title: Pleiotropic effects of the trichloroethylene-associated P81S VHL mutation on metabolism, apoptosis, and ATM-mediated DNA damage response.
abstract: BACKGROUND
  The risk relevance of the P81S von Hippel-Lindau (VHL) gene hotspot mutation identified in clear cell renal cell carcinoma from individuals exposed occupationally to trichloroethylene (TCE) is not known. VHL mutations in hereditary VHL syndrome strongly correlate with phenotypic associations, but specific sporadic mutations in VHL that uniquely alter its protein function may provide a selective growth advantage for somatic cells harboring these mutations.
  
  
  METHODS
  VHL deficient (Vhl (-/-) ) mouse embryonic stem cells were generated that stably express wild-type, P81S, or R167Q human VHL protein. Under hypoxic conditions, cell lines were examined for hypoxia-inducible transcription factor family (HIF) stabilization and E3-ubiquitin ligase complex interactions. In vivo, teratomas were examined for tumor size, proliferation, apoptosis, and immunohistochemistry and subjected to gene expression analysis. Wild-type, R167Q, and P81S VHL-expressing teratomas were also exposed to 5 Gy ionizing radiation to quantify apoptotic response. Proliferation and apoptosis and teratoma growth were analyzed by either Student t test or analysis of variance with Bonferroni correction. All statistical tests were two-sided.
  
  
  RESULTS
  The P81S VHL mutation produces deregulation of HIF factors in cell culture but exhibits a growth advantage in the tumor microenvironment, in part because of suppression of apoptosis (P81S mean = 0.9%, 95% confidence interval = 0.6 to 1.2%; WT mean = 7.6%; 95% confidence interval = 6.4 to 8.8%; P < .001) coupled with sustained proliferation. Transcriptional analysis of P81S teratomas revealed the induction of metabolic pathways, antiapoptotic genes, and global suppression of key DNA damage response genes not observed in VHL wild-type or R167Q mutants. In vivo irradiation exposure showed that P81S mutant is resistant to ionizing radiation-induced apoptosis.
  
  
  CONCLUSIONS
  The TCE-associated P81S VHL mutation can initiate a unique adaptive response required for selective tumor growth through pleiotropic effects on metabolic diversification, apoptosis suppression, and alteration of the DNA damage response.
authors.affiliation: Affiliations of authors: Department of Genetics, North Carolina State University, Raleigh, NC (MCD, DWT); Department of Genetics (WKR) and Lineberger Cancer Center, University of North Carolina, Chapel Hill, NC (WKR, DWT).
authors.forename: Michelle C
authors.initials: MC
authors.lastname: Desimone
authors.forename: W Kimryn
authors.initials: WK
authors.lastname: Rathmell
authors.forename: David W
authors.initials: DW
authors.lastname: Threadgill
mesh_terms: D000818:Animals; D017209:Apoptosis; D001120:Arginine; D064007:Ataxia Telangiectasia Mutated Proteins; D002292:Carcinoma, Renal Cell / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000378:metabolism*; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000378:metabolism*; D053595:Embryonic Stem Cells; D005973:Glutamine; D006801:Humans; D051793:Hypoxia-Inducible Factor 1 / Q000378:metabolism*; D007150:Immunohistochemistry; D007680:Kidney Neoplasms / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D016273:Occupational Exposure; D017354:Point Mutation; D011392:Proline; D040081:Protein Array Analysis; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D012694:Serine; D013724:Teratoma / Q000235:genetics*; D014241:Trichloroethylene / Q000633:toxicity*; D025521:Tumor Suppressor Proteins / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D051794:Von Hippel-Lindau Tumor Suppressor Protein / Q000235:genetics*
chemical_list: D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D051793:Hypoxia-Inducible Factor 1; D025521:Tumor Suppressor Proteins; D005973:Glutamine; D014241:Trichloroethylene; D012694:Serine; D001120:Arginine; D011392:Proline; D044767:Ubiquitin-Protein Ligases; D051794:Von Hippel-Lindau Tumor Suppressor Protein; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C576325:Atm protein, mouse; D017346:Protein Serine-Threonine Kinases; C497446:VHL protein, human