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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

Comparison of protein spots between 686Tu and 686Ln cells in rectangular area (see Figure 1(b)). (a) Enlarged images of interested area for 686Tu (Tu, left panel) and 686Ln cells (Ln, right panel). Some spots are numbered. Note that spots number 1 and 2 are absent in 686Tu cells, while number 6, 7, and 8 are missing in 686Ln. (b) 3D simulation of spot number 1 in 686Tu and 686Ln cells. (c) Comparison of pixel volumes of spots number 1 to 5 between the two cells. Pixel volumes are calculated with 3D simulation.
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fig4: Comparison of protein spots between 686Tu and 686Ln cells in rectangular area (see Figure 1(b)). (a) Enlarged images of interested area for 686Tu (Tu, left panel) and 686Ln cells (Ln, right panel). Some spots are numbered. Note that spots number 1 and 2 are absent in 686Tu cells, while number 6, 7, and 8 are missing in 686Ln. (b) 3D simulation of spot number 1 in 686Tu and 686Ln cells. (c) Comparison of pixel volumes of spots number 1 to 5 between the two cells. Pixel volumes are calculated with 3D simulation.

Mentions: We next performed a routine preparative 2D electrophoresis of proteins from 686Ln cells (Figure 4). Based on the spots distribution pattern, spots number 1, 2, 4, and 5 were excised for mass spectrometry based peptide sequencing (Figure 4). Spot number 3 was not tested due to its low quantity. Sequencing analyses demonstrated that spots number 1 and 2 were α-tubulin. Multiple proteins, including α-tubulin, β-tubulin, and others, were detected in spots number 4 and 5, probably due to their merge with protein spots of slightly higher mass (see Figure 3(a)). However, the majority of peptides matched to α-tubulin. Our previous microarray data showed that expression levels of α-tubulin transcripts are not significantly different between 686Tu and Ln cells whereas β-tubulin is one of the highly expressed genes in metastatic OSCC cells [9]. To further confirm the microarray data on α-tubulin, we performed quantitative real-time RT-PCR analysis. Quantitative RT-PCR demonstrated that copy numbers of α-tubulin mRNA in 686Ln cells are marginally higher than 686Tu cells but the difference was not significant (Figure 5). When we calculated the sums of four spots representing the α-tubulin protein in these cell lines, we found that total α-tubulin protein amount was increased by 1.27 folds in 686Ln cells (9.01 × 107 pixels, 1.27-fold) compared to 686Tu cells (7.08 × 107 pixels, see Figure 5). Although the above data revealed only marginal increase in α-tubulin expression at both mRNA and protein levels in metastatic cells compared to its parent primary tumor cells, this small quantitative difference is unlikely to play a causal role in their motility differences.


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)

Comparison of protein spots between 686Tu and 686Ln cells in rectangular area (see Figure 1(b)). (a) Enlarged images of interested area for 686Tu (Tu, left panel) and 686Ln cells (Ln, right panel). Some spots are numbered. Note that spots number 1 and 2 are absent in 686Tu cells, while number 6, 7, and 8 are missing in 686Ln. (b) 3D simulation of spot number 1 in 686Tu and 686Ln cells. (c) Comparison of pixel volumes of spots number 1 to 5 between the two cells. Pixel volumes are calculated with 3D simulation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3376782&req=5

fig4: Comparison of protein spots between 686Tu and 686Ln cells in rectangular area (see Figure 1(b)). (a) Enlarged images of interested area for 686Tu (Tu, left panel) and 686Ln cells (Ln, right panel). Some spots are numbered. Note that spots number 1 and 2 are absent in 686Tu cells, while number 6, 7, and 8 are missing in 686Ln. (b) 3D simulation of spot number 1 in 686Tu and 686Ln cells. (c) Comparison of pixel volumes of spots number 1 to 5 between the two cells. Pixel volumes are calculated with 3D simulation.
Mentions: We next performed a routine preparative 2D electrophoresis of proteins from 686Ln cells (Figure 4). Based on the spots distribution pattern, spots number 1, 2, 4, and 5 were excised for mass spectrometry based peptide sequencing (Figure 4). Spot number 3 was not tested due to its low quantity. Sequencing analyses demonstrated that spots number 1 and 2 were α-tubulin. Multiple proteins, including α-tubulin, β-tubulin, and others, were detected in spots number 4 and 5, probably due to their merge with protein spots of slightly higher mass (see Figure 3(a)). However, the majority of peptides matched to α-tubulin. Our previous microarray data showed that expression levels of α-tubulin transcripts are not significantly different between 686Tu and Ln cells whereas β-tubulin is one of the highly expressed genes in metastatic OSCC cells [9]. To further confirm the microarray data on α-tubulin, we performed quantitative real-time RT-PCR analysis. Quantitative RT-PCR demonstrated that copy numbers of α-tubulin mRNA in 686Ln cells are marginally higher than 686Tu cells but the difference was not significant (Figure 5). When we calculated the sums of four spots representing the α-tubulin protein in these cell lines, we found that total α-tubulin protein amount was increased by 1.27 folds in 686Ln cells (9.01 × 107 pixels, 1.27-fold) compared to 686Tu cells (7.08 × 107 pixels, see Figure 5). Although the above data revealed only marginal increase in α-tubulin expression at both mRNA and protein levels in metastatic cells compared to its parent primary tumor cells, this small quantitative difference is unlikely to play a causal role in their motility differences.

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