title: Hypoxia induces class III beta-tubulin gene expression by HIF-1alpha binding to its 3' flanking region.
abstract: Class III beta-tubulin (TUBB3) overexpression represents a major mechanism of drug resistance to microtubule interacting agents such as taxanes and Vinca alkaloids. Here, we tested hypoxia as a possible inducer of TUBB3. The effects of hypoxia on TUBB3 expression were monitored at mRNA and protein level in A2780, in its paclitaxel-resistant counterpart (TC1) and in HeLa cells. Hypoxia was a strong inducer of TUBB3 in A2780, but not in TC1 and HeLa cells. In A2780 HIF-1alpha was knocked down using RNA interference and TUBB3 expression was assessed in normoxia and hypoxia. The silencing abolished the hypoxia-dependent increase of TUBB3, thereby demonstrating that HIF-1alpha mediates TUBB3 induction in hypoxia. To investigate this phenomenon, the 5' flanking region of human TUBB3 was cloned upstream GFP as a reporter. This region contained the promoter gene, but activity of the reporter was unaffected by hypoxia. Thus, we looked at the 3' flanking region and, at +168 nucleotides from the stop codon, an HIF-1alpha binding site was proven to be active in hypoxia, using a construct in which the site was cloned downstream GFP as reporter gene. Deletion of the site in the construct abolished GFP enhancement upon hypoxia. Chromatin immunoprecipitation revealed the engagement by HIF-1alpha of this site in hypoxia. Methylation analysis of this 3' enhancer showed that it was free of methylation in 70% of cells in A2780, while in less than 16% in both TC1 and HeLa cells, thereby suggesting that TUBB3 increase upon hypoxia is abolished through methylation of the 3' enhancer.
authors.affiliation: Department of Oncology, Catholic University of the Sacred Heart, Campobasso, Italy.
authors.forename: Giuseppina
authors.initials: G
authors.lastname: Raspaglio
authors.forename: Flavia
authors.initials: F
authors.lastname: Filippetti
authors.forename: Silvia
authors.initials: S
authors.lastname: Prislei
authors.forename: Roberta
authors.initials: R
authors.lastname: Penci
authors.forename: Ilaria
authors.initials: I
authors.lastname: De Maria
authors.forename: Lucia
authors.initials: L
authors.lastname: Cicchillitti
authors.forename: Simona
authors.initials: S
authors.lastname: Mozzetti
authors.forename: Giovanni
authors.initials: G
authors.lastname: Scambia
authors.forename: Cristiano
authors.initials: C
authors.lastname: Ferlini
mesh_terms: D024509:3' Flanking Region; D024506:5' Flanking Region; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D019175:DNA Methylation; D019008:Drug Resistance, Neoplasm; D005786:Gene Expression Regulation; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000502:physiology*; D008870:Microtubules / Q000187:drug effects / Q000502:physiology; D017239:Paclitaxel / Q000494:pharmacology; D014404:Tubulin / Q000096:biosynthesis* / Q000235:genetics; D050257:Tubulin Modulators / Q000494:pharmacology
chemical_list: D051795:Hypoxia-Inducible Factor 1, alpha Subunit; C498585:TUBB3 protein, human; D014404:Tubulin; D050257:Tubulin Modulators; D017239:Paclitaxel