Zdena Tuháčková

Rapamycin-resistant phosphorylation of the initiation factor-4E-binding protein (4E-BP1) in v-SRC-transformed hamster fibroblasts.

Increased phosphorylation of the translational repressor protein 4E‐BP1 was found in the cell line derived from the tumor induced in Syrian hamster by Rous sarcoma virus (RSV). This was accompanied by its dissociation from the complex with initiation factor eIF4E. The ribosomal S6 protein kinase p70S6k is supposed to be regulated by the same or a closely related rapamycin‐sensitive signalling pathway to that which modulates 4E‐BP1. Phosphorylation and activity of p70S6k were found to be also increased in RSV‐transformed H19 cells that express significantly higher amounts of the Src protein (p60src) relative to the non‐transformed hamster fibroblasts NIL‐2. The increased activity and phosphorylation of p70S6k were blocked by rapamycin, indicating that the rapamycin‐sensitive pathway is involved in its regulation in v‐src‐transformed hamster fibroblasts. In agreement with this, rapamycin reduced the expression of elongation factor eEF1α (whose translation is regulated by a rapamycin‐sensitive mechanism thought to involve p70S6k) and did not affect the production of a housekeeping protein, α‐tubulin, in these cells. Synthesis of Src protein was also inhibited in cells treated with rapamycin. However, treatment of cells with a concentration of rapamycin sufficient to completely inhibit the activity and phosphorylation of p70S6k resulted in only partial de‐phosphorylation of 4E‐BP1 and its re‐association with eIF4E in the transformed cells, indicating that additional rapamycin‐insensitive mechanisms/pathways are implicated in the control of 4E‐BP1 phosphorylation in RSV‐transformed hamster fibroblasts. Over‐expression of eIF4E favours cell proliferation and can lead to a transformed phenotype, while over‐expression of 4E‐BP1 has the opposite effect. The altered signalling to the phosphorylation of 4E‐BP1 in RSV‐transformed cells, which leads to its dissociation from eIF4E and thus relief of inhibition of eIF4E function, may therefore represent an important regulatory mechanism in malignant cell growth. Int. J. Cancer 81:963–969, 1999.

Changes of E-cadherin and beta-catenin in human and mouse intestinal tumours.

An immunohistochemical analysis of E-cadherin and β-catenin was performed in human colorectal cancer as well as in surrounding normal intestinal tissue. We also analysed the expression of these two cell adhesion proteins in transgenic Apc1638N mice as a model of human familial adenometous polyposis syndrome. In the normal intestinal mucosa of both species, E-cadherin and β-catenin were localized along the lateral plasma membrances of epithelial cells. In intestinal tumour cells, however, they were also present in the cytoplasm. The expression of both proteins was reduced in human and mouse tumours. The pattern of their distribution was frequently heterogenous with strongly positive cells in a mosaic of negative ones. Further, E-cadherin and β-catenin expression did not correlate to the Dukes staging of tumours and therefore neither can be used as prognostic criteria.

Guanosine triphosphate - dependent autocatalytic phosphorylation of peptide initiation factor 2 from rat liver.

If incubated with [γ-32P]guanosine triphosphate, homogenous peptide initiation factor 2 from rat liver liberates the terminal phosphate from GTP and utilizes 25–40% of it for its own phosphorylation. This phosphorylation is apparently autocatalytic since it does not require any additional proteins. Only the γ-subunit of the factor becomes phosphorylated by an acid-labile bond and the reaction is significantly inhibited by initiator Met-tRNAF.

Initiation factor eIF2B not p70 S6 kinase is involved in the activation of the PI-3K signalling pathway induced by the v-src oncogene.

Our data show that in hamster fibroblasts transformed by Rous sarcoma virus (RSV), the phosphoinositide 3′‐kinase (PI‐3K)/Akt/glycogen synthase kinase 3 antiapoptotic pathway is upregulated and involved in increased protein synthesis through activation of initiation factor eIF2B. Upon inhibition of PI‐3K by wortmannin, phosphorylation of 70‐kDa ribosomal protein S6 kinase (p70 S6k) and its physiological substrate, ribosomal protein S6, decreased in the non‐transformed cells but not in RSV‐transformed cells. Thus PI‐3K, which is thought to be involved in regulation of p70 S6k, signals to p70 S6k in normal fibroblasts, but it does not appear to be an upstream effector of p70 S6k in fibroblasts transformed by v‐src oncogene, suggesting that changes in the PI‐3K signalling pathway upstream of p70 S6k are induced by RSV transformation.