Primary keratinocytes have an adhesion dependent S phase checkpoint that is absent in immortalized cell lines

Abstract

In order to understand the mechanism through which loss of anchorage inhibits growth, we have investigated the events that occur in murine keratinocytes upon substratum detachment utilizing both primary cells and established immortalized cell lines. Our data has revealed that while both primary and immortalized cells undergo growth arrest in suspension, the nature of this arrest is markedly di erent. Primary cells exhibit a growth arrest that is characterized by rapid cessation of DNA synthesis resulting in a static S phase population. In contrast, an immortalized non-tumorgenic cell line, Balb MK, exhibits growth arrest as measured by thymidine incorporation, but does not prevent cells that have entered S phase from continuing into G 2 /M, and accumulating as a 4N population. In contrast to both primary and MK cells, the tumorigenic SLC-1 cell line did not accumulate in a speci®c cell cycle interval and were able to undergo continuous growth in suspension. Examination of cyclin A protein and its associated activity revealed that cyclin A protein levels decreased in primary but not MK cells; suggesting the continued presence of cyclin A may allow continued DNA synthesis observed in MK cells. Furthermore, we demonstrate the accumulation of suspension cultured MK cells as a 4N population correlated with the loss of cyclin A/cdk2 kinase activity, which in turn occurred through the accumulation of p27 kip1 , whereas neither p27 kip1 accumulation nor loss of cyclin A activity was observed in SLC-1 cells. Our results clearly reveal that the process of growth inhibition in suspension cultured cells may occur in several forms with distinct characteristics that are dependent on the status of cyclin/cdk complexes and CKI proteins. Tumor derived cells in suspension did not lose cyclin A dependent kinase activity and thus continued to grow and divide.

Activation of Stat3 by cell confluence reveals negative regulation of Stat3 by cdk2

“…Indeed, recent reports that Stat3 localizes to rafts (Sehgal et al, 2002) and that stat3 undergoes endocytosis during signaling (Bild et al, 2002) indicate that Stat3 may share topographic proximity with cdk2 and cdk2 inhibitors at the cell membrane. Our finding of Stat3 upregulation by cdk2 inhibitors is consistent with the report that antisense p27Kip1 decreased Stat3 activity in keratinocytes (Hauser et al, 1998). UM-22B and PCI-15B cells were grown for up to 10 days as indicated.…”

Epidermal Growth Factor Receptor-dependent Control of Keratinocyte Survival and Bcl-xL Expression through a MEK-dependent Pathway

“…Inhibition of the EGFR tyrosine kinase moiety by use of AG1478 abrogated the effect of EGF on STAT3 and MAPK phosphorylation in A431 cells but did not affect base-line STAT3 phosphorylation in these cells. Previously, STAT3 phosphorylation was observed in primary mouse keratinocytes after placing them into forced suspension culture (27). Consistent with these earlier findings we also observed STAT3 phosphorylation in primary keratinocytes placed in suspension culture (not shown).…”

Human Papillomavirus Type 31 E5 Protein Supports Cell Cycle Progression and Activates Late Viral Functions upon Epithelial Differentiation

“…There was no change in the level of p57 expression following suspension culture in either HFKs or HPV-containing cells. These experiments confirm the induction of CKI during suspension-induced cell cycle exit in keratinocytes (2,24,42,48) but demonstrate that E5 does not influence this process. These experiments were repeated five times with similar results.…”