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The RAS oncogene induces genomic instability in thyroid PCCL3 cells via the MAPK pathway

Abstract

Activating mutations of RAS are thought to be early events in the evolution of thyroid follicular neoplasms. We used a doxycycline-inducible expression system to explore the acute effects of H-RASV12 on genomic stability in thyroid PCCL3 cells. At 2–3 days (first or second cell cycle) there was a significant increase in the frequency of micronucleation. Treatment of cells with YVAD-CHO inhibited RAS-induced apoptosis, but had no effect on micronucleation. The effects of H-RASV12 were mediated by activation of MAPK, as treatment with PD98059 at concentrations verified to selectively inhibit MEK1 reduced the frequency of prevalence of cells with micronuclei. In addition, doxycycline-inducible expression of a constitutively active MEK1, but not of a mutant RAC1, mimicked the effects of H-RASV12. The effects of H-RASV12 on genome destabilization were apparent even though the sequence of p53 in PCCL3 cells was confirmed to be wild-type. Acute activation of H-RASV12 evoked a proportional increase in both CREST negative and CREST positive micronuclei, indicating that both clastogenic and aneugenic effects were involved. H-RASV12 and activated MEK1 also induced centrosome amplification, and chromosome misalignment. Evidence that acute expression of constitutively activated RAS destabilizes the genome of PCCL3 cells is consistent with a mode of tumor initiation in which this oncogene promotes phenotypic progression by predisposing to large scale genomic abnormalities.

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Acknowledgements

The RASV12 and the RAC1V12-myc-tagged cDNA's were a generous gift from Dr. Kenji Fukasawa. The MEK1-Glu-217/Glu221 cDNA was kindly provided by Dr. Christopher Marshall. The RET/PTC1 and RET/PTC3 cDNA's were kindly provided by Dr. S.M. Jhiang. This work was supported in part by NIH grants CA50706 and CA72597 (JA Fagin), F32CA69711 (JA Knauf), F32CA80389 (JM Shirokawa), and CA65769 (PJ Stambrook).

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Saavedra, H., Knauf, J., Shirokawa, J. et al. The RAS oncogene induces genomic instability in thyroid PCCL3 cells via the MAPK pathway. Oncogene 19, 3948–3954 (2000). https://doi.org/10.1038/sj.onc.1203723

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