Table of Contents

Volume 7, Issue 21

  November 1, 2008

  Pages 3287 - 3473

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Open Access Article

Targeting a nucleolar SUMO protease for degradation: A mechanism by which ARF induces SUMO conjugation

Dimitris P. Xirodimas and David P. Lane
Pages 3287 - 3291
http://dx.doi.org/10.4161/cc.7.21.7232
Abstract | PDF

The p19^ARF (p14^ARF in humans) protein acts as a tumour suppressor through p53 dependent and independent mechanisms. A well-established role for ARF is to regulate the post-translational modification of substrate proteins with ubiquitin and ubiquitin-like molecules such as SUMO. It is now evident that induction of ARF causes a dramatic accumulation of SUMO conjugates and this has been related to the p53 independent functions of ARF. The majority of these conjugates appear to accumulate in the nucleolus where most of ARF is also found. An obvious function for ARF, which would result in increase of SUMOylation, is to act as an atypical SUMO E3-ligase. Indeed, initial studies suggested that ARF could directly interact with the SUMO E2-conjugating enzyme Ubc9 and therefore bringing the SUMO conjugation machinery in close proximity to its interacting substrates.¹ However, the highly basic charged nature of ARF makes biochemical analysis difficult and there is no clear demonstration that ARF can fulfill the criteria for an E3-ligase in vitro. Therefore, the mechanism(s) behind this phenomenon are not currently understood. As with ubiquitination, SUMO conjugation is a dynamic process controlled by E3-ligases and proteases that specifically remove SUMO from substrates. In this issue of Cell Cycle studies from the Sherr lab suggest that ARF can increase SUMO conjugation by controlling the stability of the nucleolar SUMO protease SENP3.² Recent studies have shown that SENP3 can deconjugate SUMO-2 and SUMO-3 from substrates including nucleophosmin (NPM). NPM is a nucleolar protein, which among other processes is involved in the processing of rRNA during ribosome biosynthesis. NPM interacts with ARF and this results in increased SUMOylation of NPM. SENP3 can counteract the effect of ARF by deconjugating SUMO from NPM and this appears to be critical for NPM function in rRNA processing.³ The new study now suggests that there is an opposing functional relationship between ARF and SENP3. ARF promotes phosphorylation dependent ubiquitination of SENP3, which results in SENP3 degradation and increase in NPM SUMO conjugation. In this process, NPM seems to act as a “platform” for ARF and SENP3, bringing in close proximity its two regulators. The new study suggests an interesting and complex mechanism by which ARF can control SUMOylation. It is now evident that post-translational modifications cooperate to control protein function. The new data suggest that ARF engages phosphorylation to promote ubiquitination and proteasomal degradation of a SUMO protease. This model would propose the existence of a kinase/phosphatase and an E3-ubiquitin ligase/de-ubiquitinating enzyme set which would cooperate their actions to control the stability of SENP3. Given that ARF has multiple binding partners, it would not be surprising that ARF would interact with components of the above enzymatic steps and control their activity. It would therefore be interesting to identify the role of ARF in this process. It is not clear whether degradation of SENP3 per se is sufficient to induce NPM SUMO conjugation and if this is the case which SUMO E3-ligases drive the forward reaction. Even if in this study an interaction of ARF with Ubc9 could not be demonstrated it may be the case that ARF mediates both the degradation of SENP3 and recruitment of the SUMO conjugation machinery, which will result in fast and efficient accumulation of SUMOylated NPM. Another possibility is the effect of ARF on NPM stability itself. Previous studies have shown that ARF can induce ubiquitin-mediated degradation of NPM.⁴ As NPM is important to prevent destabilisation of SENP3, ARF-mediated degradation of NPM could be part of SENP3 degradation. Another point that arises from this is the site of degradation for SENP3. Nucleoli have been suggested to be deficient for proteasomal activity, suggesting that ARF through the phosphorylation/ubiquitination events may alter the localisation/mobility of SENP3 making it susceptible to nucleoplasmic/cytoplasmic proteasomal degradation. The effect of ARF in controlling protein ubiquitination is now well established. Interaction of ARF with E3-ligases such as Mdm2 and ARF-BP1/Mule inhibits their function resulting in inhibition of p53 proteasomal degradation.^5,6 Therefore, the ability of ARF to induce ubiquitination and proteasomal degradation of SENP3 and NPM shows a complex and diverse role for ARF to control protein stability. Further experiments will show whether the ability of ARF to promote degradation of SENP3 or possibly other SUMO proteases is a general mechanism through which ARF induces SUMO conjugation of its binding partners or that the NPM/SENP3 system is a unique example.

References

1. 1. Rizos H, Woodruff S, Kefford RF. p14ARF interacts with the SUMO-conjugating enzyme Ubc9 and promotes the sumoylation of its binding partners. Cell Cycle 2005; 4:597-603.
2. 2. Kuo ML, den Besten W, Thomas MC, Sherr CJ. Arf-induced turnover of the nucleolar nucleophosmin-associated SUMO-2/3 protease Senp3. Cell Cycle 2008; 7:In this issue
3. 3. Haindl M, Harasim T, Eick D, Muller S. The nucleolar SUMO-specific protease SENP3 reverses SUMO modification of nucleophosmin and is required for rRNA processing. EMBO Rep 2008; 9:273-9
4. 4. Itahana K, Bhat KP, Jin A, Itahana Y, Hawke D, Kobayashi R, Zhang Y. Tumor suppressor ARF degrades B23, a nucleolar protein involved in ribosome biogenesis and cell proliferation. Mol Cell 2003; 12:1151-64.
5. 5. Xirodimas D, Saville MK, Edling C, Lane DP, Laín S. Different effects of p14ARF on the levels of ubiquitinated p53 and Mdm2 in vivo. Oncogene 2001; 20:4972-83.
6. 6. Chen D, Kon N, Li M, Zhang W, Qin J, Gu W. ARF-BP1/Mule is a critical mediator of the ARF tumor suppressor. Cell 2005; 121:1071-83.

 

Open Access Article

In search of a role in DNA replication: The enigmatic transcription factor Msa1

Curt Wittenberg
Pages 3287 - 3291
http://dx.doi.org/10.4161/cc.7.21.7233
Abstract | PDF

The abundance of DNA replication proteins and their regulators among proteins encoded by genes expressed during G₁ phase clearly illustrates the link between G₁-specific transcription and DNA replication.¹ However, the importance of periodic transcription for the robustness or fidelity of DNA replication remains obscure. In that context, the report by Li et al.² in this issue of Cell Cycle in which they identify MSA1 as an important factor for robust S phase entry is of particular interest. MSA1 is a cell cycle-regulated gene that interacts with the G₁-specific transcription machinery.^3,4 The current study shows that overexpression of MSA1 suppresses the growth defect caused by temperature sensitive alleles of two genes that play a critical role in the initiation of DNA replication, DRC1/SLD2 and DBP11. Conversely, MSA1 inactivation increases the detrimental effect of those mutants. This suggests that MSA1 is important when the function of those proteins is compromised and indicates that MSA1, or the regulation of genes by MSA1, is important for robust entry into S phase.

However, two observations suggest that the relationship between MSA1 and DNA replication is not straight-forward. First, in contrast to its suppression of drc1/sld2 and dpb11 mutants, overexpression of MSA1 has a detrimental effect on cell proliferation in other mutants known to affect DNA replication. However, the genes investigated primarily act prior to initiation of replication. This implies that overproduction of Msa1 is deleterious in mutants that play a role in pre-initiation events, whereas it suppresses the growth defect of mutants that are directly involved in the DNA replication complex.

Next, the identity of the targets of Msa1 identified by Li et al. based upon the convergence of genome wide chromatin immunoprecipitation and gene expression studies suggests that the transcriptional role of Msa1 is not limited to its interaction with the G₁-specific transcription factors SBF and MBF. Instead, those targets appear to fall into a number of regulatory classes and the proteins encoded by the target genes are associated with a wide variety of cellular functions. Importantly, none of the genes for which mutants were suppressed by MSA1 overexpression appear among the targets identified by that analysis. We are left wondering which, or how many, of the Msa1 targets identified by Li and colleagues are important for the suppression they observe. Only one potential target, Mcm3, is involved in DNA replication, but whether it is involved in the suppression or enhancement remains to be tested. The criterion used in this study for an Msa1 target required it to bind to upstream of a gene that’s expression is affected by Msa1 overexpression. However, a prior study⁴ found that, although Msa1 bound to SBF and MBF-regulated promoters, it influenced only the timing of transcriptional activation but not the level of expression of the target genes. Is it possible that the effect of MSA1 on DNA replication is a consequence of the effect on timing of expression of the many G₁-specific targets that encode DNA replication proteins rather than the increase in expression of a few G₁ specific targets that was observed in this study? It will be interesting to test those hypotheses.

Finally, it is striking that Msa1 has a well-conserved paralog, Msa2, that is also cell cycle regulated^2,4 and interacts with G₁-specific transcription factors.^4,5 This suggests the attractive hypothesis that the Msa1 and Msa2 are functionally redundant. However, neither the study of Li et al. nor prior studies provide support for that hypothesis. Consequently, we are left in search of a unifying hypothesis, a challenge for future research.

References

1. Bahler J. Cell-cycle control of gene expression in budding and fission yeast. Ann Rev Genet 2005; 39:69-94.
2. Li J-M, Tezlaff MT, Elledge SJ. Identification of MSA1, a cell cycle-regulated, dosage suppressor of drc1/sld2 and dpb11 mutants. Cell Cycle 2008; 7:In this issue.
3. Pramila T, Wu W, Miles S, Noble WS, Breeden LL. The Forkhead transcription factor Hcm1 regulates chromosome segregation genes and fills the S-phase gap in the transcriptional circuitry of the cell cycle. Genes Dev 2006; 20:2266-78.
4. Ashe M, de Bruin RAM, Kalashnikova T, McDonald WH, Yates JR, Wittenberg C. The SBF- and MBF-associated protein Msa1 Is required for proper timing of G1-specific transcription. in Saccharomyces cerevisiae. J Biol Chem 2008; 283:6040-9.
5. Krogan NJ, Cagney G, Yu H, Zhong G, Guo X, Ignatchenko A, Li J, et al. Global landscape of protein complexes in the yeast Saccharomyces cerevisiae. Nature 2006; 440:637-43.

Open Access Article

Mitosis poisons p53

Giovanni Blandino
Pages 3287 - 3291
http://dx.doi.org/10.4161/cc.7.21.7235
Abstract | PDF

In the current issue of Cell Cycle Sonia Lain and her collaborators provide convincing evidence of the importance of a p53 cell based assay to identify new antitumoral compounds. The reported findings represent a further development of those recently published by the same group.¹ Since its discovery in 1979 p53 has been studied intensively as a single protein and more recently as the primary member of a family of transcription factors whose main activities, such as growth arrest, apoptosis, senescence and differentiation, are closely related to tumor suppression. Within the considerable amount of published literature regarding p53 there is information on almost every one of the 393 amino acids composing p53 wild-type full length protein. Undoubtedly such advanced knowledge has the possibility of its rapid transfer to potential clinical applications thereby making p53 as a protein capable of successfully filling the gap, which still exists, from the bench to the bed of a given cancer patient.^2,3 The findings reported by Lain’s group in the current issue of Cell Cycle⁴ reflect this approach, discovering JJ78:12 as a small molecule capable of exerting antitumoral activities at very low concentrations and consequently inhibit in vivo xenograft tumor growth. By screening a collection of 30,000 compounds for their ability to trigger on p53 transcriptional activity they found that sixteen compounds lead to the accumulation of cancer cells in G₂/M phase (Fig.1A). This accumulation pairs with that obtained with nocadozole and occurs independently from wt-p53 activity. Active p53 impedes cells’ ability to enter new DNA synthesis, thus further strengthening the role of wt-p53 in preventing polyploidy. This prevention does not occur in cells whose wt-p53 activity has been severely impaired through the ectopic expression of dominant negative proteins. These findings raise a few questions that aim to investigate how widespread is the activity of JJ78:12 compound. Does JJ78:12 activate also p53 family members such as p73 and p63? If positive findings emerge from future work two important implications might be further developed from Lain’s report. First, if JJ78:12 is capable of initiating the transcriptional activity of p53 family members it will efficiently decode antitumoral signals impinging on the p53 family, independently from which a member is preferentially recruited, and consequently will maximize its effects on tumor cells (Fig. 1B). Secondly, as a consequence, JJ78:12 might affect diverse cancer cell contexts whose p53 family anti-tumoral response can be properly activated. Does JJ78:12 exert its antitumoral activities in cells carrying mutant p53 protein? It has been reported that mutant p53 cells are prone to become polyploidy through the aberrant activation of cell cycle regulated genes. This occurs mainly through the transcriptional cross-talk between mutant p53 and the transcription factor NF-Y, which cooperates with wt-p53 in repressing cell cycle regulated genes.^5,6 It can be proposed that JJ78:12 activating p73 or p63 transcriptional activity competes with gain of function mutant p53 proteins for the cooperation with specific transcriptional co-factors. Furthermore, protein complexes involving mutant p53 and p73 or p63 exist in tumor cells and result in the impairment of p53 family anticancer effects.^7,8 JJ78:12 through the transcriptional activation of p53 family members might increase the pools of free p73 and p63 and potentiate their effects over those oncogenic elicited by mutant p53 protein within the same cell context. Within this molecular scenario small molecules capable of disassembling the protein complex mutant p53/p739 (DiAgostino et al., this issue of Cell Cycle) might enhance the antitumoral effects of JJ78:12 (Fig.1C). It is reasonable to propose that small molecules whose activities impinge on specific molecular targets need to be associated with other compounds to further synergize the effects and to enlarge the spectrum of tumor response. Additional work is required to address thoroughly the questions posed by Lain’s report. We are confident that the results which emerge might hold important clues for novel anticancer approaches.

References

1. Lain S, Hollick JJ, Campbell J, Staples OD, Higgins M, Aoubala M, et al. Discovery, in vivo activity, and mechanism of action of a small-molecule of p53 activators. Cancer Cell 2008; 13:454-63.
2. Vousden KH, Lane DP. p53 in health and disease. Nat Rev Mol Cell Biol 2007; 8:275-83.
3. Bell HS, Thorsten S. Targeting the p53 family for cancer therapy-“Big brother” joins the fight. Cell Cycle 2008; 7:1726-31.
4. Staples OD, Hollick JJ, Campbell G, Higgins M, McCarthy AR, Appleyard V, et al. Characterisation, chemical optimisation and anti-tumour activity of a tubulin poison identified by a p53-based phenotypic screen. Cell Cycle 2008; 7:In this issue.
5. Di Agostino S, Strano S, Emiliozzi V, Zerbini V, Mottolese M, Sacchi A, et al. Gain of function of mutant p53: the mutant p53/NF-Y protein complex reveals an aberrant transcriptional mechanism of cell cycle regulation. Cancer Cell 2006; 10:191-202.
6. Strano S, Dell’Orso S, Di Agostino S, Fontemaggi G, Sacchi A, Blandino G. Mutant p53: an oncogenic transcription factor. Oncogene 2007; 26:2212-9.
7. Strano S, Blandino G. p73-mediated chemosensitivity: a preferential target of oncogenic mutant p53. Cell Cycle 2003; 2:348-49.
8. Li Y, Prives C. Are interactions with p63 and p73 involved in mutant p53 gain of oncogenic function? Oncogene 2007; 26:2220-5.
9. Di Agostino S, Cortese G, Monti O, Dell’Orso S, Sacchi A, Eisenstein M, et al. The disruption of the protein complex mutantp53/p73 increases selectively the response of tumor cells to anticancer drugs. Cell Cycle 2008; 7:In this issue.

Open Access Article

If p53 can't do it, ask p73

Sonia Laín
Pages 3287 - 3291
http://dx.doi.org/10.4161/cc.7.21.7236
Abstract | PDF

Promoting p53's function, without causing damage to the genome is widely accepted as a way to improve treatment of cancer. In the case of tumours where the p53 gene is not mutated or absent, there have been important advances in the recent past. Small molecules that, like the nutlins discovered by Luo Vassilev and colleagues,¹ efficiently activate wild type p53 by inhibiting the function of p53's major negative regulator mdm2 and show promising results in preclinical models as single agents or in combination with classic chemotherapeutics.² Finding compounds that instead of binding to mdm2, interact with p53 and prevent its degradation whilst still allowing its function, is also an approach under investigation.³ Restoring p53 effects in tumours that contain point mutations in the p53 gene is in some ways a bigger challenge. One possible way to achieve this is through peptides or small molecules that directly interact with mutant p53.^4,5 Because, unlike wild type p53, mutant p53 is expressed at very high levels in tumours, even if the level of mutant p53 reactivation is poor and only a small subpopulation of the mutant p53 molecules are "repaired", it is possible that this would be sufficient to exert a significant tumour cell killing effect to which normal cells carrying functional p53 would be virtually oblivious.⁶ Despite the fact that we still do not understand the mechanism of action of small molecules that like PRIMA1,⁷ selectively kill tumour cells with mutant p53, this is still an interesting approach.

In the article published by Blandino and Strano in this issue,⁸ a new possibility to exploit the differences between normal cells and cells carrying certain p53 mutations emerges. Thanks to years of thorough research on the function of p53's homologue p73 and the demonstration that inhibition of mutant p53 expression reduces tumorigenicity in animal models,⁹ there were sufficient grounds to investigate the possibility of promoting p73's function in p53-mutant tumour cells. Key to this approach is the previous finding by these authors and others that mutant p53, has the ability to interact with p73 in tumour cells.^10,11 In this paper the Blandino/Strano team have designed a series of peptides derived from the p73 DNA binding region that also interacts with p53. These small peptides (SIMPs, for Short Interfering Mutant p53 Peptides) can be introduced into cells by a standard lipofectamine transfection protocol, bind to mutant p53 and effectively disrupt the p53/p73 interaction leading to the induction of the expression of p73-dependent genes. Furthermore, at least in one tumour cell type (SKBR3 cells), SIMPs sensitise cells to the killing effect of standard chemotherapeutics such as adriamycin or cisplatin. A very important aspect of this approach is investigating the selectivity of the effects of the SIMPs in cell lines with different p53 backgrounds. In this regard, the authors fail to observe effects of the SIMPs on a cell line that is null for p53 as well as on cells that contain wild type p53. Reinforcing this selectivity further, the authors also show lack of SIMP activity in cells that bear p53 mutations other than the His175 mutation present in SKBR3 cells. In a way, the drug development approach proposed by Blandino and Strano's work hopefully resembles the initial phases in the development of the nutlins and other small molecules that inhibit mdm2's effects on p53.¹² Back in 1997 it was shown for the first time that small peptides interacting with one of the mdm2 regions that bind to p53 increase p53 levels and its activity as a transcription factor.13 Subsequently, effective biochemical assays were developed that enabled screening for small molecules that disrupt this interaction, with the discovery of the nutlins as the prime example.¹ Another groundbreaking aspect of the work by Vassilev and colleagues was the demonstration that a protein-protein interaction can be disrupted by small molecules with acceptable drug-like properties. The exciting, initial proof of concept work by Blandino and Strano opens the possibility of finding "drug-like" small molecules mimicking the effect of the SIMPs by developing a biochemical p53/p73 interaction assay that can be used as a primary screen. Hit compounds from such a screen have the potential of being selectively toxic for tumour cells with mutant p53, or at least with a particular type of p53 mutation, whilst having negligible effects on normal cells.

References

1. Vassilev LT, Vu BT, Graves B, Carvajal D, Podlaski F, Filipovic Z, et al. In vivo activation of the p53 pathway by small-molecule antagonists of MDM2. Science 2004; 300:844-8.
2. Vassilev LT. MDM2 inhibitors for cancer therapy. Trends Mol Med. 2007; 13:23-31.
3. Issaeva N, Bozko P, Enge M, Protopopova M, Verhoef LG, Masucci M, et al. Small molecule RITA binds to p53, blocks p53-HDM-2 interaction and activates p53 function in tumors. Nat Med 2004; 10:1321-8.
4. Joerger AC, Ang HC, Fersht AR. Structural basis for understanding oncogenic p53 mutations and designing rescue drugs. Proc Natl Acad Sci USA 2006; 103:15056-61.
5. Boeckler FM, Joerger AC, Jaggi G, Rutherford TJ, Veprintsev DB, Fersht AR. Targeted rescue of a destabilized mutant of p53 by an in silico screened drug. Proc Natl Acad Sci USA 2008; 105:10360-5.
6. Selivanova G, Wiman KG. Reactivation of mutant p53: molecular mechanisms and therapeutic potential. Oncogene 2007; 26:2243-54.
7. Zache N, Lambert JM, Wiman KG, Bykov VJ. PRIMA-1(MET) inhibits growth of mouse tumors carrying mutant p53. Cell Oncol 2008; 30:411-8.
8. Di Agostino S, Cortese G, Monti O, Dell'Orso S, Sacchi A, Eisenstein M, Citro G, Strano S, Blandino G. The disruption of the protein complex mutantp53/p73 increases selectively the response of tumor cells to anticancer drugs. Cell Cycle 2008; 7:In this issue.
9. Bossi G, Marampon F, Maor-Aloni R, Zani B, Rotter V, Oren M, et al. Conditional RNA interference in vivo to study mutant p53 oncogenic gain of function on tumor malignancy. Cell Cycle 2008; 7:1870-9.
10. Strano S, Munarriz E, Rossi M, Cristofanelli B, Shaul Y, Castagnoli L, et al. Physical and functional interaction between p53 mutants and different isoforms of p73. J Biol Chem 2000; 275:29503-12.
11. Gaiddon C, Lokshin M, Ahn J, Zhang T, Prives C. A subset of tumor-derived mutant forms of p53 down-regulate p63 and p73 through a direct interaction with the p53 core domain. Mol Cell Biol 2001; 21:1874-87.
12. Shangary S, Wang S. Targeting the MDM2-p53 interaction for cancer therapy. Clin Cancer Res 2008; 14:5318-24.
13. Bottger A, Bottger V, Sparks A, Liu WL, Howard SF, Lane DP. Design of a synthetic Mdm2-binding mini protein that activates the p53 response in vivo. Curr Biol 1997; 7:860-9.

Open Access Article

RHAMM in the complex p53 cell cycle network

Yosef Buganim and Varda Rotter
Pages 3287 - 3291
http://dx.doi.org/10.4161/cc.7.21.7256
Abstract | PDF

The Receptor for HA Mediated Motility (RHAMM) was initially described in 1982 as a soluble hyaluronan binding protein released by sub-confluent migrating cells.1 In 1992, RHAMM, was cloned and initially was demonstrated to play a pivotal role in locomotion. By 1995 RHAMM has already been proposed as a novel oncogene.2 Since then, many studies implicated RHAMM in several cellular processes such as motility, adhesion, wound healing, angiogenesis, migration, metastasis, invasion and growth. Furthermore, RHAMM is highly expressed in a variety of human tumors such as B-cell chronic lymphocytic leukemia, multiple myeloma, squamous cell lung carcinoma, pancreatic cancer, breast cancer, colon cancer, stomach cancer, mammary carcinoma, gastro carcinoma, endometrial carcinoma and oral squamous cell carcinoma. RHAMM is regulated by several growth-promoting factors, among them are: H-Ras, TGF-β1, FGF, β1 integrins and PKC. Although the oncogenic capabilities of RHAMM are well documented, the molecular mechanisms underling these activities are still to be elucidated. The fact that RHAMM interacts with several mitotic spindle assembly factors as well as to DNA repair genes (actin filaments, microtubles, podosomes, centrosome, calmodulin and BRCA1/BARD1) brings us closer to deciphering the function of RHAMM in cancer-related processes, such as cell motility and cell cycle progression.

In an article by Sohr and Engeland in this issue of Cell Cycle, new insights are provided regarding the expression of RHAMM during cell cycle progression.3 The authors show that RHAMM protein expression is differently regulated during cell cycle progression, reaching a peak in S phase and decrease before the maximum of RHAMM mRNA expression level is reached in G2/M. The discrepancy between RHAMM mRNA levels and protein levels led to the hypothesis that RHAMM might undergo post-translational modifications that affect its protein stabilization. This raises new questions regarding RHAMM regulation and its role in the S phase during the cell cycle.  The notion that RHAMM plays an active role in cell cycle regulation has already been proposed by others. The first evidence for this role was provided by Mohapatra and co-authors who showed that inhibition of RHAMM by several techniques induces mitotic arrest. In addition, they demonstrated that soluble RHAMM induces G2/M arrest by suppressing the expression of Cdc2/Cyclin B1, a protein kinase complex essential for mitosis.4 Maxwell and co-authors showed that a subset of cellular RHAMM localizes to the centrosome and also functions in the maintenance of spindle integrity. Deregulation of RHAMM expression inhibits mitotic progression and affects spindle architecture.5 In the study of Yang and co-authors, RHAMM expression appears to be tightly regulated during mammalian cell cycle G2/M progression and ectopic overexpression of a subset of RHAMM in 293T cells resulted in the accumulation of cells at G2/M phase predominantly in the prophase.6

One of the main cell cycle regulators is the tumor suppressor p53. p53 can transactivate cell cycle arrest genes such as p21, 14-3-3s and Gadd45, thereby leading to G1 and G2/M arrest by inhibiting the Cdc2 kinase.7  Other proteins such as reprimo, mcg10 and B99, which contribute to the G2 arrest in an unknown mechanism, are also induced by p53. In addition, p53 directly represses Cdc2, its regulator cyclinB1 and topoisomerase II to block the entry into mitosis and to strengthen the G2 arrest.7 Given that wild-type p53 is a master negative regulator of cell cycle progression, the authors examine the effect p53 might have on RHAMM. In accordance with its role in cell cycle regulation, RHAMM mRNA levels were downregulated by the wild-type p53 protein, as well as in cells treated with nutlin-3, doxorubicin or paclitaxel which stabilize p53. Although mutant forms of p53 affect some of the p53 downstream target genes,9 in this case mutant p53R175H had no impact on RHAMM expression. The authors demonstrate that p53 downregulates RHAMM expression via its promoter, which consists of both the first exon and intron, nonetheless they found no molecular mechanism that is responsible for this downregulation. It is important to mention that while activation of the p53 target genes involves direct interaction with a well known consensus DNA binding sequence, in repressed p53 target genes no such motif was discovered. By exploring the specifics of p53-dependent repression of a cluster of cell cycle-related genes, using components of the G2/M phase and mitotic spindle as an example, Tabach and co-authors found that p53-dependent repression of these components is indirect and requires the activities of p21, NFY and E2F.10 In accordance with that, it was demonstrated that p53 is associated with cyclin B2, CDC25C, and Cdc2 promoters in-vivo before and after DNA damage via the recruitment of DNA-bound NF-Y.
It is well accepted that p53 plays an important regulatory role in coordinating the gene networks controlling the cell cycle. The study by Sohr and Engeland characterized a novel negatively regulated p53 target gene that is associated with the S and G2/M checkpoints. Finding this additional branch, contributes to the understanding of the complex p53 cell cycle network and its function as a tumor suppressor.

References
1.    Turley EA. Purification of a hyaluronate-binding protein fraction that modifies cell social behavior. Biochem Biophys Res Commun 1982; 108:1016-24.
2.    Hall CL, Yang B, Yang X, Zhang S, Turley M, Samuel S, Lange LA, Wang C, Curpen GD, Savani RC, Greenberg AH, Turley EA. Overexpression of the hyaluronan receptor RHAMM is transforming and is also required for H-ras transformation. Cell 1995; 82:19-26.
3. Sohr S and Engeland K. RHAMM is differentially expressed in the cell cycle and downregulated by the tumor suppressor p53. Cell Cycle 2008; 7:In this issue.
4.    Mohapatra S, Yang X, Wright JA, Turley EA, Greenberg AH. Soluble hyaluronan receptor RHAMM induces mitotic arrest by suppressing Cdc2 and cyclin B1 expression. J Exp Med 1996; 183:1663-8.
5.    Maxwell CA, Keats JJ, Crainie M, Sun X, Yen T, Shibuya E, Hendzel M, Chan G, Pilarski LM. RHAMM is a centrosomal protein that interacts with dynein and maintains spindle pole stability. Mol Biol Cell 2003; 14:2262-76.
6.    Yang CW, Su JY, Tsou AP, Chau GY, Liu HL, Chen CH, Chien CY, Chou CK. Integrative genomics based identification of potential human hepatocarcinogenesis-associated cell cycle regulators: RHAMM as an example. Biochem Biophys Res Commun 2005; 330:489-97.
7.    Schwartz D, Rotter V. p53-dependent cell cycle control: response to genotoxic stress. Semin Cancer Biol 1998; 8:325-36.
8.    Taylor WR, Stark GR. Regulation of the G2/M transition by p53. Oncogene  2001; 20:1803-15.
9.    Buganim Y, Kalo E, Brosh R, Besserglick H, Nachmany I, Rais Y, Stambolsky P, Tang X, Milyavsky M, Shats I, Kalis M, Goldfinger N, Rotter V. Mutant p53 protects cells from 12-O-tetradecanoylphorbol-13-acetate-induced death by attenuating activating transcription factor 3 induction. Cancer Res 2006; 66:10750-9.
10.    Tabach Y, Milyavsky M, Shats I, Brosh R, Zuk O, Yitzhaky A, Mantovani R, Domany E, Rotter V, Pilpel Y. The promoters of human cell cycle genes integrate signals from two tumor suppressive pathways during cellular transformation. Mol Syst Biol 2005; 1: 2005.0022




EDITORIAL

Open Access Article

Regenerative medicine: Clinical relevance, implications, and limitations of the stem cell-based therapies

Gennadi V. Glinsky
Pages 3292 - 3293
http://dx.doi.org/10.4161/cc.7.21.6973
Abstract | PDF

Many believe that success of the concept of regenerative medicine critically depends on our unlimited access to the human embryonic stem cells for scientific and therapeutic applications. Recent ground-breaking studies seem to challenge this broadly held view by demonstrating a remarkable healing power of mammalian adult (somatic) stem cells. However, mounting evidence of involvement of stemness pathways in development of therapy-resistant metastatic cancers argue for more careful considerations of potential risk and benefits associated with the stem cell-based therapies.




EXTRA VIEWS

Open Access Article

Gene regulation by SINES and inosines: biological consequences of A-to-I editing of Alu element inverted repeats

Ling-Ling Chen and Gordon G. Carmichael
Pages 3294 - 3301
http://dx.doi.org/10.4161/cc.7.21.6927
Abstract | PDF

The Alu elements are conserved ~300 nucleotide long repeat sequences that belong to the SINE family of retrotransposons found abundantly in primate genomes. Although the vast majority of Alu elements appear to be genetically inert, it has been tempting to consider the great majority of them as “junk DNA.” However, a growing line of evidence suggests that transcribed Alu RNAs are in fact functionally involved in a number of diverse biological processes. Pairs of inverted Alu repeats in RNA can form duplex structures that lead to A-to-I editing by the ADAR enzymes. In this review we discuss the possible biological effects of Alu editing, with particular focus on the regulation of gene expression by inverted Alu repeats in the 3’-UTR regions of mRNAs.

Open Access Article

Chromatin regulation Tip(60)s the balance in embryonic stem cell self-renewal

Thomas G. Fazzio, Jason T. Huff and Barbara Panning
Pages 3302 - 3306
http://dx.doi.org/10.4161/cc.7.21.6928
Abstract | PDF

Histone modifications affect chromatin dynamics on several levels by serving as binding sites for regulatory proteins. In many cell types, including embryonic stem cells (ESCs), a subset of genes is marked with histone modifications thought to be both activating and repressing: H3 lysine 4 trimethylation (H3K4me3) and lysine 27 trimethylation (H3K27me3), respectively. As a result, genes bearing this "bivalent" mark are transcribed at low levels, but are primed for activation, should the cell receive the appropriate cues during differentiation. Recently, we found that the Tip60-p400 acetyltransferase and histone exchange complex is necessary to maintain normal self-renewal in mouse ESCs. While Tip60-p400 has histone acetyltransferase activity, which is generally associated with transcriptional activation, it acts predominantly as a repressor of genes expressed during differentiation. Surprisingly, in ESCs Tip60-p400 localizes to the promoters of genes marked by H3K4me3, which include both highly expressed genes and "bivalent" genes expressed at low levels. Tip60-p400 acetylates histones at these targets, including the promoters for developmental regulators it helps to silence in ESCs. This suggests that the effect of chromatin modifications on transcription is not always simply positive or negative. Rather, we propose that the impact of specific modifications at each promoter is determined by the chromatin context in which they are found.

Open Access Article

Fbxw7 in cell cycle exit and stem cell maintenance: Insight from gene-targeted mice

Ichiro Onoyama and Keiichi I. Nakayama
Pages 3307 - 3313
http://dx.doi.org/10.4161/cc.7.21.6931
Abstract | PDF

Regulation of the exit of cells from the cell cycle is important in the development of multicellular organisms and is also implicated in the maintenance of stem cells. Furthermore, defects in cell cycle exit are thought to be a major cause of cancer. However, the mechanisms responsible for regulation of cell cycle exit have remained largely unknown. Fbxw7 is the F-box protein subunit of an SCF-type ubiquitin ligase complex that targets positive regulators of the cell cycle—including cyclin E, c-Myc, Notch, and c-Jun—for ubiquitylation and subsequent degradation by the 26S proteasome in order to promote cell cycle exit. Consistent with such a function, mutations of the Fbxw7 gene have been detected in various human malignancies. We have recently generated conventional and conditional Fbxw7 knockout mice and examined stem cells, progenitor cells, and differentiated cells in the mutant animals for cell cycle defects. Here we summarize the pleiotropic phenotypes of Fbxw7 deficiency in various cell types including T cells, hematopoietic stem cells, and embryonic fibroblasts. Such phenotypes have provided insight into the biological roles of Fbxw7 in cell cycle exit, stem cell maintenance, and oncosuppression.

Open Access Article

Transforming human blood stem and progenitor cells: A new way forward in leukemia modeling

James C. Mulloy, Mark Wunderlich, Yi Zheng and Junping Wei
Pages 3314 - 3319
http://dx.doi.org/10.4161/cc.7.21.6951
Abstract | PDF

MLL-AF9 (MA9) is a leukemia fusion gene formed upon translocation of the AF9 gene on chromosome 9 and the MLL gene on chromosome 11. MA9 is commonly found in acute myeloid leukemia (AML) and occasionally in acute lymphoid leukemia and is associated with intermediate to poor outcome. The specific signaling pathways downstream of MA9 are still poorly understood. We have recently described a model system whereby we expressed the MA9 fusion gene in human CD34+ Umbilical Cord Blood (UCB) cells and showed that these cells transformed to acute myeloid or lymphoid leukemia when injected into immunodeficient mice. The Mixed Lineage Leukemia oncogenes are unique in this model system in that they promote full transformation of primary human blood cells, while all other leukemia-associated oncogenes tested thus far have induced only partial phenotypes. Here we provide an update on the use of this system for modeling human leukemia and its potential application for therapeutic testing of novel compounds to treat the disease. We focus specifically on the Rho family of small guanosine triphosphatases (GTPases) as potential therapeutic targets, which we have implicated in the pathogenesis of AML associated with MA9 expression.

Open Access Article

Delphinidin, a dietary anthocyanidin in pigmented fruits and vegetables: A new weapon to blunt prostate cancer growth

Bilal Bin Hafeez, Mohammad Asim, Imtiaz Ahmad Siddiqui, Vaqar Mustafa Adhami, Imtiyaz Murtaza and Hasan Mukhtar
Pages 3320 - 3326
http://dx.doi.org/10.4161/cc.7.21.6969
Abstract | PDF

In a recent publication, we have shown that delphinidin, an anthocyanidin induces apoptosis and cell cycle arrest in highly metastatic human prostate cancer (PCa) PC3 cells. Extending these studies, we provide additional evidence that delphinidin induces apoptosis and cell cycle arrest in androgen refractory human PCa 22Rn1 cells and that these effects are concomitant with inhibition of NF-kB. We observed that delphinidin treatment to 22Rn1 cells resulted in a dose-dependent (i) G2/M phase cell cycle arrest, (ii) induction of apoptosis (iii) and inhibition of NF-kB signaling. The induction of apoptosis by delphinidin was mediated via activation of caspases since a general caspase inhibitor Z-VAD-FMK significantly reversed this effect. Delphinidin treatment to cells resulted in a dose-dependent decrease in (i) phosphorylation of IKKgamma (NEMO), (ii) phosphorylation of NF-kB inhibitory protein IBα, (iii) phosphorylation of NF-kB/p65 at Ser536 and NF-kB/p50 at Ser529, (iv) NF-kB/p65 nuclear translocation, and (v) NF-kB DNA binding activity. Taken together, our data show that delphinidin induces apoptosis of both androgen independent and androgen refractory human PCa cells via activation of caspases and in addition, this effect might be due to inhibition of NF-kB signaling. We suggest that delphinidin could be developed as a novel agent against PCa.

Open Access Article

Elevated RNA polymerase III transcription drives proliferation and oncogenic transformation

Lynne Marshall
Pages 3327 - 3329
http://dx.doi.org/10.4161/cc.7.21.6970
Abstract | PDF

RNA polymerase (Pol) III is the largest of the RNA polymerases with seventeen subunits [1]. It is responsible for the production of short, untranslated RNAs that play important roles in determining the biosynthetic capacity of the cell. Pol III transcription is generally elevated in transformed cells and tumours; however, it has remained a matter for conjecture as to whether this activation is a cause or consequence of the transformation process.

Open Access Article

Beyond heterochromatin: SIR2 inhibits the initiation of DNA replication

Catherine A. Fox and Michael Weinreich
Pages 3330 - 3334
http://dx.doi.org/10.4161/cc.7.21.6971
Abstract | PDF

Over the last decade, data have accumulated that support a role for chromatin structure in regulating the initiation of DNA replication and its timing during S-phase.(1-3) However, the mechanisms underlying how chromatin structure influences replication initiation are not always understood. For example, in Drosophila histone acetylation at the ACE3 and Ori-β sequences near one of the amplified chorion loci is correlated with ORC (origin recognition complex) binding and re-replication of this locus.(4, 5) Whether histone acetylation promotes ORC binding or some later step in replication is not known. In yeast, hypo-acetylated heterochromatin and telomeric regions replicate late in S-phase(6, 7) but the mechanisms that restrict the initiation of replication at these loci are not fully understood. Nonetheless, it seems likely that histone acetylation and other types of histone modification will significantly impact DNA replication. A recent study published in Molecular Cell(8) reveals a role for the conserved NAD+-dependent histone deacetylase, Sir2(9-13), in inhibiting the assembly of the multiprotein complex necessary for the selection and activation of yeast replication origins. Here, we highlight key conclusions from this study, place them in perspective with earlier work, and outline important future questions.

Open Access Article

Signalling molecules, growth regulators and cell cycle control in Drosophila

Héctor Herranz and Marco Milán
Pages 3335 - 3337
http://dx.doi.org/10.4161/cc.7.21.6996
Abstract | PDF

During the development of a given organ or tissue within a multicellular organism, growth and patterning are controlled in a coordinated manner by the activity of a discrete number of signalling molecules and their corresponding pathways to give rise to a well formed structure with a particular size, shape and pattern. Understanding how cells of different tissues or organs translate in a context dependent manner the activity of these pathways into an activation or repression of the cell cycle machinery is one of the most intriguing questions in developmental and cancer biology nowadays. Here we revise the different roles of the signalling molecules Notch and Wingless in the regulation of cell cycle progression in the developing eye and wing imaginal discs of Drosophila and propose that depending on how growth regulators are regulated in a context dependent manner by the activity of these pathways, signalling molecules might have tumour suppressor or oncogene activity.




REVIEW

Open Access Article

Insulin and aging

Andrzej Bartke
Pages 3338 - 3343
http://dx.doi.org/10.4161/cc.7.21.7012
Abstract | PDF

In invertebrates, signaling pathways homologous to mammalian insulin and insulin-like growth factor (IGF-1) signal transduction have a major role in the control of longevity. There are numerous indications that these pathways also influence aging in mammals, but separating the role of insulin from the effects of IGF-1 and growth hormone (GH) is difficult. In mice, selective disruption of the insulin receptor in the adipose tissue extends longevity. Increases in lifespan were also reported in mice with deletion of insulin receptor substrate 1 (IRS1) in whole body or IRS2 only in the brain. GH deficiency or resistance in mutant mice leads to hypoinsulinemia and enhanced insulin sensitivity along with remarkably extended longevity. These characteristics resemble animals subjected to calorie restriction. Studies of physiological characteristics and polymorphisms of insulin-related genes in exceptionally long-lived people suggest a role of insulin signaling in the control of human aging.




CONCEPTUAL REVIEW

Open Access Article

Aging: ROS or TOR

Mikhail V. Blagosklonny
Pages 3344 - 3354
http://dx.doi.org/10.4161/cc.7.21.6965
Abstract | PDF

It is widely believed that aging is caused by the accumulation of random molecular damage due to reactive oxygen species (ROS). Here I discuss evidence for and against the ROS theory. Remarkably, even supporting evidence has an alternative explanation, consistent with the model that aging is driven by the TOR (target of rapamycin) signaling pathway.




REPORTS

Open Access Article

Growth stimulation leads to cellular senescence when the cell cycle is blocked

Zoya N. Demidenko and Mikhail V. Blagosklonny
Pages 3355 - 3361
http://dx.doi.org/10.4161/cc.7.21.6919
Abstract | PDF

We tested a hypothesis that activation of growth-promoting pathways is required for cellular senescence. In the presence of serum, induction of p21 caused senescence, characterized by beta-Galactosidase staining, cell hypertrophy, increased levels of cyclin D1 and active TOR (target of rapamycin, also known as mTOR). Serum starvation and rapamycin inhibited TOR and prevented the expression of some senescent markers, despite high levels of p21 and cell cycle arrest. In the presence of serum, p21-arrested cells irreversibly lost proliferative potential. In contrast, when cells were arrested by p21 in the absence of serum, they retained the capacity to resume proliferation upon termination of p21 induction. In normal human cells such as WI38 fibroblasts and retinal pigment epithelial (RPE) cells, serum starvation caused quiescence, which was associated with low levels of cyclin D1, inactive TOR and slim-cell morphology. In contrast, cellular senescence with high levels of TOR activity was induced by doxorubicin (DOX), a DNA damaging agent, in the presence of serum. Inhibition of TOR partially prevented senescent phenotype caused by DOX. Thus growth stimulation coupled with cell cycle arrest leads to senescence, whereas quiescence (a condition with inactive TOR) prevents senescence.

Open Access Article

Ceramide promotes apoptosis in chronic myelogenous leukemia-derived K562 cells by a mechanism involving caspase-8 and JNK

Alina Felicia Nica, Chun Chui Tsao, Julie C. Watt, Tilahun Jiffar, Svitlana Kurinna, Paul Jurasz, Marina Konopleva, Michael Andreeff, Marek W. Radomski and Peter P. Ruvolo
Pages 3362 - 3370
http://dx.doi.org/10.4161/cc.7.21.6894
Abstract | PDF

Ceramide is a sphingolipid that activates stress kinases such as p38 and c-JUN N-Terminal Kinase (JNK). Though Chronic Myelogenous Leukemia (CML) derived K562 cells resist killing by short chain C2-ceramide, we report here that longer chain C6-ceramide promotes apoptosis in these cells. C6-ceramide induces cleavage of Caspase-8 and Caspase-9, but only Caspase-8 is required for apoptosis. The sphingolipid killed CML derived KBM5 cells and, to a lesser extent, imatinib-resistant KBM5-STI cells suggesting that BCR-ABL can not completely block C6-ceramide-induced apoptosis but the kinase may regulate the process. BCR-ABL is known to suppress Protein Phosphatase 2A (PP2A) in CML cells. While C6-ceramide can activate PP2A in acute leukemia cells, the sphingolipid did not activate the phosphatase in K562 cells. C6-ceramide did not activate p38 kinase but did promote JNK activation and phosphorylation of JUN. Inhibition of JNK by pharmacological agent protected K562 cells from C6-ceramide suggesting that JNK plays an essential role in C6-ceramide mediated apoptosis. Furthermore, the sphingolipid promoted MCL-1 phosphorylation by a mechanism that, at least in part, involves JNK. The findings presented here suggest that Caspase-8, JNK, and perhaps MCL-1 may play important roles in regulating cell death and may represent new targets for therapeutic strategies for CML.

Open Access Article

Modification at HuR(S242) alters HuR localization and proliferative influence

Hyeon Ho Kim, Xiaoling Yang, Yuki Kuwano and Myriam Gorospe
Pages 3371 - 3377
http://dx.doi.org/10.4161/cc.7.21.6895
Abstract | PDF

HuR is predominantly nuclear but following exposure to stress and mitogens, it can translocate to the cytoplasm where it stabilizes target mRNAs and/or modulates their translation. Several phosphorylation sites in a central 'hinge' region of HuR have been reported to affect its nucleocytoplasmic shuttle: phosphorylation by PKC at serine (S)221 and by Cdk1 at S202. Here, we investigated if there are additional putative phosphorylation sites within the HuR hinge region capable of influencing its cytoplasmic abundance. We systematically mutated all seven serine residues within the shuttling hinge domain to the nonphosphorylatable residue alanine (A), S197A, S202A, S221A, S229A, S232A, S241A, and S242A. Using HeLa cells as the study system, we found that the HuR(S242A) mutant was more abundant in the cytoplasm in both untreated cells and in cells treated with short-wavelength ultraviolet light or with an inhibitor of Cdk1. Conversely, mutation of S242 to aspartic acid (D), rendered the phosphomimetic HuR(S242D) nuclear under all treatment conditions. S242 mutations did not influence HuR stability, but HuR(S242A) showed increased association with target cyclin A2 and cyclin B1 mRNAs. Accordingly, expression of HuR(S242A) led to increased cyclin mRNA stability and heightened cell proliferation rates. Our findings suggest that HuR phosphorylation at S242 hinders its cytoplasmic localization, its function as a posttranscriptional regulator, and its proliferative influence.

Open Access Article

Arf-induced turnover of the nucleolar nucleophosmin-associated SUMO-2/3 protease Senp3

Mei-Ling Kuo, Willem den Besten, Mary C. Thomas and Charles J. Sherr
Pages 3378 - 3387
http://dx.doi.org/10.4161/cc.7.21.6930
Abstract | PDF

The stabilization and subcellular localization of the p19Arf tumor suppressor protein and the SUMO-2/3 deconjugating protease Senp3 each depend upon their binding to the abundant nucleolar protein nucleophosmin (Npm/B23). Senp3 and p19Arf antagonize each other’s functions in regulating the SUMOylation of target proteins including Npm itself. The p19Arf protein triggers the sequential phosphorylation, polyubiquitination, and rapid proteasomal degradation of Senp3, and this ability of p19Arf to accelerate Senp3 turnover also depends on the presence of Npm. In turn, endogenous p19Arf and Senp3 are both destabilized in viable Npm-null mouse embryo fibroblasts (that also lack p53), and reintroduction of the human NPM protein into these cells reverses this phenotype. NPM mutants that retain their acidic and oligomerization domains can re-stabilize both p19Arf and Senp3 in this setting, but the nucleolar localization of NPM is not strictly required for these effects. Knockdown of Senp3 with shRNAs mimics the anti-proliferative functions of p19Arf in cells that lack p53 alone or in triple knock-out cells that lack the Arf, Mdm2 and p53 genes. These findings reinforce the hypothesis that the p53-independent tumor suppressive functions of p19Arf may be mediated by its ability to antagonize Senp3, thereby inducing cell cycle arrest by abnormally elevating the cellular levels of SUMOylated proteins.

Open Access Article

Identification of MSA1, a cell cycle-regulated, dosage suppressor of drc1/sld2 and dpb11 mutants

Ju-mei Li, Michael T. Tetzlaff and Stephen J. Elledge
Pages 3388 - 3398
http://dx.doi.org/10.4161/cc.7.21.6932
Abstract | PDF

The Dpb11 and Drc1/Sld2 proteins form a complex that is critical for the initiation of DNA replication. In this study we identify MSA1 as a high copy suppressor of a drc1-1 mutant. MSA1 overproduction can also suppress the temperature sensitivity of dpb11-1 and pol2-12 mutants. Reciprocally, msa1 deletion exacerbates the mutant phenotypes of both drc1/sld2 and dpb11 mutants and msa1 deletion alone results in a delay in S phase entry of synchronous cells indicating a positive role for MSA1 in DNA replication. Paradoxically, MSA1 overproduction is deleterious to cdc6-1, cdc7-1, cdc28-1N and cdc14-1 mutants indicating a complex relationship with DNA replication and cell cycle regulatory genes. The Msa1 protein is tightly cell cycle regulated. Msa1 and its paralog, Msa2, both accumulate in highly modified forms just as cells commit to enter S phase and then are rapidly destroyed. MSA1 represents a new cell cycle regulated gene important for S phase entry.

Open Access Article

PCNA is ubiquitinated by RNF8

Sufang Zhang, Jennifer Chea, Xiao Meng, Yajing Zhou, Ernest Y.C. Lee and Marietta Y.W.T. Lee
Pages 3399 - 3404
http://dx.doi.org/10.4161/cc.7.21.6949
Abstract | PDF

The ubiquitination of PCNA is an essential event in the operation of the DNA Damage Tolerance (DDT) pathway that is activated after DNA damage caused by UV or chemical agents during S-phase. This pathway allows the bypass of DNA damage by translesion synthesis that would otherwise cause replication fork stalling. PCNA is mono-ubiquitinated by Rad18-Rad6, and polyubiquitinated by Rad5-Ubc13/Uev1 in the DDT pathway. Mono-and polyubiquitination of PCNA are key processes in the translesion bypass and template switching sub-pathways of the DDT. DNA damage by IR causes DSBs, which trigger the DNA Damage Response (DDR). The ubiquitin ligase RNF8 has a critical role in the assembly of BRCA1 complexes at the DSBs in the DDR. We show that RNF8 readily mono-ubiquitinates PCNA in the presence of UbcH5c, and polyubiquitinates PCNA in the added presence of Ubc13/Uev1a. These reactions are the same as those performed by Rad18-Rad6 and Rad5-Ubc13. RNF8 depletion suppressed both UV and MNNG-stimulated mono-ubiquitination of PCNA, revealing that an RNF8-dependent pathway for PCNA ubiquitination is operative in vivo. These findings provide evidence that RNF8, a key E3 ligase in the DDR, may also play a role in the DDT.

Open Access Article

Cell cycle-dependent regulation of SFK, JAK1 and STAT3 signaling by the protein tyrosine phosphatase TCPTP

Ben J. Shields, Naomi W. Court, Christine Hauser, Patricia E. Bukczynska and Tony Tiganis
Pages 3405 - 3416
http://dx.doi.org/10.4161/cc.7.21.6950
Abstract | PDF

Janus-activated kinases (JAKs) and Src family kinases (SFKs) and their common substrate signal transducer and activator of transcription (STAT)-3 are frequently hyperactivated in human cancer contributing to the proliferative drive by promoting G1/S and G2/M progression. Previous studies have established that the protein tyrosine phosphatase TCPTP can dephosphorylate and inactivate the SFK and JAK protein tyrosine kinases (PTKs) to attenuate cytokine signalling in vivo. In this study we determined whether TCPTP regulates SFK and JAK signalling during the cell cycle. We used primary mouse embryonic fibroblasts (MEFs) isolated from TCPTP-/- versus +/+ mice, immortalised TCPTP-/- MEFs versus those reconstituted with physiological levels of TCPTP and HeLa cells in which TCPTP protein levels had been suppressed by RNA interference, to establish TCPTP as a negative regulator of SFK, JAK1 and STAT3 signalling during the cell cycle. We found that the progression of TCPTP-deficient MEFs after the G1 restriction point into S-phase was enhanced. We used RNA interference and pharmacological inhibitors to demonstrate that elevated SFK and downstream phosphatidylinositol 3-kinase signalling but not JAK1 or STAT3 signalling were required for the enhanced G1/S transition. These results identify TCPTP as a negative regulator of the cell cycle.

Open Access Article

Characterization, chemical optimization and anti-tumor activity of a tubulin poison identified by a p53-based phenotypic screen

Oliver D. Staples, Jonathan J. Hollick, Johanna Campbell, Maureen Higgins, Anna R. McCarthy, Virginia Appleyard, Karen E. Murray, Lee Baker, Alastair Thompson, Sebastien Ronseaux, Alex Slawin, David P. Lane, Nicholas J. Westwood and Sonia Laín
Pages 3417 - 3427
http://dx.doi.org/10.4161/cc.7.21.6982
Abstract | PDF

A robust p53 cell based assay that exploits p53's function as a transcription factor was used to screen a small molecule library and identify bioactive small molecules with potential antitumor activity. Unexpectedly, the majority of the highest ranking hit compounds from this screen arrest cells in mitosis and most of them impair polymerisation of tubulin in cells and in vitro. One of these novel compounds, JJ78:1, was subjected to structure-activity relationship studies and optimised leading to the identification of JJ78:12. This molecule is significantly more potent than the original hit JJ78:1, as it is active in cells at two-digit nanomolar concentrations and shows clear antitumor activity in a mouse xenograft model as a single agent. The effects of nocodazole, a well established tubulin poison, and JJ78:12 on p53 levels are remarkably similar, supporting that tubulin depolymerisation is the main mechanism by which JJ78:12 treatment leads to p53 activation in cells. In summary, these results identify JJ78:12 as a potential cancer therapeutic, demonstrate that screening for activators of p53 in a cell-based assay is an effective way to identify inhibitors of mitosis progression and highlights p53's sensitivity to alterations during mitosis.

Open Access Article

Rad24 truncation, coupled with altered telomere structure, promotes cdc13-1 suppression in S. cerevisiae

Vanessa Y. Small, Charles Chuang and Constance I. Nugent
Pages 3428 - 3439
http://dx.doi.org/10.4161/cc.7.21.6983
Abstract | PDF

Distinguishing telomeres from DNA double strand breaks is critical for genome stability. In S. cerevisiae, the Cdc13 single-strand telomere binding protein is critical for protecting chromosome ends. The C-rich telomere strand is lost at high temperatures in cdc13-1 strains, leading to activation of the DNA damage checkpoint and cell inviability. Through a screen performed to identify activities involved in telomere C-strand loss, we identified two new rad24 alleles. Rad24 is an alternate Rfc1 subunit, functioning to load the 9-1-1 checkpoint clamp. In each rad24 allele, a transposon inserted within the RAD24 coding region leads to expression of different carboxyl-terminal portions of Rad24, deleting or truncating the amino-terminus. We show that an intact Rad24 amino-terminus is necessary for its checkpoint function. Interestingly, the initial cdc13-1 rad24-2 strains grew at 36˚C, but the extent of suppression associated with rad24-2 weakened in serial backcrosses, and cdc13-1 segregants from these crosses showed a modest increase in temperature resistance. Moreover, while a RAD24 plasmid suppressed the checkpoint defect in the initial cdc13-1 rad24-2 strain, the temperature resistance was only partially suppressed. These data suggest that the TG1-3 amplification observed in this strain contributes to the suppression phenotype. By recreating the rad24-2 allele in a strain with normal telomeres, we find that, relative to the rad24-∆ allele, rad24-2 increases the frequency of obtaining cdc13-1 cells capable of growth at high temperatures. Our hypothesis is that the Rad24-2 truncation protein affects telomere structure or recombination in a manner distinct from rad24-∆.

Open Access Article

The disruption of the protein complex mutantp53/p73 increases selectively the response of tumor cells to anticancer drugs

Silvia Di Agostino, Giancarlo Cortese, Olimpia Monti, Stefania Dell'Orso, Ada Sacchi, Miriam Eisenstein, Gennaro Citro, Sabrina Strano and Giovanni Blandino
Pages 3440 - 3447
http://dx.doi.org/10.4161/cc.7.21.6995
Abstract | PDF

Many in vitro and in vivo evidence have shown that the status of p53 is a key determinant in the response of tumor cells to anticancer treatment. Here we provide evidence that peptide-mediated targeting of the protein complex mutantp53/p73 enhances the response of mutant p53 tumor cells to commonly used anticancer drugs. Indeed, we show that the disruption of the protein complex mutantp53/p73 and the consequent restoration of p73 transcriptional effects, through the activity of short interfering peptides, render mutant p53 cells more prone to the killing of adriamycin and cisplatin. Of note, the activity of the short interfering peptides is mutant p53 specific and causes no effects on wt-p53 and p53 null cells. Our findings highlight the protein complex mutantp53/p73 as a molecular target, whose successful overriding through the selective activity of small interfering peptides, might contribute to the optimization of mutant p53 tumor treatments.

Open Access Article

RHAMM is differentially expressed in the cell cycle and downregulated by the tumor suppressor p53

Sindy Sohr and Kurt Engeland
Pages 3448 - 3460
http://dx.doi.org/10.4161/cc.7.21.7014
Abstract | PDF

The receptor for hyaluronan-mediated motility RHAMM exerts different functions in the cell as well as on the cell membrane. RHAMM can be exported to the cell surface where it binds hyaluronic acid (HA) and interacts with the HA receptor CD44. Processes like cell motility, wound healing and invasion are modulated by RHAMM. Intracellularly, RHAMM is associated with the cytoskeleton, microtubules, centrosomes and the mitotic spindle. It participates in the control of mitotic spindle stability and integrity. Furthermore, RHAMM is overexpressed in several cancer tissues. We found that RHAMM expression is differentially regulated during the cell cycle and that cell cycle-dependent synthesis of RHAMM is controlled by its promoter on the transcriptional level. RHAMM protein levels follow mRNA expression in the early phases of the cell cycle. However, they already peak in S phase and decrease before the maximum of RHAMM mRNA expression is reached in G2/M. Furthermore, RHAMM expression is downregulated by the tumor suppressor p53. This regulation is observed in a transgenic p53-inducible cell system as well as in cells with wild-type p53 treated with nutlin-3, doxorubicin or paclitaxel. Reporter assays demonstrated that the repression by p53 is regulated on the transcriptional level by the RHAMM promoter also comprising the first exon and the first intron of the gene. In general, our data support a role of RHAMM already in S phase. Additionally, p53-dependent downregulation is consistent with an oncogenic function of RHAMM and the recently reported tumor-suppressive function of CD44 transcriptional repression by p53.

Open Access Article

Role of TRF2 in the assembly of telomeric chromatin

Roberta Benetti, Stefan Schoeftner, Purificación Muñoz and Maria A. Blasco
Pages 3461 - 3468
http://dx.doi.org/10.4161/cc.7.21.7013
Abstract | PDF

Telomeric chromatin is composed of TTAGGG repeats bound by the shelterin complex. Mammalian telomeres also contain arrays of nucleosomes enriched in histone heterochromatic marks, as well as are associated by long non-coding telomeric RNAs. In addition, the adjacent subtelomeric DNA repeats are heavily methylated. This repressive chromatin environment at telomeres is thought to suppress the expression of closely located genes, a phenomenon known as "telomere position effect" (TPE), which can be influenced by telomere length and the telomeric chromatin. Here, we address the role of TRF2, a major telomere component, in the epigenetic regulation of telomeres. To this end, we took advantage of transgenic mice that over express TRF2 under the keratin 5 (K5) promoter (K5TRF2 mice). K5TRF2 mice have very short telomeres and an increased susceptibility to develop skin cancer. Interestingly, K5TRF2 cells show an aberrant nucleosomal organization at telomeres. In particular, K5TRF2 cells display a loss of heterochromatin marks at telomeric and subtelomeric repeats, which is concomitant with decreased abundance of core histones H3 and H4 and with an increased nucleosomal spacing at telomeres. Telomere shortening in K5TRF2 mice is also accompanied by decreased abundance of telomeric transcripts. Together, these findings indicate a previously unnoticed role for TRF2 in the assembly of telomeric chromatin.




LETTERS TO THE EDITOR

Open Access Article

Distinct but complementary roles of Fas ligand and Bim in homeostatic T cell apoptosis

Daniela Kassahn, Thomas Brunner and Nadia Corazza
Pages 3469 - 3471
http://dx.doi.org/10.4161/cc.7.21.6929
PDF

Open Access Article

Role of Tcf-DNA binding and the chromatin remodeling factor Brg-1 in the modulation of Wnt activity by butyrate

Michael Bordonaro, Darina L. Lazarova and Alan C. Sartorelli
Pages 3472 - 3473
http://dx.doi.org/10.4161/cc.7.21.6933
Abstract | PDF

No abstract available.