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Confocal laser scanning microscopy of oncogene localization in rainbow trout cell lines derived from normal and tumor
C A Carter1, W W Ellington, R J Van Beneden
1Laboratory of Experimental Pathology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA.
Abstract:
We examined the localization and expression of the nuclear oncoprotein c-myc and the cytoplasmic membrane-associated oncoprotein c-ras in rainbow trout cell lines derived from both normal and tumor tissue in order to question whether c-myc and ras oncoprotein immunostaining was increased in cells derived from tumors compared to cells derived from normal tissue. Cell lines examined were derived from normal rainbow trout gonadal cells (RTG-2), a rainbow trout hepatoma (RTH-149), and a rainbow trout mesothelioma (RTM). Protein products of c-ras and c-myc were visualized in these 3 cell lines by employing fluorescein-labeled anti-mouse pan-ras and c-myc antibodies. The RTG-2 cells were used in this study as normal, control cells, and they exhibited little pan-ras and c-myc staining. The RTH-149 cell line (a tumorigenic cell line) exhibited positive pan-ras staining in regions of the membrane and cell cytoplasm. Localization of c-myc staining to perinuclear regions was punctate in RTH-149 cells. RTM cells (also a tumorigenic cell line) displayed a ras staining localization similar to the pattern seen in RTH-149 cells. RTM cells exhibit a diffuse perinuclear staining and, thus, display a more ubiquitous localization of c-myc than RTH-149 cells. Northern blot analysis indicated that c-myc expression was highest in RTM cells, whereas RTG-2 cells and RTH-149 cells expressed similar lower levels of c-myc expression. We were unable to detect significant ras expression in any of the cell lines by Northern blot analysis. In summary, the cell line derived from normal tissue, the RTG-2 cells, displayed little ras and c-myc immunostaining, whereas the cell lines derived from tumorigenic tissue, RTH and RTM cells, displayed increased immunostaining for c-myc and ras proteins.
Insights
Oncoproteins c-myc and c-ras were studied in rainbow trout cell lines. Tumorigenic cells showed increased immunostaining for these oncoproteins compared to normal cells.
Area of Science:
- Oncology
- Molecular Biology
- Aquatic Animal Health
Background:
- Oncoproteins like c-myc and c-ras play critical roles in cellular growth and differentiation.
- Aberrant expression of these oncoproteins is frequently observed in various cancers.
- Understanding oncoprotein expression in fish models can provide insights into cancer development.
Purpose of the Study:
- To investigate the expression and localization of c-myc and c-ras oncoproteins in rainbow trout cell lines.
- To determine if c-myc and c-ras oncoprotein immunostaining is elevated in tumor-derived cells compared to normal cells.
Main Methods:
- Utilized three rainbow trout cell lines: normal gonadal (RTG-2) and two tumorigenic lines (RTH-149 hepatoma, RTM mesothelioma).
- Employed immunofluorescence with fluorescein-labeled anti-mouse pan-ras and c-myc antibodies to visualize protein products.
- Performed Northern blot analysis to assess c-myc and ras gene expression levels.
Main Results:
- Normal RTG-2 cells exhibited minimal c-myc and pan-ras immunostaining.
- Tumorigenic RTH-149 and RTM cells showed increased immunostaining for both c-myc and ras proteins.
- RTH-149 cells displayed punctate perinuclear c-myc staining and membrane/cytoplasmic ras staining.
- RTM cells showed more diffuse perinuclear c-myc staining and similar ras localization to RTH-149.
- Northern blot confirmed highest c-myc expression in RTM cells, with lower, similar levels in RTG-2 and RTH-149; ras expression was undetectable by Northern blot.
Conclusions:
- Rainbow trout tumor cell lines demonstrate significantly higher immunostaining for c-myc and c-ras oncoproteins compared to normal cell lines.
- These findings suggest a potential role for c-myc and c-ras in rainbow trout tumorigenesis.
- The differential expression patterns highlight the utility of these cell lines for studying fish cancer biology.