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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

Scattergram depiction of DIGE data to generate protein expression profile of 686Tu and 686Ln cells. Each dot represents a protein detected in 686Tu and 686Ln cells. The dots are expressed as bigger pixel volume in either cell (right Y-axis), and plotted against log scale of pixel volume ratio between 686Tu and 686Ln (X axis), with 686Tu as standard. Two vertical lines indicate two-fold difference limits between the two cells. Left Y-axis is number of spots at certain pixel ranges. Related pink curve is actual distribution of spots versus their pixel volume ratio and green curve is normalized Gaussian distribution (R2 = 0.963). Arrows indicate two protein spots to be studied.
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fig3: Scattergram depiction of DIGE data to generate protein expression profile of 686Tu and 686Ln cells. Each dot represents a protein detected in 686Tu and 686Ln cells. The dots are expressed as bigger pixel volume in either cell (right Y-axis), and plotted against log scale of pixel volume ratio between 686Tu and 686Ln (X axis), with 686Tu as standard. Two vertical lines indicate two-fold difference limits between the two cells. Left Y-axis is number of spots at certain pixel ranges. Related pink curve is actual distribution of spots versus their pixel volume ratio and green curve is normalized Gaussian distribution (R2 = 0.963). Arrows indicate two protein spots to be studied.

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)

Scattergram depiction of DIGE data to generate protein expression profile of 686Tu and 686Ln cells. Each dot represents a protein detected in 686Tu and 686Ln cells. The dots are expressed as bigger pixel volume in either cell (right Y-axis), and plotted against log scale of pixel volume ratio between 686Tu and 686Ln (X axis), with 686Tu as standard. Two vertical lines indicate two-fold difference limits between the two cells. Left Y-axis is number of spots at certain pixel ranges. Related pink curve is actual distribution of spots versus their pixel volume ratio and green curve is normalized Gaussian distribution (R2 = 0.963). Arrows indicate two protein spots to be studied.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: Scattergram depiction of DIGE data to generate protein expression profile of 686Tu and 686Ln cells. Each dot represents a protein detected in 686Tu and 686Ln cells. The dots are expressed as bigger pixel volume in either cell (right Y-axis), and plotted against log scale of pixel volume ratio between 686Tu and 686Ln (X axis), with 686Tu as standard. Two vertical lines indicate two-fold difference limits between the two cells. Left Y-axis is number of spots at certain pixel ranges. Related pink curve is actual distribution of spots versus their pixel volume ratio and green curve is normalized Gaussian distribution (R2 = 0.963). Arrows indicate two protein spots to be studied.
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