Activation of the cyclin D1 gene by the E1A-associated protein p300 through AP-1 inhibits cellular apoptosis.

The adenovirus E1A protein interferes with regulators of apoptosis and growth by physically interacting with cell cycle regulatory proteins including the retinoblastoma tumor suppressor protein and the coactivator proteins p300/CBP (where CBP is the CREB-binding protein). The p300/CBP proteins occupy a pivotal role in regulating mitogenic signaling and apoptosis. The mechanisms by which cell cycle control genes are directly regulated by p300 remain to be determined. The cyclin D1 gene, which is overexpressed in many different tumor types, encodes a regulatory subunit of a holoenzyme that phosphorylates and inactivates PRB. In the present study E1A12S inhibited the cyclin D1 promoter via the amino-terminal p300/CBP binding domain in human choriocarcinoma JEG-3 cells. p300 induced cyclin D1 protein abundance, and p300, but not CBP, induced the cyclin D1 promoter. cyclin D1 or p300 overexpression inhibited apoptosis in JEG-3 cells. The CH3 region of p300, which was required for induction of cyclin D1, was also required for the inhibition of apoptosis. p300 activated the cyclin D1 promoter through an activator protein-1 (AP-1) site at -954 and was identified within a DNA-bound complex with c-Jun at the AP-1 site. Apoptosis rates of embryonic fibroblasts derived from mice homozygously deleted of the cyclin D1 gene (cyclin D1(-/-)) were increased compared with wild type control on several distinct matrices. p300 inhibited apoptosis in cyclin D1(+/+) fibroblasts but increased apoptosis in cyclin D1(-/-) cells. The anti-apoptotic function of cyclin D1, demonstrated by sub-G(1) analysis and annexin V staining, may contribute to its cellular transforming and cooperative oncogenic properties.

Modulation of interleukin-18 expression in human colon carcinoma: consequences for tumor immune surveillance.

The production in colon cancer of interferon-gamma (IFN-gamma), a type-1 T-helper (TH1) cytokine, is considered as a marker of good prognosis. We asked whether interleukin-18 (IL-18), which strongly induces IFN-gamma and regulates Fas ligand (Fas-L)-dependent cytotoxicity, may play a role in colon homeostasis, and if its expression was modulated in colon adenocarcinomas. We analyzed 14 specimens of colon adenocarcinomas, 6 of normal colon mucosa of the series, and 6 colon-tumor cell lines. The expression of IL-18, of ICE protease, involved in the processing of this cytokine, and of the downstream effectors of IL-18, IFN-gamma and Fas-L was analyzed by RT-PCR. We further performed IL-18 immunostaining of normal and tumor specimens. The results were correlated with tumor dissemination and clinical outcome. We report the synthesis of IL-18 in human normal colon, mainly by epithelial cells of the mucosa. Out of the 6 tumor cell lines, 4 expressed IL-18 transcripts, but neither ICE mRNA nor secreted forms of IL-18 were detected. We observed decreased or abolished synthesis of IL-18 in colon adenocarcinomas, as compared with normal mucosa. Thus, half of the colon-cancer tissues (7/14 cases) expressed neither IFN-gamma nor Fas-L. This feature was correlated with the existence of distant metastases (Fischer's exact test, p = 0.02) and an unfavorable outcome. These findings suggest that production of IL-18 in human colon may play a role in homeostasis and in tumor immune surveillance, by enhancing IFN-gamma production and Fas-L-dependent cytotoxicity of immune cells.

Inhibition of cyclin D1 kinase activity is associated with E2F-mediated inhibition of cyclin D1 promoter activity through E2F and Sp1.

Coordinated interactions between cyclin-dependent kinases (Cdks), their target "pocket proteins" (the retinoblastoma protein [pRB], p107, and p130), the pocket protein binding E2F-DP complexes, and the Cdk inhibitors regulate orderly cell cycle progression. The cyclin D1 gene encodes a regulatory subunit of the Cdk holoenzymes, which phosphorylate the tumor suppressor pRB, leading to the release of free E2F-1. Overexpression of E2F-1 can induce apoptosis and may either promote or inhibit cellular proliferation, depending upon the cell type. In these studies overexpression of E2F-1 inhibited cyclin D1-dependent kinase activity, cyclin D1 protein levels, and promoter activity. The DNA binding domain, the pRB pocket binding region, and the amino-terminal Sp1 binding domain of E2F-1 were required for full repression of cyclin D1. Overexpression of pRB activated the cyclin D1 promoter, and a dominant interfering pRB mutant was defective in cyclin D1 promoter activation. Two regions of the cyclin D1 promoter were required for full E2F-1-dependent repression. The region proximal to the transcription initiation site at -127 bound Sp1, Sp3, and Sp4, and the distal region at -143 bound E2F-4-DP-1-p107. In contrast with E2F-1, E2F-4 induced cyclin D1 promoter activity. Differential regulation of the cyclin D1 promoter by E2F-1 and E2F-4 suggests that E2Fs may serve distinguishable functions during cell cycle progression. Inhibition of cyclin D1 abundance by E2F-1 may contribute to an autoregulatory feedback loop to reduce pRB phosphorylation and E2F-1 levels in the cell.

p27KIP1 blocks cyclin E-dependent transactivation of cyclin A gene expression.

Cyclin E is necessary and rate limiting for the passage of mammalian cells through the G1 phase of the cell cycle. Control of cell cycle progression by cyclin E involves cdk2 kinase, which requires cyclin E for catalytic activity. Expression of cyclin E/cdk2 leads to an activation of cyclin A gene expression, as monitored by reporter gene constructs derived from the human cyclin A promoter. Promoter activation by cyclin E/cdk2 requires an E2F binding site in the cyclin A promoter. We show here that cyclin E/cdk2 kinase can directly bind to E2F/p107 complexes formed on the cyclin A promoter-derived E2F binding site, and this association is controlled by p27KIP1, most likely through direct protein-protein interaction. These observation suggest that cyclin E/cdk2 associates with E2F/p107 complexes in late G1 phase, once p27KIP1 has decreased below a critical threshold level. Since a kinase-negative mutant of cdk2 prevents promoter activation, it appears that transcriptional activation of the cyclin A gene requires an active cdk2 kinase tethered to its promoter region.

A novel simian virus 40 early-region domain mediates transactivation of the cyclin A promoter by small-t antigen and is required for transformation in small-t antigen-dependent assays.

At least three regions of the simian virus 40 small-t antigen (small-t) contribute to the protein's ability to enhance cellular transformation. As we showed previously for rat F111 cells, one region includes sequences from residues 97 to 103 that are involved in the binding and inhibition of protein phosphatase 2A. In the present study, the role of the protein phosphatase 2A binding region was confirmed in two additional small-t-dependent transformation systems. Second, small-t was found to provide a function previously identified as a large-T transformation domain. Mutations in residues 19 to 28 of large-T affected its transforming ability, but these mutations were complemented by a wild-type small-t. A third region of small-t was also required for efficient transformation. This region, the 42-47 region, is shared by large-T and small-t and contains a conserved HPDKGG hexapeptide. The 42-47 region function could be provided by either small-t or large-T in small-t-dependent systems. Mutations in the 42-47 region reduced the ability of small-t to transactivate the cyclin A promoter, of interest because small-t increased endogenous cyclin A mRNA levels in both human and monkey cells, as well as transactivating the promoter in transient assays.

Anchorage-dependent transcription of the cyclin A gene.

NIH 3T3 cells cultured in suspension fail to express cyclin A and hence cannot enter S phase and divide. We show that loss of cell adhesion to substratum abrogates cyclin A gene expression by blocking its promoter activity through the E2F site that mediates its cell cycle regulation in adherent cells. In suspended cells, G0-specific E2F complexes remain bound to the cyclin A promoter. Overexpression of cyclin D1 restores cyclin A transcription in suspended cells and rescues them from cell cycle arrest. In suspended cells, cyclin D1 and cyclin E accumulate normally upon serum stimulation, but their associated kinases remain inactive; their substrates, pRb and p107, are not hyperphosphorylated. Concomitantly, the cyclin-dependent kinase inhibitor, p27KIP1, is stabilized. Ectopic expression of p27KIP1 blocks cyclin A promoter activity through its EN binding site. These data suggest that the block to cyclin A transcription in nonadherent NIH 3T3 cells results from stabilization of p27KIP1 and subsequent inactivation of the specific E2F moiety required for its induction.

Activation of cyclin-dependent kinases by Myc mediates induction of cyclin A, but not apoptosis.

The activation of conditional alleles of Myc induces both cell proliferation and apoptosis in serum-deprived RAT1 fibroblasts. Entry into S phase and apoptosis are both preceded by increased levels of cyclin E- and cyclin D1-dependent kinase activities. To assess which, if any, cellular responses to Myc depend on active cyclin-dependent kinases (cdks), we have microinjected expression plasmids encoding the cdk inhibitors p16, p21 or p27, and have used a specific inhibitor of cdk2, roscovitine. Expression of cyclin A, which starts late in G1 phase, served as a marker for cell cycle progression. Our data show that active G1 cyclin/cdk complexes are both necessary and sufficient for induction of cyclin A by Myc. In contrast, neither microinjection of cdk inhibitors nor chemical inhibition of cdk2 affected the ability of Myc to induce apoptosis in serum-starved cells. Further, in isoleucine-deprived cells, Myc induces apoptosis without altering cdk activity. We conclude that Myc acts upstream of cdks in stimulating cell proliferation and also that activation of cdks and induction of apoptosis are largely independent events that occur in response to induction of Myc.

Adenovirus E1A activates cyclin A gene transcription in the absence of growth factors through interaction with p107.

Using the infection of quiescent human fibroblasts with adenovirus type 5 and various deletion mutants, we show that E1A can stimulate transcription of the cyclin A gene in the absence of exogenous growth factors. Required for this activity is conserved region 2 (CR2), while both the N-terminal part of E1A and CR1 are dispensable. This indicates that activation of cyclin A gene expression requires the binding of E1A to p107, while binding to either pRB or p300 is not involved in transcriptional activation. We demonstrate that p107 represses the cyclin A promoter through its cell cycle-regulatory E2F binding site and that 12S E1A can activate the cyclin A promoter, essentially by counteracting its repression by p107. Since Cr2 is required for cell transformation, transcriptional activation of the cyclin A gene by E1A appears to be important for its capacity to override control of cellular growth.

Cell cycle regulation of the cyclin A gene promoter is mediated by a variant E2F site.

Cyclin A is involved in the control of S phase and mitosis in mammalian cells. Expression of the cyclin A gene in nontransformed cells is characterized by repression of its promoter during the G1 phase of the cell cycle and its induction at S-phase entry. We show that this mode of regulation is mediated by the transcription factor E2F, which binds to a specific site in the cyclin A promoter. It differs from the prototype E2F site in nucleotide sequence and protein binding; it is bound by E2F complexes containing cyclin E and p107 but not pRB. Ectopic expression of cyclin D1 triggers premature activation of the cyclin A promoter by E2F, and this effect is blocked by the tumor suppressor protein p16INK4.

Sequential activation of cyclin E and cyclin A gene expression by human papillomavirus type 16 E7 through sequences necessary for transformation.

To investigate E7-dependent biochemical changes which are involved in cellular transformation, we analyzed the influence of human papillomavirus type 16 (HPV-16) E7 on the expression of cell cycle regulatory proteins. Expression of E7 in established rodent fibroblasts (NIH 3T3), which was shown to be sufficient for transformation of these cells, leads to constitutive expression of the cyclin E and cyclin A genes in the absence of external growth factors. Surprisingly, expression of the cyclin D1 gene, which encodes a major regulator of G1 progression, is unaltered in E7-transformed cells. In transient transfection experiments, the cyclin A gene promoter is activated by E7 via an E2F binding site. In 14/2 cells, which were used as a model system to analyze the role of HPV-16 E7 in the transformation of primary cells, we observed rapid E7-dependent activation of cyclin E gene expression, which can be uncoupled from activation of the cyclin A gene, since the latter requires additional protein synthesis. E7-driven induction of cyclin E and cyclin A gene expression was accompanied by an increase in the associated kinase activities. Two domains of the E7 oncoprotein, which are designated cd1 and cd2, are essential for transformation of rodent fibroblasts. It is shown here that growth factor-independent expression of the cyclin E gene requires cd2 but not cd1, while activation of cyclin A gene expression requires cd1 function in addition to that of cd2. These data suggest that cyclin A gene expression is controlled by two distinct negative signals, one of which also restricts expression of the cyclin E gene. The ability of E7 to separately override each of these inhibitory signals, via cd1 and cd2, cosegregates with its ability to fully transform rodent fibroblasts. Unlike serum growth factors, E7 induces S-phase entry without activating cyclin D1 gene expression, in keeping with the finding that cyclin D1 function is not required in cells transformed by DNA tumor viruses.

TGF-beta 1 and cAMP attenuate cyclin A gene transcription via a cAMP responsive element through independent pathways.

Transforming growth factor beta (TGF-beta) is a potent inhibitor of the proliferation of many cell lines. The expression of Cyclin A is down-regulated by TGF-beta 1 in Chinese hamster lung fibroblasts and most of this effect is mediated at the transcriptional level through a cAMP-responsive element (CRE), but does not require a functional cAMP-dependent protein kinase. However, activation of the cAMP pathway in these cells gives rise to a strong inhibition of proliferation, paralleled by a down-regulation of Cyclin A promoter activity. This effect requires the integrity of the CRE, suggesting a role for CRE-binding proteins in late G1/S controls.

Activation of the E2F transcription factor by cyclin D1 is blocked by p16INK4, the product of the putative tumor suppressor gene MTS1.

The oncoprotein cyclin D1 binds to and activates cyclin-dependent kinase 4 (cdk4), whose activity is inhibited by p16INK4, the product of the putative tumor suppressor gene MTS1. Cyclin D1 controls the timing of S phase onset in mammalian cells. We show that cyclin D1 acts as a positive regulator of the transcription factor E2F. In particular, cyclin D1 overexpression leads to the activation of the dihydrofolate reductase (DHFR) gene promoter. Activation depends on the E2F binding site in the DHFR promoter, known to mediate its activation at the G1/S transition in vivo. Cyclin D1 can also activate the adenovirus E2 promoter via E2F. Both promoters are repressed by p16INK4 and this repression can be released by overexpression of cdk4. The data reported here support a direct role for cyclin D1 and its associated kinase in cell cycle regulation of E2F activity and S phase-specific gene expression. In addition, we show that both E2F sites bind complexes containing the retinoblastoma protein (pRB) and that in RB-deficient cell lines overexpression of cyclin D1 fails to activate E2F-dependent transcription, indicating that pRB may be involved in promoter activation.

Structure and cell cycle-regulated transcription of the human cyclin A gene.

Cyclin A is a cell cycle regulatory protein that functions in mitotic and S-phase control in mammalian somatic cells. Its deregulated expression may have a role in cellular transformation. We have cloned and sequenced the human cyclin A gene and cDNAs representing its mRNAs and have characterized its promoter. Using synchronized cultures of NIH 3T3 cells stably transfected with cyclin A promoter/luciferase constructs, we show that the promoter is repressed during the G1 phase of the cell cycle and is activated at S-phase entry. Cell cycle regulation of the cyclin A gene promoter is mediated by sequences extending from -79 to +100 relative to the predominant transcription start site. It does not require the presence of a functional retinoblastoma protein.

Loss of the G1-S control of cyclin A expression during tumoral progression of Chinese hamster lung fibroblasts.

The expression of cyclin A, one of the key regulators of cell cycle progression in association with cdc2/cdk2 protein kinases and which undergoes cyclic accumulation during the cell cycle, has been investigated in CCL39 Chinese hamster lung fibroblasts and in two transformed variants, A71 and 39Py. Whereas A71 (selected after tumor induction in nude mice) is subject to growth arrest (less than 5% of labeled nuclei after 24 h of serum starvation), 39Py (obtained after transformation by polyoma virus) is not (more than 50% of labeled nuclei). In both cells, cyclin A expression was correlated with establishment of S phase, with a progressive deregulation of its G1 controls. This deregulation was not detected with the two early response genes c-fos and c-myc. The kinetics of accumulation of cyclin A lagged behind that of [3H]thymidine incorporation, thereby questioning a direct role for cyclin A in S phase triggering. Moreover, transforming growth factor beta 1, which is known to inhibit alpha-thrombin or fibroblast growth factor-induced mitogenicity in G0-arrested CCL39 cells, is shown here to down-regulate cyclin A expression in both CCL39 and A71 cells but has no effect on 39Py cells. These data establish cyclin A as a sensitive marker for the loss of growth factor requirement.

Mapping chromosomal breakpoints of Burkitt's t(8;14) translocations far upstream of c-myc.

To analyze the region upstream of c-myc, a number of novel probes were established. These were generated by chromosomal walking starting from the breakpoint of the chromosomal translocation of the B-cell line 380 and by cloning the breakpoint of the translocation of the Burkitt lymphoma cell line IARC/BL72. Using the newly isolated probes a detailed physical map of 500 kilobases of the region upstream of c-myc was established applying pulsed-field gel electrophoresis. The chromosomal breakpoint of IARC/BL72 cells was mapped to a site 55 kilobases 5' of c-myc. A region 20 kilobases in length and containing the breakpoints of 380, EW36, P3HR-1, and Daudi cells was identified 170-190 kilobases upstream of c-myc. In addition the HPV18 integration site in HeLa cells was located between 340 and 500 kilobases 5' of c-myc. The probes were used to define the c-myc amplification units in Colo320-HSR and HL60 cells as well as in four cases of small cell lung cancer. Evidence is provided that the amplicon of HL60 cells is discontinuously organized.

Variable breakpoints in Burkitt lymphoma cells with chromosomal t(8;14) translocation separate c-myc and the IgH locus up to several hundred kb.

In about 80% of Burkitt's lymphoma cases, the tumour cell harbours a reciprocal chromosomal translocation which invariably transposes the coding exons 2 and 3 of c-myc from chromosome 8 to the immunoglobulin heavy chain locus on chromosome 14. Those t(8;14) translocations which disrupt chromosome 8 within or close to the c-myc gene are well documented. In this study we have focussed on t(8;14) translocations with the chromosomal breakpoint far upstream of c-myc. We analyzed the breakpoint position in 44 BL cell lines with t(8;14) translocations of different geographical origin and identified 9 cell lines with the breakpoint more than 14 kb upstream of c-myc. In these cell lines the positions of the translocation junctions on the derivative chromosomes 8q- and 14q+ were mapped by pulsed field gel electrophoresis and multicolour fluorescence in situ hybridization. The breakpoints occur at distances between 55 and more than 340 kb upstream of c-myc with no preferential site on chromosome 8. On chromosome 14, however, the translocation breakpoints are clustered in a narrow region 5' of the intron enhancer of the immunoglobulin heavy chain gene. In 7 of 9 cases, the enhancer is fused to the c-myc bearing sequences of chromosome 8. In two cases, the translocation has occurred in switch mu and downstream of C mu, respectively. The impact of these results with respect to the hypothesis, that cis-regulatory sequences from the immunoglobulin heavy chain locus can deregulate c-myc expression in a manner sufficient for tumour formation, is discussed.

Modification of cyclin A expression by hepatitis B virus DNA integration in a hepatocellular carcinoma.

We have previously reported the identification of a hepatitis B virus (HBV) DNA integration in an intron of the cyclin A gene in an early hepatocellular carcinoma (HCC) and the isolation of human cyclin A cDNA. We have now constructed a cDNA library from the tumor and isolated several hybrid HBV-cyclin A cDNAs from it. The hybrid cDNAs encode an HBV-cyclin A fusion protein. In the chimeric protein, the N-terminus of cyclin A, including the signals for cyclin degradation, is deleted and replaced by viral PreS2/S sequences, transcription being initiated from the viral PreS2/S promoter. This chimeric protein is undegradable in an in vitro cyclin degradation assay. Northern blot analyses showed strong expression of the hybrid transcripts in the tumor, while cyclin A- or HBV-specific transcripts were not detected in the non-tumorous liver of the same patient. Thus, HBV DNA integration in the cyclin A gene resulted in a strong expression of hybrid HBV-cyclin A transcripts encoding a stabilized cyclin A. This chimeric protein may play an important role in the development of the tumor.

Cyclin A is required in S phase in normal epithelial cells.

We have investigated cyclin A expression in a primary culture of normal rat hepatocytes and during rat liver regeneration after partial hepatectomy. In both cases, cyclin A mRNA and protein accumulate as the cells enter S phase. To investigate the potential implication of cyclin A accumulation at S phase, we microinjected anti-sense DNA constructs for cyclin A, resulting in effective inhibition of S phase entry. These effects were specific for cyclin A since anti-sense cyclin B construct had no similar effects. These results therefore, obtained in normal epithelial cells, indicate that cyclin A is involved in S phase and thus should not be only considered as a mitotic cyclin.

Frequent activation of N-myc genes by hepadnavirus insertion in woodchuck liver tumours.

The recent finding of c-myc activation by insertion of woodchuck hepatitis virus DNA in two independent hepatocellular carcinoma has given support to the hypothesis that integration of hepatitis B viruses into the host genome, observed in most human and woodchuck liver tumours, might contribute to oncogenesis. We report here high frequency of woodchuck hepatitis virus DNA integrations in two newly identified N-myc genes: N-myc1, the homologue of known mammalian N-myc genes, and N-myc2, an intronless 'complementary DNA gene' or 'retroposon' that has retained extensive coding and transforming homology with N-myc. N-myc2 is totally silent in normal liver, but is overexpressed without genetic rearrangements in most liver tumours. Moreover, viral integrations occur within either N-myc1 or N-myc2 in about 20% of the tumours, giving rise to chimaeric messenger RNAs in which the 3' untranslated region of N-myc was replaced by woodchuck hepatitis virus sequences encompassing the viral enhancer. Insertion sites were clustered in a short sequence of the third exon that coincides with a retroviral integration hotspot within the murine N-myc gene, recently described in T-cell lymphomas induced by murine leukaemia virus. Thus, comparable mechanisms, leading to deregulated expression of N-myc genes, may operate in the development of tumours induced either by hepatitis virus or by nonacute retroviruses in rodents. Activation of myc genes by insertion of hepadnavirus DNA now emerges as a common event in the genesis of woodchuck hepatocellular carcinoma.