Irreversibility of cellular senescence: dual roles of p16(INK4a)/Rb-pathway in cell cycle control

The retinoblastoma (Rb) tumor suppressor gene product, pRb, has an established role in the implementation of cellular senescence, the state of irreversible G1 cell cycle arrest provoked by diverse oncogenic stresses. In murine cells, senescence cell cycle arrest can be reversed by subsequent inactivation of pRb, indicating that pRb is required not only for the onset of cellular senescence, but also for the maintenance of senescence program in murine cells. However, in human cells, once pRb is fully activated by p16(INK4a), senescence cell cycle arrest becomes irreversible and is no longer revoked by subsequent inactivation of pRb, suggesting that p16(INK4a)/Rb-pathway activates an alternative mechanism to irreversibly block the cell cycle in human senescent cells. Here, we discuss the molecular mechanism underlying the irreversibility of senescence cell cycle arrest and its potential towards tumor suppression

Japanese Monetary Policy during the Collapse of the Bubble Economy: A View of Policymaking under Uncertainty

Focusing on policymaking under uncertainty, we analyze the monetary policy of the Bank of Japan (BOJ) in the early 1990s, when the bubble economy collapsed. Conducting stochastic simulations with a large- scale macroeconomic model of the Japanese economy, we find that the BOJf s monetary policy at that time was essentially optimal under uncertainty about the policy multiplier. On the other hand, we also find that the BOJ's policy was not optimal under uncertainty about inflation dynamics, and that a more aggressive policy response than actually implemented would have been needed. Thus, optimal monetary policy differs greatly depending upon which type of uncertainty is emphasized. Taking into account the fact that overcoming deflation became an important issue from the latter 1990s, it is possible to argue that during the early 1990s the BOJ should have placed greater emphasis on uncertainty about inflation dynamics and implemented a more aggressive monetary policy. The result from a counterfactual simulation indicates that the inflation rate and the real growth rate would have been higher to some extent if the BOJ had implemented a more accommodative policy during the early 1990s. However, the simulation result also suggests that the effects would have been limited, and that an accommodative monetary policy itself would not have changed the overall image of the prolonged stagnation of the Japanese economy during the 1990s.Collapse of the bubble economy; Monetary policy; Uncertainty

Real-time in vivo imaging of p16Ink4a gene expression: a new approach to study senescence stress signaling in living animals

Oncogenic proliferative signals are coupled to a variety of growth inhibitory processes. In cultured primary human fibroblasts, for example, ectopic expression of oncogenic Ras or its downstream mediator initiates cellular senescence, the state of irreversible cell cycle arrest, through up-regulation of cyclin-dependent kinase (CDK) inhibitors, such as p16INK4a. To date, much of our current knowledge of how human p16INK4a gene expression is induced by oncogenic stimuli derives from studies undertaken in cultured primary cells. However, since human p16INK4a gene expression is also induced by tissue culture-imposed stress, it remains unclear whether the induction of human p16INK4a gene expression in tissue-cultured cells truly reflects an anti-cancer process or is an artifact of tissue culture-imposed stress. To eliminate any potential problems arising from tissue culture imposed stress, we have recently developed a bioluminescence imaging (BLI) system for non-invasive and real-time analysis of human p16INK4a gene expression in the context of a living animal. Here, we discuss the molecular mechanisms that direct p16INK4a gene expression in vivo and its potential for tumor suppression

The p16INK4a-RB pathway : molecular link between cellular senescence and tumor suppression

The p16INK4a tumor suppressor protein functions as an inhibitor ofCDK4andCDK6, the D-type cyclin-dependent kinases that initiate the phosphorylation of the retinoblastoma tumor suppressor protein, RB. Thus, p16INK4a has the capacity to arrest cells in the G1-phase of the cell cycle and its probable physiological role is in the implementation of irreversible growth arrest termed cellular senescence. Cellular senescence is a state of permanent growth arrest that can be induced by a variety of stresses such as DNA-damage and aberrant mitogenic signaling in human primary cells. In contrast to normal cells, the function of the p16INK4a gene or its downstream mediators is frequently deregulated in many types of human cancers, illustrating the importance of cellular senescence in tumor suppression. Here we discuss the molecular mechanisms that direct cellular senescence and reveal its potential for tumor suppression

Epstein-Barr virus LMP1 blocks p16INK4a–RB pathway by promoting nuclear export of E2F4/5

The p16INK4a–RB pathway plays a critical role in preventing inappropriate cell proliferation and is often targeted by viral oncoproteins during immortalization. Latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is often present in EBV-associated proliferative diseases and is critical for the immortalizing and transforming activity of EBV. Unlike other DNA tumor virus oncoproteins, which possess immortalizing activity, LMP1 does not bind to retinoblastoma tumor suppressor protein, but instead blocks the expression of p16INK4a tumor suppressor gene. However, it has been unclear how LMP1 represses the p16INK4a gene expression. Here, we report that LMP1 promotes the CRM1-dependent nuclear export of Ets2, which is an important transcription factor for p16INK4a gene expression, thereby reducing the level of p16INK4a expression. We further demonstrate that LMP1 also blocks the function of E2F4 and E2F5 (E2F4/5) transcription factors through promoting their nuclear export in a CRM1-dependent manner. As E2F4/5 are essential downstream mediators for a p16INK4a-induced cell cycle arrest, these results indicate that the action of LMP1 on nuclear export has two effects on the p16INK4a–RB pathway: (1) repression of p16INK4a expression and (2) blocking the downstream mediator of the p16INK4a–RB pathway. These results reveal a novel activity of LMP1 and increase an understanding of how viral oncoproteins perturb the p16INK4a–RB pathway

Crosstalk between the Rb Pathway and AKT Signaling Forms a Quiescence-Senescence Switch

SummaryCell-cycle arrest in quiescence and senescence is largely orchestrated by the retinoblastoma (Rb) tumor-suppressor pathway, but the mechanisms underlying the quiescence-senescence switch remain unclear. Here, we show that the crosstalk between the Rb-AKT-signaling pathways forms this switch by controlling the overlapping functions of FoxO3a and FoxM1 transcription factors in cultured fibroblasts. In the absence of mitogenic signals, although FoxM1 expression is repressed by the Rb pathway, FoxO3a prevents reactive oxygen species (ROS) production by maintaining SOD2 expression, leading to quiescence. However, if the Rb pathway is activated in the presence of mitogenic signals, FoxO3a is also inactivated by AKT, thus reducing SOD2 expression and consequently allowing ROS production. This situation elicits senescence through irreparable DNA damage. We demonstrate that this pathway operates in mouse liver, indicating that this machinery may contribute more broadly to tissue homeostasis in vivo

Reduction of total E2F/DP activity induces senescence-like cell cycle arrest in cancer cells lacking functional pRB and p53

E2F/DP complexes were originally identified as potent transcriptional activators required for cell proliferation. However, recent studies revised this notion by showing that inactivation of total E2F/DP activity by dominant-negative forms of E2F or DP does not prevent cellular proliferation, but rather abolishes tumor suppression pathways, such as cellular senescence. These observations suggest that blockage of total E2F/DP activity may increase the risk of cancer. Here, we provide evidence that depletion of DP by RNA interference, but not overexpression of dominant-negative form of E2F, efficiently reduces endogenous E2F/DP activity in human primary cells. Reduction of total E2F/DP activity results in a dramatic decrease in expression of many E2F target genes and causes a senescence-like cell cycle arrest. Importantly, similar results were observed in human cancer cells lacking functional p53 and pRB family proteins. These findings reveal that E2F/DP activity is indeed essential for cell proliferation and its reduction immediately provokes a senescence-like cell cycle arrest

<Abstract of Published Report>Identification of Amino Acid Residues Responsible for Difference in Substrate Specificity and Inhibitor Sensitivity Between Two Human Liver Dihydrodiol Dehydrogenase Isoenzymes by Site-directed Mutagenesis.

Human liver dihydrodiol dehydrogenase isoenzymes (DD1 and DD2), in which only seven amino acid residues are substituted, differ remarkably in specificity for steroidal substrates and inhibitor sensitivity: DD1 shows 20alpha-hydroxysteroid dehydrogenase activity and sensitivity to 1,10-phenanthroline, whereas DD2 oxidizes 3alpha-hydroxysteroids and is highly inhibited by bile acids. In the present study we performed site-directed mutagenesis of the seven residues (Thr-38, Arg-47, Leu-54, Cys-87, Val-151, Arg-170 and Gln-172) of DD1 to the corresponding residues (Val, His, Val, Ser, Met, His and Leu respectively) of DD2. Of the seven mutations, only the replacement of Leu-54 with Val produced an enzyme that had almost the same properties as DD2. No significant changes were observed in the other mutant enzymes. An additional site-directed mutagenesis of Tyr-55 of DD1 to Phe yielded an inactive protein, suggesting the catalytically important role of this residue. Thus a residue at a position before the catalytic Tyr residue might play a key role in determining the orientation of the substrates and inhibitors