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Isolation of transformation suppressor genes by cDNA subtraction: lumican suppresses transformation induced by v-src
N Yoshioka1, H Inoue, K Nakanishi
1Department of Tumor Virology, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan.
Abstract:
We have reported that suppressive factors for transformation by viral oncogenes are expressed in primary rat embryo fibroblasts (REFs). To identify such transformation suppressor genes, we prepared a subtracted cDNA library by using REFs and a rat normal fibroblast cell line, F2408, and isolated 30 different cDNA clones whose mRNA expression was markedly reduced in F2408 cells relative to that in REFs. We referred to these as TRIF (transcript reduced in F2408) clones. Among these genes, we initially tested the suppressor activity for transformation on three TRIF genes, TRIF1 (neuronatin), TRIF2 (heparin-binding growth-associated molecule), and TRIF3 (lumican) by focus formation assay and found that lumican inhibited focus formation induced by activated H-ras in F2408 cells. Colony formation in soft agar induced by v-K-ras or v-src was also suppressed in F2408 clones stably expressing exogenous lumican without disturbing cell proliferation. Tumorigenicity in nude mice induced by these oncogenes was also suppressed in these lumican-expressing clones. These results indicate that lumican has the ability to suppress transformation by v-src and v-K-ras.
Insights
Lumican, a transcript reduced in F2408 cells, suppresses oncogene-induced cell transformation. This study identifies lumican as a potential tumor suppressor by inhibiting focus formation, soft agar colony formation, and tumorigenicity.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Viral oncogenes can induce cell transformation.
- Suppressor genes for transformation are expressed in primary rat embryo fibroblasts (REFs).
Purpose of the Study:
- Identify transformation suppressor genes.
- Investigate the role of lumican in suppressing oncogene-induced transformation.
Main Methods:
- Subtracted cDNA library construction using REFs and F2408 cells.
- Focus formation assay to test transformation suppression.
- Soft agar colony formation and nude mouse tumorigenicity assays.
Main Results:
- Identified 30 TRIF (transcript reduced in F2408) clones.
- Lumican (TRIF3) inhibited focus formation induced by activated H-ras.
- Exogenous lumican suppressed colony formation and tumorigenicity induced by v-K-ras and v-src without affecting cell proliferation.
Conclusions:
- Lumican acts as a suppressor of oncogene-induced cell transformation.
- Lumican inhibits transformation mediated by v-src and v-K-ras.
- Lumican demonstrates potential as a tumor suppressor gene.