Table of Contents

Volume 7, Issue 20

  October 15, 2008

  Pages 3111 - 3282

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 Issue Catalogue (MARC XML)

NEWS

Open Access Article

Pushing cancer cells to commit suicide: Is 072RB, the new BH3 mimetic, up to the job?

Zbigniew Darzynkiewicz
Page 3111
http://dx.doi.org/10.4161/cc.7.20.7063
Abstract | PDF

It has been known since the early 90s that apoptosis is the mode of death of cancer cells during chemotherapy.1 Propensity of cells to undergo apoptosis is modulated by the balance of pro-apoptotic versus anti-apoptotic members of Bcl-2 family proteins.2 Mitochondrial outer membrane permeabilization (MOMP) which leads to release of cytochrome c and other apoptogenic factors triggering apoptosis occurs as a result of shift of this balance towards pro-apoptotic Bcl-2 proteins. Furthermore, constitutive prevalence of the anti-apoptotic Bcl-2 family proteins is considered to promote cancer development; the classic example is B-cell lymphoma. Anticancer strategies therefore, were designed that rely on promoting apoptosis of cancer cells via altering the balance among the interacting Bcl-2 proteins. One strategy involves the use of antisense oligonucleotides targeting anti-apoptotic Bcl-2 proteins. Preclinical and clinical investigations on the drugs developed along this strategy [e.g. Oblimersen (Genasense®)] are already well advanced. Another, attractive approach is to use agents that mimic the Bcl-2 homology 3 (BH3) domains of the pro-apoptotic Bcl-2 family proteins (BH3 mimetics). Their mode of action involves competitive binding to surface hydrophobic grooves of anti-apoptotic Bcl-2 members thereby releasing the pro-apoptotic Bcl-2 molecules otherwise sequestered in complexes with the anti-apoptotic ones.2-4 The most investigated BH3 mimetic ABT-737 demonstrated distinct antitumor activity in vitro and in vivo against some leukemia types and solid tumors.3,5

In the article published in this issue of Cell Cycle6 Ponassi and her collaborators describe a novel BH3 mimetic, named 072RB, constructed by replacing specific moieties of Bim-BH3 with natural and non-natural aminoacids and adding an internalizing sequence. In elegant studies the authors convincingly demonstrate internalization and mitochondrial localization of 072RB followed by suppression of growth and apoptotic death of cells of leukemia cell lines. They also observed lethal ex vivo effects of 072RB on AML leukemic cells as well as remarkable inhibition of growth of xenografted human AML cells in NOD/SCID mice with no evidence of toxicity to normal tissue. Normal human lymphocytes, whether quiescent or mitogenically stimulated, were resistant to this BH3 mimetic. An important virtue of 072RB is resistance to proteolysis conferring its stability when used in vivo.

The interplay between the pro-apoptotic and anti-apoptotic Bcl-2 family members is rather complex because depending on cell type and the agent that induces apoptosis different members interact with each other. The mechanism of these interactions is still not fully understood. According to the “different affinity” model the BH3-only proteins Bad and Bmf target Bcl-2, Bcl-w and Bcl-xL, Noxa targets Mcl-1 and A1 whereas Bim and Puma target all the above pro-survival Bcl-2 proteins with comparable affinities.3 In the “direct activator” model Bim, tBid and Puma are the most downstream molecules, directly binding to Bax/Bak and thereby preventing their release, oligomerization and MOMP. In either of these models therefore, the Bim-activating BH-3 mimetic, such as 072RB, is expected to have wider spectrum of activity towards different cell types and different inducers of apoptosis than for instance ABT-737, as the latter, because of its Bad-like structure, does not target Mcl-1.

It is too early to predict whether BH3 mimetics bestow the breakthrough in cancer therapy. Their unique mechanism of action specifically targeting apoptotic machinery raises hopes that this may be the case.3 The new BH3 mimetic 072RB described by Ponassi et al.6 has all attributes to become the leading member of this new class of anticancer drugs. 072RB definitely deserves further evaluation in clinical trials to reveal its therapeutic capabilities whether used as a single agent or in combinatorial therapy.

References

1. Gorczyca W, at al. Leukemia 1993; 7:659-70.
2. Fletcher JI, et al. Cell Cycle 2008; 7:39-44.
3. Labi V, et al. Cell Death Differ 2008; 15:977-87.
4. Wade et al., Cell Cycle 2008; 7:1973-82.
5. Konoplewa M, et al. Cancer Cell 2006; 10:375-88.
6. Ponassi R, et al. Cell Cycle 2008; In this issue.




EXTRA VIEWS

Open Access Article

MicroRNAs as regulators of epithelial-mesenchymal transition

Philip A. Gregory, Cameron P. Bracken, Andrew G. Bert and Gregory J. Goodall
Pages 3112 - 3117
http://dx.doi.org/10.4161/cc.7.20.6851
Abstract | PDF

Epithelial-mesenchymal transition (EMT) describes the molecular reprogramming and phenotypic changes involved in the conversion of polarised immotile epithelial cells to motile mesenchymal cells. This process allows the remodelling of tissues during embryonic development and is implicated in the promotion of tumor invasion and metastasis. Several recent studies have identified the miR-200 family and miR-205 as key regulators of EMT and enforcers of the epithelial phenotype. The miR-200 family participates in a signaling network with the E-cadherin transcriptional repressors ZEB1/δEF1 and ZEB2/SIP1, and TGF-β2 that is postulated to facilitate maintenance of stable epithelial or mesenchymal states but also allow reversible switching between these states in response to EMT effectors (such as TGF-β). This review summarizes these recent findings and their implications in both developmental EMT and tumor progression.

Open Access Article

mTOR signaling: PLD takes center stage

Yuting Sun and Jie Chen
Pages 3118 - 3123
http://dx.doi.org/10.4161/cc.7.20.6881
Abstract | PDF

The mammalian target of rapamycin (mTOR) assembles a signaling network that transduces nutrient signals and various other stimuli to regulate a wide range of cellular functions. Of the two distinct mTOR complexes, mTORC1 is under the control of the TSC-Rheb pathway, which serves as an integrator of multiple upstream signals. A lipid signaling cascade involving phospholipase D (PLD) and phosphatidic acid (PA) has also been known to mediate mitogenic signals upstream of mTORC1. A new study now reveals a direct connection between these two regulatory pathways and demonstrates that PLD1 is an effector of Rheb in the activation of mTORC1. A novel role of PLD as a nutrient sensor has also been suggested. In this extra-view, we discuss the emerging importance of PA and PLD in the mTORC1 signaling network and the biological processes it governs. We also consider the implications from several recent findings and propose mechanistic models of PLD-mTOR signaling to be tested in the near future.

Open Access Article

Phosphorylated HuR shuttles in cycles

Hyeon Ho Kim and Myriam Gorospe
Pages 3124 - 3126
http://dx.doi.org/10.4161/cc.7.20.6884
Abstract | PDF

HuR is a ubiquitous RNA-binding protein (RBP) that associates with many mRNAs encoding proliferative proteins. Although predominantly nuclear, HuR translocation to the cytoplasm is linked to its ability to stabilize target mRNAs and modulate their translation. We recently reported that HuR phosphorylation by Cdk1 at S202 (within the HuR hinge region that is necessary for nucleocytoplasmic shuttle) increases HuR association with 14-3-3 and contributes to its nuclear retention. In this issue of Cell Cycle we report that residue S242 also regulates HuR’s cytoplasmic localization, influences cyclin expression, and modulates cell proliferation. Together with evidence of other post-translational HuR modifications, we propose that HuR phosphorylation ensures the timely mobilization of HuR across the nuclear envelope. In turn, HuR helps to schedule gene expression programs in a cell cycle-dependent manner.

Open Access Article

Microsatellites are EWS/FLI response elements: Genomic "junk" is EWS/FLI's treasure

Kunal Gangwal and Stephen L. Lessnick
Pages 3127 - 3132
http://dx.doi.org/10.4161/cc.7.20.6892
Abstract | PDF

Ewing's sarcoma is a solid tumor of the bone that primarily occurs in children and young adults. Most cases harbor the (11;22)(q24;q12) chromosomal translocation that encodes the EWS/FLI oncoprotein. EWS/FLI is an aberrant ETS-type transcription factor that dysregulates a number of genes important in the development of Ewing's sarcoma. Because EWS/FLI is the key oncoprotein in this tumor and ETS proteins are often dysregulated in various human cancers, Ewing's sarcoma serves as a useful paradigm for ETS-mediated oncogenesis. We recently showed that EWS/FLI interacts with a GGAA-microsatellite to regulate some of its target genes, including NR0B1, an EWS/FLI-regulated gene that is required for the oncogenic phenotype of Ewing's sarcoma. While microsatellites typically have no ascribed function, and are sometimes considered "junk" DNA, our findings provide a unique role for microsatellites in cancer development. Furthermore, these findings may indicate a novel mechanism for normal ETS protein function as well. Finally, it is tempting to speculate that microsatellite polymorphisms may confer differences in susceptibility to Ewing's sarcoma, both between individuals and between populations, and other diseases mediated by ETS transcription factors. The observation of microsatellites as transcriptional response elements for EWS/FLI suggest that these elements may not be "junk" after all.

Open Access Article

Resist or die: FOXO transcription factors determine the cellular response to chemotherapy

Ana R. Gomes, Jan J. Brosens and Eric W.-F. Lam
Pages 3133 - 3136
http://dx.doi.org/10.4161/cc.7.20.6920
Abstract | PDF

FOXO transcription factors are important regulators of cell fate decisions, capable of inducing cell death as well as promoting cell survival and resistance to environmental stress. As is the case for many cancers, apoptosis of leukaemic cells in response to chemotherapeutic drugs such as doxorubicin is dependent upon FOXO activation. Surprisingly, prolonged FOXO activity paradoxically promotes drug-resistance in leukaemia by enhancing the expression of critical signal intermediates that drive the activity of the Class 1A PI3-K/Akt survival pathway. Additionally, under continuous stress, FOXO factors also induce the expression of genes important for drug efflux, antioxidant defence, and DNA damage repair. Thus, the same effector molecules, FOXOs, are responsible for not only the initial therapeutic response to cancer drugs but also the subsequent acquisition of resistance. This emerging evidence demonstrates that FOXO factors have a dual role in stress response and function at the axis of cancer drug sensitivity and resistance. Nevertheless, the mechanism that allows FOXO to escape the negative control of the PI3-K/Akt pathway in response to persistent cytotoxic stress and to switch its control from pro-apoptotic to pro-survival target genes is not well understood but likely to involve a series of defined post-translational protein modifications.

Open Access Article

Combinatorial microRNAs: Working together to make a difference

Irena Ivanovska and Michele A. Cleary
Pages 3137 - 3142
http://dx.doi.org/10.4161/cc.7.20.6923
Abstract | PDF

MicroRNAs regulate gene networks and therefore are inherently complex. MicroRNAs themselves function in networks of other microRNAs, some of which are co-expressed from the same locus. To better understand the interplay among microRNAs that underlies their functions, we examined the potential of combinatorial effects of endogenously and exogenously co-expressed microRNAs. In this review, we first distill the similarities and differences between three microRNA families that function in cell division, miR-16, miR-34a and miR-106b, with emphasis on their exquisite phenotypic diversity. Given that the microRNAs affect cell cycle progression via distinct targets, we tested for phenotypic synergism among them. Furthermore, we investigate target regulation by individual and pooled microRNAs to gain insight into interactions among microRNAs co-expressed from the same chromosomal locus. The ability of microRNAs to modulate multiple genes within a molecular pathway engenders a novel way of thinking about targeting pathways: instead of a one-inhibitor-one-target model, multiple components in a pathway can be modulated by a microRNA resulting in a potent yet reversible inhibition of the pathway. To fully realize this potential, we need to understand how microRNAs function singly and in concert with each other.




PERSPECTIVES

Open Access Article

Control of cell proliferation pathways by microRNAs

María José Bueno, Ignacio Pérez de Castro and Marcos Malumbres
Pages 3143 - 3148
http://dx.doi.org/10.4161/cc.7.20.6833
Abstract | PDF

microRNAs (miRNAs) are small non-coding RNAs that regulate a large variety of cellular processes including differentiation, apoptosis and proliferation. Several miRNAs display defective expression patterns in human tumors with the consequent alteration of target oncogene or tumor suppressor genes. Many of these miRNAs modulate the major proliferation pathways through direct interaction with critical regulators such as RAS, PI3K/PTEN or ABL, as well as members of the retinoblastoma pathway, Cyclin-CDK complexes or cell cycle inhibitors of the INK4 or Cip/Kip families. A complex interplay between miRNAs and MYC or E2F family members also exists to modulate cell cycle-dependent transcription during normal or tumoral proliferation. The ability of miRNAs to modulate these proliferation pathways may have relevant implications not only in physiological or developmental processes but also in tumor progression or cancer therapy.

Open Access Article

Regulation of p53: The next generation

Christine Blattner
Pages 3149 - 3153
http://dx.doi.org/10.4161/cc.7.20.6921
Abstract | PDF

The p53 protein is one of the most important tumor suppressor proteins. The most prevailing property of this tumor suppressor protein is its activation in response to DNA damage which counteracts the propagation of genetic alterations to daughter cells under conditions that provoke mutagenesis. In response to DNA damage and some other kinds of cellular stress the turnover of p53 is reduced or completely switched-off, which leads to a strong increase in the amount of the p53 protein and subsequently to the implementation of cell cycle arrest and apoptosis. Although post-translational modifications of p53 certainly contribute to the activation of p53 under physiologic conditions, an increase in the amount of the protein e.g. after overexpression, is sufficient for p53's deadly activities. This makes this tumor suppressor protein an interesting target for cancer therapy. This article summarizes the most important principles for the regulation of p53, with a particular focus on recent findings. Furthermore, open questions and possible future directions shall be discussed.

Open Access Article

The Cullin7 E3 ubiquitin ligase: A novel player in growth control

Antonio Sarikas, Xinsong Xu, Loren J. Field and Zhen-Qiang Pan
Pages 3154 - 3161
http://dx.doi.org/10.4161/cc.7.20.6922
Abstract | PDF

Cullin7 (CUL7) is a molecular scaffold that organizes an E3 ubiquitin ligase containing the F-box protein Fbw8, Skp1 and the ROC1 RING finger protein. Dysregulation of the CUL7 E3 Ligase has been directly linked to hereditary human diseases as cul7 germline mutations were found in patients with autosomal-recessive 3-M and Yakuts short stature syndromes, which are characterized by profound pre- and postnatal growth retardation. In addition, genetic ablation of CUL7 in mice resulted in intrauterine growth retardation and perinatal lethality, underscoring its importance for growth regulation. The recent identification of insulin receptor substrate 1, a critical mediator of insulin and insulin-like growth factor-1 signaling, as the proteolytic target of the CUL7 E3 ligase, provided a molecular link between CUL7 and a well-established growth regulatory pathway. This result, coupled with other studies demonstrating interactions between CUL7 and the p53 tumor suppressor protein, as well as the simian virus 40 large T antigen oncoprotein, further implicated CUL7 as a novel player in growth control and suggested pathomechanistic insights into CUL7-linked growth retardation syndromes.




REVIEW

Open Access Article

FoxO1 integrates insulin signaling to VLDL production

Adama Kamagate and H. Henry Dong
Pages 3162 - 3170
http://dx.doi.org/10.4161/cc.7.20.6882
Abstract | PDF

Very low-density lipoproteins (VLDL) are triglyceride-rich particles. VLDL is synthesized in hepatocytes and secreted from the liver in a pathway that is tightly regulated by insulin. Hepatic VLDL production is stimulated in response to reduced insulin action, resulting in increased release of VLDL into the blood under fasting conditions. Circulating VLDL serves as a vehicle for transporting lipids to peripheral tissues for energy homeostasis. Conversely, hepatic VLDL production is suppressed in response to increased insulin release after meals. This effect is critical for preventing prolonged excursion of postprandial plasma lipid profiles in normal individuals. In subjects with obesity and type 2 diabetes, the ability of insulin to regulate VLDL production becomes impaired due to insulin resistance in the liver, resulting in excessive VLDL secretion and accumulation of triglyceride-rich particles in the blood. Such abnormality in lipid metabolism characterizes the pathogenesis of hypertriglyceridemia and accounts for increased risk of coronary artery disease in obesity and type 2 diabetes. Nevertheless, the molecular basis that links insulin resistance to VLDL overproduction remains poorly understood. Our recent studies illustrate that the forkhead transcription factor FoxO1 acts in the liver to integrate hepatic insulin action to VLDL production. Augmented FoxO1 activity in insulin resistant livers promotes hepatic VLDL overproduction and predisposes to the development of hypertriglyceridemia. These new findings raise an important question: Is FoxO1 a therapeutic target for ameliorating hypertriglyceridemia? Here we discuss this question in the context of recent advances toward our understanding of the pathophysiology of hypertriglyceridemia.




REPORTS

Open Access Article

14-3-3 gamma is required to enforce both the incomplete S phase and G2 DNA damage checkpoints

Amol S. Hosing, Samrat T. Kundu and Sorab N. Dalal
Pages 3171 - 3179
http://dx.doi.org/10.4161/cc.7.20.6812
Abstract | PDF

Checkpoint pathways inhibit mitotic progression by inducing the phosphorylation of serine 216 in cdc25C resulting in the generation of a 14-3-3 binding site on cdc25C. Two 14-3-3 isoforms, 14-3-3ε and 14-3-3γ form a complex with cdc25C and inhibit cdc25C function. To examine the contribution of 14-3-3γ to checkpoint regulation, the expression of 14-3-3γ was inhibited in HCT116 cells using vector based RNA interference. A transient reduction in the expression of 14-3-3γ in HCT116 cells resulted in an override of both the incomplete S phase and the G2 DNA damage checkpoint. A 14-3-3γ knockdown clone also showed an override of both checkpoint pathways. These phenotypes were reversed upon expression of a shRNA resistant 14-3-3γ cDNA. Override of the G2 DNA damage checkpoint pathway was accompanied by a decrease in the levels of inhibitory phosphorylation on cdc25C and cdk1. However, there was no difference in the γ-H2AX foci formation and levels of phospho-chk1 and phospho-chk2, suggesting that activation of the DNA damage checkpoint response and subsequent activation of the checkpoint kinases Chk1 and Chk2 was not perturbed. These results suggest that the override of checkpoint observed in 14-3-3γ knockdown cells is due to failure to inhibit cdc25C function.

Open Access Article

PDIP38 is a novel mitotic spindle-associated protein that affects spindle organization and chromosome segregation

Esther Klaile, Alexander Kukalev, Björn Öbrink and Mario M. Müller
Pages 3180 - 3186
http://dx.doi.org/10.4161/cc.7.20.6813
Abstract | PDF

In order to maintain genomic integrity during mitosis, cells assemble the mitotic spindle to separate sister chromosomes to the two daughter cells. A variety of motor- and non motor-proteins are involved in the organization and regulation of this complex apparatus. DNA polymerase δ-interacting protein 38 (PDIP38) is a highly conserved protein and has so far been shown to be a cytoplasmic and nuclear protein. Cell cycle dependent nuclear localization and the interaction with DNA polymerase δ and proliferating cell nuclear antigen (PCNA) indicate a role for PDIP38 in DNA modification and/or proliferation. Here, we show for the first time that PDIP38 localizes to the mitotic spindle throughout mitosis. Using anti-PDIP38 antibody injections and siRNA silencing, we demonstrate that PDIP38 loss-of-function causes problems with spindle organization, aberrant chromosome segregation, and multinucleated cells. Taken together, the data indicate different roles for PDIP38 in safeguarding a proper cell division at various stages of the cell cycle, including DNA synthesis and repair, organization of the mitotic spindle and chromosome segregation.

Open Access Article

Cell cycle dependent degradation of MCAK: Evidence against a role in anaphase chromosome movement

Anutosh Ganguly, Rajat Bhattacharya and Fernando Cabral
Pages 3187 - 3193
http://dx.doi.org/10.4161/cc.7.20.6814
Abstract | PDF

MCAK, a kinesin related motor protein with microtubule depolymerizing activity, is known to play an important role in spindle assembly and correcting errors in mitotic chromosome alignment. Experiments to determine how cellular levels of the protein are regulated demonstrate that MCAK accumulates during cell cycle progression, reaches a maximum at G2/M phase, and is rapidly degraded by the proteasome during mitosis. Immunofluorescence microscopy further indicates that MCAK largely disappears from kinetochores and spindle poles at the metaphase to anaphase transition. A phosphorylated form of MCAK appears during mitosis and seems to be preferentially degraded, but degradation does not appear to depend on Aurora B, a kinase reported to be involved in regulating the error correcting activity of the protein. These studies indicate that MCAK activity is limited during the latter stages of mitosis by protein degradation, and argue against a role for the protein in anaphase chromosome movement.

Open Access Article

Never-in-mitosis related Kinase 1 functions in DNA damage response and checkpoint control

Yumay Chen, Phang-Lang Chen, Chi-Fen Chen, Xianzhi Jiang and Daniel J. Riley
Pages 3194 - 3201
http://dx.doi.org/10.4161/cc.7.20.6815
Abstract | PDF

Nek1, the first mammalian ortholog of the fungal protein kinase never in mitosis A, is involved early in the DNA damage sensing/repair pathway after ionizing radiation. Here we extend this finding by showing that Nek1 localizes to nuclear foci of DNA damage in response to many different types of damage in addition to IR. Untransformed cells established from kat2J/Nek1 -/- mice fail to arrest properly at G1/S and M-phase checkpoints in response to DNA damage. G1-S-phase checkpoint control can be rescued by ectopically overexpressing wild-type Nek1. In Nek1-/- murine cells and in human cells with Nek1 expression silenced by siRNA, the checkpoint kinases Chk1 and Chk2 fail to be activated properly in response to ionizing or UV radiation. In cells without functional Nek1, DNA is not repaired properly, double-stranded DNA breaks persist long after low dose IR, and excessive numbers of chromosome breaks are observed. These data show that Nek1 is important for efficient DNA damage checkpoint control and for proper DNA damage repair.

Open Access Article

Epigenetic down-regulation of the suppressor of cytokine signaling 1 (Socs1) gene is associated with the STAT3 activation and development of hepatocellular carcinoma induced by methyl-deficiency in rats

Tetyana V. Bagnyukova, Volodymyr P. Tryndyak, Levan Muskhelishvili, Sharon A. Ross, Frederick A. Beland and Igor P. Pogribny
Pages 3202 - 3210
http://dx.doi.org/10.4161/cc.7.20.6816
Abstract | PDF

The members of the platelet-derived growth factor (PDGF) and the transforming growth factor-beta (TGF-β) pathways are important in the induction of liver fibrosis and cirrhosis; however, their role in the subsequent progression to hepatocellular carcinoma (HCC) remains elusive. Our study provides new insights into mechanisms of dysregulation of PDGFs, TGF-β and signal transducer and activator of transcription (STAT) pathways in the pathogenesis of methyl-deficient rodent liver carcinogenesis, a remarkably relevant model to the development of HCC in humans. We demonstrated a progressive increase in the Pdgfs and TGF-β expression in preneoplastic tissue and liver tumors indicating their promotional role in carcinogenesis, particularly in progression of liver fibrosis and cirrhosis. However, activation of the STAT3 occurred only in fully developed HCC and was associated with down-regulation of the Socs1 gene. The inhibition of the Socs1 expression in HCC was associated with an increase in histone H3 lysine 9, H3 lysine 27, and H4 lysine 20 trimethylation at the Socs1 promoter, but not with promoter methylation. The results of our study suggest the following model of events in hepatocarcinogenesis: during early stages, over-expression of the Socs1 effectively inhibits TGF-β- and PDGF-induced STAT3 activation, whereas, during the advanced stages of hepatocarcinogenesis, the Socs1 down-regulation resulted in loss of its ability to attenuate the signal from the up-regulated TGF-β and PDGFs leading to oncogenic STAT3 activation and malignant cell transformation. This model illustrates that the Socs1 acts as classic tumor suppressor by preventing activation of the STAT3 and down-regulation of Socs1 and consequent activation of STAT3 may be a crucial events leading to formation of HCC.

Open Access Article

A novel Bim-BH3-derived Bcl-XL inhibitor: Biochemical characterization, in vitro, in vivo and ex-vivo anti-leukemic activity

Raffaella Ponassi and 27 others
Pages 3211 - 3224
http://dx.doi.org/10.4161/cc.7.20.6830
Abstract | PDF

BH3-only members of the Bcl-2 family exert a fundamental role in apoptosis induction. This work focuses on the development of a novel peptidic molecule based on the BH3 domain of Bim. The antiapoptotic molecule Bcl-XL, involved in cancer development/progression and tumour resistance to cytotoxic drugs, is a target for Bim. According to a rational study of the structural interactions between wt Bim-BH3 and Bcl-XL, we replaced specific residues of Bim-BH3 with natural and non-natural aminoacids and added an internalizing sequence, thus increasing dramatically the inhibitory activity of our modified Bim-BH3 peptide, called 072RB. Confocal microscopy and flow cytometry demonstrated cellular uptake and internalization of 072RB, followed by co-localization with mitochondria. Multiparameter flow cytometry demonstrated that the 072RB dose-dependent growth inhibition of leukaemia cell lines was due to apoptotic cell death. No effect was observed when cells were treated with the internalizing vector alone or a mutated control peptide (single aminoacid substitution L94A). Ex-vivo derived leukemic cells from acute myeloid leukaemia (AML) patients underwent cell death when cultured in vitro in the presence of 072RB. Conversely, no significant cytotoxic effect was observed when 072RB was administered to cultures of peripheral blood mononuclear cells, either resting or PHA-stimulated, and bone marrow cells of normal donors. Xenografts of human AML cells in NOD/SCID mice displayed a significant delay of leukemic cell growth upon treatment with 072RB administered intravenously (15 mg/Kg three times, 48 hours after tumour cell injection). Altogether, these observations support the therapeutic potentials of this novel BH3 mimetic.

Open Access Article

Voltage-dependent anion channels (VDAC) in the plasma membrane play a critical role in apoptosis in differentiated hippocampal neurons but not in neural stem cells

Nesar Akanda, Roshan Tofighi, Johan Brask, Christoffer Tamm, Fredrik Elinder and Sandra Ceccatelli
Pages 3225 - 3234
http://dx.doi.org/10.4161/cc.7.20.6831
Abstract | PDF

microRNAs (miRNAs) are small non-coding RNAs that regulate a large variety of cellular processes including differentiation, apoptosis and proliferation. Several miRNAs display defective expression patterns in human tumors with the consequent alteration of target oncogene or tumor suppressor genes. Many of these miRNAs modulate the major proliferation pathways through direct interaction with critical regulators such as RAS, PI3K/PTEN or ABL, as well as members of the retinoblastoma pathway, Cyclin-CDK complexes or cell cycle inhibitors of the INK4 or Cip/Kip families. A complex interplay between miRNAs and MYC or E2F family members also exists to modulate cell cycle-dependent transcription during normal or tumoral proliferation. The ability of miRNAs to modulate these proliferation pathways may have relevant implications not only in physiological or developmental processes but also in tumor progression or cancer therapy.

Open Access Article

Simultaneous inhibition of the constitutively activated nuclear factor κB and of the Interleukin-6 pathways is necessary and sufficient to completely overcome apoptosis resistance of human U266 myeloma cells

Natalia Malara, Domenico Focà, Francesca Casadonte, Maria Francesca Sesto, Lorenza Macrina, Lucia Santoro, Monica Scaramuzzino, Rosa Terracciano and Rocco Savino
Pages 3235 - 3245
http://dx.doi.org/10.4161/cc.7.20.6832
Abstract | PDF

Elevated Nuclear Factor κB (NF-κB) levels have been reported in multiple myeloma cells derived from patients relapsing after chemotherapy. In the search of an in vitro a model with molecular features similar to relapsing lesions, we focused our attention on an IL-6 autocrine human myeloma cell line (U266), characterized by apoptosis resistance due to up-regulation of two constitutive signaling pathways: NF-κB and STAT-3. NF-κB activity was inhibited with proteasome inhibitory agents, such as PS-341 and Withaferin A, with an IKK inhibitor (Wedelolactone) or with the adenoviral vector HD IκBαmut-IRES-EGFP encoding a mutant IκBα protein, resistant to proteasomal degradation. We observed that the NF-κB intracellular dislocation at the beginning of the treatment affected therapeutic effectiveness of PS-341, Withaferin A and Wedelolactone; interestingly, the adenoviral vector was highly effective in inducing apopotosis even with NF-κB being predominantly nuclear at the time of infection. We also observed that U266 treated with the Interleukin-6 antagonist Sant7 exhibited reduced STAT3 activity and preferential cytoplasmic NF-κB location; moreover they became capable of undergoing apoptosis mainly from the G1 phase. Adenoviral vector treated U266 have NF-κB localized completely in the cytoplasm and also showed down-regulation of nuclear phospho STAT-3. Finally, combined targeting of NF-κB and STAT3 signaling pathways was the most effective treatment in inducing apoptosis. These findings suggest that combined NF-κB κB and STAT3 targeting warrants further investigations in other apoptosis resistant MM cell lines as well as in suitable MM animal models.

Open Access Article

Underlying principles of cell fate determination during G1 phase of the mammalian cell cycle

Benjamin Pfeuty, Thérèse David-Pfeuty and Kunihiko Kaneko
Pages 3246 - 3257
http://dx.doi.org/10.4161/cc.7.20.6853
Abstract | PDF

Upon their exit from mitosis, mammalian cells enter a G1 phase during which they acutely sense all sorts of environmental stimuli. On the basis of these signals that they first need to decipher and integrate, they decide whether to undergo division, differentiation, senescence or apoptosis. We questioned whether, despite the complexity of the G1 regulatory network, simple organizing principles might be identified that could explain how specific input signals are converted into appropriate cell fates. For this purpose, we formulated a mathematical model of the G1 regulatory network using a simplified description of activities linked to signal transduction, cell growth, cell division and cell death. Bifurcation analysis of the model revealed the existence of multistability between several attractor states corresponding to G0-arrest, G1-arrest, S-phase entry and apoptosis cell fates. We further unravelled interlinked feedback and feedforward loops within the G1 regulatory network that drive the signal-dependent transition between G0 arrest and the other cell fates. Initially, exit from G0 and progression in early G1 entail growth factor-dependent activation of an upstream positive feedback loop that activates the cell-growth machinery. Once ribosome synthesis is restored in G1, a competition develops between a downstream positive feedback loop, which, upon activation, triggers S-phase entry, and stress-activated pathways that promote G1 arrest. If S-phase entry prevails over G1 arrest, cells are sensitized to apoptosis due to stress-induced activation of pro-apoptotic pathways or repression of pro-survival pathways. Thus, the choice between the four possible cell fates in the G1 phase relies on the flexibly interlinked growth-activatory and division-activatory modules, certain components of which have antagonistic effects on pathways involved in driving apoptosis and G1 arrest. The final outcome ultimately depends on the context-dependent coordination between the cell-growth and cell-division processes.

Open Access Article

The cytotoxic ribonuclease onconase targets RNA interference (siRNA)

Hong Zhao, Barbara Ardelt, Wojciech Ardelt, Kuslima Shogen and Zbigniew Darzynkiewicz
Pages 3258 - 3261
http://dx.doi.org/10.4161/cc.7.20.6855
Abstract | PDF

Onconase (Onc), a ribonuclease from oocytes of Northern Leopard frogs (Rana pipiens) is cytostatic and cytotoxic to a variety of tumor lines in vitro, inhibits growth of tumors in animal in vivo models and enhances sensitivity of tumor cells to a number of other cytotoxic agents with diverse mechanism of action. In Phase III clinical trials Onc demonstrated significant efficacy in patients with malignant mesothelioma that failed prior chemotherapy. We previously postulated that the antitumor activity of Onc and the observed synergisms with other antitumor modalities at least in part may be mediated by targeting RNA interference (RNAi). In the present study we observed that the silencing of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene in human lung adenocarcinoma A549 cells by siRNA was effectively prevented by Onc. While transfection of cells with GAPDH siRNA reduced expression of this protein by nearly 70%, the expression was restored in the cells exposed to 0.8 µM Onc for 48 or 72 h. The data thus provide evidence that one of the targets of Onc is siRNA, likely within the RNA-induced silencing complex (RISC). In light of the findings that microRNAs are involved in tumor pathogenesis as well as in enhancing cell resistance to anticancer therapy the present data may provide explanation for both, the antitumor Onc activity and its propensity to enhance effectiveness of cytotoxic drugs.

Open Access Article

The Polo-like kinase Cdc5 interacts with FEAR network components and Cdc14

Rami Rahal and Angelika Amon
Pages 3262 - 3272
http://dx.doi.org/10.4161/cc.7.20.6852
Abstract | PDF

Exit from mitosis in Saccharomyces cerevisiae is triggered by activation of the phosphatase Cdc14. Throughout interphase and early mitosis, Cdc14 is sequestered in the nucleolus by its inhibitor Cfi1/Net1. In anaphase, the Cdc Fourteen Early Anaphase Release (FEAR) network and the Mitotic Exit Network (MEN) coordinately trigger the release of Cdc14 from the nucleolus. Here we show that the FEAR network component Cdc5 physically associates with two other members of the pathway, the Separase Esp1 and the Esp1-binding protein Slk19. Furthermore, we find that Cdc5 physically interacts with Cdc14 and that this association is mediated by Cdc5's Polo-box domain, a phospho-serine/phospho-threonine binding domain. Finally, we present evidence that the Cdc5-Cdc14 association is direct, further supporting the central role of Cdc5 in Cdc14 localization.

Open Access Article

IRES-mediated translational control of AMAP1 expression during differentiation of monocyte U937 cells

Mariko Miyata, Jennifer F. Raven, Dionissios Baltzis, Antonis E. Koromilas and Hisataka Sabe
Pages 3273 - 3281
http://dx.doi.org/10.4161/cc.7.20.6883
Abstract | PDF

Global control of mRNA translation plays key roles in cell regulation, including growth, differentiation and apoptosis. Human monocyte-like U937 cells differentiate into macrophage-like cells upon 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment, a process which is known to be accompanied with a large decrease in general protein synthesis. Here, we found that protein levels of AMAP1 (also called ASAP1 or DDEF1), a GTPase-activating protein for Arf GTPases, increase several fold during U937 cell differentiation. This increase was not accompanied with a notable increase in the AMAP1 gene transcript, nor seemed to be due to 5’-Cap-dependent mRNA translational activities in differentiated U937 cells. We identified the 5’-untranslated region (5’-UTR) of AMAP1 mRNA, and found that this 5’-UTR exhibits significant internal ribosome entry site (IRES)-dependent translational activity in differentiated U937 cells, but not in undifferentiated cells. Our results indicate that monocyte differentiation involves enhancement of IRES activity, by which protein levels of AMAP1 are primarily upregulated.




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Open Access Article

Random versus non-random DNA strand segregation difference between cancer and normal stem cells might provide an opportunity to specific targeting the former by radiotherapy

Feng Zhang, Jing Ye, Zengshan Li, Ruian Wang, Jing Zhang, Shoujing Yang, Zhe Wang and Qing Li
Page 3282
http://dx.doi.org/10.4161/cc.7.20.6893
Abstract | PDF

No abstract