Furthermore, coincubation with L365,260, a gastrin receptor (CCK2) antagonist, attenuated the upregulation of namely ��-catenin by greater than 50%, suggesting that the increase in ��-catenin was specific (Figure 1C). An induction of ��-catenin was consistently detected, and the results were reproduced on four separate occasions. Although the magnitude of the change in total ��-catenin protein varied within individual experiments, a 3�C4-fold increase in total ��-catenin protein levels was detected when bands were quantified by densitometry and normalised to ��-actin levels (Figure 1D). Figure 1 (A) Gastrin does not affect ��-catenin mRNA levels, as demonstrated by Northern blot analysis. MC-26 cells were incubated for 4h in the presence of increasing concentrations of G-17 (10�C100nM; upper panel).

28S and 18S … As mentioned above, nuclear accumulation of ��-catenin represents a key event in CRC progression. To examine whether G-17 can enhance nuclear ��-catenin in MC-26 cells, cells were treated with G-17 for 4h and nuclear extracts were prepared. In all, 20 and 50nM G-17 induced approximately a two-fold increase in nuclear ��-catenin levels (Figure 1E), suggesting that G-17 promotes nuclear translocation of ��-catenin. Expression of Sp1, a ubiquitously expressed transcription factor, was used as a loading control for nuclear extracts. Gastrin-17 increases LEF-1-dependent transcriptional activity To examine whether the increase in nuclear ��-catenin protein is also associated with the activation of LEF-1, LEF-1-dependent reporter assays were performed.

The pGL3-LEF-1 luciferase construct (kindly provided by Dr R Grosschedl, Munich, Germany) contains eight repeats of the LEF binding site that is activated only in the presence of an exogenous LEF-1 construct. The level of LEF-1-dependent transcription is also dependent on nuclear ��-catenin levels, as ��-catenin is a known coactivator for TCF/LEF transcription factors. As we speculated that an increase in ��-catenin protein by G-17 might be functionally important for the transcriptional activation of its target genes, LEF-1-dependent reporter assays were performed in the absence and presence of G-17. We observed that 20 and 50nM G-17 induced a concentration-dependent increase in LEF-1-dependent transcriptional activity (P0.005) (Figure 2).

In addition, the effects of gastrin on cyclin D1, one of the target genes of ��-catenin-dependent transcription, were analysed. In response to the inclusion of G-17 in the culture medium, both cyclin D1 protein levels and promoter activity were increased (Figure 3). Specifically, 50nM G-17 significantly enhanced the activity of the Dacomitinib full-length cyclin D1 promoter (?1745) when compared to either the empty or minimal promoter (?66) (Figure 3B, P0.01). Figure 2 Gastrin-17 enhances LEF-1-dependent transcriptional activity.