Molecular analysis of TGF-(beta)s and their receptors in human keratinocyte cell lines of different biological behaviour.
We investigated three clonally related human keratinocyte cell lines of different biological behaviour, HaCaT (non-tumorigenic), A5 (benign, tumorigenic) and II-4RT (malignant, tumorigenic), with regard to the expression of TGF-beta-isoforms -1, -2 and -3 and that of the TGF-beta-cell-receptors TBR-I, -II and -III. In addition, we amplified and sequenced the genome of TBR-II which is known to be a target for mutations in several types of malignant tumors including squamous cell carcinomas. In all three cell lines, TGF-beta1 and -beta3 were present only in very low amounts. Western blots provided no evidence for differences in TGF-beta1 between the cell lines. However, in immunohistochemistry more cells were slightly positive for this cytokine in HaCaT than in A5 and II-4RT cells. In contrast, a significantly variable expression of TGF-beta2 was seen by both Western blot and immunohistochemistry. Thereby, the non-tumorigenic HaCaT-cells contained significantly more TGF-beta2 than the tumorigenic, benign A5 cells and the malignant II-4RT cells. TBR-I, -II and -III were present in all three cell lines. While most cells were positive for TBR-I, only part of the cells contained TBR-II and -III, however, without obvious differences between the three cell lines. The molecular analysis of all 7 exons of TBR-II by PCR amplification and direct sequencing revealed in all three cell lines correct sequences without evidence for mutations. Our study indicates differences in the expression of TGF-beta in a human model of keratinocytes of varying tumorigenicity, but presents no evidence for mutations in the functionally most important TGF-beta-receptor TBR-II. This suggests a dysregulation of cytokine control on the level of TGF-beta expression, which may be responsible for the biological behaviour.
Bachmeier
Beatrice E
BE
Department of Clinical Chemistry and Clinical Biochemistry, Ludwig-Maximilians-University, Munich, Germany. bachmeier@clinbio.med.uni-muenchen.de
Ruoss
Isabell
I
Hagedorn
Hjalmar G
HG
Nerlich
Andreas G
AG
D015153:Blotting, Western; D006801:Humans; D007150:Immunohistochemistry; D015603:Keratinocytes / Q000378:metabolism*; D018125:Receptors, Transforming Growth Factor beta / Q000235:genetics* / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000235:genetics* / Q000378:metabolism
D018125:Receptors, Transforming Growth Factor beta; D016212:Transforming Growth Factor beta