Assessment of TGF-beta1-mediated growth inhibition of HPV-16- and HPV-18-transfected foreskin keratinocytes during and following immortalization.
The responsiveness to transforming growth factor-beta1 (TGF-beta1) of two human keratinocyte cell lineages (FK16A and FK18B) generated after transfection with HPV-16 and HPV-18, respectively, was investigated. Both cell lineages revealed loss of heterozygosity (LOH) at 18q and/or 3p associated with the acquisition of the immortal phenotype. These loci harbour genes (TGF-beta receptor II gene at 3p, and Smad2 and Smad4 genes at 18q) encoding products involved in the TGF-beta1 signalling pathway. Mortal and early immortal stages of both cell lineages displayed growth reduction upon exposure to TGF-beta1 concentrations in the range 100 pg/ml to 1 ng/ml. However, the late immortal stages were resistant to TGF-beta1 at concentrations up to 10 ng/ml. TGF-beta1 receptors type I and II were expressed at all stages in both cell lineages. Moreover, mRNA levels of Smad2 and Smad4 genes were nearly constant throughout. TGF-beta1 expression and secretion, which were demonstrated in all analysed stages, may provide selective conditions underlying unresponsiveness to TGF-beta1 upon prolonged monolayer culturing. Thus, LOH at 3p and/or 18q seen during HPV-mediated immortalization of human keratinocytes was not associated with resistance to TGF-beta1-mediated growth inhibition or a marked reduction in TGF- beta1 receptors and mRNA levels of Smad2 or Smad4. Therefore, alternative events are likely to underlie unresponsiveness to TGF- beta1 in late-passage FK16A and FK18B cells.
Department of Pathology, Section of Molecular Pathology, Vrije Universiteit Medical Center, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands. ingo.nindl@charite.de
Ingo
I
Nindl
Renske D M
RD
Steenbergen
Josef O
JO
Schurek
Chris J L M
CJ
Meijer
Paul
P
van der Valk
Peter J F
PJ
Snijders
D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002461:Cell Line, Transformed; D002472:Cell Transformation, Viral; D004268:DNA-Binding Proteins / Q000235:genetics; D004351:Drug Resistance; D006801:Humans; D015603:Keratinocytes / Q000166:cytology* / Q000187:drug effects*; D019656:Loss of Heterozygosity; D008297:Male; D027383:Papillomaviridae / Q000235:genetics*; D010413:Penis; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D012867:Skin; D051899:Smad2 Protein; D051901:Smad4 Protein; D015534:Trans-Activators / Q000235:genetics; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D053773:Transforming Growth Factor beta1
D004268:DNA-Binding Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; C497782:SMAD2 protein, human; C497796:SMAD4 protein, human; D051899:Smad2 Protein; D051901:Smad4 Protein; C508960:TGFB1 protein, human; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1