Farnesylated RhoB inhibits radiation-induced mitotic cell death and controls radiation-induced centrosome overduplication

Our previous results demonstrated that expressing the GTPase ras homolog gene family, member B (RhoB) in radiosensitive NIH3T3 cells increases their survival following 2 Gy irradiation (SF2). We have first demonstrated here that RhoB expression inhibits radiation-induced mitotic cell death. RhoB is present in both a farnesylated and a geranylgeranylated form in vivo. By expressing RhoB mutants encoding for farnesylated (RhoB-F cells), geranylgeranylated or the CAAX deleted form of RhoB, we have then shown that only RhoB-F expression was able to increase the SF2 value by reducing the sensitivity of these cells to radiation-induced mitotic cell death. Moreover, RhoB-F cells showed an increased G2 arrest and an inhibition of centrosome overduplication following irradiation mediated by the Rho-kinase, strongly suggesting that RhoB-F may control centrosome overduplication during the G2 arrest after irradiation. Overall, our results for the first time clearly implicate farnesylated RhoB as a crucial protein in mediating cellular resistance to radiation-induced nonapoptotic cell death.     

Cdc20 control of cell fate during prolonged mitotic arrest

The fate of cells arrested in mitosis by antimitotic compounds is complex but is influenced by competition between pathways promoting cell death and pathways promoting mitotic exit. As components of both of these pathways are regulated by Cdc20-dependent degradation, I hypothesize that variations in Cdc20 protein levels, rather than mutations in checkpoint genes, could affect cell fate during prolonged mitotic arrest. This hypothesis is supported by experiments where manipulation of Cdc20 levels affects the response to antimitotic compounds. The observed differences in Cdc20 levels between cell lines likely reflects differences in the rate of synthesis or degradation of the protein; therefore, understanding these pathways at a molecular level could pave the way for modulating the activity of Cdc20, in turn presenting novel therapeutic possibilities.     

p38 Mitogen-activated protein kinase mediates cell death and p21-activated kinase mediates cell survival during chemotherapeutic drug-induced mitotic arrest

Activation of the mitotic checkpoint by chemotherapeutic drugs such as taxol causes mammalian cells to arrest in mitosis and then undergo apoptosis. However, the biochemical basis of chemotherapeutic drug-induced cell death is unclear. Herein, we provide new evidence that both cell survival and cell death-signaling pathways are concomitantly activated during mitotic arrest by microtubule-interfering drugs. Treatment of HeLa cells with chemotherapeutic drugs activated both p38 mitogen-activated protein kinase (MAPK) and p21-activated kinase (PAK). p38 MAPK was necessary for chemotherapeutic drug-induced cell death because the p38 MAPK inhibitors SB203580 or SB202190 suppressed cell death. Dominant-active MKK6, a direct activator of p38 MAPK, also induced cell death by stimulating translocation of Bax from the cytosol to the mitochondria in a p38 MAPK-dependent manner. Dominant active PAK suppressed this MKK6-induced cell death. PAK seems to mediate cell survival by phosphorylating Bad, and inhibition of PAK in mitotically arrested cells reduced Bad phosphorylation and increased apoptosis. Our results suggest that therapeutic strategies that suppress PAK-mediated survival signals may improve the efficacy of current cancer chemotherapies by enhancing p38 MAPK-mediated cell death.     

Involvement of p53 in cell death following cell cycle arrest and mitotic catastrophe induced by rotenone

In order to investigate the cell death-inducing effects of rotenone, a plant extract commonly used as a mitochondrial complex I inhibitor, we studied cancer cell lines with different genetic backgrounds. Rotenone inhibits cell growth through the induction of cell death and cell cycle arrest, associated with the development of mitotic catastrophe. The cell death inducer staurosporine potentiates the inhibition of cell growth by rotenone in a dose-dependent synergistic manner. The tumor suppressor p53 is involved in rotenone-induced cell death, since the drug treatment results in increased expression, phosphorylation and nuclear localization of the protein. The evaluation of the effects of rotenone on a p53-deficient cell line revealed that although not required for the promotion of mitotic catastrophe, functional p53 appears to be essential for the extensive cell death that occurs afterwards. Our results suggest that mitotic slippage also occurs subsequently to the rotenone-induced mitotic arrest and cells treated with the drug for a longer period become senescent. Treatment of mtDNA-depleted cells with rotenone induces cell death and cell cycle arrest as in cells containing wild-type mtDNA, but not formation of reactive oxygen species. This suggests that the effects of rotenone are not dependent from the production of reactive oxygen species. This work highlights the multiple effects of rotenone in cancer cells related to its action as an anti-mitotic drug.     

Mitotic kinesin inhibitors induce mitotic arrest and cell death in Taxol-resistant and -sensitive cancer cells

Taxanes are powerful chemotherapy agents that target the microtubule cytoskeleton, leading to mitotic arrest and cell death; however, their clinical efficacy has been hampered due to the development of drug resistance. Therefore, other proteins involved in spindle assembly are being examined as potential targets for anticancer therapy. The mitotic kinesin, Eg5 is critical for proper spindle assembly; as such, inhibition of Eg5 leads to mitotic arrest making it a potential anticancer target. We wanted to validate Eg5 as a therapeutic target and determine if Eg5 inhibitors retain activity in Taxol-resistant cells. Using affinity chromatography we first show that the compound HR22C16 is an Eg5 inhibitor and does not interact with other microtubule motor proteins tested. Furthermore, HR22C16 along with its analogs, inhibit cell survival in both Taxol-sensitive and -resistant ovarian cancer cells with at least 15-fold greater efficacy than monastrol, the first generation Eg5 inhibitor. Further analysis with HR22C16-A1, the most potent HR22C16 analog, showed that it retains efficacy in PgP-overexpressing cells, suggesting that it is not a PgP substrate. We further show that HR22C16-A1 induces cell death following mitotic arrest via the intrinsic apoptotic pathway. Interestingly, the combination of HR22C16-A1 with Taxol results in an antagonistic antiproliferative and antimitotic effect, possibly due to the abrogation of Taxol-induced mitotic spindles by HR22C16-A1. Taken together, our results show that Eg5 inhibitors have promising anticancer activity and can be potentially used to overcome Taxol resistance in the clinical setting.     

Regulation of caspase-3 processing by cIAP2 controls the switch between pro-inflammatory activation and cell death in microglia

The activation of microglia, resident immune cells of the central nervous system, and inflammation-mediated neurotoxicity are typical features of neurodegenerative diseases, for example, Alzheimer''s and Parkinson''s diseases. An unexpected role of caspase-3, commonly known to have executioner role for apoptosis, was uncovered in the microglia activation process. A central question emerging from this finding is what prevents caspase-3 during the microglia activation from killing those cells? Caspase-3 activation occurs as a two-step process, where the zymogen is first cleaved by upstream caspases, such as caspase-8, to form intermediate, yet still active, p19/p12 complex; thereafter, autocatalytic processing generates the fully mature p17/p12 form of the enzyme. Here, we show that the induction of cellular inhibitor of apoptosis protein 2 (cIAP2) expression upon microglia activation prevents the conversion of caspase-3 p19 subunit to p17 subunit and is responsible for restraining caspase-3 in terms of activity and subcellular localization. We demonstrate that counteracting the repressive effect of cIAP2 on caspase-3 activation, using small interfering RNA targeting cIAP2 or a SMAC mimetic such as the BV6 compound, reduced the pro-inflammatory activation of microglia cells and promoted their death. We propose that the different caspase-3 functions in microglia, and potentially other cell types, reside in the active caspase-3 complexes formed. These results also could indicate cIAP2 as a possible therapeutic target to modulate microglia pro-inflammatory activation and associated neurotoxicity observed in neurodegenerative disorders.     

Programmed cell death in plants: distinguishing between different modes

Programmed cell death (PCD) in plants is a crucial componentof development and defence mechanisms. In animals, differenttypes of cell death (apoptosis, autophagy, and necrosis) havebeen distinguished morphologically and discussed in these morphologicalterms. PCD is largely used to describe the processes of apoptosisand autophagy (although some use PCD and apoptosis interchangeably)while necrosis is generally described as a chaotic and uncontrolledmode of death. In plants, the term PCD is widely used to describemost instances of death observed. At present, there is a vastarray of plant cell culture models and developmental systemsbeing studied by different research groups and it is clear fromwhat is described in this mass of literature that, as with animals,there does not appear to be just one type of PCD in plants.It is fundamentally important to be able to distinguish betweendifferent types of cell death for several reasons. For example,it is clear that, in cell culture systems, the window of timein which ‘PCD’ is studied by different groups varieshugely and this can have profound effects on the interpretationof data and complicates attempts to compare different researcher'sdata. In addition, different types of PCD will probably havedifferent regulators and modes of death. For this reason, inplant cell cultures an apoptotic-like PCD (AL-PCD) has beenidentified that is fairly rapid and results in a distinct corpsemorphology which is visible 4–6 h after release of cytochromec and other apoptogenic proteins. This type of morphology, distinctfrom autophagy and from necrosis, has also been observed inexamples of plant development. In this review, our model systemand how it is used to distinguish specifically between AL-PCDand necrosis will be discussed. The different types of PCD observedin plants will also be discussed and the importance of distinguishingbetween different forms of cell death will be highlighted. Key words: Apoptosis, apoptosis-like programmed cell death (AL-PCD), Arabidopsis, autophagy, mitochondria, necrosis, programmed cell death (PCD) Received 5 June 2007; Revised 13 September 2007 Accepted 20 September 2007     

Density-dependent tumor cell death and reversible cell cycle arrest: mutually exclusive modes of monocyte-mediated growth control

The population development of five human tumor cell lines is examined under the influence of elutriator-prepared human monocytes in a serum-free hormone- and growth factor-supplemented medium. Analysis was performed by electronic counting and sizing of tumor cell nuclei and flow cytometric detection of cell cycle phases. Tumor cell death is triggered at rather low monocyte:tumor cell ratios (1:2 to 1:4) whereas it is strongly reduced at high monocyte densities. Furthermore, it is shown that confluence of the target cell population is a necessary prerequisite for lysis. The data suggest that in monocyte/tumor cell cocultures the decision on target cell lysis is not made by the effector cell, but rather by the target cell and that the criterion for this decision is the target cell's ability or inability to respond to a monocyte challenge by arresting the cell cycle in G1. Interactions between target cells play an important role in determining the result of this decision process. A common basis is suggested for this kind of density-dependent monocyte-triggered lysis and density-dependent cell death in 3T3 cell cultures as described previously.     

MCL1 meets its end during mitotic arrest

Evidence for the destruction of the anti-apoptotic protein MCL1 during prolonged mitotic arrest comes from three papers, one in The EMBO Journal and two in Nature, thus shedding light on the mechanism of apoptosis induction under these conditions.EMBO Rep (2011) advance online publication. doi:10.1038/nature09732EMBO Rep (2011) advance online publication. doi:10.1038/nature09779EMBO Rep (2011) advance online publication. doi:10.1038/emboj.2010.112During mitosis, eukaryotic cells have to properly align their chromosomes. Only after the kinetochore of each chromosome is attached to a polar microtubule can a cell satisfy the ‘spindle assembly checkpoint'', which prevents the mis-segregation of chromosomes. Failure to correctly segregate chromosomes before cell division might contribute to chromosome instability and tumorigenesis. To counteract chromosome aberrations, the cell initiates the apoptotic programme. It has become clear through the use of microtubule-poisoning, chemotherapeutic agents—such as paclitaxel and vincristine—that prolonged activation of the spindle checkpoint can induce mitotic arrest and, subsequently, programmed cell death. The molecular mechanisms responsible for initiating apoptosis during mitotic arrest have remained poorly defined. Two recent papers in Nature (Inuzuka et al, 2011; Wertz et al, 2011) and a report published by the Clarke group last year in The EMBO Journal (Harley et al, 2010) highlight the destruction of MCL1 during prolonged mitotic arrest and shed light on the mechanisms of apoptosis induction.Myeloid cell leukaemia 1 (MCL1) is an anti-apoptotic member of the B-cell lymphoma 2 (BCL2) family of proteins. MCL1, like BCL2 and BCLxL, prevents the downstream activation of BAX and BAK, which are responsible for mitochondrial outer-membrane permeabilization, initiation of the caspase cascade and induction of apoptosis (Youle & Strasser, 2008). Ubiquitination and proteolysis of MCL1 have been reported, but a mechanism for MCL1 degradation following spindle checkpoint activation remains unknown. Now, the studies referenced above suggest that degradation of MCL1 during prolonged mitotic arrest is essential for the induction of apoptosis. Given its prominent role in driving the cell cycle, as well as in safeguarding the fidelity of this process, it is not surprising that the ubiquitin-proteasome system (UPS) has a key role in dictating the activation of the intrinsic apoptotic pathway in cells arrested in mitosis. However, it is surprising that two E3 ubiquitin ligase complexes simultaneously facilitate this degradation event.Harley and colleagues describe the regulation of MCL1 by APC/C^Cdc20(anaphase-promoting complex/cyclosome and its activator Cdc20). This multi-subunit RING E3 ubiquitin ligase is active in mitosis, and ubiquitinates substrates such as securin and cyclin B, thereby allowing progression into anaphase. In their report, Harley and co-workers (2010) demonstrate a Cdk1/cyclin-B-mediated, site-specific phosphorylation (Thr 92 in humans) of MCL1 upon mitotic arrest, followed by its proteolytic destruction by APC/C^Cdc20. Thus, like the sand of an hourglass flipped at each entry into mitosis, the level of MCL1 steadily decreases. If time ‘runs out'' due to a prolonged mitotic arrest (that is, if MCL1 is completely destroyed), then apoptosis is initiated (Fig 1A). Both phosphorylation at Thr 92 and the presence of a conserved destruction or ‘D''-box motif (a characteristic of APC/C substrates) are required for MCL1 proteolysis, although the precise role of phosphorylation in promoting degradation remains unclear.Open in a separate windowFigure 1Two models for proteolytic destruction of MCL1 during mitotic arrest. (A) Schematic illustration of the effects of APC/C^Cdc20 and Cdk1/cyclin B on the degradation of MCL1 during prolonged arrest in mitosis. (B) Schematic illustration of the effects of SCFFbw7, JNK/p38/CKII and Cdk1/cyclin B on MCL1 degradation during mitotic arrest. PP2A is a protein phosphatase that is reported to associate with MCL1. Dashed lines represent inactive processes. Question marks denote unknown mechanisms. APC/C^Cdc20, anaphase-promoting complex/cyclosome and its activator Cdc20; SAC, spindle assembly checkpoint.Interestingly, the stability of MCL1 in asynchronous cells seems to be unaffected when the ability of APC/C^Cdc20 to target MCL1 is compromised by knockdown of Cdc20, or when phosphorylation at Thr 92 is ablated. Although the spindle assembly checkpoint is believed to inhibit APC/C^Cdc20 activity, the degradation of some targets, such as the CDK-inhibitor p21 and cyclin A, is not affected. Consequently, it is possible that MCL1 can be destroyed through Cdc20 during mitotic arrest.More recently, in two reports in Nature (Inuzuka et al, 2011; Wertz et al, 2011), it is shown that MCL1 interacts with another E3 ubiquitin ligase, SCF^Fbxw7. Similarly to the APC/C, the SCF (Skp1/Cul1/F-box protein) is a multi-subunit, RING E3 ubiquitin ligase. The F-box protein provides the specificity for target recognition, often by using specific interaction domains to bind to substrates. In the case of Fbxw7 (also known as Fbw7 and hCdc4), a series of WD40 domains form a pocket that dictates the binding of several substrates. For all known substrates, one or two phosphorylated degradation motifs (phospho-degrons) are recognized by Fbxw7 (Welcker & Clurman, 2008), and MCL1 seems to follow this trend. Briefly, two Fbxw7 degrons—Ser 121/Glu 125 and Ser 159/Thr 163—with different binding affinities were identified in MCL1. Inuzuka and colleagues report that these sites are phosphorylated in a GSK3-dependent manner, supporting a previous report that demonstrated a role for GSK3 in controlling MCL1 degradation (Maurer et al, 2006). They also demonstrate that Fbxw7 affects MCL1 stability during the DNA damage response. Wertz and co-workers provide evidence that, during mitotic arrest, the degrons in MCL1 are instead phosphorylated by JNK, p38 and CKII. Interestingly, when Wertz and colleagues investigated the degradation of MCL1 during mitotic arrest, they discovered a dependence on Fbxw7 similar to that reported for Cdc20 (Fig 1B). Furthermore, a functional Fbxw7–MCL1 interaction was required for the induction of apoptosis in ovarian cancer and T-ALL cell lines treated with microtubule-targeting chemotherapies. This observation presents a dilemma. Which ubiquitin ligase complex—APC/C^Cdc20 or SCF^Fbxw7—targets MCL1 for destruction during mitotic arrest? Do they compete or cooperate?There are several approaches that could be taken to investigate these questions. Perhaps the most promising direction is through understanding the role of various MCL1 phosphorylation events, particularly phosphorylation of Thr 92. The reports collectively demonstrate that Thr 92 and the Fbxw7 degrons are phosphorylated in mitotic cells. It is interesting that Thr 92 phosphorylation is specifically induced at mitosis, and Wertz and colleagues suggest that this event might drive the dissociation of a phosphatase to allow Fbxw7 degron phosphorylation (Fig 1B). However, the results so far are preliminary, and a more complete understanding of the mechanism by which Cdk1/cyclin B phosphorylation of MCL1 promotes proteolysis, and whether this is through Cdc20 and/or Fbxw7, is essential. Although MCL1 degradation after mitotic arrest is unlikely to be associated with the activity of GSK3, is there an induction of GSK3-dependent phosphorylation of MCL1 under other conditions? This important question has been studied previously, but it requires further investigation. Perhaps additional ‘priming'' kinases are involved, as is suspected to be the case for cyclin E and c-Myc, two other substrates of Fbxw7.The concept of a protein being targeted by two ubiquitin ligases is not new. For example, similarly to MCL1, p21 and MLL are targeted by both APC/C^Cdc20 and an SCF complex (SCF^Skp2). Several APC/C^Cdh1 substrates (for example, Cdc25A and claspin) are also degraded via SCFβ^TrCP (Frescas & Pagano, 2008). However, in these instances, APC/C and SCF target the substrates at different phases of the cell cycle. The case of MCL1 is less clear. Wertz and colleagues show that mitotic arrest specifically induces binding of MCL1 to Fbxw7. Conversely, Inuzuka and colleagues provide data suggesting that Fbxw7 loss affects the non-mitotic stability of MCL1. Additionally, in an earlier paper, the Fbxw7 degron was reported to be phosphorylated by GSK3 during cytokine withdrawal (Maurer et al, 2006). Thus, we are left with a picture in which Fbxw7 targets MCL1 during mitotic arrest, but it might also target MCL1 at other points during the cell cycle or in response to external stimuli. With regard to Cdc20-mediated degradation of MCL1, mutation of the D-box seems to stabilize MCL1 only during mitotic arrest, although Cdc20 remains bound to MCL1 in non-mitotic cells. Thus, there might be differences in the conditions for recognition by either Fbxw7 or Cdc20 that merit further investigation. It is also worth mentioning that deubiquitinating enzymes (DUBs) might counteract the activity of Fbxw7, Cdc20, or both. In fact, the DUB USP9X was found to associate with MCL1 (Schwickart et al, 2010).Assuming that both E3 ligases target the same pool of MCL1 at the same time during mitotic arrest, why are there two modes of regulation? It could be that the ligases cooperate to lower MCL1 levels. It is possible that Fbxw7 and Cdc20 together deplete MCL1 to a point at which apoptosis can be initiated; if either ligase is compromised, apoptotic induction is inefficient. Alternatively, there might be a particularly relevant growth condition or cell-type specificity that favours the activity of one complex over the other. For example, it is possible that in tissues that give rise to human cancers harbouring Fbxw7 mutations (for example, T-ALLs or ovarian carcinomas), SCF^Fbxw7 acts as the predominant ligase. Finally, there could be redundancy or competition between the different E3 ligases. Perhaps untransformed cells maintain both systems, to protect against apoptosis evasion in the face of spindle dysfunction. Alternatively, one or both of these systems might be compromised in the cell-culture models. Notably, the situation is further complicated by reports indicating that other ligases seem to affect MCL1 stability: Mule/Huwe1 (Zhong et al, 2005) and SCF^βTrCP (Ding et al, 2007). Silencing of Mule stabilizes MCL1, although Wertz and colleagues did not observe dramatic changes in MCL1 stability after Mule depletion during mitotic arrest. Instead, three groups did not observe stabilization of MCL1 after βTrCP silencing (Wertz et al, 2011; Inuzuka et al, 2011; Dehan et al, 2009). Moreover, the interaction between MCL1 and βTrCP seems to be mediated by BimEL (a βTrCP substrate), as indicated by increased binding under conditions when BimEL is degraded (rather than under conditions when MCL1 is degraded) and by the fact that some BimEL mutants lose their ability to bind to βTrCP, regardless of their binding to MCL1 (Dehan et al, 2009).Although the details of MCL1 regulation at mitotic arrest have only begun to unfold, it is clear that this pathway holds promise for furthering our understanding of the regulation of apoptosis. Microtubule-poisoning agents have historically been reliable chemotherapeutics, so, identifying cellular components that regulate MCL1 degradation during mitotic arrest is not only a way to stratify patients for a positive response to such drugs, but might also lead to the identification of novel targets for pharmacological intervention.     

Autocrine beta-related interferon controls c-myc suppression and growth arrest during hematopoietic cell differentiation

Different hematopoietic cells produce minute amounts of beta-related interferon (IFN) following induction of differentiation by chemical or natural inducers. The endogenous IFN binds to type I cell surface receptors and modulates gene expression in the producer cells. We show that self-induction of two members of the IFN-induced gene family differs in the dose response sensitivity and the prolonged kinetics of mRNA accumulation from the response to exogenous IFN-beta 1. Production and response to endogenous IFN are also detected when bone marrow precursor cells differentiate to macrophages after exposure to colony stimulating factor 1. In M1 myeloid cells induced to differentiate by lung-conditioned medium, addition of antibodies against IFN-beta partially abrogates the reduction of c-myc mRNA and the loss in cell proliferative activity, which both occur during differentiation. The endogenous IFN therefore functions as an autocrine growth inhibitor that participates in controlling c-myc suppression and the specific G0/G1 arrest during terminal differentiation of hematopoietic cells.     

Amyloid fibrils formed by the programmed cell death regulator Bcl-xL

The accumulation of amyloid fibers due to protein misfolding is associated with numerous human diseases. For example, the formation of amyloid deposits in neurodegenerative pathologies is correlated with abnormal apoptosis. We report here the in vitro formation of various types of aggregates by Bcl-xL, a protein of the Bcl-2 family involved in the regulation of apoptosis. Bcl-xL forms aggregates in three states, micelles, native-like fibrils, and amyloid fibers, and their biophysical characterization has been performed in detail. Bcl-xL remains in its native state within micelles and native-like fibrils, and our results suggest that native-like fibrils are formed by the association of micelles. Formation of amyloid structures, that is, nonnative intermolecular β-sheets, is favored by the proximity of proteins within fibrils at the expense of the Bcl-xL native structure. Finally, we provide evidence of a direct relationship between the amyloid character of the fibers and the tertiary-structure stability of the native Bcl-xL. The potential causality between the accumulation of Bcl-xL into amyloid deposits and abnormal apoptosis during neurodegenerative diseases is discussed.     

Increased phosphorylation rate of intermediate filaments during mitotic arrest

A detergent insoluble 58 000 D polypeptide is phosphorylated at an increased rate during mitotic arrest in cultured Chinese hamster ovary cells. Using one-dimensional peptide mapping, this polypeptide was identified as a phosphorylated form of intermediate filament protein (IFP). The increased rate of phosphorylation is observed with either colcemid- or vinblastine-treated mitotic cells, but is not seen with similarly treated interphase cells.     

Parvovirus infection-induced cell death and cell cycle arrest

The cytopathic effects induced during parvovirus infection have been widely documented. Parvovirus infection-induced cell death is often directly associated with disease outcomes (e.g., anemia resulting from loss of erythroid progenitors during parvovirus B19 infection). Apoptosis is the major form of cell death induced by parvovirus infection. However, nonapoptotic cell death, namely necrosis, has also been reported during infection of the minute virus of mice, parvovirus H-1 and bovine parvovirus. Recent studies have revealed multiple mechanisms underlying the cell death during parvovirus infection. These mechanisms vary in different parvoviruses, although the large nonstructural protein (NS)1 and the small NS proteins (e.g., the 11 kDa of parvovirus B19), as well as replication of the viral genome, are responsible for causing infection-induced cell death. Cell cycle arrest is also common, and contributes to the cytopathic effects induced during parvovirus infection. While viral NS proteins have been indicated to induce cell cycle arrest, increasing evidence suggests that a cellular DNA damage response triggered by an invading single-stranded parvoviral genome is the major inducer of cell cycle arrest in parvovirus-infected cells. Apparently, in response to infection, cell death and cell cycle arrest of parvovirus-infected cells are beneficial to the viral cell lifecycle (e.g., viral DNA replication and virus egress). In this article, we will discuss recent advances in the understanding of the mechanisms underlying parvovirus infection-induced cell death and cell cycle arrest.     

Bcl-xL and UVRAG cause a monomer-dimer switch in Beclin1

Beclin1 has a key regulatory role in the initiation of autophagy and is a tumor suppressor. We have examined the interplay between viral or human Bcl-2-like proteins and UVRAG and their opposite effects on Beclin1. We show that Beclin1 forms a dimer in solution via its coiled-coil domain both in vivo and in vitro. Viral Bcl-2 binds independently to two sites on the Beclin1 dimer, one with high affinity and one with lower affinity, whereas human Bcl-x(L) binds both sites equally with relatively low affinity. UVRAG disrupts the Beclin1-dimer interface, forming a heterodimer with Beclin1, suggesting that this is how UVRAG causes its effects on Beclin1 to activate autophagy. Both Bcl-2-like proteins reduce the affinity of UVRAG for Beclin1 approximately 4-fold, suggesting that they stabilize the Beclin1 dimer. Moreover, coimmunoprecipitation assays show that UVRAG substantially reduces Beclin1 dimerization in vivo. These data explain the concentration-dependent interplay between Bcl-2, UVRAG, and Beclin1, as both tumor suppressors, UVRAG and Beclin1, have single-copy mutations in human cancers. Furthermore, our data suggest that an alternative strategy for developing anti-cancer compounds would be to disrupt the Beclin1-dimer interface.