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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

686Tu and 686Ln cells expressed comparable levels of α-tubulin. (a) Quantitative RT-PCR detection of mRNA for the genes as indicated in 686Tu and 686Ln cells. The quantities of mRNA are expressed as relative abundance using GAPDH in 686Tu as 100%. (b) Total pixel volumes of α-tubulin in 686Tu and 686Ln cells.
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fig5: 686Tu and 686Ln cells expressed comparable levels of α-tubulin. (a) Quantitative RT-PCR detection of mRNA for the genes as indicated in 686Tu and 686Ln cells. The quantities of mRNA are expressed as relative abundance using GAPDH in 686Tu as 100%. (b) Total pixel volumes of α-tubulin in 686Tu and 686Ln cells.

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)

686Tu and 686Ln cells expressed comparable levels of α-tubulin. (a) Quantitative RT-PCR detection of mRNA for the genes as indicated in 686Tu and 686Ln cells. The quantities of mRNA are expressed as relative abundance using GAPDH in 686Tu as 100%. (b) Total pixel volumes of α-tubulin in 686Tu and 686Ln cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig5: 686Tu and 686Ln cells expressed comparable levels of α-tubulin. (a) Quantitative RT-PCR detection of mRNA for the genes as indicated in 686Tu and 686Ln cells. The quantities of mRNA are expressed as relative abundance using GAPDH in 686Tu as 100%. (b) Total pixel volumes of α-tubulin in 686Tu and 686Ln cells.
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