Chaperones/heat shock proteins (HSPs) of the HSP90 and HSP70 families show elevated levels in proliferating mammalian cells and a cell cycle-dependent expression. They transiently associate with key molecules of the cell cycle control system such as Cdk4, Wee-1, pRb, p53, p27/Kip1 and are involved in the nuclear localization of regulatory proteins. They also associate with viral oncoproteins such as SV40 super T, large T and small t antigen, polyoma large and middle S antigen and EpsteinBarr virus nuclear antigen. This association is based on a J-domain in the viral proteins and may assist their targeting to the pRb/E2F complex. Small HSPs and their state of phosphorylation and oligomerization also seem to be involved in proliferation and differentiation. Chaperones/HSPs thus play important roles within cell cycle processes. Their exact functioning, however, is still a matter of discussion. HSP90 in particular, but also HSP70 and other chaperones associate with proteins of the mitogen-activated signal cascade, particularly with the Src kinase, with tyrosine receptor kinases, with Raf and the MAP-kinase activating kinase (MEK). This apparently serves the folding and translocation of these proteins, but possibly also the formation of large immobilized complexes of signal transducing molecules (scaffolding function).

The adenomatous polyposis coli (APC) tumor suppressor is a nucleocytoplasmic protein. The nuclear accumulation of APC was recently found to vary depending on cell density, suggesting that putative APC function(s) in the nucleus is controlled by the establishment of cell contacts. We report here that the density-dependent redistribution of APC between nucleus and cytoplasm prevails in 6/6 thyroid and colorectal carcinoma cell lines. Moreover, mutated APC lacking known nuclear localization sequences had the similar distribution pattern as the fulllength protein. APC invariably accumulated in the nuclei of Ki-67 expressing cells, but was largely cytoplasmic when cell cycle exit was induced by serum starvation or at high cell density. APC colocalized with b-catenin in the nucleus only in one cell line (SW480). Also, APC maintained a predominantly nuclear position in early confluent states when cytoplasmic b-catenin was recruited to newly formed adherens-like junctions. The results indicate that nuclear targeting of APC is driven by cell cycle entry rather than altered cell-cell contact. The ability of C-terminally truncated APC to accumulate in the nucleus suggests that nuclear import signals other than NLS1 APC and NLS2 APC are functionally important. Residual function(s) of N-terminal APC fragments in tumor cells carrying APC mutations might be beneficial to tumor growth and survival.

beta-Catenin is a structural component of the adherens junctions. Outside the adherens junctions a complex consisting of glycogen synthase kinase 3beta, the tumor suppressor adenomatous polyposis coli, and axin constantly targets beta-Catenin for degradation to keep levels of free beta-Catenin low. Free beta-Catenin is able to bind to transcription factors of the T cell factor/lymphoid-enhancing factor family and to stimulate transcription of target genes. This signaling function of beta-Catenin is activated by extracellular Wnt factors that bind to Frizzled receptors and induce inhibition of beta-Catenin degradation. By RT-PCR and subcloning, we observed the expression of five Wnt factors, three members of the Frizzled receptor family, and all known Disheveled isoforms in thyroid cells. Immunoprecipitation studies demonstrated the formation of the complex targeting beta-Catenin for degradation. Introduction of a degradation resistant beta-Catenin into the thyroid carcinoma cell line WRO induced appearance of monomeric beta-Catenin as shown by size fractionation and nuclear beta-Catenin immunostaining. Reporter gene assays demonstrated a stimulation of T cell factor/lymphoid-enhancing factor-mediated transcription in these cells. In ARO cells, a thyroid carcinoma cell line carrying a mutated adenomatous polyposis coli gene, monomeric beta-Catenin and nuclear immunostaining were observed. In summary, our data indicate that elements of the Wnt signaling pathway are expressed in thyroid cells and that this pathway is functionally active.

Analysis of constitutive heat shock protein 70 (HSC70) concentration in unstressed proliferating and differentiated rat C6 glioma cells revealed a striking reduction in the amount of HSC70 in differentiated cells. Proliferating cells showed a significantly higher HSC70 concentration, particularly observable during S phase in synchronous cultures. The activity of the cAMP/PKA signaling pathway was enhanced in differentiated cells. cAMP-elevating treatments both inhibited growth and reduced HSC70 concentration. Inactivation of PKA by H-89 upregulated the reduced HSC70 expression in differentiated cells and stimulated proliferation. Treatment with an inhibitor of MAP kinase activation (PD98059) reduced the HSC70 concentration. We assume that cAMP does not directly inhibit HSC70 expression by transcriptional repression, but by its inhibitory effect on the MAP kinase pathway.