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Updated: Aug 9, 2026
Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene
Published on: December 31, 2014
Generation of myc/fos transfectant Balb-3T3 cell lines
M C Armelin1, C A Joazeiro, K M Rocha
1Instituto de Química, Universidade de Sao Paulo, Brasil.
Abstract:
Early and transient expression of proto-oncogenes c-fos and c-myc is involved in the mitogenic response to PDGF (platelet-derived growth factor). We used DNA-mediated transfection to approach the role played by these genes in cell growth control by PDGF and in growth deregulation (neoplasia). Cloned pFBJ-2 (v-fos) and glucocorticoid-inducible mouse c-myc were co-transfected with a neo genetic marker to allow a neutral selection on the basis of resistance to the neomycin derivative geneticin G418. pFBJ-2 transfection was found to interfere with the number of G418-resistant (G418r) colonies. By using a v-fos-deleted pFBJ-2 construct, the deleterious effect was attributed to v-fos coding sequences. Cellular fos gene disruption, by homologous recombination with exogenous v-fos, is proposed as the basis for the deleterious effect. Co-transfection with MMTV-H3-c-myc effectively counteracts the negative effects of v-fos. Different from the parental line or single myc or fos transfectants, double myc/fos transfectants are morphologically transformed. Double transfectants still retain the PDGF requirement for growth in monolayer cultures.
Insights
Proto-oncogenes c-fos and c-myc regulate cell growth. Introducing viral fos disrupted cell growth, but co-expression with c-myc induced transformation while retaining platelet-derived growth factor (PDGF) dependence.
Area of Science:
- Molecular biology
- Cellular biology
- Oncology
Background:
- Proto-oncogenes c-fos and c-myc are crucial for the mitogenic response to platelet-derived growth factor (PDGF).
- Understanding their role in PDGF-mediated cell growth and neoplastic transformation is essential.
Purpose of the Study:
- To investigate the specific roles of c-fos and c-myc in PDGF-induced cell proliferation and growth deregulation.
- To explore the potential of manipulating these oncogenes for therapeutic strategies.
Main Methods:
- DNA-mediated transfection of cloned pFBJ-2 (v-fos) and a glucocorticoid-inducible mouse c-myc construct.
- Co-transfection with a neo genetic marker for selection of G418-resistant cells.
- Utilizing a v-fos-deleted construct to identify the specific oncogene sequences responsible for observed effects.
Main Results:
- Transfection with pFBJ-2 (v-fos) negatively impacted the number of G418-resistant colonies, an effect attributed to v-fos coding sequences.
- Cellular fos gene disruption via homologous recombination with exogenous v-fos was proposed as the mechanism for this negative effect.
- Co-transfection with MMTV-H3-c-myc counteracted the deleterious effects of v-fos.
- Double myc/fos transfectants exhibited morphological transformation, unlike parental or single-oncogene transfectant lines.
- These double transfectants maintained their requirement for PDGF for growth in monolayer cultures.
Conclusions:
- The interplay between v-fos and c-myc is critical in driving cellular transformation.
- While oncogene co-expression can induce transformation, the PDGF signaling pathway remains essential for sustained growth in this model.
- This study provides insights into oncogene cooperation and potential therapeutic targets in neoplasia.
