Loss of transforming growth factor-beta (TGF-beta) receptor type I mediates TGF-beta resistance in human papillomavirus type 16-transformed human keratinocytes at late stages of in vitro progression.
Human keratinocytes (HKc) immortalized by human papillomavirus type 16 DNA (HKc/HPV16) progress toward malignancy through growth factor-independent (HKc/GFI) and differentiation-resistant stages (HKc/DR). This progression is associated with a loss of sensitivity to growth inhibition by both all-trans-retinoic acid (RA) and transforming growth factor-beta (TGF-beta). In the accompanying article (Borger et al., 2000, Virology 270, 397-407), we demonstrate that RA resistance in HKc/HPV16 arises despite functional nuclear retinoid receptors and that TGF-beta mediates growth inhibition by RA. To investigate the basis for the loss of TGF-beta sensitivity during in vitro progression of HKc/HPV16, we explored the expression of TGF-beta receptors type I and type II in independently derived HKc/HPV16 lines and their corresponding HKc/GFI and HKc/DR derivatives. While TGF-beta receptor type II mRNA levels were unchanged during progression, mRNA levels for TGF-beta receptor type I decreased dramatically as the cells became TGF-beta resistant. At the HKc/DR stage, loss of TGF-beta receptor type I mRNA, compared to low-passage cells, ranged from 55 to 87% in four HKc/HPV16 lines examined. Immunohistochemistry, using anti-TGF-beta receptor type I antibodies, confirmed a loss of TGF-beta receptor type I expression in HKc/DR. Reintroduction of the TGF-beta-receptor type I into TGF-beta-resistant HKc/DR completely restored growth inhibition by TGF-beta. Southern blot analysis of DNA extracted from normal HKc, HKc/HPV16, and HKc/DR ruled out any gross changes in the TGF-beta receptor type I gene. The activity of the TGF-beta receptor type I promoter, cloned upstream of a luciferase reporter gene, was decreased in HKc/DR, to an extent comparable to the decrease in mRNA levels for the TGF-beta receptor type I. Thus, TGF-beta resistance at late stages of HPV16-mediated transformation of HKc is the result of a loss of expression of TGF-beta receptor type I.
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Children's Cancer Research Laboratory, University of South Carolina, Columbia, SC 29208, USA.
Borger
D R
DR
Fernandes
P R
PR
Pirisi
L
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Creek
K E
KE
D002472:Cell Transformation, Viral; D002478:Cells, Cultured; D015536:Down-Regulation; D004351:Drug Resistance; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism / Q000473:pathology / Q000821:virology*; D027383:Papillomaviridae; D012333:RNA, Messenger / Q000378:metabolism; D018125:Receptors, Transforming Growth Factor beta / Q000378:metabolism*; D013997:Time Factors; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
D012333:RNA, Messenger; D018125:Receptors, Transforming Growth Factor beta; D016212:Transforming Growth Factor beta