amanitins and Osteosarcoma

Aberrant centrosome numbers are detected in virtually all human cancers where they can contribute to chromosomal instability by promoting mitotic spindle abnormalities. Despite their widespread occurrence, the molecular mechanisms that underlie centrosome amplification are only beginning to emerge. Here, we present evidence for a novel regulatory circuit involved in centrosome overduplication that centers on RNA polymerase II (pol II). We found that human papillomavirus type 16 E7 (HPV-16 E7)- and hydroxyurea (HU)-induced centriole overduplication are abrogated by alpha-amanitin, a potent and specific RNA pol II inhibitor. In contrast, normal centriole duplication proceeded undisturbed in alpha-amanitin-treated cells. Centriole overduplication was significantly reduced by siRNA-mediated knock down of CREB-binding protein (CBP), a transcriptional co-activator. We identified cyclin A2 as a key transcriptional target of RNA pol II during HU-induced centriole overduplication. Collectively, our results show that ongoing RNA pol II transcription is required for centriole overduplication whereas it may be dispensable for normal centriole duplication. Given that many chemotherapeutic agents function through inhibition of transcription, our results may help to develop strategies to target centrosome-mediated chromosomal instability for cancer therapy and prevention.

Topics: Amanitins; Bone Neoplasms; Centrosome; CREB-Binding Protein; Cyclin A; Cyclin A2; Enzyme Inhibitors; G2 Phase; Humans; Hydroxyurea; Oncogene Proteins, Viral; Osteosarcoma; Papillomavirus E7 Proteins; RNA Polymerase II; RNA, Small Interfering; Transcription, Genetic; Tumor Cells, Cultured

The stimulation of bone gamma-carboxyglutamic acid protein (BGP) synthesis by 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) in clonal osteosarcoma cell culture has been analyzed using specific inhibitors of RNA and protein synthesis. Addition of actinomycin D or alpha-amanitin simultaneously with 1,25-(OH)2D3 prevented hormonal elevation of BGP levels but did not affect basal BGP synthesis. Neither transcriptional inhibitor had any effect on BGP synthesis in cultures which had already been fully stimulated by 1,25-(OH)2D3. In contrast, the protein synthesis inhibitor cycloheximide blocked BGP synthesis in both untreated and 1,25-(OH)2D3-treated cells. Inhibitor effects on media BGP levels corresponded in all cases to effects on the rapidly turned over intracellular BGP pool. These results support a model of transcriptional control for the action of 1,25-(OH)2D3 and suggest that the hormone elicits a transient burst of transcription of the BGP gene.

Topics: Amanitins; Animals; Calcitriol; Calcium-Binding Proteins; Cell Line; Clone Cells; Cycloheximide; Dactinomycin; Kinetics; Osteocalcin; Osteosarcoma; Rats; Transcription, Genetic