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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 686Ln and primary 686Tu cells show a different cellular distribution pattern of α-tubulin. 686Tu (Tu, upper row) or 686Ln cells (Ln, lower row) were stained for α-tubulin (red) and β actin (green). The cells were counterstained by DAPI. Note that 686Ln cells show defuse α-tubulin distribution, in contrast to filament network in 686Tu cells ×600.
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fig6: Metastatic 686Ln and primary 686Tu cells show a different cellular distribution pattern of α-tubulin. 686Tu (Tu, upper row) or 686Ln cells (Ln, lower row) were stained for α-tubulin (red) and β actin (green). The cells were counterstained by DAPI. Note that 686Ln cells show defuse α-tubulin distribution, in contrast to filament network in 686Tu cells ×600.

Mentions: Next, we examined whether the immunofluorescence staining patterns of α-tubulin protein in this pair of cell lines would reveal any remarkable quantitative and/or qualitative differences. Overall, 686Ln cells showed a higher intensity of anti-α-tubulin immunofluorescence staining compared to 686Tu cells (Figure 6). Both cell lines demonstrated a higher density of α-tubulin expression in their perinuclear region. A comparison of the staining patterns of α-tubulin in 686Tu and Ln cells showed significant qualitative differences in the α-tubulin filament distribution. In 686Tu cells, α-tubulin proteins formed a network composed of coarse filaments, probably associated with cytoskeleton. Pattern of this network was similar to those in many other cell types including nontransformed cells [13]. In contrast, α-tubulin in 686Ln cells exhibited only partial cytoskeletal distribution with majority the protein diffusely distributed within the cytosol. As a result, a uniform filament network as seen in 686Tu cells was not evident in 686Ln cells (Figure 6). Examination of the cells under higher magnification revealed that the cytoskeleton of 686Ln cells is composed of a network of much finer α-tubulin filaments (Figure 6). It should be noted that the 686Tu and Ln cells exhibit distinguishable morphologies, especially after migrating through the barrier membrane during motility and invasion assays. Almost all 686Tu cells displayed a round-flattened shape (Figure 1(a)). In contrast, more than 80% of 686Ln cells were elongated and branched, with only a small fraction to be in round spread shape (Figure 1(a)). Hence, we asked whether the unusual α-tubulin network in 686Ln cells was due to overlapping of multiple layer of the network because of their elongated and rounded morphology. We selectively examined a number of 686Ln cells with flattened morphology and found that none of these cells exhibited α-tubulin staining pattern similar to that of 686Tu cells. This finding suggests that the observed difference in the α-tubulin networks between 686Ln and 686Tu cells was not an artefact related to the cellular morphology. Moreover, we compared the immunofluorescences staining patterns of α-actin and β-actin in these two cell lines and showed that their staining patterns are indistinguishable between 686Tu and 686Ln cells (Figure 6). The sizes of α- and β-actin filaments are similar between these two cell lines, except for minor differences in their distribution patterns. These findings strongly suggest that the altered α-tubulin filament distribution pattern in 686Ln cells is most likely due to its posttranslational modification resulting in altered polymerization and/or increased solubility.


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 686Ln and primary 686Tu cells show a different cellular distribution pattern of α-tubulin. 686Tu (Tu, upper row) or 686Ln cells (Ln, lower row) were stained for α-tubulin (red) and β actin (green). The cells were counterstained by DAPI. Note that 686Ln cells show defuse α-tubulin distribution, in contrast to filament network in 686Tu cells ×600.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig6: Metastatic 686Ln and primary 686Tu cells show a different cellular distribution pattern of α-tubulin. 686Tu (Tu, upper row) or 686Ln cells (Ln, lower row) were stained for α-tubulin (red) and β actin (green). The cells were counterstained by DAPI. Note that 686Ln cells show defuse α-tubulin distribution, in contrast to filament network in 686Tu cells ×600.
Mentions: Next, we examined whether the immunofluorescence staining patterns of α-tubulin protein in this pair of cell lines would reveal any remarkable quantitative and/or qualitative differences. Overall, 686Ln cells showed a higher intensity of anti-α-tubulin immunofluorescence staining compared to 686Tu cells (Figure 6). Both cell lines demonstrated a higher density of α-tubulin expression in their perinuclear region. A comparison of the staining patterns of α-tubulin in 686Tu and Ln cells showed significant qualitative differences in the α-tubulin filament distribution. In 686Tu cells, α-tubulin proteins formed a network composed of coarse filaments, probably associated with cytoskeleton. Pattern of this network was similar to those in many other cell types including nontransformed cells [13]. In contrast, α-tubulin in 686Ln cells exhibited only partial cytoskeletal distribution with majority the protein diffusely distributed within the cytosol. As a result, a uniform filament network as seen in 686Tu cells was not evident in 686Ln cells (Figure 6). Examination of the cells under higher magnification revealed that the cytoskeleton of 686Ln cells is composed of a network of much finer α-tubulin filaments (Figure 6). It should be noted that the 686Tu and Ln cells exhibit distinguishable morphologies, especially after migrating through the barrier membrane during motility and invasion assays. Almost all 686Tu cells displayed a round-flattened shape (Figure 1(a)). In contrast, more than 80% of 686Ln cells were elongated and branched, with only a small fraction to be in round spread shape (Figure 1(a)). Hence, we asked whether the unusual α-tubulin network in 686Ln cells was due to overlapping of multiple layer of the network because of their elongated and rounded morphology. We selectively examined a number of 686Ln cells with flattened morphology and found that none of these cells exhibited α-tubulin staining pattern similar to that of 686Tu cells. This finding suggests that the observed difference in the α-tubulin networks between 686Ln and 686Tu cells was not an artefact related to the cellular morphology. Moreover, we compared the immunofluorescences staining patterns of α-actin and β-actin in these two cell lines and showed that their staining patterns are indistinguishable between 686Tu and 686Ln cells (Figure 6). The sizes of α- and β-actin filaments are similar between these two cell lines, except for minor differences in their distribution patterns. These findings strongly suggest that the altered α-tubulin filament distribution pattern in 686Ln cells is most likely due to its posttranslational modification resulting in altered polymerization and/or increased solubility.

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