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 Dl triggers premature activation of the cyclin A promoter by E2F, and this effect is blocked by the tumor suppressor protein p16INK4.Progression through the mammalian cell cycle is controlled by cyclins and cyclin-dependent kinases (cdk) (1). Cyclin gene expression is tightly regulated in a phase-specific manner. Expression of cyclin Dl precedes that of cyclin E in the G1 phase of the cell cycle (2); both proteins are required and are rate-limiting for passage through G, (3)(4)(5)(6)(7). Cyclin A is first expressed at the GI/S transition; it is required for S and M phases (8-10). Cyclin A may be a component of the DNA replication machinery (11,12) and may have a role in transcriptional control during S phase (13,14). Constitutive expression of cyclin A has been associated with tumorigenesis (15, 16); inversely, abolishment of cyclin A gene expression was recently found to cause the growth arrest of adhesiondependent cells grown in suspension (17). Overexpression of cyclin Dl (18) as well as of its partner kinase cdk4 (19) is linked to tumorigenesis, and the gene MTS1, coding for p16INK4, a cellular kinase inhibitor for cdk4, is found inactivated in a large variety of human tumor cell types (20,21). We report here a regulatory link between the expression of cyclins Dl and A. Phase-specific transcription of the human cyclin A gene (22) is mediated by a binding site for the transcription factor E2F (23). Cyclin Dl can activate cyclin A transcription through this element, and this signal is antagonized by p16INK4. MATERIALS AND METHODSReporter Plasmids and Expression Vectors. cDNAs encoding human cyclin A (15), cyclin Bi (24), cyclin Dl (3), cyclin E (25), cdk4 (26), cdc2 (27), and p16'NK4 (28) were subcloned by standard techniques in the cytomegalovirus (CMV)-based expression vector pX (10). Cyclin A promoter/reporter genes were constructed as described (22). Point mutation of the E2F site was performed by PCR and verified by sequence analysis after cloning. The inducible expression vector CMV/T was constructed by inserting the simian virus 40 polyadenylylation sequence upstream of the tetracycline-controlled promoter of plasmid pUHD10-3 (29). Insertion of cDNA coding for firefly luciferase (30), cyclin Dl, and cyclin A into CMV/T yielded plasmids luc/T, cycDl/T, and cycA/T, respectively.Cell Culture and Transfection. NIH 3T3 cells and human diploid fibroblasts from foreskin were cultured and starvation synchronized as described (22). Trans...

Hepatitis B virus (HBV) DNA frequently integrates into the genome of human primary liver cancer cells, but the significance of this integration in liver carcinogenesis is still unclear. Here we report the cloning of a single HBV integration site in a human hepatocellular carcinoma at an early stage of development, and of its germline counterpart. The normal locus was found to be transcribed into two polyadenylated messenger RNA species of 1.8 and 2.7 kilobases. We have isolated a complementary DNA clone from a normal adult human liver cDNA library which has an open reading frame with a coding capacity for a protein of 432 amino acids and relative molecular mass 48,536. The strong homology of the C-terminal half of the protein to the A-type cyclins of clam and Drosophila identifies it as a human cyclin A. The cyclin A gene has several exons, and the HBV integration occurs within an intron. As cyclins are important in the control of cell division, the disruption of a cyclin A gene by viral insertion might contribute to tumorigenesis.

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 G, 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.Cyclins control the transitions between the phases of the eukaryotic cell cycle as regulatory subunits of the cyclindependent kinases (cdk) (1). Phase-specific activation of the cdk is in part regulated by phase-specific expression of their cyclin component. In mammalian somatic cells, the G1 cyclins ofthe D and E type regulate the passage of G1 (2), cyclin A is required for S phase and passage through G2 (3), and the kinase composed of a B-type cyclin and p34cdc2 is the key regulator of mitosis (4). Cyclin A was first identified in sea urchin embryos (5). It can bind and activate the kinases cdc2 and cdk2 (6, 7) and is involved in the control of mitosis and S phase as well. Thus, injection of cyclin A mRNA drives G2-arrested Xenopus oocytes into meiotic metaphase (8), and degradation of the protein is necessary for the exit from mitosis (9). Cyclin A mutant Drosophila embryos undergo cell cycle arrest in G2 (10), and somatic mammalian cells are blocked in G2 upon ablation of cyclin A (11). In addition to its mitotic functions, cyclin A may have a role in the dependence of mitosis on S-phase completion (12). Cyclin A is required for S-phase passage in normal mammalian cells, while cyclin B is not (3,12,13). Cyclin A may indeed be directly involved in DNA replication: it colocalizes with the DNA replication complex (14, 15) and was found in a cellular activity that promotes large tumor-antigen-dependent simian virus 40 DNA replication in Gl-phase cell extracts (16); cyclin A/cdk2 may phosphorylate and activate the cellular DNA replication factor RPA (17, 18). During S phase, cyclin A forms a complex containing cdk2, the pRB-related protein p107, and the transcription factor E2F (19,20). E2F regulates a number of growth-promoting genes (21) and is negatively controlled during G1 by binding to pRB (22). In adenovirus 5-infected cells, the viral ElA oncoprotein dissociates the pRB/E2F complex (23,24) and binds to cyclin A via p107 in S phase (25,26). A possible role for cyclin A in cellular transformation was further suggested by our previous finding of a hepatitis B virus integration in the human cyclin A gene in a primary liver carcinoma (27). In this tumor, cyclin A-specific mRNA is exclusively transcribed from a viral promoter (28), indicating that deregu...