okadaic-acid and Uterine-Cervical-Neoplasms

Exon 2 of the Bcl-x gene undergoes alternative splicing in which the Bcl-xS splice variant promotes apoptosis in contrast to the anti-apoptotic splice variant Bcl-xL. In this study, the regulation of the alternative splicing of pre-mRNA of Bcl-x was examined in response to emetine. Treatment of different types of cancer cells with emetine dihydrochloride downregulated the level of Bcl-xL mRNA with a concomitant increase in the mRNA level of Bcl-xS in a dose- and time-dependent manner. Pretreatment with calyculin A, an inhibitor of protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A), blocked emetine-induced alternative splicing in contrast to okadaic acid, a specific inhibitor of PP2A in cells, demonstrating a PP1-mediated mechanism. Our finding on the regulation of RNA splicing of members of the Bcl-2 family in response to emetine presents a potential target for cancer treatment.

Topics: Alternative Splicing; bcl-X Protein; Breast Neoplasms; Cell Line, Tumor; Cycloheximide; Down-Regulation; Emetine; Enzyme Inhibitors; Female; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Male; Marine Toxins; Okadaic Acid; Oxazoles; Prostatic Neoplasms; Protein Phosphatase 1; Protein Phosphatase 2; Up-Regulation; Uterine Cervical Neoplasms

Human papillomavirus (HPV) DNA sequences are found in most carcinomas originating from the uterine cervix. HPV E6 and E7 oncogenes have been shown to cooperate with ras oncogenes to fully transform human epithelial cells. We investigated the effect of the Ha-ras oncogene on the transcriptional activity of HPV-18 and found that it induced the transcriptional activity of the viral promoter, whereas the normal gene had only a minimal effect. However, transfection of the normal Ha-ras gene and simultaneous inhibition of protein phosphatase sensitive to okadaic acid (OA) resulted in a cooperative transactivation of the viral promoter. When cloned upstream of a minimal promoter, the AP-1 binding sites present in the viral promoter conferred transcriptional responsiveness to Ha-ras and OA. Furthermore, HeLa cell clones permanently expressing the Ha-ras oncogene or high levels of the normal gene exhibited a twofold to threefold increase in E6*E7/E1 and E6*E7 transcripts. We propose that both Ha-ras and a protein phosphatase sensitive to OA regulate HPV oncogene expression through modulation of AP-1 activity and suggest that increased levels of E6 and E7, resulting from activated viral transcription in the presence of ras oncogenes, may in part explain the observed cooperation between these viral and cellular oncogenes in the transformation of human cells.

Topics: DNA-Binding Proteins; Female; Genes, ras; Genes, Viral; HeLa Cells; Humans; Okadaic Acid; Oncogene Proteins, Viral; Papillomaviridae; Phosphoprotein Phosphatases; Promoter Regions, Genetic; Transcription Factor AP-1; Transcriptional Activation; Transfection; Tumor Cells, Cultured; Uterine Cervical Neoplasms