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Does ABCG2 need a heterodimer partner? Expression and functional evaluation of ABCG2 (Arg 482)*
Megumi Yoshikawa1, Shiho Kasamatsu, Masa Yasunaga
1Department of Biomolecular Engineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology.
Abstract:
Accumulating evidence suggests that several ATP-binding cassette (ABC) transporters mediate the elimination of anticancer drugs from cancer cells and thereby confer drug resistance. SN-38-selected PC-6/SN2-5H human lung carcinoma cells were shown to overexpress ABCG2 with the reduced intracellular accumulation of SN-38, the active metabolite of irinotecan. We have recently demonstrated that plasma membrane vesicles prepared from those cells transported SN-38 in an ATP-dependent manner, and it was suggested that ABCG2 is involved in the active extrusion of SN-38 from cancer cells. In the present study, we have cloned the cDNA of ABCG2 from PC-6/SN2-5H human lung carcinoma cells, expressed ABCG2 in Sf9 insect cells, and characterized its function. Sequence analysis has revealed that the cloned ABCG2 has an arginine at the amino acid position 482, as does the wild type. Expression of the cloned ABCG2 in Sf9 cell membranes was detected by immunoblotting with the BXP-21 antibody. Contrary to our expectation, however, ATPase activity in the cell membranes expressing ABCG2 was stimulated by neither SN-38 nor rhodamine 123. It is suggested that there is a partner protein of ABCG2 required for heterodimer formation to exhibit transport activity toward SN-38.
Insights
ATP-binding cassette (ABC) transporters like ABCG2 can cause anticancer drug resistance. This study found that ABCG2 alone may not transport SN-38, suggesting a partner protein is needed for its function.
Area of Science:
- Molecular Biology
- Cancer Research
- Biochemistry
Background:
- ATP-binding cassette (ABC) transporters contribute to anticancer drug resistance by exporting drugs from cancer cells.
- Overexpression of ABCG2 in SN-38-selected lung carcinoma cells correlates with reduced intracellular SN-38 accumulation.
- Previous studies indicated ABCG2's role in SN-38 extrusion, but its precise function remained unclear.
Purpose of the Study:
- To clone and characterize the function of ABCG2 from SN-38-resistant human lung carcinoma cells.
- To investigate the direct transport activity of ABCG2 towards SN-38.
- To explore the potential requirement of a partner protein for ABCG2-mediated transport.
Main Methods:
- Cloning of ABCG2 cDNA from SN-38-selected PC-6/SN2-5H cells.
- Expression of ABCG2 in Sf9 insect cells.
- Immunoblotting to confirm ABCG2 expression using the BXP-21 antibody.
- Assessing ATPase activity in membranes expressing ABCG2 with and without SN-38 or rhodamine 123.
Main Results:
- The cloned ABCG2 sequence from PC-6/SN2-5H cells was identical to wild-type ABCG2 at amino acid position 482.
- ABCG2 expression in Sf9 cell membranes was confirmed via immunoblotting.
- No significant stimulation of ATPase activity was observed in ABCG2-expressing membranes when treated with SN-38 or rhodamine 123.
Conclusions:
- The cloned ABCG2, despite being expressed, did not exhibit direct SN-38 or rhodamine 123 stimulated ATPase activity.
- These findings suggest that ABCG2 may require a partner protein for heterodimerization to mediate the transport of SN-38.
- Further research is needed to identify potential interacting partners of ABCG2 involved in drug resistance mechanisms.
