Proteomic Analysis Reveals a Role for Bcl2-associated Athanogene 3 and Major Vault Protein in Resistance to Apoptosis in Senescent Cells by Regulating ERK1/2 Activation

Senescence is a prominent solid tumor response to therapy in which cells avoid apoptosis and instead enter into prolonged cell cycle arrest. We applied a quantitative proteomics screen to identify signals that lead to therapy-induced senescence and discovered that Bcl2-associated athanogene 3 (Bag3) is up-regulated after adriamycin treatment in MCF7 cells. Bag3 is a member of the BAG family of co-chaperones that interacts with Hsp70. Bag3 also regulates major cell-signaling pathways. Mass spectrometry analysis of the Bag3 Complex revealed a novel interaction between Bag3 and Major Vault Protein (MVP). Silencing of Bag3 or MVP shifts the cellular response to adriamycin to favor apoptosis. We demonstrate that Bag3 and MVP contribute to apoptosis resistance in therapy-induced senescence by increasing the level of activation of extracellular signal-regulated kinase1/2 (ERK1/2). Silencing of either Bag3 or MVP decreased ERK1/2 activation and promoted apoptosis in adriamycin-treated cells. An increase in nuclear accumulation of MVP is observed during therapy-induced senescence and the shift in MVP subcellular localization is Bag3-dependent. We propose a model in which Bag3 binds to MVP and facilitates MVP accumulation in the nucleus, which sustains ERK1/2 activation. We confirmed that silencing of Bag3 or MVP shifts the response toward apoptosis and regulates ERK1/2 activation in a panel of diverse breast cancer cell lines. This study highlights Bag3-MVP as an important complex that regulates a potent prosurvival signaling pathway and contributes to chemotherapy resistance in breast cancer.Cellular senescence plays an important role in determining the response of tumors to cancer therapy (1). Senescence is regulated by the p53 and p16-pRB tumor suppressor pathways and characterized by irreversible cell cycle arrest and expression of the lysosomal protein, senescence associated beta galactosidase (SA-β-gal)¹. Additional characteristics of senescent cells include the presence of senescence-associated heterochromatic foci, and a senescence associated secretory phenotype (SASP) (2). Because of the SASP of senescent cells, therapy-induced senescence (TIS) may be harmful in cancer and the quantitative elimination of senescent cells could prove to be therapeutically beneficial. A recent study demonstrated that pharmacologically targeting the metabolic pathways of TIS in vivo prompted tumor regression and improved treatment outcomes (3).A characteristic of senescent cells is their ability to resist apoptosis although the responsible mechanism is poorly understood. Impairment of apoptosis in senescent cells is associated with a poor outcome in cancer (4). Manipulation of the apoptotic machinery may serve as a therapeutic means of eliminating senescent cells with harmful SASP. It has been proposed that in senescent cells, p53 may preferentially activate genes that arrest proliferation, rather than those that facilitate apoptosis. Alternatively, resistance to apoptosis may be caused by altered expression of proteins that inhibit, promote, or mediate apoptotic cell death, such as Bcl2.Bcl2 associated athanogene 3 (Bag3) is a member of the BAG family of chaperones that interacts with the ATPase domain of heat shock protein-70 (Hsp70). In addition to its BAG domain, Bag3 contains a WW domain and a proline-rich (PXXP) repeat, which mediates binding to partners other than Hsp70. Bag3 is expressed in response to cellular stress under the induction of HSF1 and is known to suppress apoptosis and regulate autophagy (5–6). Suppression of apoptosis may be partially explained by the ability of Bag3 to protect Bcl2 family members against proteasomal degradation (7). In normal cells, Bag3 is constitutively expressed in only a few cell types, including cardiomyocytes (8). Bag3 is overexpressed in leukemia and several solid tumors where it has been reported to sustain cell survival, induce resistance to therapy, and promote metastasis. The pleiotropic functions of Bag3 may reflect its ability to assemble scaffolding complexes, which participate in multiple signal transduction pathways (9).In this study, we describe a role for Bag3 in regulating cancer chemotherapy induced senescence in breast cancer cell. Using a quantitative SILAC approach, we show that Bag3 is up-regulated in TIS. Mass spectrometry analysis reveals that Bag3 binds to the Major Vault Protein (MVP) complex, a protein complex strongly associated with chemotherapy resistance. We also show that Bag3 and MVP contribute to apoptosis resistance by regulating ERK1/2 signaling in senescent MCF7 and ZR751 cells.     

Low fruit consumption and folate deficiency are associated with LINE-1 hypomethylation in women of a cancer-free population

Several dietary agents, such as micronutrient and non-nutrient components, the so-called bioactive food components, have been shown to display anticancer properties and influence genetic processes. The most common epigenetic change is DNA methylation. Hypomethylation of long interspersed elements (LINE-1) has been associated with an increased risk of several cancers, although conflicting findings have also been observed. The aim of the present study was to test the hypothesis that a low adherence to the Mediterranean diet (MD) and folate deficiency may cause LINE-1 hypomethylation in blood leukocytes of healthy women, and thus genomic instability. One hundred and seventy-seven non-pregnant women were enrolled. Mediterranean diet score (MDS) and folate intake were calculated using a food frequency questionnaire. LINE-1 methylation level was measured by pyrosequencing analysis in three CpG sites of LINE-1 promoter. According to MDS, only 9.6 % of subjects achieved a high adherence to MD. Taking into account the use of supplements, there was a high prevalence of folate deficiency (73.4 %). Women whose consumption of fruit was below the median value (i.e., <201 gr/day) were 3.7 times more likely to display LINE-1 hypomethylation than women whose consumption was above the median value (OR 3.7; 95 % CI 1.4–9.5). Similarly, women with folate deficiency were 3.6 times more likely to display LINE-1 hypomethylation than women with no folate deficiency (OR 3.6; 95 % CI 1.1–12.1). A dietary pattern characterized by low fruit consumption and folate deficiency is associated with LINE-1 hypomethylation and with cancer risk.     

Should reference conditions be drawn from a single 10 ha plot? Assessing representativeness in a 10,000 ha old‐growth European beech forest

The management targets of modern forestry are often dictated by a desire to restore natural conditions, largely considered to be those found in contemporary reference sites. Beech reference sites are usually subjectively placed plots located in old‐growth reserves. Given the inherent variability in old‐growth, the validity of using a single such plot to guide restoration efforts is questionable. We therefore applied 3 methods to assess the representativeness of a 10 ha research plot in an old‐growth European beech (Fagus sylvatica L.) forest in Ukraine compared to an inventory of the entire 10,282 ha forest reserve. We compared the research plot to the 500‐m² inventory plots using (1) permutation tests of structure metrics, (2) synthetic multivariate structural condition, and (3) functional condition via the proportion of area assigned to 8 forest development phases. Despite up to 82% distributional overlap for some metrics, both the averages and distributions of individual structural metrics (e.g. basal area, tree diameters) differed significantly between the RP and the inventory, as did the synthetic structural condition and the proportion of late optimal and decay phases. Extrapolations from this subjectively placed plot to the surrounding old‐growth matrix would overestimate several stereotypical “old‐growth” structures. These results support the need to draw on multiple reference sites and metrics and to select restoration target conditions that account for the variability associated with naturally dynamic ecosystems. A lack of absolute representativeness does not, however, necessarily preclude the generalizability of process‐based dynamics from old‐growth remnants.     

MiR‐133a regulates collagen 1A1: Potential role of miR‐133a in myocardial fibrosis in angiotensin II‐dependent hypertension

MicroRNAs play an important role in myocardial diseases. MiR‐133a regulates cardiac hypertrophy, while miR‐29b is involved in cardiac fibrosis. The aim of this study was to evaluate whether miR‐133a and miR‐29b play a role in myocardial fibrosis caused by Angiotensin II (Ang II)‐dependent hypertension. Sprague–Dawley rats were treated for 4 weeks with Ang II (200 ng/kg/min) or Ang II + irbesartan (50 mg/kg/day in drinking water), or saline by osmotic minipumps. At the end of the experimental period, cardiac miR‐133a and miR‐29b expression was measured by real‐time PCR, and myocardial fibrosis was evaluated by morphometric analysis. A computer‐based prediction algorithm led to the identification of collagen 1a1 (Col1A1) as a putative target of miR‐133a. A reporter plasmid bearing the 3′‐untranslated regions (UTRs) of Col1A1 mRNA was constructed and luciferase assay was performed. MiR‐133a suppressed the activity of luciferase when the reporter gene was linked to a 3′‐UTR segment of Col1A1 (P < 0.01). Mutation of miR‐133a binding sites in the 3′‐UTR of Col1A1 mRNA abolished miR‐133a‐mediated repression of reporter gene activity, showing that Col1A1 is a real target of miR‐133a. In vivo, Ang II caused an increase in systolic blood pressure (P < 0.0001, tail cuff) and myocardial fibrosis in presence of a decrease in miR‐133a (P < 0.01) and miR‐29b (P < 0.01), and an increase in Col1A1 expression (P < 0.01). These effects were abolished by Ang II administration + irbesartan. These data demonstrate a relationship between miR‐133a and Col1A1, suggesting that myocardial fibrosis occurring in Ang II‐dependent hypertension is regulated by the down‐regulation of miR‐133a and miR‐29b through the modulation of Col1A1 expression. J. Cell. Physiol. 227: 850–856, 2012. © 2011 Wiley Periodicals, Inc.     

Auxin transport at cellular level: new insights supported by mathematical modelling

The molecular basis of cellular auxin transport is still not fully understood. Although a number of carriers have been identified and proved to be involved in auxin transport, their regulation and possible activity of as yet unknown transporters remain unclear. Nevertheless, using single-cell-based systems it is possible to track the course of auxin accumulation inside cells and to specify and quantify some auxin transport parameters. The synthetic auxins 2,4-dichlorophenoxyacetic acid (2,4-D) and naphthalene-1-acetic acid (NAA) are generally considered to be suitable tools for auxin transport studies because they are transported specifically via either auxin influx or efflux carriers, respectively. Our results indicate that NAA can be metabolized rapidly in tobacco BY-2 cells. The predominant metabolite has been identified as NAA glucosyl ester and it is shown that all NAA metabolites were retained inside the cells. This implies that the transport efficiency of auxin efflux transporters is higher than previously assumed. By contrast, the metabolism of 2,4-D remained fairly weak. Moreover, using data on the accumulation of 2,4-D measured in the presence of auxin transport inhibitors, it is shown that 2,4-D is also transported by efflux carriers. These results suggest that 2,4-D is a promising tool for determining both auxin influx and efflux activities. Based on the accumulation data, a mathematical model of 2,4-D transport at a single-cell level is proposed. Optimization of the model provides estimates of crucial transport parameters and, together with its validation by successfully predicting the course of 2,4-D accumulation, it confirms the consistency of the present concept of cellular auxin transport.     

Autocrine TNF is critical for the survival of human dendritic cells by regulating BAK, BCL-2, and FLIPL

The life span of dendritic cells (DCs) is determined by the balance of pro- and antiapoptotic proteins. In this study, we report that serum-free cultured human monocyte-derived DCs after TLR stimulation with polyinosinic acid-polycytidylic acid or LPS underwent apoptosis, which was correlated with low TNF production. Apoptosis was prevented by the addition of exogenous TNF or by concomitant stimulation with R-848, which strongly amplified endogenous TNF production. Neutralization of TNF confirmed that DC survival was mediated by autocrine TNF induced either by stimulation with R-848 or by ligation of CD40. DCs stimulated by polyinosinic acid-polycytidylic acid or IFN-β, another known inducer of DC apoptosis, were characterized by high levels and activation of the proapoptotic protein BAK. The ratio of antiapoptotic BCL-2 to BAK correlated best with the survival of activated DCs. Addition of TNF increased this ratio but had little effect on BAX and XIAP. Knockdown experiments using small interfering RNAs confirmed that the survival of activated and also of immature DCs was regulated by BAK and showed that TNF was protective only in the presence of FLIP(L). Together, our data demonstrate that the survival of DCs during differentiation and activation depends on autocrine TNF and that the inhibition of BAK plays an important role in this process.     

BLyS-mediated modulation of naive B cell subsets impacts HIV Env-induced antibody responses

Neutralizing Abs provide the protective effect of the majority of existing human vaccines. For a prophylactic vaccine against HIV-1, broadly neutralizing Abs targeting conserved epitopes of the viral envelope glycoproteins (Env) are likely required, because the pool of circulating HIV-1 variants is extremely diverse. The failure to efficiently induce broadly neutralizing Abs by vaccination may be due to the use of suboptimal immunogens or immunization regimens, or it may indicate that B cells specific for broadly neutralizing Env determinants are selected against during peripheral checkpoints, either before or after Ag encounter. To investigate whether perturbation of B cell subsets prior to immunization with recombinant Env protein affects the vaccine-induced Ab response in mice, we used B lymphocyte stimulator (BLyS), a cytokine that regulates survival and selection of peripheral B cells. We show that the transient BLyS treatment used in this study substantially affected naive B cell populations; in particular, it resulted in more B cells surviving counter-selection at the transitional stages. We also observed more mature naive B cells, especially marginal zone B cells, in BLyS-treated mice. Intriguingly, provision of excess BLyS prior to immunization led to a consistent improvement in the frequency and potency of HIV-1 Env vaccine-induced neutralizing Ab responses, without increasing the number of Env-specific Ab-secreting cells or the Ab-binding titers measured after boosting. The results presented in this article suggest that an increased understanding of BLyS-regulated processes may help the design of vaccine regimens aimed at eliciting improved neutralizing Ab responses against HIV-1.