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Conditional BRAF^V600E Expression Induces DNA Synthesis, Apoptosis, Dedifferentiation, and Chromosomal Instability in Thyroid PCCL3 Cells

Division of Endocrinology and Metabolism, University of Cincinnati College of Medicine, Cincinnati, Ohio

Requests for reprints: James A. Fagin, Division of Endocrinology and Metabolism, University of Cincinnati College of Medicine, 3125 Eden Avenue, P.O. Box 670547, Cincinnati, OH 45267-0547. Phone: 513-558-4444; Fax: 513-558-8581; E-mail: james.fagin{at}uc.edu.

The activating mutation BRAF^T1796A is the most prevalent genetic alteration in papillary thyroid carcinomas (PTC). It is associated with advanced PTCs, suggesting that this oncoprotein confers thyroid cancers with more aggressive properties. BRAF^T1796A is also observed in thyroid micropapillary carcinomas and may thus be an early event in tumor development. To explore its biological consequences, we established doxycycline-inducible BRAF^V600E-expressing clonal lines derived from well-differentiated rat thyroid PCCL3 cells. Expression of BRAF^V600E did not induce growth in the absence of thyrotropin despite increasing DNA synthesis, which is likely explained because of a concomitant increase in apoptosis. Thyrotropin-dependent cell growth and DNA synthesis were reduced by BRAF^V600E because of decreased thyrotropin responsiveness associated with inhibition of thyrotropin receptor gene expression. These results are similar to those obtained following conditional expression of RET/PTC. However, in contrast to RET/PTC, BRAF activation did not impair key activation steps distal to the thyrotropin receptor, such as forskolin-induced adenylyl cyclase activity or cyclic AMP–induced DNA synthesis. We reported previously that acute RET/PTC expression in PCCL3 cells did not induce genomic instability. By contrast, induction of BRAF^V600E expression increased the frequency of micronuclei by both clastogenic and aneugenic events. These data indicate that BRAF^V600E expression confers thyroid cells with little growth advantage because of concomitant activation of DNA synthesis and apoptosis. However, in contrast to RET/PTC, BRAF^V600E may facilitate the acquisition of secondary genetic events through induction of genomic instability, which may account for its aggressive properties.

Key Words: BRAF • mutation • MAPK • thyroid cancer • chromosomal instability

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