Related Experiment Videos
Functionally active homodimer of P-glycoprotein in multidrug-resistant tumor cells
Abstract:
P-glycoprotein plays a key role in multidrug resistance of tumor cells. In order to elucidate the possible quarternary structure/function relationship of P-glycoprotein, we treated multidrug-resistant human leukemia K562/ADM cells with the crosslinking reagent, disuccinimidyl suberate. In addition to 180K P-glycoprotein, a 340K protein was immunoprecipitated with an anti-P-glycoprotein monoclonal antibody, MRK-16. The 340K protein is most probably a dimeric P-glycoprotein, since only the 180K P-glycoprotein was immunoprecipitated with MRK-16 when K562/ADM cells were treated with the cleavable crosslinking reagent, dithiobis(succinimidylpropionate), and analysed under reduced conditions. The dimeric P-glycoprotein was photolabeled with [3H]azidopine like the 180K monomeric P-glycoprotein and the photolabeling was inhibited by excess amount of vincristine and verapamil. The dimeric P-glycoprotein could be a functionally active form of the protein involved in the transport of antitumor agents.
Insights
Researchers investigated the structure of P-glycoprotein in multidrug-resistant leukemia cells. They found evidence that P-glycoprotein can form dimers, which appear to be functionally active in transporting antitumor agents.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- P-glycoprotein is crucial in tumor cell multidrug resistance.
- Understanding P-glycoprotein's quaternary structure is key to its function.
Purpose of the Study:
- To investigate the quaternary structure and function of P-glycoprotein.
- To determine if P-glycoprotein exists as a dimer in multidrug-resistant cells.
Main Methods:
- Treatment of K562/ADM cells with crosslinking reagents (disuccinimidyl suberate and dithiobis(succinimidylpropionate)).
- Immunoprecipitation using anti-P-glycoprotein monoclonal antibody (MRK-16).
- Photolabeling with [3H]azidopine and inhibition studies with vincristine and verapamil.
Main Results:
- A 340K protein, likely a dimeric P-glycoprotein, was detected alongside the 180K monomer.
- Dimeric P-glycoprotein was confirmed using a cleavable crosslinking reagent under reduced conditions.
- Dimeric P-glycoprotein exhibited photolabeling activity, inhibited by known P-glycoprotein substrates.
Conclusions:
- P-glycoprotein exists in a dimeric form in multidrug-resistant leukemia cells.
- This dimeric form is likely functionally active in the transport of anticancer drugs.
- Findings provide insights into the mechanism of multidrug resistance and potential therapeutic targets.