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.
Translation initiation in eukaryotes is a rate-limiting step in protein synthesis. It is a complicated process that involves many eukaryotic initiation factors (eIFs). Altering the expression level or the function of eIFs may influence the synthesis of some proteins and consequently cause abnormal cell growth and malignant transformation. P170, the largest putative subunit of eIF3, has been found elevated in human breast, cervical, esophageal, and lung cancers, suggesting that p170 may have a potential role in malignant transformation and/or cell growth control. Our recent studies suggested that p170 is likely a translational regulator and it may mediate the effect of mimosine on the translation of a subset mRNAs. Mimosine, a plant nonprotein amino acid, inhibits mammalian DNA synthesis, an essential event of cell growth. The rate-limiting step in DNA synthesis is the conversion of the ribonucleotides to their corresponding deoxyribonucleotides catalysed by ribonucleotide reductase of which the activity is regulated by the level of its M2 subunit. It has been reported that inhibiting the activity of M2 also inhibits cell growth. To understand the relationship between protein and DNA synthesis and between p170 and cell growth control, we investigated in this study whether p170 regulates the synthesis of M2 and, thus, cell growth. We found that altering the expression level of p170 changes the synthesis rate of both M2 and DNA. Decreasing p170 expression in human lung cancer cell line H1299 and breast cancer cell line MCF7 significantly reversed their malignant growth phenotype. However, the overall [ 35 S]methionine incorporation following dramatic decrease in p170 expression was only B25% less than the control cells. These observations, together with our previous findings, suggest that p170 may regulate the translation of a subset mRNAs and its elevated expression level may be important for cancer cell growth and for maintaining their malignant phenotype.
l-Mimosine, a plant amino acid, can reversibly block mammalian cells at late G1 phase and has been suggested to affect translation of mRNAs such as p27, the CDK inhibitor. However, the mechanism of this effect is not known. Regulation of translation generally occurs at the initiation step that, in mammalian cells, is a complex process that requires multiple eukaryotic initiation factors (eIFs) and ribosome. The effects of mimosine on initiation factors or regulators consequently will influence translation initiation. P170, a putative subunit of eIF3, has been suggested to be nonessential for eIF3 function to form preinitiation complexes and it may function as a regulator for translation of a subset of mRNAs. In this article, we tested this hypothesis and investigated whether eIF3 p170 mediates mimosine effect on mRNA translation. We found that p170 translation was dramatically reduced by mimosine due to its iron-chelating function. The decreased expression of p170 by mimosine caused diminished de novo synthesis of tyrosinated ␣-tubulin and elevated translation of p27 before cell cycle arrest. These observations suggest that p170 is likely an early response gene to mimosine treatment and a mediator for mimosine effect on mRNA translation. The effect of p170 on the synthesis of tyrosinated ␣-tubulin and p27 in a reciprocal manner also suggests that p170 functions as a regulator for mRNA translation. INTRODUCTIONl-Mimosine, a plant amino acid derived from seeds of Leucaena leucocephala or Mimosa pudica, can specifically and reversibly block mammalian cells at late G1 phase (Lalande, 1990;Mosca et al., 1992). Mimosine is commonly used as a synchronizing agent for investigating cell cycle progression in mammalian cells (Wang et al., 1995;Hughes and Cook, 1996). However, the molecular mechanism of mimosine action is ill-defined. It has been reported that the cells treated with mimosine lack proteins required for initiation and elongation of DNA replication (Kalejta and Hamlin, 1997). Although mimosine does not affect mRNA synthesis in general (Tsai and Ling, 1971), a subset of mRNAs was found to relocate to the mono-ribosome pool at the expense of polyribosome fraction after mimosine treatment. On mimosine removal, they reappeared in poly-ribosome pool, indicating that the mimosine effect is reversible (Hanauske-Abel et al., 1995). A recent study showed that expression of the cyclindependent kinase inhibitor p27 was elevated at the translational level by mimosine (Wang et al., 2000). These findings suggest that mimosine may modulate the synthesis of some proteins, which in turn causes inhibition of DNA replication and cell cycle arrest. One possible mechanism for such effect is that mimosine affects the synthesis and/or the function of proteins controlling mRNA translation.The rate-limiting step in mRNA translation is the initiation step. In eukaryotes, 11 factors have been identified that participate in the translation initiation step (Hershey and Merrick, 2000). Among these factors, eIF3 is the largest and the most ...
Signal transducer and activator of transcription 3 (STAT3) plays important roles in multiple aspects of cancer aggressiveness including migration, invasion, survival, self-renewal, angiogenesis, and tumor cell immune evasion by regulating the expression of multiple downstream target genes. STAT3 is constitutively activated in many malignant tumors and its activation is associated with high histological grade and advanced cancer stages. Thus, inhibiting STAT3 promises an attracting strategy for treatment of advanced and metastatic cancers. Herein, we identified a STAT3 inhibitor, inS3-54, by targeting the DNA-binding domain of STAT3 using an improved virtual screening strategy. InS3-54 preferentially suppresses proliferation of cancer over non-cancer cells and inhibits migration and invasion of malignant cells. Biochemical analyses show that inS3-54 selectively inhibits STAT3 binding to DNA without affecting the activation and dimerization of STAT3. Furthermore, inS3-54 inhibits expression of STAT3 downstream target genes and STAT3 binding to chromatin in situ. Thus, inS3-54 represents a novel probe for development of specific inhibitors targeting the DNA-binding domain of STAT3 and a potential therapeutic for cancer treatments.
Survivin, the smallest member of the inhibitor of apoptosis protein (IAP) family, is overexpressed in cells of almost all cancers but not in most normal tissues in adults. Survivin expression is required for cancer cell survival and knocking down its expression or inhibiting its function using molecular approaches results in spontaneous apoptosis. Thus, survivin is an attractive and perhaps ideal target for cancer drug discovery. However, a US Food and Drug Administration (FDA)-approved drug targeting survivin has yet to emerge. In this Foundation Review, we examine and evaluate various strategies that have been used to target survivin and the stages of each survivin inhibitor to help understand this lack of success. We also provide future perspectives moving forward in targeting survivin for drug discovery.
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