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Cellular and molecular changes in 3T3 cells transformed spontaneously or by DNA transfection
Abstract:
Transformed cell lines have been selected following exposure of NIH 3T3 cells to a calcium phosphate precipitate containing DNA from: the human colon carcinoma cell line, SW 480; the cloned Harvey sarcoma virus ras gene; the parental NIH 3T3 cell line; or no DNA (a spontaneous transformant). Unlike the parental 3T3 cells, each of these lines readily formed malignant spindle cell tumors in Swiss nu/mu mice. Southern blots confirmed the presence of the human homologue of the Kirsten ras gene in the cells transformed by SW480 DNA, and the Harvey ras gene in the cells transformed with that cloned sequence. The morphology of each of the lines was different, the cells transformed with the human and viral ras genes being the most aberrant (but not identical) and forming the most extensive foci in culture. These ras transformed lines also exhibited anchorage-independent growth, while the two other transformed lines did not. Both of the ras transformed lines, as well as the spontaneously transformed line, exhibited a pronounced disruption of actin microfilament structure. Two-dimensional gel electrophoresis of 35S-methionine-labeled peptides revealed that these three lines also had a marked decrease in an acidic peptide of 32K daltons.
Insights
Transformed NIH 3T3 cells, including those with ras genes, formed tumors in mice. Ras-transformed cells showed distinct morphologies, anchorage independence, and altered actin structure, indicating oncogenic potential.
Area of Science:
- Molecular Biology
- Oncology
- Cell Biology
Background:
- NIH 3T3 cells were exposed to DNA from various sources, including human colon carcinoma cells (SW 480) and cloned Harvey sarcoma virus ras gene.
- Transformed cell lines were established and characterized for their tumorigenic and phenotypic properties.
Purpose of the Study:
- To investigate the oncogenic potential of ras genes and other DNA sources in NIH 3T3 cells.
- To compare the biological and molecular characteristics of transformed cell lines.
Main Methods:
- NIH 3T3 cells were transfected with DNA using calcium phosphate precipitation.
- Tumorigenicity was assessed in Swiss nu/mu mice.
- Southern blotting was used to confirm gene integration.
- Cell morphology, focus formation, and anchorage-independent growth were evaluated.
- Actin microfilament structure and protein expression were analyzed using gel electrophoresis.
Main Results:
- All transformed cell lines, including those with ras genes, formed malignant tumors in mice.
- Ras-transformed cells exhibited aberrant morphology and anchorage-independent growth.
- Disruption of actin microfilament structure and decreased expression of a 32K acidic peptide were observed in ras-transformed and spontaneously transformed cells.
Conclusions:
- The Harvey and Kirsten ras genes confer potent oncogenic transformation capabilities to NIH 3T3 cells.
- Ras transformation leads to significant alterations in cell morphology, growth properties, and cytoskeletal organization.
- A common set of molecular changes, including actin disruption and decreased 32K peptide levels, are associated with ras-mediated transformation and spontaneous transformation.