Université de Montréal, Département de Biochimie, Montréal, Canada

Correspondence to: G Ferbeyre, Université de Montréal, Département de Biochimie, E-515, CP 6128, Succ Centre-Ville, Montréal, Qc H3C 3J7, Canada; Fax: 514 343-2210

PML is the most frequent fusion partner of the RAR in the specific translocations associated with acute promyelocytic leukemia (APL). Models to explain the origin of this leukemia propose a block in cell differentiation due to aberrant repression of retinoic acid responsive genes and/or disruption of the function of the PML-containing nuclear bodies. Recently, PML has been identified as a regulator of replicative senescence and the premature senescence that occurs in response to oncogenic ras. This review discusses the idea that senescence is a general tumor suppressor mechanism related to terminal differentiation and disrupted during the establishment of APL and other cancers. According to this idea the PML-RAR fusion protein promotes leukemogenesis not only through repression of retinoic acid responsive genes, but also by way of interfering with several tumor suppressor proteins that cooperate to establish senescence. Retinoids and other drugs effective against APL do so by re-establishment of the senescence program, which also includes features of cell differentiation.

Leukemia (2002) 16, 1918-1926. doi:10.1038/sj.leu.2402722

senescence; PML; differentiation; retinoic acid; ras

Introduction

Acute promyelocytic leukemia (APL) is a type of myeloid leukemia clinically characterized by a bleeding diathesis, abnormal promyelocytes and a remarkable remission in response to retinoic acid (RA) (reviewed in Refs 1 and 2). A hallmark of the disease is the presence of a balanced reciprocal translocation. As a result, APL cells express fusion proteins where one of the partners is always the retinoic acid receptor (RAR), and the other is in most cases the promyelocytic leukemia (PML) gene. The RAR is a transcription factor that dimerizes with the retinoic X receptor (RXR) in order to regulate gene expression.³ In the absence of retinoic acid the RAR/RXR heterodimer inhibits transcription by recruitment of several repressor complexes to the promoters of RA responsive genes. These repressor complexes contain histone deacetylases that block transcription by changing the acetylation pattern of the chromatin.^4,5,6 The ligand RA induces transcription through disruption of the repressor complexes and recruitment of histone acetylases and subsequently RNA polymerase.⁷ The functions of PML are less understood but it has been linked to the regulation of cell growth, apoptosis and more recently cellular senescence. This review discusses the implications of the PML/senescence connection for APL and other cancers.

Models for APL origin

The identification of the fusion proteins that characterize APL has offered molecular insights into the pathogenesis of this leukemia. In most cases of APL, the RAR gene fuses to the PML gene, but in patients with related leukemias the fusion partner of the RAR gene is either the promyelocytic leukemia zinc finger (PLZF) gene, the nucleophosmin (NPM) gene, the nuclear mitotic apparatus (NuMA) gene or the STAT5b gene (reviewed in Refs 1 and 2). The role of these translocations in APL and related diseases has been examined by generating transgenic mice expressing the RAR fusion proteins in the promyelocytic cell compartment.^8,9,10,11,12 All transgenic mice developed leukemia with APL features, a fact that is consistent with the idea that the fusion proteins initiate tumorigenesis. However, expression of the RAR fusion proteins was not sufficient for leukemogenesis since in every one of these transgenic models, the leukemia displayed incomplete penetrance and was preceded by a long pre-leukemic phase characterized by the accumulation of immature myeloid precursors in the bone marrow and spleen. Clearly, additional mutations are required for the development of leukemic disease in these transgenic mice.

The models proposed to explain how the RAR fusion proteins induce APL are influenced by the well-known fact that APL patients respond to treatment with all-trans retinoic acid (ATRA). The aberrant repression model postulates that the RAR fusion proteins block promyelocytic cell differentiation by repressing a set of RA-responsive genes required for maturation in the myeloid lineage.^6,13 Consistent with this idea all RAR fusion proteins bind and repress promoters containing the RARE (retinoic acid response elements)¹⁴ and block the differentiation of myeloid cell lines in response to physiological concentrations of RA.¹ As stated by a recent version of this model, the main role of the fusion partners of the RAR in APL is to provide a dimerization domain that allows the formation of homodimers of the fusion proteins which bind transcriptional co-repressors with higher affinity than the normal RAR/RXR heterodimers.^15,16 According to this model, high doses of ATRA convert the PML-RAR fusion protein from a repressor into an activator by stimulating the release of co-repressors, allowing the expression of the RA-responsive genes supposed to mediate granulocyte differentiation.^1,17 The aberrant repression model successfully explains why patients bearing the PLZF-RAR display an APL-like disease resistant to RA therapy. It turned out that the PLZF-RAR fusion protein contains a RA-resistant binding site for co-repressors. Inhibition of the histone deacetylase activity required for the functions of the corepressors switches the transcriptional effects of PLZF-RAR from being an inhibitor to an activator of the RA signaling pathway promoting the maturation of APL cells.⁶ Thus, the aberrant repression model proposes that genes essential for granulocyte differentiation are repressed by the RAR fusion proteins in APL and are activated by RA alone or in combination with HDAC inhibitors.

There are still some facts not explained by this aberrant repression model of APL. The main problem is that in vivo the RAR is apparently not required for granulocyte differentiation, as RAR^-/- mice have a normal granulocyte compartment and do not develop APL.^18,19 In fact, vitamin A deficiency increases myeloid differentiation in vivo suggesting that retinoids might suppress rather than promote granulocyte maturation.²⁰ In addition the nature of the RA-responsive genes controlling granulocyte maturation is still a mystery. Candidates include the p21^WAF1 cyclin-dependent kinase inhibitor,²¹ the myeloid transcription factor CEBP²² and perhaps some of the genes identified through microarray analysis of ATRA-treated APL cell lines.^3,23 To explain these discrepancies it has been proposed that the block in cell differentiation that characterizes APL is a gain-of-function phenotype, possibly the result of recognition of new sequences by the RAR fusion proteins.¹⁷ However, in vitro studies showed that the fusion proteins have a restricted rather than an expanded ability to recognize variant RAREs.²⁴ Clearly, additional studies are required to establish whether the fusion proteins block cell differentiation through direct repression of RAREs-containing genes.

Another important point is that the model reduces the participation of the fusion protein to simply providing a dimerization domain. An attractive hypothesis is that the fusion protein disrupts the functions of both the RAR and its fusion partner.²⁵ Consistently, PML ablation dramatically increased the incidence of leukemia in PML-RAR transgenic mice²⁶ and PLZF ablation turned the leukemia observed in PLZF-RAR transgenic mice into an APL-like disease.²⁷ Since PML is the main component of the PML oncogenic domains (PODs) or nuclear bodies (NBs) it is possible that impaired NBs functions contribute to leukemogenesis. This idea is supported by the fact that most APL cases have a disruption of the nuclear bodies and a redistribution of PML to a nuclear micro speckle pattern.^28,29 In addition, PLZF, the partner of the RAR in an APL-like disease might also be a component of the NBs.³⁰ However, so far, the other partners of the RAR in other leukemias that resemble APL, NuMA, nucleophosmin or Stat5B have not been associated with the functions of the NBs. It is nevertheless intriguing that all these fusion proteins are associated with a similar type of leukemia suggesting that they normally function in the same biochemical pathway.

The nuclear bodies

The NBs were first identified by electron microscopy and subsequently characterized as novel nuclear structures stained by a variety of antibodies (reviewed in Ref. 31). The discovery that PML, a gene altered in promyelocytic leukemia, was a component of NBs hinted at the possibility that they were involved in the regulation of cell growth and differentiation and raised the interest for their study.²⁸ NBs are regulated by interferon^32,33 and are particularly prominent in tissues regulated by steroid hormones like the breast ductal epithelium, the endometrium and the testicular Leydig cells.³⁴ Therefore the NBs come out as a highly dynamic and regulated compartment rather than a fixed and static part of the nucleus.

PML and the nuclear bodies have been proposed to regulate the basic mechanisms of gene expression (ie transcription, translation, RNA transport and protein degradation^35,36). Several models have been advanced to explain such a wide regulatory activity. One proposal is that they serve as a transient storage site or nuclear depot for regulatory molecules.³⁷ This idea is consistent with the fact that several components of the NBs are also found in the nucleoplasm.³⁸ A variant of this model proposes that proteins are modified and/or assembled into high molecular weight complexes in the NBs and then redistributed to their functional locations.^39,40,41,42 A particular interesting case is that of the transcriptional co-activator CBP, which constantly moves in and out of the NBs perhaps to form complexes with some transcription factors.^40,43 Another idea suggests that the NBs are sites for sequestering regulatory molecules out of the functional pool in the nucleoplasm. Therefore, the sequestration of repressors would explain some of the positive effects of PML on transcription, while the sequestration of activators possibly explains some of its negative effects.⁴⁴ Finally, it has been proposed that transcription itself occurs in the nuclear bodies⁴⁵ or in their immediate vicinity,⁴⁶ so the effects of PML would be associated to the formation of appropriate microenvironments for either transcriptional repression or activation.¹⁷

PML and the NBs have been implicated in the regulation of cell growth,^{47,48,49,50,51,52} apoptosis,^53,54 antigen presentation,^55,56,57 genomic stability⁵⁸ and senescence.^39,40,59 It is remarkable that the dissimilar biochemical activities of PML seem to converge to promote different anti-tumor mechanisms. For instance, the regulation of cell growth and senescence by PML involves the ability of PML to stimulate the p53 and the RB tumor suppressor proteins^39,40 and its ability to repress Sp1-dependent promoters.⁶⁰ Moreover, in human cells these activities are not sufficient to explain PML-induced senescence³⁹ suggesting that PML controls other regulators of cell proliferation and senescence.

Senescence as a tumor suppressor mechanism

Senescence can be defined as a stable arrest of cell proliferation that occurs in response to a variety of stresses and is characterized by metabolically active cells with a unique gene expression pattern and distinctive flat cell morphology. The term was originally used to describe the fact that normal human fibroblasts proliferate in culture for a finite number of cell divisions.⁶¹ This replicative senescence is due to the stress of having short telomeres (free DNA ends).^62,63 A similar arrest can be induced when normal fibroblasts are forced to express certain oncogenes^64,65,66,67 or treated either with histone deacetylase inhibitors⁶⁸ or DNA damaging agents.^69,70,71 These induced types of senescence arise shortly after exposing the cells to the inducing agent and do not depend on telomere shortening. They are also known as premature senescence⁶⁴ or stress-induced premature senescence (SIPS).⁷² The senescent arrest is characterized by accumulation of the cells in the G1 phase of the cell cycle, a flat and extended cell morphology and a specific gene expression pattern. The genes commonly used to identify senescence include the tumor suppressors p53, PML, p21 and p16INK4a, the plasminogen activator inhibitor-1 (PAI-1) and the senescence associated -galactosidase (SA--gal).^39,73 Since this phenotype can be triggered by a variety of stressors, it has been hypothesized that senescence is a general response of the cells to damage.^39,74 It can be compared to apoptosis, which is also triggered by a variety of stressors and involves a specific pattern of gene expression (Figure 1).

It has been proposed that senescence evolved as a mechanism of tumor suppression. Accordingly, senescence is induced by tumorigenic stimuli including activated oncogenes and is mediated by a variety of tumor suppressor genes.^39,64,73 Senescence induced by oncogenes is conserved in mouse and humans, while the ability to count cell generations and induce senescence through telomere shortening is a recent evolutionary acquisition of human cells. The coupling between telomere shortening and the senescence program evolved in humans perhaps as a consequence of their large size and long life span.⁷⁵ Apparently, tumor suppressor mechanisms based on premature senescence and apoptosis are good enough to protect the smaller and short-living mice from the accumulation of mutations and cancer. Human cells acquired this additional, telomere-based, tumor suppression mechanism by restraining the expression of telomerase in most human somatic cells. This mechanism is lost in human tumor cells, which often reactivate telomerase expression.⁷⁶ In agreement, reintroduction of telomerase in normal human cells not only inhibits replicative senescence, but also cooperates with other genetic changes to induce transformation.⁷⁷ Thus, senescence functions as a general tumor suppressor mechanism by inhibiting the proliferation of cells bearing activated oncogenes, DNA damage or short telomeres.

The different stressors that trigger the senescence program do so by activating distinct signaling pathways. Accordingly, the genes initiating senescence are probably specific for every stressful stimuli. For instance, oncogenic ras induces senescence through the MAP kinase pathway, (65) while the same pathway is down-regulated during the replicative senescence triggered by short telomeres.⁷⁸ However, since the senescence arrest is similar in response to a variety of stimuli, all these signaling pathways should finally converge into a common set of senescence regulators. Candidate genes controlling senescence include the tumor suppressors p53, Rb, and PML, the p53 modulators p19^ARF and p33^ING1 and the cyclin-dependent kinase inhibitors p21 and p16^INK4a.⁷³ Although these tumor suppressor genes control premature senescence both in human and mouse cells, there are significant differences between these two species. In mouse cells inactivation of the tumor suppressor pathways controlled by the pocket proteins (Rb, p107 and p130), p53 or PML is sufficient to override premature senescence. This is not the case in human cells where a tumor suppressor network controls premature senescence³⁹ (Figure 2). Hence, to inhibit premature senescence in human cells it is necessary to disrupt simultaneously several tumor suppressor pathways. This is remarkably accomplished by several viral oncoproteins such as T antigen from SV40 and E1A from adenovirus.^39,64,77 (Figure 2a). Certainly, having a more robust premature senescence mechanism and a telomere barrier to excessive proliferation makes human cells much more difficult to transform than mouse cells.

So far premature senescence in response to oncogenes has been demonstrated in human and mouse fibroblasts,⁶⁴ mouse primary keratinocytes,⁷⁹ and human primary thyroid epithelial cells.⁸⁰ It is then important to find out whether oncogenes can trigger a senescence response in other cell types. Primitive mouse hematopoietic stem cells decline in function with age in vivo and genes on chromosomes 7, 11 and 12 may regulate this mouse stem cell senescence.^81,82,83 Mouse cells do not undergo replicative senescence,^84,85,86 so it is reasonable to suppose that the loss of division potential of mouse hematopoietic cells in vivo is due to stresses, perhaps oncogenic mutations, that trigger premature cellular senescence. The ability of hematopoietic cells to enter a senescence cell cycle arrest was recently demonstrated in an in vivo model of murine lymphoma. In this important work senescence was induced by chemotherapy and was characterized by expression of SA--gal and an increase in PML bodies.⁸⁷ This work stresses the importance of characterizing senescence in the hematopoietic compartment, as well as its role as a tumor suppressor mechanism for different kinds of leukemias and lymphomas.

Senescence and terminal differentiation

To a certain extent, the process of senescence and terminal differentiation largely overlap. Terminal differentiation is the final post-mitotic state in the life of a cell and is regulated under physiological conditions by a variety of extracellular factors in interaction with particular genetic programs. Genes expressed in terminally differentiated cells are cell type specific and perform functions in harmony with tissue physiology and homeostasis. Cell differentiation is the hallmark of development and tissue renewal in vivo and it has been extensively studied and characterized in vitro using a variety of cell culture techniques. Since senescence is also a post mitotic state, it shares with terminal differentiation the expression of genes preventing cell proliferation and the down-regulation of genes promoting proliferation. In addition senescent cells might also display features of terminal differentiation because the stressors that elicit senescence activate the same signaling pathways used by the inducers of cell differentiation (Figure 3). Hence terminal differentiation and senescence might often come together in non-differentiated stem cells in response to stressors.

For example, prolonged exposure to high levels of cAMP or treatment with melanocyte stimulating hormone (-MSH) or cholera toxin (CT) results in terminal differentiation of human melanocytes characterized by augmented melanogenesis, but also several senescence features like an increase in the cyclin-dependent kinase inhibitors (CDK-Is) p27 (KIP1) and p16 (INK4); failure to phosphorylate the retinoblastoma protein (pRB); decreased expression of E2F proteins and phenotypic changes characteristic of senescent cells.^88,89 The link between senescence and terminal differentiation is also observed in cultures of primary normal human oral keratinocytes, which undergo differentiation in the presence of calcium and senescence after serial subculture. Notably, both calcium treatment and serial subculture induced differentiation features such as elevated cellular involucrin and inhibited telomerase activity. On the other hand continued sub-culture was associated with an increase in SA--gal-positive cells and p16INKA, but such changes were not observed in keratinocytes exposed to calcium.⁹⁰ Remarkably oncogenic ras induced a cell cycle arrest in primary mouse keratinocytes accompanied by differentiation features like increased involucrin expression, but also involving an increase in the tumor suppressors p19^ARF, p16INK4a and p53 which was not observed in calcium-treated cells.⁷⁹ Thus, in keratinocytes, senescence induced by serial sub-culture or oncogenic ras displays features of terminal differentiation in addition to changes specific for the senescence phenotype.

The myeloid cell line HL60 and the monocytic cell line U937 are among the best-characterized models of in vitro differentiation.⁹¹ Phorbol esters, topoisomerase inhibitors and a large variety of agents including thermal stress induce the differentiation of these cell lines into macrophage-like cells.^92,93 This differentiation is not entirely normal,^94,95 perhaps reflecting the altered genome of these transformed cells. However, it is also possible that the agents used to trigger differentiation also initiate a stress response and senescence. Intriguingly, the oncogenes mos and ras that activate the MAP kinase pathway are able to induce differentiation and growth arrest in U937 cells.^96,97 As mentioned before, oncogenic ras induces senescence in human diploid fibroblasts and mouse primary fibroblasts, suggesting that U937 cells and fibroblasts respond to sustained MAP kinase activation triggering a similar permanent growth arrest program. Consistent with this idea, blockade of MAP kinase pathway with the Mek inhibitor PD098059 prevents both phorbol ester-induced morphological differentiation of U937 cells and ras-induced senescence of human diploid fibroblasts.^65,98 In addition, differentiated U937 cells display up to 100-fold increase in PAI-1⁹⁹ a hallmark of replicative senescence and ras-induced senescence of human diploid fibroblasts.⁶⁴ Also the tumor suppressor p53, which regulates senescence in fibroblasts is also able to induce the differentiation of U937 cells¹⁰⁰ and HL60 cells.¹⁰¹ In summary, senescence and terminal differentiation are both post-mitotic cellular states. Terminal differentiation occurs in response to physiological stimulus in non-differentiated stem cells, while senescence is a general response to stress of many cell types. Both phenotypes overlap in a number of traits but they can be distinguished. Senescent cells express genes associated to stress response and tumor suppression that might interfere with normal differentiation (Figure 3).

Is APL the result of a failure of senescence as a tumor suppressor mechanism?

As discussed above the current model of the origin of APL is largely based on the proposal that RAR fusion proteins repress RA-responsive genes required for myeloid cell differentiation. The discovery that PML regulates senescence suggests a new scenario for the development of APL that does not exclude previously proposed models of the disease (Figure 2b). In this model a mutation or an abnormal bone marrow microenvironment¹⁰² might initiate uncontrolled proliferation, which eventually can trigger the induction of premature senescence. In most individuals this mechanism will suffice to remain free of leukemic disease. However, in a few cases mutated cells will escape senescence in a process associated to chromosomal abnormalities including the balanced reciprocal translocations observed in all cases of APL. This scenario is consistent with recent studies where mammary epithelial cells in culture escaped senescence in association with chromosomal aberrations.¹⁰³ It is also consistent with results showing multiple and non-random chromosome abnormalities in APL cells from transgenic mice expressing PML-RAR.¹⁰⁴ In conjunction, the chromosomal aberrations, including the translocations, will provide mechanisms to by-pass the normal barriers to malignant transformation. The PML-RAR fusion for instance could inhibit senescence and promote transformation in the same way as the viral oncoproteins E1A and Tag, that is, by interfering with multiple tumor suppressor pathways (Figure 2b). In fact the oncoprotein PML-RAR can inhibit the functions of the NBs and p53,^40,105 Rb¹⁰⁶ and the retinoic acid receptor⁶ (Figure 2). These activities of the PML-RAR act in conjunction with its transcriptional repression functions to promote tumorigenesis. However, as discussed before, bypassing premature senescence and blocking granulocyte maturation is not enough for full malignant transformation and additional mutations should be required to overcome replicative senescence due to telomere shortening and provide a proliferative advantage to mutated cells. Clearly, the hunt for oncogenic mutations in APL is not yet finished.

The 'escape from senescence' model of APL suggests that RA might inhibit the proliferation of APL cells by restoring the functions of PML bodies and activating signaling pathways that promote senescence, although it remains to be demonstrated whether RA induce other features of senescence (ie high p16, PAI-1 and SA--gal) in APL cells (Figure 3). According to this model the terminal differentiation in response to pharmacological doses of ATRA is not the reflection of a normal function of RA in granulocyte maturation. The terminal differentiation observed in response to RA might be the result of triggering a stress response that simultaneously activates the signaling pathways linked to the granulocyte maturation and the senescence program. Consistently, RA induces cellular senescence in normal mammary epithelial cells, in the human breast cancer cell line MCF7 and in neuroblastoma cells.^107,108,109 An intriguing connection between retinoids and the senescence response is the fact that retinoids promote the formation of reactive oxygen species (ROS) in cells.¹¹⁰ High levels of ROS can be detected in several types of senescence and treatment with hydrogen peroxide or culturing cells on high oxygen also trigger senescence (for a review see Ref. 111). Furthermore, RA can down-regulate telomerase activity in APL cells independently of its differentiation effect, a situation that can trigger senescence via short telomeres.¹¹² Additional evidence to support the idea that RA induces senescence in APL cells comes from the comparison of the patterns of gene expression of ATRA treated APL cells (NB4 cell line) and senescent human diploid fibroblasts. Both expression patterns include the up-regulation of proteasome components, genes that arrest cell proliferation like p21^WAF1, and genes that block apoptosis. They also have as a feature the down-regulation of genes promoting cell proliferation.^3,21,23,39 However, more work is needed to demonstrate that these genes are regulated as the result of the establishment of a senescence program in these cells.

Another intriguing connection between ATRA-induced maturation of APL cells and ras-induced senescence take account of the interferon response. Interferon enhances the response to retinoids in APL cells and patients.¹¹³ Conversely, ATRA induces interferon synthesis and activation of Stat-1 in APL cells.^114,115 On the other hand, ras-induced senescence also displays an interferon-like response including the expression of the NBs components PML and Sp100.³⁹ The viral oncoprotein E1A that efficiently overrides Ras-induced senescence prevents the induction of PML by Ras³⁹ perhaps by inhibiting signaling pathways common to Ras-senescence and interferon.^116,117 Taken together, this evidence suggest that interferon-regulated genes have a role in both ras-induced senescence and the response of APL cells to ATRA.

The idea that RA-induced terminal differentiation is accompanied by senescence features is consistent with observations that RA induces an abnormal granulocyte maturation in APL cells, including nuclear filamentous connections and/or nuclear blebs and the absence of neutrophil secondary granules.¹¹⁸ Again this abnormal differentiation might simply reflect a highly deregulated control of transcriptional programs in tumor cells. However, it is intriguing that a similarly abnormal differentiation was a hallmark of the senescent arrest of primary mouse keratinocytes in response to oncogenic ras.⁷⁹ The abnormal differentiation of APL blasts in response to RA therapy might explain the so-called ATRA syndrome characterized by fever, leukocytosis and respiratory distress due to the release of cytokines by the differentiated cells. Intriguingly, a similar increase in the expression of cytokine genes was observed during the premature senescence of human diploid fibroblasts in response to oncogenic ras.³⁹ An additional parallel between ras-induced senescence and the effects of retinoids on APL cells is that in both cases a proliferation phase precedes the growth arrest.^39,65,119 The leukocytosis that characterizes the ATRA syndrome is perhaps the result of this initial proliferative response to ATRA. Of note, the symptoms described in APL patients treated with ATRA are not observed in patients treated with G-CSF after bone marrow transplantation or allogenic transplantation of peripheral blood stem cells^120,121 presumably because G-CSF triggers normal differentiation in granulocytes precursors.

The idea that the 'maturation therapy' in APL is also a 'senescent therapy' is consistent with the positive response of APL patients to HDAC inhibitors.⁶ According to the aberrant repression model of APL, HDAC inhibitors are required to convert ATRA insensitive fusion proteins from repressors of retinoic acid responsive genes into transcriptional activators.^6,11,122 Conversely, HDAC inhibitors are also able to trigger senescence in human diploid fibroblasts⁶⁸ suggesting that they might have a similar effect on APL cells. In fact HDAC inhibitors could trigger a p53 response by promoting the acetylation of p53 increasing its transcriptional activity.¹²³ Taken together, these data suggest that APL cells have genetic changes that bypass senescence and that treatment with retinoids, HDAC inhibitors and perhaps chemotherapy inhibits tumor cell proliferation by re-establishing the senescence program. This senescence program might include features of terminal differentiation depending on the agent used and the cell type (Figure 3).

This concept of therapy-induced senescence in APL has wide implications for the treatment of other cancers. Tumors might develop mechanisms to by-pass senescence usually through acquired mutations that disable the signaling pathways from activated oncogenes to the senescence machinery. Drugs might restore the program by activating alternative signaling pathways that trigger senescence and/or terminal differentiation. Understanding the mechanisms that regulate senescence will allow the search and the design of new drugs against APL and other cancers.

Acknowledgements

GF is a fellow of the Canadian Institute of Health and Research. I thank Drs Clemens Schmitt and Ari Melnick for valuable comments on this manuscript and V Bourdeau for help with the figures. The ideas presented in this review were motivated in the excellent scientific environment of the laboratory of Scott Lowe at Cold Spring Harbor. I thank Dr Lowe and the members of his laboratory for discussions.

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Figure 1 Senescence and apoptosis as general stress response mechanisms. Multiple stressors initiate cellular programs aimed to avoid the expansion of cells bearing mutations that can disturb tissue physiology. Apoptosis or programmed cell death leads to an immediate physical elimination of the cells, while senescence is a permanent arrest program where the cells remain viable. Complete elimination of senescent cells might require the action of immune/inflammatory cells. We do not understand how either program is chosen. However, cells bearing mutations in oncogenes that induce anti-apoptotic pathways like ras should be obviously eliminated from the cycling population by cellular senescence. On the other hand, cells bearing mutations in immortalizing oncogenes, like myc or E1A, are better eliminated through apoptosis. ROS means reactive oxygen species.

Figure 2 Tumor suppressor networks. In normal human cells, several tumor suppressor genes are activated by oncogenic damages. The emergence of malignant cells requires mutations of several components of the tumor suppressor network or the action of dominant acting oncoproteins that block simultaneously several tumor suppressor proteins. (a) Tumor suppressor network activated by oncogenic ras and inhibited by E1A in human diploid fibroblasts. (b) A model of tumorigenesis in promyelocytes. Oncogenic mutations activate a tumor suppressor network involving PML and RA-responsive genes. The oncoprotein PML-RAR blocks the network at multiple points promoting tumorigenesis.

Figure 3 Terminal differentiation and senescence. Stimuli that trigger senescence might initiate a terminal differentiation response in non-differentiated stem cells because they stimulate the same signaling pathways activated by normal differentiation inducers. Both processes share a post-mitotic gene expression profile with up-regulation of growth suppressor genes like p21 and the down-regulation of proliferation genes like E2F1. TD means terminal differentiation and S means senescence.