Cytotoxic effect of 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR) on childhood acute lymphoblastic leukemia (ALL) cells: implication for targeted therapy

Acute lymphoblastic leukemia (ALL) is the most common hematological malignancy affecting children. Despite significant progress and success in the treatment of ALL, a significant number of children continue to relapse and for them, outcome remains poor. Therefore, the search for novel therapeutic approaches is warranted. The aim of this study was to investigate the AMP activated protein kinase (AMPK) as a potential target in childhood acute lymphoblastic leukemia (ALL) subtypes characterized by non-random translocation signature profiles. We evaluated the effects of the AMPK activator AICAR on cell growth, cell cycle regulators and apoptosis of various childhood ALL cells. We found that treatment with AICAR inhibited cell proliferation, induced cell cycle arrest in G1-phase, and apoptosis in CCRF-CEM (T-ALL), NALM6 (Bp-ALL), REH (Bp-ALL, TEL/AML1) and SupB15 (Bp-ALL, BCR/ABL) cells. These effects were abolished by treatment with the adenosine kinase inhibitor 5'-iodotubericidin prior to addition of AICAR indicating that AICAR's cytotoxicity is mediated through AMPK activation. Moreover, we determined that growth inhibition exerted by AICAR was associated with activation of p38-MAPK and increased expression of the cell cycle regulators p27 and p53. We also demonstrated that AICAR mediated apoptosis through the mitochondrial pathway as revealed by the release of cytochrome C and cleavage of caspase 9. Additionally, AICAR treatment resulted in phosphorylation of Akt suggesting that activation of the PI3K/Akt pathway may represent a compensatory survival mechanism in response to apoptosis and/or cell cycle arrest. Combined treatment with AICAR and the mTOR inhibitor rapamycin resulted in additive anti-proliferative activity ALL cells. AICAR-mediated AMPK activation was found to be a proficient cytotoxic agent in ALL cells and the mechanism of its anti-proliferative and apoptotic effect appear to be mediated via activation of p38-MAPK pathway, increased expression of cell cycle inhibitory proteins p27 and p53, and downstream effects on the mTOR pathway, hence exhibiting therapeutic potential as a molecular target for the treatment of childhood ALL. Therefore, activation of AMPK by AICAR represents a novel approach to targeted therapy, and suggests a role for AICAR in combination therapy with inhibitors of the PI3K/Akt/mTOR pathways for the treatment of childhood in ALL

Analysis of folylpoly-γ-glutamate synthetase gene expression in human B-precursor ALL and T-lineage ALL cells

BACKGROUND: Expression of folylpoly-γ-glutamate synthetase (FPGS) gene is two- to three-fold higher in B-precursor ALL (Bp- ALL) than in T-lineage ALL (T-ALL) and correlates with intracellular accumulation of methotrexate (MTX) polyglutamates and lymphoblast sensitivity to MTX. In this report, we investigated the molecular regulatory mechanisms directing FPGS gene expression in Bp-ALL and T-ALL cells. METHODS: To determine FPGS transcription rate in Bp-ALL and T-ALL we used nuclear run-on assays. 5'-RACE was used to uncover potential regulatory regions involved in the lineage differences. We developed a luciferase reporter gene assay to investigate FPGS promoter/enhancer activity. To further characterize the FPGS proximal promoter, we determined the role of the putative transcription binding sites NFY and E-box on FPGS expression using luciferase reporter gene assays with substitution mutants and EMSA. RESULTS: FPGS transcription initiation rate was 1.6-fold higher in NALM6 vs. CCRF-CEM cells indicating that differences in transcription rate led to the observed lineage differences in FPGS expression between Bp-ALL and T-ALL blasts. Two major transcripts encoding the mitochondrial/cytosolic and cytosolic isoforms were detected in Bp-ALL (NALM6 and REH) whereas in T-ALL (CCRF-CEM) cells only the mitochondrial/cytosolic transcript was detected. In all DNA fragments examined for promoter/enhancer activity, we measured significantly lower luciferase activity in NALM6 vs. CCRF-CEM cells, suggesting the need for additional yet unidentified regulatory elements in Bp-ALL. Finally, we determined that the putative transcription factor binding site NFY, but not E-box, plays a role in FPGS transcription in both Bp- and T-lineage. CONCLUSION: We demonstrated that the minimal FPGS promoter region previously described in CCRF-CEM is not sufficient to effectively drive FPGS transcription in NALM6 cells, suggesting that different regulatory elements are required for FPGS gene expression in Bp-cells. Our data indicate that the control of FPGS expression in human hematopoietic cells is complex and involves lineage-specific differences in regulatory elements, transcription initiation rates, and mRNA processing. Understanding the lineage-specific mechanisms of FPGS expression should lead to improved therapeutic strategies aimed at overcoming MTX resistance or inducing apoptosis in leukemic cells

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

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

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

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

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

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