title: Partial loss of Smad signaling during in vitro progression of HPV16-immortalized human keratinocytes.
abstract: BACKGROUND
  Disruption of the transforming growth factor-beta (TGF-β) signaling pathway is observed in many cancers, including cervical cancer, resulting in TGF-β resistance. While normal human keratinocytes (HKc) and human papillomavirus type 16-immortalized HKc (HKc/HPV16) are sensitive to the growth inhibitory effects of TGF-β, HKc/HPV16 develop resistance to TGF-β1 as they progress in vitro to a differentiation resistant phenotype (HKc/DR). The loss of sensitivity to the antiproliferative effects of TGF-β1 in HKc/DR is due, at least partially, to decreased expression of the TGF-β receptor type I. In the present study, we explored in detail whether alterations in Smad protein levels, Smad phosphorylation, or nuclear localization of Smads in response to TGF-β could contribute to the development of TGF-β resistance during in vitro progression of HKc/HPV16, and whether TGF-β induction of a Smad-responsive reporter gene was altered in HKc/DR.
  
  
  METHODS
  Western blot analysis was used to assess Smad protein levels. In order to study Smad nuclear localization we performed indirect immunofluorescence. In addition, we determined Smad-mediated TGF-β signaling using a luciferase reporter construct.
  
  
  RESULTS
  We did not find a decrease in protein levels of Smad2, Smad3 or Smad4, or an increase in the inhibitory Smad7 that paralleled the loss of sensitivity to the growth inhibitory effects of TGF-β1 observed in HKc/DR. However, we found diminished Smad2 phosphorylation, and delayed nuclear Smad3 localization in response to TGF-β1 in HKc/DR, compared to normal HKc and TGF-β sensitive HKc/HPV16. In addition, we determined that TGF-β1 induction of a Smad responsive promoter is reduced by about 50% in HKc/DR, compared to HKc/HPV16.
  
  
  CONCLUSIONS
  These results demonstrate that alterations in Smad protein levels are not associated with the loss of response to the antiproliferative effects of TGF-β in HKc/DR, but that diminished and delayed Smad phosphorylation and nuclear localization, and decreased Smad signaling occur in response to TGF-β in HKc/DR.
authors.affiliation: Department of Drug Discovery and Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolina, Columbia, SC, USA. creekk@sccp.sc.edu.
authors.forename: Diego
authors.initials: D
authors.lastname: Altomare
authors.forename: Rupa
authors.initials: R
authors.lastname: Velidandla
authors.forename: Lucia
authors.initials: L
authors.lastname: Pirisi
authors.forename: Kim E
authors.initials: KE
authors.lastname: Creek
mesh_terms: D002461:Cell Line, Transformed; D002467:Cell Nucleus / Q000378:metabolism; D052162:Human papillomavirus 16 / Q000502:physiology*; D006801:Humans; D015603:Keratinocytes / Q000187:drug effects / Q000378:metabolism* / Q000821:virology*; D010766:Phosphorylation / Q000187:drug effects; D021381:Protein Transport; D015398:Signal Transduction; D051785:Smad Proteins / Q000378:metabolism*; D051899:Smad2 Protein / Q000378:metabolism; D051900:Smad3 Protein / Q000378:metabolism; D051901:Smad4 Protein / Q000378:metabolism; D051906:Smad7 Protein / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology
chemical_list: C497782:SMAD2 protein, human; C497790:SMAD3 protein, human; C497796:SMAD4 protein, human; C497806:SMAD7 protein, human; D051785:Smad Proteins; D051899:Smad2 Protein; D051900:Smad3 Protein; D051901:Smad4 Protein; D051906:Smad7 Protein; D053773:Transforming Growth Factor beta1
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