Microenvironmental topographic cues influence migration dynamics of nasopharyngeal carcinoma cells from tumour spheroids.

Tumour metastasis is a complex process that strongly influences the prognosis and treatment of cancer. Apart from intracellular factors, recent studies have indicated that metastasis also depends on microenvironmental factors such as the biochemical, mechanical and topographical properties of the surrounding extracellular matrix (ECM) of tumours. In this study, as a proof of concept, we conducted tumour spheroid dissemination assay on engineered surfaces with micrograting patterns. Nasopharyngeal spheroids were generated by the 3D culture of nasopharyngeal carcinoma (NPC43) cells, a newly established cell line that maintains a high level of Epstein-Barr virus, a hallmark of NPC. Three types of collagen I-coated polydimethylsiloxane (PDMS) substrates were used, with 15 µm deep "trenches" that grated the surfaces: (a) 40/10 µm ridges (R)/trenches (T), (b) 18/18 µm (R/T) and (c) 50/50 µm (R/T). The dimensions of these patterns were designed to test how various topographical cues, different with respect to the size of tumour spheroids and individual NPC43 cells, might affect dissemination behaviours. Spreading efficiencies on all three patterned surfaces, especially 18/18 µm (R/T), were lower than that on flat PDMS surface. The outspreading cell sheets on flat and 40/10 µm (R/T) surfaces were relatively symmetrical but appeared ellipsoid and aligned with the main axes of the 18/18 µm (R/T) and 50/50 µm (R/T) grating platforms. Focal adhesions (FAs) were found to preferentially formed on the ridges of all patterns. The number of FAs per spheroid was strongly influenced by the grating pattern, with the least FAs on the 40/10 µm (R/T) and the most on the 50/50 µm (R/T) substrate. Taken together, these data indicate a previously unknown effect of surface topography on the efficiency and directionality of cancer cell spreading from tumour spheroids, suggesting that topography, like ECM biochemistry and stiffness, can influence the migration dynamics in 3D cell culture models.

Quantitative nucleolar proteomics reveals nuclear re-organization during stress- induced senescence in mouse fibroblast.

BACKGROUND: Nucleolus is the most prominent mammalian organelle within the nucleus which is also the site for ribosomal biogenesis. There have been many reports indicating the involvement of nucleolus in the process of aging. Several proteins related to aging have been shown to localize in the nucleolus, which suggests the role of this organelle in senescence. RESULTS: In this study, we used quantitative mass spectrometry to map the flux of proteins into and out of the nucleolus during the induction of senescence in cultured mammalian cells. Changes in the abundance of 344 nucleolar proteins in sodium butyrate-induced senescence in NIH3T3 cells were studied by SILAC (stable isotope labeling by amino acids in cell culture)-based mass spectrometry. Biochemically, we have validated the proteomic results and confirmed that B23 (nucleophosmin) protein was down-regulated, while poly (ADP-ribose) polymerase (PARP) and nuclear DNA helicase II (NDH II/DHX9/RHA) were up-regulated in the nucleolus upon treatment with sodium butyrate. Accumulation of chromatin in the nucleolus was also observed, by both proteomics and microscopy, in sodium butyrate-treated cells. Similar observations were found in other models of senescence, namely, in mitoxantrone- (MTX) treated cells and primary fibroblasts from the Lamin A knockout mice. CONCLUSION: Our data indicate an extensive nuclear organization during senescence and suggest that the redistribution of B23 protein and chromatin can be used as an important marker for senescence.

In situ labeling of transcription sites in marine medaka.

Transcription factories have been characterized in cultured mammalian cells, but little is known about the regulation of these nuclear structures in different primary cell types. Using marine medaka, we observed transcription sites labeled by the metabolic incorporation of 5-fluorouridine (5-FU) into nascent RNA. Medaka was permeable to 5-FU in ambient water and became fully labeled within 4 hr of incubation. The incorporation of 5-FU was inhibited by the transcription inhibitor actinomycin D. The 5-FU incorporation sites were detected in the cell nucleus, and could be abolished by RNase digestion. The tissue distribution of 5-FU incorporation was visualized by immunocytochemistry on whole-mount specimens and histological sections. The 5-FU labeling appeared highly cell type specific, suggesting a regulation of the overall transcription activities at tissue level. Mapping of transcription factories by 5-FU incorporation in fish provides a useful and physiologically relevant model for studying the control of gene expression in the context of the functional organization of the cell nucleus. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Proteomics analysis of the nucleolus in adenovirus-infected cells.

Adenoviruses replicate primarily in the host cell nucleus, and it is well established that adenovirus infection affects the structure and function of host cell nucleoli in addition to coding for a number of nucleolar targeted viral proteins. Here we used unbiased proteomics methods, including high throughput mass spectrometry coupled with stable isotope labeling by amino acids in cell culture (SILAC) and traditional two-dimensional gel electrophoresis, to identify quantitative changes in the protein composition of the nucleolus during adenovirus infection. Two-dimensional gel analysis revealed changes in six proteins. By contrast, SILAC-based approaches identified 351 proteins with 24 proteins showing at least a 2-fold change after infection. Of those, four were previously reported to have aberrant localization and/or functional relevance during adenovirus infection. In total, 15 proteins identified as changing in amount by proteomics methods were examined in infected cells using confocal microscopy. Eleven of these proteins showed altered patterns of localization in adenovirus-infected cells. Comparing our data with the effects of actinomycin D on the nucleolar proteome revealed that adenovirus infection apparently specifically targets a relatively small subset of nucleolar antigens at the time point examined.

Promoter hypermethylation mediates downregulation of thiamine receptor SLC19A3 in gastric cancer.

As an important way to inactivate tumor suppressor genes (TSGs) during cancer development, promoter hypermethylation can be used to define novel TSGs and identify biomarkers for cancer diagnosis. SLC19A3 (solute carrier family 19, member 3) was found to be such a biomarker. SLC19A3 expression was downregulated in gastric cancer cell lines (71%, 5/7) and restored after pharmacological demethylation. Notably, hypermethylation of SLC19A3 promoter was detected in gastric cancer cell lines (57%, 4/7), primary gastric carcinoma tissues (51%, 52/101) and precancerous lesion (intestinal metaplasia) tissues (32%, 8/25). Exogenous SLC19A3 expression caused growth inhibition of gastric cancer cells. In summary, SLC19A3 was epigenetically downregulated in gastric cancer. Methylation of SLC19A3 promoter could be a novel biomarker for early gastric cancer development.

ABC50 promotes translation initiation in mammalian cells.

ABC50 is an ATP-binding cassette (ABC) protein, which, unlike most ABC proteins, does not possess membrane-spanning domains. ABC50 interacts with eukaryotic initiation factor 2 (eIF2), which plays a key role in translation initiation and its control. ABC50 binds to ribosomes, and this interaction requires both the N-terminal domain and at least one ABC domain. Knockdown of ABC50 by RNA interference impaired translation of both cap-dependent and -independent reporters, consistent with a positive role for ABC50 in the function of eIF2, which is required for both types of translation initiation. Mutation of the Walker box A or B motifs in both ABC regions of ABC50 yielded a mutant protein that exerted a dominant-interfering phenotype with respect to protein synthesis and translation initiation. Importantly, although dominant-interfering mutants of ABC50 impaired cap-dependent translation, translation driven by certain internal ribosome entry segments was not inhibited. ABC50 is located in the cytoplasm and nucleoplasm but not in the nucleolus. Thus, ABC50 is not likely to be directly involved in early ribosomal biogenesis, unlike some other ABC proteins. Taken together, the present data show that ABC50 plays a key role in translation initiation and has functions that are distinct from those of other non-membrane ABC proteins.

Nucleolar proteome dynamics.

The nucleolus is a key organelle that coordinates the synthesis and assembly of ribosomal subunits and forms in the nucleus around the repeated ribosomal gene clusters. Because the production of ribosomes is a major metabolic activity, the function of the nucleolus is tightly linked to cell growth and proliferation, and recent data suggest that the nucleolus also plays an important role in cell-cycle regulation, senescence and stress responses. Here, using mass-spectrometry-based organellar proteomics and stable isotope labelling, we perform a quantitative analysis of the proteome of human nucleoli. In vivo fluorescent imaging techniques are directly compared to endogenous protein changes measured by proteomics. We characterize the flux of 489 endogenous nucleolar proteins in response to three different metabolic inhibitors that each affect nucleolar morphology. Proteins that are stably associated, such as RNA polymerase I subunits and small nuclear ribonucleoprotein particle complexes, exit from or accumulate in the nucleolus with similar kinetics, whereas protein components of the large and small ribosomal subunits leave the nucleolus with markedly different kinetics. The data establish a quantitative proteomic approach for the temporal characterization of protein flux through cellular organelles and demonstrate that the nucleolar proteome changes significantly over time in response to changes in cellular growth conditions.