当前位置: 首页 > 期刊 > 《新英格兰医药杂志》 > 2006年第10期 > 正文
编号:11332668
Oncogene-Induced Cell Senescence — Halting on the Road to Cancer
http://www.100md.com 《新英格兰医药杂志》
     In many tissues, there are numerous small and inconspicuous neoplastic lesions that rarely become overt cancers. In these small lesions, clonality and oncogenic mutations have been identified. These lesions include established benign tumors such as melanocytic nevi,1 some that were previously regarded to be of a "reactive" nature,2 and even groups of cells that are histologically only marginally abnormal.3 Once they have grown to a certain size, such lesions stop growing appreciably and do not become more aggressive over many years or even decades. For instance, few moles, which are benign tumors of cutaneous melanocytes, grow larger than 1 cm, and fewer than 1 per 1000 ever progresses to melanoma.

    Several factors may account for the biologic indolence of these small neoplasms and their failure to grow. Progression to a malignant phenotype requires multiple oncogenic mutations, which are rare occurrences. In addition, lesions without the ability to induce vascular stroma cannot grow bigger than a few cubic millimeters. Moreover, apoptotic cell death may be triggered by oncogene-driven cellular proliferation, either directly4,5 or by means of the activation of nearby natural killer cells and other immune cells.6

    Other mechanisms must play a role. Additional rare mutations are required in order for the cells to transform to a fully malignant phenotype, but this does not explain the cessation of growth of the initial lesion. The absence of stroma induction cannot prevent the growth of flat-surface neoplasms. Apoptotic cell death would need to be closely coordinated with proliferation to maintain a lesion with stable or nearly stable size. In time, a slight increase in cell death would annihilate the lesion, whereas a small deficit in apoptosis would result in its continued growth.

    A related phenomenon that has long puzzled pathologists is the absence of mitotic figures in many benign tumors such as melanocytic nevi and lipomas. Like cancers and minute neoplastic lesions, benign tumors are clonal proliferations of cells harboring one or several oncogenic mutations. Thus, at some stage, the oncogene-driven mitogenic signaling in these benign tumors must cease to evoke a proliferative response.

    The elucidation of the causes of these phenomena will have immediate clinical importance. Defects in the mechanisms that block proliferation of early neoplastic cells and prevent neoplastic progression to cancer are likely to result in susceptibility to cancer. Even in malignant tumors, many tumor cells exit the proliferative pool, and this incompletely understood phenomenon has an obvious effect on cancer kinetics. Investigations of the effects of oncogene-driven mitogenic signaling may have diagnostic and prognostic applications, may lead to a better understanding of the mechanisms halting neoplastic growth, and may identify targets for new strategies of cancer prevention and treatment.

    Oncogene-Induced Proliferative Arrest

    Recent work has revealed an intriguing mechanism contributing to the cessation of growth of premalignant or benign neoplasms; its role in the prevention of overt cancer is probably substantial. The first indication of this mechanism came from in vitro studies, which revealed that under certain circumstances, oncogenic signaling can elicit, paradoxically, a potent growth-arrest response.7 This arrested growth is associated with a change in a cellular phenotype that is generally referred to as "senescent." These in vitro findings quickly led to speculation about possible in vivo correlates that might provide protection against cancer and account for the phenomenon of proliferative arrest in benign tumors.8,9,10,11,12 It has proved difficult to obtain direct evidence of this phenomenon in laboratory animals and humans. However, recent work13,14,15,16 has uncovered substantial support for the notion that oncogene-induced senescence is an in vivo mechanism that contributes to protection against cancer.

    Aberrant Proliferative Signaling and Disruption of Cell-Cycle Checkpoints

    The structural and functional stability of the tissues of multicellular organisms requires precise regulation of cellular proliferation and cell death. To avert the untoward effects of excessive cellular proliferation, a complex machinery of antiproliferative signaling allows proliferation only under particular circumstances. Specific cell-cycle checkpoints are in place to preserve genomic integrity and cell survival. Cellular differentiation and apoptotic cell death are similarly subject to strict regulation. When the homeostatic regulation of cell proliferation and cell loss is disrupted or overwhelmed, cells may enter and progress on the road to cancer. Oncogenic mutations, the root cause of cancer, generally result in enhanced growth-promoting signals, disrupted antiproliferative signals, or defective proapoptotic signaling.

    Telomeres, Replicative Senescence, and Disease

    In vitro, nontransformed cells spontaneously stop proliferating after a certain number of cell divisions. This phenomenon is called the Hayflick limit.17 Cultured cells that reach the Hayflick limit become large and flat, are often vacuolated, and commonly activate senescence-associated acidic -galactosidase, which has recently been suggested to correspond to lysosomal -D-galactosidase.18,19 Senescence-associated acidic -galactosidase activity can be visualized by means of a cytochemical reaction resulting in blue precipitate (Figure 1A) and is a widely used marker of cellular senescence and other types of cellular stress.19 Such altered cells that have lost the ability to proliferate are senescent. So-called senescence-associated heterochromatin foci, which are stretches of transcriptionally silenced DNA associated with a specific modification of histones, are also characteristic of senescent cells.20

    Figure 1. Senescence in Vitro and in Vivo.

    Panel A shows primary human fibroblasts cultured in vitro and subjected to senescence-associated acidic -galactosidase staining, resulting in a blue precipitate. Young cells (left) proliferate, are small, and contain few cells that are positive for senescence-associated acidic -galactosidase. In contrast, cells that have reached the end of their life span typically appear flat and large. They are often multinucleated and are positive for senescence-associated acidic -galactosidase. Panel B shows histologic sections of a human nevus (all images from the same lesion). Melan A (brown) identifies the nevus cells. Proliferation marker Ki-67 (brown) is not identified in the intradermal nevus cells that are positive for Melan A. Some basal epidermal keratinocytes are positive. The p16INK4A antibody detects p16INK4A (brown), with positive results in some nevus cells, but not in others. For senescence-associated acidic -galactosidase staining, frozen sections were used. Blue areas in the dermis represent groups of nevus cells; this is especially evident in the combined staining with hematoxylin and eosin and senescence-associated acidic -galactosidase.

    The Hayflick limit results from the fact that at each round of DNA replication, telomeres become slightly shorter and are eventually dysfunctional.21 When they reach a critical minimal length, a DNA damage response is triggered. This response is associated with chromosomal instability, senescence, and loss of cell viability. A causal role for telomeres in senescence became evident from the discovery of telomerase, a ribonucleoprotein that elongates telomeres, thereby halting or even reversing the progressive shortening of the telomeres of proliferating cells.22,23 Telomerase activity allows various cell types to proliferate beyond the usual maximum number of cellular divisions.

    Cellular senescence was originally defined according to in vitro observations, but evidence is accumulating that replicative (telomere-associated) senescence limits the regenerative capacity of tissues in intact organisms as well; this limitation plays crucial roles in aging and in various proliferative diseases, including cancer. Cells with a senescent phenotype accumulate in the skin of elderly people,19 and the regenerative hyperproliferation of hepatocytes in cirrhosis results in the emergence of cells with features of senescence, including telomere shortening and senescence-associated acidic -galactosidase activity.24 Presumably, these hepatic cells have exhausted their proliferative capacity. In line with this notion, liver regeneration is impaired after partial hepatectomy in telomerase-deficient mice.25

    Telomere shortening probably constitutes a generic way to avoid the growth of cancer, since it emerges whenever a cell has used up a maximum number of "allowed" divisions. Indeed, proliferation-associated telomere shortening would make cancer a self-limiting disease, but for the inappropriate activation of telomerase26 or an alternative mechanism that similarly results in telomere lengthening.27 Whereas short telomeres can suppress tumorigenesis in mice,28 forced expression of telomerase contributes to malignant transformation of primary cells in vitro in the presence of cooperating oncogenes.29 Collectively, these findings show a strong link between the loss of telomere-associated senescence and cancer.

    Other Mechanisms of Cellular Senescence

    Cellular senescence involves much more than dysfunctional telomeres. In addition to telomere exhaustion, cellular senescence can be triggered by various cellular stresses, including DNA damage; by most artificial culture conditions; and by unscheduled oncogene activation. Approximately 20 years ago, it was noted that in untransformed fibroblasts, an activated mutant of the RAS gene induced the arrest of cell growth rather than oncogenic transformation.30,31 However, if the cells already harbored certain oncogenic mutations, the same mutant RAS gene contributed to oncogenic transformation.31 It took more than a decade for this paradox to be understood.

    In 1997, Serrano and colleagues noticed that the type of proliferative arrest elicited in young diploid fibroblasts by mutant RAS exhibited many features of replicative (telomere-associated) cellular senescence, including stable maintenance of cell-cycle arrest.7 Thus, it appeared that senescence could be induced in nonmalignant cells "prematurely" — that is, before telomeric shortening could account for it.7 Such "premature senescence," as the phenomenon came to be called, met with much excitement because it showed that a hitherto unknown mechanism may abrogate the emergence of cancer. Premature, or oncogene-induced, senescence is totally or largely irreversible, unlike the normal reversible departure from the cell cycle that results from, for example, growth-factor depletion. It also differs from the growth arrest that accompanies normal cellular differentiation, which is thought to depend on physiological cues.

    Oncogene-induced senescence is essentially different from these normal processes in that it is triggered by unscheduled signaling within the cell by the protein products of oncogenes and is accompanied by the activation of a tumor-suppressor network. Indeed, in cultured cells, premature senescence is brought about by the activation of a set of tumor-suppressor genes that are often inactivated in human cancer: INK4A (inhibitor of cyclin-dependent kinase 4), ARF (alternative reading frame), p53, and RB (the retinoblastoma tumor-suppressor gene). The INK4A protein p16INK4A inhibits the activation of cyclin-dependent kinases (CDKs). These enzymes stimulate progression of the cell cycle by phosphorylating the RB protein (pRB). As a result of the action of p16INK4A, however, unphosphorylated pRB remains tightly bound to its major effectors, called E2F transcription factors, thereby preventing them from stimulating replication of DNA. In this way, p16INK4A blocks entry of the cell into the S phase of the cell cycle.

    The principle that emerges from these observations is that oncogene-induced senescence involves the activation of a set of well-known tumor suppressors. Indeed, various cancer-susceptibility syndromes can be directly linked to the same genes that become activated in the senescence response. For example, inherited susceptibility to melanoma can result from germ-line mutations in the genes encoding p16,32,33 CDK4,34 or the p53 activator p14ARF.35,36 In vitro, functional disruption of ARF, p53, or pRB-family proteins abrogates the senescence response, resulting in unlimited cell proliferation and sometimes even oncogenic transformation.37,38,39,40,41 The current thinking is that inactivation of the p53 and pRB tumor-suppressor pathways is a sine qua non for tumorigenesis in humans.42 For this reason, oncogene-induced senescence has become the subject of intense research in various contexts, including genome-wide functional screens to identify new human oncogenes and tumor-suppressor genes.43,44,45

    Skeptics of the idea that oncogene-induced cell senescence is a potential anticancer mechanism have correctly pointed out the many differences between in vitro and in vivo conditions.46 For example, the microenvironment (in particular, the relatively high oxygen tension) of cultured cells, which differs markedly from the in vivo microenvironment, can cause a phenomenon called "culture stress," and this might be an important confounder.

    Recently, however, several investigations that used various ways to transmit hyperproliferative signals to target cells in animals or unmanipulated intact human tissues have provided substantial support for the notion that oncogene-induced senescence is a physiologic mechanism for protection against cancer.13,14,15,16,47 Four of these studies were performed on the basis of transgenic mouse models. Serrano and colleagues15 used a "knock-in" mouse model in which oncogenic RAS was expressed at a physiologic level.48 Unexpectedly, most mutant RAS-expressing cells in these mice remained normal for long periods, with no apparent defects in differentiation or signs of unscheduled proliferation. Lung adenomas did emerge, but only infrequently and after a long latency period. In these adenomas, a low proliferative index was associated with elevated senescence-associated acidic -galactosidase activity and increased expression of a set of newly identified senescence markers.15 Adenocarcinomas also emerged at low frequency; they had considerable proliferative activity and were negative for all senescence markers. Schmitt and colleagues13 studied RAS-driven mouse T-cell lymphomas that entered senescence after drug therapy, when apoptosis was blocked. The senescence response was dependent on the enzyme catalyzing the specific histone modification of the senescence-associated heterochromatin foci mentioned above. Pandolfi and colleagues14 found that cell senescence can be triggered in vivo not only by oncogene activation, but also by inactivation of tumor suppressors. Loss of the tumor-suppressor PTEN (phosphatase and tensin homologue), an enzyme that participates in the cessation of cell division and apoptosis, was associated with the development of prostate tumors that were characterized by activation of senescence-associated tumor suppressors, including the p53 gene. The activation of p53 appeared to be causally involved, because disruption of p53 resulted in the emergence of aggressive prostatic adenocarcinomas. Lazzerini Denchi et al.47 reported a similar activation of senescence, including induction of p16INK4A and p19ARF, in pituitary gland tumors that were driven by forced expression of E2F3 (one of the growth-promoting E2Fs released on phosphorylation of pRB). The first direct evidence of cellular senescence in a growth-arrested human neoplasm was reported for the melanocytic nevus.16

    Nevi, Melanomas, p16INK4A, and Senescence

    In humans, cutaneous melanocytes reside largely in the epidermis and hair follicles. They are long-lived cells, despite exposure to potentially mutagenic insults such as ultraviolet radiation. Melanocytic nevi are exceedingly common and generally harbor an activating BRAFE600 mutation or, less commonly, an NRAS or HRAS mutation.1,49,50 The BRAF mutation is also common in melanomas, where its silencing eliminates the transformed state.51,52 Indeed, mutant BRAF has attracted much attention as a potential therapeutic agent in melanoma and other cancers.53 However, in spite of the activation of a MAP kinase pathway (RAS–RAF–MEK–ERK) that in principle mediates a potent proliferative signal, benign nevi eventually lose all proliferative activity, and their growth remains arrested for decades, until they gradually disappear.54,55

    Michaloglou and coworkers16 showed that overexpression of BRAFE600 in cultured human melanocytes causes growth arrest. Furthermore, they observed that human congenital nevi invariably activate senescence-associated acidic -galactosidase, in addition to p16INK4A, which was reported previously by others (Figure 1B).56,57 Thus, human melanocytic nevi that have reached the end of their growth phase display the four established hallmarks of oncogene-induced cell senescence: expression of an activated oncogene (BRAFE600), stable and total or near-total proliferative arrest, up-regulated levels of a tumor suppressor (p16INK4A), and emergence of senescence-associated acidic -galactosidase. Fluorescence in situ hybridization analysis of telomeres in tissue sections revealed no significant difference in telomere fluorescence between congenital nevi and surrounding tissues; this finding was in line with previous observations regarding common acquired nevi and Spitz nevi.58 These results, which recently received independent confirmation in a study by Bennett et al.,59 favor an active oncogene-driven senescence process (Figure 2), rather than senescence triggered by exhaustion of replicative potential resulting from telomere shortening. The fact that nevi arise at all indicates that the senescence response takes time to develop, allowing an initial proliferative phase, but eventually producing a benign tumor with stable growth arrest.

    Figure 2. A Working Model for Tumor Progression.

    When it acquires an oncogenic mutation or mutations, a nonmalignant cell may engage in three types of response. An antiproliferative response can be activated, leading to either programmed cell death (apoptosis) or senescence. Alternatively, in the absence of an immediate response, the mutation-driven cell proliferation may produce a lesion. At this stage, both apoptosis and senescence programs might be activated or gain the upper hand, resulting in cell death or senescence. In the absence of appropriate defense mechanisms, continued growth with additional genetic events may lead to a malignant lesion. If cells undergo a senescence response, they may do so for decades, which appears to be the case for melanocytic nevi. Cells may infrequently escape from such a senescent state and undergo malignant transformation.

    The elevation of p16INK4A levels does not appear to be a strict prerequisite for senescence in human nevi, since varying numbers of nevus cells that are negative for p16INK4A occur among those that are positive.16 This finding is remarkable, because most nevus cells, regardless of p16INK4A immunoreactivity, are devoid of any proliferative activity. This result seems to indicate that p16INK4A levels are not linked to the arrest of nevus growth. The induction of senescence by means of physiologic levels of BRAFE600, irrespective of p16INK4A status, can be recapitulated in vitro, at least in cultured nonmalignant human fibroblasts.16 The in vivo observations are in line with the fact that the growth arrest of nevi also occurs in patients with the INK4A-Leiden dysplastic nevus syndrome, who are homozygous for p16INK4A deficiency.32 However, the nevi in these patients are more numerous and larger than those in persons with normal p16INK4A levels; this indicates that in some way, p16INK4A does limit the proliferative potential of melanocytic nevi. This phenomenon might reflect a contribution of p16INK4A at another level — for example, to the maintenance of growth arrest60 — thereby collaborating with other factors that block proliferation in response to senescence signals. Various mouse models also support an important role for p16INK4A in melanomagenesis.61,62,63,64

    A causal role of BRAF in the development of nevi is further supported by observations in zebrafish that carry a BRAFE600 transgene.65 The expression of mutant (but not wild-type) BRAF resulted in the formation of large melanocytic patches that were called "fish nevi." Of note, BRAFE600 triggered the development of invasive melanoma only in the context of p53 deficiency, suggesting that BRAFE600 alone is insufficient to drive oncogenic transformation. Other model systems also suggest that additional molecular defects are required for tumorigenesis mediated by BRAFE600.66

    Telomeric Insufficiency, DNA Damage, and Oncogenic Stress

    Two recent reports described activation of the DNA damage response in small and early neoplastic human lesions of various types, including dysplastic melanocytic nevi.4,5 This observation provides an interesting link to the induction of senescence, since it has long been known that a senescence response also can result from DNA double-strand breaks.67 Collectively, these observations suggest that early in tumorigenesis (before genomic instability emerges and results in multiple additional genetic aberrations), mammalian cells respond to inappropriate mitogenic signaling, DNA-replication stress, or telomere dysfunction by activating protective cellular networks that abrogate cancer progression (Figure 3).

    Figure 3. Types of Stress That Activate Tumor-Suppressor Networks.

    Various stresses lead to activation of the two major established tumor-suppressor networks. Cells raise the levels of the CDK inhibitor p16INK4A. As a result, the retinoblastoma tumor-suppressor protein (pRB) accumulates in its hypophosphorylated, active state. In this form, pRB binds to various downstream effectors, including E2F transcription factors, which are thus unable to activate target genes required for DNA replication. In contrast, pRB–E2F complexes repress transcription, which is associated with the nuclear accumulation of senescence-associated heterochromatin foci. The second major tumor-suppressor pathway activated by stress is the p53 network. Critical targets for this transcription factor include antiproliferative genes (such as p21) and various proapoptotic genes. The genetic context dictates the outcome of p53 activation. For example, abundant levels of the survival factors BCL2 and Slug (found in melanocytes) tip the balance in favor of senescence.

    Senescence and Apoptosis

    Why has the oncogene-induced senescence response emerged in evolution? What advantage might it confer when there is an effective alternative: oncogene-induced apoptosis? Perhaps there are evolutionary pressures to preserve cutaneous melanocytes, which are exposed to mutagenic influences, yet are long-lived and presumably cannot be replaced in large numbers. A response to oncogenic stress that blocks proliferation (and the resultant oncogenic threat) but allows the cell to live on and perform its physiologic function could thus be beneficial. Normal skin melanocytes produce abundant levels of the antiapoptotic protein BCL2, which promotes melanocyte survival.68 Senescent fibroblasts resist apoptosis and also express high BCL2 levels, and69 mouse lymphomas that express BCL2 undergo drug-induced senescence.70 In addition to BCL2, melanocytes express Slug,71 which can protect cells from p53-dependent apoptosis.72 Mutations in the p53 gene itself are uncommon in melanoma.73 Thus, the choice between a senescence response and apoptosis is probably decided by the genetic makeup of a cell, with a primary role for the relative abundance of proapoptotic and antiapoptotic factors such as BCL2 and Slug (Figure 3). The choice probably also depends on the oncogenic trigger. For example, in contrast to signaling oncogenes such as RAS, the MYC oncogene product at elevated levels lowers the apoptotic threshold.74

    There may be a dark side to the senescence response. Growing evidence shows that its beneficial anticancer effect may occur at the cost of a gradual accumulation of long-lived senescent cells, the negative effects of which are manifested with advancing age.75,76 A study of polymorphisms of codon 72 in human p53 showed that the best cancer protection is conferred by alleles associated with early emergence of symptoms of aging and decreased longevity.77 This p53-dependent link between tumor protection and reduced life span can be reproduced in transgenic mice.78 It is ironic that senescence-related diseases include many of the common types of cancer. Senescent stromal cells might contribute to the malignant transformation of nearby epithelial cells by secreting matrix metalloproteinases and inflammatory cytokines that stimulate proliferation of preneoplastic epithelial cells.79 Although in principle the senescence response promotes survival by providing protection against cancer, it could interfere with long-term survival by promoting the accumulation of senescent cells.

    Future Prospects

    Despite recent progress, many questions remain. How common is oncogene-induced senescence? Is it part of our daily life? Circumstantial findings suggest that it may be. Some tissues of the body harbor many minute neoplastic lesions that do not grow appreciably, despite the presence of activating mutations in oncogenes.80,81 Additional work will be required to reveal the contribution of oncogene-induced senescence to the cessation of neoplastic progression in such lesions. This contribution is probably substantial. A prime challenge is to identify more and better-defined markers of in vivo senescence.

    Could germ-line mutations or polymorphisms that influence oncogene-induced senescence play a role in the risk of cancer? The dysplastic nevus syndrome caused by the germ-line INK4A Leiden mutation is an example. There might well be more such syndromes.

    From a therapeutic perspective, studies of mouse lymphomas13 and human breast cancer82 show that malignant transformation may not completely abrogate the ability to mount a senescence response after cytotoxic therapy. However, the abrogation of apoptosis, an intrinsic part of the senescence response, may enhance tumor-cell survival during therapy. A transient or incomplete therapy-induced senescence response could cause relapse of the tumor. Along these lines, acute inactivation of tumor-suppressor genes can abrogate oncogene-induced senescence in vitro,60,83,84 but it remains to be determined whether this represents a primary mechanism for the transformation of a nevus into melanoma. The clinical implications of all these findings will be the focus of intense investigation in the years to come. Oncogene-induced senescence has wide ramifications for our understanding of cancer risk, cancer kinetics, and cancer treatment.

    Supported by grants from the Dutch Cancer Society, the Netherlands Organization for Scientific Research (to Dr. Peeper), and the European Molecular Biology Organization Young Investigator Program (to Dr. Peeper).

    No potential conflict of interest relevant to this article was reported.

    We are indebted to Dr. C. Michaloglou and to Dr. R. van Doorn for helpful contributions.

    Source Information

    From the Department of Pathology, Vrije University Medical Center (W.J.M.), and the Division of Molecular Genetics, Netherlands Cancer Institute (D.S.P.) — both in Amsterdam.

    Address reprint requests to Dr. Mooi at the Department of Pathology, Vrije University Medical Center, De Boelelaan 1117, 1081 HV Amsterdam, the Netherlands, or at wj.mooi@vumc.nl.

    References

    Pollock PM, Harper UL, Hansen KS, et al. High frequency of BRAF mutations in nevi. Nat Genet 2003;33:19-20.

    Vanni R, Fletcher CD, Sciot R, et al. Cytogenetic evidence of clonality in cutaneous benign fibrous histiocytomas: a report of the CHAMP study group. Histopathology 2000;37:212-217.

    Jonason AS, Kunala S, Price GJ, et al. Frequent clones of p53-mutated keratinocytes in normal human skin. Proc Natl Acad Sci U S A 1996;93:14025-14029.

    Bartkova J, Horejsi Z, Koed K, et al. DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis. Nature 2005;434:864-870.

    Gorgoulis VG, Vassiliou LV, Karakaidos P, et al. Activation of the DNA damage checkpoint and genomic instability in human precancerous lesions. Nature 2005;434:907-913.

    Gasser S, Orsulic S, Brown EJ, Raulet DH. The DNA damage pathway regulates innate immune system ligands of the NKG2D receptor. Nature 2005;436:1186-1190.

    Serrano M, Lin AW, McCurrach ME, Beach D, Lowe SW. Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a. Cell 1997;88:593-602.

    Bennett DC. Human melanocyte senescence and melanoma susceptibility genes. Oncogene 2003;22:3063-3069.

    Mooi WJ, Peeper DS. Pathogenesis of melanocytic naevi: growth arrest linked with cellular senescence? Histopathology 2002;41:Suppl 2:139-143.

    Campisi J. Cellular senescence as a tumor-suppressor mechanism. Trends Cell Biol 2001;11:S27-S31.

    Bringold F, Serrano M. Tumor suppressors and oncogenes in cellular senescence. Exp Gerontol 2000;35:317-329.

    Lowe SW, Sherr CJ. Tumor suppression by Ink4a-Arf: progress and puzzles. Curr Opin Genet Dev 2003;13:77-83.

    Braig M, Lee S, Loddenkemper C, et al. Oncogene-induced senescence as an initial barrier in lymphoma development. Nature 2005;436:660-665.

    Chen Z, Trotman LC, Shaffer D, et al. Crucial role of p53-dependent cellular senescence in suppression of Pten-deficient tumorigenesis. Nature 2005;436:725-730.

    Collado M, Gil J, Efeyan A, et al. Tumour biology: senescence in premalignant tumours. Nature 2005;436:642-642.

    Michaloglou C, Vredeveld LC, Soengas MS, et al. BRAFE600-associated senescence-like cell cycle arrest of human naevi. Nature 2005;436:720-724.

    Hayflick L. The limited in vitro lifetime of human diploid cell strains. Exp Cell Res 1965;37:614-636.

    Lee BY, Han JA, Im JS, et al. Senescence-associated beta-galactosidase is lysosomal beta-galactosidase. Aging Cell 2006;5:187-195.

    Dimri GP, Lee X, Basile G, et al. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci U S A 1995;92:9363-9367.

    Narita M, Nunez S, Heard E, et al. Rb-mediated heterochromatin formation and silencing of E2F target genes during cellular senescence. Cell 2003;113:703-716.

    Shay JW, Wright WE. Hayflick, his limit, and cellular ageing. Nat Rev Mol Cell Biol 2000;1:72-76.

    Harley CB, Futcher AB, Greider CW. Telomeres shorten during ageing of human fibroblasts. Nature 1990;345:458-460.

    Meyerson M, Counter CM, Eaton EN, et al. hEST2, the putative human telomerase catalytic subunit gene, is up-regulated in tumor cells and during immortalization. Cell 1997;90:785-795.

    Wiemann SU, Satyanarayana A, Tsahuridu M, et al. Hepatocyte telomere shortening and senescence are general markers of human liver cirrhosis. FASEB J 2002;16:935-942.

    Satyanarayana A, Wiemann SU, Buer J, et al. Telomere shortening impairs organ regeneration by inhibiting cell cycle re-entry of a subpopulation of cells. EMBO J 2003;22:4003-4013.

    Counter CM, Hirte HW, Bacchetti S, Harley CB. Telomerase activity in human ovarian carcinoma. Proc Natl Acad Sci U S A 1994;91:2900-2904.

    Muntoni A, Reddel RR. The first molecular details of ALT in human tumor cells. Hum Mol Genet 2005;14:Spec No. 2:R191-T196.

    Gonzalez-Suarez E, Samper E, Flores JM, Blasco MA. Telomerase-deficient mice with short telomeres are resistant to skin tumorigenesis. Nat Genet 2000;26:114-117.

    Hahn WC, Counter CM, Lundberg AS, Beijersbergen RL, Brooks MW, Weinberg RA. Creation of human tumour cells with defined genetic elements. Nature 1999;400:464-468.

    Franza BR Jr, Maruyama K, Garrels JI, Ruley HE. In vitro establishment is not a sufficient prerequisite for transformation by activated ras oncogenes. Cell 1986;44:409-418.

    Land H, Parada LF, Weinberg RA. Tumorigenic conversion of primary embryo fibroblasts requires at least two cooperating oncogenes. Nature 1983;304:596-602.

    Gruis NA, van der Velden PA, Sandkuijl LA, et al. Homozygotes for CDKN2 (p16) germline mutation in Dutch familial melanoma kindreds. Nat Genet 1995;10:351-353.

    Kamb A, Gruis NA, Weaver-Feldhaus J, et al. A cell cycle regulator potentially involved in genesis of many tumor types. Science 1994;264:436-440.

    Wolfel T, Hauer M, Schneider J, et al. A p16INK4a-insensitive CDK4 mutant targeted by cytolytic T lymphocytes in a human melanoma. Science 1995;269:1281-1284.

    Randerson-Moor JA, Harland M, Williams S, et al. A germline deletion of p14(ARF) but not CDKN2A in a melanoma-neural system tumour syndrome family. Hum Mol Genet 2001;10:55-62.

    Rizos H, Puig S, Badenas C, et al. A melanoma-associated germline mutation in exon 1beta inactivates p14ARF. Oncogene 2001;20:5543-5547.

    Palmero I, Pantoja C, Serrano M. p19ARF links the tumour suppressor p53 to Ras. Nature 1998;395:125-126.

    Tanaka N, Ishihara M, Kitagawa M, et al. Cellular commitment to oncogene-induced transformation or apoptosis is dependent on the transcription factor IRF-Cell 1994;77:829-39.

    Peeper DS, Dannenberg JH, Douma S, te Riele H, Bernards R. Escape from premature senescence is not sufficient for oncogenic transformation by Ras. Nat Cell Biol 2001;3:198-203.

    Dannenberg JH, van Rossum A, Schuijff L, te Riele H. Ablation of the retinoblastoma gene family deregulates G(1) control causing immortalization and increased cell turnover under growth-restricting conditions. Genes Dev 2000;14:3051-3064.

    Sage J, Mulligan GJ, Attardi LD, et al. Targeted disruption of the three Rb-related genes leads to loss of G(1) control and immortalization. Genes Dev 2000;14:3037-3050.

    Sherr CJ, McCormick F. The RB and p53 pathways in cancer. Cancer Cell 2002;2:103-112.

    Berns K, Hijmans EM, Mullenders J, et al. A large-scale RNAi screen in human cells identifies new components of the p53 pathway. Nature 2004;428:431-437.

    Peeper DS, Shvarts A, Brummelkamp T, et al. A functional screen identifies hDRIL1 as an oncogene that rescues RAS-induced senescence. Nat Cell Biol 2002;4:148-153.

    Rowland BD, Bernards R, Peeper DS. The KLF4 tumour suppressor is a transcriptional repressor of p53 that acts as a context-dependent oncogene. Nat Cell Biol 2005;7:1074-1082.

    Sherr CJ, DePinho RA. Cellular senescence: mitotic clock or culture shock? Cell 2000;102:407-410.

    Lazzerini Denchi E, Attwooll C, Pasini D, Helin K. Deregulated E2F activity induces hyperplasia and senescence-like features in the mouse pituitary gland. Mol Cell Biol 2005;25:2660-2672.

    Guerra C, Mijimolle N, Dhawahir A, et al. Tumor induction by an endogenous K-ras oncogene is highly dependent on cellular context. Cancer Cell 2003;4:111-120.

    Saldanha G, Purnell D, Fletcher A, Potter L, Gillies A, Pringle JH. High BRAF mutation frequency does not characterize all melanocytic tumor types. Int J Cancer 2004;111:705-710.

    Bastian BC, LeBoit PE, Pinkel D. Mutations and copy number increase of HRAS in Spitz nevi with distinctive histopathological features. Am J Pathol 2000;157:967-972.

    Hingorani SR, Jacobetz MA, Robertson GP, Herlyn M, Tuveson DA. Suppression of BRAF(V599E) in human melanoma abrogates transformation. Cancer Res 2003;63:5198-5202.

    Davies H, Bignell GR, Cox C, et al. Mutations of the BRAF gene in human cancer. Nature 2002;417:949-954.

    Tuveson DA, Weber BL, Herlyn M. BRAF as a potential therapeutic target in melanoma and other malignancies. Cancer Cell 2003;4:95-98.

    Maldonado JL, Timmerman L, Fridlyand J, Bastian BC. Mechanisms of cell-cycle arrest in Spitz nevi with constitutive activation of the MAP-kinase pathway. Am J Pathol 2004;164:1783-1787.

    Kuwata T, Kitagawa M, Kasuga T. Proliferative activity of primary cutaneous melanocytic tumours. Virchows Arch A Pathol Anat Histopathol 1993;423:359-364.

    Sparrow LE, Eldon MJ, English DR, Heenan PJ. p16 and p21WAF1 protein expression in melanocytic tumors by immunohistochemistry. Am J Dermatopathol 1998;20:255-261.

    Wang YL, Uhara H, Yamazaki Y, Nikaido T, Saida T. Immunohistochemical detection of CDK4 and p16INK4 proteins in cutaneous malignant melanoma. Br J Dermatol 1996;134:269-275.

    Miracco C, Margherita De Santi M, Schurfeld K, et al. Quantitative in situ evaluation of telomeres in fluorescence in situ hybridization-processed sections of cutaneous melanocytic lesions and correlation with telomerase activity. Br J Dermatol 2002;146:399-408.

    Gray-Schopfer VC, Cheong SC, Chong H, et al. Cellular senescence in naevi and immortalisation in melanoma: a role for p16? Br J Cancer (in press).

    Beausejour CM, Krtolica A, Galimi F, et al. Reversal of human cellular senescence: roles of the p53 and p16 pathways. EMBO J 2003;22:4212-4222.

    Chin L, Pomerantz J, Polsky D, et al. Cooperative effects of INK4a and ras in melanoma susceptibility in vivo. Genes Dev 1997;11:2822-2834.

    Chin L, Merlino G, DePinho RA. Malignant melanoma: modern black plague and genetic black box. Genes Dev 1998;12:3467-3481.

    Krimpenfort P, Quon KC, Mooi WJ, Loonstra A, Berns A. Loss of p16Ink4a confers susceptibility to metastatic melanoma in mice. Nature 2001;413:83-86.

    Sharpless NE, Bardeesy N, Lee KH, et al. Loss of p16Ink4a with retention of p19Arf predisposes mice to tumorigenesis. Nature 2001;413:86-91.

    Patton EE, Widlund HR, Kutok JL, et al. BRAF mutations are sufficient to promote nevi formation and cooperate with p53 in the genesis of melanoma. Curr Biol 2005;15:249-254.

    Chudnovsky Y, Adams AE, Robbins PB, Lin Q, Khavari PA. Use of human tissue to assess the oncogenic activity of melanoma-associated mutations. Nat Genet 2005;37:745-749.

    Di Leonardo A, Linke SP, Clarkin K, Wahl GM. DNA damage triggers a prolonged p53-dependent G1 arrest and long-term induction of Cip1 in normal human fibroblasts. Genes Dev 1994;8:2540-2551.

    McGill GG, Horstmann M, Widlund HR, et al. Bcl2 regulation by the melanocyte master regulator Mitf modulates lineage survival and melanoma cell viability. Cell 2002;109:707-718.

    Wang E. Senescent human fibroblasts resist programmed cell death, and failure to suppress bcl2 is involved. Cancer Res 1995;55:2284-2292.

    Schmitt CA, Fridman JS, Yang M, et al. A senescence program controlled by p53 and p16INK4a contributes to the outcome of cancer therapy. Cell 2002;109:335-346.

    Gupta PB, Kuperwasser C, Brunet JP, et al. The melanocyte differentiation program predisposes to metastasis after neoplastic transformation. Nat Genet 2005;37:1047-1054.

    Wu WS, Heinrichs S, Xu D, et al. Slug antagonizes p53-mediated apoptosis of hematopoietic progenitors by repressing puma. Cell 2005;123:641-653.

    Gwosdz C, Scheckenbach K, Lieven O, et al. Comprehensive analysis of the p53 status in mucosal and cutaneous melanomas. Int J Cancer 2006;118:577-582.

    Lowe SW, Cepero E, Evan G. Intrinsic tumour suppression. Nature 2004;432:307-315.

    Campisi J. Senescent cells, tumor suppression, and organismal aging: good citizens, bad neighbors. Cell 2005;120:513-522.

    Kirkwood TB, Austad SN. Why do we age? Nature 2000;408:233-238.

    Van Heemst D, Mooijaart SP, Beekman M, et al. Variation in the human TP53 gene affects old age survival and cancer mortality. Exp Gerontol 2005;40:11-15.

    Tyner SD, Venkatachalam S, Choi J, et al. p53 Mutant mice that display early ageing-associated phenotypes. Nature 2002;415:45-53.

    Krtolica A, Parrinello S, Lockett S, Desprez PY, Campisi J. Senescent fibroblasts promote epithelial cell growth and tumorigenesis: a link between cancer and aging. Proc Natl Acad Sci U S A 2001;98:12072-12077.

    Alrawi SJ, Schiff M, Carroll RE, et al. Aberrant crypt foci. Anticancer Res 2006;26:107-119.

    Maitra A, Wistuba II, Washington C, et al. High-resolution chromosome 3p allelotyping of breast carcinomas and precursor lesions demonstrates frequent loss of heterozygosity and a discontinuous pattern of allele loss. Am J Pathol 2001;159:119-130.

    te Poele RH, Okorokov AL, Jardine L, Cummings J, Joel SP. DNA damage is able to induce senescence in tumor cells in vitro and in vivo. Cancer Res 2002;62:1876-1883.

    Dirac AM, Bernards R. Reversal of senescence in mouse fibroblasts through lentiviral suppression of p53. J Biol Chem 2003;278:11731-11734.

    Sage J, Miller AL, Perez-Mancera PA, Wysocki JM, Jacks T. Acute mutation of retinoblastoma gene function is sufficient for cell cycle re-entry. Nature 2003;424:223-228.(W.J. Mooi, M.D., and D.S.)