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Growth in serum-free medium of NIH3T3 cells transformed by the EJ-H-ras oncogene: evidence for multiple autocrine
M Pironin1, G Clément, O Benzakour
1Unité 1443 du CNRS, Institut Curie, Orsay, France.
Abstract:
Rodent fibroblastic cells transformed by ras oncogenes can grow in serum-free (S-) medium. We have studied clonal lines of mouse NIH3T3 fibroblasts transfected with the EJ-H-ras oncogene, and observed that practically all become independent of exogenous growth and attachment factors shortly after transfection. Moreover, all the clones tested soon form anchorage-independent (AI) colonies in S- medium, and most give rise to spheroids able to grow in suspension. The cell-conditioned S- medium of the transformed (TR) cells stimulates autocrinally the AI and anchored growth of these cells, in the absence of serum, and it contains growth factors related to TGF-alpha (or EGF), PDGF and bFGF, and other uncharacterized factors. Some of these factors are not found, or are found only in very small amounts, in the S- medium of non-transformed NIH3T3 cells, which also stimulates the growth of the TR cells, in the absence of serum. In addition, the TR cells contain 4-6 times more cell-associated bFGF than the non-transformed cells and release more latent TGF-beta activatable by acid treatments. However, no active TGF-beta is secreted by either cell type. Activated TGF-beta and pure TGF-beta 1 stimulate the growth of the anchored TR and NIH3T3 cells, but inhibit the AI growth of the TR cells. Another inhibitor of this growth is also found in the concentrated medium of the NIH3T3 cells.
Insights
Ras-transformed cells gain independence from growth factors and form colonies in serum-free conditions. Their conditioned medium contains growth factors, while TGF-beta influences both anchored and suspension growth.
Area of Science:
- Oncology
- Cell Biology
- Molecular Biology
Background:
- Ras oncogenes are crucial in cell transformation and cancer development.
- Cellular transformation often leads to altered growth factor dependency and anchorage independence.
- Understanding growth factor signaling in transformed cells is key to cancer research.
Purpose of the Study:
- To investigate the growth factor requirements of ras-oncogene-transformed rodent fibroblasts in serum-free conditions.
- To identify and characterize growth factors present in the conditioned medium of transformed cells.
- To explore the role of TGF-beta in the growth of transformed and non-transformed cells.
Main Methods:
- Transfection of NIH3T3 fibroblasts with EJ-H-ras oncogene.
- Culturing cells in serum-free (S-) medium and assessing anchorage-independent (AI) growth.
- Analysis of conditioned medium for growth factor content (TGF-alpha, PDGF, bFGF, TGF-beta).
- Assessing the effects of activated TGF-beta on cell growth.
Main Results:
- Ras-transformed (TR) cells exhibit independence from exogenous growth and attachment factors.
- TR cells form AI colonies and spheroids in S- medium.
- TR cell-conditioned medium contains TGF-alpha, PDGF, bFGF, and other factors promoting autocrine growth.
- TR cells show increased cell-associated bFGF and release activatable TGF-beta.
- Activated TGF-beta stimulates anchored growth but inhibits AI growth of TR cells.
Conclusions:
- Ras transformation confers serum and growth factor independence to fibroblasts.
- Autocrine growth factor production is a key mechanism in ras-transformed cells.
- TGF-beta exhibits differential effects on anchored vs. anchorage-independent growth of transformed cells.