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Acquiring Metastatic Competence by Oral Squamous Cell Carcinoma Cells Is Associated with Differential Expression of α-Tubulin Isoforms.

Lou B, Engler D, Dubinsky W, Wu J, Vigneswaran N - J Oncol (2012)

Bottom Line: Thus, two distinct isoforms of α-tubulin, probably due to posttranslational modification, were associated with metastatic 686Ln cells.Immunofluorescence demonstrated remarkable differences in the cytosolic α-tubulin distribution patterns between the two cells.Since α-tubulin is critical for cell shape and mobility, our finding suggests a role of α-tubulin isoforms in acquisition of metastatic phenotype and represents potential target for therapeutic intervention.

View Article: PubMed Central - PubMed

Affiliation: New York Medical College, 40 Sunshine Cottage Road, Valhalla, NY 10595, USA.

ABSTRACT
We performed comparative global proteomics analyses of patient-matched primary (686Tu) and metastatic (686Ln) OSCC cells. The metastatic OSCC 686Ln cells showed greater in vitro migratory/invasive potential and distinct cell shape from their parental primary 686Tu cells. Ettan DIGE analysis revealed 1316 proteins spots in both cell lines with >85% to be quantitatively similar (<2 folds) between the two cell lines. However, two protein spots among four serial spots were highly dominant in 686Ln cells. Mass spectrometry sequencing demonstrated all four spots to be α-tubulin isotypes. Further analysis showed no significant quantitative difference in the α-tubulin between the two cell lines either at mRNA or protein levels. Thus, two distinct isoforms of α-tubulin, probably due to posttranslational modification, were associated with metastatic 686Ln cells. Immunofluorescence demonstrated remarkable differences in the cytosolic α-tubulin distribution patterns between the two cells. In 686Tu cells, α-tubulin proteins formed a normal network composed of filaments. In contrast, α-tubulin in 686Ln cells exhibited only partial cytoskeletal distribution with the majority of the protein diffusely distributed within the cytosol. Since α-tubulin is critical for cell shape and mobility, our finding suggests a role of α-tubulin isoforms in acquisition of metastatic phenotype and represents potential target for therapeutic intervention.

No MeSH data available.


Related in: MedlinePlus

Metastatic OSSC 686Ln cells have higher invasion potentials than primary 686Tu cells. (a) Morphology of migrated OSSC 686Tu (Tu, left panel) and 686Ln cells (Ln, right panel) on the filter after H-E staining during migration assay. Pores on the filter are visible: ×300. (b) Summary of invasion potentials of 686Tu (Tu) and 686Ln (Ln) cells in migration and invasion assays as indicated at the top of each graph. Invasion potentials are expressed as numbers of migrated cells in each well.
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fig1: Metastatic OSSC 686Ln cells have higher invasion potentials than primary 686Tu cells. (a) Morphology of migrated OSSC 686Tu (Tu, left panel) and 686Ln cells (Ln, right panel) on the filter after H-E staining during migration assay. Pores on the filter are visible: ×300. (b) Summary of invasion potentials of 686Tu (Tu) and 686Ln (Ln) cells in migration and invasion assays as indicated at the top of each graph. Invasion potentials are expressed as numbers of migrated cells in each well.

Mentions: Previously, we analyzed the global gene expression profiles of 686Tu and 686Ln and demonstrated that expression levels of >90% of the genes in cell lines derived from the primary and metastatic tumors of the same patient were identical to each other than the two metastatic cell lines from two different patients [9]. In line with the published reports, our study confirmed that the gene expression pattern of primary tumor cells are mostly preserved in their metastatic counterpart except for a few differentially expressed genes which are implicated in promoting metastasis [9, 12]. We performed cell motility and invasion assays to characterize the phenotypic differences between 686Tu and Ln cells. In the cell motility assay performed using the cell culture insets without Matrigel coating, 686Ln cells revealed enhanced migration which was 5.5 fold higher than that of 686Tu cells (left panel in Figure 1(b)). In the invasion assay performed using the cell culture inserts coated with three-dimensional Matrigel, invasion of both cell lines were greatly decreased. However, the reductions rates were significantly different between 686Tu and 686Ln cells. The 686Ln cells showed only 3-fold reduction as compared to 686Tu cells with 7.1-fold reduction (Figure 1(c)). As a result, only a 3-fold difference was observed between 686Ln and 686Tu cells when invading through Matrigel, as compared to 5.5-fold difference when migrating through noncoated membrane. This data suggests that the molecular machinery that underlies cell motility is more critical for metastatic phenotype than their extracellular matrix degrading molecules which are essential for invasion through the Matrigel. Tumor cells are known to change their phenotype during culture. However, our result showed that the metastatic 686Ln cells demonstrate higher motility and invasive potential than their parent primary tumor cells and indicate that the phenotypic features acquired in vivo are also preserved in cultured cells. Therefore, this pair of cell lines represents an ideal model to characterize proteomic signature that is causal for metastatic phenotype.


Acquiring Metastatic Competence by Oral Squamous Cell Carcinoma Cells Is Associated with Differential Expression of α-Tubulin Isoforms.

Lou B, Engler D, Dubinsky W, Wu J, Vigneswaran N - J Oncol (2012)

Metastatic OSSC 686Ln cells have higher invasion potentials than primary 686Tu cells. (a) Morphology of migrated OSSC 686Tu (Tu, left panel) and 686Ln cells (Ln, right panel) on the filter after H-E staining during migration assay. Pores on the filter are visible: ×300. (b) Summary of invasion potentials of 686Tu (Tu) and 686Ln (Ln) cells in migration and invasion assays as indicated at the top of each graph. Invasion potentials are expressed as numbers of migrated cells in each well.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3376782&req=5

fig1: Metastatic OSSC 686Ln cells have higher invasion potentials than primary 686Tu cells. (a) Morphology of migrated OSSC 686Tu (Tu, left panel) and 686Ln cells (Ln, right panel) on the filter after H-E staining during migration assay. Pores on the filter are visible: ×300. (b) Summary of invasion potentials of 686Tu (Tu) and 686Ln (Ln) cells in migration and invasion assays as indicated at the top of each graph. Invasion potentials are expressed as numbers of migrated cells in each well.
Mentions: Previously, we analyzed the global gene expression profiles of 686Tu and 686Ln and demonstrated that expression levels of >90% of the genes in cell lines derived from the primary and metastatic tumors of the same patient were identical to each other than the two metastatic cell lines from two different patients [9]. In line with the published reports, our study confirmed that the gene expression pattern of primary tumor cells are mostly preserved in their metastatic counterpart except for a few differentially expressed genes which are implicated in promoting metastasis [9, 12]. We performed cell motility and invasion assays to characterize the phenotypic differences between 686Tu and Ln cells. In the cell motility assay performed using the cell culture insets without Matrigel coating, 686Ln cells revealed enhanced migration which was 5.5 fold higher than that of 686Tu cells (left panel in Figure 1(b)). In the invasion assay performed using the cell culture inserts coated with three-dimensional Matrigel, invasion of both cell lines were greatly decreased. However, the reductions rates were significantly different between 686Tu and 686Ln cells. The 686Ln cells showed only 3-fold reduction as compared to 686Tu cells with 7.1-fold reduction (Figure 1(c)). As a result, only a 3-fold difference was observed between 686Ln and 686Tu cells when invading through Matrigel, as compared to 5.5-fold difference when migrating through noncoated membrane. This data suggests that the molecular machinery that underlies cell motility is more critical for metastatic phenotype than their extracellular matrix degrading molecules which are essential for invasion through the Matrigel. Tumor cells are known to change their phenotype during culture. However, our result showed that the metastatic 686Ln cells demonstrate higher motility and invasive potential than their parent primary tumor cells and indicate that the phenotypic features acquired in vivo are also preserved in cultured cells. Therefore, this pair of cell lines represents an ideal model to characterize proteomic signature that is causal for metastatic phenotype.

Bottom Line: Thus, two distinct isoforms of α-tubulin, probably due to posttranslational modification, were associated with metastatic 686Ln cells.Immunofluorescence demonstrated remarkable differences in the cytosolic α-tubulin distribution patterns between the two cells.Since α-tubulin is critical for cell shape and mobility, our finding suggests a role of α-tubulin isoforms in acquisition of metastatic phenotype and represents potential target for therapeutic intervention.

View Article: PubMed Central - PubMed

Affiliation: New York Medical College, 40 Sunshine Cottage Road, Valhalla, NY 10595, USA.

ABSTRACT
We performed comparative global proteomics analyses of patient-matched primary (686Tu) and metastatic (686Ln) OSCC cells. The metastatic OSCC 686Ln cells showed greater in vitro migratory/invasive potential and distinct cell shape from their parental primary 686Tu cells. Ettan DIGE analysis revealed 1316 proteins spots in both cell lines with >85% to be quantitatively similar (<2 folds) between the two cell lines. However, two protein spots among four serial spots were highly dominant in 686Ln cells. Mass spectrometry sequencing demonstrated all four spots to be α-tubulin isotypes. Further analysis showed no significant quantitative difference in the α-tubulin between the two cell lines either at mRNA or protein levels. Thus, two distinct isoforms of α-tubulin, probably due to posttranslational modification, were associated with metastatic 686Ln cells. Immunofluorescence demonstrated remarkable differences in the cytosolic α-tubulin distribution patterns between the two cells. In 686Tu cells, α-tubulin proteins formed a normal network composed of filaments. In contrast, α-tubulin in 686Ln cells exhibited only partial cytoskeletal distribution with the majority of the protein diffusely distributed within the cytosol. Since α-tubulin is critical for cell shape and mobility, our finding suggests a role of α-tubulin isoforms in acquisition of metastatic phenotype and represents potential target for therapeutic intervention.

No MeSH data available.


Related in: MedlinePlus