No abstract

SummaryThe cell-cycle regulating gene, p16 INK4A, encoding an inhibitor of cyclin-dependent kinases 4 and 6, is considered to play an important role in cellular aging and in premature senescence.

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...

Aging is a multifactorial process where deterioration of body functions is driven by stochastic damage while counteracted by distinct genetically encoded repair systems. To better understand the genetic component of aging, many studies have addressed the gene and protein expression profiles of various aging model systems engaging different organisms from yeast to human. The recently identified small non-coding miRNAs are potent post-transcriptional regulators that can modify the expression of up to several hundred target genes per single miRNA, similar to transcription factors. Increasing evidence shows that miRNAs contribute to the regulation of most if not all important physiological processes, including aging. However, so far the contribution of miRNAs to age-related and senescence-related changes in gene expression remains elusive. To address this question, we have selected four replicative cell aging models including endothelial cells, replicated CD8+ T cells, renal proximal tubular epithelial cells, and skin fibroblasts. Further included were three organismal aging models including foreskin, mesenchymal stem cells, and CD8+ T cell populations from old and young donors. Using locked nucleic acid-based miRNA microarrays, we identified four commonly regulated miRNAs, miR-17 down-regulated in all seven; miR-19b and miR-20a, down-regulated in six models; and miR-106a down-regulated in five models. Decrease in these miRNAs correlated with increased transcript levels of some established target genes, especially the cdk inhibitor p21/CDKN1A. These results establish miRNAs as novel markers of cell aging in humans.