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| 2. |
- Klionsky, Daniel J., et al.
(författare)
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Guidelines for the use and interpretation of assays for monitoring autophagy
- 2012
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Ingår i: Autophagy. - : Informa UK Limited. - 1554-8635 .- 1554-8627. ; 8:4, s. 445-544
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Forskningsöversikt (refereegranskat)abstract
- In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
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| 3. |
- Aftab, Obaid, 1984-, et al.
(författare)
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Label-free detection and dynamic monitoring of drug-induced intracellular vesicle formation enabled using a 2-dimensional matched filter
- 2014
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Ingår i: Autophagy. - : Informa UK Limited. - 1554-8627 .- 1554-8635. ; 10:1, s. 57-69
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Tidskriftsartikel (refereegranskat)abstract
- Analysis of vesicle formation and degradation is a central issue in autophagy research and microscopy imaging is revolutionizing the study of such dynamic events inside living cells. A limiting factor is the need for labeling techniques that are labor intensive, expensive, and not always completely reliable. To enable label-free analyses we introduced a generic computational algorithm, the label-free vesicle detector (LFVD), which relies on a matched filter designed to identify circular vesicles within cells using only phase-contrast microscopy images. First, the usefulness of the LFVD is illustrated by presenting successful detections of autophagy modulating drugs found by analyzing the human colorectal carcinoma cell line HCT116 exposed to each substance among 1266 pharmacologically active compounds. Some top hits were characterized with respect to their activity as autophagy modulators using independent in vitro labeling of acidic organelles, detection of LC3-II protein, and analysis of the autophagic flux. Selected detection results for 2 additional cell lines (DLD1 and RKO) demonstrate the generality of the method. In a second experiment, label-free monitoring of dose-dependent vesicle formation kinetics is demonstrated by recorded detection of vesicles over time at different drug concentrations. In conclusion, label-free detection and dynamic monitoring of vesicle formation during autophagy is enabled using the LFVD approach introduced.
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| 4. |
- De Milito, Angelo
(författare)
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Immune activation during HIV-1 infection : implication for B cell dysfunctions and therapy monitoring
- 2002
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Aberrant activation of cells of the immune system plays a major role in the pathogenesis of HIV infection. The major aims of the thesis were: (a) to measure the effects of current antiretroviral therapy on immune activation through the analysis of soluble activation markers; (b) to evaluate the extent of T and B cell immune activation in relation to immunological and virological parameters; (c) to analyse the T-B cell molecular pathway leading to hyperactivation of B cells and loss of B cell function. The goals of highly active antiretroviral therapy (HAART) are to suppress viral replication and to reduce immune activation, thereby stimulating recovery of immune functions We evaluated two soluble molecules (Fas and CD27) as markers of immune activation during HAART. In a longitudinal study of 69 patients we observed a minimal decrease of the sFas plasma levels after one year of therapy. No significant changes in sFas were observed between virological responders and non-responders. These findings suggested that long-term HAART may be required to norm~ the dysregulation of the Fas pathway. Analysis of plasma sCD27 was done on 26 HIV-1-infected patients undergoing HAART during 24 months of therapy. HAART induced a significant reduction, but not a normalization, of plasma sCD27 after 24 months. A full normalization of plasma sCD27 was observed in virological responders and in patients with moderate immunodeficiency at baseline. Baseline sCD27 levels were predictive of a greater increase in CD4+ T cell count during the follow-up. Discontinuation of therapy resulted in a rapid increase of sCD27 plasma levels associated with viremia rebound and drop in CD4+ T cell count. Our findings suggested that plasma sCD27 may represent an alternative and simple marker to monitor immune activation during potent antiretroviral therapy. Polyclonal B cell activation is a major hallmark of the B cell dysfunction in HIV-1-infected patients. The phenotype and function of different B cell subsets were analysed. The population of memory (CD27+) B lymphocytes was dramatically reduced in HIV-1-infected patients. A positive correlation was found between the memory B cells and the CD70+ T cells, suggesting that T-B cell interaction through CD70-CD27 is upregulated and may chronically lead to terminal differentiation of memory B cells. Both naive and memory B lymphocytes from HIV-1-infected patients expressed high levels of Fas and Fas ligand although this pathway did not appear to be involved in apoptosis of memory B cells during HIV infection. Detection of NGF, a survival factor for memory B cells, was significantly reduced in the patients with low memory B cells. Furthermore, addition of NGF in culture resulted in decreased cell death of memory B cells from 2 of 4 infected subjects, indicating that deprivation of NGF may partly account for the loss of memory B cells. Despite the loss of memory B cells, total B lymphocytes from infected individuals showed a normal to high responsiveness to in vitro stimulation since they produced high amount of IgG upon activation with either agonistic anti-CD40 mAb or co-culture with a CD70-transfected cell line. We also evaluated the relationship between specific humoral immunity and loss of memory B cells in infected patients. Among the HIV-1-infected subjects, we found a decrease in antibody levels to tetanus, measles and HIV-1 in patients with very low memory B cells. Despite the selective loss of antibodies, hypergammaglobulinemia, was detected in all patients. These observations suggest that polyclonal B cell activation is accompanied by loss of specific antibodies during HIV-1 infection. Together with the loss of memory B cells, these defects might account for the increased susceptibility to secondary/opportunistic infections and to poor response to immunisation against non HIV antigens observed in HIV-1-infected subjects. We also analysed the expression of CD70 and LAIR-1 on naive and memory B cells from HIV-1-infected and uninfected subjects. We observed that in the HIV-1-infected individuals naive B cells showed increased levels of CD70 that is otherwise barely detectable in healthy subjects. Since only CD70+ B cells produce antibodies in a T-cell dependent fashion, activated naive B cells might represent one possible source of antibody production in HIV-1 infection. On the other hand, memory B cells from infected subjects showed a significantly reduced expression of LAIR-1, whose expression is normally downregulated during terminal differentiation.
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| 5. |
- Dyczynski, Matheus, et al.
(författare)
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Targeting autophagy by small molecule inhibitors of vacuolar protein sorting 34 (Vps34) improves the sensitivity of breast cancer cells to Sunitinib
- 2018
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Ingår i: Cancer Letters. - : Elsevier. - 0304-3835 .- 1872-7980. ; 435, s. 32-43
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Tidskriftsartikel (refereegranskat)abstract
- Resistance to chemotherapy is a challenging problem for treatment of cancer patients and autophagy has been shown to mediate development of resistance. In this study we systematically screened a library of 306 known anti-cancer drugs for their ability to induce autophagy using a cell-based assay. 114 of the drugs were classified as autophagy inducers; for 16 drugs, the cytotoxicity was potentiated by siRNA-mediated knock-down of Atg7 and Vps34. These drugs were further evaluated in breast cancer cell lines for autophagy induction, and two tyrosine kinase inhibitors, Sunitinib and Erlotinib, were selected for further studies. For the pharmacological inhibition of autophagy, we have characterized here a novel highly potent selective inhibitor of Vps34, SB02024. SB02024 blocked autophagy in vitro and reduced xenograft growth of two breast cancer cell lines, MDA-MB-231 and MCF-7, in vivo. Vps34 inhibitor significantly potentiated cytotoxicity of Sunitinib and Erlotinib in MCF-7 and MDA-MB-231 in vitro in monolayer cultures and when grown as multicellular spheroids. Our data suggests that inhibition of autophagy significantly improves sensitivity to Sunitinib and Erlotinib and that Vps34 is a promising therapeutic target for combination strategies in breast cancer.
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| 6. |
- Hernlund, Emma, et al.
(författare)
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The phosphoinositide 3-kinase/mammalian target of rapamycin inhibitor NVP-BEZ235 is effective in inhibiting regrowth of tumour cells after cytotoxic therapy
- 2012
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Ingår i: European Journal of Cancer. - : Elsevier BV. - 0959-8049 .- 1879-0852. ; 48:3, s. 396-406
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Tidskriftsartikel (refereegranskat)abstract
- PURPOSE:Regrowth of tumour cells between cycles of chemotherapy is a significant clinical problem. Treatment strategies where antiproliferative agents are used to inhibit tumour regrowth between chemotherapy cycles are attractive, but such strategies are difficult to test using conventional monolayer culture systems.METHODS:We used the in vitro tumour spheroid model to study regrowth of 3-D colon carcinoma tissue after cytotoxic therapy. Colon carcinoma cells with wild-type or mutant phosphatidyl inositol 3-kinase catalytic subunit (PI3KCA) or KRAS alleles were allowed to form multicellular spheroids and the effects of different pharmacological compounds were studied after sectioning and staining for relevant markers of cell proliferation and apoptosis.RESULTS:Studies using colon cancer cells with gene disruptions suggested that the phosphoinositide 3-kinase (PI3K)-mammalian target of rapamycin (mTOR) pathway was essential for proliferation in 3-D culture. The dual PI3K-mTOR inhibitor NVP-BEZ235, currently in clinical trials, was found to inhibit phosphorylation of the mTOR target 4EBP1 in 3-D cultured cells. The ability of NVP-BEZ235 to inhibit tumour cell proliferation and to induce apoptosis was markedly more pronounced in 3-D cultures compared to monolayer cultures. It was subsequently found that NVP-BEZ235 was effective in inhibiting regrowth of 3-D cultured cells after treatment with two cytotoxic inhibitors of the ubiquitin-proteasome system (UPS), methyl-13-hydroxy-15-oxokaurenoate (MHOK) and bortezomib (Velcade®).CONCLUSIONS:The dual PI3K-mTOR inhibitor NVP-BEZ235 was found to reduce cell proliferation and to induce apoptosis in 3-D cultured colon carcinoma cells, NVP-BEZ235 is a promising candidate for use in sequential treatment modalities together with cytotoxic drugs to reduce the cell mass of solid tumours.
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| 7. |
- Kolosenko, Iryna, et al.
(författare)
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Cell crowding induces interferon regulatory factor 9, which confers resistance to chemotherapeutic drugs
- 2015
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Ingår i: International Journal of Cancer. - : Wiley. - 0020-7136 .- 1097-0215. ; 136:4, s. E51-E61
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Tidskriftsartikel (refereegranskat)abstract
- The mechanism of multicellular drug resistance, defined as the reduced efficacy of chemotherapeutic drugs in solid tumors is incompletely understood. Here we report that colon carcinoma cells cultured as 3D microtissues (spheroids) display dramatic increases in the expression of a subset of type I interferon-(IFN)-stimulated genes (ISGs). A similar gene signature was associated previously with resistance to radiation and chemotherapy, prompting us to examine the underlying biological mechanisms. Analysis of spheroids formed by different tumor cell lines and studies using knock-down of gene expression showed that cell crowding leads to the induction of IFN regulatory factor-9 (IRF9) which together with STAT2 and independently of IFNs, is necessary for ISG upregulation. Increased expression of IRF9 alone was sufficient to induce the ISG subset in monolayer cells and to confer increased resistance to clinically used cytotoxic drugs. Our data reveal a novel mechanism of regulation of a subset of ISGs, leading to drug resistance in solid tumors. What's new? Drug resistance remains a major challenge in the management of cancer patients. Using a 3D model of tumor cells the authors identify cell crowding and the interferon response as important mediators of drug resistance. They demonstrate that interferon regulatory factor 9 (IRF9) and a panel of interferon-stimulated genes are induced by cell crowding in this model. These results link unexpected new molecular mechanisms with the therapy resistance of solid tumors.
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| 8. |
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| 9. |
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| 10. |
- Pellegrini, Paola, et al.
(författare)
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A drug screening assay on cancer cells chronically adapted to acidosis
- 2018
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Ingår i: Cancer Cell International. - : BMC. - 1475-2867. ; 18
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Tidskriftsartikel (refereegranskat)abstract
- Background: Drug screening for the identification of compounds with anticancer activity is commonly performed using cell lines cultured under normal oxygen pressure and physiological pH. However, solid tumors are characterized by a microenvironment with limited access to nutrients, reduced oxygen supply and acidosis. Tumor hypoxia and acidosis have been identified as important drivers of malignant progression and contribute to multicellular resistance to different forms of therapy. Tumor acidosis represents an important mechanism mediating drug resistance thus the identification of drugs active on acid-adapted cells may improve the efficacy of cancer therapy. Methods: Here, we characterized human colon carcinoma cells (HCT116) chronically adapted to grow at pH 6.8 and used them to screen the Prestwick drug library for cytotoxic compounds. Analysis of gene expression profiles in parental and low pH-adapted cells showed several differences relating to cell cycle, metabolism and autophagy. Results: The screen led to the identification of several compounds which were further selected for their preferential cytotoxicity towards acid-adapted cells. Amongst 11 confirmed hits, we primarily focused our investigation on the benzoporphyrin derivative Verteporfin (VP). VP significantly reduced viability in low pH-adapted HCT116 cells as compared to parental HCT116 cells and normal immortalized epithelial cells. The cytotoxic activity of VP was enhanced by light activation and acidic pH culture conditions, likely via increased acid-dependent drug uptake. VP displayed the unique property to cause light-dependent cross-linking of proteins and resulted in accumulation of polyubiquitinated proteins without inducing inhibition of the proteasome. Conclusions: Our study provides an example and a tool to identify anticancer drugs targeting acid-adapted cancer cells.
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