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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

DIGE gel images of primary OSSC 686Tu and metastatic 686Ln cells. (a) Cy5 image (red, left panel) and Cy3 image (green, right panel) for 686Tu (Tu) and 686Ln (Ln), respectively. (b) Merged Cy5 and Cy3 image. Rectangular area, outlined by white line, would be further discussed. Note that some spots in the area appear pure green or red color, suggesting their uneven quantities in the two cells.
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fig2: DIGE gel images of primary OSSC 686Tu and metastatic 686Ln cells. (a) Cy5 image (red, left panel) and Cy3 image (green, right panel) for 686Tu (Tu) and 686Ln (Ln), respectively. (b) Merged Cy5 and Cy3 image. Rectangular area, outlined by white line, would be further discussed. Note that some spots in the area appear pure green or red color, suggesting their uneven quantities in the two cells.

Mentions: Proteins from the two cell lines were used for DIGE 2D gel electrophoresis. The scanned Cy3 and Cy5 images for 686Ln and 686Tu, respectively, displayed similar spot distribution patterns (Figure 2(a)). In line with our gene expression profiling data, the metastatic OSCC cells and its parent primary tumor cells revealed almost identical cellular protein profiles in CyDy DIGE analysis (Figure 2(a)). Based on our previous testing, over 5-fold difference in a protein of two sources would generate obvious green or red spot. In combined image, dots with obvious green or red color were visible (Figure 2(b)). Squared area in Figure 1(b) is an example to show different colors. With 2 × 104 pixels as background, the software detected 1316 protein spots in combined image for 686Tu (Cy5) and 686Ln proteins (Cy3), see Figure 3. Pixel volume for each spot was calculated and compared between 686Ln and 686Tu cells using 686Tu as standard. Plot with log volume ratios against spot numbers fit well with Gaussian distribution (R2 = 0.9629) with 0.032 ± 0.16 in Log scale or 1.033 ± 1.176 folds (Figure 3). To estimate how closely the two cell lines were related, we used 2-fold differences in pixel volume as threshold. Calculation demonstrated that 686Ln and 686Tu cells showed similar volumes in 1159 spots (88.1%), suggesting their close relationship. In addition, 686Ln cells showed increased volume in 70 spots (5.3%) and decreased volume in 87 spots (8.6%), as compared to those of 686Tu cells. However, most spots with different volumes between the two cells clustered between 2 to 4 folds (Figure 3).


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)

DIGE gel images of primary OSSC 686Tu and metastatic 686Ln cells. (a) Cy5 image (red, left panel) and Cy3 image (green, right panel) for 686Tu (Tu) and 686Ln (Ln), respectively. (b) Merged Cy5 and Cy3 image. Rectangular area, outlined by white line, would be further discussed. Note that some spots in the area appear pure green or red color, suggesting their uneven quantities in the two cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig2: DIGE gel images of primary OSSC 686Tu and metastatic 686Ln cells. (a) Cy5 image (red, left panel) and Cy3 image (green, right panel) for 686Tu (Tu) and 686Ln (Ln), respectively. (b) Merged Cy5 and Cy3 image. Rectangular area, outlined by white line, would be further discussed. Note that some spots in the area appear pure green or red color, suggesting their uneven quantities in the two cells.
Mentions: Proteins from the two cell lines were used for DIGE 2D gel electrophoresis. The scanned Cy3 and Cy5 images for 686Ln and 686Tu, respectively, displayed similar spot distribution patterns (Figure 2(a)). In line with our gene expression profiling data, the metastatic OSCC cells and its parent primary tumor cells revealed almost identical cellular protein profiles in CyDy DIGE analysis (Figure 2(a)). Based on our previous testing, over 5-fold difference in a protein of two sources would generate obvious green or red spot. In combined image, dots with obvious green or red color were visible (Figure 2(b)). Squared area in Figure 1(b) is an example to show different colors. With 2 × 104 pixels as background, the software detected 1316 protein spots in combined image for 686Tu (Cy5) and 686Ln proteins (Cy3), see Figure 3. Pixel volume for each spot was calculated and compared between 686Ln and 686Tu cells using 686Tu as standard. Plot with log volume ratios against spot numbers fit well with Gaussian distribution (R2 = 0.9629) with 0.032 ± 0.16 in Log scale or 1.033 ± 1.176 folds (Figure 3). To estimate how closely the two cell lines were related, we used 2-fold differences in pixel volume as threshold. Calculation demonstrated that 686Ln and 686Tu cells showed similar volumes in 1159 spots (88.1%), suggesting their close relationship. In addition, 686Ln cells showed increased volume in 70 spots (5.3%) and decreased volume in 87 spots (8.6%), as compared to those of 686Tu cells. However, most spots with different volumes between the two cells clustered between 2 to 4 folds (Figure 3).

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