Table of Contents

Volume 7, Issue 19

  October 1, 2008

  Pages 2947 - 3106

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NEWS

Open Access Article

p53 in autophagy control

Wei-Xing Zong and Ute Moll
Pages 2947 - 2948
http://dx.doi.org/10.4161/cc.7.19.7008
Abstract | PDF

p53 is a well known tumor suppressor recognized for its ability to cause cell senescence and apoptosis.1 The pro-senescence function of p53 is mainly mediated by its transcriptional activation of cell cycle inhibitors. The proapoptotic function of p53 is carried out at two levels: one is through the transcriptional activation of proapoptotic proteins such as Puma, Noxa and Bax, the other is independent of its nuclear function. It physically and functionally interacts with the multi-domain members of the Bcl-2 family of proteins at mitochondria to cause mitochondrial membrane permeabilization. Stimuli such as genotoxic, hypoxic, and oncogenic stress can cause transcriptional activation as well as cytosolic accumulation with mitochondrial localization of wild-type p53 that leads to rapid mitochondrial apoptosis. Both the cytostatic and cytocidal functions of p53 contribute to its tumor suppressor nature.

Adding to its tumor suppressing activity, p53 has recently been found to also regulate autophagy.2,3 Autophagy is the process of 'self-eating' that functions to rid the cell of damaged intracellular macromolecules and organelles via degradative autophagic vacuoles, and to provide cells with energy under nutrient-starved conditions. It has been found that autophagy can be induced in a p53-dependent manner in response to genotoxins. One mechanism for p53-induced autophagy is through activation of AMPK kinase, with subsequent activation of TSC1 and TSC2 kinases, which leads to the acute inhibition of mTOR kinase (mammalian target of rapamycin), an anabolic node which controls the translation machinery and cell growth. In addition to this rapidly induced inhibition of mTOR, p53 activation also leads to upregulation of PTEN and TSC2 at the transcriptional level, which may contribute to the long-term suppression of mTOR. Another mechanism of p53-induced autophagy is through transcriptional activation of DRAM, a p53 target and a lysosomal protein. Autophagy induced by p53 may facilitate p53's cell cycle arrest activities, such that autophagy mediates the selective degradation of damaged molecules and organelles in order to provide an energy source for the damage repair process and promote 'cell healing.' Alternatively, when the extent of damage is beyond repair, autophagy may act to synergize with accelerated cell death in response to p53 activation.

Adding to these versatile functions of p53, a series of exciting recent papers from Guido Kroemer's group including two in this issue of Cell Cycle report yet another novel role of p53, namely its ability to inhibit autophagy.4,5,6 The authors find that inhibition of endogenous p53 either by genetic deletion, RNAi knockdown, or pharmacological inhibitors results in autophagy in human, mouse and nematode cells.4 They also report that in cells, several autophagy-inducing stress conditions such as nutrient starvation, ER (endoplasmic reticulum) stress, and mTOR inhibition by rapamycin all result in proteasome-mediated p53 degradation that leads to the de-repression of an autophagy program.5 Furthermore, autophagy that is induced by p53 inhibition/elimination cannot be further stimulated by ER stress, indicating that under normal (nutrient) conditions p53 maximally suppresses autophagy. The canonical nutrient sensing autophagy pathway (AMPK-mTOR-Atgs) is required for autophagy induced by p53 inhibition. Moreover, the autophagy suppression activity of p53 is strictly cell cycle-dependent, i.e., p53 inhibition triggers autophagy mostly in the G1 and less so in the S phase but never in the G2/M phase of the cycle.5 p53 degradation in response to nutrient deprivation and ER stress induces autophagy and leads to high levels of intracellular ATP. This is associated with better cell viability, and may account for cancer cell survival in the often nutrient-poor environment encountered in a 'cancer bed.' Together, these observations place p53 at the center of autophagy regulation, such that both p53 activation and suppression can lead to autophagy in response to a wide variety of cell damage signals that are either of a genotoxic or a metabolic nature, respectively.

While these observations on the p53-suppressed autophagy program might point to a role in tumor promotion, we do not yet understand its biological significance. Using retransfection of p53-/- HCT116 colon carcinoma cells with either WTp53 or a large series of p53 missense point mutants that naturally occur in human cancers, Kroemer et al find that cytoplasmic but not nuclear p53 exerts the autophagy-inhibiting function.4,6 Thus, in contrast to the autophagy-promoting activity that requires p53's nuclear transactivation activity, the autophagy-suppressing function of p53 is solely cytoplasmic and transcription-independent. Accordingly, gene expression arrays failed to identify autophagy-related genes when comparing isogenic wild-type and p53-/- HCT116 colon cancer cells. In addition, the cytoplasmic autophagy-suppressing function of p53 was found to be uncoupled from its cytoplasmic apoptosis-inducing function, since several mutants unable to interact with Bcl-2/Bcl-xL and unable to induce mitochondrial apoptosis can still inhibit autophagy. Thus, cytoplasmic p53 inhibits autophagy in a manner that is drastically distinct from both its classic transcriptional function in the nucleus and its proapoptotic function at mitochondria. And even more puzzling, the autophagy-inhibiting activity of cytoplasmic p53 does not seem to correlate with p53 tumor suppressor activity, as both tumor suppression competent and deficient, or even oncogenic, p53 mutants can inhibit autophagy, as long as they are localized in the cytoplasm.6 The coming years will likely unravel how the autophagy-suppressing function of cytoplasmic p53 is regulated, what physiologic role it plays in normal vs in nascent and established tumor cells, and how their respective status in p53 function, metabolic stress, apoptotic threshold and cell cycle progression/checkpoint influences the outcome. Finally, interesting insights can be expected once we understand the biochemical mechanism behind this novel cytoplasmic p53 function, possibly based on protein-protein interactions with autophagy regulators. Stay tuned.

References

1. Levine AJ, Hu W, Feng Z. The P53 pathway: what questions remain to be explored? Cell Death Differ 2006; 13:1027-36.
2. Jin S. p53, Autophagy and tumor suppression. Autophagy 2005; 1:171-3.
3. Crighton D, Wilkinson S, Ryan KM. DRAM links autophagy to p53 and programmed cell death. Autophagy 2007; 3:72-4.
4. Tasdemir E, Maiuri MC, Galluzzi L, Vitale I, Djavaheri-Mergny M, et al. Regulation of autophagy by cytoplasmic p53. Nat Cell Biol 2008; 10:676-87.
5. Tasdemir E, Maiuri MC, Orhon I, Kepp O, Morselli E, Criollo A, Kroemer G. p53 represses autophagy in a cell cycle-dependent fashion. Cell Cycle 2008; In this issue.
6. Morselli E, Tasdemir E, Maiuri MC, Galluzzi L, Kepp O, Criollo A, Vicencio JM, Soussi T, Kroemer G. Mutant p53 protein localized in the cytoplasm inhibits autophagy. Cell Cycle 2008; In this issue.

Open Access Article

SIRT1 protein levels in cancer: Tuning SIRT1 to the needs of a cancer cell

Wolfgang Deppert
Pages 2947 - 2948
http://dx.doi.org/10.4161/cc.7.19.7010
Abstract | PDF

SIRT1 (silent information regulator 1) is a member of the highly conserved gene family of sirtuins, discovered in yeast as factors required for gene silencing, and classified as enzymes harboring NAD+-dependent deacetylase and ADP-ribosyltransferase activities. SIRT1 is able to deacetylate histones and histone methyl-transferase SUV39H1, thereby initiating and coordinating facultative heterochromatin formation. SIRT1 also deacetylates a number of non-histone target proteins, including the tumor suppressor p53, the retinoblastoma protein, the transcription factors of the FoxO family, MyoD and RelA/p65 (NFκB), the DNA repair protein Ku70, and the transcriptional co-activators PCG-1α and p300. SIRT1 co-ordinates cellular responses to caloric restriction (CR) including enhanced metabolic efficiency, is involved in the biogenesis of ribosomes and mitochondria, and in metabolic endocrine signaling 1. In this way, SIRT1 regulates important cellular processes like aging and longevity, acts anti-apoptotic and protects neurons from degeneration. Given the diversity of SIRT1 functions, it is not surprising that SIRT1 is also implicated in a variety of pathologic conditions, including diabetes, neurodegeneration, Parkinson’s disease, and cancer 2. The beneficial or detrimental roles of SIRT1 strongly depend on SIRT1 activity levels; therefore regulation of its activity is of pivotal importance. SIRT1 deacetylase activity can be regulated by interacting proteins, like AROS which enhances 3, and DBC1 (deleted in breast cancer-1) which inhibits SIRT1 activity 4. Alternatively or in addition, changes in SIRT1 protein levels will also change SIRT1 activity.

In cancer cells, SIRT1 protein levels are abnormally elevated, indicative for higher SIRT1 activity in such cells. SIRT1 functions as a survival factor in cancer cells, as demonstrated by RNAi-induced silencing of SIRT1, as SIRT1 depletion resulted in apoptosis of epithelial cancer cells, but not of non-cancer epithelial cells 5. In this issue of Cell Cycle, Milner and her colleagues provide convincing evidence that elevated SIRT1 protein levels in cancer cells are not attributable to increased SIRT1 mRNA levels, but instead correlate with increased SIRT1 protein stability. Protein stability often is regulated by post-translational modification. SIRT1 is a phosphoprotein, with two potential phosphorylation sites at serines 27 (S27P) and 47 (S47P). Using phosphopeptide-specific antibodies, Milner and colleagues found that SIRT1 protein levels in normal and in cancer cells strongly correlated with the presence of S47P, indicating that S47 is constitutively phosphorylated. In contrast, S27P levels were variable and did not correlate with SIRT1 proteins levels: S27P was hardly detectable in normal cells, but strongly present in cancer cells. Thus S27P phosphorylation correlated with the enhanced SIRT1 protein levels in cancer cells, and indicated that phosphorylation of SIRT1 at S27 enhances its metabolic stability. Increased SIRT1 protein stability is dependent, directly or indirectly, upon the presence of JNK2, as silencing of JNK2, but not of JNK1, with RNAi decreased SIRT1 protein stability while SIRT1 mRNA levels remained unaffected. The JNK2 connection links SIRT1 with the MAP kinase system.

A very interesting effect was observed, when colon cancer HCT116 cells were treated with the commonly used anti-cancer drug 5-fluorouracil (5FU). 5FU treatment also induced a decline in SIRT1 protein levels, but only in parental HCT116 cells expressing wild-type p53 and not in their p53-negative derivatives (HCT116 p53-/- cells). Thus control of SIRT1 levels is also dependent on a functional wild-type p53, creating yet another link between SIRT1 and p53. However, p53 influences SIRT1 protein levels not via SIRT1 turnover, but via control of SIRT1 translation. In a very recent paper Yamakuchi et al. 6 demonstrated that miR-34a, a bona fide transcriptional target of p53, represses SIRT1 expression. As p53 loss of function is common to many cancer cells, cancer cells can achieve increased SIRT1 protein levels by two independent means. The finding that SIRT1 protein levels can be regulated at the translational (via miR-34a) and at the post-translational level (via JNK2), and that both effects are additive, should provide novel approaches for a combinatorial targeting of SIRT1 not only in cancer therapy. Given the diverse roles of SIRT1 and its links with diseases including cancer understanding the various means of regulating SIRT1 protein levels and activity are extremely important.

References

1. Jiang J, Huang Z, Zhao Q, Feng W, Belikova NA, Kagan VE. Interplay between bax, reactive oxygen species production, and cardiolipin oxidation during apoptosis. Biochem Biophys Res Commun 2008; 368:145-50.
2. Guarente L. Sirtuins in aging and disease. Cold Spring Harb Symp Quant Biol 2007; 72:483-8.
3. Verdin E. AROuSing SIRT1: identification of a novel endogenous SIRT1 activator. Mol Cell 2007; 28:354-6.
4. Zhao W, Kruse JP, Tang Y, Jung SY, Qin J, Gu W. Negative regulation of the deacetylase SIRT1 by DBC1. Nature 2008; 451:587-90.
5. Ford J, Jiang M, Milner J. Cancer-specific functions of SIRT1 enable human epithelial cancer cell growth and survival. Cancer Res 2005; 65:10457-63.
6. Yamakuchi M, Ferlito M, Lowenstein CJ. miR-34a repression of SIRT1 regulates apoptosis. Proc Natl Acad Sci USA 2008; 105:13421-6.




EXTRA VIEWS

Open Access Article

The insulin-like growth factor system as a potential therapeutic target in gastrointestinal stromal tumors

Martin G. Belinsky, Lori Rink, Kathy Q. Cai, Michael F. Ochs, Burton Eisenberg, Min Huang, Margaret von Mehren and Andrew K. Godwin
Pages 2949 - 2955
http://dx.doi.org/10.4161/cc.7.19.6760
Abstract | PDF

The majority of gastrointestinal stromal tumors (GISTs) are characterized by oncogenic gain-of-function mutations in the receptor tyrosine kinase (RTK) c-KIT with a minority in PDGFRa. Therapy for GISTs has been revolutionized by the use of the selective tyrosine kinase inhibitor imatinib mesylate (IM). For the subset (~10-15%) of GISTs that lack oncogenic mutations in these receptors, the genetic changes driving tumorigenesis are unknown. We recently reported that the gene encoding the insulin-like growth factor 1 receptor (IGF-1R) is amplified in a subset of GISTs, and the IGF-1R protein is over-expressed in WT and pediatric GISTs. In this report we present a more complete picture of the involvement of components of the insulin-like growth factor-signaling pathway in the pathogenesis of GISTs. We also discuss how the IGF pathway may provide additional molecular targets for the treatment of GISTs that respond poorly to IM therapy.

Open Access Article

Control of senescence by CXCR2 and its ligands

Juan C. Acosta, Ana Loghlen, Ana Banito, Selina Raguz and Jesús Gil
Pages 2956 - 2959
http://dx.doi.org/10.4161/cc.7.19.6780
Abstract | PDF

Senescence is an irreversible growth arrest with important physiological implications as it contributes to tumour suppression and may have a role in aging. During senescence, cells suffer profound phenotypic changes affecting amongst others cell morphology and chromatin structure. Senescent cells also undergo significant transcriptional changes, such as the increased production of a plethora of different secreted factors, which are the basis of the so-called senescence-associated secretory phenotype. While some of these factors have been previously shown to possess different pro-tumorigenic activities, we recently demonstrated that the secretion of CXCR2-binding chemokines (such as IL-8 or GROα) by senescent cells contribute to reinforce senescence via activation of the p53 pathway. Importantly, our data adds to that presented by several groups suggesting that also other factors secreted during senescence (such as PAI-1, IGFBP-7 or IL-6) contribute to the senescent response. Here, we discuss our findings in the context of the emerging role for secreted factors in regulating senescence through paracrine and/or autocrine mechanisms

Open Access Article

Consorting kinases, end of destruction and birth of a spindle

Karen Crasta, Hong Hwa Lim, Tao Zhang, Saurabh Nirantar and Uttam Surana
Pages 2960 - 2966
http://dx.doi.org/10.4161/cc.7.19.6783
Abstract | PDF

Centrosomes (spindle pole body in yeast) constitute the two poles of the bipolar mitotic spindle and play a prominent role in the segregation of chromosomes during mitosis. Like chromosomes, the centrosome inherited from the progenitor cell duplicates once in each division cycle, following which the sister centrosomes segregate away from each other to assemble a short spindle upon initiation of mitosis. Cdh1, an activator of the E3 ubiquitin ligase APC (Anaphase Promoting Complex), is a potent inhibitor of centrosome segregation and suppresses spindle assembly during S phase by mediating proteolytic destruction of the microtubule associated proteins (MAPs) required for centrosome separation. A recent study in yeast suggests that concerted action by two prominent kinases Cdk1 and polo are required to bring this destruction to a halt by inactivating Cdh1 and to facilitate spindle assembly. This is an effective strategy for the modulation of the activities of cell cycle regulators that require multiple phosphorylation. The control circuit involving Cdh1, Cdk1, Polo and MAPs may be also targeted by other cellular networks in contexts that demand the restraining of spindle dynamics.

Open Access Article

MdmX regulates transformation and chromosomal stability in p53-deficient cells

Zdenka Matijasevic, Anna Krzywicka-Racka, Greenfield Sluder and Stephen N. Jones
Pages 2967 - 2973
http://dx.doi.org/10.4161/cc.7.19.6797
Abstract | PDF

The cellular homologues Mdm2 and MdmX play critical roles in regulating the activity of the p53 tumor suppressor in damaged and non-damaged cells and during development in mice. Recently, we have utilized genetically defined primary cells and mice to reveal that endogenous levels of MdmX can also suppress multipolar mitosis and transformation in hyperploid p53-deficient cells and tumorigenesis in p53-deficient mice. These MdmX functions are not shared by Mdm2, and are distinct from the well-established ability of MdmX to complex with and inhibit p53 activity. Here we discuss some of the ramifications of MdmX loss in p53-deficient cells and mice, and we explore further the fate of MdmX/p53-double null embryonic fibroblasts undergoing multi-polar cell division using time-lapse video microscopy. We also discuss the relationship between chromosomal loss, cell proliferation, and the tumorigenic potential of p53-deficient cells lacking MdmX.

Open Access Article

T cell-mediated help against tumors

Thomas Wieder, Heidi Braumüller, Manfred Kneilling, Bernd Pichler and Martin Röcken
Pages 2974 - 2977
http://dx.doi.org/10.4161/cc.7.19.6798
Abstract | PDF

Adoptive transfer of tumor antigen-specific T helper (Th) cells is a surprisingly potent anti-tumor therapy. Even in RIP1-Tag2 mice with a rapidly growing, aggressive endogenous beta cell tumor Th can significantly extend life time and are more efficient than any other therapy studied. The therapeutic effect of Th cells seems to be independent of tumor cell destruction. It critically relies on three principles: (i) inhibition of tumor angiogenesis, (ii) inhibition of beta cell proliferation, and (iii) induction of tumor dormancy. As tumor cell destruction by cytotoxic CD8+ T cells (CTL) largely failed in tumor therapy, induction of tumor dormancy by Th cell-mediated immune responses represents a novel therapeutic option that may be combined with other cytotoxic regimens, e. g. radio- and/or chemotherapy, as it is established for bone marrow transplantation. Importantly, Th cell efficacy strictly requires interferon-gamma (IFN-gamma) signaling, and, in the absence of IFN-gamma, Th cells may even worsen tumor diseases. Therefore, using the immune system to control tumor dormancy represents a novel approach, especially as therapy of tumors resistant to conventional therapies. Yet, it is important to underline that Th cell-based antitumor effects critically depend on a functional cytokine network, especially appropriate IFN-gamma signaling.

Open Access Article

Dissecting the role of the 3-phosphoinositide-dependent protein kinase-1 (PDK1) signalling pathways

Jose R. Bayascas
Pages 2978 - 2982
http://dx.doi.org/10.4161/cc.7.19.6810
Abstract | PDF

The 3-phosphoinositide-dependent protein kinase-1 (PDK1) mediates the cellular effect of insulin and growth factors by activating a group of kinases including PKB/Akt, S6K, RSK, SGK and PKC isoforms. PDK1 possesses two regulatory domains namely a Pleckstrin Homology (PH) domain that binds to the phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] second messenger, and a substrate binding site termed the PIF-pocket. Employing a combination of biochemical, structural and mouse knock-in approaches we have been able to define the roles that the regulatory domains on PDK1 play. We have established that binding of PDK1 to PtdIns(3,4,5)P3 is essential for efficient activation of PKB isoforms as well as for maintaining normal cell size and insulin sensitivity. In contrast, the PIF-substrate binding pocket of PDK1 is not required for PKB activation, but is necessary for PDK1 to activate all of its other substrates.




PERSPECTIVES

Open Access Article

SCFFbx4/αB-crystallin E3 ligase: When one is not enough

Olena Barbash and J Alan Diehl
Pages 2983 - 2986
http://dx.doi.org/10.4161/cc.7.19.6775
Abstract | PDF

Cell cycle progression is determined by the balance of positive regulators, cyclin-dependent-protein kinases (cdks) relative to negative regulators, cyclin-dependent kinase inhibitors (ckis). D-type cyclins, (D1, D2, D3) are expressed in a tissue-specific manner and are the first cyclins to be expressed during the cell cycle. Of the three D-type cyclins, cyclin D1 is most frequently overexpressed in human cancer. The mechanisms of cyclin D1 overexpression can be attributed to gene amplification, transcriptional activation and altered protein degradation; of these, inhibition of the ubiquitin-dependent proteolysis of cyclin D1 is thought to be a primary mechanism of cyclin D1 overexpression in human tumors. Because the identity of the regulators of cyclin D1 proteolysis were largely undefined until recently, it had not been possible to determine whether this regulatory network was directly targeted in primary cancer. Cyclin D1 proteolysis requires phosphorylation by GSK3β at Thr-286; additional work recently established that p286-D1 is a substrate for the SCFFbx4/αB-crystallin E3 ligase. This discovery has facilitated an analysis of SCFFbx4/αB-crystallin ligase in human cancers. This recent work revealed that Fbx4 is subject to mutational inactivation in human cancer, resulting in the accumulation of cyclin D1. Molecular analysis of this ligase has revealed striking regulatory features that contribute to regulated cyclin D1 accumulation and support the idea that Fbx4 is a bona fide tumor suppressor.

Open Access Article

Targeting cancer cells by synthetic lethality

Denise A. Chan and Amato J. Giaccia
Pages 2987 - 2990
http://dx.doi.org/10.4161/cc.7.19.6776
Abstract | PDF

Standard cytotoxic agents for treating cancer were developed based on their effectiveness to kill rapidly dividing cells, not on their ability to selectively kill cancer cells and spare normal tissue. Much of contemporary cancer research is aimed at identifying specific molecular features of cancers to directly target tumor cells with the hope of reducing or eliminating unwanted side effects. Targeted therapy for the treatment of cancer can be divided into two main categories: monoclonal antibodies and small molecules. In this Perspective, we review the approach of synthetic lethality to target cancer, specifically renal cell carcinoma. The concept of synthetic lethality is used to describe a genetic interaction of two non-allelic and non-lethal genes that when mutated simultaneously results in cell death. Recently, we identified a compound, STF-62247, that functions in a synthetic lethal manner to the loss of VHL, a mutation found in the majority of renal cell carcinomas.




REVIEWS

Open Access Article

Metastasis and AKT activation

Meng Qiao, Shijie Sheng and Arthur B. Pardee
Pages 2991 - 2996
http://dx.doi.org/10.4161/cc.7.19.6784
Abstract | PDF

Metastasis is responsible for 90% of cancer patient deaths. More information is needed about the molecular basis for its potential detection and treatment. The activated AKT kinase is necessary for many events of the metastatic pathway including escape of cells from the tumor's environment, into and then out of the circulation, activation of proliferation, blockage of apoptosis, and activation of angiogenesis. A series of steps leading to metastatic properties can be initiated upon activation of AKT by phosphorylation on Ser-473. These findings lead to the question of how this activation is connected to metastasis. Activated AKT phosphorylates GSK-3β causing its proteolytic removal. This increases stability of the negative transcription factor SNAIL, thereby decreasing transcription of the transmembrane protein E-cadherin that forms adhesions between adjacent cells, thereby permitting their detachment. How is AKT hyperactivated in metastatic cells? Increased PI3K or TORC2 kinase activity- or decreased PHLPP phosphatase could be responsible. Furthermore, a positive feedback mechanism is that the decrease of E-cadherin lowers PTEN and thereby increases PIP3, further activating AKT and metastasis.

Open Access Article

Molecular insights into mechanisms regulating faithful chromosome separation in female meiosis

Shen Yin, Xiao-Fang Sun, Heide Schatten and Qing-Yuan Sun
Pages 2997 - 3005
http://dx.doi.org/10.4161/cc.7.19.6809
Abstract | PDF

The faithful segregation of chromosomes into daughter cells in meiosis is crucial to produce healthy progeny. In gametogenesis, two consecutive rounds of chromosome separation occur with only one round of DNA replication, and the chromosome number is reduced to half to produce haploid gametes. Here, we discuss the molecular mechanisms underlying faithful chromosome separation in meiosis from three aspects: Spindle checkpoint, two-step releases of cohesion, and the specific space-time protection of cohesin.




REPORTS

Open Access Article

p53 represses autophagy in a cell cycle-dependent fashion

Ezgi Tasdemir, Maria Chiara Maiuri, Idil Orhon, Oliver Kepp, Eugenia Morselli, Alfredo Criollo and Guido Kroemer
Pages 3006 - 3011
http://dx.doi.org/10.4161/cc.7.19.6702
Abstract | PDF

Autophagy is one of the principal mechanisms of cellular defense against nutrient depletion and damage to cytoplasmic organelles. When p53 is inhibited by a pharmacological antagonist (cyclic pifithrin-), depleted by a specific small interfering RNA (siRNA) or deleted by homologous recombination, multiple signs of autophagy are induced. Here, we show by epistatic analysis that p53 inhibition results in a maximum level of autophagy that cannot be further enhanced by a variety of different autophagy inducers including lithium, tunicamycin-induced stress of the endoplasmic reticulum (ER) or inhibition of Bcl-2 and Bcl-XL with the BH3 mimetic ABT737. Chemical inducers of autophagy (including rapamycin, lithium, tunicamycin and ABT737) induced rapid depletion of the p53 protein. The absence or the inhibition of p53 caused autophagy mostly in the G1 phase, less so in the S phase and spares the G2/M phase of the cell cycle. The possible pathophysiological implications of these findings are discussed.

Open Access Article

Localization of Aurora A and Aurora B kinases during interphase: Role of the N-Terminal domain

Yoann Rannou, Marie-Bérengère Troadec, Clotilde Petretti, Fabienne Hans, Stéphanie Dutertre, Stefan Dimitrov and Claude Prigent
Pages 3012 - 3020
http://dx.doi.org/10.4161/cc.7.19.6718
Abstract | PDF

Aurora kinases possess a conserved catalytic domain (CD) and a N-terminal domain (ND) that varies in size and sequence. We have previously reported that the N-terminal domain of AuroraA (AurA) participates in the localization of the kinase to the centrosome in interphase. AuroraB (AurB) is a chromosome passenger protein and its N-terminal domain is not necessary for its localization or function during mitosis. Using various combinations of GFP-AurA and AurB protein domains we show that AurB N-terminal domain is required for nuclear localization in Xenopus XL2 cells in interphase. In human cells, however, we found both AurA and AurB kinases in the nucleus, AurA being mainly cytoplasmic and AurB mainly nuclear. Both proteins are actively excluded from the nucleus by a CRM1 dependent pathway. Interestingly, at a functional level, in interphase, every combination of Aurora kinase domains (ND-CD) rescues histone H3 Serine10 phosphorylation defect induced by AurB knockdown. This clearly indicates the presence of a functional AurA in the nucleus. However, the chimera ND-AurA/CD-AurB was much more efficient than the ND-AurB/CD-AurA to rescue multinucleation also induced by AurB knockdown. This indicates that the catalytic domain of AurB is required to fulfill specific functions during mitosis that cannot be fulfilled by the catalytic domain of AurA, probably for localization reasons during mitosis.

Open Access Article

Co-expression of fatty acid synthase and caveolin-1 in pancreatic ductal adenocarcinoma: Implications for tumor progression and clinical outcome

Agnieszka K. Witkiewicz, Katherine Nguyen, Abhijit Dasgupta, Eugene P. Kennedy, Charles J. Yeo, Michael P. Lisanti and Jonathan R. Brody
Pages 3021 - 3025
http://dx.doi.org/10.4161/cc.7.19.6719
Abstract | PDF

Pancreatic cancer is a devastating disease that afflicts over 35,000 Americans every year. Since therapeutic options are limited, understanding the molecular aspects of this disease is critical for moving towards targeted treatment of this aggressive form of cancer. Caveolin-1 (Cav-1) and fatty acid synthase (FASN) are two proteins that have been shown to be dysregulated in a number of cancers. Functionally these proteins have been shown to be involved in the process of tumorigenesis. We thus surveyed the expression of both these critical proteins in a series of pancreatic precancerous lesions (pancreatic intraepithelial neoplasia, PanINs) and pancreatic cancers. Cav-1 and FASN expression correlated predominantly with clinical characteristics, such as histologic grade and advanced tumor stage (e.g. high Cav-1 and FASN expression correlated with poor differentiation status) and a significant survival advantage was found in patients with low co-expressing FASN and Cav-1 tumors. Cav-1 and FASN expression was absent in PanIN lesions and the normal ducts and acini. Of note, Cav-1 expression was detected in the fibroblasts of the desmoplastic pancreatic cancer stroma, but not in stromal cells of the normal pancreas. Mechanistically, these data support the notion that these proteins are co-regulated either directly or indirectly by another factor. Importantly, the co-expression of these proteins significantly correlates with clinical features and survival status of pancreatic cancer patients. Thus, Cav-1 and FASN may functionally cooperate in the process of pancreatic tumorigenesis, and as such, may be good candidate prognostic markers and targets for therapeutic intervention.

Open Access Article

X-ray structure of the complex of regulatory subunits of human DNA polymerase delta

Andrey G. Baranovskiy, Nigar D. Babayeva, Victoria G. Liston, Igor B. Rogozin, Eugene V. Koonin, Youri I. Pavlov, Dmitry G. Vassylyev and Tahir H. Tahirov
Pages 3026 - 3036
http://dx.doi.org/10.4161/cc.7.19.6720
Abstract | PDF

The eukaryotic DNA polymerase delta (Pol delta) participates in genome replication, homologous recombination, DNA repair and damage tolerance. Regulation of the plethora of Pol delta functions depends on the interaction between the second (p50) and third (p66) non-catalytic subunits. We report the crystal structure of p50p66N complex featuring oligonucleotide binding and phosphodiesterase domains in p50 and winged helix-turn-helix N-terminal domain in p66. Disruption of the interaction between the yeast orthologs of p50 and p66 by strategic amino acid changes leads to cold-sensitivity, sensitivity to hydroxyurea and to reduced UV mutagenesis, mimicking the phenotypes of strains where the third subunit of Pol delta is absent. The second subunits of all B-family replicative DNA polymerases in archaea and eukaryotes, except Pol delta, share a three-domain structure similar to p50p66N, raising the possibility that a portion of the gene encoding p66 was derived from the second subunit gene relatively late in evolution.

Open Access Article

Speedy/Ringo C regulates S and G₂ phase progression in human cells

Aiyang Cheng and Mark J. Solomon
Pages 3037 - 3047
http://dx.doi.org/10.4161/cc.7.19.6736
Abstract | PDF

Cyclin-dependent kinases (CDKs) control cell cycle transitions and progression. In addition to their activation via binding to cyclins, CDKs can be activated via binding to an unrelated class of cell cycle regulators termed Speedy/Ringo (S/R) proteins. Although mammals contain at least five distinct Speedy/Ringo homologues, the specific functions of members of this growing family of CDK activators remain largely unknown. We investigated the cell cycle roles of human Speedy/Ringo C in HEK293 cells. Down-regulation of Speedy/Ringo C by RNA interference delayed S and G2 progression whereas ectopic expression had the opposite effect, reducing S and G2/M populations. Double thymidine arrest and release experiments showed that overexpression of Speedy/Ringo C promoted late S phase progression. Using a novel three-color FACS protocol to determine the length of G2 phase, we found that the suppression of Speedy/Ringo C by RNAi prolonged G2 phase by ~30 min whereas ectopic expression of Speedy/Ringo C shortened G2 phase by ~25 min. In addition, overexpression of Speedy/Ringo C disrupted the G2 DNA damage checkpoint, increased cell death and caused a cell cycle delay at the G1-to-S transition. These observations indicate that CDK-Speedy/Ringo C complexes positively regulate cell cycle progression during the late S and G2 phases of the cell cycle.

Open Access Article

Phosphorylation of p53 on Ser15 during cell cycle and caused by Topo I and Topo II inhibitors in relation to ATM and Chk2 activation

Hong Zhao, Frank Traganos and Zbigniew Darzynkiewicz
Pages 3048 - 3055
http://dx.doi.org/10.4161/cc.7.19.6750
Abstract | PDF

The DNA topoisomerase I (topo1) inhibitor topotecan (TPT) and topo2 inhibitor mitoxantrone (MXT) damage DNA inducing formation of DNA double-strand breaks (DSBs). We have recently examined the kinetics of ATM and Chk2 activation as well as histone H2AX phosphorylation, the reporters of DNA damage, in individual human lung adenocarcinoma A549 cells treated with these drugs. Using a phospho-specific Ab to tumor suppressor protein p53 phosphorylated on Ser15 (p53-Ser15P) combined with an Ab that detects p53 regardless of the phosphorylation status and multiparameter cytometry we correlated the TPT- and MXT- induced p53-Ser15P with ATM and Chk2 activation as well as with H2AX phosphorylation in relation to the cell cycle phase. In untreated interphase cells, p53-Ser15P had "patchy" localization throughout the nucleoplasm while mitotic cells showed strong p53-Ser15P cytoplasmic immunofluorescence (IF). The intense phosphorylation of p53-Ser15, combined with activation of ATM and Chk2 (involving centrioles) as well as phosphorylation of H2AX seen in the untreated mitotic cells, suggest mobilization of the DNA damage detection/repair machinery in controlling cytokinesis. In the nuclei of cells treated with TPT or MXT, the expression of p53-Ser15P appeared as closely packed foci of intense IF. Following TPT treatment, the induction of p53-Ser15P was most pronounced in S-phase cells while no significant cell cycle phase differences were seen in cells treated with MXT. The maximal increase in p53-Ser15P expression, rising up to 2.5-fold above the level of its constitutive expression, was observed in cells treated with TPT or MXT for 4 - 6 h. This maximum expression of p53-Ser15P coincided in time with the peak of Chk2 activation but not with ATM activation and H2AX phosphorylation, both of which crested 1-2 h after the treatment with TPT or MXT. The respective kinetics of p53-Ser15 phosphorylation versus ATM and Chk2 activation suggest that in response to DNA damage by TPT or MXT, Chk2 rather than ATM mediates p53 phosphorylation.

Open Access Article

Mutant p53 protein localized in the cytoplasm inhibits autophagy

Eugenia Morselli, Ezgi Tasdemir, Maria Chiara Maiuri, Lorenzo Galluzzi, Oliver Kepp, Alfredo Criollo, Jose Miguel Vicencio, Thierry Soussi and Guido Kroemer
Pages 3056 - 3061
http://dx.doi.org/10.4161/cc.7.19.6751
Abstract | PDF

The knockout, knockdown or chemical inhibition of p53 stimulates autophagy. Moreover, autophagy-inducing stimuli such as nutrient depletion, rapamycin or lithium cause the depletion of cytoplasmic p53, which in turn is required for the induction of autophagy. Here, we show that retransfection of p53-/- HCT 116 colon carcinoma cells with wild type p53 decreases autophagy down to baseline levels. Surprisingly, one third among a panel of 22 cancer-associated p53 single amino acid mutants also inhibited autophagy when transfected into p53-/- cells. Those variants of p53 that preferentially localize to the cytoplasm effectively repressed autophagy, whereas p53 mutants that display a prominently nuclear distribution failed to inhibit autophagy. The investigation of a series of deletion mutants revealed that removal of the DNA-binding domain from p53 fails to interfere with its role in the regulation of autophagy. Altogether, these results identify the cytoplasmic localization of p53 as the most important feature for p53-mediated autophagy inhibition. Moreover, the structural requirements for the two biological activities of extranuclear p53, namely induction of apoptosis and inhibition of autophagy, are manifestly different.

Open Access Article

Microarray and pathway analysis reveals decreased CDC25A and increased CDC42 associated with slow growth of Bcl-2-over-expressing immortalized breast cell line

Jacquelyn M. Long, Charles W. Bell, W. Samuel Fagg IV, Mary E. Kushman, Kevin G. Becker, James A. McCubrey and Mary A. Farwell
Pages 3062 - 3073
http://dx.doi.org/10.4161/cc.7.19.6761
Abstract | PDF

Bcl-2 is an anti-apoptotic protein that is frequently over-expressed in cancer cells but its role in carcinogenesis is not clear. We are interested in how Bcl-2 expression affects non-cancerous breast cells and its role in the cell cycle. We prepared an MCF10A breast epithelial cell line that stably over-expressed Bcl-2. We analyzed the cells by flow cytometry after synchronization, and used cDNA microarrays with quantitative reverse-transcription PCR (qRT-PCR) to determine differences in gene expression. The microarray data was subjected to two pathway analysis tools, parametric analysis of gene set enrichment (PAGE) and ingenuity pathway analysis (IPA), and western analysis was carried out to determine the correlation between mRNA and protein levels. The MCF10A/Bcl-2 cells exhibited a slow-growth phenotype compared to control MCF10A/Neo cells that we attributed to a slowing of the G1-S cell cycle transition. A total of 363 genes were differentially expressed by at least two-fold, 307 up-regulated and 56 down-regulated. PAGE identified 22 significantly changed gene sets. The highest ranked network of genes identified by IPA contained 24 genes. Genes that were chosen for further analysis were confirmed by qRT-PCR, however, the western analysis did not always confirm differential expression of the proteins. Down-regulation of the phosphatase CDC25A could solely be responsible for the slow growth phenotype in MCF10A/Bcl-2 cells. Increased levels of GTPase Cdc42 could be adding to this effect. PAGE and IPA are valuable tools for microarray analysis, but protein expression results do not always follow mRNA expression results.

Open Access Article

Expression of Arl2 is associated with p53 localization and chemosensitivity in a breast cancer cell line

Anne Beghin, Eva-Laure Matera, Stephanie Brunet-Manquat and Charles Dumontet
Pages 3074 - 3082
http://dx.doi.org/10.4161/cc.7.19.6777
Abstract | PDF

In mammalian cells, ADP ribosylation factor like 2 (Arl2) has been shown to form a complex with tubulin binding cofactor D (TBC-D) and the tumor suppressor protein phosphatase 2A (PP2A). We have previously shown that alterations in Arl2 protein content were associated with corresponding modifications of the tumor suppressor PP2Ac protein content in breast cancer cells. Here, we show that modified Arl2 expression level influences sensitivity to various anticancer compounds such as taxol, navelbine, gemcitabine and doxorubicin in MCF7 derived cell lines. Modifications of Arl2 expression levels were also associated with an altered phosphorylation status and/or cellular sublocalization of certain PP2A targets such as p53, a key mediator of chemotherapy-induced apoptosis. A decreased level of Arl2 expression was associated with an increase of phospho-ser15-p53, a form which was found to be preferentially bound to microtubules. Assays using okadaic and cantharidic acid, two different PP2A inhibitors, showed an increase in microtubule-bound phospho-p53 and reduced sensitivity to chemotherapy. Our results suggest that Arl2 could, via PP2A, influence p53 phosphorylation status. Certain forms of phosphorylated p53 demonstrating increased binding to microtubules appear to be less prone to nuclear translocation after exposure to chemotherapeutic agents, thereby possibly contributing to reduced chemosensitivity.

Open Access Article

The let-7 microRNA interfaces extensively with the translation machinery to regulate cell differentiation

Xavier C. Ding, Frank J. Slack and Helge Großhans
Pages 3083 - 3090
http://dx.doi.org/10.4161/cc.7.19.6778
Abstract | PDF

MicroRNAs (miRNAs) are noncoding RNAs that regulate numerous target genes through a posttranscriptional mechanism and thus control major developmental pathways. The phylogenetically conserved let-7 miRNA regulates cell proliferation and differentiation, thus functioning as a key regulator of developmental timing in C. elegans and a tumor suppressor gene in humans. Using a reverse genetic screen, we have identified genetic interaction partners of C. elegans let-7, including known and novel potential target genes. Initial identification of several translation initiation factors as suppressors of a let-7 mutation led us to systemically examine genetic interaction between let-7 and the translational machinery, which we found to be widespread. In the presence of wild-type let-7, depletion of the translation initiation factor eIF3 resulted in precocious cell differentiation, suggesting that developmental timing is translationally regulated, possibly by let-7. As overexpression of the eIF3 in humans promotes translation of mRNAs that are also targets of let-7-mediated repression, we suggest that eIF3 may directly or indirectly oppose let-7 activity. This might provide an explanation for the opposite functions of let-7 and eIF3 in regulating tumorigenesis.

Open Access Article

JNK2-dependent regulation of SIRT1 protein stability

Jack Ford, Shafiq Ahmed, Simon Allison, Ming Jiang and Jo Milner
Pages 3091 - 3097
http://dx.doi.org/10.4161/cc.7.19.6799
Abstract | PDF

Mammalian SIRT1 is an NAD-dependent deacetylase with critical roles in the maintenance of homeostasis and cell survival. Elevated levels of SIRT1 protein are evident in cancer in which SIRT1 can function as a cancer-specific survival factor. Here we demonstrate that elevated SIRT1 protein in human cells is not attributable to increased SIRT1 mRNA levels but, instead, reflects SIRT1 protein stability. RNAi-mediated depletion of JNK2 reduced the half-life of SIRT1 protein from > 9h to < 2h and this correlated with lack of SIRT1 protein phosphorylation at serine 27. In contrast, depletion of JNK1 had no effect upon SIRT1 protein stability and SIRT1 phosphorylation at serine 47 showed no correlation with SIRT1 protein stability. Thus we show that JNK2 is linked, directly or indirectly, with SIRT1 protein stability and that this function is coupled with SIRT1 phosphorylation at serine 27. Our observations identify a route for therapeutic modulation of SIRT1 protein levels in SIRT1-linked diseases including cancer, neurodegeneration and diabetes.

Open Access Article

Mdm2 exerts pro-apoptotic activities by antagonizing insulin-like growth factor-I-mediated survival

Pascal Froment, Jacques Dupont and Jean Christophe-Marine
Pages 3098 - 3103
http://dx.doi.org/10.4161/cc.7.19.6807
Abstract | PDF

The Mdm2 oncoprotein is an E3 ubiquitin ligase required to maintain the p53 protein at low levels in embryonic and adult tissues. It also contributes to tumor formation by antagonizing p53 tumor suppressor activity when amplified and/or overexpressed. Importantly, p53-independent role for Mdm2 has been suggested by transfection studies. Among the growing list of putative Mdm2-regulated proteins are several proteins playing a key role in the control of cell proliferation such as pRb, E2F1/DP1, Numb, Smads, Lats2 or IGF-1R. Consistent with the ability of Mdm2 to promote ubiquitylation and proteasome destruction of IGFR-I independently of p53, we show herein that loss of Mdm2 leads to a significant increase in IGF1-R-β protein levels both in cells lacking or expressing p53. Interestingly, IGF-1 protects cells from DNA-damage-induced apoptosis only in absence of Mdm2. These data therefore further highlight a physiological role for Mdm2 in the control of IGF1 signalling and provide genetic evidence for a p53-independent proapoptotic function of Mdm2. The consequences of these findings for the development of p53-Mdm2-targeted anti-cancer therapies are discussed.




LETTER TO THE EDITOR

Open Access Article

Identification of a synaptosome- associated form of BAG3 protein

Anna Paola Bruno, Michela Festa, Fabrizio Dal Piaz, Alessandra Rosati, Maria Caterina Turco, Antonio Giuditta and Liberato Marzullo
Pages 3104 - 3105
http://dx.doi.org/10.4161/cc.7.19.6774
PDF




ERRATUM

Open Access Article

Erratum to Wendy Roeb et al., Cell Cycle Volume 7, Issue 7; pp. 837-841

Wendy Roeb, Antonia Boyer, Webster K. Cavenee and Karen C. Arden
Page 3106
http://dx.doi.org/10.4161/cc.7.19.6946
Abstract | PDF

In the paper, Guilt by association:PAX3-FOXO1 regulates gene expression through selective destabilization of the EGR1 transcription factor by Wendy Roeb, Antonia Boyer, Webster K. Cavenee and Karen C. Arden (Cell Cycle 2008; 7:837-41), Figure 1 and Figure 2 were mislabeled and published alongside the incorrect figure legends. Figures 1 and 2 appear with their correct figure legends below.