DCA does not influence the protein expression levels of any of these MAPKs. Mek1 2 and MKK3 6 are the respective upstream activa tors of Erk1 2 and p38. The PD98059 and SB203580 compounds, known specific inhibitors selleck products of Mek1 2 and MKK3 6 were used to verify activation of Erk1 2 and p38 in response to DCA. SKGT4 cells were incubated with 50 M PD98059 or 2 M of the SB203580 for 30 minutes prior to the addition of 300 M DCA. PD98059 completely abolishes basal, PdBu and DCA induced Erk1 2 activity at all the time points tested. Similarly, the SB203580 compound inhibits the DCA for 6 hr and DNA affinity precipitation assays were performed. Pre treatment of SKGT4 cells with 10 M PD98059 impairs and diminishes DCA induced activa tion of Fra 1 and JunB, respectively.

The SB203580 compound completely abolishes DCA induced Fra 1 DNA binding while having no effect on DCA induced JunB DNA binding. These data indi cate that both Raf Mek1 2 Erk1 2 and MKK3 6 p38 are involved in DCA induced Fra 1 activation, while only Raf Mek1 2 Erk1 2 is upstream of JunB activation. DCA induces a decrease in cell proliferation that is accompanied by low levels of apoptosis Bile acids, in particular DCA, inhibit proliferation and are potent inducers of apoptosis in several cell types includ ing, hepatocytes and colonic cells. Activation of AP 1 can have both anti apoptotic and pro apoptotic functions depending on the cellular context. Since DCA induces sustained activation of AP 1 in SKGT4 cells, its possible contribution to deregulated cell survival and apoptosis was examined.

SKGT4 cells were stimulated with 300 M DCA or 300 M ursodeoxycholic acid for 0 6 hr. We have previ ously shown that UDCA, in contrast to DCA, does not induce AP 1 transcription factor activation in colon cancer cells. In fact, it inhibits interleukin 1 beta and deoxycholic acid induced activation of NF kappaB and AP 1 in these cells. Cell proliferation was assessed using the MTT assay. DCA induces a dose and time dependent decrease in cellular proliferation, which is initially observed within the first hour of treatment, remains at similar levels up to 8 hr and is more pronounced at 12 and 24 hr. This decrease is clear at 300 M DCA and higher concen trations, being statistically significant at 400 500 M. Dramatic morphological changes indicative of apoptosis are also observed at 6 hr of DCA treatment at concentrations in excess of 300 M.

In comparison, cells stimulated with UDCA show identical proliferation patterns and morphology as compared to untreated cells at all times and concentra tions tested. DNA fragmentation and PARP cleavage, two of the hall marks of apoptosis, GSK-3 were respectively assessed by quanti fying cytoplasmic histone associated DNA fragments by ELISA and Western blotting using a specific antibody that recognizes the 85 kDa cleaved PARP fragment.