The Cytosolic pH of Individual Saccharomyces cerevisiae Cells Is a Key Factor in Acetic Acid Tolerance

It was shown recently that individual cells of an isogenic Saccharomyces cerevisiae population show variability in acetic acid tolerance, and this variability affects the quantitative manifestation of the trait at the population level. In the current study, we investigated whether cell-to-cell variability in acetic acid tolerance could be explained by the observed differences in the cytosolic pHs of individual cells immediately before exposure to the acid. Results obtained with cells of the strain CEN.PK113-7D in synthetic medium containing 96 mM acetic acid (pH 4.5) showed a direct correlation between the initial cytosolic pH and the cytosolic pH drop after exposure to the acid. Moreover, only cells with a low initial cytosolic pH, which experienced a less severe drop in cytosolic pH, were able to proliferate. A similar correlation between initial cytosolic pH and cytosolic pH drop was also observed in the more acid-tolerant strain MUCL 11987-9. Interestingly, a fraction of cells in the MUCL 11987-9 population showed initial cytosolic pH values below the minimal cytosolic pH detected in cells of the strain CEN.PK113-7D; consequently, these cells experienced less severe drops in cytosolic pH. Although this might explain in part the difference between the two strains with regard to the number of cells that resumed proliferation, it was observed that all cells from strain MUCL 11987-9 were able to proliferate, independently of their initial cytosolic pH. Therefore, other factors must also be involved in the greater ability of MUCL 11987-9 cells to endure strong drops in cytosolic pH.     

肿瘤微环境应激因子乳酸在肿瘤发展中的作用

肿瘤微环境应激主要包括:缺氧、胞外酸环境、葡萄糖缺乏等。乳酸堆积也是肿瘤微环境应激中一个重要特征。长期以来,乳酸一直被认为是代谢废物,但随着研究深入,发现乳酸作为癌代谢物与肿瘤增殖、转移、血管生成、免疫逃逸以及放化疗效果等肿瘤生物学功能密切相关,此外乳酸还可促进缺氧诱导因子稳定、作为"替代燃料"供能等进一步促进肿瘤恶性进展。因此,本文就肿瘤微环境中乳酸代谢与调控、乳酸对肿瘤生物学功能的影响等方面作一综述,旨在为针对乳酸这一异常代谢特征的抗肿瘤药物开发及临床治疗提供必要依据。     

Pigment Epithelium-Derived Factor Is a Survival Factor for Cerebellar Granule Cells in Culture

Abstract: Pigment epithelium-derived factor (PEDF), purified from human fetal retinal pigment epithelium cell culture medium, was shown to potentiate the differentiation of human Y-79 retinoblastoma cells. To investigate potential neurotrophic effects of PEDF on neurons other than those of retinal derivation, we used cultures of cerebellar granule cells. The number of cerebellar granule cells was significantly larger in the presence of PEDF, as demonstrated by an assay for viable cells that uses 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H -tetrazolium, inner salt, conversion, by cell count, and by immunocytochemistry. The effect of PEDF showed a dose-response relationship, with a larger effect in chemically defined medium than in serum-containing medium [ED₅₀ = 30 ng/ml (0.70 n M ) in chemically defined medium and 100 ng/ml (2.3 n M ) in serum-containing medium]. PEDF had no effect on incorporation of bromodeoxyuridine (cell proliferation) or on neurofilament content (neurite outgrowth) measured by an enzyme-linked immunoadsorbent assay. These results demonstrate that PEDF has a neurotrophic survival effect on cerebellar granule cells in culture and suggest the possibility that it may affect other CNS neurons as well.     

Granuloma Encapsulation Is a Key Factor for Containing Tuberculosis Infection in Minipigs

A transthoracic infection involving a low dose of Mycobacterium tuberculosis has been used to establish a new model of infection in minipigs. The 20-week monitoring period showed a marked Th1 response and poor humoral response for the whole infection. A detailed histopathological analysis was performed after slicing the formalin-fixed whole lungs of each animal. All lesions were recorded and classified according to their microscopic aspect, their relationship with the intralobular connective network and their degree of maturity in order to obtain a dissemination ratio (DR) between recent and old lesions. CFU counts and evolution of the DR with time showed that the proposed model correlated with a contained infection, decreasing from week 9 onwards. These findings suggest that the infection induces an initial Th1 response, which is followed by local fibrosis and encapsulation of the granulomas, thereby decreasing the onset of new lesions. Two therapeutic strategies were applied in order to understand how they could influence the model. Thus, chemotherapy with isoniazid alone helped to decrease the total number of lesions, despite the increase in DR after week 9, with similar kinetics to those of the control group, whereas addition of a therapeutic M. tuberculosis fragment-based vaccine after chemotherapy increased the Th1 and humoral responses, as well as the number of lesions, but decreased the DR. By providing a local pulmonary structure similar to that in humans, the mini-pig model highlights new aspects that could be key to a better understanding tuberculosis infection control in humans.     

Serotonin Is a Key Factor for Mouse Red Blood Cell Survival

Serotonin (5-HT) is a monoamine originally purified from blood as a vasoactive agent. In nonneuronal tissues, its presence is linked with the expression of tryptophan hydroxylase 1 (TPH1) that catalyzes the rate-limiting step of its synthesis. Targeted disruption in mice of the TPH1 gene results in very low levels of circulating 5-HT. Previous analysis of the TPH1 knockout (TPH1^−/−) mouse revealed that they develop a phenotype of macrocytic anemia with a reduced half-life of their circulating red blood cells (RBC). In this study, to establish whether the observed reduced half-life of TPH1^−/− RBC is an intrinsic or an extrinsic characteristic, we compared their survival to RBC isolated from wild-type mice. Both in vivo and in vitro data converge to demonstrate an extrinsic protective effect of 5-HT since presence of 5-HT in the RBC environment protects RBC from senescence. The protective effect played by 5-HT is not mediated through activation of a classical pharmacological pathway as no 5-HT receptors were detected on isolated RBC. Rather, 5-HT acts as an effective antioxidant since reduction of 5-HT circulating levels are associated with a decrease in the plasma antioxidant capacity. We further demonstrate a link between oxidation and the removal of damaged RBC following transfusion, as supplementation with 5-HT improves RBC post-transfusion survival in a mouse model of blood banking.     

Leukemia Inhibitory Factor Is an Autocrine Survival Factor for Schwann Cells

Abstract: Schwann cells play a major role in promoting nerve survival and regeneration after injury. Their activities include providing neurotrophic factors and increasing the production of extracellular matrix components and cell surface adhesion molecules to promote axon regeneration. Following nerve transection, leukemia inhibitory factor (LIF) is up-regulated by Schwann cells at the injury site. LIF receptors are also up-regulated at the nerve injury site, but their cellular localization and function have not been fully characterized. We demonstrate that Schwann cells express mRNAs for LIF and the LIF receptor components LIF receptor subunit β and glycoprotein 130 in vitro. We also show that although LIF is not required for the genesis of Schwann cells, it can potentiate the survival of differentiated Schwann cells in the context of neuregulin support. Not only does exogenous LIF promote survival under these conditions, but addition of the soluble LIF receptor (LIF binding protein) and anti-LIF antibodies significantly reduced cell survival, suggesting that LIF exerts autocrine effects. These results suggest that Schwann cell survival following nerve injury is potentially modulated by LIF.     

Protein S, an Antithrombotic Factor, Is Synthesized and Released by Neural Tumor Cells

Abstract: Protein S, an anticoagulant factor in the protein C antithrombotic pathway, was found to be synthesized and released by six tumor cell lines of neural origin by western blotting and ELISA. The rate of synthesis ranged from three-to 11-fold higher than that of a microvascular endothelial cell line and 36–144% that of a hepatoma cell line. The secreted protein S displayed specific anticoagulant activity similar to that of purified plasma protein S, implying that it was fully γ-carboxylated. Ten primary brain tumor tissues also expressed protein S antigen, as shown by western blot analysis. Expression of anticoagulantly active protein S by neural cells raises important questions concerning possible physiologic roles for this multidomain protein beyond its function in control of thrombosis.     

Phosphatidylethanolamine Is a Key Regulator of Membrane Fluidity in Eukaryotic Cells

Adequate membrane fluidity is required for a variety of key cellular processes and in particular for proper function of membrane proteins. In most eukaryotic cells, membrane fluidity is known to be regulated by fatty acid desaturation and cholesterol, although some cells, such as insect cells, are almost devoid of sterol synthesis. We show here that insect and mammalian cells present similar microviscosity at their respective physiological temperature. To investigate how both sterols and phospholipids control fluidity homeostasis, we quantified the lipidic composition of insect SF9 and mammalian HEK 293T cells under normal or sterol-modified condition. As expected, insect cells show minimal sterols compared with mammalian cells. A major difference is also observed in phospholipid content as the ratio of phosphatidylethanolamine (PE) to phosphatidylcholine (PC) is inverted (4 times higher in SF9 cells). In vitro studies in liposomes confirm that both cholesterol and PE can increase rigidity of the bilayer, suggesting that both can be used by cells to maintain membrane fluidity. We then show that exogenously increasing the cholesterol amount in SF9 membranes leads to a significant decrease in PE:PC ratio whereas decreasing cholesterol in HEK 293T cells using statin treatment leads to an increase in the PE:PC ratio. In all cases, the membrane fluidity is maintained, indicating that both cell types combine regulation by sterols and phospholipids to control proper membrane fluidity.     

外泌体靶向递药在肿瘤治疗中的进展

外泌体是由细胞分泌、粒径为30～ 150 nm的纳米囊泡.外泌体具有优越的生物相容性、良好的载药功能以及便于修饰的膜表面,是一种具有潜力的药物递送载体.在肿瘤治疗研究中,可利用具有靶向识别功能的外泌体来降低脱靶效应,减少不良反应,达到增强治疗效果的目的 .归纳了用不同修饰方法增强外泌体靶向性的研究进展,总结了近五年来利...     

BMP9 Is a Proliferative and Survival Factor for Human Hepatocellular Carcinoma Cells

TGF-β family members play a relevant role in tumorigenic processes, including hepatocellular carcinoma (HCC), but a specific implication of the Bone Morphogenetic Protein (BMP) subfamily is still unknown. Although originally isolated from fetal liver, little is known about BMP9, a BMP family member, and its role in liver physiology and pathology. Our results show that BMP9 promotes growth in HCC cells, but not in immortalized human hepatocytes. In the liver cancer cell line HepG2, BMP9 triggers Smad1,5,8 phosphorylation and inhibitor of DNA binding 1 (Id1) expression up- regulation. Importantly, by using chemical inhibitors, ligand trap and gene silencing approaches we demonstrate that HepG2 cells autocrinely produce BMP9 that supports their proliferation and anchorage independent growth. Additionally, our data reveal that in HepG2 cells BMP9 triggers cell cycle progression, and strikingly, completely abolishes the increase in the percentage of apoptotic cells induced by long-term incubation in low serum. Collectively, our data unveil a dual role for BMP9, both promoting a proliferative response and exerting a remarkable anti-apoptotic function in HepG2 cells, which result in a robust BMP9 effect on liver cancer cell growth. Finally, we show that BMP9 expression is increased in 40% of human HCC tissues compared with normal human liver as revealed by immunohistochemistry analysis, suggesting that BMP9 signaling may be relevant during hepatocarcinogenesis in vivo. Our findings provide new clues for a better understanding of BMPs contribution, and in particular BMP9, in HCC pathogenesis that may result in the development of effective and targeted therapeutic interventions.     

Natural Killer Cells Control Tumor Growth by Sensing a Growth Factor

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LARP4 Is Regulated by Tumor Necrosis Factor Alpha in a Tristetraprolin-Dependent Manner

LARP4 is a protein with unknown function that independently binds to poly(A) RNA, RACK1, and the poly(A)-binding protein (PABPC1). Here, we report on its regulation. We found a conserved AU-rich element (ARE) in the human LARP4 mRNA 3′ untranslated region (UTR). This ARE, but not its antisense version or a point-mutated version, significantly decreased the stability of β-globin reporter mRNA. We found that overexpression of tristetraprolin (TTP), but not its RNA binding mutant or the other ARE-binding proteins tested, decreased cellular LARP4 levels. RNA coimmunoprecipitation showed that TTP specifically associated with LARP4 mRNA in vivo. Consistent with this, mouse LARP4 accumulated to higher levels in TTP gene knockout (KO) cells than in control cells. Stimulation of WT cells with tumor necrosis factor alpha (TNF-α), which rapidly induces TTP, robustly decreased LARP4 with a coincident time course but had no such effect on LARP4B or La protein or on LARP4 in the TTP KO cells. The TNF-α-induced TTP pulse was followed by a transient decrease in LARP4 mRNA that was quickly followed by a subsequent transient decrease in LARP4 protein. Involvement of LARP4 as a target of TNF-α–TTP regulation provides a clue as to how its functional activity may be used in a physiologic pathway.     

Neuregulin-4 Is a Survival Factor for Colon Epithelial Cells both in Culture and in Vivo

Expression of the ErbB4 tyrosine kinase is elevated in colonic epithelial cells during inflammatory bowel disease, whereas ErbB4 overexpression in cultured colonocytes blocks TNF-induced apoptosis in a ligand-dependent manner. Together, these observations suggest that ErbB4 induction may be a protective response. However, the effects of ErbB4 signaling in the colonic epithelium in vivo are not known. Furthermore, previous work on ErbB4 used ligands shared with other receptors, raising the question of whether the observed responses are explicitly due to ErbB4. In this study, we used the ErbB4-specific ligand neuregulin-4 (NRG4) to activate ErbB4 and define its role in colonocyte biology. NRG4 treatment, either in cultured cells or in mice, blocked colonic epithelial apoptosis induced by TNF and IFN-γ. It was also protective in a murine experimental colitis model. NRG4 stimulated phosphorylation of ErbB4 but not other ErbB receptors, indicating that this is a specific response. Furthermore, in contrast to related ligands, NRG4 enhanced cell survival but not proliferation or migration, and stimulated phosphorylation of the anti-apoptotic mediator Akt but not ERK MAPK. Pharmacological inhibition of PI3K/Akt signaling reversed the anti-apoptotic effects of NRG4, confirming the role of this cascade in NRG4-induced cell survival. With regard to the potential clinical importance of this pathway, NRG4 expression was decreased in human inflammatory bowel disease samples and mouse models of colitis, suggesting that activation of ErbB4 is altered in disease. Thus, exogenous NRG4 may be beneficial for disorders in which epithelial apoptosis is part of the pathology.     

Trio Is a Key Guanine Nucleotide Exchange Factor Coordinating Regulation of the Migration and Morphogenesis of Granule Cells in the Developing Cerebellum

Orchestrated regulation of neuronal migration and morphogenesis is critical for neuronal development and establishment of functional circuits, but its regulatory mechanism is incompletely defined. We established and analyzed mice with neural-specific knock-out of Trio, a guanine nucleotide exchange factor with multiple guanine nucleotide exchange factor domains. Knock-out mice showed defective cerebella and severe signs of ataxia. Mutant cerebella had no granule cells in the internal granule cell layer due to aberrant granule cell migration as well as abnormal neurite growth. Trio-deficient granule cells showed reduced extension of neurites and highly branched and misguided processes with perturbed stabilization of actin and microtubules. Trio deletion caused down-regulation of the activation of Rac1, RhoA, and Cdc42, and mutant granule cells appeared to be unresponsive to neurite growth-promoting molecules such as Netrin-1 and Semaphorin 6A. These results suggest that Trio may be a key signal module for the orchestrated regulation of neuronal migration and morphogenesis during cerebellar development. Trio may serve as a signal integrator decoding extrinsic signals to Rho GTPases for cytoskeleton organization.     

Tumor Microenvironmental Changes Induced by the Sulfamate Carbonic Anhydrase IX Inhibitor S4 in a Laryngeal Tumor Model

Background and Purpose

Carbonic anhydrase IX (CAIX) plays a pivotal role in pH homeostasis, which is essential for tumor cell survival. We examined the effect of the CAIX inhibitor 4-(3′(3″,5″-dimethylphenyl)-ureido)phenyl sulfamate (S4) on the tumor microenvironment in a laryngeal tumor model by analyzing proliferation, apoptosis, necrosis, hypoxia, metabolism and CAIX ectodomain shedding.

Methods

SCCNij202 tumor bearing-mice were treated with S4 for 1, 3 or 5 days. CAIX ectodomain shedding was measured in the serum after therapy. Effects on tumor cell proliferation, apoptosis, necrosis, hypoxia (pimonidazole) and CAIX were investigated with quantitative immunohistochemistry. Metabolic transporters and enzymes were quantified with qPCR.

Results

CAIX ectodomain shedding decreased after treatment with S4 (p<0.01). S4 therapy did neither influence tumor cell proliferation nor the amount of apoptosis and necrosis. Hypoxia (pimonidazole) and CAIX expression were also not affected by S4. CHOP and MMP9 mRNA as a reference of intracellular pH did not change upon treatment with S4. Compensatory mechanisms of pH homeostasis at the mRNA level were not observed.

Conclusion

As the clinical and biological meaning of the decrease in CAIX ectodomain shedding after S4 therapy is not clear, studies are required to elucidate whether the CAIX ectodomain has a paracrine or autocrine signaling function in cancer biology. S4 did not influence the amount of proliferation, apoptosis, necrosis and hypoxia. Therefore, it is unlikely that S4 can be used as single agent to influence tumor cell kill and proliferation, and to target primary tumor growth.     

Hsp60 Is Actively Secreted by Human Tumor Cells

Background

Hsp60, a Group I mitochondrial chaperonin, is classically considered an intracellular chaperone with residence in the mitochondria; nonetheless, in the last few years it has been found extracellularly as well as in the cell membrane. Important questions remain pertaining to extracellular Hsp60 such as how generalized is its occurrence outside cells, what are its extracellular functions and the translocation mechanisms that transport the chaperone outside of the cell. These questions are particularly relevant for cancer biology since it is believed that extracellular chaperones, like Hsp70, may play an active role in tumor growth and dissemination.

Methodology/Principal Findings

Since cancer cells may undergo necrosis and apoptosis, it could be possible that extracellular Hsps are chiefly the result of cell destruction but not the product of an active, physiological process. In this work, we studied three tumor cells lines and found that they all release Hsp60 into the culture media by an active mechanism independently of cell death. Biochemical analyses of one of the cell lines revealed that Hsp60 secretion was significantly reduced, by inhibitors of exosomes and lipid rafts.

Conclusions/Significance

Our data suggest that Hsp60 release is the result of an active secretion mechanism and, since extracellular release of the chaperone was demonstrated in all tumor cell lines investigated, our observations most likely reflect a general physiological phenomenon, occurring in many tumors.     

DEPTOR Is a Stemness Factor That Regulates Pluripotency of Embryonic Stem Cells

The mammalian target of rapamycin (mTOR) pathway regulates stem cell regeneration and differentiation in response to growth factors, nutrients, cellular energetics, and various extrinsic stressors. Inhibition of mTOR activity has been shown to enhance the regenerative potential of pluripotent stem cells. DEPTOR is the only known endogenous inhibitor of all known cellular mTOR functions. We show that DEPTOR plays a key role in maintaining stem cell pluripotency by limiting mTOR activity in undifferentiated embryonic stem cells (ESCs). DEPTOR levels dramatically decrease with differentiation of mouse ESCs, and knockdown of DEPTOR is sufficient to promote ESC differentiation. A strong decrease in DEPTOR expression is also observed during human ESCs differentiation. Furthermore, reduction in DEPTOR level during differentiation is accompanied by a corresponding increase in mTOR complex 1 activity in mouse ESCs. Our data provide evidence that DEPTOR is a novel stemness factor that promotes pluripotency and self-renewal in ESCs by inhibiting mTOR signaling.