A study of the prenyl transferase reactions was performed by fluorescence using rat brain cytosol fractions as an enzyme source. Four dansylated peptides corresponding to the C-terminal sequence of Ras isoforms were synthesised. The effects of different detergents on the farnesylation or geranylgeranylation of the four peptides were evaluated. Dose-dependent effects of dodecyl-maltoside, a non-ionic detergent, on the farnesyl transferase or geranylgeranyl transferase activities were observed with all peptide substrates. Additionally, the effect of temperature was investigated and these assays were applied to determine Michaelis-Menten constants (K(m)) of the substrates: dansyl-GCVLS (1.8 microM), dansyl-GCVVM (3.2 microM), dansyl-CVIM (3.4 microM) and dansyl-GCVLL (8.4 microM) and FPP (22.6 microM) for FTase activity. Using GGPP as co-substrate, GGTase activity was measured with K(m) values superior to 50 microM for all the three substrate dansyl-GCVLS, dansyl-GCVVM, or dansyl-CVIM, whereas values of 7.6 and 5.4 microM were calculated for the dansyl-GCVLL sequence and GGPP co-substrate, respectively. IC50 values of selective prenyl transferase inhibitors, B-581, FTI 276 and GGTI 287 have been measured to 34, 0.8 and 18 nM, respectively, using dansyl-GCVLS as substrate (FTase inhibition). When dansyl-GCVLL is used as substrate (GGTase inhibition) the IC50 values are 5100, 75 and 5 nM for B-581, FTI 276 and GGTI 287, respectively. Then, this developed method allowed to evaluate the selectivity of all the three inhibitors tested.

01 Aug 2004·Journal of Pharmacology and Experimental TherapeuticsQ3 · MEDICINE

Acetoacetate Activation of Extracellular Signal-Regulated Kinase 1/2 and p38 Mitogen-Activated Protein Kinase in Primary Cultured Rat Hepatocytes: Role of Oxidative Stress

Q3 · MEDICINE

Article

Author: Raymond F. Novak ; Kimberley J. Woodcroft ; Mohamed A. Abdelmegeed ; Sang K. Kim

Diabetes is characterized by elevated levels of ketone bodies acetoacetate (AA) and 3-hydroxybutyrate (3HB). High levels of ketone bodies have been implicated in generation of cellular oxidative stress. Ketone body activation of cellular signaling pathways associated with oxidative stress, however, has not been established. Thus, ketone body effects on kinase activation in primary cultured rat hepatocytes have been examined. Treatment with AA increased the phosphorylation of extracellular signal-regulated kinase 1/2 (Erk1/2) and p38 mitogen-activated protein kinase (MAPK), maximally by approximately 2.5- and 4-fold, respectively. AA failed to activate c-Jun NH(2)-terminal kinase. AA-mediated Erk1/2 and p38 MAPK activation was detectable at 3 h post-treatment with maximal activation occurring at 12 h. In contrast, 3HB failed to activate any of these kinases. Elevated phosphorylation of Raf and MKK3/6 also occurred in response to AA. Bisindolylmaleimide, a generalized protein kinase C (PKC) inhibitor, and B581, a Ras farnesylation inhibitor, inhibited AA-mediated activation of Erk1/2 and p38 MAPK, suggesting a role for PKC and Ras in mediating such activation. Interestingly, the tyrosine kinase inhibitor genistein prevented the AA-mediated phosphorylation of Erk1/2, but not p38 MAPK. AA treatment resulted in the generation of reactive oxygen species (ROS) and the depletion of cellular glutathione levels, which was ameliorated by the antioxidants N-Acetyl-l-cysteine (NAC) and Trolox (6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid). NAC and Trolox also ameliorated AA-mediated Erk1/2 and p38 MAPK activation, suggesting that this activation is associated with ROS and oxidative stress.

15 Mar 2001·BloodQ1 · MEDICINE

Cell-cycle–dependent activation of mitogen-activated protein kinase kinase (MEK-1/2) in myeloid leukemia cell lines and induction of growth inhibition and apoptosis by inhibitors of RAS signaling

Q1 · MEDICINE

Article

Author: Morgan, Michael A. ; Reuter, Christoph W. M. ; Dolp, Oliver

Disruption of the RAS–to–mitogen-activated protein kinase (MAPK/ERK) signaling pathway, either directly through activatingRAS gene mutations or indirectly through other genetic aberrations, plays an important role in the molecular pathogenesis of myeloid leukemias. Constitutive activation of ERK-1/2 and MEK-1/2, which elicit oncogenic transformation in fibroblasts, has recently been observed in acute myeloid leukemias (AML). In this study, the activation of the RAS-to-MAPK cascade in 14 AML and 5 chronic myeloid leukemia (CML) cell lines is examined and correlated with the effects of a panel of 9 RAS signaling inhibitors on cell viability, colony formation, cell-cycle progression, and induction of apoptosis. Activation of MEK, ERK, and the transcription factors CREB-1, ATF-1, and c-Myc is demonstrated in the majority of the cell lines (9 of 14 AML and 2 of 5 CML cell lines). Although activation of the ERK cascade did not always correlate with the presence of activatingRAS mutations or BCR-Abl, it is linked to the G0/G1 and the G2/M phase of the cell cycle. In contrast to most inhibitors (eg, B581, Cys-4-Abs-Met, FPT-2, FTI-276, and FTS), a significant growth inhibition was only observed for FTI-277 (19 of 19), FPT-3 (10 of 19), and the MEK inhibitors U0126 (19 of 19) and PD098059 (8 of 19). Treatment of NB-4 cells with FTI-277 primarily resulted in a G2/M block, whereas treatment with FPT-3 and U0126 led to induction of apoptosis. FTI-277 revealed strong toxicity toward normal purified CD34+ cells. The results suggest differences in the mechanisms of action and support a potential therapeutic usefulness of these inhibitors in the treatment of myeloid leukemias.

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