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
Low oxygen tension influences tumor progression by enhancing angiogenesis; and histone deacetylases (HDAC) are implicated in alteration of chromatin assembly and tumorigenesis. Here we show induction of HDAC under hypoxia and elucidate a role for HDAC in the regulation of hypoxia-induced angiogenesis. Overexpressed wild-type HDAC1 downregulated expression of p53 and von Hippel-Lindau tumor suppressor genes and stimulated angiogenesis of human endothelial cells. A specific HDAC inhibitor, trichostatin A (TSA), upregulated p53 and von Hippel-Lindau expression and downregulated hypoxia-inducible factor-1alpha and vascular endothelial growth factor. TSA also blocked angiogenesis in vitro and in vivo. TSA specifically inhibited hypoxia-induced angiogenesis in the Lewis lung carcinoma model. These results indicate that hypoxia enhances HDAC function and that HDAC is closely involved in angiogenesis through suppression of hypoxia-responsive tumor suppressor genes.
Microvesicles (MV) are membrane vesicles secreted from the plasma and endosomal membrane compartment by various cell types such as hematopoietic, epithelial, and tumor cells. Actively growing tumor cells shed MV, and the rate of shedding increases in malignant tumors. Although recent progress in this area has revealed that tumor-derived MV play multiple roles in tumor growth and metastasis via immune escape, tumor invasion, and angiogenesis, the mechanism of vesicle formation and the biological roles of tumor-derived MV are not understood. Here, we report the first global proteomic analysis of highly purified MV from human colorectal cancer cells. Using 1D SDS gel electrophoresis and nano-LC-MS/MS analyses, we identified a total of 547 microvesicular proteins from three independent experiments with high confidence; 416 proteins were identified at least in two trials, including 181 as yet unreported proteins. We identified 49 proteins involved in the biogenesis of MV, including annexins, ADP-ribosylation factors, and Rab proteins. We also identified 28 proteins that may function in tumorigenesis via promotion of migration, invasion, and growth of tumor cells, immune modulation, metastasis, and angiogenesis. Taken together with previously reported results, our observations suggest that tumor-derived MV may act as communicasomes, that is, extracellular organelles that play diverse roles in intercellular communication. This information will help elucidate the biogenesis and functions of tumor-derived MV, and aid in the development of effective vaccines for various cancers, including colorectal cancer.
Although histone deacetylases (HDACs) appear to play a crucial role in carcinogenesis, the expression status of HDACs in primary human cancer tissues has not yet been reported. In this study, we investigated the expression level of HDAC1 in 25 paired primary human gastric cancer (GC) tissues and corresponding normal tissues through semi-quantitative RT-PCR and immunoblot analysis. The HDAC1 expression pattern was also topologically examined through immunohistochemistry. Overexpression of HDAC1 mRNA was detected in 68% of GC tissues (17 of 25), and the relative density of HDAC1 mRNA in GC tissue was increased 1.8-fold versus the normal counterpart (P < < < <0.01). Elevated expression of HDAC1 protein was also detected in 61% of GC samples (11 of 18), which also showed an increased mRNA level of HDAC. Immunohistochemically, overexpression of HDAC1 was predominantly localized in the nuclei of most neoplastic cells, including embolic tumor cells, whereas normal glandular epithelial cells revealed only weak HDAC1 expression that was focal in distribution. Thus, the present study clearly demonstrates that HDAC1 is overexpressed in GC and probably plays a significant role in gastric carcinogenesis.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.