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Preclinical cellular pharmacology of LY231514 (MTA): a comparison with methotrexate, LY309887 and raltitrexed for their effects on intracellular folate and nucleoside triphosphate pools in CCRF-CEM cells.

ABSTRACT: LY231514 (N-[4-[2-(2-amino-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethy l]-benzoyl]-L-glutamic acid) is a new folate-based antimetabolite currently in broad phase II clinical evaluation. Previous in vitro studies (C. Shih et al, CancerRes 57: 1116-1123, 1997) have suggested that LY231514 could be a multitargeted antifolate (MTA) capable of inhibiting thymidylate synthase (TS), dihydrofolate reductase (DHFR) and glycinamide ribonucleotide formyltransferase (GARFT). The present study compared LY231514 with methotrexate, raltitrexed and a glycinamide ribonucleotide formyltransferase inhibitor, LY309887, at 300, 100, 30 and 100 nM, respectively, for their effects on intracellular folate and at 100, 66, 20 and 30 nM respectively, for their effects on nucleoside triphosphate pools in CCRF-CEM cells. Methotrexate induced an accumulation of dihydrofolate species, together with a rapid depletion of ATP, GTP and all of the deoxynucleoside triphosphates. LY309887 caused an accumulation of 10-formyltetrahydrofolate, a rapid loss of ATP, GTP and dATP, but a slower loss in dCTP, dTTP and dGTP. Both LY231514 and raltitrexed had minimal effects on folate pools. In contrast, they caused rapid depletion of dTTP, dCTP and dGTP, but induced an accumulation of dATP at different rates, with raltitrexed doing so about 2.5 times faster. Most of the observed metabolic changes could be understood on the basis of current knowledge of folate and nucleotide metabolism. We concluded that LY231514 was distinct from methotrexate, LY309887 and raltitrexed based on their metabolic effects in CCRF-CEM cells, and that in this cell line the inhibitory effects of LY231514 were exerted primarily against the thymidylate cycle and secondarily against de novo purine biosynthesis.

SUBMITTER: Chen VJ

PROVIDER: S-EPMC2062799 | biostudies-literature | 1998

REPOSITORIES: biostudies-literature

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Preclinical cellular pharmacology of LY231514 (MTA): a comparison with methotrexate, LY309887 and raltitrexed for their effects on intracellular folate and nucleoside triphosphate pools in CCRF-CEM cells.

Chen V J VJ Bewley J R JR Andis S L SL Schultz R M RM Iversen P W PW Shih C C Mendelsohn L G LG Seitz D E DE Tonkinson J L JL

British journal of cancer 19980101

LY231514 (N-[4-[2-(2-amino-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethy l]-benzoyl]-L-glutamic acid) is a new folate-based antimetabolite currently in broad phase II clinical evaluation. Previous in vitro studies (C. Shih et al, CancerRes 57: 1116-1123, 1997) have suggested that LY231514 could be a multitargeted antifolate (MTA) capable of inhibiting thymidylate synthase (TS), dihydrofolate reductase (DHFR) and glycinamide ribonucleotide formyltransferase (GARFT). The present study com ...[more]

PMID: 9717988

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Project description:Glucocorticoids play an essential part in anti-leukemic therapies, but resistance is a crucial event for the prognosis of the disease. Glucocorticoids influence the metabolic properties of leukemic cells. The inherent plasticity of clinically evolving cancer cells justifies the characterization of drug-induced early oncogenic pathways, which represent a likely source of detrimental secondary effects. The present work aims to investigate the effect of glucocorticoids in metabolic pathways in the CCRF-CEM leukemic cells. Metabolic factors and gene expression profiles were examined in order to unravel the possible mechanisms of the CCRF-CEM leukemic cell growth dynamics. CCRF-CEM cells were used as a model. Cells were treated with prednisolone with concentrations 0-700 μM. Cell culture supernatants were used for glucose, lactic acid, LDH, Na+, K+ and Ca++ measurements. Cytotoxicity was determined with flow cytometry. Microarray analysis was performed using two different chips of 1.2 k and 4.8 k genes. Gene Ontology enrichment analysis was applied to find metabolism- and GC-related genes. Higher prednisolone concentrations inhibited glucose uptake, without exhibiting any cytotoxic effects. Glucose consumption did not correlate with the total cell population, or the viable population, indicating that growth is not directly proportional to glucose consumption. Neither of the subpopulations, i.e., viable, necrotic, or apoptotic cells, contributed to this. Different types of leukemic cells seem to exhibit different patterns of glucose metabolism. Both resistant and sensitive CCRF-CEM cells followed the aerobic pathway of glycolysis. There is probably a rapid change in membrane permeability, causing a general shutdown towards everything that is outside the cell. This could in part also explain the observed resistance. Glucocorticoids do not enter the cell passively anymore and therefore no effects are observed. Based on our observations, ion concentrations are measurable factors both in vitro and in vivo, which makes them possible markers of glucocorticoid cytotoxic action.

Project description:Background: Glucocorticoids are important pharmaceutical agents in the treatment of acute lymphoblastic leukemia in children. Resistance or sensitivity to glucocorticoids is considered to be of crucial importance for disease prognosis. Prednisolone is a first-line chemotherapeutic agent in the treatment of acute lymphoblastic leukemia. Here, the effects of prednisolone on the resistant CCRF-CEM leukemic cell line were studied. Methods: Prednisolone’s cytotoxic and cell cycle effects were studied with flow cytometry. NF-κB translocation was studied with Western Blotting and differential gene expression was studied with cDNA microarrays. Results: Prednisolone exerted a delayed biphasic effect, necrotic at low doses and apoptotic at higher doses. At low doses, prednisolone exerted a pre-dominant mitogenic effect despite its induction on total cell death, while at higher doses, prednisolone’s mitogenic and cell death effects were counterbalanced. NF-κB was constitutively present in the nucleus. Early gene microarray analysis revealed 40 differentially expressed genes upon 4 hours of prednisolone’s exposure. Notable differences in gene regulation were observed between the lowest and the highest glucocorticoid doses. Prednisolone activated genes related to apoptosis/tumor suppression, cell cycle progression, metabolism and intra-/ extra-cellular signaling pathways. Conclusions: The mitogenic/biphasic effects of prednisolone are of clinical importance in the case of resistant leukemic cells. This approach might lead to the identification of gene candidates for future molecular drug targets in combination therapy with glucocorticoids, along with early markers for glucocorticoid resistance. Elucidation of the mechanisms of GC action may lead to identification of gene targets responsible for GC resistance. Key tools in this process are high-throughput technologies such as microarray-based gene expression analysis. For this purpose, the parental CCRF-CEM cell line was chosen as the system of study for the effects of prednisolone treatment. This is a T-cell leukemia cell line characterized by a mutation (L753F) on one GR gene allele that impairs ligand binding (Thompson and Johnson 2003). It is known that both the DNA and ligand binding domains of the GR are required in order to repress NF-κB transactivation (Wissink, van Heerde et al. 1997). Interestingly, concerning the question whether this mutation would affect GC resistance, it has been reported previously that both the GC-resistant as well as the GC-sensitive CCRF-CEM subclones express heterogeneous populations of the GR (GRwt/GRL753F) (Palmer and Harmon 1991; Powers, Hillmann et al. 1993). The CCRF-CEM cell line has been reported to be resistant to GCs, presumably due to the accumulation of more resistant variants after long periods of prolonged culture (Norman and Thompson 1977). It is possible that these cells are clonally inhomogeneous, as possibly the cells obtained in vivo by patients. Moreover, the large number of the CCRF-CEM subclone studies in the literature makes it difficult to choose an appropriate resistant cell model. In addition, the utilization of an in vitro system for this study offered reproducibility, an opportunity to closely examine intracellular signals and avoid interference from other in vivo-participating systems. Thus, the cell line used for this study was considered to be useful in studying GC action and resistance in leukemic cells. The aim of this work was to determine the cytotoxic, cell cycle phase distribution and early cancer-specific gene expression effects of prednisolone in CCRF-CEM cells, as an in vitro model of ALL resistance to glucocorticoids. The early gene expression profile allowed identification of genes initiating pivotal, early onset regulatory mechanisms activated by GC and excluded ensuing feedback responses and further downstream signals. Samples tested were in the form of a loop-design i.e. control vs. 10nM prednisolone (designated 0vs1) , 10nM prednisolone vs.700uM prednisolone (designated 1vs3) and control vs. 700uM prednisolone (designated 0vs3), the sum of the logarithms of the first two should equal the third. In other words if Rj,i is the ratio of the ith gene in the jth experiment then R0vs1,i+R1vs3,i=R0vs3,i. We tested this using a statistical test. We have applied an intensity-dependent z-score where the sum of the ratios was compared to the ratio of the third experiment. If the difference was significant genes, in a standard deviation threshold of ±1.5 in relative units, were rejected from further analysis (Kerr and Churchill 2001; Kerr and Churchill 2001; Altman and Hua 2006; Kerr and Churchill 2007).

Dataset Information

Preclinical cellular pharmacology of LY231514 (MTA): a comparison with methotrexate, LY309887 and raltitrexed for their effects on intracellular folate and nucleoside triphosphate pools in CCRF-CEM cells.

Publications

Preclinical cellular pharmacology of LY231514 (MTA): a comparison with methotrexate, LY309887 and raltitrexed for their effects on intracellular folate and nucleoside triphosphate pools in CCRF-CEM cells.

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