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Transformation-resistant mos revertant is unable to activate MAP kinase kinase in response to v-mos or v-raf
1Laboratory of Molecular Oncology, National Cancer Institute, Frederick, Maryland 21702.
Abstract:
To study the mechanism by which v-mos induces cell transformation, we generated a transformed rat cell line (DTM) containing two functional copies of mos, one encoding the p37v-mos of the m1 wild-type strain of Moloney murine sarcoma virus (Mo-MuSV) and the other the p85gag-mos fusion protein of the ts110 mutant of Moloney murine sarcoma virus. Subsequently, we isolated a revertant cell line (F-1) following transfection of DTM with a mutant retroviral construct (pIC4Neo) carrying a selectable marker. Like DTM, the F-1 revertant contained two integrated copies of v-mos, expressed mos containing viral RNA, and contained rescuable transforming viruses. The revertant did not grow in soft agar, showed a greatly reduced ability to form tumors in nude mice, and exhibited organized tubulin and actin structures similar to those found in normal cells. Revertant cells were resistant to retransformation by v-mos and v-raf but could be retransformed by v-ras. MAP kinase (ERK-2) and MAP kinase kinase (MKK-1) activity, which are constitutively elevated in v-mos- and v-raf-transformed cells, exhibits levels in the F-1 revertant similar to those seen in nontransformed cells. F-1 and normal REF-1 cells express elevated levels of protein phosphatases in comparison to DTM cells. In vivo treatment with okadaic acid, a potent protein phosphatase inhibitor, leads to an increase in MKK-1 and MAP kinase activity in F-1 cells but not in REF-1. The results support the hypothesis that mos acts through the MAP kinase cascade (MKK-1 and ERK-2) to induce cell transformation and that blocking v-mos activation of that cascade (possibly because of increased levels of phosphatase) prevents transformation.
Insights
The v-mos oncogene transforms cells by activating the MAP kinase cascade (MKK-1 and ERK-2). Increased protein phosphatase levels in revertant cells block this activation, preventing transformation and restoring normal cell structure.
Area of Science:
- Molecular Biology
- Cell Biology
- Oncology
Background:
- The v-mos oncogene is known to induce cell transformation.
- The precise molecular mechanisms underlying v-mos-induced cell transformation require further elucidation.
Purpose of the Study:
- To investigate the mechanism by which v-mos induces cell transformation.
- To identify key signaling pathways and cellular components involved in v-mos-mediated transformation.
Main Methods:
- Generation of a v-mos-transformed rat cell line (DTM) and isolation of a revertant cell line (F-1).
- Analysis of cellular morphology, tumor formation in nude mice, and resistance to retransformation.
- Assay of MAP kinase (ERK-2) and MAP kinase kinase (MKK-1) activity.
- Assessment of protein phosphatase levels and inhibition studies with okadaic acid.
Main Results:
- The F-1 revertant cells, despite containing v-mos, exhibited normal cell structure and reduced tumor formation.
- F-1 cells were resistant to v-mos and v-raf retransformation but sensitive to v-ras.
- MAP kinase cascade activity (MKK-1 and ERK-2) was normalized in F-1 cells compared to DTM cells.
- F-1 cells showed elevated protein phosphatase levels, which could be inhibited by okadaic acid, leading to increased MKK-1 and ERK-2 activity.
Conclusions:
- v-mos likely induces cell transformation by activating the MAP kinase cascade (MKK-1 and ERK-2).
- Elevated protein phosphatase activity in revertant cells appears to block v-mos activation of this cascade, preventing transformation.
- These findings provide insights into the signaling pathways governing oncogene-induced cell transformation.