74 research outputs found
Depletion of Kinesin 5B Affects Lysosomal Distribution and Stability and Induces Peri-Nuclear Accumulation of Autophagosomes in Cancer Cells
Background: Enhanced lysosomal trafficking is associated with metastatic cancer. In an attempt to discover cancer relevant
lysosomal motor proteins, we compared the lysosomal proteomes from parental MCF-7 breast cancer cells with those from
highly invasive MCF-7 cells that express an active form of the ErbB2 (DN-ErbB2).
Methodology/Principal Findings: Mass spectrometry analysis identified kinesin heavy chain protein KIF5B as the only
microtubule motor associated with the lysosomes in MCF-7 cells, and ectopic DN-ErbB2 enhanced its lysosomal association.
KIF5B associated with lysosomes also in HeLa cervix carcinoma cells as analyzed by subcellular fractionation. The depletion
of KIF5B triggered peripheral aggregations of lysosomes followed by lysosomal destabilization, and cell death in HeLa cells.
Lysosomal exocytosis in response to plasma membrane damage as well as fluid phase endocytosis functioned, however,
normally in these cells. Both HeLa and MCF-7 cells appeared to express similar levels of the KIF5B isoform but the death
phenotype was weaker in KIF5B-depleted MCF-7 cells. Surprisingly, KIF5B depletion inhibited the rapamycin-induced
accumulation of autophagosomes in MCF-7 cells. In KIF5B-depleted cells the autophagosomes formed and accumulated in
the close proximity to the Golgi apparatus, whereas in the control cells they appeared uniformly distributed in the
cytoplasm.
Conclusions/Significance: Our data identify KIF5B as a cancer relevant lysosomal motor protein with additional functions in
autophagosome formatio
Mycobacterium tuberculosis Induces an Atypical Cell Death Mode to Escape from Infected Macrophages
BACKGROUND: Macrophage cell death following infection with Mycobacterium tuberculosis plays a central role in tuberculosis disease pathogenesis. Certain attenuated strains induce extrinsic apoptosis of infected macrophages but virulent strains of M. tuberculosis suppress this host response. We previously reported that virulent M. tuberculosis induces cell death when bacillary load exceeds approximately 20 per macrophage but the precise nature of this demise has not been defined.
METHODOLOGY/PRINCIPAL FINDINGS: We analyzed the characteristics of cell death in primary murine macrophages challenged with virulent or attenuated M. tuberculosis complex strains. We report that high intracellular bacillary burden causes rapid and primarily necrotic death via lysosomal permeabilization, releasing hydrolases that promote Bax/Bak-independent mitochondrial damage and necrosis. Cell death was independent of cathepsins B or L and notable for ultrastructural evidence of damage to lipid bilayers throughout host cells with depletion of several host phospholipid species. These events require viable bacteria that can respond to intracellular cues via the PhoPR sensor kinase system but are independent of the ESX1 system.
CONCLUSIONS/SIGNIFICANCE: Cell death caused by virulent M. tuberculosis is distinct from classical apoptosis, pyroptosis or pyronecrosis. Mycobacterial genes essential for cytotoxicity are regulated by the PhoPR two-component system. This atypical death mode provides a mechanism for viable bacilli to exit host macrophages for spreading infection and the eventual transition to extracellular persistence that characterizes advanced pulmonary tuberculosis
Concanavalin A/IFN-Gamma Triggers Autophagy-Related Necrotic Hepatocyte Death through IRGM1-Mediated Lysosomal Membrane Disruption
Interferon-gamma (IFN-Ξ³), a potent Th1 cytokine with multiple biological functions, can induce autophagy to enhance the clearance of the invading microorganism or cause cell death. We have reported that Concanavalin A (Con A) can cause autophagic cell death in hepatocytes and induce both T cell-dependent and -independent acute hepatitis in immunocompetent and immunodeficient mice, respectively. Although IFN-Ξ³ is known to enhance liver injury in Con A-induced hepatitis, its role in autophagy-related hepatocyte death is not clear. In this study we report that IFN-Ξ³ can enhance Con A-induced autophagic flux and cell death in hepatoma cell lines. A necrotic cell death with increased lysosomal membrane permeabilization (LMP) is observed in Con A-treated hepatoma cells in the presence of IFN-Ξ³. Cathepsin B and L were released from lysosomes to cause cell death. Furthermore, IFN-Ξ³ induces immunity related GTPase family M member 1(IRGM1) translocation to lysosomes and prolongs its activity in Con A-treated hepatoma cells. Knockdown of IRGM1 inhibits the IFN-Ξ³/Con A-induced LMP change and cell death. Furthermore, IFN-Ξ³β/β mice are resistant to Con A-induced autophagy-associated necrotic hepatocyte death. We conclude that IFN-Ξ³ enhances Con A-induced autophagic flux and causes an IRGM1-dependent lysosome-mediated necrotic cell death in hepatocytes
Sensitization of interferon-Ξ³ induced apoptosis in human osteosarcoma cells by extracellular S100A4
BACKGROUND: S100A4 is a small Ca(2+)-binding protein of the S100 family with metastasis-promoting properties. Recently, secreted S100A4 protein has been shown to possess a number of functions, including induction of angiogenesis, stimulation of cell motility and neurite extension. METHODS: Cell cultures from two human osteosarcoma cell lines, OHS and its anti-S100A4 ribozyme transfected counterpart II-11b, was treated with IFN-Ξ³ and recombinant S100A4 in order to study the sensitizing effects of extracellular S100A4 on IFN-Ξ³ mediated apoptosis. Induction of apoptosis was demonstrated by DNA fragmentation, cleavage of poly (ADP-ribose) polymerase and Lamin B. RESULTS: In the present work, we found that the S100A4-expressing human osteosarcoma cell line OHS was more sensitive to IFN-Ξ³-mediated apoptosis than the II-11b cells. S100A4 protein was detected in conditioned medium from OHS cells, but not from II-11b cells, and addition of recombinant S100A4 to the cell medium sensitized II-11b cells to apoptosis induced by IFN-Ξ³. The S100A4/IFN-Ξ³-mediated induction of apoptosis was shown to be independent of caspase activation, but dependent on the formation of reactive oxygen species. Furthermore, addition of extracellular S100A4 was demonstrated to activate nuclear factor-ΞΊB (NF-ΞΊB). CONCLUSION: In conclusion, we have shown that S100A4 sensitizes osteosarcoma cells to IFN-Ξ³-mediated induction of apoptosis. Additionally, extracellular S100A4 activates NF-ΞΊB, but whether these events are causally related remains unknown
Stat3 controls cell death during mammary gland involution by regulating uptake of milk fat globules and lysosomal membrane permeabilization.
We have previously demonstrated that Stat3 regulates lysosomal-mediated programmed cell death (LM-PCD) during mouse mammary gland involution in vivo. However, the mechanism that controls the release of lysosomal cathepsins to initiate cell death in this context has not been elucidated. We show here that Stat3 regulates the formation of large lysosomal vacuoles that contain triglyceride. Furthermore, we demonstrate that milk fat globules (MFGs) are toxic to epithelial cells and that, when applied to purified lysosomes, the MFG hydrolysate oleic acid potently induces lysosomal leakiness. Additionally, uptake of secreted MFGs coated in butyrophilin 1A1 is diminished in Stat3-ablated mammary glands and loss of the phagocytosis bridging molecule MFG-E8 results in reduced leakage of cathepsins in vivo. We propose that Stat3 regulates LM-PCD in mouse mammary gland by switching cellular function from secretion to uptake of MFGs. Thereafter, perturbation of lysosomal vesicle membranes by high levels of free fatty acids results in controlled leakage of cathepsins culminating in cell death.This work was
supported by a grant from the Medical Research Council programme grant no. MR/J001023/1 (T.J.S. and B. L-L.)
and a Cancer Research UK Cambridge Cancer Centre PhD studentship (H.K.R.).This is the accepted manuscript. The final version is available from Nature Publishing at http://www.nature.com/ncb/journal/vaop/ncurrent/full/ncb3043.html
Decreased expression of the mannose 6- phosphate/insulin-like growth factor-II receptor promotes growth of human breast cancer cells
BACKGROUND: Loss or mutation of the mannose 6-phosphate/insulin-like growth factor-II receptor (M6P/IGF2R) has been found in breast cancer. However, whether or not decreased levels of functional M6P/IGF2R directly contribute to the process of carcinogenesis needs to be further verified by functional studies. METHODS: In this study, using viral and ribozyme strategies we reduced the expression of M6P/IGF2R in human breast cancer cells and then examined the effect on growth and apoptosis of these cells. RESULTS: Our results showed that infection of MCF-7 cells with the adenovirus carrying a ribozyme targeted against the M6P/IGF2R mRNA dramatically reduced the level of transcripts and the functional activity of M6P/IGF2R in these cells. Accordingly, cells treated with the ribozyme exhibited a higher growth rate and a lower apoptotic index than control cells (infected with a control vector). Furthermore, decreased expression of M6P/IGF2R enhanced IGF-II-induced proliferation and reduced cell susceptibility to TNF-induced apoptosis. CONCLUSIONS: These results suggest that M6P/IGF2R functions as a growth suppressor and its loss or mutation may contribute to development and progression of cancer. This study also demonstrates that adenoviral delivery of the ribozyme provides a useful tool for investigating the role of M6P/IGF2R in regulation of cell growth
Cathepsin-B and cathepsin-L expression levels do not correlate with sensitivity of tumour cells to TNF-Ξ±-mediated apoptosis
Deep-Sequencing Analysis of the Mouse Transcriptome Response to Infection with Brucella melitensis Strains of Differing Virulence
Brucella melitensis is an important zoonotic pathogen that causes brucellosis, a disease that affects sheep, cattle and occasionally humans. B. melitensis strain M5-90, a live attenuated vaccine cultured from B. melitensis strain M28, has been used as an effective tool in the control of brucellosis in goats and sheep in China. However, the molecular changes leading to attenuated virulence and pathogenicity in B. melitensis remain poorly understood. In this study we employed the Illumina Genome Analyzer platform to perform genome-wide digital gene expression (DGE) analysis of mouse peritoneal macrophage responses to B. melitensis infection. Many parallel changes in gene expression profiles were observed in M28- and M5-90-infected macrophages, suggesting that they employ similar survival strategies, notably the induction of anti-inflammatory and antiapoptotic factors. Moreover, 1019 differentially expressed macrophage transcripts were identified 4 h after infection with the different B. melitensis strains, and these differential transcripts notably identified genes involved in the lysosome and mitogen-activated protein kinase (MAPK) pathways. Further analysis employed gene ontology (GO) analysis: high-enrichment GOs identified endocytosis, inflammatory, apoptosis, and transport pathways. Path-Net and Signal-Net analysis highlighted the MAPK pathway as the key regulatory pathway. Moreover, the key differentially expressed genes of the significant pathways were apoptosis-related. These findings demonstrate previously unrecognized changes in gene transcription that are associated with B. melitensis infection of macrophages, and the central signaling pathways identified here merit further investigation. Our data provide new insights into the molecular attenuation mechanism of strain M5-90 and will facilitate the generation of new attenuated vaccine strains with enhanced efficacy
Potentiation of apoptosis by histone deacetylase inhibitors and doxorubicin combination: cytoplasmic cathepsin B as a mediator of apoptosis in multiple myeloma
Cell Cycle- and Cancer-Associated Gene Networks Activated by Dsg2: Evidence of Cystatin A Deregulation and a Potential Role in Cell-Cell Adhesion
This work was supported by grants from
the National Institutes of Health (Mahoney,
R01AR056067; Riobo, RO1 GM088256). The
funders had no role in study design, data collection
and analysis, decision to publish, or preparation of
the manuscript
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