Abstract
Objective: A human pancreatic adenocarcinoma cell line (A818-6) — which shows all typical genetic alterations of malignant cells — develops single layer epithelial hollow spheres resembling normal pancreatic ductal structures in vitro. In this highly differentiation state of hollow sphere formation, A818-6 cells are showing growth arrest and a decrease of telomerase activity. This differentiation status of hollow sphere formation is reversible. Material and methods: A818-6 cells were cultivated as monolayer and under three-dimensional growth conditions. To examine how growth inhibition/reduction of telomerase activity was mediated in hollow spheres, we checked expression/phosphorylation of members of MAP-kinase pathway by western-blotting in hollow spheres in comparison to monolayer. The same experiments were carried out with hTERT (catalytic subunit of telomerase)-transduced A818-6 cells and constitutively active TGFβ-receptor I-transduced A818-6 cells. Additionally, relative telomerase activity (TRAP-assay) and expression/activity of members of MAP-kinase pathway (Western blot) were tested in constitutively active TGFβ-receptor I-transduced A818-6 cells. Results: A818-6, A818-6/hTERT and ASIS-6/constitutively active TGFβ-receptor I cells showed under three-dimensional growth conditions a strong decrease of phospho-ERKl and phospho-ERK2-expression. Additionally we observed in A818-6 cells growing under three-dimensional growth conditions a decrease of c-myc and raf expression. A818-6 monolayer cells transduced with constitutively active TGFs-receptor I showed a decrease of ERK1/2 phophorylation and a 4-fold reduction of telomerase activity. Conclusions: The structure of A818-6 hollow spheres dominates the genetic background: Under three-dimensional growth conditions A818-6 cells are able to bypass constitutively active ras on the level of raf, which implicates that alternative entrance possibilities into MAPK-kinase pathway must exist. Because a decrease of telomerase activity is combined with a decrease of ERK1/2 phosphorylation, it is likely that an interaction of MAP-kinases and SMAD proteins exist, which may present a new form of tumor dormancy.
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