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A novel method to establish glucocorticoid resistant acute lymphoblastic leukemia cell lines.

ABSTRACT: BACKGROUND:Drug-resistant cell lines, established from drug-sensitive cell lines by drug exposure in vitro, are the most useful cancer models in studies on the mechanism of chemoresistance. However, the success rate of the traditional approaches to construct such cell lines is low because a long time is required for the addition of drugs. METHODS:A cell culture technique was used to establish the drug-resistant cell lines from their parental cells. Molecular and cellular biological techniques including flow cytometry, MTT assay, western blotting, and DNA fingerprinting analysis were used to characterize the drug-resistant cell lines. Nude mice were used for xenograft studies. RESULTS:We established novel glucocorticoid (GC)-resistant cell lines from 3 GC-sensitive acute lymphoblastic leukemia (ALL) cell lines. First, we established a novel GC-resistant T-ALL cell line, CEM-C7/HDR, by mimicking the microenvironment of the bone marrow and culturing GC-sensitive CEM-C7-14 cells under hypoxia for 5?weeks with a single dexamethasone (Dex) treatment. The CEM-C7/HDR cells had been cultured continuously in drug-free medium under normoxia for 1?year. The IC50 and resistance index (RI) to Dex were maintained at 60~70??M and 1500~1800, respectively, which is in consistent with the IC50 and RI of GC-resistant CEM-C1-15 cells. To clarify the reliability of the method, we subcloned CEM-C7-14 cells, and obtained Dex-resistant cell lines, CEM-C7-SC2/HDR and CEM-C7-SC14/HDR, from 2 monoclonal cells of CEM-C7-14 by the same method. Moreover, we obtained two additional Dex-resistant B-ALL cell lines, NALM-6/HDR and HXEX-ALL1/HDR, from NALM-6 and HXEX-ALL1 cells with the same approach. CONCLUSIONS:CEM-C7/HDR, NALM-6/HDR and HXEX-ALL1/HDR cell lines may serve as useful GC-resistant ALL models for both in vitro and in vivo studies. Culturing under hypoxic condition with a single Dex treatment is a novel and convenient approach for generating stable GC resistant cell lines.

SUBMITTER: Gu L

PROVIDER: S-EPMC6585113 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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A novel method to establish glucocorticoid resistant acute lymphoblastic leukemia cell lines.

Gu Ling L Zhang Ge G Zhang Yanle Y

Journal of experimental & clinical cancer research : CR 20190620 1

<h4>Background</h4>Drug-resistant cell lines, established from drug-sensitive cell lines by drug exposure in vitro, are the most useful cancer models in studies on the mechanism of chemoresistance. However, the success rate of the traditional approaches to construct such cell lines is low because a long time is required for the addition of drugs.<h4>Methods</h4>A cell culture technique was used to establish the drug-resistant cell lines from their parental cells. Molecular and cellular biologica ...[more]

PMID: 31221196

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Project description:Gene expression data of glucocorticoid resistant and sensitive acute lymphoblastic leukemia cell lines for the article: Expression, regulation and function of phosphofructo-kinase/fructose-biphosphatases (PFKFBs) in glucocorticoid-induced apoptosis of acute lymphoblastic leukemia cells Glucocorticoids (GCs) cause apoptosis and cell cycle arrest in lymphoid cells and constitute a central component in the therapy of lymphoid malignancies, most notably childhood acute lymphoblastic leukemia (ALL). PFKFB2 (6-phosphofructo-2-kinase/fructose-2,6-biphosphatase-2), a kinase controlling glucose metabolism, was identified by us previously as GC response gene in expression profiling analyses performed in children with ALL during initial systemic GC mono-therapy. Since deregulation of glucose metabolism has been implicated in apoptosis induction, this gene and its relatives PFKFB1, 3, and 4 were further analyzed. Expression analyses in additional ALL children, non-leukemic individuals and leukemic cell lines confirmed frequent PFKFB2 induction by GC in most systems sensitive to GC-induced apoptosis, particularly in T-ALL cells. The 3 other family members, in contrast, were not or weakly expressed (PFKFB1 and 4) or not induced by GC (PFKFB3). Conditional PFKFB2 over-expression in the CCRF-CEM T-ALL in vitro model revealed that its 2 splice variants (15A and 15B) did not have any detectable effect on survival or cell cycle progression. Moreover, neither PFKFB2 splice variant significantly affected sensitivity to, or kinetics of, GC-induced apoptosis. Our data suggest that, at least in the model system investigated, PFKFB2 is not an essential upstream regulator of the anti-leukemic effects of GC. Generation of the GC sensitive and resistant clones is described in Parson et al. FASEB J 2005 (Pubmed id 15637111). In brief GC sensitive clones were generated by limiting dilution subcloning from the GC sensitive T-ALL cell line CCRF-CEM-C7H2. To generate GC resistant clones the CCRF-CEM-C7H2 cell line was clutured in the presence of 10E-7 M dexametasone. Gene expression profiles of glucocorticoid (GC) resistant and sensitive T-ALL cells during GC treatment and corresponding control samples (cells treated with carrier control). GC induced regulation of PFKFB2 was determined in the various cell lines based on the expression intensities of the corresponding probe sets in GC treated and control samples.

Project description:BackgroundAcquired glucocorticoid (GC) resistance remains the main obstacle in acute lymphoblastic leukemia (ALL) therapy. The aim of the present study was to establish a novel GC-resistant B-ALL cell line and investigate its biological characteristics.MethodsA cell culture technique was used to establish the GC-resistant cell line from the parental cell, NALM-6. Molecular and cellular biological techniques including flow cytometry, MTT assay, western blotting, DNA fingerprinting analysis and whole transcriptome sequencing (WTS) were used to characterize the GC-resistant cell lines. Nude mice were used for xenograft studies.ResultsThe GC-resistant cell line, NALM-6/HDR, was established by culturing NALM-6 cells under hypoxia for 5 weeks with a single dexamethasone (Dex) treatment. We subcloned the NALM-6/HDR cell lines, and got 6 monoclone Dex-resistant cell lines, NALM-6/HDR-C1, C3, C4, C5, C6 and C9 with resistance index (RI) ranging from 20,000-50,000. NALM-6/HDR and its monoclone cell line, NALM-6/HDR-C5, exhibited moderate (RI 5-15) to high resistance (RI > 20) to Ara-c; low or no cross-resistance to L-Asp, VCR, DNR, and MTX (RI < 5). STR analysis confirmed that NALM-6/HDR and NALM-6/H were all derived from NALM-6. All these cells derived from NALM-6 showed similar morphology, growth curves, immunophenotype, chromosomal karyotype and tumorigenicity. WTS analysis revealed that the main metabolic differences between NALM-6 or NALM-6/H (GC-sensitive) and NALM-6/HDR (GC-resistant) were lipid and carbohydrates metabolism. Western blotting analysis showed that NALM-6/HDR cells had a low expression of GR and p-GR. Moreover, AMPK, mTORC1, glycolysis and de novo fatty acid synthesis (FAS) pathway were inhibited in NALM-6/HDR when compared with NALM-6.ConclusionsNALM-6/HDR cell line may represent a subtype of B-ALL cells in patients who acquired GC and Ara-c resistance during the treatment. These patients may get little benefit from the available therapy target of AMPK, mTORC1, glycolysis and FAS pathway.

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A novel method to establish glucocorticoid resistant acute lymphoblastic leukemia cell lines.

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A novel method to establish glucocorticoid resistant acute lymphoblastic leukemia cell lines.

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