The biochemical signaling mechanisms involved in transducing the effects of tumor necrosis factor alpha (TNF alpha) and gamma-interferon (gamma-IFN) on leukemia cell differentiation are poorly defined. Recent studies established the existence of a sphingomyelin cycle that operates in response to the action of vitamin D3 on HL-60 cells and that may transduce the effects of vitamin D3 on cell differentiation (Okazaki, T., Bell, R., and Hannun, Y. (1989) J. Biol. Chem. 264, 19076-19080). The effects of TNF alpha and gamma-IFN on sphingomyelin turnover were determined, and the specificity and role of sphingomyelin hydrolysis in HL-60 human promyelocytic leukemia cells with 20% hydrolysis of sphingomyelin at 15 min, 40% hydrolysis at 30-60 min, and return to base line at 2 h. The hydrolyzed sphingomyelin (18 pmol/nmol total phospholipid) was accompanied by the concomitant generation of ceramide (11.2 pmol/nmol total phospholipid). gamma-IFN also caused reversible hydrolysis of sphingomyelin with onset at 1 h and peak effect at 2 h. This sphingomyelin cycle appeared to be specific to the monocytic pathway of HL-60 differentiation, since it was not activated by retinoic acid or dibutyryl cAMP, inducers of granulocytic differentiation, nor with phorbol myristate acetate, an inducer of macrophage-like differentiation. Addition of synthetic ceramide or bacterial sphingomyelinase induced monocytic differentiation of HL-60 cells. Cell-permeable ceramide also caused prompt down-regulation of mRNA for the c-myc protooncogene. The time course of c-myc down-regulation was consistent with the action of ceramide as the mediator of TNF alpha action. These results suggest that sphingomyelin turnover may be an important signaling mechanism transducing the actions of TNF alpha and gamma-IFN with specific function in cell differentiation.

Prediction of gene regulatory sequences using phylogenetic footprinting has advanced considerably but lacks experimental validation. Here, we report whether transcription factor binding sites predicted by dot plotting or web-based Trafac analysis could be validated by chromatin immunoprecipitation assays. MYC overexpression enhances glycolysis without hypoxia and hence may contribute to altered tumor metabolism. Because the full spectrum of glycolytic genes directly regulated by Myc is not known, we chose Myc as a model transcription factor to determine whether it binds target glycolytic genes that have conserved canonical Myc binding sites or E boxes (5'-CACGTG-3'). Conserved canonical E boxes in ENO1, HK2, and LDHA occur in 31- to 111-bp islands with high interspecies sequence identity (>65%). Trafac analysis revealed another region in ENO1 that corresponds to a murine region with a noncanonical E box. Myc bound all these conserved regions well in the human P493-6 B lymphocytes. We also determined whether Myc could bind nonconserved canonical E boxes found in the remaining human glycolytic genes. Myc bound PFKM, but it did not significantly bind GPI, PGK1, and PKM2. Binding to BPGM, PGAM2, and PKLR was not detected. Both GAPD and TPI1 do not have conserved E boxes but are induced and bound by Myc through regions with noncanonical E boxes. Our results indicate that Myc binds well to conserved canonical E boxes, but not nonconserved E boxes. However, the binding of Myc to unpredicted genomic regions with noncanonical E boxes reveals a limitation of phylogenetic footprinting. In aggregate, these observations indicate that Myc is an important regulator of glycolytic genes, suggesting that MYC plays a key role in a switch to glycolytic metabolism during cell proliferation or tumorigenesis.

The hepatitis C virus (HCV) H strain polyprotein is cleaved to produce at least nine distinct products: NH2-C-E1-E2-NS2-NS3-NS4A-NS4B-NS5A-NS5B-CO OH. In this report, a series of C-terminal truncations and fusion with a human c-myc epitope tag allowed identification of a tenth HCV-encoded cleavage product, p7, which is located between the E2 and NS2 proteins. As determined by N-terminal sequence analysis, p7 begins with position 747 of the HCV H strain polyprotein. p7 is preceded by a hydrophobic sequence at the C terminus of E2 which may direct its translocation into the endoplasmic reticulum, allowing cleavage at the E2/p7 site by host signal peptidase. This hypothesis is supported by the observation that cleavage at the E2/p7 and p7/NS2 sites in cell-free translation studies was dependent upon the addition of microsomal membranes. However, unlike typical cotranslational signal peptidase cleavages, pulse-chase experiments indicate that cleavage at the E2/p7 site is incomplete, leading to the production of two E2-specific species, E2 and E2-p7. Possible roles of p7 and E2-p7 in the HCV life cycle are discussed.

Recent genomewide association studies have found multiple genetic variants on chromosome 8q24 that are significantly associated with an increased susceptibility to prostate, colorectal, and breast cancer. These risk loci are located in a "gene desert," a few hundred kilobases telomeric to the Myc gene. To date, the biological mechanism(s) underlying these associations remain unclear. It has been speculated that these 8q24 genetic variant(s) might affect Myc expression by altering its regulation or amplification status. Here, we show that multiple enhancer elements are present within this region and that they can regulate transcription of Myc. We also demonstrate that one such enhancer element physically interacts with the Myc promoter via transcription factor Tcf-4 binding and acts in an allele specific manner to regulate Myc expression.

The generation of induced pluripotent stem cells (iPSC) has enormous potential for the development of patient-specific regenerative medicine. Human embryonic stem cells (hESC) are able to defend their genomic integrity by maintaining low levels of reactive oxygen species (ROS) through a combination of enhanced removal capacity and limited production of these molecules. Such limited ROS production stems partly from the small number of mitochondria present in hESC; thus, it was important to determine that human iPSC (hiPSC) generation is able to eliminate the extra mitochondria present in the parental fibroblasts (reminiscent of "bottleneck" situation after fertilization) and to show that hiPSC have antioxidant defenses similar to hESC. We were able to generate seven hiPSC lines from adult human dermal fibroblasts and have fully characterized two of those clones. Both hiPSC clones express pluripotency markers and are able to differentiate in vitro into cells belonging to all three germ layers. One of these clones is able to produce fully differentiated teratoma, whereas the other hiPSC clone is unable to silence the viral expression of OCT4 and c-MYC, produce fully differentiated teratoma, and unable to downregulate the expression of some of the pluripotency genes during the differentiation process. In spite of these differences, both clones show ROS stress defense mechanisms and mitochondrial biogenesis similar to hESC. Together our data suggest that, during the reprogramming process, certain cellular mechanisms are in place to ensure that hiPSC are provided with the same defense mechanisms against accumulation of ROS as the hESC.

The far upstream element (FUSE) of the human c-myc proto-oncogene stimulates expression in undifferentiated cells. A FUSE-binding protein (FBP) is present in undifferentiated but not differentiated cells. Peptide sequences from the purified protein allowed cloning of cDNAs encoding FBP. Expression of FBP mRNA declined upon differentiation, suggesting transcriptional regulation of FBP. Features in the FBP cDNA suggest that FBP is also regulated by RNA processing, translation, and post-translational mechanisms. Both cellular and recombinant FBP form sequence-specific complexes with a single strand of FUSE. Transfection of FBP into human leukemia cells stimulated c-myc-promoter-driven expression from a reporter plasmid in a FUSE-dependent manner. Deletion and insertion mutagenesis of FBP defined a novel single-strand DNA-binding domain. Analysis of the primary and predicted secondary structure of the amino acid sequence reveals four copies of a reiterated unit comprised of a 30-residue direct repeat and an amphipathic alpha-helix separated by an 18- to 21-residue spacer. The third and fourth copies of this repeat-helix unit constitute the minimum single-stranded DNA-binding domain. To determine whether the FUSE site, in vivo, possesses single-strand conformation, and therefore could be bound by FBP, cells were treated with potassium permanganate (KMnO4) to modify unpaired bases. Modification of genomic DNA in vivo revealed hyperreactivity associated with single-stranded DNA in the FUSE sequence and protection on the strand that binds FBP in vitro. The role of single-stranded DNA and single-strand binding proteins in c-myc regulation is discussed.

c-Myc stimulates angiogenesis in tumors through mechanisms that remain incompletely understood. Recent work indicates that c-Myc upregulates the miR-17∼92 microRNA cluster and downregulates the angiogenesis inhibitor thrombospondin-1, along with other members of the thrombospondin type 1 repeat superfamily. Here, we show that downregulation of the thrombospondin type 1 repeat protein clusterin in cells overexpressing c-Myc and miR-17∼92 promotes angiogenesis and tumor growth. However, clusterin downregulation by miR-17∼92 is indirect. It occurs as a result of reduced transforming growth factor-β (TGFβ) signaling caused by targeting of several regulatory components in this signaling pathway. Specifically, miR-17-5p and miR-20 reduce the expression of the type II TGFβ receptor and miR-18 limits the expression of Smad4. Supporting these results, in human cancer cell lines, levels of the miR-17∼92 primary transcript MIR17HG negatively correlate with those of many TGFβ-induced genes that are not direct targets of miR-17∼92 (e.g., clusterin and angiopoietin-like 4). Furthermore, enforced expression of miR-17∼92 in MIR17HG(low) cell lines (e.g., glioblastoma) results in impaired gene activation by TGFβ. Together, our results define a pathway in which c-Myc activation of miR-17∼92 attenuates the TGFβ signaling pathway to shut down clusterin expression, thereby stimulating angiogenesis and tumor cell growth.

The prognosis of hepatocellular carcinoma (HCC) still remains dismal, although many advances in its clinical study have been made. It is important for tumor control to identify the factors that predispose patients to death. With new discoveries in cancer biology, the pathological and biological prognostic factors of HCC have been studied quite extensively. Analyzing molecular markers (biomarkers) with prognostic significance is a complementary method. A large number of molecular factors have been shown to associate with the invasiveness of HCC, and have potential prognostic significance. One important aspect is the analysis of molecular markers for the cellular malignancy phenotype. These include alterations in DNA ploidy, cellular proliferation markers (PCNA, Ki-67, Mcm2, MIB1, MIA, and CSE1L/CAS protein), nuclear morphology, the p53 gene and its related molecule MD M2, other cell cycle regulators (cyclin A, cyclin D, cyclin E, cdc2, p27, p73), oncogenes and their receptors (such as ras, c-myc, c-fms, HGF, c-met, and erb-B receptor family members), apoptosis related factors (Fas and FasL), as well as telomerase activity. Another important aspect is the analysis of molecular markers involved in the process of cancer invasion and metastasis. Adhesion molecules (E-cadherin, catenins, serum intercellular adhesion molecule-1, CD44 variants), proteinases involved in the degradation of extracellular matrix (MMP-2, MMP-9, uPA, uPAR, PAI), as well as other molecules have been regarded as biomarkers for the malignant phenotype of HCC, and are related to prognosis and therapeutic outcomes. Tumor angiogenesis is critical to both the growth and metastasis of cancers including HCC, and has drawn much attention in recent years. Many angiogenesis-related markers, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived endothelial cell growth factor (PD-ECGF), thrombospondin (TSP), angiogenin, pleiotrophin, and endostatin (ES) levels, as well as intratumor microvessel density (MVD) have been evaluated and found to be of prognostic significance. Body fluid (particularly blood and urinary) testing for biomarkers is easily accessible and useful in clinical patients. The prognostic significance of circulating DNA in plasma or serum, and its genetic alterations in HCC are other important trends. More attention should be paid to these two areas in future. As the progress of the human genome project advances, so does a clearer understanding of tumor biology, and more and more new prognostic markers with high sensitivity and specificity will be found and used in clinical assays. However, the combination of some items, i.e., the pathological features and some biomarkers mentioned above, seems to be more practical for now.

We present a detailed analysis of strand-specific transcription in different regions of the murine c-myc locus. In normal and transformed cell lines, RNA polymerase II directed transcription occurs in the sense and anti-sense direction. Three noncontiguous regions show a high level of transcription in the anti-sense orientation: upstream of the first exon, within the first intron and in the 3' part of the gene (intron 2 and exon 3). In a cell line carrying a c-myc amplification (54c12), anti-sense transcription is not uniformly increased throughout the locus and is differentially affected by inhibition of protein synthesis. These results suggest that anti-sense transcription in various parts of the locus is independently regulated. In the sense orientation, transcriptional activity is higher in the first exon than in the rest of the gene indicating that transcription pauses near the 3' end of the first exon. The extent of this intragenic pausing varies among different cell lines and is most severe in cells with a c-myc amplification. Transcription initiation and pausing are both negatively regulated by labile proteins.

The role of hSWI/SNF complexes in transcriptional activation is well characterized; however, little is known about their function in transcriptional repression. We have previously shown that subunits of the mSin3A/histone deacetylase 2 (HDAC2) corepressor complex copurify with hSWI/SNF complexes. Here we show that the type II arginine-specific methyltransferase PRMT5, which is involved in cyclin E repression, can be found in association with Brg1 and hBrm-based hSWI/SNF complexes. We also show that hSWI/SNF-associated PRMT5 can methylate hypoacetylated histones H3 and H4 more efficiently than hyperacetylated histones H3 and H4. Protein-protein interaction studies indicate that PRMT5 and mSin3A interact with the same hSWI/SNF subunits as those targeted by c-Myc. These observations prompted us to examine the expression profile of the c-Myc target genes, carbamoyl-phosphate synthase-aspartate carbamoyltransferase-dihydroorotase (cad) and nucleolin (nuc). We found that cad repression is altered in cells that express inactive Brg1 and in cells treated with the HDAC inhibitor depsipeptide. Using chromatin immunoprecipitation assays, we found that Brg1, mSin3A, HDAC2, and PRMT5 are directly recruited to the cad promoter. These results suggest that hSWI/SNF complexes, through their ability to interact with activator and repressor proteins, control expression of genes involved in cell growth and proliferation.

The development of nontoxic natural agents with chemopreventive activity against colon cancer is the focus of investigation in many laboratories. Curcumin (feruylmethane), a natural plant product, possesses such chemopreventive activity, but the mechanisms by which it prevents cancer growth are not well understood. In the present study, we examined the mechanisms by which curcumin treatment affects the growth of colon cancer cells in vitro. Results showed that curcumin treatment causes p53- and p21-independent G(2)/M phase arrest and apoptosis in HCT-116(p53(+/+)), HCT-116(p53(-/-)) and HCT-116(p21(-/-)) cell lines. We further investigated the association of the beta-catenin-mediated c-Myc expression and the cell-cell adhesion pathways in curcumin-induced G(2)/M arrest and apoptosis in HCT-116 cells. Results described a caspase-3-mediated cleavage of beta-catenin, decreased transactivation of beta-catenin/Tcf-Lef, decreased promoter DNA binding activity of the beta-catenin/Tcf-Lef complex, and decreased levels of c-Myc protein. These activities were linked with decreased Cdc2/cyclin B1 kinase activity, a function of the G(2)/M phase arrest. The decreased transactivation of beta-catenin in curcumin-treated HCT-116 cells was unpreventable by caspase-3 inhibitor Z-DEVD-fmk, even though the curcumin-induced cleavage of beta-catenin was blocked in Z-DEVD-fmk pretreated cells. The curcumin treatment also induced caspase-3-mediated degradation of cell-cell adhesion proteins beta-catenin, E-cadherin and APC, which were linked with apoptosis, and this degradation was prevented with the caspase-3 inhibitor. Our results suggest that curcumin treatment impairs both Wnt signaling and cell-cell adhesion pathways, resulting in G(2)/M phase arrest and apoptosis in HCT-116 cells.

The application of RNA interference (RNAi) to mammalian systems has the potential to revolutionize genetics and produce novel therapies. Here we investigate whether RNAi applied to a well-characterized gene can stably suppress gene expression in hematopoietic stem cells and produce detectable phenotypes in mice. Deletion of the Trp53 tumor suppressor gene greatly accelerates Myc-induced lymphomagenesis, resulting in highly disseminated disease. To determine whether RNAi suppression of Trp53 could produce a similar phenotype, we introduced several Trp53 short hairpin RNAs (shRNAs) into hematopoietic stem cells derived from E(mu)-Myc transgenic mice, and monitored tumor onset and overall pathology in lethally irradiated recipients. Different Trp53 shRNAs produced distinct phenotypes in vivo, ranging from benign lymphoid hyperplasias to highly disseminated lymphomas that paralleled Trp53-/- lymphomagenesis in the E(mu)-Myc mouse. In all cases, the severity and type of disease correlated with the extent to which specific shRNAs inhibited p53 activity. Therefore, RNAi can stably suppress gene expression in stem cells and reconstituted organs derived from those cells. In addition, intrinsic differences between individual shRNA expression vectors targeting the same gene can be used to create an 'epi-allelic series' for dissecting gene function in vivo.

The c-Myc protein is up-regulated in many different types of cancer, suggesting that a detailed understanding of Myc function is an important goal. Our previous studies have focused on determining the mechanism by which Myc activates transcription using the target gene cad as an experimental model. Previously, we found that Myc activates cad transcription at a post-RNA polymerase II recruitment step and that the Myc transactivation domain interacts with a number of cdk-cyclin complexes. We now extend these studies to determine the role of these cyclin-cdk complexes in Myc-mediated transactivation. We have found that cyclin T1 binding to Myc localizes to the highly conserved Myc Box I, whereas cdk8 binding localizes to the amino-terminal 41 amino acids of the Myc transactivation domain. We showed that recruitment of cdk8 is sufficient for activation of a synthetic promoter construct. In contrast, the ability of Myc to activate transcription of the cad promoter correlates with binding of cyclin T1. Furthermore, recruitment of cyclin T1 to the cad promoter via a Gal4 fusion protein or through protein-protein interaction with the HIV-1 Tat protein can also activate cad transcription. These results suggest that Myc activates transcription by stimulating elongation and that P-TEFb is a key mediator of this process.

Burkitt lymphoma cells carrying either a rearranged or unrearranged c-myc oncogene were examined with the use of probes from the 5' exon and for the second and third exon of the oncogene. The results indicate that the normal c-myc gene on chromosome 8 and the 5' noncoding and 3' coding segments of the c-myc oncogene separated by the chromosomal translocation are under different transcriptional control in the lymphoma cells. Burkitt lymphoma cells carrying a translocated but unrearranged c-myc oncogene express normal c-myc transcripts. In contrast, lymphoma cells carrying a c-myc gene rearranged head to head with the immunoglobulin constant mu region gene express c-myc transcripts lacking the normal untranslated leader.

We have prepared an antiserum against a synthetic dodecapeptide whose sequence corresponds to the C terminus of the MC29 v-myc protein. This antiserum (anti-v-myc 12C) specifically precipitates the known gag-myc fusion proteins produced by the defective leukemia viruses MC29, CMII, and OK10, but does not react with gag-precursor or product proteins. In addition, proteins of 62 kd and 61/63 kd are precipitated by anti-v-myc 12C from OK10 and MH2 transformants, respectively. The serum also recognizes comigrating 62 kd proteins from three chicken bursal lymphoma cell lines and from the products of in vitro translation of c-myc-specific mRNA. All of these myc-related proteins are phosphorylated and all appear to be localized in the cell nucleus. In uninfected quail cells, anti-v-myc 12C also recognizes a candidate c-myc protein of 60 kd, which does not appear to be phosphorylated and is present in low levels relative to v-myc and lymphoma c-myc proteins.

The stability of certain mRNAs is known to be affected by translation. Some mRNAs appear to be protected from rapid degradation by translation, whereas degradation is coupled to translation for other mRNAs. The molecular determinants of this selective effect of translation are unknown. One example of this effect is the induction of early-response gene mRNAs in the presence of translation inhibitors. To define the molecular basis of induction of early-response gene mRNA expression by inhibitors of protein synthesis, we have performed a mutational analysis of one member of the early response gene family, the c-myc gene. We find that induction by cycloheximide is due to stabilization of c-myc transcripts. The requirements for increased expression of c-myc mRNA by cycloheximide are the presence of the sequence encoding c-myc amino acids 335-439 on a mRNA that can be translated; all other portions of the c-myc gene are dispensable, and this sequence can confer induction of mRNA expression by protein synthesis inhibitors on a heterologous gene. By direct measurement of mRNA turnover in the absence of transcription-blocking drugs, we show that this sequence can function as a selective mRNA destabilizing element, that turnover mediated by this element is translation dependent, and turnover mediated by this element is inhibited by actinomycin D. Our results support the hypothesis that degradation of c-myc mRNA is coupled to translation, that the sequences specifying this form of degradation are contained in the protein-coding sequence, and that translation inhibitors induce expression of c-myc mRNA by blocking turnover mediated by this element.

In contrast to other human tumors in which the c-myc gene and its transcript are greatly amplified, careful analysis of t(8;14) Burkitt cell lines indicates that the c-myc transcript is marginally, and in some cases not at all, increased by comparison to control lymphoblastoid cell lines. Instead, there is a more subtle alteration in the expression of the translocated c-myc gene characterized by a shift in promoter utilization and an apparent insensitivity to the regulation that inactivates the normal c-myc allele within these same cells. In some Burkitt cell lines, such deregulation might be because of the loss of a putative control region through removal of the large dual promoter/leader segment of the c-myc gene. In other cell lines, however, this deregulation may be explained by somatic mutations that occur within the putative control region even though it is located many hundreds of bases from the translocation breakpoint.

To identify new diagnostic markers and drug targets for pancreatic cancer, we compared the gene expression patterns of pancreatic cancer cell lines growing in tissue culture with those of normal pancreas using cDNA microarray analysis. Fluorescently (cyanine 5) labeled cDNA probes, made individually from mRNA samples of nine pancreatic cell lines, were each combined with fluorescently (cyanine 3) labeled universal reference mRNA. The mixed probes of each sample were then hybridized with 5760 cDNA arrays (5289 unique cDNA sequences) printed on individual microscope slides. Fluorescently (cyanine 5) labeled normal pancreas mRNA was also compared with the same universal reference mRNA reference pool. The expression ratios of neoplastic versus normal pancreas cells were then calculated by multiplying the ratio of cancer versus the universal reference mRNA and the ratio of the universal reference mRNA cell versus normal pancreas. For 5289 different genes interrogated by the arrays, 30 of them showed an expression ratio 2 SD from the mean in at least three of the nine pancreatic cell lines studied. To confirm the expression profiles of these genes, quantitative reverse transcription-PCR and Northern blot were carried out for 25 of the overexpressed genes. To verify the overexpression in patient samples, two of the overexpressed genes, c-Myc and Rad51, were selected to undergo analysis by reverse transcription-PCR in frozen tumor tissues and by immunostaining in paraffin-embedded tissue section microarrays. The results of these experiments are in agreement with the microarray data. Potential up-regulated targets of note from this study include urokinase-type plasminogen activator receptor, serine/threonine kinase 15, thioredoxin reductase, and CDC28 protein kinase 2, as well as several others.

A link between ABL oncogenes and MYC is suggested by the transformation synergy that is observed when MYC is expressed at high levels. Dominant negative MYC proteins were overexpressed in fibroblasts to determine if MYC complements ABL oncogene transformation or is essential for this process. Transformation by both v-abl and BCR-ABL oncogenes was reduced 5- to 10-fold, whereas transformation by the serine/threonine kinase oncogene v-mos was unaffected. Using a retrovirus construct modified to express BCR-ABL and MYC genes simultaneously, we show that dominant negative MYC suppressed transformation of primary mouse bone marrow pre-B cells by BCR-ABL. These observations demonstrate that c-MYC is essential for transformation and help define the pathway by which these proteins cause transformation.

The ability of a transcription factor (TF) to regulate its targets is modulated by a variety of genetic and epigenetic mechanisms, resulting in highly context-dependent regulatory networks. However, high-throughput methods for the identification of proteins that affect TF activity are still largely unavailable. Here we introduce an algorithm, modulator inference by network dynamics (MINDy), for the genome-wide identification of post-translational modulators of TF activity within a specific cellular context. When used to dissect the regulation of MYC activity in human B lymphocytes, the approach inferred novel modulators of MYC function, which act by distinct mechanisms, including protein turnover, transcription complex formation and selective enzyme recruitment. MINDy is generally applicable to study the post-translational modulation of mammalian TFs in any cellular context. As such it can be used to dissect context-specific signaling pathways and combinatorial transcriptional regulation.

Recent studies indicate that the expression of ER beta in breast cancer is lower than in the normal breast, suggesting that ER beta could play an important role in carcinogenesis. To investigate this hypothesis, we engineered ER-negative MDA-MB-231 (human breast cancer cells) to reintroduce either ER alpha or ER beta protein with an adenoviral vector. In these cells, ER beta (as ER alpha) expression was monitored using RT-PCR and Western blot. ER beta protein was localized in the nucleus (immunocytochemistry) and able to transactivate estrogen-responsive reporter constructs in the presence of E2. ER beta and ER alpha induced the expression of several endogenous genes such as pS2, TGF alpha, or the cyclin kinase inhibitor p21 but, in contrast to ER alpha, ER beta was unable to regulate c-myc proto-oncogene expression. The pure antiestrogen ICI 164, 384 completely blocked ER alpha and ER beta estrogen-induced activities. ER beta inhibited MDA-MB-231 cell proliferation in a ligand-independent manner, whereas ER alpha inhibition of proliferation is hormone dependent. Moreover, ER beta and ER alpha decreased cell motility and invasion. Our data bring the first evidence that ER beta is an important modulator of proliferation and invasion of breast cancer cells and support the hypothesis that the loss of ER beta expression could be one of the events leading to the development of breast cancer.

The c-Myc protein (Myc) is a transcription factor, and deregulated expression of the c-myc gene (myc) is frequently found in tumours. In Burkitt's lymphoma (BL), myc is transcriptionally activated by chromosomal translocation. We have used a B-cell line called P493-6 that carries a conditional myc allele to elucidate the role of Myc in the proliferation of BL cells. Regulation of proliferation involves the coordination of cell growth (accumulation of cell mass) and cell division [1] [2] [3]. Here, we show that division of P493-6 cells was strictly dependent on the expression of the conditional myc allele and the presence of foetal calf serum (FCS). More importantly, cell growth was regulated by Myc without FCS: Myc alone induced an increase in cell size and positively regulated protein synthesis. An increase in protein synthesis is thought to be one of the causes of cell mass increase. Furthermore, Myc stimulated metabolic activities, as indicated by the acidification of culture medium and the activation of mitochondrial enzymes. Our results confirm the model that Myc is involved in the regulation of cell growth [4] and provide, for the first time, direct evidence that Myc induces cell growth, that is, an increase in cell size, uncoupled from cell division.

We assessed tumor cell DNA content (ploidy) and N-myc gene copy number as predictors of long-term disease-free survival in 298 children with neuroblastoma. Diploid tumor stem lines were identified in 101 patients (34%), clonal hyperdiploid abnormalities in 194 (65%), and hypodiploid stem lines in three (1%). In children with widely disseminated tumors at diagnosis (stage D), ploidy had a highly age-dependent influence on prognosis. Among infants (less than 12 months) treated with cyclophosphamide-doxorubicin, hyperdiploidy was closely associated with long-term disease-free survival (greater than 90% of cases), while diploidy invariably predicted early treatment failure (P less than .001). Similarly, in children 12 to 24 months of age who were treated with cisplatin-teniposide and cyclophosphamide-doxorubicin, diploidy uniformly predicted early failure, whereas half of the children with hyperdiploidy achieved long-term disease-free survival (P less than .001). There was no relationship between ploidy and treatment outcome in children older than 24 months with stage D tumors who had a very low probability of long-term disease-free survival (less than 10%). N-myc gene amplification was detected in 37 (25%) of the 147 tumors tested, with the remainder showing single-copy levels of the gene. N-myc gene amplification was more frequent in diploid than in hyperdiploid tumors (23 of 57 v 14 of 87, P = .001) and predicted a high likelihood of early treatment failure. In children younger than 2 years with disseminated neuroblastoma, tumor cell ploidy and N-myc gene copy number provide complementary prognostic information that will distinguish patients who can be cured on current regimens from those who require new treatment strategies.

Estrogen, progesterone, and HER2 receptor-negative triple-negative breast cancers encompass the most clinically challenging subtype for which targeted therapeutics are lacking. We find that triple-negative tumors exhibit elevated MYC expression, as well as altered expression of MYC regulatory genes, resulting in increased activity of the MYC pathway. In primary breast tumors, MYC signaling did not predict response to neoadjuvant chemotherapy but was associated with poor prognosis. We exploit the increased MYC expression found in triple-negative breast cancers by using a synthetic-lethal approach dependent on cyclin-dependent kinase (CDK) inhibition. CDK inhibition effectively induced tumor regression in triple-negative tumor xenografts. The proapoptotic BCL-2 family member BIM is up-regulated after CDK inhibition and contributes to this synthetic-lethal mechanism. These results indicate that aggressive breast tumors with elevated MYC are uniquely sensitive to CDK inhibitors.