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Survivin, a homodimeric member of the Inhibitor of Apoptosis Protein (IAP) family, is required for cancer cell survival and overexpressed in almost all solid tumors. However, targeting survivin has been challenging due to its "undruggable" nature. Recently, we used a novel approach to target the dimerization interface and identified inhibitors of two scaffolds that can directly bind to and inhibit survivin dimerization. One of the scaffolds, represented by the compound LQZ-7, contains an undesirable labile hydrazone linker and a potentially nonfunctional furazanopyrazine ring that we attempted to eliminate in this study. We found one compound, 7I , that is more active than the parent compound, LQZ-7, and when given orally effectively inhibits xenograft tumor growth and induces survivin loss in tumors. These findings indicate that 7I with a stable linker and a quinoxaline ring can be used as a lead for further optimization of this novel class of survivin inhibitors.

eIF3a is a putative subunit of the eukaryotic translation initiation factor 3 complex. Accumulating evidence suggests that eIF3a may have a translational regulatory function by suppressing translation of a subset of mRNAs while accelerating that of other mRNAs. Albeit the suppression of mRNA translation may derive from eIF3a binding to the 5'-UTRs of target mRNAs, how eIF3a may accelerate mRNA translation remains unknown. In this study, we show that eIF3a up-regulates translation of Chk1 but not Chk2 mRNA by interacting with HuR, which binds directly to the 3'-UTR of Chk1 mRNA. The interaction between eIF3a and HuR occurs at the 10-amino-acid repeat domain of eIF3a and the RNA recognition motif domain of HuR. This interaction may effectively circularize Chk1 mRNA to form an end-to-end complex that has recently been suggested to accelerate mRNA translation. Together with previous findings, we conclude that eIF3a may regulate mRNA translation by directly binding to the 5'-UTR to suppress or by interacting with RNA-binding proteins at 3'-UTRs to accelerate mRNA translation., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

ATP-binding cassette (ABC) subfamily G member 2 (ABCG2) belongs to the ABC transporter superfamily and has been implicated in multidrug resistance of cancers. Although the structure and function of ABCG2 have been extensively studied, little is known about its biogenesis and the regulation thereof. In this study, using mutagenesis and several biochemical analyses, we show that the positive charges in the vicinity of the RKR motif downstream of the ABC signature drive trafficking of nascent ABCG2 out of the endoplasmic reticulum (ER) onto plasma membranes. Substitutions of and naturally occurring single-nucleotide polymorphisms within these positively charged residues disabled the trafficking of ABCG2 out of the ER. A representative ABCG2 variant in which the RKR motif had been altered underwent increased ER stress-associated degradation. We also found that unlike WT ABCG2, genetic ABCG2 RKR variants have disrupted normal maturation and do not reduce accumulation of the anticancer drug mitoxantrone and no longer confer resistance to the drug. We conclude that the positive charges downstream of the ABC signature motif critically regulate ABCG2 trafficking and maturation. We propose that single-nucleotide polymorphisms of these residues reduce ABCG2 expression via ER stress-associated degradation pathway and may contribute to reduced cancer drug resistance, improving the success of cancer chemotherapy., Competing Interests: The authors declare that they have no conflicts of interest with the contents of this article., (© 2019 Zhang et al.)

CC-115, a triazole-containing compound, is a dual mammalian target of rapamycin (mTOR)/DNA-dependent protein kinase (DNA-PK) inhibitor currently in clinical trials. To develop this compound further, we investigated factors that may affect cellular response to CC-115. Previously, fatty acid synthase (FASN) was shown to upregulate DNA-PK activity and contribute to drug resistance; therefore, we hypothesized that FASN may affect cellular response to CC-115. Instead, however, we showed that CC-115 is a substrate of ATP-binding cassette G2 (ABCG2), a member of the ATP-binding cassette transporter superfamily, and that expression of ABCG2, not FASN, affects the potency of CC-115. ABCG2 overexpression significantly increases resistance to CC-115. Inhibiting ABCG2 function, using small-molecule inhibitors, sensitizes cancer cells to CC-115. We also found that CC-115 may be a substrate of ABCB1, another known ABC protein that contributes to drug resistance. These findings suggest that expression of ABC transporters, including ABCB1 and ABCG2, may affect the outcome in clinical trials testing CC-115. Additionally, the data indicate that ABC transporters may be used as markers for future precision use of CC-115. SIGNIFICANCE STATEMENT: In this article, we report our findings on the potential mechanism of resistance to CC-115, a dual inhibitor of mTOR and DNA-PK currently in clinical trials. We show that CC-115 is a substrate of ABCG2 and can be recognized by ABCB1, which contributes to CC-115 resistance. These findings provide novel information and potential guidance on future clinical testing of CC-115., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

Constitutive activation of signal transducer and activator of transcription 3 (STAT3) plays important roles in oncogenic occurrence and transformation by regulating the expression of diverse downstream target genes important for tumor growth, metastasis, angiogenesis and immune evasion. Feasibility of targeting the DNA-binding domain (DBD) of STAT3 has been proven previously. With the aid of 3D shape- and electrostatic-based drug design, we identified a new STAT3 inhibitor, LC28, and its five analogs, based on the pharmacophore of a known STAT3 DBD inhibitor. Microscale thermophoresis assay shows that these compounds inhibits STAT3 binding to DNA with a K i value of 0.74-8.87 μM. Furthermore, LC28 and its analogs suppress survival of cisplatin-resistant ovarian cancer cells by inhibiting STAT3 signaling and inducing apoptosis. Therefore, these compounds may serve as candidate compounds for further modification and development as anticancer therapeutics targeting the DBD of human STAT3 for treatment of cisplatin-resistant ovarian cancer., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Signal transducer and activator of transcription 3 (STAT3) is constitutively activated in malignant tumors and plays important roles in multiple aspects of cancer aggressiveness. Thus, targeting STAT3 promises to be an attractive strategy for the treatment of advanced metastatic tumors. Bisindolylmaleimide alkaloid (BMA) has been shown to have anti-cancer activities and was thought to suppress tumor cell growth by inhibiting protein kinase C. In this study, we show that a newly synthesized BMA analog, BMA097, is effective in suppressing tumor cell and xenograft growth and in inducing spontaneous apoptosis. We also provide evidence that BMA097 binds directly to the SH2 domain of STAT3 and inhibits STAT3 phosphorylation and activation, leading to reduced expression of STAT3 downstream target genes. Structure activity relationship analysis revealed that the hydroxymethyl group in the 2,5-dihydropyrrole-2,5-dione prohibits STAT3 inhibitory activity of BMA analogs. Altogether, we conclude that the synthetic BMA analogs may be developed as anti-cancer drugs by targeting and binding to the SH2 domain of STAT3 and inhibiting the STAT3 signaling pathway.

eIF3a is the largest subunit of eIF3, which is a key player in all steps of translation initiation. During the past years, eIF3a is recognized as a proto-oncogene, which is an important discovery in this field. It is widely reported to be correlated with cancer occurrence, metastasis, prognosis, and therapeutic response. Recently, the mechanisms of eIF3a action in the carcinogenesis are unveiled gradually. A number of cellular, physiological, and pathological processes involving eIF3a are identified. Most importantly, it is emerging as a new potential drug target in the eIF family, and some small molecule inhibitors are being developed. Thus, we perform a critical review of recent advances in understanding eIF3a physiological and pathological functions, with specific focus on its role in cancer and anticancer drug targets., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Translation initiation is the rate-limiting step of protein synthesis and highly regulated. Eukaryotic initiation factor 3 (eIF3) is the largest and most complex initiation factor consisting of 13 putative subunits. A growing number of studies suggest that eIF3 and its subunits may represent a new group of proto-oncogenes and associates with prognosis. They regulate translation of a subset of mRNAs involved in many cellular processes including proliferation, apoptosis, DNA repair, and cell cycle. Therefore, unveiling the mechanisms of eIF3 action in tumorigenesis may help identify attractive targets for cancer therapy. Here, we describe a series of methods used in the study of eIF3 function in regulating protein synthesis, tumorigenesis, and cellular response to therapeutic treatments.

Fatty acid synthase (FASN), the sole cytosolic mammalian enzyme for de novo lipid synthesis, is crucial for cancer cell survival and associates with poor prognosis. FASN overexpression has been found to cause resistance to genotoxic insults. Here we tested the hypothesis that FASN regulates DNA repair to facilitate survival against genotoxic insults and found that FASN suppresses NF-κB but increases specificity protein 1 (SP1) expression. NF-κB and SP1 bind to a composite element in the poly(ADP-ribose) polymerase 1 (PARP-1) promoter in a mutually exclusive manner and regulate PARP-1 expression. Up-regulation of PARP-1 by FASN in turn increases Ku protein recruitment and DNA repair. Furthermore, lipid deprivation suppresses SP1 expression, which is able to be rescued by palmitate supplementation. However, lipid deprivation or palmitate supplementation has no effect on NF-κB expression. Thus, FASN may regulate NF-κB and SP1 expression using different mechanisms. Altogether, we conclude that FASN regulates cellular response against genotoxic insults by up-regulating PARP-1 and DNA repair via NF-κB and SP1., Competing Interests: The authors declare no conflict of interest.

Many proteins exist and function as homodimers. Understanding the detailed mechanism driving the homodimerization is important and will impact future studies targeting the "undruggable" oncogenic protein dimers. In this study, we used 14-3-3σ as a model homodimeric protein and performed a systematic investigation of the potential roles of amino acid residues in the interface for homodimerization. Unlike other members of the conserved 14-3-3 protein family, 14-3-3σ prefers to form a homodimer with two subareas in the dimeric interface that has 180° symmetry. We found that both subareas of the dimeric interface are required to maintain full dimerization activity. Although the interfacial hydrophobic core residues Leu(12) and Tyr(84) play important roles in 14-3-3σ dimerization, the non-core residue Phe(25) appears to be more important in controlling 14-3-3σ dimerization activity. Interestingly, a similar non-core residue (Val(81)) is less important than Phe(25) in contributing to 14-3-3σ dimerization. Furthermore, dissociating dimeric 14-3-3σ into monomers by mutating the Leu(12), Phe(25), or Tyr(84) dimerization residue individually diminished the function of 14-3-3σ in resisting drug-induced apoptosis and in arresting cells at G2/M phase in response to DNA-damaging treatment. Thus, dimerization appears to be required for the function of 14-3-3σ.

eIF3a (eukaryotic translation initiation factor 3a), one of the core subunits of the eIF3 complex, has been implicated in regulating translation of different mRNAs and in tumorigenesis. A subcomplex consisting of eIF3a, eIF3b, eIF3g, and eIF3i (eIF3(a:b:i:g)) has also been identified. However, how eIF3a participates in translational regulation and in formation of the eIF3(a:b:i:g) subcomplex remain to be solved. In this study, we used the tandem affinity purification approach in combination with tandem MS/MS and identified the spectrin domain of eIF3a as the docking site for the formation of eIF3(a:b:i:g) subcomplex. Although eIF3b and eIF3i bind concurrently to the spectrin domain of eIF3a within ∼10-15 amino acids apart, eIF3g binds to eIF3a indirectly via binding to the carboxyl-terminal domain of eIF3b. The binding of eIF3b to the spectrin domain of eIF3a occurs in its RNA recognition motif domain where eIF3j also binds in a mutually exclusive manner. Together, we conclude that the spectrin domain of eIF3a is responsible for the formation of eIF3(a:b:i:g) subcomplex and, because of mutually exclusive nature of bindings of eIF3a and eIF3j to eIF3b, different subcomplexes of eIF3 likely exist and may perform noncanonical functions in translational regulation.

Fatty acid synthase (FASN) is a key enzyme in the synthesis of palmitate, the precursor of major nutritional, energetic, and signaling lipids. FASN expression is upregulated in many human cancers and appears to be important for cancer cell survival. Overexpression of FASN has also been found to associate with poor prognosis and higher risk of recurrence of human cancers. Indeed, elevated FASN expression has been shown to contribute to drug resistance. However, the mechanism of FASN-mediated drug resistance is currently unknown. In this study, we show that FASN overexpression causes resistance to multiple anticancer drugs via inhibiting drug-induced ceramide production, caspase 8 activation, and apoptosis. We also show that FASN overexpression suppresses tumor necrosis factor-α production and nuclear factor-κB activation as well as drug-induced activation of neutral sphingomyelinase. Thus, TNF-α may play an important role in mediating FASN function in drug resistance.

Human ABCG2 is a member of the ATP-binding cassette (ABC) transporter superfamily and is known to contribute to multidrug resistance (MDR) in cancer chemotherapy. Among ABC transporters that are known to cause MDR, ABCG2 is particularly interesting for its potential role in protecting cancer stem cells and its complex oligomeric structure. Recent studies have also revealed that the biogenesis of ABCG2 could be modulated by small molecule compounds. These modulators, upon binding to ABCG2, accelerate the endocytosis and trafficking to lysosome for degradation and effectively reduce the half-life of ABCG2. Hence, targeting ABCG2 stability could be a new venue for therapeutic discovery to sensitize drug resistant human cancers. In this report, we review recent progress on understanding the structure, function, biogenesis, as well as physiological and pathophysiological functions of ABCG2.

Objective: To compare the behavioral effects of psychoactive drugs between two strains of mice., Methods: The Kunming (KM) and ICR mice were injected intraperitoneally with caffeine (3, 10, 30, 100 mg/kg), ephedrine (3, 10, 30, 100 mg/kg), diazepam (1, 3,1 0 mg/kg) and chloral hydrate (10, 30, 100 mg/kg), respectively. Ten min after injection, the locomotor activity in the open field was recorded for 2 h. The total distance, the distance ratio to total distance and the time in central region were analyzed for each drugs. Thirty min after injection, the latent time in the passive avoidance test was measured in a shuttle box., Results: Caffeine and diazepam prolonged the latent time, and ephedrine and chloral hydrate decreased the latent time, but there were no differences between the two strains. The two strains of mice exhibited significant differences in the total distance after injection of ephedrine 10 mg/kg, diazepam 3 mg/kg and chloral hydrate 100 mg/kg. Compared to KM mice, ICR mice exhibited an increase in the distance ratio and the time in central region after injection of ephedrine 10-100 mg/kg, but a decrease after diazepam 3-10 mg/kg., Conclusion: KM and ICR mice show no differences in latent time, but significant differences in the total distance, the distance ratio and the time in central region in the locomotor activity. Therefore, selection of mouse strains is important in the study of psychoactive drugs.

Translational control is one of primary regulation mechanisms of gene expression. Eukaryotic translational control mainly occurs at the initiation step, the speed-limiting step, which involves more than ten translation initiation factors [eIFs (eukaryotic initiation factors)]. Changing the level or function of these eIFs results in abnormal translation of specific mRNAs and consequently abnormal growth of cells that leads to human diseases, including cancer. Accumulating evidence from recent studies showed that the expression of many eIFs was associated with malignant transformation, cancer prognosis, as well as gene expression regulation. In the present paper, we perform a critical review of recent advances in understanding the role and mechanism of eIF action in translational control and cancer as well as the possibility of targeting eIFs for therapeutic development.

Human fatty acid synthase (FASN) is a homo-dimeric protein with multi-enzymatic activity responsible for the synthesis of palmitate. FASN expression has been found to be up-regulated in multiple types of human cancers and its expression correlates with poor prognosis possibly by causing treatment resistance. In this study, we tested if FASN expression is up-regulated in human pancreatic cancers and if its higher expression level in pancreatic cancers causes intrinsic resistance to gemcitabine and radiation. We found that FASN expression is significantly up-regulated in human pancreatic cancer tissues without any correlation to age, sex, race, and tumor stage. Knocking down or over-expressing FASN significantly down- or up-regulate resistance of pancreatic cancer cell lines to both gemcitabine and radiation treatments. These findings imply that the elevated FASN expression in pancreatic cancers may contribute to unsuccessful treatments of pancreatic cancers by causing intrinsic resistance to both chemotherapy and radiation therapy.

Human fatty acid synthase (FASN) is a 270-kDa cytosolic dimeric enzyme that is responsible for palmitate synthesis. FASN is slowly emerging and rediscovered as a marker for diagnosis and prognosis of human cancers. Recent studies showed that FASN is an oncogene and inhibition of FASN effectively and selectively kill cancer cells. With recent publications of the FASN crystal structure and the new development of FASN inhibitors, targeting FASN opens a new window of opportunity for metabolically combating cancers. In this article, we will review critically the recent progresses in understanding the structure, function, and the role of FASN in cancers and pharmacologically targeting FASN for human cancer treatment.

14-3-3sigma is a member of a highly conserved family of 14-3-3 proteins that are present in all eukaryotic organisms. 14-3-3sigma has been considered as a tumor suppressor with reduced expression in some human cancers while its increased expression causes resistance to anticancer agents and radiation that cause DNA damages. The increased expression of 14-3-3sigma may also predict poor prognosis in some human cancers. Thus, 14-3-3sigma may play an important role as a double-edged sword in human cancers, which may attribute to its property as a molecular chaperone by binding to various protein ligands important to many cellular processes such as cell cycle checkpoint regulation and apoptosis in response to DNA damages. In this article, we will review recent studies and progresses in understanding 14-3-3sigma as a double-edged sword in human cancers.

Drug resistance is a major problem in successful cancer chemotherapy. Many molecular mechanisms that are responsible for drug resistance are known whereas others have yet to be discovered. Determining the exact mechanism activated in a particular case (clinical or laboratory) is a difficult task. Recently, proteomics has been applied to investigate drug resistance mechanisms in model cancer cell lines. As a result, novel mechanisms of resistance have been discovered and known mechanisms of resistance confirmed. In this paper, we wish to review recent developments and progresses in the application of proteomic tools to identify known and novel drug resistance mechanisms in drug-selected model cancer cell lines. Our combined analyses of multiple proteomic studies of various drug resistant cancer cell lines revealed that many mechanisms of resistance likely exist in any given drug-selected cancer cell line and that common mechanisms of resistance may be selected in a spectrum of cancer cell lines. These observations suggest that combination therapies targeting multiple mechanisms to sensitize drug resistant cancers may be necessary to eradicate cancers in the future.

Eukaryotic initiation factor 3a (eIF3a) has been suggested to play a regulatory role in mRNA translation. Decreased eIF3a expression has been observed in differentiated cells while higher levels have been observed in cancer cells. However, whether eIF3a plays any role in differentiation and development is currently unknown. Here, we investigated eIF3a expression during mouse development and its role in differentiation of colon epithelial cells. We found that eIF3a expression was higher in fetal tissues compared with postnatal ones. Its expression in intestine, stomach, and lung abruptly stopped on the 18th day in gestation but persisted in liver, kidney, and heart throughout the postnatal stage at decreased levels. Similarly, eIF3a expression in colon cancer cell lines, HT-29 and Caco-2, drastically decreased prior to differentiation. Enforced eIF3a expression inhibited while knocking it down using small interference RNA promoted Caco-2 differentiation. Thus, eIF3a may play some roles in development and differentiation and that the decreased eIF3a expression may be a pre-requisite of intestinal epithelial cell differentiation.

ABCG2 is a half ATP-binding cassette (ABC) transporter that facilitates efflux of a wide variety of substrates ranging from natural products to synthetic anticancer drugs. Over-expression of ABCG2 has been shown to cause multidrug resistance in both laboratory model cell lines and in clinical settings. The polymorphism in ABCG2 has also been suggested to affect the function and clinical outcome in cancer patients. More recently, ABCG2 has been suggested to play a protective role for cancer and normal stem cells. Thus, ABCG2 is an ideal target for therapeutic development to chemo-sensitize drug resistant cancers. In this paper, the recent progress on understanding the structure, function, and pharmacology of ABCG2 and its role in drug resistance and cancer stem cells will be reviewed.

Overexpression of some ATP-binding cassette (ABC) membrane transporters such as ABCB1/P-glycoprotein/MDR1 and ABCC1/MRP1 causes multidrug resistance in cancer chemotherapy. It has been thought that half-ABC transporters with one nucleotide-binding domain and one membrane-spanning domain (MSD) likely work as dimers, whereas full-length transporters with two nucleotide-binding domains and two or three MSDs function as monomers. In this study, we examined the oligomeric status of the human full-length ABC transporter ABCC1/MRP1 using several biochemical approaches. We found 1) that it is a homodimer, 2) that the dimerization domain is located in the amino-terminal MSD0L0 (where L0 is loop 0) region, and 3) that MSD0L0 has a dominant-negative function when coexpressed with wild-type ABCC1/MRP1. These findings suggest that ABCC1/MRP1 may exist and function as a dimer and that MSD0L0 likely plays some structural and regulatory functions. It is also tempting to propose that the MSD0L0-mediated dimerization may be targeted for therapeutic development to sensitize ABCC1/MRP1-mediated drug resistance in cancer chemotherapy.

Human ABCG2, a member of the ATP-binding cassette transporter superfamily which transports a wide variety of substrates, is highly expressed in placental syncytiotrophoblasts, in the canalicular membranes of liver, in the apical membrane of the small intestine epithelium, and at the luminal surface of the endothelial cells of human brain micro vessels. This strategic tissue localization indicates that ABCG2 plays an important role in absorption, distribution, and elimination of xenobiotics and drugs. High ABCG2 expression has also been detected in many hematological malignancies and solid tumors, indicating that ABCG2 is likely responsible also for the multidrug resistance in cancer chemotherapy. Indeed, ABCG2 can actively transport structurally diverse conjugated- or unconjugated-organic molecules and various anticancer drugs. Many chemo-sensitizing agents have been discovered, which can be developed for increasing drug adsorption and reversing drug resistance in cancer chemotherapy by inhibiting ABCG2 function or expression. This review summarizes current knowledge on ABCG2, its relevance to multidrug resistance and drug disposition, and its ever-growing numbers of substrates and inhibitors.

Multidrug resistance is a serious problem in successful cancer chemotherapy. Studies using model cell lines have demonstrated that overexpression of some members of the ATP-binding cassette (ABC) transporter superfamily, such as ABCC1, causes enhanced efflux and, thus, decreased accumulation of multiple anticancer drugs, which leads to increased cell survival. Unlike most other ABC transporters, ABCC1 has an additional membrane-spanning domain (MSD0) with a putative extracellular amino terminus of 32 amino acids. However, the function of MSD0 and the role of the extracellular amino terminus are largely unknown. In this study, we examined the structural folding and the function of the amino terminus. We found that it has a U-shaped folding with the bottom of the U-structure facing cytoplasm and both ends in extracellular space. We also found that this U-shaped amino terminus probably functions as a gate to regulate the drug transport activity of human ABCC1.

One important regulation of gene expression in eukaryotes occurs at the level of mRNA translation, specifically at the step of translational initiation. Deregulation at this step will cause abnormal gene expression, leading to altered cell growth and possibly cancer. Translational initiation is controlled by multiple eIFs and one of these, eIF3, is the most complex and important factor for regulation of translation. Various subunits of eIF3 have recently been implicated to play important roles in regulating translation of specific mRNAs encoding proteins important for cell growth control. The expression of these eIF3 subunits has also been found altered in various human tumors and their altered expression may cause cancer and/or affect prognosis. Although the importance of translational regulation in cell growth control and oncogenesis is being slowly recognized, more vigorous studies on the role of eIFs in oncogenesis and cancer will likely benefit diagnosis, prognosis, and treatment of human cancers.

p27kip1 regulates cell proliferation by binding to and inhibiting the activity of cyclin-dependent kinases and its expression oscillates with cell cycle. Recently, it has been suggested from studies using the traditional dicistronic DNA assay that the expression of p27kip1 is regulated by internal ribosome entry site (IRES)-mediated translation initiation, and several RNA-binding protein factors were thought to play some role in this regulation. Considering the inevitable drawbacks of the dicistronic DNA assay, which could mislead a promoter activity or alternative splicing to IRES as previously demonstrated, we decided to reanalyze the 5'-untranslated region (5'-UTR) sequence of p27kip1 and test whether it contains an IRES element or a promoter using more stringent methods, such as dicistronic RNA and promoterless dicistronic and monocistronic DNA assays. We found that the 5'-UTR sequence of human p27kip1 does not have any significant IRES activity. The previously observed IRES activities are likely generated from the promoter activities present in the 5'-UTR sequences of p27kip1. The findings in this study indicate that transcriptional regulation likely plays an important role in p27kip1 expression, and the mechanism of regulation of p27 expression by RNA-binding factors needs to be re-examined. The findings in this study also further enforce the importance that more stringent studies, such as promoterless dicistronic and monocistronic DNA and dicistronic RNA tests, are required to safeguard any future claims of cellular IRES.

L-mimosine, a plant amino acid, can reversibly block mammalian cells at late G1 phase and has been found to affect translation of mRNAs of the cyclin-dependent kinase inhibitor p27, eIF3a (eIF3 p170), and ribonucleotide reductase M2. The effect of mimosine on the expression of these genes may be essential for the G1 phase arrest. To determine additional genes that may be early respondents to the mimosine treatment, we performed two-dimensional gel electrophoretic analysis of [35S]methionine-labeled cell lysates followed by identification of the altered protein spots by LC-tandem mass spectrometry. In this study, the synthesis of two protein spots (MIP42 and MIP17) was found to be enhanced by mimosine, whereas the formation of another protein spot (MSP17) was severely blocked following mimosine treatment. These protein spots, MIP42, MIP17, and MSP17, were identified to be differentiation-related gene 1 (Drg-1; also called RTP, cap43, rit42, Ndrg-1, and PROXY-1), deoxyhypusine-containing eIF5A intermediate, and mature hypusine-containing eIF5A, respectively. The effect of mimosine on eIF5A maturation was due to inhibition of deoxyhypusine hydroxylase, the enzyme catalyzing the final step of hypusine biosynthesis in eIF5A. The mimosine-induced expression of Drg-1 was mainly attributable to increased transcription likely by the c-Jun/AP-1 transcription factor. Because induction of Drg-1 is an early event after mimosine treatment and is observed before a notable reduction in the steady-state level of mature eIF5A, eIF5A does not appear to be involved in the modulation of Drg-1 expression.

Ribonucleotide reductase catalyzes a rate-limiting reaction in DNA synthesis by converting ribonucleotides to deoxyribonucleotides. It consists of two subunits and the small one, M2 (or R2), plays an essential role in regulating the enzyme activity and its expression is finely controlled. Changes in the M2 level influence the dNTP pool and, thus, DNA synthesis and cell proliferation. M2 gene has two promoters which produce two major mRNAs with 5'-untranslated regions (5'-UTRs) of different lengths. In this study, we found that the M2 mRNAs with the short (63 nt) 5'-UTR can be translated with high efficiency whereas the mRNAs with the long (222 nt) one cannot. Examination of the long 5'-UTR revealed four upstream AUGs, which are in the same reading frame as the unique physiological translation initiation codon. Further analysis demonstrated that these upstream AUGs act as negative cis elements for initiation at the downstream translation initiation codon and their inhibitory effect on M2 translation is eIF4G dependent. Based on the findings of this study, we conclude that the expression of M2 is likely regulated by fine tuning the translation from the mRNA with a long 5'-UTR during viral infection and during the DNA replication phase of cell proliferation.

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.

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 [35S]methionine incorporation following dramatic decrease in p170 expression was only approximately 25% 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.

The long and GC-rich 5'-untranslated region (5'-UTR) of the known 3.8-kb platelet-derived growth factor B (PDGF-B)/c-sis mRNA is highly conserved and inhibits its own translation. It has been thought that this 5'-UTR functions by regulating translation possibly using an internal ribosome entry site (IRES)-mediated mechanism. However, in the present study we found no evidence that the 5'-UTR sequence of PDGF-B mRNA contains any IRES activity. Instead, we found that the 5'-UTR sequence of PDGF-B functions as a promoter both constitutively and upon induction in a variety of cell lines. The 5'-UTR sequence contains two promoters (termed P1 and P2) when only the 5'-UTR sequence is analyzed. In the presence of the upstream TATA-box-containing promoter (P0), P1 and P0 promoters are integrated into one promoter, whereas the P2 promoter still functions. The full promoter with combined P0, P1, and P2 produced two transcripts, with the major one having the full-length 5'-UTR and the minor one the short 5'-UTR. The integrated P0/P1 promoter and P2 promoter are likely responsible for producing the endogenous 3.8- and 2.8-kb PDGF-B mRNAs that are detected in cultured human renal microvascular endothelial cells, a few tumor cells, and rat brain tissues. Furthermore, we detected the 2.8-kb PDGF-B mRNA in erythroleukemia K562 cells upon 12-O-tetradecanoylphorbol-13-acetate-induced differentiation. Considering that the 5'-UTR in the 3.8-kb mRNA contains no IRES activity and inhibits cap-dependent translation, we believe that the endogenous 2.8-kb mRNA produced from the 5'-UTR promoter is likely the major template responsible for protein production both constitutively and upon induction. We also found that the transcription from the 5'-UTR P2 promoter might be coordinated by the major upstream P0 promoter upon stimulation. Based on these observations, we propose that the TATA-containing P0 promoter and the 5'-UTR promoter work together to tightly control the expression of PDGF-B.

Multidrug resistance-associated protein 1 (MRP1) is a member of the ATP-binding cassette membrane transport superfamily and is responsible for multidrug resistance in cancer cells. Currently, there are nine known human MRPs. Distinct from many other members of the ATP-binding cassette superfamily, human MRP1 and four other MRPs have an additional membrane-spanning domain (MSD) with a putative extracellular amino terminus. The functional significance of this additional MSD (MSD1) is currently unknown. To understand the role of MSD1 in human MRP1 structure and function, we studied the amino-terminal 33 amino acids. We found that the amino terminus of human MRP1 has two cysteine residues (Cys(7) and Cys(32)) that are conserved among the five human MRPs that have MSD1. Mutation analyses of the two cysteines in human MRP1 revealed that the Cys(7) residue is critical for the MRP1-mediated drug resistance and leukotriene C(4) transport activity. On the other hand, mutation of Cys(32) reduced only moderately the MRP1 function. The effect of Cys(7) mutation on MRP1 activity appears to be due to the 5-7-fold decrease in the maximal transport rate V(max). We also found that mutation of Cys(7) changed the amino-terminal conformation of MRP1. This conformational change is likely responsible for the decrease in V(max) of LTC(4) transport mediated by the mutant MRP1. Based on these studies, we conclude that the amino terminus of human MRP1 is important and that the Cys(7) residue plays a critical role in maintaining the proper structure and function of human MRP1.

XPA plays an important role in the DNA damage recognition during human nucleotide excision repair. Here we report that the XPA is a homodimer either in the free state or as a complex with human RPA in solution under normal conditions. The human XPA protein purified from baculovirus-infected sf21 insect cells has a molecular mass of 36 317 Da, as determined by mass spectroscopy. However, the apparent molecular mass of XPA determined by the native gel filtration chromatography was about 71 kDa, suggesting that XPA is a dimer. This observation was supported by a native PFO-PAGE and fluorescence spectroscopy analysis. XPA formed a dimer (XPA2) in a broad range of XPA and NaCl concentrations, and the dimerization was not due to the disulfide bond formation. Furthermore, a titration analysis of the binding of XPA to the human RPA indicated that it was the XPA2 that formed the complex with RPA. Finally, the difference between the mass spectrometric and the calculated masses of XPA implies that the protein contains posttranslational modifications. Taken together, our data suggest that the dimerization of XPA may play an important role in the DNA damage recognition of nucleotide excision repair.

Human multidrug resistance protein 1 (MRP1) is a member of the ATP-binding cassette (ABC) transport superfamily which also includes human multidrug resistance 1 (MDR1) gene product P-glycoprotein (Pgp). Overexpression of MRP1 or Pgp causes multidrug resistance in cancer cells. Different from Pgp, MRP1 contains an extra membrane-spanning domain (MSD1) with a putative extracellular amino terminus in addition to the core structure of two MSDs and two NBDs (nucleotide-binding domains). The structural and functional significance of the additional MSD1 in MRP1 remains elusive. In this study, we generated an IgG1 subclass monoclonal antibody, IU2H10, specific to the amino terminus of human MRP1 and mapped its epitope to 10 amino acids (S8ADGSDPLWD17). It can be used for Western blot, immunoprecipitation, and indirect immunofluorescence studies of human MRP1. However, surprisingly we found that IU2H10 cannot react with MRP1 unless cells are permeabilized. Furthermore, the IU2H10 epitope is exposed extracellularly when the carboxyl-terminal core domain of human MRP1 is deleted. Examination of the amino-terminal sequence of human MRP1 suggests that it consist of mainly coiled structures. These observations provide evidence for a model that is different from the prevailing extracellular location of the amino terminus of human MRP1. It is possible that part of the amino terminus of human MRP1, following exposure to the lumen of the endoplasmic reticulum, is retracted to the cytoplasm.