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Environment-dependent growth inhibition of human epidermal keratinocytes by recombinant human transforming growth
B J Rollins1, T M O'Connell, G Bennett
1Division of Medicine, Dana-Farber Cancer Institute, Boston, MA.
Abstract:
Transforming growth factor-beta (TGF-beta) purified from platelets is a potent growth inhibitor of several normal epithelial cell types in culture. In contrast, some carcinoma cell lines derived from tumors of these same tissues are resistant to this factor. Using recombinant human TGF-beta, the authors have confirmed these results with six normal human epidermal keratinocyte strains and four human epidermal squamous carcinoma cell lines. However, the sensitivity of normal cells to TGF-beta was found to depend on the culture conditions. When grown in a specialized nutrient medium supplemented with pituitary extract, keratinocytes were completely inhibited by the addition of 0.3 ng/ml TGF-beta. In contrast, when their growth was supported by cocultivation with 3T3 fibroblast feeder cells, 30- to 100-fold higher concentrations of TGF-beta were required to achieve comparable growth inhibition. This differential sensitivity occurred despite the fact that in both culture systems TGF-beta in the culture medium had a half-life of about 50 minutes, becoming tightly bound to the surface of the culture dish. Bound TGF-beta proved to be biologically active and stable for about a week in the absence of 3T3 feeder cells. Incubating 3T3 cells on TGF-beta-coated dishes, however, resulted in nearly quantitative removal and degradation of the TGF-beta within 2 days, permitting normal rates of keratinocyte growth. The binding of TGF-beta to surfaces and the ability of fibroblasts to attenuate its inhibitory activity for epithelial cells must be considered when evaluating in vitro models and in planning strategies for the use of this factor in vivo.
Insights
Transforming growth factor-beta (TGF-beta) inhibits normal epithelial cell growth, but cancer cells are resistant. Culture conditions, particularly the presence of fibroblasts, significantly alter normal cell sensitivity to TGF-beta.
Area of Science:
- Cell Biology
- Molecular Biology
- Cancer Research
Background:
- Transforming growth factor-beta (TGF-beta) is a potent inhibitor of normal epithelial cell proliferation.
- Carcinoma cell lines often exhibit resistance to TGF-beta, unlike their normal counterparts.
- Understanding TGF-beta's role in cell growth regulation is crucial for cancer research.
Purpose of the Study:
- To investigate the differential sensitivity of normal keratinocytes and squamous carcinoma cells to TGF-beta.
- To determine the influence of culture conditions on TGF-beta's inhibitory effects.
- To explore the mechanisms by which fibroblasts modulate TGF-beta activity.
Main Methods:
- Utilized recombinant human TGF-beta for experiments.
- Compared growth inhibition in normal human epidermal keratinocytes and human epidermal squamous carcinoma cell lines.
- Assessed TGF-beta sensitivity under different culture conditions: specialized medium vs. 3T3 fibroblast co-culture.
- Measured TGF-beta half-life, binding to culture dishes, and degradation by fibroblasts.
Main Results:
- Normal keratinocytes were highly sensitive to TGF-beta in specialized medium but required significantly higher concentrations when co-cultured with 3T3 fibroblasts.
- TGF-beta bound to culture dishes and remained active, but 3T3 fibroblasts efficiently removed and degraded it.
- Fibroblast co-culture attenuated TGF-beta's inhibitory effect on keratinocyte growth.
Conclusions:
- The sensitivity of normal epithelial cells to TGF-beta is highly dependent on the culture microenvironment.
- Fibroblasts play a critical role in modulating TGF-beta's biological activity.
- These findings have implications for in vitro model interpretation and in vivo therapeutic strategies involving TGF-beta.