The irreversible G 1 arrest in senescent human diploid fibroblasts is probably caused by inactivation of the G 1 cyclin-cyclin-dependent kinase (Cdk) complexes responsible for phosphorylation of the retinoblastoma protein (pRb). We show that the Cdk inhibitor p21 Sdi1,Cip1,Waf1, which accumulates progressively in aging cells, binds to and inactivates all cyclin E-Cdk2 complexes in senescent cells, whereas in young cells only p21-free Cdk2 complexes are active. Furthermore, the senescent-cell-cycle arrest occurs prior to the accumulation of the Cdk4-Cdk6 inhibitor p16Ink4a , suggesting that p21 may be sufficient for this event. Accordingly, cyclin D1-associated phosphorylation of pRb at Ser-780 is lacking even in newly senescent fibroblasts that have a low amount of p16. Instead, the cyclin D1-Cdk4 and cyclin D1-Cdk6 complexes in these cells are associated with an increased amount of p21, suggesting that p21 may be responsible for inactivation of both cyclin E-and cyclin D1-associated kinase activity at the early stage of senescence. Moreover, even in the late stage of senescence when p16 is high, cyclin D1-Cdk4 complexes are persistent, albeit reduced by <50% compared to young cells. We also provide new evidence that p21 may play a role in inactivation of the DNA replication factor proliferating cell nuclear antigen during early senescence. Finally, because p16 accumulates in parallel with the increases in senescence-associated ␤-Gal activity and cell volume that characterize the senescent phenotype, we suggest that p16 upregulation may be part of a differentiation program that is turned on in senescent cells. Since p21 decreases after senescence is achieved, this upregulation of p16 may be essential for maintenance of the senescent-cell-cycle arrest.Human diploid fibroblasts (HDF) have a finite proliferative lifespan, at the end of which they are unable to enter S phase in response to mitogenic stimuli. Senescent HDF are also enlarged and flattened and synthesize an altered repertoire of cell-type-specific proteins, suggesting that they have differentiated as well as aged (5). Serum-stimulated senescent HDF fail to phosphorylate the retinoblastoma protein (pRb) (51), an event that is necessary for the release of E2F transcription factors that promote the expression of late G 1 genes whose products are required for S-phase initiation and progression (39, 55). The inhibition of DNA synthesis in senescent nuclei can be overcome by factors or treatments that block or inactivate the inhibitory activity of pRb and its family of related proteins. For example, transfection or microinjection of simian virus 40 (SV40) T antigen into senescent HDF induces DNA synthesis, but this effect is lost when SV40 T antigen deficient in pRb binding is used (22). These data suggest that failure to phosphorylate pRb is a key mechanism for the cell cycle arrest of senescent cells.Phosphorylation of pRb during G 1 phase is carried out by cyclin D-Cdk4 and cyclin D-Cdk6 (cyclin D-Cdk4/6) and cyclin E-Cdk2 complexes (44,50,55). In quiesce...

Noncoding RNAs (ncRNAs) have recently been discovered to regulate mRNA transcription in trans, a role traditionally reserved for proteins. The breadth of ncRNAs as transacting transcriptional regulators and the diversity of signals to which they respond are only now becoming recognized. Here we show that human Alu RNA, transcribed from short interspersed elements (SINEs), is a transacting transcriptional repressor during the cellular heat shock response. Alu RNA blocks transcription by binding RNA polymerase II (Pol II) and entering complexes at promoters in vitro and in human cells. Transcriptional repression by Alu RNA involves two loosely structured domains that are modular, a property reminiscent of classical protein transcriptional regulators. Two other SINE RNAs, human scAlu RNA and mouse B1 RNA, also bind Pol II but do not repress transcription in vitro. These studies provide an explanation for why mouse cells harbor two major classes of SINEs, whereas human cells contain only one.

Senescent human diploid fibroblasts are unable to enter S phase in response to mitogenic stimulation. One of the key deficiencies in mitogen-stimulated senescent ceUls is their failure to phosphorylate the retinoblastoma protein, which acts as an inhibitor of entry into S phase in its unphosphorylated form. Recent data suggest that cyclin-dependent kinases (Cdks) regulated by G1 cyclins (D type and E) are responsible for the primary phosphorylation of the retinoblastoma protein prior to the G1/S boundary. Surprisingly, we found 10-to 15-fold higher constitutive amounts of both cyclin E and cyclin Dl in senescent cells compared to quiescent early-passage ceUs. Nevertheless, cyclin E-associated kinase activity in senescent cells was very low and did not increase signficantly upon mitogenic stimulation even though cyclin E-Cdk2 complexes were abundant. In contrast to earlypassage cells in late G, phase, senescent cells contained mainly underphosphorylated cyclin E and proportionally more unphosphorylated and inactive Cdk2, perhaps accounting for the low kinase activity. We also show that a majority of the Cdk2 in senescent cells, but not in early-passage cells, was complexed with cyclin Dl. Cyclin Dl-Cdk2 complexes, severalfold enriched in senescent cells, contained exclusively unphosphorylated Cdk2. Amounts of cyclin A, which ordinarily accumulates in S and G2 phases, were extremely low in stimulated senescent ceUls. We suggest that the failure to activate cyclin E-Cdk2 kinase activity in senescent cells may account for the inability ofthese cells to phosphorylate the retinoblastoma protein in late

Irreversible G 1 arrest in senescent human fibroblasts is mediated by two inhibitors of cyclin-dependent kinases (Cdks), p21Cip1/SDI1/WAF1 and p16 Ink4A . To determine the physiological and molecular events that specifically require p21, we studied senescence in human diploid fibroblasts expressing the human papillomavirus type 16 E6 oncogene, which confers low p21 levels via enhanced p53 degradation. We show that in late-passage E6 cells, high Cdk activity drives the cell cycle, but population expansion is slowed down by crisis-like events, probably owing to defective cell cycle checkpoints. At the end of lifespan, terminal-passage E6 cells exhibited several aspects of the senescent phenotype and accumulated unphosphorylated pRb and p16. However, both replication and cyclin-Cdk2 kinase activity were still not blocked, demonstrating that phenotypic and replicative senescence are uncoupled in the absence of normal p21 levels. At this stage, E6 cells also failed to upregulate p27 and inactivate cyclin-Cdk complexes in response to serum deprivation. Eventually, irreversible G 1 arrest occurred coincident with inactivation of cyclin E-Cdk2 owing to association with p21. Similarly, when p21 ؊/؊ mouse embryo fibroblasts reached the end of their lifespan, they had the appearance of senescent cells yet, in contrast to their wild-type counterparts, they were deficient in downregulating bromodeoxyuridine incorporation, cyclin E-and cyclin A-Cdk2 activity, and inhibiting pRb hyperphosphorylation. These data support the model that the critical event ensuring G 1 arrest in senescence is p21-dependent Cdk inactivation, while other aspects of senescent phenotype appear to occur independently of p21.Human diploid fibroblasts (HDFs) have a finite proliferative lifespan, at the end of which they cease irreversibly to divide and they undergo a series of phenotypic changes that distinguish senescence from quiescence (26). These phenotypic changes include altered morphology, increased cell volume, expression of a neutral senescence-associated ␤-galactosidase activity (SA-␤-Gal), and increased production of extracellular matrix degradative enzymes such as collagenase and stromelysin (26,40,61). It is now generally accepted that two inhibitors of cyclin-dependent kinases (Cdks), p16Ink4a (p16) and p21Cip1/ Waf1/Sdi1 (p21), whose amounts increase with age, have an essential role in inactivating Cdks in senescent fibroblasts (1,24,42,44,60). Cdk inactivation, in turn, allows the accumulation of unphosphorylated retinoblastoma protein (pRb) (59), a growth suppressor whose function is modulated by Cdks. Unphosphorylated pRb exerts negative regulation of cell cycle progression by forming complexes with members of the E2F transcription factor family (23, 28). In spite of their undisputed role in mediating senescence, the precise contribution of each Cdk inhibitor (CKI) is not fully established. The CKI p21 binds to and inactivates most cyclin-Cdk complexes, whereas p16 blocks cyclin D-Cdk activation by binding specifically to Cdk4 and Cdk6,...

Senescent human diploid fibroblasts (HDF) (2), which identified the human homologue of cdc2, showed that the human cdc2 gene can supply the G1/S and G2/M functions of S. pombe cdc2 in mutants of that organism. Finally, Lee et al. (8) have shown that the increase in cdc2 RNA and protein narrowly precedes the increase in DNA synthesis in serum-stimulated quiescent human diploid fibroblasts (HDF). On the other hand, there are reports that injection of rat fibroblasts with antisera to p34`1`did not block entry into S phase (9) and that mouse cells with a temperature defect in p34cdc2 arrested in G2 but not in G' at the nonpermissive temperature (10); however, both studies discuss the possibility that a G1/S function of p34CdC2 was not affected in these experiments.Cell proliferation in HDF is regulated at three levels: passage through the mitotic cycle, entry into and exit from quiescence, and cessation of proliferation owing to cellular senescence. In senescence and quiescence, the cells are rrested with G1 phase DNA contents and can be maintained in the nonreplicative state for many months (11,12). When serum stimulated, senescent HDF express many of the same cell-cycle-regulated genes as do serum-stimulated quiescent HDF-e.g., expression of c-myc, c-jun, and c-Ha-ras (13-15). However, in contrast to serum-stimulated quiescent HDF, serum-stimulated senescent HDF fail to express c-fas (15) 11012The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

RNA polymerase II (Pol II) is a well-characterized DNAdependent RNA polymerase, which has also been reported to have RNA-dependent RNA polymerase (RdRP) activity. Natural cellular RNA substrates of mammalian Pol II, however, have not been identified and the cellular function of the Pol II RdRP activity is unknown. We found that Pol II can use a non-coding RNA, B2 RNA, as both a substrate and a template for its RdRP activity. Pol II extends B2 RNA by 18 nt on its 3 0 -end in an internally templated reaction. The RNA product resulting from extension of B2 RNA by the Pol II RdRP can be removed from Pol II by a factor present in nuclear extracts. Treatment of cells with a-amanitin or actinomycin D revealed that extension of B2 RNA by Pol II destabilizes the RNA. Our studies provide compelling evidence that mammalian Pol II acts as an RdRP to control the stability of a cellular RNA by extending its 3 0 -end.

At eukaryotic promoters, multi-faceted protein-protein and protein-DNA interactions can result in synergistic transcriptional activation. NFAT and AP-1 proteins induce interleukin-2 (IL-2) transcription in stimulated T cells, but the contributions of individual members of these activator families to synergistically activating IL-2 transcription is not known. To investigate the combinatorial regulation of IL-2 transcription we tested the ability of different combinations of NFATc2, NFATc1, cJun, and cFos to synergistically activate transcription from the IL-2 promoter. We found that NFATc2 and cJun are exclusive in their ability to synergistically activate human IL-2 transcription. Protein-protein interaction assays revealed that in the absence of DNA, NFATc2, but not NFATc1, bound directly to cJun/cJun dimers, but not to cFos/cJun heterodimers. A region of NFATc2 C-terminal of the DNA binding domain was necessary and sufficient for interaction with cJun in the absence of DNA, and this same region of NFATc2 was required for the synergistic activation of IL-2 transcription in T cells. Moreover, expression of this C-terminal region of NFATc2 specifically repressed the synergistic activation of IL-2 transcription. These studies show that a previously unidentified interaction between human NFATc2 and cJun is necessary for synergistic activation of IL-2 transcription in T cells.

Transcription of interleukin-2 (IL-2), a pivotal cytokine in the mammalian immune response, is induced by NFAT and AP-1 transcriptional activators in stimulated T cells. NFATc2 and cJun drive high levels of synergistic human IL-2 transcription, which requires a unique interaction between the C-terminal activation domain of NFATc2 and cJun homodimers. Here we studied the mechanism by which this interaction contributes to synergistic activation of IL-2 transcription. We found that NFATc2 can recruit cJun homodimers to the −45 NFAT element, which lacks a neighboring AP-1 site. The bZip domain of cJun is sufficient to interact with the C-terminal activation domain of NFATc2 in the absence of DNA and this interaction is inhibited by AP-1 DNA. When the −45 NFAT site was replaced by either a NFAT/AP-1 composite site or a single AP-1 site the specificity for cJun homodimers in synergistically activating IL-2 transcription was lost, and cJun/cFos heterodimers strongly activated transcription. These studies support a model in which IL-2 transcriptional synergy is mediated by the unique recruitment of a cJun homodimer to the −45 NFAT site by NFATc2, where it acts as a co-activator for IL-2 transcription.