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p21Ras downstream effectors are increased in activity or expression in mouse liver tumors but do not differ between
A Kalkuhl1, J Troppmair, A Buchmann
1Institute of Toxicology, University of Tübingen, Germany.
Abstract:
Mouse liver tumors frequently harbor activating ras gene mutations. Downstream effector molecules of p21Ras include Raf-1 kinase which mediates external signals via kinase signaling pathways to nuclear transcription factors including c-Fos and c-Jun. Mouse liver tumors with differing ras-mutational status were analyzed for alterations in Ras/Raf-1 signal transduction. Tumors were characterized with respect to the presence of base substitutions in the 3 known hot-spot positions at codons 12, 13, and 61 of Ha-ras, Ki-ras, and N-ras. Ha-ras codon 61 or Ki-ras codon 13 mutations, but no N-ras mutations, were detected in 23 out of 33 tumors analyzed, while no ras-mutations were found in 10 of the tumors. There was no significant difference in the expression of p21RaS proteins between ras-mutated tumors and tumors without detectable ras mutations. To allow for determination of Raf-1 kinase activity in tumors, a sensitive and specific assay was developed for measurements with tissue homogenates. Raf-1 kinase activity was increased about four-fold in liver tumors as compared with normal liver tissue. No significant differences in kinase activity, however, were evident between ras-mutated and ras-wild-type tumors. The same was true with respect to the levels of c-fos and c-jun mRNAs. Moreover, there were no significant differences in cell division (5-bromo-2'-deoxyuridine-labeling indices) of hepatocytes from ras-mutated and ras-wild-type tumors. The similar degree of constitutive activation of the Ras/Raf-1 signaling pathway in liver tumors, with and without detectable ras mutations, suggests that other molecules within the signaling pathway may substitute for ras-mutations during oncogenic conversion of ras-wild-type hepatocytes.
Insights
Activating ras gene mutations are common in mouse liver tumors. However, the Ras/Raf-1 signaling pathway shows similar activation in both mutated and non-mutated tumors, suggesting alternative pathways contribute to liver cancer development.
Area of Science:
- Oncology
- Molecular Biology
- Signal Transduction
Background:
- Activating ras gene mutations are frequently observed in mouse liver tumors.
- The Ras/Raf-1 signaling pathway, involving molecules like p21Ras, Raf-1 kinase, c-Fos, and c-Jun, plays a crucial role in mediating external signals to nuclear transcription factors.
- Understanding alterations in this pathway is key to deciphering liver oncogenesis.
Purpose of the Study:
- To investigate alterations in the Ras/Raf-1 signal transduction pathway in mouse liver tumors with varying ras mutational status.
- To correlate ras mutational status with Raf-1 kinase activity, c-fos and c-jun mRNA levels, and hepatocyte proliferation.
Main Methods:
- Analysis of ras gene mutations (Ha-ras, Ki-ras, N-ras) at hot-spot codons (12, 13, 61) in 33 mouse liver tumors.
- Development of a sensitive assay to measure Raf-1 kinase activity in tissue homogenates.
- Quantification of p21Ras protein expression, c-fos and c-jun mRNA levels, and hepatocyte proliferation (5-bromo-2'-deoxyuridine-labeling indices).
Main Results:
- Mutations in Ha-ras codon 61 or Ki-ras codon 13 were detected in 23 of 33 tumors; no N-ras mutations were found.
- Raf-1 kinase activity was significantly increased (approximately four-fold) in all liver tumors compared to normal liver tissue.
- No significant differences in Raf-1 kinase activity, c-fos/c-jun mRNA levels, or hepatocyte proliferation were observed between ras-mutated and ras-wild-type tumors.
Conclusions:
- The Ras/Raf-1 signaling pathway is constitutively activated in mouse liver tumors, irrespective of detectable ras mutations.
- These findings suggest that alternative upstream or downstream molecules may compensate for the absence of ras mutations, driving oncogenic conversion in ras-wild-type hepatocytes.
- Further research is warranted to identify these alternative signaling components in liver cancer development.