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...

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, G 0 -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, p27 KIP1 , is stabilized. Ectopic expression of p27 KIP1 blocks cyclin A promoter activity through its E2F binding site. These data suggest that the block to cyclin A transcription in nonadherent NIH 3T3 cells results from stabilization of p27 KIP1 and subsequent inactivation of the specific E2F moiety required for its induction.

BackgroundPatients with metastatic melanoma have a poor median rate of survival. It is therefore necessary to increase our knowledge about melanoma cell dissemination which includes extravasation, where cancer cells cross the endothelial barrier. Extravasation is well understood during travelling of white blood cells, and involves integrins such as LFA-1 (composed of two chains, CD11a and CD18) expressed by T cells, while ICAM-1 is induced during inflammation by endothelial cells. Although melanoma cell lines cross endothelial cell barriers, they do not express LFA-1. We therefore hypothesized that melanoma-endothelial cell co-culture might induce the LFA-1/ICAM ligand/receptor couple during melanoma transmigration.MethodsA transwell approach has been used as well as blocking antibodies against CD11a, CD18 and ICAM-1. Data were analyzed with an epifluorescence microscope. Fluorescence intensity was quantified with the ImageJ software.ResultsWe show here that HUVEC-conditioned medium induce cell-surface expression of LFA-1 on melanoma cell lines. Similarly melanoma-conditioned medium activates ICAM-1 expression in endothelial cells. Accordingly blocking antibodies of ICAM-1, CD11a or CD18 strongly decrease melanoma transmigration. We therefore demonstrate that melanoma cells can cross endothelial monolayers in vitro due to the induction of ICAM-1 and LFA-1 occurring during the co-culture of melanoma and endothelial cells. Our data further suggest a role of LFA-1 and ICAM-1 in the formation of melanoma cell clumps enhancing tumor cell transmigration.ConclusionMelanoma-endothelial cell co-culture induces LFA-1 and ICAM-1 expression, thereby favoring in vitro melanoma trans-migration.