Direct activation of TERT transcription by c-MYC.

The MYC proto-oncogene encodes a ubiquitous transcription factor (c-MYC) involved in the control of cell proliferation and differentiation. Deregulated expression of c-MYC caused by gene amplification, retroviral insertion, or chromosomal translocation is associated with tumorigenesis. The function of c-MYC and its role in tumorigenesis are poorly understood because few c-MYC targets have been identified. Here we show that c-MYC has a direct role in induction of the activity of telomerase, the ribonucleoprotein complex expressed in proliferating and transformed cells, in which it preserves chromosome integrity by maintaining telomere length. c-MYC activates telomerase by inducing expression of its catalytic subunit, telomerase reverse transcriptase (TERT). Telomerase complex activity is dependent on TERT, a specialized type of reverse transcriptase. TERT and c-MYC are expressed in normal and transformed proliferating cells, downregulated in quiescent and terminally differentiated cells, and can both induce immortalization when constitutively expressed in transfected cells. Consistent with the recently reported association between MYC overexpression and induction of telomerase activity, we find here that the TERT promoter contains numerous c-MYC-binding sites that mediate TERT transcriptional activation. c-MYC-induced TERT expression is rapid and independent of cell proliferation and additional protein synthesis, consistent with direct transcriptional activation of TERT. Our results indicate that TERT is a target of c-MYC activity and identify a pathway linking cell proliferation and chromosome integrity in normal and neoplastic cells.

Burkitt lymphoma in the mouse.

Chromosomal translocations juxtaposing the MYC protooncogene with regulatory sequences of immunoglobulin (Ig) H chain or kappa (Ig kappa) or lambda (Ig lambda) L chain genes and effecting deregulated expression of MYC are the hallmarks of human Burkitt lymphoma (BL). Here we report that lymphomas with striking similarities to BL develop in mice bearing a mutated human MYC gene controlled by a reconstructed Ig lambda locus encompassing all the elements required for establishment of locus control in vitro. Diffusely infiltrating lymphomas with a typical starry sky appearance occurred in multiple founders and an established line, indicating independence from positional effects. Monoclonal IgM(+)CD5(-)CD23(-) tumors developed from an initially polyclonal population of B cells. These results demonstrate that the phenotype of B lineage lymphomas induced by MYC dysregulation is highly dependent on cooperativity among the regulatory elements that govern expression of the protooncogene and provide a new system for studying the pathogenesis of BL.

Coordinated regulation of iron-controlling genes, H-ferritin and IRP2, by c-MYC.

The protein encoded by the c-MYC proto-oncogene is a transcription factor that can both activate and repress the expression of target genes, but few of its transcriptional targets have been identified. Here, c-MYC is shown to repress the expression of the heavy subunit of the protein ferritin (H-ferritin), which sequesters intracellular iron, and to stimulate the expression of the iron regulatory protein-2 (IRP2), which increases the intracellular iron pool. Down-regulation of the expression of H-ferritin gene was required for cell transformation by c-MYC. These results indicate that c-MYC coordinately regulates genes controlling intracellular iron concentrations and that this function is essential for the control of cell proliferation and transformation by c-MYC.

Control of cell growth by c-Myc in the absence of cell division.

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.

Activation of c-myc promoter P1 by immunoglobulin kappa gene enhancers in Burkitt lymphoma: functional characterization of the intron enhancer motifs kappaB, E box 1 and E box 2, and of the 3' enhancer motif PU.

Deregulated expression of the proto-oncogene c- myc in Burkitt lymphoma (BL) cells carrying a t(2;8) translocation is mediated by a synergistic interaction of the translocated immunoglobulin (Ig) kappa gene intron (kappaEi) and 3' (kappaE3') enhancers and characterized by a strong activation of the promoter P1. We have investigated the functional role of distinct kappa enhancer sequence motifs in P1 activation on both mini-chromosomes and reporter gene constructs. Stable and transient transfections of BL cells revealed critical roles of the kappaEi and kappaE3' elements kappaB and PU, respectively. Joint mutation of kappaB and PU completely abolished P1 activity, implying that an interaction of kappaB- and PU-binding factors is essential for the enhancer synergism. Mutation of the E box 1 and E box 2 motifs markedly decreased P1 activity in transient but not in stable transfection experiments. Co-expression of the NF-kappaB subunit p65(RelA) and Sp1, an essential factor for P1 transcription, in Drosophila melanogaster SL2 cells synergistically enhanced promoter activity. Our results support a model which proposes cross-talk between promoter and enhancer binding factors as the basic mechanism for kappa enhancer-mediated c- myc activation in BL cells.

The transcriptional program of a human B cell line in response to Myc.

The proto-oncogene c-myc (myc) encodes a transcription factor (Myc) that promotes growth, proliferation and apoptosis. Myc has been suggested to induce these effects by induction/repression of downstream genes. Here we report the identification of potential Myc target genes in a human B cell line that grows and proliferates depending on conditional myc expression. Oligonucleotide microarrays were applied to identify downstream genes of Myc at the level of cytoplasmic mRNA. In addition, we identified potential Myc target genes in nuclear run-on experiments by changes in their transcription rate. The identified genes belong to gene classes whose products are involved in amino acid/protein synthesis, lipid metabolism, protein turnover/folding, nucleotide/DNA synthesis, transport, nucleolus function/RNA binding, transcription and splicing, oxidative stress and signal transduction. The identified targets support our current view that myc acts as a master gene for growth control and increases transcription of a large variety of genes.

The block of elongation in c-myc exon 1 is abolished in Burkitt's lymphoma cell lines with variant translocation.

Dimethylsulfoxide (DMSO) induces a block of c-myc RNA elongation in the human B cell line BJAB. In Burkitt's lymphoma cell lines with variant translocations, which are characterized by mutations in and around the first c-myc exon, DMSO is not capable of inducing the RNA elongation block. The action of DMSO is, however, not restricted to regulation at the level of RNA elongation. In the cell line BL2 with a t(8;22) translocation c-myc steady-state RNA decreased about 20 fold 1 and 2 h after DMSO treatment, followed by an increase to approximately initial levels after 4 h. During the time course of the experiment the usage of the dual promoter P1/P2 shifted from the ratio 3:1 in untreated cells to the ratio of 1:5 in BL2 cells treated with DMSO for 4 h. This promoter shift is presumably regulated at the transcriptional level. In BJAB cells an isolated intragenic transcription was detected at the boundary of intron 1 and exon 2. This transcription appeared 2 to 4 h after addition of DMSO when expression of the c-myc gene was downregulated by blocking RNA elongation at the end of exon 1.

Transcription of c-myc in human mononuclear cells is regulated by an elongation block.

Human mononuclear cells (MNC) isolated from tonsils express very low amounts of c-myc RNA. High expression of the c-myc gene is achieved by stimulation of MNC with pokeweed mitogen (PWM). Nuclear run-on transcription experiments revealed activation of the c-myc gene on the transcriptional level. In unstimulated cells a high transcriptional activity is observed on exon 1, but not on exon 2 and 3. After PWM stimulation, however, a high transcription rate is detected on the entire c-myc gene. Therefore, we conclude that c-myc gene expression in MNC is regulated by an RNA elongation block within the first exon. The analysis of the c-myc chromatin structure revealed that DNAaseI hypersensitive site II upstream of the gene is present in unstimulated as well as stimulated MNC, indicating that accessibility of this site is an essential but not sufficient requirement for c-myc gene expression.

Two antisense promoters in the immunoglobulin mu-switch region drive expression of c-myc in the Burkitt's lymphoma cell line BL67.

In the Burkitt's lymphoma (BL) cell line BL67 the first exon of the c-myc gene is fused to the mu-switch region of the immunoglobulin heavy-chain gene (IgH). BL67 cells express IgH/c-myc hybrid RNAs which are initiated in the immunoglobulin locus, transcribed across the chromosomal breakpoint into the first exon of c-myc and spliced using the physiological splice donor and acceptor sites of the c-myc gene. We have isolated cDNAs of these hybrid RNAs and characterized the start points in the Ig heavy-chain gene. Two promoters were identified in the mu-switch region of BL67 cells which give rise to antisense transcription of the mu-gene. These promoters are also active in other BL cell lines, in B cells without Ig translocation and in a T-cell line. Both promoters co-localize with DNAase I-hypersensitive sites, HNF and HSW, in the mu-switch region. The structures of IgH/c-myc hybrid RNAs and of the corresponding promoters are described.

Absence of a paused transcription complex from the c-myc P2 promoter of the translocation chromosome in Burkitt's lymphoma cells: implication for the c-myc P1/P2 promoter shift.

We have shown recently that pausing of RNA polymerase II (pol II) at the transcription start site regulates expression from the P2 promoter of the proto-oncogene c-myc. RNAs initiated at the P2 promoter usually contribute > 80% to steady-state c-myc RNA levels in normal cells. In Burkitt's lymphoma (BL) cells c-myc is chromosomally translocated to an immunoglobulin (Ig) gene and preferentially transcribed from the upstream P1 promoter. We have studied the activity of c-myc promoters in two BL cell lines with high expression of P1 RNA. Kinetic nuclear run-on experiments show that the initiation rate at the c-myc P1 promoter in BL2 and BL60 cells is not increased compared with control BJAB cells, whereas the number of paused polymerases at the P2 promoter is greatly diminished. The translocation c-myc gene of BL60 cells was cloned and stably transfected into the BL cell line Raji. The transfected c-myc gene regained the ability to form a paused transcription complex at the c-myc P2 promoter. The data suggest that a paused polymerase at the c-myc P2 promoter impedes transcription from the upstream P1 promoter on a normal c-myc gene. The c-myc gene on the translocation chromosome in BL cells has lost the ability to retain pol II at the P2 promoter, probably by interaction with elements of the adjacent Ig gene locus.

Expression of P0- and P3-RNA from the normal and translocated c-myc allele in Burkitt's lymphoma cells.

We have studied the allele specific expression of c-myc P0- and P3-RNA in Burkitt's lymphoma (BL) cells. The steady state levels of P0-RNA show considerable variations in BL cells. Expression of P0-RNA was found to be restricted to the translocated allele, but could be induced by TPA from the normal allele. P0-transcription was particularly sensitive to inhibitors of protein synthesis compared to expression of P1-, P2- and P3-RNA. Transcription of P3-RNA is initiated in the first intron of the c-myc gene and has previously been described to be specific for translocated c-myc alleles in BL cells broken within exon 1 or intron 1. Here we show that P3-RNA is also expressed from an unrearranged c-myc gene. In the BL cell line Raji, substantial amounts of c-myc RNA are derived from the P3-promoter of the normal allele. This indicates that repression of the normal allele in BL cells does not include the P3-promoter. The potential coding capacity of P3-RNA is discussed.

The intron enhancer of the immunoglobulin kappa gene activates c-myc but does not induce the Burkitt-specific promoter shift.

In Burkitt's lymphoma cells the c-myc gene locus is consistently fused to the constant region of one of the immunoglobulin genes by chromosomal translocation. The translocated c-myc gene is transcriptionally activated and preferentially transcribed from the P1 promoter whenever the exon-intron structure of c-myc remains intact. In order to define elements involved in this promoter shift we have cloned the translocated c-myc allele from Burkitt's lymphoma cell line BL60, which is characterized by several point mutations. The mutated c-myc allele of BL60 was stably introduced into baby hamster kidney and Burkitt's lymphoma cells. S1 nuclease and RNAase protection mapping experiments demonstrated that the mutated c-myc allele was expressed at a low level and with a normal promoter usage (P2 greater than P1) in Burkitt's lymphoma and baby hamster kidney cells. Furthermore, we have studied the expression of a construct consisting of the mutated c-myc allele, part of the bvr1 (Burkitt's variant rearranging region 1) locus, the human immunoglobulin kappa constant region, and the kappa intron enhancer after stable transfection into Burkitt's lymphoma cells. Although c-myc expression was about fivefold increased, the transcripts still initiated predominantly at promoter P2. This indicates that 5 kb of the constant kappa light-chain locus including the kappa intron enhancer is not sufficient to induce the Burkitt's lymphoma-specific promoter shift.

The role of immunoglobulin kappa elements in c-myc activation.

Burkitt's lymphoma cells are characterized by chromosomal translocations involving the proto-oncogene c-myc on chromosome 8 and one of the immunoglobulin gene loci on chromosome 2, 14 or 22. The translocated c-myc allele is transcriptionally activated, shows a preferential usage of promoter P1 over P2 (promoter shift) and lacks the ability to retain the transcription complex at the P2 promoter. In order to define the elements of the immunoglobulin kappa gene involved in deregulation of c-myc in a t(2;8) translocation, we designed constructs consisting of c-myc and different parts of the immunoglobulin kappa gene locus. Chromatin analysis of these stably transfected constructs revealed DNase I hypersensitive sites within the c-myc 5' region characteristic for an actively transcribed c-myc gene and three sites within the immunoglobulin kappa locus corresponding to the matrix attachment region, the intron and 3' enhancers, respectively. These three regulatory elements were necessary and sufficient for maximal c-myc activation and formation of the promoter shift. Kinetic nuclear run on experiments were performed to study the distribution of transcription complexes on c-myc exon 1 on constructs with and without the immunoglobulin kappa regulatory elements. The absence of a pausing polymerase complex at the c-myc P2 promoter could be demonstrated for constructs consisting of c-myc and the two kappa enhancers. Therefore the two enhancers are sufficient to relief the elongational block at the P2 promoter, however, the matrix attachment region is additionally required for maximal c-myc activation observed in Burkitt's lymphoma cells.

c-myc expression is activated by the immunoglobulin kappa-enhancers from a distance of at least 30 kb but not by elements located within 50 kb of the unaltered c-myc locus in vivo.

50 kb of contiguous DNA sequences covering the human c-myc coding region and approximately 20 kb of flanking upstream and downstream sequences were cloned onto a prokaryotic F-factor derived plasmid, which also contains a selectable marker and the plasmid origin of DNA replication oriP of Epstein Barr virus (EBV). Since these plasmids replicate extrachromosomally after stable transfection into EBV-positive B-cell lines, the gene regulation of c-myc can be analysed independent from chromosomal integration positions. Despite the presence of all known c-myc regulatory elements on these constructs, expression from the stably transfected c-myc gene was barely detectable in either cell line. Hypermethylation of these plasmids could be excluded as a mechanism for the lack of gene expression. Insertion of the immunoglobulin kappa-intron and 3' enhancers, however, activated c-myc transcription, when placed adjacent to or separated from the c-myc promoters by as far as 30 kb. These results indicate that transcription of c-myc in vivo requires additional and still unidentified control elements located outside this 50 kb fragment, and experimentally demonstrate long range enhancer function in vivo.

Deregulation of the proto-oncogene c-myc through t(8;22) translocation in Burkitt's lymphoma.

In Burkitt's lymphoma (BL) cells the proto-oncogene c-myc is juxtaposed to one of the immunoglobulin (Ig) loci on chromosomes 2, 14, or 22. The c-myc gene becomes transcriptionally activated as a consequence of the chromosomal translocation and shows preferential usage of promoter P1 over P2, a phenomenon referred to as promoter shift. In order to define the responsible regulatory elements within the Ig lambda locus, we studied the effect of the human Ig lambda enhancer (HuE lambda) on c-myc expression after stable transfection into BL cells. A 12 kb genomic fragment encompassing HuE lambda, but not HuE lambda alone, strongly activated c-myc expression and induced the promoter shift. To identify additional elements involved in c-myc deregulation, we mapped DNaseI hypersensitive sites within the 12 kb lambda fragment on the construct. Besides one hypersensitive site corresponding to HuE lambda, three additional sites were detected. Two of these elements displayed enhancer activity after transient transfection. The third element did not activate c-myc transcription, but was required for full c-myc activation and promoter shift. Deletion analyses of the c-myc promoter identified the immediate promoter region as sufficient for activation by the Ig lambda. locus, but also revealed that induction of the promoter shift requires additional upstream elements.

Molecular cloning, structural organization, sequence, chromosomal assignment, and expression of the mouse alpha-N-acetylgalactosaminidase gene.

Alpha-N-acetylgalactosaminidase (2-acetamido-2-deoxy-alpha-d-galactoside acetamidodeoxy-galactohydrolase, NAGA; EC 3.2.1.49) deficiency is a recently recognized autosomal recessive lysosomal disease. As a prerequisite for the generation of an animal model, the mouse NAGA gene was cloned and characterized. The NAGA gene was assigned to mouse chromosome 15 band E3, syntenic to the region encompassing the human gene, and NAGA-deficient mutant human cells transfected with the cosmid clone containing the mouse NAGA gene expressed NAGA activity. Comparison of the mouse NAGA nucleotide sequence with the human NAGA sequence predicted that the mouse NAGA gene contains an open reading frame of 1245bp, comprising nine coding exons and spanning a genomic region of 8258bp, and a 3' untranslated region of 0.5kb. The 5' untranslated region was determined in primer extension studies to be 235bp in length. Nucleotide identity between the human and mouse NAGA exons ranged from 67.4 to 89.5%, with better matches for exons 1-7 than for 8 and 9. The overall amino acid identity between the mouse and human deduced NAGA polypeptides was 82.0%, between those of mouse and chicken 72.9%. Homology was found to only one other mouse gene, i.e. the alpha-galactosidase A (GALA; EC 3.2.1. 22) gene. The amino acid identity ranged from 51.6 to 62.1% in the polypeptide regions corresponding to NAGA exons 2-7 and GALA exons 1-6, but little, if any, in the remainder. These analyses gave emphasis to the strong conservation of the NAGA gene and its origin from an ancestor common with the GALA gene, with NAGA exons 8 and 9 and GALA exon 7 being the most divergent regions in the evolution of the two genes.

A complete set of overlapping cosmid clones of M-ABA virus derived from nasopharyngeal carcinoma and its similarity to other Epstein-Barr virus isolates.

DNA of the transforming, nondefective Epstein-Barr virus (EBV) strain M-ABA, which is derived from nasopharyngeal carcinoma cells, was cloned as large overlapping pieces into the cosmid pHC79 . The termini were cloned from closed circular virus DNA molecules out of M-ABA cell DNA in phage lambda L47 . The large overlapping clones were used to prepare a library of subclones with inserts of 1-15 kb. A detailed restriction enzyme map of M-ABA virus DNA reveals the close similarity to isolates from other sources. The high number of tandem repeats in EBV DNA stresses the importance of using cloning vectors that can be propagated in recA- Escherichia coli hosts.

Tumor promoters in commercial indoor-plant cultivars of the Euphorbiaceae.

Certain decorative indoor-plant cultivars are derived from toxic wild plant species. Native members of the Euphorbiaceae (spurge) contain highly irritating and tumor-promoting diterpene esters. Plant breeders and gardeners are constantly searching for less toxic cultivars of the popular Euphorbiaceae indoor plants. In this investigation, 22 commercial cultivars of Euphorbiaceae indoor plants were examined for tumor promoter contents by high-performance liquid chromatography (HPLC). Cultivars of E. milii (E. lomii hybrids), and in particular E. leuconeura, contained ingenol derivatives, whereas cultivars of E. pulcherrima and Codiaeum variegatum were devoid of these compounds. Tumor-promoting activity was assessed by induction of a luciferase reporter gene, which was placed under the control of an Epstein-Barr virus early antigen promoter. The response was closely correlated with ingenol ester content; the latex of the two E. leuconeura cultivars tested gave the strongest response. The HPLC and bioassay methods used in this study provide a basis for the development of nontoxic indoor-plant cultivars and perhaps for consumer-oriented labeling.

TATA box and Sp1 sites mediate the activation of c-myc promoter P1 by immunoglobulin kappa enhancers.

In Burkitt's lymphoma (BL) cells the proto-oncogene c-myc is transcriptionally activated by chromosomal translocation to the immunoglobulin (Ig) gene loci. This activation is characterized by preferential transcription from the c-myc promoter P1 and accomplished by juxtaposed Ig enhancer elements. To identify promoter elements required for enhancer-activated P1 transcription, we studied the activation of c-myc reporter gene constructs by the Ig kappa intron and 3' enhancers. Deletion analysis defined the core promoter with a TATA box and two adjacent GC/GT boxes upstream sufficient for basal and enhancer-activated transcription. Gel retardation assays revealed Sp1's binding affinity to the GC/GT box proximal to the TATA box to be higher than to the distal one. This difference correlated well with the resulting levels of transcription mediated by Sp1 in contransfection experiments in BL and Sp1-deficient SL2 cells. Sp3 also bound to the core promoter in vitro, but failed to transactivate in vivo. Mutation of the distal Sp1 site moderately affected basal transcription concomitant with a modest decrease in enhancer stimulation. Mutation of the proximal Sp1 site almost entirely abolished basal as well as enhanced transcription. A considerable level of basal transcription was maintained upon mutation of the TATA box, whereas enhancer-activated transcription largely was abolished. Stable transfection of the BL cell line Raji with constructs containing core promoter mutations confirmed that the proximal Sp1 site and the TATA box are essential for the activation of promoter P1 by the Ig kappa enhancers.

Comparison of models describing the sorption of nitroglycerin and diazepam by plastic infusion systems: diffusion and compartment models.

The time course of sorption of diazepam and nitroglycerin from aqueous solutions into plastic materials has been represented by the diffusion and compartmental models for a variety of storage conditions. The diffusion model seemed to be the more satisfactory model in respect to both description and prediction of the drug uptake for all conditions. The compartment model appeared to be useful for describing the drug uptake at earlier times, giving a satisfactory fit to the data and reliable final parameter estimates. However, that model was not able to describe the loss as equilibrium was approached or accurately predict the disappearance profiles for these solutes with alterations in solution volume or infusion bag size. Approximations of the diffusion model gave parameter estimates consistent with those obtained by nonlinear regression using the full equations.