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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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- Bhaskaran, Nimesh, et al.
(författare)
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Comparative proteome profiling of MCF10A and 184A1 human breast epithelial cells emphasized involvement of CDK4 and cyclin D3 in cell proliferation
- 2009
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Ingår i: Proteomics Clinical Applications. - : Wiley. - 1862-8354 .- 1862-8346. ; 3:1, s. 68-77
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Tidskriftsartikel (refereegranskat)abstract
- Acquiring high proliferation rate is crucial for carcinogenic transformation of cells. We reporthere proteome profiling of human breast epithelial cells with low (184A1) and high (MCF10A)proliferation rates.We identified 183 proteins in 184A1 and 318 proteins in MCF10A cells. Thesedatasets provide the most comprehensive proteome annotations of 184A1 and MCF10A cells.Proteins were taken for identification from 2-D gels in a systematic and unbiased way. Functionalclustering of the identified proteins showed similarities in distribution of proteins to the samefunctional domains, indicating similarities in proteomes of 184A1 and MCF10A cells. Amongobserved differences in protein expression, we validated correlation of expression of endogenouscyclin-dependent kinase 4 (CDK4), cyclin D3, cdc25B, and p38g with cell proliferation. Furthermore,down-regulation of CDK4 and cyclin D3 with specific siRNA inhibited cell proliferation,which emphasized the role of CDK4 and cyclin D3 in enhancement of cell proliferation rate ofhuman breast epithelial cells.
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| 4. |
- Lin, Kah Wai, et al.
(författare)
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Phosphorylation of eEF1A1 at Ser300 by T beta R-I Results in Inhibition of mRNA Translation
- 2010
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Ingår i: Current Biology. - : Elsevier BV. - 0960-9822 .- 1879-0445. ; 20:18, s. 1615-1625
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Tidskriftsartikel (refereegranskat)abstract
- Background: Transforming growth factor beta (TGF-beta) is a potent inhibitor of cell proliferation that regulates cell functions by activating specific serine/threonine kinase receptors on the cell surface. Type I TGF-beta receptor (T beta R-I) is essential for TGF-beta signaling, and substrates of T beta R-I provide insights into molecular mechanisms of TGF-beta signaling. Results: Here we identify eukaryotic elongation factor 1A1 (eEF1A1) as a novel substrate of T beta R-I. We show that T beta R-I phosphorylates eEF1A1 at Ser300 in vitro and in vivo. Ser300 was found to be important for aminoacyl-tRNA (aa-tRNA) binding to eEF1A1. Ser300 phosphorylation or mutations of Ser300 correlate with inhibition of protein synthesis in vitro and in vivo. We show that mimicking eEF1A1 phosphorylation at Ser300 results in inhibition of cell proliferation, and that mutations of Ser300 affect TGF-beta dependency in inhibition of protein synthesis and cell proliferation. Increased expression of eEF1A has been reported to enhance carcinogenesis. An analysis of human breast cancer cases revealed a decrease of eEF1A1 phosphorylation at Ser300 in malignant tumor cells as compared to epithelial cells in noncancerous tissues. Conclusions: Phosphorylation of eEF1A1 by T beta R-I is a novel regulatory mechanism that provides a direct link to regulation of protein synthesis by TGF-beta, as an important component in the TGF-beta-dependent regulation of protein synthesis and cell proliferation.
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| 5. |
- Lin, Kah Wai
(författare)
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Proteomics of invasiveness of human breast epithelial cells
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Mechanisms of malignant transformation and cancer invasion and factors controlling them in response to various stimuli remain elusive. We used proteomics and systems biology to explore these mechanisms in human breast epithelial cells. The objective has been to identify a set of biomarkers for diagnostics and prognostics of breast cancer. Acquiring of high proliferation by cells is a major hallmark of malignant transformation. Using global expression proteome profiling approach, we identified a set of proteins associated with high proliferation rate of human breast epithelial cells upon carcinogenic transformation (paper I). In this study, we described a proteome signature of cells with enhanced proliferation rate, and observed that deregulation of CDK4 and cyclin D3 may be among the early malignant transformation events. Distal metastasis is the leading cause of death among breast cancer patients, and invasion of cancer cells is the first step in metastatic process. We established a highly invasive clone of MCF7 cells from non-invasive MCF7 cells (paper II). Using proteome profiling, we identified key regulators of invasiveness. Systemic analysis suggested that the invasive-specific network has features of a scale-free network, with TGFβ, EGFRB, TAF1, HNF4α, MYC and RB1 as key nodes. Analysis of TGFβ and EGF-centered network showed more than 30 key nodes which may define how TGFβ and EGF cooperate. Among these nodes were identified insulin, VEGF, HNF4α and NFκB. This result indicates that the insulin signaling disturbance may interfere with the invasiveness, thus explain the clinical observation of the increased risk of breast cancer metastasis in diabetes patients. The correlation between protein translation and breast cancer is crucial in understanding of breast carcinogenesis. In paper III, we identified eukaryortic elongation factor 1 A1 (eEF1A1) as a direct substrate of type I transforming growth factor-β-receptor (TβRI). We showed that the phosphorylation of eEF1A1 at Ser300 by TβR-I mediates a direct inhibitory effect of TGFβ on protein synthesis, and contributes to effects on cell proliferation, anchorage-dependent and anchorage-independent cell growth. Furthermore, we showed that the phosphorylation of Ser300 is decreased in human breast tumors. In paper IV, we showed that eEF1A1 itself contributed to the increased proliferation of human breast epithelial cells by promoting transition of cells through the S- and G2/M-phases of the cell cycle. Therefore, our identification of eEF1A1 as a substrate of TβR-I unveiled novel translation-related regulatory pathway downstream of TβR-I, which is involved in breast tumorigenesis. Breast cancer metastatic suppressor I (BRMS1) was identified by us as an invasiveness-related protein. In paper V, we showed that expression of BRMS1 resulted in a shift to epithelial morphology of otherwise mesenchymal morphology MDA-MB-231 cells. Our study concluded that TGFβ and EGF may modulate BRMS1-dependent breast cancer invasion by regulating focal adhesion and cytoskeletal rearrangement, and that Smad2 and Erk1/2 phosphorylation are involved in molecular mechanisms engaged by BRMS1. Thus, presented here studies delivered a proteome signature of invasiveness and enhanced proliferation, and explored roles of eEF1A1 and BRMS1 in breast tumorigenesis. We described proteome signatures and proteins which may be considered as markers for diagnostics and prognostics of human breast cancer.
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