Published ahead of print on September 15, 2005, doi:10.1165/rcmb.2005-0213OC Am. J. Respir. Cell Mol. Biol., Volume 33, Number 6, December 2005, 589-600 A more recent version of this article appeared on December 1, 2005
Submitted on June 9, 2005 Molecular Mechanisms of Hexavalent Chromium-induced Apoptosis in Human Bronchoalveolar CellsPatrizia A Russo1*,1 Department of Integrated Medical Oncology (DOMI), Laboratory of Translational Research B (Lung Cancer), National Cancer Institute, Genoa, Italy, 2 Department of Biology (DIBIO), University of Genoa, Genoa, Italy, 3 Department of Surgical Science, Division of General Thoracic Surgery, Catholic University, Rome, Italy; Clinical Respiratory and Pathology Translational Laboratory, IRCCS San Raffaele, Rome, Italy, 4 Center of Thoracic Surgery, University of Insubria, Varese, Italy, 5 Department of Internal Medicine Sciences, IRCCS San Raffaele, Rome, Italy, 6 Department of Surgical Science, Division of General Thoracic Surgery, Catholic University, Rome, Italy * To whom correspondence should be addressed. E-mail: patrizia.russo{at}istge.it.
Rationale: Hexavalent chromium [Cr(VI)] is classified by the International Agency for Research on Cancer as a group I carcinogen. Although the U.S. Occupational Safety and Health Administration obliged to reduce the permissible exposure limit (PEL), it was reported that U.S. workers continue to be exposed to dangerously high Cr(VI) levels. Objectives: In this work we examined the role of p53 and target genes in a bronchoalveolar carcinoma isogenic cell line system and in primary human bronchial epithelial cells. Methods: p53-negative parental H358 cell line and the same line in which the wild type p53 expression vector (pC53-SN3) was introduced and cells obtained from biopsies of human bronchus were exposed to K2CrO4. Induction of DNA-strand breaks were evaluated by alkaline elution assay, apoptosis was analyzed by gel ladder, Annexin V-PI staining and ELISA whereas p53 and target genes were evaluated by western blots. Results: Although Cr(VI) induced DNA-strands breaks in both H358 cell clones, apoptosis was present only in the p53 transfected (H358p53+/+). In these cells Cr(VI)-induced apoptosis is mediated by p53 up-regulation of PUMA, Bax translocation to mitochondria, cytochrome c release and caspase-3 activation. In primary human bronchial epithelial cells, expressing functional p53, Cr(VI) induced expression of PUMA and Noxa, which promote apoptosis through BAX. Conclusions: This result establishes p53 as the "necessary" player in Cr(VI)-induced apoptosis. To the best of our knowledge this is the first report indicating strict correlation of Cr(VI)-apoptosis to PUMA induction on primary human bronchoalveolar cells in short term cultures.
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