Generation of cell lines with tetracycline-regulated autophagy and a role for autophagy in controlling cell size

Autophagy is an intracellular bulk degradation system. We established mouse fibroblast lines coupling the Tet-off system with an Atg5(-/-) mouse embryonic fibroblast line to artificially regulate autophagic ability. In the presence of doxycycline (Dox), Atg5 expression was completely suppressed and these cells were autophagy-defective. After removal of Dox, autophagic ability was restored within 6h. Very low levels of Atg5 could induce an autophagy competent state. We applied this novel system to examine the contribution of autophagy to controlling cell size. Cell size reduction in response to starvation was significantly inhibited in cells unable to undergo autophagy. The generated cell lines will be useful reagents for future mechanistic studies into the regulation and physiologic significance of autophagy.     

Digest this! A role for autophagy in controlling pathogens

Autophagy provides a mechanism for cells to conserve nutrients, but was recently associated with immunity to intracellular pathogens. Here, Zhao et al. (2008) present direct in vivo evidence that autophagy is linked to macrophage control of Toxoplasma gondii and Listeria monocytogenes and highlights that this process intersects with cytokine-mediated antimicrobial responses.     

c-di-AMP is a new second messenger in Staphylococcus aureus with a role in controlling cell size and envelope stress

The cell wall is a vital and multi-functional part of bacterial cells. For Staphylococcus aureus, an important human bacterial pathogen, surface proteins and cell wall polymers are essential for adhesion, colonization and during the infection process. One such cell wall polymer, lipoteichoic acid (LTA), is crucial for normal bacterial growth and cell division. Upon depletion of this polymer bacteria increase in size and a misplacement of division septa and eventual cell lysis is observed. In this work, we describe the isolation and characterization of LTA-deficient S. aureus suppressor strains that regained the ability to grow almost normally in the absence of this cell wall polymer. Using a whole genome sequencing approach, compensatory mutations were identified and revealed that mutations within one gene, gdpP (GGDEF domain protein containing phosphodiesterase), allow both laboratory and clinical isolates of S. aureus to grow without LTA. It was determined that GdpP has phosphodiesterase activity in vitro and uses the cyclic dinucleotide c-di-AMP as a substrate. Furthermore, we show for the first time that c-di-AMP is produced in S. aureus presumably by the S. aureus DacA protein, which has diadenylate cyclase activity. We also demonstrate that GdpP functions in vivo as a c-di-AMP-specific phosphodiesterase, as intracellular c-di-AMP levels increase drastically in gdpP deletion strains and in an LTA-deficient suppressor strain. An increased amount of cross-linked peptidoglycan was observed in the gdpP mutant strain, a cell wall alteration that could help bacteria compensate for the lack of LTA. Lastly, microscopic analysis of wild-type and gdpP mutant strains revealed a 13-22% reduction in the cell size of bacteria with increased c-di-AMP levels. Taken together, these data suggest a function for this novel secondary messenger in controlling cell size of S. aureus and in helping bacteria to cope with extreme membrane and cell wall stress.     

A novel role for autophagy in T cell education

During T cell development in the thymus, scanning of peptide/major histocompatibility (MHC) molecule complexes on the surface of thymic epithelial cells ensures that only useful (self-MHC restricted) and harmless (self-tolerant) thymocytes survive. In recent years, a number of distinct cell-biological features of thymic epithelial cells have been unraveled that may have evolved to render these cells particularly suited for T cell selection, e.g., cortical epithelial cells use unique proteolytic enzymes for the generation of MHC/peptide complexes, whereas medullary epithelial cells "promiscuously" express otherwise tissue-restricted self-antigens. We recently showed that macroautophagy in thymic epithelial cells contributes to CD4 T cell selection and is essential for the generation of a self-tolerant T cell repertoire. We propose that the unusually high constitutive levels of autophagy in thymic epithelial cells deliver endogenous proteins to MHC class II molecules for both positive and negative selection of developing thymocytes.     

自体吞噬在细胞死亡中的角色

一直以来人们认为自主性细胞死亡就是凋亡。然而 ,最近的研究表明 ,自主性细胞死亡不仅包括凋亡 ,还包括自体吞噬。自体吞噬通过与凋亡不同的降解机制 (膜包被降解 )和分子机制 (特定基因的激活 ) ,使细胞在某些特殊环境如饥饿、发育、分化等条件下自主死亡。在自主性细胞死亡中 ,自体吞噬与凋亡是两个既相互独立又紧密相关的过程。对自体吞噬机制的了解 ,必将为自主性细胞死亡机制的阐明及其相关疾病的治疗提供新的思路。     

Generation of reference cell lines,media, and a process platform for CHO cell biomanufacturing

Due to the favorable attributes of Chinese hamster ovary (CHO) cells for therapeutic proteins and antibodies biomanufacturing, companies generate proprietary cells with desirable phenotypes. One key attribute is the ability to stably express multi-gram per liter titers in chemically defined media. Cell, media, and feed diversity has limited community efforts to translate knowledge. Moreover, academic, and nonprofit researchers generally cannot study “industrially relevant” CHO cells due to limited public availability, and the time and knowledge required to generate such cells. To address these issues, a university-industrial consortium (Advanced Mammalian Biomanufacturing Innovation Center, AMBIC) has acquired two CHO “reference cell lines” from different lineages that express monoclonal antibodies. These reference cell lines have relevant production titers, key performance outcomes confirmed by multiple laboratories, and a detailed technology transfer protocol. In commercial media, titers over 2 g/L are reached. Fed-batch cultivation data from shake flask and scaled-down bioreactors is presented. Using productivity as the primary attribute, two academic sites aligned with tight reproducibility at each site. Further, a chemically defined media formulation was developed and evaluated in parallel to the commercial media. The goal of this work is to provide a universal, industrially relevant CHO culture platform to accelerate biomanufacturing innovation.     

The role of autophagy in plasma cell ontogenesis

We have identified in plasma cells a novel ATG5-dependent selective negative control on the secretory pathway, which restricts antibody production, sustaining energy metabolism. Revealing new immune functions, autophagy is required in vivo for antibody responses and to maintain the memory plasma cell compartment.     

Generation and characterization of human insulin-releasing cell lines

Background  

The in vitro culture of insulinomas provides an attractive tool to study cell proliferation and insulin synthesis and secretion. However, only a few human beta cell lines have been described, with long-term passage resulting in loss of insulin secretion. Therefore, we set out to establish and characterize human insulin-releasing cell lines.     

FTY720 induces necrotic cell death and autophagy in ovarian cancer cells: a protective role of autophagy

FTY720, a sphingosine analog, is a novel immunosuppressant currently undergoing multiple clinical trials for the prevention of organ transplant rejection and treatment of various autoimmune diseases. Recent studies indicate an additional cytotoxic effect of FTY720 and its preclinical efficacy in a variety of cancer models, yet the underlying mechanisms remain unclear. We demonstrate here for the first time that FTY720 exhibits a potent, dose- and time-dependent cytotoxic effect in human ovarian cancer cells, even in the cells that are resistant to cisplatin, a commonly prescribed chemotherapeutic drug for treatment of ovarian cancer. In contrast to the previously reported cytotoxicity of FTY720 in many other cancer cell types, FTY720 kills ovarian cancer cells independent of caspase 3 activity and induces cellular swelling and cytoplasmic vacuolization with evident features of necrotic cell death. Furthermore, the presence of autophagic hallmarks, including an increased number of autophagosomes and the formation and accumulation of LC3-II, are observed in FTY720-treated cells before cell death. FTY720 treatment enhances autophagic flux as reflected in the increased LC3 turnover and p62 degradation. Notably, blockade of autophagy by either specific chemical inhibitors or siRNAs targeting Beclin 1 or LC3 resulted in aggravated necrotic cell death in response to FTY720, suggesting that FTY720-induced autophagy plays a self-protective role against its own cytotoxic effect. Thus, our findings not only demonstrate a new death pathway underlying the cytotoxic effect of FTY720, but also suggest that targeting autophagy could augment the anticancer potency, providing the framework for further development of FTY720 as a new chemotherapeutic agent for ovarian cancer treatment.     

Generation of rabbit pluripotent stem cell lines

Pluripotent stem cells have the capacity to divide indefinitely and to differentiate into all somatic cells and tissue lines. They can be genetically manipulated in vitro by knocking genes in or out, and therefore serve as an excellent tool for gene function studies and for the generation of models for some human diseases. Since 1981, when the first mouse embryonic stem cell (ESC) line was generated, many attempts have been made to generate pluripotent stem cell lines from other species. Comparative characterization of ESCs from different species would help us to understand differences and similarities in the signaling pathways involved in the maintenance of pluripotency and the initiation of differentiation, and would reveal whether the fundamental mechanism controlling self-renewal of pluripotent cells is conserved across different species. This report gives an overview of research into embryonic and induced pluripotent stem cells in the rabbit, an important nonrodent species with considerable merits as an animal model for specific diseases. A number of putative rabbit ESC and induced pluripotent stem cell lines have been described. All of them expressed stem cell-associated markers and maintained apparent pluripotency during multiple passages in vitro, but none have been convincingly proven to be fully pluripotent in vivo. Moreover, as in other domestic species, the markers currently used to characterize the putative rabbit ESCs are suboptimal because recent studies have revealed that they are not always specific to the pluripotent inner cell mass. Future validation of rabbit pluripotent stem cells would benefit greatly from a validated panel of molecular markers specific to pluripotent cells of the developing rabbit embryos. Using rabbit-specific pluripotency genes may improve the efficiency of somatic cell reprogramming for generating induced pluripotent stem cells and thereby overcome some of the challenges limiting the potential of this technology.     

Wild-type rabies virus induces autophagy in human and mouse neuroblastoma cell lines

Different rabies virus (RABV) strains have their own biological characteristics, but little is known about their respective impact on autophagy. Therefore, we evaluated whether attenuated RABV HEP-Flury and wild-type RABV GD-SH-01 strains triggered autophagy. We found that GD-SH-01 infection significantly increased the number of autophagy-like vesicles, the accumulation of enhanced green fluorescent protein (EGFP)-LC3 fluorescence puncta and the conversion of LC3-I to LC3-II, while HEP-Flury was not able to induce this phenomenon. When evaluating autophagic flux, we found that GD-SH-01 infection triggers a complete autophagic response in the human neuroblastoma cell line (SK), while autophagosome fusion with lysosomes was inhibited in a mouse neuroblastoma cell line (NA). In these cells, GD-SH-01 led to apoptosis and mitochondrial dysfunction while triggering autophagy, and apoptosis could be decreased by enhancing autophagy. To further identify the virus constituent causing autophagy, 5 chimeric recombinant viruses carrying single genes of HEP-Flury instead of those of GD-SH-01 were rescued. While the HEP-Flury virus carrying the wild-type matrix protein (M) gene of RABV triggered LC3-I to LC3-II conversion in SK and NA cells, replacement of genes of nucleoprotein (N), phosphoprotein (P) and glycoprotein (G) produced only minor autophagy. But no one single structural protein of GD-SH-01 induced autophagy. Moreover, the AMPK signaling pathway was activated by GD-SH-01 in SK. Therefore, our data provide strong evidence that autophagy is induced by GD-SH-01 and can decrease apoptosis in vitro. Furthermore, the M gene of GD-SH-01 may cooperatively induce autophagy.     

A role of autophagy in Trypanosoma brucei cell death

The early branching eukaryote Trypanosoma brucei contains functional autophagy machinery that allows regulated degradation of its own cellular components. In this study, we examined the function of two Atg8 genes, TbAtg8.1 and TbAtg8.2, in starvation-induced autophagosome formation and cell death in procyclic T. brucei. Upon starvation, both TbAtg8.1 and TbAtg8.2 localize to punctate structures characteristic of autophagosomes as shown by fluorescence and electron microscopy, and wortmannin and chloroquine treatments. While TbAtg8.1 depletion has no detectable effects on TbAtg8.2 recruitment to autophagosomes, TbAtg8.2 depletion greatly reduced the autophagosome relocation of TbAtg8.1. Depletion of TbAtg8.1 and 8.2, individually or together, promote cell survival under starvation conditions. Taken together, these observations confirm the presence of an autophagy-related cell death pathway in T. brucei, where TbAtg8.1 and TbAtg8.2 play essential but distinct roles in autophagosome formation and cell death.     

Generation and regulation of developing immortalized neural cell lines

The genetic and environmental signals that regulate progressive lineage elaboration in the mammalian brain are poorly understood. In addition, characterization of the developmental profiles of early central nervous system (CNS) stem/ progenitor cells and analysis of the mechanisms involved in their clonal expansion, lineage restriction, and cellular maturation have been fragmentary and elusive. These seminal neurodevelopmental issues have been examined using a series of clonally derived neural stem/progenitor cell lines established by retroviral transduction of embryonic (E16.5-E17.5) murine hippocampal and cerebellar cells using temperature-sensitive alleles (A58/U19) of the simian virus (SV) 40 large tumor (T) antigen. Under conditions permissive for T-antigen expression (33 degrees C), single neural stem cells exhibited self-renewal, clonal expansion, and both symmetric and asymmetric modes of cell division. By contrast, at the nonpermissive temperature for T-antigen expression (39 degrees C), specific sets of cytokines potentiated the progressive elaboration of neuronal, oligodendroglial, and astroglial lineage species. These observations demonstrate that a spectrum of genetic and epigenetic signals and distinct cellular processes are involved in orchestrating the evolution of individual neural lineages from regional CNS stem/progenitor species. Further, the availability of conditionally immortalized neural cell lines that can be transplanted back into the mammalian brain may represent an important experimental resource for the detailed characterization of cellular and molecular mechanisms involved in the developmental sculpting, plasticity, and regeneration of the mammalian CNS.     

Type I interferons induce autophagy in certain human cancer cell lines

Autophagy is an evolutionarily conserved cellular recycling mechanism that occurs at a basal level in all cells. It can be further induced by various stimuli including starvation, hypoxia, and treatment with cytokines such as IFNG/IFNγ and TGFB/TGFβ. Type I IFNs are proteins that induce an antiviral state in cells. They also have antiproliferative, proapoptotic and immunomodulatory activities. We investigated whether type I IFN can also induce autophagy in multiple human cell lines. We found that treatment with IFNA2c/IFNα2c and IFNB/IFNβ induces autophagy by 24 h in Daudi B cells, as indicated by an increase of autophagy markers MAP1LC3-II, ATG12–ATG5 complexes, and a decrease of SQSTM1 expression. An increase of MAP1LC3-II was also detected 48 h post-IFNA2c treatment in HeLa S3, MDA-MB-231, T98G and A549 cell lines. The presence of autophagosomes in selected cell lines exposed to type I IFN was confirmed by electron microscopy analysis. Increased expression of autophagy markers correlated with inhibition of MTORC1 in Daudi cells, as well as inhibition of cancer cell proliferation and changes in cell cycle progression. Concomitant blockade of either MTOR or PI3K-AKT signaling in Daudi and T98G cells treated with IFNA2c increased the level of MAP1LC3-II, indicating that the PI3K-AKT-MTORC1 signaling pathway may modulate IFN-induced autophagy in these cells. Taken together, our findings demonstrated a novel function of type I IFN as an inducer of autophagy in multiple cell lines.     

雷帕霉素对二种鳞翅目昆虫细胞自噬和凋亡的影响

以2种鳞翅目昆虫细胞为材料,采用雷帕霉素进行处理,初步研究自噬作用与昆虫细胞凋亡的关系。结果表明:雷帕霉素能够提高家蚕细胞系BMN-e细胞的自噬水平,并能诱导BMN-e细胞发生凋亡;自噬抑制剂3-甲基腺嘌呤能抑制雷帕霉素诱导的BMN-e细胞凋亡。相反,雷帕霉素虽能诱导斜纹夜蛾细胞系SL-HP细胞的自噬水平提高,但不能诱导斜纹夜蛾Spodoptera litura(Fabricius)细胞发生凋亡;雷帕霉素的预处理能抑制放线菌素D诱导的斜纹夜蛾细胞系SL-HP细胞发生凋亡;自噬抑制剂3-甲基腺嘌呤对放线菌素D诱导的细胞凋亡没有影响。因此家蚕Bombyx mori细胞自噬水平的提高与细胞凋亡具有正相关性,而斜纹夜蛾细胞自噬水平的提高与细胞凋亡不相关,相反还对细胞凋亡的诱导具有一定的抑制作用。