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Selection for transformation and met protooncogene amplification in NIH 3T3 fibroblasts using tumor necrosis factor
R M Hudziak1, G D Lewis, W E Holmes
1Department of Developmental Biology, Genentech, Inc., South San Francisco, California 94080.
Abstract:
Alterations in the structure and expression of protein tyrosine kinases are associated with cellular transformation and tumorigenicity. These enzymes may contribute to tumor progression by interfering with host mechanisms of antitumor surveillance. In the present work, we show that selection of NIH 3T3 fibroblasts for resistance to the cytotoxic effects of tumor necrosis factor alpha (TNF-alpha), a major mediator of macrophage-induced tumor cell cytotoxicity, leads to enrichment in the remaining cells for those with transformed morphology and is often associated with amplified copy number and expression of the met protooncogene. We further substantiate the relationship between met protooncogene amplification and resistance to TNF-alpha by showing that spontaneous (non-TNF-alpha-selected) NIH 3T3 cell transformants which have amplified met protooncogene copy number have increased resistance to the growth-inhibitory activity of this cytokine. These results provide evidence for one mechanism by which the activated macrophage may select for tumor cells within a developing focus with properties associated with tumor progression. In addition, our ability to select cells with such properties, as demonstrated using the met protooncogene as a model system, may also provide a unique means (i.e., selection with TNF-alpha) for identifying other gene products, including other tyrosine kinases, associated with aggressive tumor growth.
Insights
Tumor necrosis factor alpha (TNF-alpha) resistance in NIH 3T3 cells selects for transformed cells with amplified met protooncogene. This suggests a mechanism by which macrophages may promote tumor progression and offers a method to identify genes linked to aggressive tumors.
Area of Science:
- Oncology
- Molecular Biology
- Immunology
Background:
- Protein tyrosine kinases alterations drive cellular transformation and tumorigenicity.
- These kinases can impede host antitumor surveillance mechanisms.
- Tumor necrosis factor alpha (TNF-alpha) is a key mediator of macrophage-induced tumor cell killing.
Purpose of the Study:
- To investigate the relationship between TNF-alpha resistance and cellular transformation.
- To explore the role of the met protooncogene in TNF-alpha resistance and tumor progression.
- To establish a selection method for identifying genes associated with aggressive tumor growth.
Main Methods:
- NIH 3T3 fibroblasts were selected for resistance to TNF-alpha.
- Analysis of cellular morphology, met protooncogene copy number, and expression levels.
- Assessment of TNF-alpha resistance in spontaneous transformants with amplified met protooncogene.
Main Results:
- Selection for TNF-alpha resistance enriched NIH 3T3 cells with transformed morphology.
- Transformed cells frequently exhibited amplified copy number and expression of the met protooncogene.
- Spontaneous NIH 3T3 transformants with amplified met protooncogene showed increased resistance to TNF-alpha's growth-inhibitory effects.
Conclusions:
- Activated macrophages may select for tumor cells with progression-associated properties via TNF-alpha.
- Met protooncogene amplification confers resistance to TNF-alpha, linking it to tumor progression.
- TNF-alpha selection can serve as a method to identify other gene products, including tyrosine kinases, associated with aggressive tumor growth.