direct evidence for such P-gp activity is lacking. However, the fact that tunicamycin also blocks the glycosylation of several proteins, including P-gp, in P-gp-positive cells (as shown in Figure 2 and described elsewhere [27,33,34]) is contradictory to this speculation. Moreover, resistance of R and T cells to tunicamycin could not be reversed by verapamil (a known P-gp inhibitor) even if this substance completely blocked P-gp efflux activity [27].
- (b)
- Tunicamycin induced the global inhibition of glycoprotein synthesis (Hiss et al., 2007) associated with the elevation of immature proteins cell content leading to endoplasmic reticulum stress and apoptosis [35]. P-gp-positive cells with altered regulation of apoptosis initiation and progression [13,17] could survive better under this condition.
- (c)
- The application of tunicamycin increases the cellular level of UDP-N-acetylglucosamine, associated with different features of cell damage visible using electron microscopy, namely, in the membranes of the endoplasmic reticulum and nuclei [34]. Significantly decreased intracellular levels of UDP-sugars and UDP-glucose have been detected in P-gp-positive R and T cells compared with P-gp-negative S cells [14,23]. Therefore, the resistance of P-gp-positive L1210 cells to tunicamycin may reflect the decreased levels of UDP-sugars in these cells.
4. Materials and Methods
4.1. Cell Culture Conditions
4.2. Western and Eastern Blot Procedures
4.3. Deglycosydation of Membrane Proteins with PNGase and Endo H
4.4. Detection of ConA and GNA Binding to the Surface of S, R and T Cells by Flow Cytometry
4.5. Immunoprecipitation of Proteins by Antiubiquitin Antibody
4.6. Detection of Ubiquitin on Cell Surface of S, R and T Cells by Confocal Microscopy and Flow Cytometry
5. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Pavlikova, L.; Seres, M.; Hano, M.; Bohacova, V.; Sevcikova, I.; Kyca, T.; Breier, A.; Sulova, Z. L1210 Cells Overexpressing ABCB1 Drug Transporters Are Resistant to Inhibitors of the N- and O-glycosylation of Proteins. Molecules 2017, 22, 1104. https://doi.org/10.3390/molecules22071104
Pavlikova L, Seres M, Hano M, Bohacova V, Sevcikova I, Kyca T, Breier A, Sulova Z. L1210 Cells Overexpressing ABCB1 Drug Transporters Are Resistant to Inhibitors of the N- and O-glycosylation of Proteins. Molecules. 2017; 22(7):1104. https://doi.org/10.3390/molecules22071104
Chicago/Turabian StylePavlikova, Lucia, Mario Seres, Milan Hano, Viera Bohacova, Ivana Sevcikova, Tomas Kyca, Albert Breier, and Zdena Sulova. 2017. "L1210 Cells Overexpressing ABCB1 Drug Transporters Are Resistant to Inhibitors of the N- and O-glycosylation of Proteins" Molecules 22, no. 7: 1104. https://doi.org/10.3390/molecules22071104