Branched-chain aminotransferase 1 (BCAT1) has been proposed to drive proliferation and invasion of isocitrate dehydrogenase (IDH) wild-type glioblastoma cells. However, the Cancer Genome Atlas (TCGA) dataset shows considerable variation in the expression of this enzyme in glioblastoma. The aim of this study was to determine the role of BCAT1 in driving the proliferation and invasion of glioblastoma cells and xenografts that have widely differing levels of BCAT1 expression and the mechanism responsible.

The activity of BCAT1 was modulated in IDH wild-type patient-derived glioblastoma cell lines, and in orthotopically implanted tumors derived from these cells, to examine the effects of BCAT1 expression on tumor phenotype.

In cells with constitutively high BCAT1 expression and a glycolytic metabolic phenotype, inducible shRNA knockdown of the enzyme resulted in reduced proliferation and invasion by increasing the concentration of α-ketoglutarate, leading to reduced DNA methylation, HIF-1α destabilization, and reduced expression of the transcription factor Forkhead box protein M1 (FOXM1). Conversely, overexpression of the enzyme increased HIF-1α expression and promoted proliferation and invasion. However, in cells with an oxidative phenotype and very low constitutive expression of BCAT1 increased expression of the enzyme had no effect on invasion and reduced cell proliferation. This occurred despite an increase in HIF-1α levels and could be explained by decreased TCA cycle flux.

There is a wide variation in BCAT1 expression in glioblastoma and its role in proliferation and invasion is dependent on tumor subtype.

Significance: Unique to the branched-chain aminotransferase (BCAT) proteins is their redox-active CXXC motif. Subjected to post-translational modification by reactive oxygen species and reactive nitrogen species, these proteins have the potential to adopt numerous cellular roles, which may be fundamental to their role in oncogenesis and neurodegenerative diseases. An understanding of the interplay of the redox regulation of BCAT with important cell signaling mechanisms will identify new targets for future therapeutics. Recent Advances: The BCAT proteins have been assigned novel thiol oxidoreductase activity that can accelerate the refolding of proteins, in particular when S-glutathionylated, supporting a chaperone role for BCAT in protein folding. Other metabolic proteins were also shown to have peroxide-mediated redox associations with BCAT, indicating that the cellular function of BCAT is more diverse. Critical Issues: While the role of branched-chain amino acid metabolism and its metabolites has dominated aspects of cancer research, less is known about the role of BCAT. The importance of the CXXC motif in regulating the BCAT activity under hypoxic conditions, a characteristic of tumors, has not been addressed. Understanding how these proteins operate under various cellular redox conditions will become important, in particular with respect to their moonlighting roles. Future Directions: Advances in the quantification of thiols, their measurement, and the manipulation of metabolons that rely on redox-based interactions should accelerate the investigation of the cellular role of moonlighting proteins such as BCAT. Given the importance of cross talk between signaling pathways, research should focus more on these "housekeeping" proteins paying attention to their wider application. Antioxid. Redox Signal. 34, 1048-1067.

The developmental plasticity of embryonic stem cells (ESCs) is mainly controlled by well-characterized transcription factors, but additional factors, especially those related to metabolism that modulate this intrinsic program remain elusive. Here, using whole transcriptome analysis, we identified branched-chain amino acid aminotransferase-1(Bcat1) as highly-expressed in mouse ESCs and dramatically down-regulated upon differentiation. Bcat1 deletion impaired pluripotency and self-renewal in mouse ESCs, while Bcat1 overexpression resulted in robust ESC self-renewal and inhibition of differentiation. Whole genome bisulfite sequencing (WGBS) analysis showed that Bcat1 deletion altered whole genome methylation levels and hence gene expression in multiple pathways. Specifically, Bcat1 deletion increased expression of RAS protein activator like 1(Rasal1), leading to inactivation of Ras-Erk/MAPK signaling, while Rasal1 inhibition rescued defects seen in Bcat1 deleted cells. In summary, we demonstrate that Bcat1 is essential for mouse ESC self-renewal and pluripotency and that this effect is mediated by DNA methylation and the Ras signaling pathway.

Oncogenic c-Myc-induced metabolic reprogramming triggers cellular dependency on exogenous glucose and glutamine. Understanding how nutrients are used may provide new target for therapeutic intervention. We previously provided an alternate route to c-Myc-driven glucose metabolism via the repression of thioredoxin-interacting protein (TXNIP), which is a potent negative regulator of glucose uptake. Herein, we demonstrate that c-Myc suppression of TXNIP is predominantly through the activation of glutaminolysis via glutaminase (GLS1) in prostate cancer cells. Glutamine depletion blocked c-Myc-dependent reductions of TXNIP and its principal regulator MondoA transcriptional activity. Further, GLS1 inhibition by either siRNA or CB-839 resumed TXNIP expression that was repressed by c-Myc. The TXNIP promoter with mutant E-Box region, which was recognized by MondoA, failed to respond to c-Myc or GLS1, indicating c-Myc repression of TXNIP by GLS1 is predominantly through the blockage of MondoA activity. Especially, ectopic TXNIP expression decreased c-Myc-induce glucose uptake and lead to a broad range of glycolytic target gene suppressions. Thus TXNIP is a key adaptor for c-Myc-driven aerobic glycolysis. Supporting the biological significance of c-Myc and TXNIP, their reciprocal relationship are correlates with patient outcome and contributes to the aggressive phenotype in PCAs.

Both p53-related p63 and c-Myc are transcription factors playing key roles in cell proliferation, survival, development and tumorigenesis. In the present study, we identified that MM1, a c-Myc inhibitor, specifically binds to C-termini of p63α (including ΔNp63α and TAp63α). Further study demonstrates that p63α facilitates MM1 protein degradation via proteasomal pathway, resulting in elevation of c-Myc transactivation activity. Knockdown of ΔNp63α leads to decrease in c-Myc protein levels, concomitant with reduced expression of CDK4 and Cyclin D1, and impaired cell cycle progression, both of which are effectively reversed by simultaneous knockdown of MM1. Moreover, expression of p63 and CDK4 is concomitantly up-regulated in B-cell acute lymphoblastic leukemia. Together, this study reveals a novel crosstalk between p63 and c-Myc that may play an important role in cell cycle progression and tumorigenesis.

As one form of branched-chain amino-acid transaminase (BCAT) enzymes, It has been found that up-regulation of BCAT1 is associated with poor prognosis in numerous types of tumors, but studies on the role of BCAT1 expression in gastric cancer (GC) are rare. The aims of this study were to detect BCAT1 expression in GC and to analyze its association with prognosis of GC patients. Microarray experiments were performed on the Affymetrix U133 plus 2.0 GeneChip Array. The protein and messenger RNA levels of BCAT1 were validated by immunohistochemistry and real-time quantitative polymerase chain reaction in GC tissues and adjacent noncancerous tissues. Our study shows that the expression of BCAT1 significantly increased in human GC. Furthermore, it can also be found that BCAT1 overexpression was associated with TNM stage (P < .05), local invasion (P < .05), Lauren type (P < .05), tumor classification (P < .05), lymph node metastasis (P < .05), and presence of distant metastasis (P < .05). Kaplan-Meier survival analysis revealed that high BCAT1 expression predicted significantly worse overall survival (P < .05), whereas multivariate Cox regression analysis showed that BCAT1 affects GC independently. In conclusion, up-regulation of BCAT1 indicated a poor survival rate of GC and may serve as a useful marker for predicting the outcome of patients with GC.

MicroRNAs have been reported to be associated with chemosensitivity of several types of cancers. However, the underlying molecular mechanisms are poorly understood. In this study, we explored miR-218 increased the chemosensitivity to cis-diaminedichloroplatinum treatment of prostate cancer. We found that the expression level of miR-218 was down-regulated in the human prostate cancer specimens. Moreover, overexpression of miR-218 inhibited cell viability, migration, and invasion in PC3 and DU145 cells. Furthermore, we demonstrated that the tumor suppressive role of miR-218 was mediated by negatively regulating branched-chain amino acid transaminase 1 (BCAT1) protein expression. Importantly, overexpression of BCAT1 decreased the chemosensitivity to CDDP treatment of PC3 and DU145 cells. Our study is the first to identify the positive role of miR-218 in chemosensitivity, which will facilitate the development of novel therapeutic strategies for prostate cancer in the future.

BCAT1 initiates the catabolism of branched-chain amino acids. Here, we investigated the function of BCAT1 and its transcriptional regulatory mechanism in hepatocellular carcinoma (HCC).

RNASeq was used to evaluate BCAT1 mRNA levels in HCC and normal matched specimens. After the exogenous expression of BCAT1 in BEL-7404 cells and the suppression of endogenous BCAT1 expression with shRNA in HepG2 cells, the cell proliferation, clone-forming ability and cell-cycle changes were measured with MTT assay, colony-forming assay and flow cytometry respectively. A xenograft model was used to investigate the effect of BCAT1 on cancer growth in vivo. Chromatin immunoprecipitation and luciferase reporter technologies were used to confirm the transcriptional regulation of the BCAT1 gene by MYC. The expression of the BCAT1 and MYC proteins in 122 HCC tissues was determined with an immunohistochemical analysis.

BCAT1 mRNA was clearly increased in HCC tissues and hepatomas. The ectopic expression of BCAT1 in BEL-7404 cells enhanced their proliferation, clone formation, tumourigenic properties, S-G₂ /M phase transition and chemoresistance to cisplatin. The suppression of BCAT1 expression in HepG2 cells significantly inhibited their proliferation, clone formation, and S-G₂ /M phase transition and caused their chemosensitization to cisplatin. MYC affected the transcriptional regulation of BCAT1. Clinical data showed that BCAT1 expression correlated with a significantly poorer prognosis.

BCAT1 plays a pathogenic role in HCC by causing cell proliferation and chemoresistance. The MYC transcription factor is involved in regulating the transcriptional activity of BCAT1. BCAT1 expression has prognostic significance for the survival of patients with HCC.

The Myc-Max heterodimer is a transcription factor that regulates expression of a large number of genes. Genome occupancy of Myc-Max is thought to be driven by Enhancer box (E-box) DNA elements, CACGTG or variants, to which the heterodimer binds in vitro.

By analyzing ChIP-Seq datasets, we demonstrate that the positions occupied by Myc-Max across the human genome correlate with the RNA polymerase II, Pol II, transcription machinery significantly better than with E-boxes. Metagene analyses show that in promoter regions, Myc is uniformly positioned about 100 bp upstream of essentially all promoter proximal paused polymerases with Max about 15 bp upstream of Myc. We re-evaluate the DNA binding properties of full length Myc-Max proteins. Electrophoretic mobility shift assay results demonstrate Myc-Max heterodimers display significant sequence preference, but have high affinity for any DNA. Quantification of the relative affinities of Myc-Max for all possible 8-mers using universal protein-binding microarray assays shows that sequences surrounding core 6-mers significantly affect binding. Compared to the in vitro sequence preferences,Myc-Max genomic occupancy measured by ChIP-Seq is largely, although not completely, independent of sequence specificity.

We quantified the affinity of Myc-Max to all possible 8-mers and compared this with the sites of Myc binding across the human genome. Our results indicate that the genomic occupancy of Myc cannot be explained by its intrinsic DNA specificity and suggest that the transcription machinery and associated promoter accessibility play a predominant role in Myc recruitment.

Powerful analytical tools are vital for characterizing the complex molecular changes underlying oncogenesis and cancer treatment. This is particularly true, if information is to be collected in vivo by noninvasive approaches. In the recent past, hyperpolarized (13)C magnetic resonance (MR) spectroscopy has been employed to quickly collect detailed spectral information on the chemical fate of tracer molecules in different tissues at high sensitivity. Here, we report a preclinical study showing that alpha-ketoisocaproic acid (KIC) can be used to assess molecular signatures of tumors with hyperpolarized MR spectroscopy. KIC is metabolized to leucine by the enzyme branched chain amino acid transferase (BCAT), which is found upregulated in some tumors. BCAT is a putative marker for metastasis and a target of the proto-oncogene c-myc. Very different fluxes through the BCAT-catalyzed reaction can be detected for murine lymphoma (EL4) and rat mammary adenocarcinoma (R3230AC) tumors in vivo. EL4 tumors show a more than 7-fold higher hyperpolarized (13)C leucine signal relative to the surrounding healthy tissue. In R3230AC tumor on the other hand branched chain amino acid metabolism is not enhanced relative to surrounding tissues. The distinct molecular signatures of branched chain amino acid metabolism in EL4 and R3230AC tumors correlate well with ex vivo assays of BCAT activity.

Overexpression of the oncogene c-Myc sensitizes many apoptotic signals through the activation of mitochondrial apoptosis pathway. However, the underling mechanism has not been clearly defined. Here, we investigated the effect of c-Myc expression on histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA)-induced apoptosis in rat fibroblast cells possessing various c-Myc levels. In Rat 1a cells overexpressing c-Myc, SAHA-induced enhanced the cell death response relative to the parental cells; whereas Rat 1a cells lacking c-Myc were refractory to SAHA treatment. We demonstrated that SAHA selectively induced the expression of pro-apoptotic BH3-only protein Bim, leading to Bax activation in c-Myc-expressing cells. Where c-Myc was absent, Bim, despite its induction by SAHA, failed to activate Bax and was unable to induce apoptosis. These results indicate that c-Myc is dispensable for Bim induction by SAHA, but is required for subsequent Bax activation. We further show that the expression levels of anti-apoptotic Bcl-2/Bcl2-xL were much elevated in Myc-null cells compared with the c-Myc-expressing cells; furthermore, depletion of Bcl-2/Bcl-xL in these cells restored the ability of SAHA to induce apoptosis by enhancing Bax activation. These data indicate that SAHA induces apoptosis through Bim-triggered Bax activation and that c-Myc regulates this process by modulating Bcl-2/Bcl-xL. Our results provide novel insight into the mechanism whereby Myc sensitizes the apoptotic signals; furthermore, our data suggest that cancer cells with deregulated Myc might be more sensitive to SAHA treatment.

The N-myc downstream-regulated gene 1 (ndrg1) is highly expressed in N-myc knock-out mice through an unknown regulatory mechanism. As one member of the human NDRG gene family, NDRG2 encodes a protein highly homologous to Ndrg1. However, it is uncertain whether the expression of human NDRG2 is regulated by Myc because mouse ndrg2 and -3 are not affected by Myc. In this study, we provide the novel evidence that the expression of human NDRG2 is down-regulated by Myc via transcriptional repression. A high level of NDRG2 was observed as Myc expression was reduced in differentiated cells, whereas a low level of NDRG2 was shown following increased Myc expression upon serum stimulation. The ectopic expression of c-Myc dramatically reduces the cellular Ndrg2 protein and mRNA level. We further identified the core promoter region of NDRG2 that is required for Myc repression on NDRG2 transcription, and we verified the interaction of Myc with the core promoter region both in vitro and in vivo. Moreover, the c-Myc-mediated repression of NDRG2 requires association with Miz-1, and possibly the recruitment of other epigenetic factors, such as histone deacetylases, to the promoter. The regulatory function of Myc on NDRG2 gene expression implicated the role of the Ndrg2 in regulating cell differentiation.

To investigate the possible role of polysaccharide-K (PSK) -related markers in predicting distant metastasis and in the clinical outcome of colorectal cancer (CRC).

Firstly, we used protein microarrays to analyze the in vitro expression profiles of potential PSK-related markers in the human colorectal adenocarcinoma cell line SW480, which carries a mutant p53 gene. Then, we investigated the clinical implications of these markers in the prognosis of CRC patients.

ECA39, a direct target of c-Myc, was identified as a candidate protein affected by the anti-metastatic effects of PSK. Immunohistochemistry revealed that ECA39 was expressed at significantly higher levels in tumor tissues with distant metastases compared to those without (P<0.00001). Positive ECA39 expression was shown to be highly reliable for the prediction of distant metastases (sensitivity: 86.7%, specificity: 90%, positive predictive value: 86.7%, negative predictive value: 90%). A significantly higher cumulative 5-yr disease free survival rate was observed in the ECA39-negative patient group (77.3%) compared with the ECA39-positive patient group (25.8%) (P<0.05).

Our results suggest that ECA39 is a dominant predictive factor for distant metastasis in patients with advanced CRC and that its suppression by PSK might represent a useful application of immunotherapy as part of a program of integrated medicine.

Recently determined structures of a number of Myc family proteins have provided significant insights into the molecular nature of complex assembly and DNA binding. These structures illuminate the details of specific interactions that govern the assembly of nucleoprotein complexes and, in doing so, raise more questions regarding Myc biology. In this review, we focus on the lessons provided by these structures toward understanding (1) interactions that govern transcriptional repression by Mad via the Sin3 pathway, (2) homodimerization of Max, (3) heterodimerization of Myc-Max and Mad-Max, and (4) DNA recognition by each of the Max-Max, Myc-Max, and Mad-Max dimers.

Pulmonary adenoma susceptibility 1 (Pas1), located on chromosome 6, is the major locus affecting inherited predisposition to lung tumor development in mice. We have fine mapped the Pas1 locus to a region of approximately 0.5 megabases by using congenic strains of mice, constructed by placing the Pas1 region of chromosome 6 from A/J mice onto the genetic background of C57BL/6J mice. Systematic characterization of Pas1 candidates establishes the Las1 (lung adenoma susceptibility 1) and Kras2 (Kirsten rat sarcoma oncogene 2) genes as primary candidates for the Pas1 locus. Clearly, Kras2 affects lung tumor progression only, and Las1 is likely to affect lung tumor multiplicity.

Expression of the laminin-binding alpha7 integrin is tightly regulated during myogenic differentiation, reflecting required functions that range from cell motility to formation of stable myotendinous junctions. However, the exact mechanism controlling alpha7 expression in a tissue- and differentiation-specific manner is poorly understood. This report provides evidence that alpha7 gene expression during muscle differentiation is regulated by the c-Myc transcription factor. In myoblasts, alpha7 is expressed at basal levels, but following conversion to myotubes the expression of the integrin is strongly elevated. The increased alpha7 mRNA and protein levels following myogenic differentiation are inversely correlated with c-Myc expression. Transfection of myoblasts with the c-Myc transcription factor down-regulated alpha7 expression, whereas overexpression of Madmyc, a dominant-negative c-Myc chimera, induced elevated alpha7 expression. Functional analysis with site-specific deletions identified a specific double E-box sequence in the upstream promoter region (-2.0 to -2.6 kb) that is responsible for c-Myc-induced suppression of alpha7 expression. DNA-protein binding assays and supershift analysis revealed that c-Myc forms a complex with this double E-box sequence. Our results suggest that the interaction of c-Myc with this promoter region is an important regulatory element controlling alpha7 integrin expression during muscle development and myotendinous junction formation.

Three type 1 diabetes associated regions on distal mouse chromosome 6 have recently been defined by the construction and analysis of a series of congenic strains, carrying C3H/HeJ genomic material on a NOD/Lt genetic background. Whilst NOD/Lt alleles at the most distal locus Idd6 confer susceptibility, C3H/HeJ alleles confer resistance to diabetes. Idd6 overlaps with a locus controlling low rates of proliferation in immature NOD-thymocytes, suggesting that Idd6 could be controlling diabetes development through an effect on T cell proliferation rates. Candidates for Idd6 therefore include genes, which are implicated in the immune system and/or in the control of cell proliferation rates, such as Lrmp (Jaw1), Bcat1 and Kras2 that map to the Idd6 candidate region. In the present study, we have undertaken an expression and mutational analysis of all three genes. A surprisingly large number of polymorphisms and amino acid changes were identified in both Lrmp and Bcat1 indicating that they are candidates for Idd6. The two genes are located within a genomic interval of about 3 Mb that contains a large number of single nucleotide polymorphisms (SNP) and which has possibly been derived from distinct ancestral haplotypes in the C3H/HeJ and NOD/Lt strains.

All invasive testicular germ cell tumors of adolescents and adults (TGCTs), that is, seminomas and nonseminomas, show gain of 12p sequences, mostly as isochromosomes. Although several candidate genes have been suggested, the relevant gene(s) have not been identified yet. About 10% of testicular seminomas, however, show a more restricted amplification of the 12p11.2-p12.1 region, in which the various amplicons show an apparent overlap, allowing for the shortest region of amplification overlap approach, aiming at the identification of pathogenetically relevant sequences residing in this region. Here we report on a high-resolution 12p-amplicon architecture analysis using microarray-based comparative genomic hybridization, the results of which were subsequently confirmed by fluorescent in situ hybridization studies. The 12p-specific microarray contained 63 positionally selected BAC clones, which are more or less evenly distributed over the short arm of chromosome 12 (average spacing: less than 500 Kb), including 20 clones within the region of amplification. Out of a series of 17 seminomas, seven seminomas showed amplification of the whole amplicon region, of which three showed a dip in T/R value in the center of the amplified area. A more complex amplification pattern was found in the other 10 seminomas: three showed predominant amplification at the centromeric border; one mainly at the telomeric border; six showed a balanced amplification of both the centromeric and telomeric regions. The only nonseminoma investigated showed a structure in which the centromeric border was only amplified. These data support a mechanistic model in which at least two 12p genes, situated at the border regions of the amplicon, are positional candidates capable of actively supporting tumor progression in TGCTs.

Transgenic mice with engineered disruptions in bidirectional endocrine signaling between the pituitary and gonad have shed light on the specific effects of the loss of function of gonadotropins and inhibins. These models are valuable tools for studying ovarian biology because they phenocopy specific pathological states and have variations in ovarian tissue composition that allow us to identify genes expressed in specific cell types. We have used emerging mRNA expression profiling technologies to gain a more comprehensive view of genes that are expressed in the mammalian ovary and adrenal gland in the FSHbeta and inhibin alpha knockout mouse models. Oligonucleotide array hybridization experiments using Affymetrix GeneChip technology and NIA 15K murine cDNA microarray studies identified hundreds of transcripts differentially expressed compared with wild type, over 30 of which were selected for further characterization by Northern blot analyses. Additionally, we performed in situ hybridization studies to localize 10 mRNAs, melanocyte-specific gene 1, amino acid transporter SN2, overexpressed and amplified in teratocarcinoma (Bcat1), Forkhead box protein FOXO1, 24p3, vascular cell adhesion molecule, epiregulin, Bcl2-like10, PC3B, and retinoblastoma binding protein 7. These 10 genes have expression patterns and postulated functions suggesting that they mediate important processes in the physiology and pathology of ovarian and adrenal tissue.

Gain of 12p material is invariably associated with testicular germ cell tumors (TGCTs) of adolescents and adults, most usually as an isochromosome 12p. We analyzed TGCTs with i(12p) using a global approach to expression profiling targeting chromosomes (comparative expressed sequence hybridization, CESH). This indicated overexpression of genes from 12p11.2-p12.1 relative to testis tissue and fibroblasts. The nonseminoma subtype showed higher levels of expression than seminomas. Notably, 12p11.2-p12.1 is amplified in about 10% of TGCTs and CESH analysis of such amplicon cases showed high levels of overexpression from this region. Microarray analysis, including cDNA clones representing most UniGene clusters from 12p11.2-p12.1, was applied to DNA and RNA from 5 TGCTs with amplification of 12p11.2-p12.1 and seven TGCTs with gain of the entire short arm of chromosome 12. Expression profiles were consistent with the CESH data and overexpression of EST595078, MRPS35 and LDHB at 12p11.2-p12.1 was detected in most TGCTs. High-level overexpression of BCAT1 was specific to nonseminomas and overexpression of genes such as CMAS, EKI1, KRAS2, SURB7 and various ESTs correlated with their amplification. Genes such as CCND2, GLU3, LRP6 and HPH1 at 12p13 were also overexpressed. The overexpressed sequences identified, particularly those in the region amplified, represent candidate genes for involvement in TGCT development.

The activated product of the myc oncogene deregulates both cell growth and death check points and, in a permissive environment, rapidly accelerates the affected clone through the carcinogenic process. Advances in understanding the molecular mechanism of Myc action are highlighted in this review. With the revolutionary developments in molecular diagnostic technology, we have witnessed an unprecedented advance in detecting activated myc in its deregulated, oncogenic form in primary human cancers. These improvements provide new opportunities to appreciate the tumor subtypes harboring deregulated Myc expression, to identify the essential cooperating lesions, and to realize the therapeutic potential of targeting Myc. Knowledge of both the breadth and depth of the numerous biological activities controlled by Myc has also been an area of progress. Myc is a multifunctional protein that can regulate cell cycle, cell growth, differentiation, apoptosis, transformation, genomic instability, and angiogenesis. New insights into Myc's role in regulating these diverse activities are discussed. In addition, breakthroughs in understanding Myc as a regulator of gene transcription have revealed multiple mechanisms of Myc activation and repression of target genes. Moreover, the number of reported Myc regulated genes has expanded in the past few years, inspiring a need to focus on classifying and segregating bona fide targets. Finally, the identity of Myc-binding proteins has been difficult, yet has exploded in the past few years with a plethora of novel interactors. Their characterization and potential impact on Myc function are discussed. The rapidity and magnitude of recent progress in the Myc field strongly suggests that this marvelously complex molecule will soon be unmasked.

The functions of thyroid hormone receptors (TRs) are regulated by a host of co-regulatory proteins. Tissue-specific expression of these co-regulators leads to distinct expression patterns and regulation of thyroid hormone (T3) target genes in tissues. Previously we have found that human colon carcinoma RKO cells exhibit strong T3-independent transcriptional activity. We therefore searched for co-regulatory proteins in RKO cells using a yeast two-hybrid system with the intact TRbeta1 as bait. One of the three positive clones, designated as P3, was identified to be an isoform of human mitochondria branched-chain aminotransferase (BCATm). P3 was a spliced variant of BCATm with an internal 12-amino acid deletion near the carboxyl-terminal region and was abundantly expressed in RKO cells. The expressed protein localized both to the mitochondria and the nucleus of transfected CV1 cells. P3 physically interacted with TRbeta1 in a T3-independent manner that led to the inhibition in binding of TRbeta1 to thyroid hormone-responsive element. P3 not only enhanced the repressor activity of the unliganded TR but also repressed the ligand-dependent activation of TR. This repression was reversed by treatment of cells with trichostatin A, suggesting that in addition to the inhibition of DNA binding, the repression activity of P3 on TR may also be mediated by histone deacetylase activity. Thus, unlike the currently known co-repressors, P3 is a novel ligand-independent co-repressor for TR.

Overexpression and inhibitor studies have suggested that the c-Myc target gene for ornithine decarboxylase (ODC), the enzyme which converts ornithine to putrescine, plays an important role in diverse biological processes, including cell growth, differentiation, transformation, and apoptosis. To explore the physiological function of ODC in mammalian development, we generated mice harboring a disrupted ODC gene. ODC-heterozygous mice were viable, normal, and fertile. Although zygotic ODC is expressed throughout the embryo prior to implantation, loss of ODC did not block normal development to the blastocyst stage. Embryonic day E3.5 ODC-deficient embryos were capable of uterine implantation and induced maternal decidualization yet failed to develop substantially thereafter. Surprisingly, analysis of ODC-deficient blastocysts suggests that loss of ODC does not affect cell growth per se but rather is required for survival of the pluripotent cells of the inner cell mass. Therefore, ODC plays an essential role in murine development, and proper homeostasis of polyamine pools appears to be required for cell survival prior to gastrulation.

Primitive neuroectodermal tumors (PNETs) such as human medulloblastomas are genetically heterogeneous and therefore poorly understood. In a rat model the SV40 large T antigen was used to induce neoplasms with characteristic features of PNETs. Tumor development requires a latency period of 8-11 months implicating secondary genetic alterations. To identify such secondary alterations we performed comparative analyses of two phenotypically identical PNET-derived cell lines. Indeed, these cell lines displayed distinct high-level amplification sites. Using a combination of subtractive cDNA analysis and radiation hybrid mapping we have now identified genes in the amplicon regions of the two cell lines. Interestingly, one of these genes encodes the rat homolog of a cytosolic branched chain aminotransferase (BCAT(C)) previously shown to be amplified in a mouse teratocarcinoma cell line. We propose that this simple cloning strategy may serve as a powerful tool for the isolation of genes implicated in known chromosomal aberrations in primary tumors and tumor cell lines.

The Myc/Max/Mad network comprises a group of transcription factors whose distinct interactions result in gene-specific transcriptional activation or repression. A great deal of research indicates that the functions of the network play roles in cell proliferation, differentiation, and death. In this review we focus on the Myc and Mad protein families and attempt to relate their biological functions to their transcriptional activities and gene targets. Both Myc and Mad, as well as the more recently described Mnt and Mga proteins, form heterodimers with Max, permitting binding to specific DNA sequences. These DNA-bound heterodimers recruit coactivator or corepressor complexes that generate alterations in chromatin structure, which in turn modulate transcription. Initial identification of target genes suggests that the network regulates genes involved in the cell cycle, growth, life span, and morphology. Because Myc and Mad proteins are expressed in response to diverse signaling pathways, the network can be viewed as a functional module which acts to convert environmental signals into specific gene-regulatory programs.

The HMG-I/Y gene encodes the HMG-I and HMG-Y proteins, which function as architectural chromatin binding proteins important in the transcriptional regulation of several genes. Although increased expression of the HMG-I/Y proteins is associated with cellular proliferation, neoplastic transformation, and several human cancers, the role of these proteins in the pathogenesis of malignancy remains unclear. To better understand the role of these proteins in cell growth and transformation, we have been studying the regulation and function of HMG-I/Y. The HMG-I/Y promoter was cloned, sequenced, and subjected to mutagenesis analysis. A c-Myc-Max consensus DNA binding site was identified as an element important in the serum stimulation of HMG-I/Y. The oncoprotein c-Myc and its protein partner Max bind to this site in vitro and activate transcription in transfection experiments. HMG-I/Y expression is stimulated by c-Myc in a Myc-estradiol receptor cell line in the presence of the protein synthesis inhibitor cycloheximide, indicating that HMG-I/Y is a direct c-Myc target gene. HMG-I/Y induction is decreased in Myc-deficient fibroblasts. HMG-I/Y protein expression is also increased in Burkitt's lymphoma cell lines, which are known to have increased c-Myc protein. Like Myc, increased expression of HMG-I protein leads to the neoplastic transformation of both Rat 1a fibroblasts and CB33 cells. In addition, Rat 1a cells overexpressing HMG-I protein form tumors in nude mice. Decreasing HMG-I/Y proteins using an antisense construct abrogates transformation in Burkitt's lymphoma cells. These findings indicate that HMG-I/Y is a c-Myc target gene involved in neoplastic transformation and a member of a new class of potential oncogenes.

The human Surf-1 and Surf-2 genes are divergently transcribed and share a single bi-directional promoter. The addition of serum growth factors to serum-starved cells activates transcription in the Surf-1 direction, but has no effect on transcription in the Surf-2 direction. Mutations that block the binding of YY1 to a site immediately downstream of the major Surf-1 transcription start point abolish this response to serum factors. Here we show that over-expression of mitogen-activated protein (MAP) kinase phosphatase MKP-1, an inhibitor of the MAP kinase cascade, also blocks the response the Surf-1 promoter to serum factors. YY1 has previously been shown to interact with several transcription factors including Myc. We show that although the Surf-1/Surf-2 promoter does not contain Myc binding sites (E-boxes), Myc over-expression, or the activation of a Myc-oestrogen receptor fusion protein, activates transcription in the Surf-1 direction and that this response to Myc requires a functional YY1 binding site. Our data suggest that the MAP kinase cascade is required for the stimulation of Surf-1 promoter activity and that the Myc-YY1 interaction mediates this response.

The c-myc protooncogene product (c-Myc) is a transcription factor and is rapidly induced in resting cells following various mitogenic stimuli. c-Myc is thus suggested to play an important role in the transition from quiescence to proliferation. Despite numerous studies, including those on the connection between cyclin E/cyclin-dependent kinase 2 and c-Myc, little has been clarified about c-Myc in terms of the cell cycle regulation. Here we show that c-Myc can directly bind to the carboxyl-terminal region of the cyclin-dependent kinase inhibitor p21(cip1/waf1/sdi1) and thus partially relieves the p21 of the inhibitory effect on DNA synthesis directed by the proliferating cell nuclear antigen-dependent DNA polymerase delta. As for transcription, on the other hand, the p21 binding to the Myc box II region of c-Myc blocks c-Myc-Max complex formation on the E-box and thereby suppresses the transcriptional activation from the E-box by c-Myc. These results suggest that c-Myc activates DNA replication via inactivation of p21 and that p21, vice versa, represses the transcriptional activity of c-Myc. The balance of the reciprocal inactivation between c-Myc and p21 may determine the course of cellular processes such as cell proliferation, differentiation, and apoptosis.

MSSPs, myc single strand binding proteins, were originally identified as proteins recognizing a putative replication origin/transcriptional enhancer in the human c-Myc gene. The cDNAs encoding four of the family proteins, MSSP-1, MSSP-2, Scr2 and Scr3, were cloned. These proteins carry two copies of the putative RNA binding domains, RNP-A and RNP-B, and have been suggested to participate in DNA replication and cell cycle progression from the G1 to the S phase.

We report that MSSP-1 and MSSP-2 bound directly to the C-terminal portion of c-Myc, along with Max, side by side. MSSP, c-Myc and Max formed a ternary complex in vivo, although MSSP did not directly associate with Max. The MSSP/Myc/Max ternary complex lost the binding activity to the E-box sequence-the recognition sequence of c-Myc/Max complex-thereby abrogating the E-box-dependent transcription activity of c-Myc. MSSP specifically stimulated the cooperative transforming activity of c-myc with ras, in a manner dependent upon the RNP sequences, while mssp itself showed no transforming activity in mouse NIH3T3 cells. The NIH3T3 transformants, together with ras, myc and mssp, grew to form very large colonies in soft agar, as compared to those with ras plus myc or ras alone.

MSSP is a modulator of c-Myc and the c-Myc/MSSP complex may deregulate cell cycle controls and lead cells towards transforming pathways.

Human c-myc cDNA was fused with the hormone-binding domain (HBD) cDNA of murine estrogen receptor gene and the chimeric gene was introduced into the CHO cells. The fusion protein, c-MycER, becomes activated when the synthetic steroid, 4-hydroxy-tamoxifen (OHT), binds HBD. Activated c-MycER, likely c-Myc, can induce quiescent CHO cells reentry into S phase and subsequent cell death under serum-free condition. In addition, the expression of some proposed c-myc target genes such as ODC, MrDb, cad, rcc1 and rcl were found to increase upon OHT induction before S phase entry and apoptosis, indicating that these target genes are involved in cell cycle regulation and/or apoptosis control. However, the mutant D106-143c-MycER protein does not have above activities.

The c-myc gene and the expression of the c-Myc protein are frequently altered in human cancers. The c-myc gene encodes the transcription factor c-Myc, which heterodimerizes with a partner protein, termed Max, to regulate gene expression. Max also heterodimerizes with the Mad family of proteins to repress transcription, antagonize c-Myc, and promote cellular differentiation. The constitutive activation of c-myc expression is key to the genesis of many cancers, and hence the understanding of c-Myc function depends on our understanding of its target genes. In this review, we attempt to place the putative target genes of c-Myc in the context of c-Myc-mediated phenotypes. From this perspective, c-Myc emerges as an oncogenic transcription factor that integrates the cell cycle machinery with cell adhesion, cellular metabolism, and the apoptotic pathways.

A suppression subtractive hybridization technique was used to identify reactive oxygen species (ROS)-regulated genes in rat vascular smooth muscle cells. Three genes out of 89 clones, identified as fibronectin, p105 coactivator and ECA39, showed increased expression after treatment with H(2)O(2). The mRNA expressions of these three genes were induced in a time- and dose-dependent manner, independent of protein kinase C activation. Immunohistochemical staining showed that the p105 coactivator expression was markedly induced in the neointima of balloon-injured rat carotid arteries. These results suggest that ROS may play an important role in the development of atherosclerosis by regulating the gene expressions we identified in this study.

The branched-chain amino acid aminotransferase, Bcat1/Eca39, catalyzes the first step of branched-chain amino acid catabolism. Bcat1/Eca39 was originally isolated from a c-myc-induced tumor and was proven to be a direct target for c-Myc regulation. The gene is highly conserved in evolution and disruption of its yeast homolog affects cell growth. To assess the role of Bcat1/Eca39 in mammalian cells, we overexpressed Bcat1/Eca39 in murine cells and studied effects on cell growth. Overexpression of Bcat1/Eca39 had no apparent effect on the proliferation of cells grown with high serum concentrations, but under serum deprivation conditions, led to a decrease in cell viability. Cell death under these conditions displayed apoptotic features. The branched-chain keto acid, alpha-ketoisocaproate, a metabolite of leucine catabolism produced by BCAT1/ECA39, was previously found to inhibit cell growth. We show that alpha-ketoisocaproate can induce rapid apoptotic cell death. This observation suggests that the growth inhibitory effect of BCAT1/ECA39 and its apoptosis promoting effect may be mediated by the levels of the products of BCAT1/ECA39 activity, namely, branched-chain keto acids.

Much recent research on c-Myc has focused on how it drives apoptosis. c-Myc is widely known as a crucial regulator of cell proliferation in normal and neoplastic cells, but until relatively recently its apoptotic properties, which appear to be intrinsic, were not fully appreciated. Its death-dealing aspects have gained wide attention in part because of their potential therapeutic utility in advanced malignancy, where c-Myc is frequently deregulated and where novel modalities are badly needed. Although its exact function remains obscure, c-Myc is a transcription factor and advances have been made in characterizing target genes which may mediate its apoptotic properties. Candidate regulators and effectors are also emerging. Among recent findings are connections to the CD95/Fas and TNF pathways and roles for the tumor suppressor p19ARF and the c-Myc-interacting adaptor protein Binl in mediating cell death. In this review I summarize the data establishing a role for c-Myc in apoptosis in diverse settings and present a modified dual signal model for c-Myc function. It is proposed that c-Myc induces apoptosis through separate 'death priming' and 'death triggering' mechanisms in which 'death priming' and mitogenic signals are coordinated. Investigation of the mechanisms that underlie the triggering steps may offer new therapeutic opportunities.

Mutations which disrupt the regulation or expression level of the c-myc gene are among the most common found in human and animal cancers (reviewed in ref. Cole, 1986; Henriksson and Luscher, 1996; Marcu et al., 1992). Ectopic expression studies define numerous biological activities of the c-myc gene, including transformation, immortalization, blockage of cell differentiation and induction of apoptosis (Askew et al., 1991; Cole, 1986; Evan and Littlewood, 1993; Freytag et al., 1990; Henriksson and Luscher, 1996; Marcu et al., 1992). Furthermore, c-myc is required for efficient progression through the cell cycle (Goruppi et al., 1994; Prochownik et al., 1988; Yokoyama and Imamoto, 1987), although recent studies indicate that it is not absolutely essential (Mateyak et al., 1997). This fascinating array of biological activities makes the c-myc gene one of the most intriguing oncogenes and presents the challenging question of how a single gene can manifest so many different effects. The c-Myc protein exhibits sequence-specific DNA binding when dimerized with its partner Max, and DNA binding is mediated through the basic region, which recognizes the core sequence CACGTG (Berberich et al., 1992; Blackwell et al., 1993; Blackwood and Eisenman, 1991; Prendergast and Ziff, 1991; Prendergast et al., 1991), but exhibits somewhat higher affinity for the more extended sequence ACCACGTGGT (Berberich et al., 1992; Blackwell et al., 1993; Halazonetis and Kandil, 1991). There are three closely related Myc family proteins (c-Myc, N-Myc and L-Myc), each with documented oncogenic potential (Birrer et al., 1988; Schwab et al., 1985; Yancopoulos et al., 1985) and similar DNA binding properties (Mukherjee et al., 1992). For simplicity, we will use the term Myc to refer to all three proteins, but delineate any distinct activities where they apply. The goal of this review is to discuss Myc as a transcriptional activator and critically evaluate the evidence for the transactivation of specific target genes as direct downstream effectors. Since excellent comprehensive reviews on Myc have been published recently (Facchini and Penn, 1998; Henriksson and Luscher, 1996), we will focus on the latest observations that offer mechanistic insight into transactivation and oncogenic transformation.

To identify genes regulated by N-myc, subtraction of whole embryo cDNA was carried out between wild type and N-myc-deficient mutant mice. Six cDNA clones were isolated as representing genes expressed higher in the mutant embryos and two as those expressed lower. One of them, Ndr1, coding for 43 kDa cytoplasmic protein was studied in detail. The Ndr1 gene was augmented 20-fold in the mutant embryos at 10.5 days post coitus which is indicative of repression by N-myc. An inverse relationship actually existed between the expression of N-myc and Ndr1 in various developing tissues of the wild type embryos. In the early stage of differentiation of these tissues when N-myc expression was high Ndr1 expression was low or undetectable, and later when N-myc activity diminished Ndr1 expression was augmented concomitantly with the occurrence of terminal differentiation. To establish the direct link between N-myc activity and the Ndr1 regulation, the Ndr1 gene was cloned and analyzed. The Ndr1 promoter activity was down-regulated by N-myc, and more strongly by the combination of N-myc and Max in the cotransfection assay. This repressive effect was mediated by the promoter region within 52 base pairs from the transcription start site but direct binding of N-myc:Max to the promoter sequence was not demonstrated, which is analogous to the cases recently reported for transcriptional repression by c-myc. c-myc also repressed Ndr1 promoter activity similarly to N-myc. The effect of N-myc:Max was sensitive to Trichostatin A, indicating involvement of histone deacetylase activity in repression of the Ndr1 promoter. The strategy we adopted in identifying target genes of a transcription factor should prove widely applicable when mutant animals are available.

The c-Myc oncoprotein induces cell proliferation and transformation through its activity as a transcription factor. Uncovering the genes regulated by c-Myc is an essential step for understanding these processes. We recently isolated the tumor-associated membrane protein gene, Tmp, from a c-myc-induced mouse brain tumor. Here we show that Tmp is specifically highly expressed in mammary tumors and T-cell lymphomas which develop in c-myc transgenic mice, suggesting that Tmp expression is a general characteristic of c-Myc-induced tumors. In addition, Tmp expression is induced upon serum stimulation of fibroblasts as shown in a time course closely correlated with c-myc expression. We have isolated the Tmp promoter region and identified a putative c-Myc binding element, CACGTG, located in the first intron of the gene. We show here that constructs containing the Tmp regulatory region fused to a reporter gene are activated by c-Myc through this CACGTG element and that the c-Myc-Max protein complex can bind to this element. Moreover, an inducible form of c-Myc, the MycER fusion protein, can activate the endogenous Tmp gene. We also show that Tmp-overexpressing fibroblasts induce rapidly growing tumors when injected into nude mice, suggesting that Tmp may possess a tumorigenic activity. Thus, TMP, a member of a novel family of membrane glycoproteins with a suggested role in cellular contact, is a c-Myc target and is possibly involved in c-Myc-induced transformation.

We report here that the expression of virtually all proposed c-Myc target genes is unchanged in cells containing a homozygous null deletion of c-myc. Two noteworthy exceptions are the gene cad, which has reduced log phase expression and serum induction in c-myc null cells, and the growth arrest gene gadd45, which is derepressed by c-myc knockout. Thus, cad and gadd45 are the only proposed targets of c-Myc that may contribute to the dramatic slow growth phenotype of c-myc null cells. Our results demonstrate that a loss-of-function approach is critical for the evaluation of potential c-Myc target genes.

Uncertainty as to which member of a family of DNA-binding transcription factors regulates a specific promoter in intact cells is a problem common to many investigators. Determining target gene specificity requires both an analysis of protein binding to the endogenous promoter as well as a characterization of the functional consequences of transcription factor binding. By using a formaldehyde crosslinking procedure and Gal4 fusion proteins, we have analyzed the timing and functional consequences of binding of Myc and upstream stimulatory factor (USF)1 to endogenous cellular genes. We demonstrate that the endogenous cad promoter can be immunoprecipitated with antibodies against Myc and USF1. We further demonstrate that although both Myc and USF1 can bind to cad, the cad promoter can respond only to the Myc transactivation domain. We also show that the amount of Myc bound to the cad promoter fluctuates in a growth-dependent manner. Thus, our data analyzing both DNA binding and promoter activity in intact cells suggest that cad is a Myc target gene. In addition, we show that Myc binding can occur at many sites in vivo but that the position of the binding site determines the functional consequences of this binding. Our data indicate that a post-DNA-binding mechanism determines Myc target gene specificity. Importantly, we have demonstrated the feasibility of analyzing the binding of site-specific transcription factors in vivo to single copy mammalian genes.

We have isolated the cDNA encoding a novel c-Myc-binding protein, MM-1, by the yeast two-hybrid screening of a human HeLa cell cDNA library. The protein deduced from the cDNA comprises 167 amino acids and was localized in the nucleus of introduced COS-I cells. The MM-1 mRNA was highly expressed in human pancreas and skeletal muscle and moderately in other tissues. As for the c-Myc binding, glutathione S-transferase MM-1 expressed in Escherichia coli bound in vitro to c-Myc translated in reticulocyte lysate, and almost whole, the MM-1 molecule was necessary for the binding in the yeast two-hybrid system. The mammalian two-hybrid assays in hamster CHO cells revealed that MM-1 interacts in vivo with the N-terminal domain covering the myc box 2, a transcription-activating domain, of c-Myc. Furthermore, MM-1 repressed the activation of E-box-dependent transcription by c-Myc.

Cellular levels of the rapidly degraded c-myc protein play an important role in determining the proliferation status of cells. Increased levels of c-myc are frequently associated with rapidly proliferating tumor cells. We show here that myc boxes I and II, found in the N termini of all members of the myc protein family, function to direct the degradation of the c-myc protein. Both myc boxes I and II contain sufficient information to independently direct the degradation of otherwise stably expressed proteins to which they are fused. At least part of the myc box-directed degradation occurs via the proteasome. The mechanism of myc box-directed degradation appears to be conserved between yeast and mammalian cells. Our results suggest that the myc boxes may play an important role in regulating the level and activity of the c-myc protein.

The c-myc proto-oncogene has been suggested to play key roles in cell proliferation, differentiation, transformation and apoptosis. A variety of functions of C-MYC, the product of c-myc, are attributed to protein-protein interactions with various cellular factors including Max, YY1, p107, Bin1 and TBP. Max and YY1 bind to the C-terminal region of C-MYC, while p107, Bin1 and TBP bind to the N-terminal region covering myc boxes. The N-terminal region is involved in all the biological functions of C-MYC, and different proteins are therefore thought to interact with the N-terminal region of C-MYC to display different functions.

We cloned two cDNAs which encode a novel C-MYC-binding protein of 11 kDa, designated AMY-1 (Associate of C-MYC). The two cDNAs, AMY-1L and AMY-1S, derived from alternative usage of polyadenylation signals, code for the same protein of 11 kDa. AMY-1 was bound via its C-terminal region to the N-terminal region of C-MYC (amino acids nos 58-148) corresponding to the transactivation domain. AMY-1 was localized in the cytoplasm in cells expressing c-myc at low levels, but in the nucleus in the cells of a high c-myc expression in transiently transfected cells. A similar difference in endogenous AMY-1 localization was observed during the cell cycle: AMY-1 translocated from cytoplasm to nucleus during the S phase when c-myc expression was increased. AMY-1 by itself did not recognize the E-box element, the MYC/Max binding sequence, nor did it transactivate via the element, but stimulated the activation of E-box-regulated transcription by MYC/Max. FISH analyses revealed that the amy-1 gene was located at 1p32.2-1p33 in human genome.

AMY-1 is a 11 kDa protein which binds to the N-terminal region of C-MYC and stimulates the activation of E-box-dependent transcription by C-MYC. AMY-1, which mostly localizes in the cytoplasm, translocates into the nucleus in the S phase of the cell cycle upon an increase of c-myc expression, and may thus control the transcriptional activity of C-MYC.