衰老或肿瘤：癌基因诱导的双向性

在大部分的肿瘤中都发现有癌基因的活化,癌基因的活化被认为是导致肿瘤发生的重要原因．然而,在野生型细胞内,癌基因的活化可以诱导细胞衰老,称为癌基因诱导的细胞衰老(oncogene-induced senescence, OIS),从而抑制进一步的肿瘤发生．因而,癌基因的活化具有诱导衰老或肿瘤的双向性．DNA损伤调控反应(DNA damage checkpoint response, DDR)是细胞应对DNA损伤时感应损伤,从而延迟或阻滞细胞周期进程的一种分子信号传递通路,是诱导细胞衰老的重要机制．癌基因的活化可以引发DNA损伤信号的产生,从而激活DDR,诱导细胞衰老．在DDR异常时,癌基因的激活可引发DNA的过度复制与细胞的过度增殖,并导致基因组不稳定性的积累,最终导致肿瘤发生．DDR的完整性决定了癌基因诱导的双向性．DDR在癌基因诱导中的重要作用,提示了保持和恢复DDR的完整性可以作为肿瘤预防和治疗的新方向．     

Subversion of autophagy by Kaposi's sarcoma-associated herpesvirus impairs oncogene-induced senescence

Acute oncogenic stress can activate autophagy and facilitate permanent arrest of the cell cycle through a failsafe mechanism known as oncogene-induced senescence (OIS). Kaposi's sarcoma-associated herpesvirus (KSHV) proteins are known to subvert autophagic pathways, but the link to Kaposi's sarcoma pathogenesis is unclear. We find that oncogenic assault caused by latent KSHV infection elicits DNA damage responses (DDRs) characteristic of OIS, yet infected cells display only modest levels of autophagy and fail to senesce. These aberrant responses result from the combined activities of tandemly expressed KSHV v-cyclin and v-FLIP proteins. v-Cyclin deregulates the cell cycle, triggers DDRs, and if left unchecked can promote autophagy and senescence. However, during latency v-FLIP blocks v-cyclin-induced autophagy and senescence in a manner that requires intact v-FLIP ATG3-binding domains. Together, these data reveal a coordinated viral gene expression program that usurps autophagy, blocks senescence, and facilitates the proliferation of KSHV-infected cells.     

细胞衰老与肿瘤发生

细胞衰老（cell senescence）是指细胞在信号转导作用下不可逆地脱离细胞周期并丧失增殖能力后进入的一种相对稳定的状态。细胞衰老有增殖衰老与早熟衰老两种形式：增殖衰老由端粒缩短激发的信号转导激发,与TP53/CDKN1a（p21^WAF-1/Cip1）/pRB/E2F信号通路密切相关;早熟衰老由细胞内在或外在急慢性应激信号引发,与TP53/CDKN1a（p21^WAF-1/Cip1）/pRB/E2F或CDKN2a（p16^ink4A）/pRB/E2F信号通路相关。目前研究已经证实早熟衰老是细胞在癌变过程中的天然屏障,是继DNA修复、细胞凋亡后的第三大细胞内在抗癌机制,在机体防止肿瘤形成中起重要作用。     

Two faces of p53: aging and tumor suppression

The p53 tumor suppressor protein, often termed guardian of the genome, integrates diverse physiological signals in mammalian cells. In response to stress signals, perhaps the best studied of which is the response to DNA damage, p53 becomes functionally active and triggers either a transient cell cycle arrest, cell death (apoptosis) or permanent cell cycle arrest (cellular senescence). Both apoptosis and cellular senescence are potent tumor suppressor mechanisms that irreversibly prevent damaged cells from undergoing neoplastic transformation. However, both processes can also deplete renewable tissues of proliferation-competent progenitor or stem cells. Such depletion, in turn, can compromise the structure and function of tissues, which is a hallmark of aging. Moreover, whereas apoptotic cells are by definition eliminated from tissues, senescent cells can persist, acquire altered functions, and thus alter tissue microenvironments in ways that can promote both cancer and aging phenotypes. Recent evidence suggests that increased p53 activity can, at least under some circumstances, promote organismal aging. Here, we discuss the role of p53 as a key regulator of the DNA damage responses, and discuss how p53 integrates the outcome of the DNA damage response to optimally balance tumor suppression and longevity.     

p21-Mediated nuclear retention of cyclin B1-Cdk1 in response to genotoxic stress

G2 arrest of cells suffering DNA damage in S phase is crucial to avoid their entry into mitosis, with the concomitant risks of oncogenic transformation. According to the current model, signals elicited by DNA damage prevent mitosis by inhibiting both activation and nuclear import of cyclin B1-Cdk1, a master mitotic regulator. We now show that normal human fibroblasts use additional mechanisms to block activation of cyclin B1-Cdk1. In these cells, exposure to nonrepairable DNA damage leads to nuclear accumulation of inactive cyclin B1-Cdk1 complexes. This nuclear retention, which strictly depends on association with endogenous p21, prevents activation of cyclin B1-Cdk1 by Cdc25 and Cdk-activating kinase as well as its recruitment to the centrosome. In p21-deficient normal human fibroblasts and immortal cell lines, cyclin B1 fails to accumulate in the nucleus and could be readily detected at the centrosome in response to DNA damage. Therefore, in normal cells, p21 exerts a dual role in mediating DNA damage-induced cell cycle arrest and exit before mitosis. In addition to blocking pRb phosphorylation, p21 directly prevents mitosis by inactivating and maintaining the inactive state of mitotic cyclin-Cdk complexes. This, with subsequent degradation of mitotic cyclins, further contributes to the establishment of a permanent G2 arrest.     

Telomere shortening triggers senescence of human cells through a pathway involving ATM, p53, and p21(CIP1), but not p16(INK4a)

Cellular senescence can be triggered by telomere shortening as well as a variety of stresses and signaling imbalances. We used multiparameter single-cell detection methods to investigate upstream signaling pathways and ensuing cell cycle checkpoint responses in human fibroblasts. Telomeric foci containing multiple DNA damage response factors were assembled in a subset of senescent cells and signaled through ATM to p53, upregulating p21 and causing G1 phase arrest. Inhibition of ATM expression or activity resulted in cell cycle reentry, indicating that stable arrest requires continuous signaling. ATR kinase appears to play a minor role in normal cells but in the absence of ATM elicited a delayed G2 phase arrest. These pathways do not affect expression of p16, which was upregulated in a telomere- and DNA damage-independent manner in a subset of cells. Distinct senescence programs can thus progress in parallel, resulting in mosaic cultures as well as individual cells responding to multiple signals.     

Oncogenic ras and p53 cooperate to induce cellular senescence

Oncogenic activation of the mitogen-activated protein (MAP) kinase cascade in murine fibroblasts initiates a senescence-like cell cycle arrest that depends on the ARF/p53 tumor suppressor pathway. To investigate whether p53 is sufficient to induce senescence, we introduced a conditional murine p53 allele (p53(val135)) into p53-null mouse embryonic fibroblasts and examined cell proliferation and senescence in cells expressing p53, oncogenic Ras, or both gene products. Conditional p53 activation efficiently induced a reversible cell cycle arrest but was unable to induce features of senescence. In contrast, coexpression of oncogenic ras or activated mek1 with p53 enhanced both p53 levels and activity relative to that observed for p53 alone and produced an irreversible cell cycle arrest that displayed features of cellular senescence. p19(ARF) was required for this effect, since p53(-/-) ARF(-/-) double-null cells were unable to undergo senescence following coexpression of oncogenic Ras and p53. Although the levels of exogenous p53 achieved in ARF-null cells were relatively low, the stabilizing effects of p19(ARF) on p53 could not explain the cooperation between oncogenic Ras and p53 in promoting senescence. Hence, enforced p53 expression without oncogenic ras in p53(-/-) mdm2(-/-) double-null cells produced extremely high p53 levels but did not induce senescence. Taken together, our results indicate that oncogenic activation of the MAP kinase pathway in murine fibroblasts converts p53 into a senescence inducer through both quantitative and qualitative mechanisms.     

Activation of the DNA damage response by telomere attrition: a passage to cellular senescence

Critical telomere shortening induces senescence in many normal human cell types grown in culture. Recent data have revealed that dysfunctional telomeres can resemble certain forms of DNA damage, and point to a role for DNA damage signaling in the establishment and maintenance of telomere-initiated senescence. Here, we review these new observations and highlight potential avenues of future research. We consider the identities of the key DNA damage response factors involved in senescence and discuss a model for the molecular events occurring in pre-senescent cells that ultimately lead to a permanent cell cycle arrest phenotype.     

Jailbreak: Oncogene-induced senescence and its evasion

Aberrant oncogenic signals are typically counteracted by anti-proliferative mechanisms governed principally by the p53 and Rb tumour-suppressor proteins. Apoptosis is firmly established as a potent anti-proliferative mechanism to prevent tumour growth but it is only in recent years that oncogene-induced senescence has achieved similar recognition. Senescence is defined as an irreversible cell-cycle arrest suggesting that entry of oncogene-expressing cells into this static yet viable state is permanent. However, tumours do develop and express the very same oncogenes that landed them in jail. We ask whether this is because rogue incipient cancer cells find ways to escape this imposed imprisonment or otherwise entirely avoid capture by senescence gate-keepers.     

Mitochondrial Dysfunction Contributes to Oncogene-Induced Senescence

The expression of oncogenic ras in normal human cells quickly induces an aberrant proliferation response that later is curtailed by a cell cycle arrest known as cellular senescence. Here, we show that cells expressing oncogenic ras display an increase in the mitochondrial mass, the mitochondrial DNA, and the mitochondrial production of reactive oxygen species (ROS) prior to the senescent cell cycle arrest. By the time the cells entered senescence, dysfunctional mitochondria accumulated around the nucleus. The mitochondrial dysfunction was accompanied by oxidative DNA damage, a drop in ATP levels, and the activation of AMPK. The increase in mitochondrial mass and ROS in response to oncogenic ras depended on intact p53 and Rb tumor suppression pathways. In addition, direct interference with mitochondrial functions by inhibiting the expression of the Rieske iron sulfur protein of complex III or the use of pharmacological inhibitors of the electron transport chain and oxidative phosphorylation was sufficient to trigger senescence. Taking these results together, this work suggests that mitochondrial dysfunction is an effector pathway of oncogene-induced senescence.Mitochondria are central to cell metabolism and energy production. High-energy electrons coming from the oxidation of different carbon sources such as glucose and fatty acids enter the mitochondrial electron transport chain as reduced equivalents, and their energy gradually is converted into a proton gradient. Mitochondria use this gradient to synthesize ATP that later is used for biosynthetic reactions (9, 30). Mitochondria also control decisions for life and death. Changes in mitochondrial membrane permeability lead to the release of proapoptotic mediators that can kill cells with DNA damage or activated oncogenes (16). In this way, mitochondria control one of the major tumor suppressor responses: apoptosis (27). Some oncogenes, such as RasV12, STAT5, and Bcl2, have antiapoptotic activity, and some cell types have a high apoptosis threshold. Another tumor suppressor response, called cellular senescence, serves as a fail-safe mechanism against the transforming activity of antiapoptotic oncogenes (29, 40, 43). However, currently it is unknown whether mitochondria also can play a role in oncogene-induced senescence (OIS).OIS is phenotypically similar to the senescence response triggered by short telomeres, also known as replicative senescence (6). Replicative senescence is, in essence, the consequence of a DNA damage response triggered by short telomeres (11). OIS also involves the DNA damage response (2, 15, 28), but the mechanism of DNA damage and the contribution of mitochondria to it are unclear. It has been demonstrated that mitochondria play a critical role in replicative senescence, and several mitochondrial changes, including an increase in the production of reactive oxygen species (ROS), were reported in cells with short telomeres (34, 35). Mitochondrion-derived ROS contribute to the senescent phenotype by damaging the DNA (35) and therefore amplifying the DNA damage signals originally caused by short telomeres. We reasoned that a similar amplifying mechanism involving the mitochondria could operate in cells expressing oncogenes.Here, we use Ha-RasV12, an oncogenic allele of Ha-Ras, to study the role of mitochondria in OIS. RasV12 is a very important human oncogene and was the first linked to the senescence program (43). We report that oncogenic ras induces an increase in mitochondrial mass, mitochondrial DNA, and mitochondrial superoxide production before any sign of senescent cell cycle arrest. With time, these mitochondrial changes evolved into a severe mitochondrial dysfunction characterized by a further increase in ROS production, the accumulation of depolarized mitochondria around the cell nucleus, a decrease in ATP, and the activation of AMPK. The mechanism of the increase in mitochondrial mass and ROS in response to oncogenic ras was found to be dependent on either p53 or Rb. In addition, direct interference with mitochondrial functions by downregulating the mitochondrial Rieske iron sulfur protein (RISP) or by using pharmacological inhibitors of oxidative phosphorylation induced senescence. We suggest that the senescence effector mechanism acting downstream of p53 and Rb involves mitochondrial dysfunction.     

The PAL-Mechanism of Chromosome Maintenance: Causes and Consequences

It is generally accepted that cells with extensive, un-repaired DNA damage canescape cell cycle arrest only by disabling checkpoint pathways and they usuallyperish, after several divisions, presumably due to catastrophic events on theirchromosomes. Our recently discovered PAL-mechanism opens a new perspective,that some eukaryotic cells with short chromosome ends (telomeres), usually detectedas DNA damage, can escape permanent cell cycle arrest (senescence) under specialconditions, despite having intact checkpoints and even immortalize, despite lackingtelomerase or other telomere-elongation mechanisms. Here we present the firstevidence that telomerase-lacking, senescent cells generate DNA damage (singlestranded DNA) at internal chromosomal regions, while the telomere proximal singlestranded DNA appears to be either lost or repaired. This first evidence is from thebudding yeast model system. We also discuss the possible involvement of the PALmechanismin carcinogenesis.     

Activation of the DNA Damage Response by Telomere Attrition: A Passage to Cellular Senescence

Critical telomere shortening induces senescence in many normal human cell types grown in culture. Recentdata have revealed that dysfunctional telomeres can resemble certain forms of DNA damage, and point to a role for DNA damage signaling in the establishment and maintenance of telomere-initiated senescence. Here, we review these new observations and highlight potential avenues of future research. We consider the identities of the key DNA damage response factors involved in senescence and discuss a model for the molecular events occurring in pre-senescent cells that ultimately lead to a permanent cell cycle arrest phenotype.     

Distinct roles for p107 and p130 in Rb-independent cellular senescence

Telomere attrition, DNA damage and constitutive mitogenic signaling can all trigger cellular senescence in normal cells and serve as a defense against tumor progression. Cancer cells may circumvent this cellular defense by acquiring genetic mutations in checkpoint proteins responsible for regulating permanent cell cycle arrest. A small family of tumor suppressor genes encoding the retinoblastoma susceptibility protein family (Rb, p107, p130) exerts a partially redundant control of entry into S phase of DNA replication and cellular proliferation. Here we report that activation of the p53-dependent DNA damage response has been found to accelerate senescence in human prostate cancer cells lacking a functional Rb protein. This novel form of irradiation-induced premature cellular senescence reinforces the notion that other Rb family members may compensate for loss of Rb protein in the DNA damage response pathway. Consistent with this hypothesis, depletion of p107 potently inhibits the irradiation-induced senescence observed in DU145 cells. In contrast, p130 depletion triggers a robust and unexpected form of premature senescence in unirradiated cells. The dominant effect of depleting both p107 and p130, in the absence of Rb, was a complete blockade of irradiation-induced cellular senescence. Onset of the p107-dependent senescence was temporally associated with p53-mediated stabilization of the cyclin-dependent kinase inhibitor p27 and decreases in c-myc and cks1 expression. These results indicate that p107 is required for initiation of accelerated cellular senescence in the absence of Rb and introduces the concept that p130 may be required to prevent the onset of terminal growth arrest in unstimulated prostate cancer cells lacking a functional Rb allele.     

Regulation of cellular senescence by p53.

Many normal cells respond to potentially oncogenic stimuli by undergoing cellular senescence, a state of irreversibly arrested proliferation and altered differentiated function. Cellular senescence very likely evolved to suppress tumorigenesis. In support of this idea, it is regulated by several tumor suppressor genes. At the heart of this regulation is p53. p53 is essential for the senescence response to short telomeres, DNA damage, oncogenes and supraphysiological mitogenic signals, and overexpression of certain tumor suppressor genes. Despite the well-documented central role for p53 in the senescence response, many questions remain regarding how p53 senses senescence-inducing stimuli and how it elicits the senescent phenotype.     

Phosphoinositide 3-kinase signaling overrides a G2 phase arrest checkpoint and promotes aberrant cell cycling and death of hematopoietic cells after DNA damage

DNA damage activates arrest checkpoints to halt cell cycle progression in G1 and G2 phases. These checkpoints can be overridden in hematopoietic cells by cytokines, such as erythropoietin, through the activation of a phosphoinositide 3-kinase (PI3K) signaling pathway. Here, we show that PI3K activity specifically overrides delayed mechanisms effecting permanent G1 and G2 phase arrests, but does not affect transient checkpoints arresting cells up to 10 hours after gamma-irradiation. Assessing the status of cell cycle regulators in hematopoietic cells arrested after gamma-irradiation, we show that Cdk2 activity is completely inhibited in both G1 and G2 arrested cells. Despite the absence of Cdk2 activity, cells arrested in G2 phase did retain detectable levels of Cdk1 activity in the absence of PI3K signaling. However, reactivation of PI3K promoted robust increases in both Cdk1 and Cdk2 activity in G2-arrested cells. Reactivation of Cdks was accompanied by a resumption of cell cycling, but with strikingly different effectiveness in G1 and G2 phase arrested cells. Specifically, G1-arrested cells resumed normal cell cycle progression with little loss in viability when PI3K was activated after gamma-irradiation. Conversely, PI3K activation in G2-arrested cells promoted endoreduplication and death of the entire population. These observations show that cytokine-induced PI3K signaling pathways promote Cdk activation and override permanent cell cycle arrest checkpoints in hematopoietic cells. While this activity can rescue irradiated cells from permanent G1 phase arrest, it results in aberrant cell cycling and death when activated in hematopoietic cells arrested at the G2 phase DNA damage checkpoint.     

Combined experimental and computational analysis of DNA damage signaling reveals context‐dependent roles for Erk in apoptosis and G1/S arrest after genotoxic stress

Following DNA damage, cells display complex multi‐pathway signaling dynamics that connect cell‐cycle arrest and DNA repair in G1, S, or G2/M phase with phenotypic fate decisions made between survival, cell‐cycle re‐entry and proliferation, permanent cell‐cycle arrest, or cell death. How these phenotypic fate decisions are determined remains poorly understood, but must derive from integrating genotoxic stress signals together with inputs from the local microenvironment. To investigate this in a systematic manner, we undertook a quantitative time‐resolved cell signaling and phenotypic response study in U2OS cells receiving doxorubicin‐induced DNA damage in the presence or absence of TNFα co‐treatment; we measured key nodes in a broad set of DNA damage signal transduction pathways along with apoptotic death and cell‐cycle regulatory responses. Two relational modeling approaches were then used to identify network‐level relationships between signals and cell phenotypic events: a partial least squares regression approach and a complementary new technique which we term ‘time‐interval stepwise regression.’ Taken together, the results from these analysis methods revealed complex, cytokine‐modulated inter‐relationships among multiple signaling pathways following DNA damage, and identified an unexpected context‐dependent role for Erk in both G1/S arrest and apoptotic cell death following treatment with this commonly used clinical chemotherapeutic drug.     

Differential Oncogenic Ras Signaling and Senescence in Tumor Cells

Several studies have shown that forced expression of oncogenic H-ras can induce a senescence-like permanent growth arrest in normal cells. Here we report that expression of oncogenic H-ras in human osteosarcoma U2OS cells also resulted in a senescence-like flat and enlarged cell morphology and permanent growth arrest. In contrast to normal human fibroblasts, U2OS cells were arrested independently of the p16 and ARF tumor suppressors. Treatment with a MEK inhibitor or a p38MAPK inhibitor interrupted oncogenic H-ras-induced growth arrest in U2OS cells, suggesting that activation of MAPK pathways is important. To further determine whether this process is unique to oncogenic H-ras signaling, we examined the effect of oncogenic K-ras on normal cells and human osteosarcoma cells. Similar to oncogenic H-ras, oncogenic K-ras also induced senescence in normal fibroblasts, while transforming immortalized mouse fibroblasts. However, in contrast to oncogenic H-ras, oncogenic K-ras failed to induce a permanent growth arrest in osteosarcoma U2OS cells. Additionally, cells transduced with oncogenic K-ras exhibited distinguishable cellular changes compared to those transduced with oncogenic H-ras. In summary, we report for the first time that oncogenic H-ras signaling can trigger a senescence-like growth arrest in tumor cells, independent of the p16 and ARF tumor suppressors. This result suggests that tumor cells may harbor a senescence-like program that can be activated by ras signaling. Moreover, our study uncovered a cell type-dependent differential response to oncogenic K-ras, as compared to oncogenic H-ras.     

Stable Cellular Senescence Is Associated with Persistent DDR Activation

The DNA damage response (DDR) is activated upon DNA damage generation to promote DNA repair and inhibit cell cycle progression in the presence of a lesion. Cellular senescence is a permanent cell cycle arrest characterized by persistent DDR activation. However, some reports suggest that DDR activation is a feature only of early cellular senescence that is then lost with time. This challenges the hypothesis that cellular senescence is caused by persistent DDR activation. To address this issue, we studied DDR activation dynamics in senescent cells. Here we show that normal human fibroblasts retain DDR markers months after replicative senescence establishment. Consistently, human fibroblasts from healthy aged donors display markers of DDR activation even three years in culture after entry into replicative cellular senescence. However, by extending our analyses to different human cell strains, we also observed an apparent DDR loss with time following entry into cellular senescence. This though correlates with the inability of these cell strains to survive in culture upon replicative or irradiation-induced cellular senescence. We propose a model to reconcile these results. Cell strains not suffering the prolonged in vitro culture stress retain robust DDR activation that persists for years, indicating that under physiological conditions persistent DDR is causally involved in senescence establishment and maintenance. However, cell strains unable to maintain cell viability in vitro, due to their inability to cope with prolonged cell culture-associated stress, show an only-apparent reduction in DDR foci which is in fact due to selective loss of the most damaged cells.     

The molecular control of DNA damage-induced cell death

Because of the singular importance of DNA for genetic inheritance, all organisms have evolved mechanisms to recognize and respond to DNA damage. In metazoans, cells can respond to DNA damage either by undergoing cell cycle arrest, to facilitate DNA repair, or by undergoing cell suicide. Cell death can either occur by activation of the apoptotic machinery or simply be a consequence of irreparable damage that prevents further cell division. In germ cells, mechanisms for limiting alterations to the genome are required for faithful propagation of the species whereas in somatic cells, responses to DNA damage prevent the accumulation of mutations that might lead to aberrant cell proliferation or behavior. Several of the genes that regulate cellular responses to DNA damage function as tumor suppressors. The clinical use of DNA damaging agents in the treatment of cancer can activate these tumor suppressors and exploits the cellular suicide and growth arrest mechanisms that they regulate. It appears that in some but not all types of tumors the propensity to undergo apoptosis is a critical determinant of their sensitivity to anti-cancer therapy. This review describes current understanding of the molecular control of DNA damage-induced apoptosis with particular attention to its role in tumor suppression and cancer therapy.