title: Functional analysis of the p53 pathway in response to ionizing radiation in uveal melanoma.
abstract: OBJECTIVE
  Uveal melanomas are notoriously radioresistant and thus necessitate treatment with extremely high radiation doses that often cause ocular complications. The p53 tumor suppressor pathway is a major mediator of the cellular response to radiation-induced DNA damage, suggesting that this pathway may be defective in uveal melanoma. The current study was conducted to analyze the functional integrity of the p53 pathway in primary uveal melanoma cells.
  
  
  METHODS
  The p53 gene was sequenced in three primary uveal melanoma cells lines. Cultured primary uveal melanoma cells (MM28, MM50, Mel202, Mel270, and Mel290), MCF7 breast carcinoma cells, normal uveal melanocytes (UM47), and normal human diploid fibroblasts (NHDFs) were irradiated at 250 kVp and 12 mA at a dose rate of 1.08 Gy/min for a total dose of up to 20 Gy. Cell viability was analyzed with trypan blue exclusion. Western blot analysis was used to analyze the expression of p53, p53-phospho-Ser15, p21, Bax, PUMA, and Bcl-x(L).
  
  
  RESULTS
  No p53 gene mutations were found in MM28, MM50, or Mel270 cells. Upstream signaling to p53 was intact, with normal induction of p53 and phosphorylation of p53-Ser15, in all five cell lines. Radiation-induced downstream activation of p21 was defective in MM28 and MM50 cells, and activation of Bax was defective in MM50 and Mel290 cells. MM28, MM50, and Mel202 cells failed to deamidate Bcl-x(L) in response to radiation-induced DNA damage. Overall, four of the five uveal melanoma cell lines exhibited at least one downstream defect in the p53 pathway.
  
  
  CONCLUSIONS
  Expression of p53 and upstream signaling to p53 in response to radiation-induced DNA damage appear to be intact in most uveal melanomas. In contrast, functional defects in the p53 pathway downstream of p53 activation appear to be common. Further elucidation of p53 pathway abnormalities in uveal melanoma may allow therapeutic interventions to increase the radiosensitivity of the tumors.
authors.affiliation: Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, MO, USA.
authors.forename: Young
authors.initials: Y
authors.lastname: Sun
authors.forename: Binh N
authors.initials: BN
authors.lastname: Tran
authors.forename: Lori A
authors.initials: LA
authors.lastname: Worley
authors.forename: Rachel B
authors.initials: RB
authors.lastname: Delston
authors.forename: J William
authors.initials: JW
authors.lastname: Harbour
mesh_terms: D051017:Apoptosis Regulatory Proteins; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000378:metabolism / Q000532:radiotherapy; D018797:Cell Cycle Proteins / Q000378:metabolism; D002470:Cell Survival; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004252:DNA Mutational Analysis; D005347:Fibroblasts / Q000378:metabolism / Q000528:radiation effects; D016158:Genes, p53 / Q000235:genetics; D006801:Humans; D008545:Melanoma / Q000378:metabolism / Q000532:radiotherapy*; D009154:Mutation; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D011839:Radiation, Ionizing; D015398:Signal Transduction / Q000502:physiology*; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism / Q000502:physiology*; D014604:Uveal Neoplasms / Q000378:metabolism / Q000532:radiotherapy*; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein
chemical_list: D051017:Apoptosis Regulatory Proteins; C494826:BAX protein, human; C438378:BBC3 protein, human; C494811:BCL2L1 protein, human; C493956:CDKN1A protein, human; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein