Partial loss of Smad signaling during in vitro progression of HPV16-immortalized human keratinocytes

BMC Cancer

Diego Altomare¹  Rupa Velidandla¹  Lucia Pirisi²  Kim E Creek¹ 

[1] Department of Drug Discovery and Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolina, Columbia, SC, USA

[2] Department of Pathology, Microbiology & Immunology, University of South Carolina School of Medicine, Columbia, SC, USA

关键词: Human keratinocytes;    HPV-mediated transformation;    Smads;    TGF-β signaling;   

Others  :  1079566
DOI  :  10.1186/1471-2407-13-424

 received in 2013-03-03, accepted in 2013-09-13,  发布年份 2013

【 摘 要 】

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.

【 授权许可】

   
2013 Altomare et al.; licensee BioMed Central Ltd.

【 图 表 】

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