Activation of AMPK is required for phosphorylation of p38-MAPK in AICAR-treated ALL cells

<p><b>Copyright information:</b></p><p>Taken from "Cytotoxic effect of 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR) on childhood acute lymphoblastic leukemia (ALL) cells: implication for targeted therapy"</p><p>http://www.molecular-cancer.com/content/6/1/46</p><p>Molecular Cancer 2007;6():46-46.</p><p>Published online 10 Jul 2007</p><p>PMCID:PMC1948012.</p><p></p> () Immunoblot of p38-MAPK phosphorylation (P-p38-MAPK, Thr180/Tyr182) and β-actin (loading control) expressed in CCRF-CEM and NALM6 cells treated with 0.1% DMSO (Control), 0.5 mM AICAR alone, 0.1 mM iodotubericidin alone (Iodo), or both agents together (Iodo + AICAR). () Phosphorylation status of AMPK (P-AMPK, Thr172) from CCRF-CEM and NALM6 cells incubated with either 0.5 mM AICAR alone, 10 μM SB 202190 alone (SB) or both inhibitors together (SB + AICAR). Level of β-actin was used as loading controls. Density value of each band was normalized to their respective β-actin level and expressed relative to control. The immunoblots shown are representative of 3 independent experiments, which produced similar results

AICAR induces upregulation of the p53 and the cyclin dependent kinase inhibitor p27 in ALL cells

<p><b>Copyright information:</b></p><p>Taken from "Cytotoxic effect of 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR) on childhood acute lymphoblastic leukemia (ALL) cells: implication for targeted therapy"</p><p>http://www.molecular-cancer.com/content/6/1/46</p><p>Molecular Cancer 2007;6():46-46.</p><p>Published online 10 Jul 2007</p><p>PMCID:PMC1948012.</p><p></p> Western blot analyses of p53, p27, and p21 were done using cell extracts from CCRF-CEM, NALM6, REH, and SupB15 cells treated with the indicated concentrations of AICAR (0 – 2 mM) for 48 h. Equivalent amount of proteins (50 μg) were separated by SDS-PAGE and immunodetected with antibodies against p53, p27, and p21. Membranes were stripped and reprobed with anti-β-actin antibody to confirm equal amount of proteins loaded in all lanes. Density value of each band was normalized to their respective β-actin level and expressed relative to control (untreated). The data shown are representative of 3 experiments producing similar results

AICAR treatment inhibits proliferation of human childhood leukemia ALL cells

<p><b>Copyright information:</b></p><p>Taken from "Cytotoxic effect of 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR) on childhood acute lymphoblastic leukemia (ALL) cells: implication for targeted therapy"</p><p>http://www.molecular-cancer.com/content/6/1/46</p><p>Molecular Cancer 2007;6():46-46.</p><p>Published online 10 Jul 2007</p><p>PMCID:PMC1948012.</p><p></p> The ALL cell lines CCRF-CEM (T-lineage), NALM6 (Bp-lineage), REH (Bp-ALL expressing the TEL/AML1 fusion protein), and SupB15 (Bp-ALL expressing the BCR/ABL fusion protein) were treated for 24 h with various concentrations of AICAR (0.25 – 2 mM), and cell growth analyzed by thymidine ribotide ([H]TdR) incorporation into DNA. The results are expressed as percentage of [H]thymidine uptake (%) relative to control values (mean ± SEM, n = 3). #, < 0.001 for AICAR-treated cells control

Anti-proliferative action of AICAR on ALL cells is associated with downstream AMPK-dependent activation of p38-MAPK

<p><b>Copyright information:</b></p><p>Taken from "Cytotoxic effect of 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR) on childhood acute lymphoblastic leukemia (ALL) cells: implication for targeted therapy"</p><p>http://www.molecular-cancer.com/content/6/1/46</p><p>Molecular Cancer 2007;6():46-46.</p><p>Published online 10 Jul 2007</p><p>PMCID:PMC1948012.</p><p></p> () CCRF-CEM, NALM6, REH, and SupB15 ALL cells treated with 0.25 mM AICAR for the indicated times (0 – 24 h) were analyzed by Western blot for phosphorylated p38-MAPK protein (P-p38-MAPK, Thr180/Tyr182). β-actin was used as a loading control. Density value of each band was normalized to their respective β-actin level and expressed relative to control (untreated). () Cell proliferation assays of CCRF-CEM, NALM6, REH, and SupB15 cells treated with 0.25 mM AICAR alone, 10 μM of the p38-MAPK inhibitor SB 202190 alone (SB), or both agents together (SB + AICAR). The cell proliferation values are expressed as a percentage relative to those obtained with untreated control cells (mean ± SEM, n = 3). Data are representative of at least three independent experiments. *, < 0.01 for AICAR SB + AICAR; #, < 0.05 for AICAR SB + AICAR

AICAR-activation of AMPK mediates apoptosis in ALL cells via the mitochondrial pathway

<p><b>Copyright information:</b></p><p>Taken from "Cytotoxic effect of 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR) on childhood acute lymphoblastic leukemia (ALL) cells: implication for targeted therapy"</p><p>http://www.molecular-cancer.com/content/6/1/46</p><p>Molecular Cancer 2007;6():46-46.</p><p>Published online 10 Jul 2007</p><p>PMCID:PMC1948012.</p><p></p> () DNA fragmentation assay. CCRF-CEM, NALM6, REH, and SupB15 cells were treated for 48 h with increased concentrations of AICAR (0 – 2 mM), and nuclear DNA was analyzed by electrophoresis on a 1.6% Tris-Borate-EDTA agarose gel. () Western blot analysis of cytochrome C and caspase 9 expression in CCRF-CEM and NALM6 cells treated for 48 h with 0.5 mM AICAR alone, 0.1 μM iodotubericidin alone (Iodo), or both agents together (Iodo + AICAR). Equal amount of loaded protein (50 μg) was confirmed by immunoblotting with anti-β-actin antibody. The data shown are representative of 3 experiments

The anti-proliferative effect of AICAR on ALL cells is mediated via activation of AMPK

<p><b>Copyright information:</b></p><p>Taken from "Cytotoxic effect of 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR) on childhood acute lymphoblastic leukemia (ALL) cells: implication for targeted therapy"</p><p>http://www.molecular-cancer.com/content/6/1/46</p><p>Molecular Cancer 2007;6():46-46.</p><p>Published online 10 Jul 2007</p><p>PMCID:PMC1948012.</p><p></p> () Western blot analysis of phosphorylated AMPK (P-AMPK, Thr172) expression in CCRF-CEM, NALM6, REH, and SupB15 cells treated with various concentrations of AICAR (0 – 2.0 mM). Total protein was extracted from AICAR-treated cells and AMPK and P-AMPK were immunodetected using specific antibodies. Equal amounts of protein (50 μg) were loaded per lane as confirmed by β-actin level. Density value of P-AMPK bands were normalized to level of AMPK and expressed relative to control. () Cell proliferation assays of ALL cells treated for 18 h with AICAR alone (0.25 mM for CCRF-CEM, NALM6, REH, and 1.0 mM for SupB15), the adenosine kinase inhibitor iodotubericidin alone (Iodo, 0.1 μM), or both agents together (Iodo + AICAR). Growth inhibition was determined using the tetrazolium (MTS) reduction assay. Values are expressed as a percentage relative to those obtained with untreated control cells (mean ± SEM). Data are representative of at least three independent experiments. #, < 0.001 for AICAR Iodo + AICAR