Mediation of c-Myc-induced apoptosis by p53.

The cellular proto-oncogene c-myc is involved in cell proliferation and transformation but is also implicated in the induction of programmed cell death (apoptosis). The same characteristics have been described for the tumor suppressor gene p53, the most commonly mutated gene in human cancer. In quiescent mouse fibroblasts expressing wild-type p53 protein, activation of c-Myc was found to induce apoptosis and cell cycle reentry, preceded by stabilization of p53. In contrast, in quiescent p53-null fibroblasts, activation of c-Myc induced cell cycle reentry but not apoptosis. These results suggest that p53 mediates apoptosis as a safeguard mechanism to prevent cell proliferation induced by oncogene activation.

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.

From initiation to elongation: comparison of transcription by prokaryotic and eukaryotic RNA polymerases.

Multisubunit RNA polymerases in prokaryotes and eukaryotes share an evolutionarily conserved core. Here, we compare the processes of promoter recognition, transcription initiation and transcript elongation by human RNA polymerase II and by the RNA polymerase of the eubacterium Escherichia coli. Although these two polymerases have diverged widely in structure, important functions have been conserved, suggesting that the basic mechanisms of RNA transcription are similar in eukaryotes and prokaryotes.

Nucleosomal structures of c-myc promoters with transcriptionally engaged RNA polymerase II.

Organization of DNA into chromatin has been shown to contribute to a repressed state of gene transcription. Disruption of nucleosomal structure is observed in response to gene induction, suggesting a model in which RNA polymerase II (pol II) is recruited to the promoter upon reorganization of nucleosomes. Here we show that induction of c-myc transcription correlates with the disruption of two nucleosomes in the upstream promoter region. This nucleosomal disruption, however, is not necessary for the binding of pol II to the promoter. Transcriptionally engaged pol II complexes can be detected when the upstream chromatin is in a more closed configuration. Thus, upstream chromatin opening is suggested to affect activation of promoter-bound pol II rather than entry of polymerases into the promoter. Interestingly, pol II complexes are detectable in both sense and antisense transcriptional directions, but only complexes in the sense direction respond to activation signals resulting in processive transcription.

Multiple single-stranded cis elements are associated with activated chromatin of the human c-myc gene in vivo.

Transcription activation and repression of eukaryotic genes are associated with conformational and topological changes of the DNA and chromatin, altering the spectrum of proteins associated with an active gene. Segments of the human c-myc gene possessing non-B structure in vivo located with enzymatic and chemical probes. Sites hypertensive to cleavage with single-strand-specific S1 nuclease or the single-strand-selective agent potassium permanganate included the major promoters P1 and P2 as well as the far upstream sequence element (FUSE) and CT elements, which bind, respectively, the single-strand-specific factors FUSE-binding protein and heterogeneous nuclear ribonucleoprotein K in vitro. Active and inactive c-myc genes yielded different patterns of S1 nuclease and permanganate sensitivity, indicating alternative chromatin configurations of active and silent genes. The melting of specific cis elements of active c-myc genes in vivo suggested that transcriptionally associated torsional strain might assist strand separation and facilitate factor binding. Therefore, the interaction of FUSE-binding protein and heterogeneous nuclear ribonucleoprotein K with supercoiled DNA was studied. Remarkably, both proteins recognize their respective elements torsionally strained but not as liner duplexes. Single-strand- or supercoil-dependent gene regulatory proteins may directly link alterations in DNA conformation and topology with changes in gene expression.

Transition from initiation to promoter proximal pausing requires the CTD of RNA polymerase II.

The C-terminal domain (CTD) of mammalian RNA polymerase II consists of 52 repeats of the consensus hepta-peptide YSPTSPS, and links transcription to the processing of pre-mRNA. Although Pol II with a CTD shortened to five repeats (Pol II Delta5) is transcriptionally inactive on chromatin templates, it is not clear whether CTD is required for promoter recognition in vivo. Here, we demonstrate that in the context of chromatin, Pol II Delta5 can bind to the c-myc promoter with the same efficiency as wild type Pol II. However, Pol II Delta5 does not form a stable initiation complex, and does not transcribe promoter proximal sequences. Fluorescence recovery after photobleaching (FRAP) experiments with cells expressing enhanced green fluorescent protein (EGFP)-tagged Delta5 or wildtype Pol II revealed a single, highly mobile Pol II Delta5 fraction whereas wildtype Pol II yielded less mobile fractions. These data suggest that CTD is not required for promoter recognition, but rather for subsequent formation of a stable initiation complex and isomerization to an elongation competent complex.

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.

Analysis of cell cycle arrest in adipocyte differentiation.

Confluent 3T3-L1 preadipocytes differentiate to adipocytes in the presence of insulin, dexamethasone, and isobutylmethylxanthine (IDI). A transient increase of DNA synthesis is induced in 3T3-L1 cells 18 h after addition of IDI, followed by an arrest in the G1 phase of the cell cycle. Growth arrested cells express the proto-oncogene c-myc and the gene for the CCAAT/enhancer binding protein (C/EBPalpha) between day 2 and 5. While c-Myc is strongly implicated in cell proliferation, C/EBPalpha: is a differentiation-specific transcription factor with antiproliferative activity. Here we have characterized the cell cycle arrest in differentiating 3T3-L1 cells. Arrested cells express the Cdk inhibitors p21 and p27, but, at the same time, show hyperphosphorylation of Rb and expression of the E2F-regulated thymidine kinase gene. The addition of new serum to arrested cells resulted in cyclin A expression and Cdk2 activity, but not in DNA synthesis. Simian virus 40 large tumor antigen (LTAg) is a potent mitogen. The mutant LTAg-K1, deficient in binding of pocket proteins and unable to induce DNA synthesis in serum-starved 3T3-L1 cells, efficiently induced DNA synthesis in differentiating 3T3-L1 cells. This indicates that pocket proteins are probably not involved in the control of the cell cycle arrest during 3T3-L1 cell differentiation. Our data suggest that the differentiation-specific cell cycle block in 3T3-L1 cells is resistant to high levels of c-Myc, inactivation of pocket proteins, upregulation of cyclin A levels, and Cdk2 activation, but can be abolished by a function of LTAg that is independent of binding to pocket proteins.

Elongation and premature termination of transcripts initiated from c-fos and c-myc promoters show dissimilar patterns.

RNA polymerase II seems to be prone to stop at intrinsic pause sites, thus introducing a further potential level of regulation. It was recently shown that RNA polymerase II was held at the P2 promoter of c-myc gene. We confirmed the presence of engaged polymerases in the murine fibroblastic Ltk- and pre-B lymphoid 70Z3 cell lines. High resolution run-on analysis and in vivo permanganate-dependent footprinting showed that this holds true for the c-fos gene in unstimulated cells where a strong block to transcription elongation was evidenced. In contrast to what was observed in the c-myc gene, an even more intense signal was observed in run-on experiments downstream to the promoter, on a c-fos oligonucleotide including position +385 where an in vitro transcription arrest site was previously mapped. Genomic footprinting of DNA from intact cells and isolated nuclei confirmed the involvement of several thymidines belonging to a T-rich stretch in a melted region which was not detected upon polymerase release. In order to observe a short abortive c-fos transcript accumulating in vivo we resorted to microinjection of c-fos templates in Xenopus oocytes where transcripts were stable.

Early down-regulation of c-myc in dimethylsulfoxide-induced mouse erythroleukemia (MEL) cells is mediated at the P1/P2 promoters.

A block of RNA elongation in exon 1 of the murine c-myc gene has been described for normal mouse fibroblasts, lymphoid and myeloid cell lines and mouse erythroleukemia (MEL) cells. MEL cells differentiate after induction with the chemical agent dimethylsulfoxide (DMSO). The rapid initial down-regulation of c-myc that occurs after treatment with DMSO has been explained by an increase in the block of RNA elongation within the 3' part of c-myc exon 1. In contrast to these reports, we find that down-regulation of c-myc in DMSO-induced MEL cells occurs at the c-myc P1 and P2 promoters. The P1 promoter is repressed by inhibition of initiation, whereas transcription of P2 RNA is blocked by retention of RNA polymerase II at or close to the P2 promoter. The earlier described block of RNA elongation at a run of five thymidines in the 3' part of c-myc exon 1 was not observed.

The pRb-related protein p130 is a possible effector of transforming growth factor beta 1 induced cell cycle arrest in keratinocytes.

Transforming growth factor beta 1 (TGF-beta 1) is known to inhibit epithelial cell growth by inducing a G1 cell cycle arrest. We have studied the effect of TGF-beta 1 on protein binding to a transcription factor E2F consensus element in extracts from early passage human keratinocytes (HFKs) and a permanent human keratinocyte cell line (HaCaT). Treatment of these cells with TGF-beta 1 resulted in the formation of a DNA binding complex between the pRb-related protein p130 and E2F. Formation of the E2F-p130 complex correlated with inhibition of cell cycle progression in G1 and suppression of the E2F-regulated cdc2 gene. While p130 mRNA and protein levels were not influenced by TGF-beta 1, the activity of cyclin-dependent kinase 2 (Cdk2) towards p130 in vitro was inhibited. The results identify p130 as a downstream target of TGF-beta 1 and a possible mediator of the G1 cell cycle arrest.

Ongoing mutations in the N-terminal domain of c-Myc affect transactivation in Burkitt's lymphoma cell lines.

A panel of 18 Burkitt's lymphoma (BL) and nine other cell lines was examined for mutations in the N-terminal transactivation domain of c-Myc. Mutations leading to exchange of amino acids were detected in 13 BL but in none of the control cell lines. Mutations in c-Myc clustered between amino acid positions 57 and 62. Thr-58 and Ser-62 are known phosphorylation sites of c-Myc in vivo. BL cell lines derived from the same tumour revealed different mutations. Mutant cDNAs of the BL cell line Raji differed at 14 positions indicating ongoing mutation of the translocated c-myc during long-term propagation in cell culture. The effect of mutations on transactivation by c-Myc was tested by expression of GAL4/c-Myc fusion proteins in the BL cell line Raji. Mutants with an amino acid exchange at positions 58 or 60 transactivated a reporter gene two- to fivefold weaker than wildtype c-Myc. Thr-58 and Ser-62 were replaced by aspartic acid to mimic constitutively phosphorylated forms of c-Myc. These mutants transactivated two- to three-fold weaker than wildtype c-Myc indicating that a negative charge at positions 58 and/or 62 per se does not enhance transactivation. We propose that mutations in the N-terminal domain of c-Myc correlate with reduced transactivation and provide a growth advantage for BL cells.

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.

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.

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.

Abrogation of p53-induced cell cycle arrest by c-Myc: evidence for an inhibitor of p21WAF1/CIP1/SDI1.

The tumor-suppressor p53 inhibits cell cycle progression by direct transactivation of the p21WAF1/CIP1/SDI1 gene, which encodes a universal inhibitor of cyclin dependent kinases (cdk). The proto-oncogene product c-Myc induces cell cycle progression and, in the absence of survival factors, apoptosis. However, a direct link between the cell cycle machinery and c-Myc has not yet been established. We show that c-Myc has not yet been established. We show that c-Myc abrogates a p53-induced G1-arrest without elevating the expression of cdks or cyclins involved in the G1/S-transition. Instead, the results suggest that c-Myc interferes with the inhibitory action of p21 on cdk/cyclin-complexes by inducing a heat-labile inhibitor of p21. The inactivation of p21 and related cdk-inhibitors may explain several of the oncogenic actions of c-Myc, including the induction of proliferation, immortalisation and the inhibition of differentiation. Modulation of cdk activity by the induction of an inhibitor of cdk-inhibitors represents a novel mechanism of cell cycle regulation in mammalian cells.

A complex between E2F and the pRb-related protein p130 is specifically targeted by the simian virus 40 large T antigen during cell transformation.

The p130 protein is a recently cloned member of the retinoblastoma protein family. We show here that transformation of NIH3T3-L1 fibroblasts (L1 cells) by the simian virus 40 large T antigen (LTAg) depends on the disruption of DNA binding complexes between transcription factor E2F and p130. LTAg binds to the pocket region of p130 in vivo and disrupts the E2F-p130 complexes. E2F-p130 complexes are present only in quiescent L1 cells and disappear at the G1/S phase boundary concomitantly to induction of DNA synthesis and expression of the E2F-regulated cdc2 gene. p130 is a substrate of cyclin-dependent kinase 2 (Cdk2) in vitro and associates with a Cdk in vivo which is activated upon serum stimulation in late G1. Overexpression of p130 inhibits cdc2 promoter activity and entry of quiescent L1 cells into S phase. The results demonstrate that p130 is negative regulator of cell cycle progression which is specifically targeted by LTAg during cell transformation.

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.

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.