Human ATP-binding cassette G2 (ABCG2, also known as mitoxantrone resistance protein, breast cancer-resistance protein, ABC placenta) is a member of the superfamily of ATP-binding cassette (ABC) transporters that have a wide variety of substrates. Overexpression of human ABCG2 in model cancer cell lines causes multidrug resistance by actively effluxing anticancer drugs. Unlike most of the other ABC transporters which usually have two nucleotide-binding domains and two transmembrane domains, ABCG2 consists of only one nucleotide-binding domain followed by one transmembrane domain. Thus, ABCG2 has been thought to be a half-transporter that may function as a homodimer. In this study, we characterized the oligomeric feature of human ABCG2 using non-denaturing detergent perfluoro-octanoic acid and Triton X-100 in combination with gel filtration, sucrose density gradient sedimentation, and gel electrophoresis. Unexpectedly, we found that human ABCG2 exists mainly as a tetramer, with a possibility of a higher form of oligomerization. Monomeric and dimeric ABCG2 did not appear to be the major form of the protein. Further immunoprecipitation analysis showed that the oligomeric ABCG2 did not contain any other proteins. Taken together, we conclude that human ABCG2 likely exists and functions as a homotetramer.

Multidrug resistance is a major problem in successful cancer chemotherapy. Various mechanisms of resistance, such as ABC transporter-mediated drug efflux, have been discovered using established model cancer cell lines. While characterizing a drug-resistant breast cancer cell line, MCF7/AdVp3000, we found that fatty acid synthase (FASN) is overexpressed. In this study, we showed that ectopic overexpression of FASN indeed causes drug resistance and that reducing the FASN expression increased the drug sensitivity in breast cancer cell lines MCF7 and MDA-MB-468 but not in the normal mammary epithelial cell line MCF10A1. Use of FASN inhibitor, Orlistat, at low concentrations also sensitized cells with FASN overexpression to anticancer drugs. The FASNmediated drug resistance appears to be due to a decrease in drug-induced apoptosis from an overproduction of palmitic acid by FASN. Together with previous findings of FASN as a poor prognosis marker for breast cancer patients, our results suggest that FASN overexpression is a new mechanism of drug resistance and may be an ideal target for chemosensitization in breast cancer chemotherapy.

As an alternative to the scanning mechanism of initiation, the direct-internal-initiation mechanism postulates that the translational machinery assembles at the AUG start codon without traversing the entire 5 untranslated region (5-UTR) of the mRNA. Although the existence of internal ribosome entry sites (IRESs) in viral mRNAs is considered to be well established, the existence of IRESs in cellular mRNAs has recently been challenged, in part because when testing is carried out using a conventional dicistronic vector, Northern blot analyses might not be sensitive enough to detect low levels of monocistronic transcripts derived via a cryptic promoter or splice site. To address this concern, we created a new promoterless dicistronic vector to test the putative IRES derived from the 5-UTR of an mRNA that encodes the translation initiation factor eIF4G. Our analysis of this 5-UTR sequence unexpectedly revealed a strong promoter. The activity of the internal promoter relies on the integrity of a polypyrimidine tract (PPT) sequence that had been identified as an essential component of the IRES. The PPT sequence overlaps with a binding site for transcription factor C/EBP␤. Two other transcription factors, Sp1 and Ets, were also found to bind to and mediate expression from the promoter in the 5-UTR of eIF4G mRNA. The biological significance of the internal promoter in the eIF4G mRNA might lie in the production of an N-terminally truncated form of the protein. Consistent with the idea that the cryptic promoter we identified underlies the previously reported IRES activity, we found no evidence of IRES function when a dicistronic mRNA containing the eIF4G sequence was translated in vitro or in vivo. Using the promoterless dicistronic vector, we also found promoter activities in the long 5-UTRs of human Sno and mouse Bad mRNAs although monocistronic transcripts were not detectable on Northern blot analyses. The promoterless dicistronic vector might therefore prove useful in future studies to examine more rigorously the claim that there is IRES activity in cellular mRNAs.