Real-time RT-PCR analysis of mRNA decay: half-life of Beta-actin mRNA in human leukemia CCRF-CEM and Nalm-6 cell lines

BACKGROUND: We describe an alternative method to determine mRNA half-life (t(1/2)) based on the Real-Time RT-PCR procedure. This approach was evaluated by using the β-actin gene as a reference molecule for measuring of mRNA stability. RESULTS: Human leukemia Nalm-6 and CCRF-CEM cells were treated with various concentrations of Actinomycin D to block transcription and aliquots were removed periodically. Total RNA was isolated and quantified using the RiboGreen(®) fluorescent dye with the VersaFluor Fluorometer System. One μg of total RNA was reverse transcribed and used as template for the amplification of a region of the β-actin gene (231 bp). To generate the standard curve, serial ten-fold dilutions of the pBactin-231 vector containing the cDNA amplified fragment were employed, β-actin mRNAs were quantified by Real-Time RT-PCR using the SYBR(®) Green I fluorogenic dye and data analyzed using the iCycle iQ system software. Using this method, the β-actin mRNA exhibited a half-life of 6.6 h and 13.5 h in Nalm-6 and CCRF-CEM cells, respectively. The t(1/2) value obtained for Nalm-6 is comparable to those estimated from Northern blot studies, using normal human leukocytes (5.5 h). CONCLUSIONS: We have developed a rapid, sensitive, and reliable method based on Real-Time RT-PCR for measuring mRNA half-life. Our results confirm that β-actin mRNA half-life can be affected by the cellular growth rate

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

Abstract 2736: PIM2 is up-regulated in response to metformin-induced cell death triggered by ER stress/UPR in ALL lymphoblasts

Abstract Acute lymphoblastic leukemia (ALL) is the most common malignancy in children, and despite significant overall improvements in cure rates, outcome for patients diagnosed with resistant phenotypes or those who relapse is dismal. We investigated the mechanism of cell death induced by metformin (MET) in ALL cell models. We showed that MET induced significant growth inhibition and apoptosis in CCRF-CEM (T-ALL) and NALM6 (Bp-ALL) cells. Western blots revealed that MET activated p-AMPK, p-ACC, p-Akt, and p-4EBP1. The latter suggested that regulation of protein translation may be an important determinant in MET-induced cell death. Indeed, inhibition of mTOR/protein translation with rapamycin (RAPA) rescued MET-induced cell death in ALL cells. In addition, knockdown of AMPK expression in ALL cells using shRNA (shAMPK) abrogated MET-induced apoptosis as compared to control cells expressing scramble shRNA (shCTRL), indicating that AMPK mediated MET's cytotoxicity. Western blots demonstrated that shAMPK cells expressed lower level of total AMPK, p-p38MAPK, p-mTOR, and p-4EBP1 compared to shCTRL cells, indicating that AMPK and protein translation are critical in MET sensitivity. Indeed, pulse labeled 35S-methionine experiments demonstrated increase incorporation confirming the importance of protein synthesis is MET-induced cytotoxicity. More important, we uncovered that MET-induced apoptosis correlated with induction of ER stress as evidenced by up-regulation of IRE1α and CHOP although GRP78 decreased significantly. We further demonstrated that RAPA rescued MET-treated cells by relieving ER stress/UPR mediated cell death. We previously showed that the unfolded protein response (UPR) in ALL cells is regulated by the contextual crosstalk between AMPK and Akt (Mol Cancer Ther 10:437, 2011). Therefore, we evaluated the effects of co-targeting Akt and AMPK using the Akt inhibitor X/perifosine + MET and found these combinations to be synergistic. To further investigate the relationship between protein translation and ER stress/UPR in MET-induced cell death, we examined the role of PIM1/2 kinases. We found that expression of PIM2 was increased in MET-treated ALL with concomitant decreased in the expression of IRE1α, ATF6, and CHOP, suggesting that PIM2 maybe up-regulated as a compensatory survival mechanism aimed at relieving MET-induced ER stress/UPR mediated cell death. To test this hypothesis, we co-treated ALL cells with a PIM1/2 kinase inhibitor + MET and found that PIM2 inhibition synergistically sensitized ALL cells to MET (CI=0.28). Taken together, our data indicate that PIM2 plays a role in buffering cell death in MET-treated cells, and that regulation of protein translation modulates ER stress/UPR induced apoptosis in ALL cells. Consequently, our data support strategies targeting these synthetic lethal interactions as suitable for clinical translation in patients with ALL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2736. doi:1538-7445.AM2012-2736</jats:p

Histone Deacetylase Inhibitors Induce FPGS mRNA Expression and Intracellular Accumulation of Long-Chain Methotrexate Polyglutamates in Childhood Acute Lymphoblastic Leukemia: Implications for Combination Therapy

Abstract Folate cofactors are essential components of one carbon metabolism and are required for the biosynthesis of purines, pyrimidines, serine and methionine. The classical folate antagonist methotrexate (MTX) continues to be a universal component of most ALL treatment regimens. MTX is retained within cells as long-chain polyglutamates (MTX-PGs) after metabolism by the enzyme folylpoly-γ -glutamate synthetase (FPGS). Intracellular retention of MTX-PGs results in enhanced cytotoxicity due to prolonged inhibition of dihydrofolate reductase (DHFR) and thymidylate synthetase (TS). The FPGS gene is regulated by the transcription factors NFY and Sp1. Using DNaseI assays we identified a hypersensitive site mapping closely upstream of exon 1, suggesting that chromatin remodeling may contribute to FPGS gene regulation. To investigate the role of histone modifications and chromatin remodeling on FPGS expression and uncover interactions between NFY, Sp1 and HDAC1, we performed co-immunoprecipitation and Western blotting. Our results demonstrate that HDAC1 complexes with NFY and Sp1 transcription factors in both B- and T-ALL cells. DNA affinity precipitation assays (DAPA) revealed that HDAC1 is recruited by NFY and Sp1 to the FPGS promoter. These findings suggest that transcription of the FPGS gene may be regulated by NFY and Sp1 factors interacting with HDAC1, and leading to chromatin remodeling. We then examined the effect of the histone deacetylase inhibitors (HDACIs) sodium butyrate (NaBu) and suberoylanilide hydroxamic acid (SAHA) on the expression of FPGS and other folate-related genes in NALM6 (Bp-ALL), REH (TEL/AML1+, Bp-ALL), SupB15 (BCR/ABL+, Bp-ALL), and CCRF-CEM (T-ALL) cells using qRT-PCR. In all cell lines examined, treatment with HDACIs increased FPGS mRNA expression by 2- to 5-fold, whereas the level of DHFR and TS mRNA expression were decreased. On this basis, we hypothesized that induction of FPGS expression by HDACIs, results in higher accumulation of MTX-PG and enhanced MTX cytotoxicity in ALL cells. Further, the concomitant decrease in the expression of the MTX-PG target enzymes DHFR and TS, would enhance the cytotoxicity of the combination of HDACIs plus MTX in ALL cells. To test this hypothesis, NALM6, REH, and SupB15 cells were treated with MTX (4h) + SAHA (24h), and cell viability assessed. We determined that SAHA increased the intracellular accumulation of long chain MTX-PGs (n ≥3 Glu) in ALL cells, correlating with the upregulation of FPGS expression in SAHA-treated cells. Treatment with MTX + SAHA increased cytotoxicity by ~30% with a calculated combination index of ≤ 0.8 indicating synergy. Analysis of apoptosis using AnnexinV/PI staining revealed a 2 to 3-fold increase in apoptotic cell death in all cell lines treated with this combination. Our data suggest HDACIs enhance MTX cytotoxicity by upregulation of FPGS expression, increased accumulation of MTXPG and downregulation of DHFR and TS. The synergism exhibited by the combination of MTX and SAHA suggests it should be tested in ALL patients, in particular those who exhibit phenotypes with de novo or acquired resistance to MTX

AMPK and Akt determine apoptotic cell death following perturbations of one-carbon metabolism by regulating ER stress in Acute Lymphoblastic Leukemia

AICAr is a cell-permeable nucleotide that has been used in vivo and in vitro to activate AMPK. Our previous findings have shown that AICAr as a single agent induces dose- and time-dependent growth inhibition in acute lymphoblastic leukemia (ALL) cell lines. In addition, the combination of AICAr with antifolates (methotrexate (MTX) or pemetrexed) has been shown to further potentiate AMPK activation and to lead to greater cytotoxicity and growth inhibition in leukemia and other malignant cell types. Our data presented herein demonstrate that sustained ER stress is the predominant mechanism behind the synergistic induction of cell death by the combination of AICAr plus the inhibitor of one-carbon metabolism, MTX, in Bp- and T-ALL, as evidenced by induction of several unfolded protein response markers leading to apoptosis. We also show for the first time that AICAr in combination with MTX significantly induces Akt phosphorylation in ALL. Under these conditions, the concomitant inhibition of Akt, a cellular antagonist of AMPK, leads to further up-regulation of AMPK activity and alleviates AICAr plus MTX-induced ER stress and apoptosis. Therefore, we also demonstrate that the concomitant activation of AMPK actually rescues the cells from AICAr plus MTX-induced ER stress and apoptosis. Our data suggest that the effects of AMPK activation on cell death or survival differ contextually depending on its signaling alterations with related oncogenic pathways and provide insight into the reported paradoxical pro-apoptotic vs. pro-survival effects of AMPK activation

PIM2 Upregulation Leads to Physiological Buffering of Metformin-Induced Cell Death Mediated by ER Stress/UPR in Acute Lymphoblastic Leukemia

Abstract Abstract 3496 Acute Lymphoblastic Leukemia (ALL) is the most common malignancy in children and adolescents. Despite significant overall improvements in cure rates, outcome remains dismal for patients with resistant phenotypes or after relapse. Therefore, novel treatment strategies are warranted. Recently, we identified the AMP activated protein kinase (AMPK), a regulator of energy homeostasis in eukaryotic cells, as a potential target for ALL therapy due to its effects on cell growth, proliferation, and cell cycle regulation, as well as its crosstalk with critical metabolic and oncogenic pathways. We showed that activation of AMPK using metformin (1-5 mM) induced significant cell growth inhibition and apoptosis in CCRF-CEM (T-ALL) and NALM6 (Bp-ALL) cell line models. Western blot analysis revealed that metformin led to activation of p-AMPK (Thr172) and its downstream target p-ACC (Ser79), the cell proliferation regulator p-Akt (Ser473), and the protein translation regulator p-4EBP1 (Thr70), suggesting that protein translation may be an important determinant in the mechanism of metformin-induced cell death. Indeed, we demonstrated that blocking protein translation with the mTOR inhibitor rapamycin (1 μg/ml) rescued ALL cells from metformin-induced cell death (p < 0.01). In addition, knockdown of AMPK1α expression using shRNAs (shAMPK) abrogated metformin-induced growth inhibition and apoptosis in ALL cells as compared to control cells expressing scramble shRNAs (shCTRL), indicating that AMPK mediates metformin's cytotoxicity in our models. Western blots demonstrated that ALL cells expressing shAMPK exhibit decreased expression of total AMPK, p-p38MAPK (Thr180), p-mTOR (Ser2448), and p-4EBP1 (Thr70) compared to shCTRL cells, implicating regulation of protein translation in the mechanism of cell death induced by metformin. In addition, metformin-induced p-Akt (Ser473) activation observed in shCTRL cells is blocked in shAMPK expressing cells, suggesting that the contextual crosstalk between AMPK and Akt is relevant for metformin's cytotoxicity. Indeed, experiments co-targeting Akt and AMPK using perifosine (6 μM) or the Akt inhibitor X (AIX, 5 μM) plus metformin (5 mM) for 72 h induced synergistic cell death in NALM6 cells (Combination Index (CI) values of 0.21 for perifosine + metformin, and 0.19 for AIX + metformin). Our studies uncovered that apoptotic death in NALM6 and CCRF-CEM cells treated with metformin correlated with metformin's induction of ER stress/UPR in ALL cells, as demonstrated by increased expression of the UPR markers IRE1α and CHOP. More important, rapamycin rescued metformin-treated ALL cells by relieving ER stress/UPR as demonstrated by decreased IRE1α and CHOP. These observations support our previous findings that ER stress/UPR mediates cell death in ALL cells under metabolic stress, and is tightly coupled to regulation of protein translation (Mol Cancer Ther 10:437, 2011). To further investigate the relationship between protein translation and ER stress/UPR, we examined the role of PIM1/2 kinases, particularly PIM2 known to regulate CAP protein translation, in metformin-induced ALL cell death. Our results indicate that expression of PIM2 is significantly increased in NALM6 cells treated with metformin (5–10 mM) for 72 h. We also observed concomitant decrease in the expression of the UPR markers IRE1α, ATF6, and CHOP, raising the possibility that PIM2 upregulation may be a compensatory survival mechanism to regulate protein translation and suppress metformin-induced ER stress/UPR. To test this hypothesis, we co-treated NALM6 cells with the small molecule PIM1/2 kinase inhibitor V (80 μM) and metformin (5 mM) and found that inhibition of PIM2 in metformin-treated NALM6 cells induced synergistic cell death (CI = 0.28). Taken together, our data indicate that PIM2 plays a role in buffering cell death in metformin treated cells, and that regulation of protein translation modulates ER stress/UPR induced apoptosis in ALL cells. Consequently, our data support strategies that exploit synthetic lethality by combining activators of AMPK such as metformin and compounds that target regulation of protein translation or protein degradation as suitable for clinical translation in patients with ALL. Disclosures: No relevant conflicts of interest to declare