scholarly journals Mild phenotype of knockouts of the major apurinic/apyrimidinic endonuclease APEX1 in a non-cancer human cell line

PLoS ONE ◽  
2021 ◽  
Vol 16 (9) ◽  
pp. e0257473
Author(s):  
Daria V. Kim ◽  
Liliya M. Kulishova ◽  
Natalia A. Torgasheva ◽  
Vasily S. Melentyev ◽  
Grigory L. Dianov ◽  
...  
Keyword(s):  
Dna Repair ◽  
Cell Line ◽  
Cell Lines ◽  
Wild Type ◽  
Single Base ◽  
293Ft Cell ◽  
Ap Sites ◽  
Insertion Allele ◽  
Ap Site ◽  
Base Insertion

The major human apurinic/apyrimidinic (AP) site endonuclease, APEX1, is a central player in the base excision DNA repair (BER) pathway and has a role in the regulation of DNA binding by transcription factors. In vertebrates, APEX1 knockouts are embryonic lethal, and only a handful of knockout cell lines are known. To facilitate studies of multiple functions of this protein in human cells, we have used the CRISPR/Cas9 system to knock out the APEX1 gene in a widely used non-cancer hypotriploid HEK 293FT cell line. Two stable knockout lines were obtained, one carrying two single-base deletion alleles and one single-base insertion allele in exon 3, another homozygous in the single-base insertion allele. Both mutations cause a frameshift that leads to premature translation termination before the start of the protein’s catalytic domain. Both cell lines totally lacked the APEX1 protein and AP site-cleaving activity, and showed significantly lower levels of the APEX1 transcript. The APEX1-null cells were unable to support BER on uracil- or AP site-containing substrates. Phenotypically, they showed a moderately increased sensitivity to methyl methanesulfonate (MMS; ~2-fold lower EC50 compared with wild-type cells), and their background level of natural AP sites detected by the aldehyde-reactive probe was elevated ~1.5–2-fold. However, the knockout lines retained a nearly wild-type sensitivity to oxidizing agents hydrogen peroxide and potassium bromate. Interestingly, despite the increased MMS cytotoxicity, we observed no additional increase in AP sites in knockout cells upon MMS treatment, which could indicate their conversion into more toxic products in the absence of repair. Overall, the relatively mild cell phenotype in the absence of APEX1-dependent BER suggests that mammalian cells possess mechanisms of tolerance or alternative repair of AP sites. The knockout derivatives of the extensively characterized HEK 293FT cell line may provide a valuable tool for studies of APEX1 in DNA repair and beyond.

scholarly journals Molecular analysis of ethyl methanesulfonate-induced reversion of a chromosomally integrated mutant shuttle vector gene in mammalian cells.

1988 ◽  
Vol 8 (10) ◽  
pp. 4185-4189 ◽  
Author(s):  
J A Greenspan ◽  
F M Xu ◽  
R L Davidson
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Dna Sequences ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Shuttle Vector ◽  
Ethyl Methanesulfonate ◽  
Wild Type ◽  
Single Base ◽  
Type Sequence

The molecular mechanisms of ethyl methanesulfonate-induced reversion in mammalian cells were studied by using as a target a gpt gene that was integrated chromosomally as part of a shuttle vector. Murine cells containing mutant gpt genes with single base changes were mutagenized with ethyl methanesulfonate, and revertant colonies were isolated. Ethyl methanesulfonate failed to increase the frequency of revertants for cell lines with mutant gpt genes carrying GC----AT transitions or AT----TA transversions, whereas it increased the frequency 50-fold to greater than 800-fold for cell lines with mutant gpt genes carrying AT----GC transitions and for one cell line with a GC----CG transversion. The gpt genes of 15 independent revertants derived from the ethyl methanesulfonate-revertible cell lines were recovered and sequenced. All revertants derived from cell lines with AT----GC transitions had mutated back to the wild-type gpt sequence via GC----AT transitions at their original sites of mutation. Five of six revertants derived from the cell line carrying a gpt gene with a GC----CG transversion had mutated via GC----AT transition at the site of the original mutation or at the adjacent base in the same triplet; these changes generated non-wild-type DNA sequences that code for non-wild-type amino acids that are apparently compatible with xanthine-guanine phosphoribosyltransferase activity. The sixth revertant had mutated via CG----GC transversion back to the wild-type sequence. The results of this study define certain amino acid substitutions in the xanthine-guanine phosphoribosyltransferase polypeptide that are compatible with enzyme activity. These results also establish mutagen-induced reversion analysis as a sensitive and specific assay for mutagenesis in mammalian cells.

Molecular analysis of ethyl methanesulfonate-induced reversion of a chromosomally integrated mutant shuttle vector gene in mammalian cells

1988 ◽  
Vol 8 (10) ◽  
pp. 4185-4189
Author(s):  
J A Greenspan ◽  
F M Xu ◽  
R L Davidson
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Dna Sequences ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Shuttle Vector ◽  
Ethyl Methanesulfonate ◽  
Wild Type ◽  
Single Base ◽  
Type Sequence

The molecular mechanisms of ethyl methanesulfonate-induced reversion in mammalian cells were studied by using as a target a gpt gene that was integrated chromosomally as part of a shuttle vector. Murine cells containing mutant gpt genes with single base changes were mutagenized with ethyl methanesulfonate, and revertant colonies were isolated. Ethyl methanesulfonate failed to increase the frequency of revertants for cell lines with mutant gpt genes carrying GC----AT transitions or AT----TA transversions, whereas it increased the frequency 50-fold to greater than 800-fold for cell lines with mutant gpt genes carrying AT----GC transitions and for one cell line with a GC----CG transversion. The gpt genes of 15 independent revertants derived from the ethyl methanesulfonate-revertible cell lines were recovered and sequenced. All revertants derived from cell lines with AT----GC transitions had mutated back to the wild-type gpt sequence via GC----AT transitions at their original sites of mutation. Five of six revertants derived from the cell line carrying a gpt gene with a GC----CG transversion had mutated via GC----AT transition at the site of the original mutation or at the adjacent base in the same triplet; these changes generated non-wild-type DNA sequences that code for non-wild-type amino acids that are apparently compatible with xanthine-guanine phosphoribosyltransferase activity. The sixth revertant had mutated via CG----GC transversion back to the wild-type sequence. The results of this study define certain amino acid substitutions in the xanthine-guanine phosphoribosyltransferase polypeptide that are compatible with enzyme activity. These results also establish mutagen-induced reversion analysis as a sensitive and specific assay for mutagenesis in mammalian cells.

Growth Properties of Neural Cell Lines Immortalized with the Tsa58 Allele of Sv40 Large T Antigen

1997 ◽  
Vol 6 (3) ◽  
pp. 231-238 ◽  
Author(s):  
M.E. Truckenmiller ◽  
Ora Dillon-Carter ◽  
Carlo Tornatore ◽  
Henrietta Kulaga ◽  
Hidetoshi Takashima ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cell Line ◽  
Cell Lines ◽  
T Antigen ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Large T Antigen ◽  
Wild Type ◽  
Sv40 Large T Antigen ◽  
Growth Properties

In vitro growth properties of three CNS-derived cell lines were compared under a variety of culture conditions. The M213-20 and J30a cell lines were each derived from embryonic CNS culture with the temperature-sensitive (ts) allele of SV40 large T antigen, tsA58, while the A7 cell line was immortalized using wild-type SV40 large T antigen. Cells immortalized with tsA58 SV40 large T proliferate at the permissive temperature, 33° C, while growth is expected to be suppressed at the nonpermissive temperature, 39.5°C. Both the M213-20 and J30a cell lines were capable of proliferating at 39.5°C continuously for up to 6 mo. All three cell lines showed no appreciable differences in growth rates related to temperature over a 7-day period in either serum-containing or defined serum-free media. The percentage of cells in S-phase of the cell cycle did not decrease or was elevated at 39.5°C for all three cell lines. After 3 wk at 39.5°C, the three cell lines also showed positive immunostaining using two monoclonal antibodies reacting with different epitopes of SV40 large T antigen. Double strand DNA sequence analyses of a 300 base pair (bp) fragment of the large T gene from each cell line, which included the ts locus, revealed mutations in both the J30a and M213-20 cell lines. The J30a cell line ts mutation had reverted to wild type, and two additional loci with bp substitutions with predicted amino acid changes were also found. While the ts mutation of the M213-20 cells was retained, an additional bp substitution with a predicted amino acid change was found. The A7 cell line sequence was identical to the reference wild-type sequence. These findings suggest that (a) nucleic acid sequences in the temperature-sensitive region of the tsA58 allele of SV40 large T are not necessarily stable, and (b) temperature sensitivity of cell lines immortalized with tsA58 is not necessarily retained.

Chromosome-mediated gene transfer of hydroxyurea resistance and amplification of ribonucleotide reductase activity

1983 ◽  
Vol 3 (6) ◽  
pp. 1053-1061
Author(s):  
W H Lewis ◽  
P R Srinivasan
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Ribonucleotide Reductase ◽  
Chinese Hamster ◽  
Fold Increase ◽  
Resistant Cell ◽  
Plating Efficiency ◽  
Wild Type Cell ◽  
Wild Type ◽  
Metaphase Chromosomes

Metaphase chromosomes purified from a hydroxyurea-resistant Chinese hamster cell line were able to transform recipient wild-type cells to hydroxyurea resistance at a frequency of 10(-6). Approximately 60% of the resulting transformant clones gradually lost hydroxyurea resistance when cultivated for prolonged periods in the absence of drug. One transformant was subjected to serial selection in higher concentrations of hydroxyurea. The five cell lines generated exhibited increasing relative plating efficiency in the presence of the drug and a corresponding elevation in their cellular content of ribonucleotide reductase. The most resistant cell line had a 163-fold increase in relative plating efficiency and a 120-fold increase in enzyme activity when compared with the wild-type cell line. The highly hydroxyurea-resistant cell lines had strong electron paramagnetic resonance signals characteristic of an elevated level of the free radical present in the M2 subunit of ribonucleotide reductase. Two-dimensional electrophoresis of cell-free extracts from one of the resistant cell lines indicated that a 53,000-dalton protein was present in greatly elevated quantities when compared with the wild-type cell line. These data suggest that the hydroxyurea-resistant cell lines may contain an amplification of the gene for the M2 subunit of ribonucleotide reductase.

scholarly journals Function of wild-type or mutant Rac2 and Rap1a GTPases in differentiated HL60 cell NADPH oxidase activation

Blood ◽  
1995 ◽  
Vol 85 (3) ◽  
pp. 804-811 ◽  
Author(s):  
TG Gabig ◽  
CD Crean ◽  
PL Mantel ◽  
R Rosli
Keyword(s):  
Plasma Membrane ◽  
Nadph Oxidase ◽  
Cell Line ◽  
Cell Lines ◽  
Wild Type ◽  
Free System ◽  
Hl60 Cells ◽  
Cell Free System ◽  
Nadph Oxidase Activation ◽  
O2 Production

Studies of neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation in a cell-free system showed that the low molecular-weight guanosine triphosphatase (GTPase) Rac was required, and that Rap1a may participate in activation of the catalytic complex. Full-length posttranslationally modified Rac2 was active, whereas only the 1–166 truncated form of Rap1a was functional in the cell-free system, and thus, clarification of the function of Rap1a and Rac2 in intact human phagocytes is needed to provide further insight into their roles as signal transducers from plasma membrane receptors. In the present studies, oligonucleotide-directed mutagenesis was used to introduce a series of mutations into human rap1a or rac2 in the mammalian expression vector pSR alpha neo. HL60 cells transfected with wild-type or mutated rac2 or rap1a cDNA constructs and control HL60 cells transfected with the pSR alpha neo vector containing no inserted cDNA were selected in G418-containing media, then subclones were isolated. Compared with the parent HL60 cells, each of the stable transfected cell lines differentiated similarly into neutrophil-like cells and expressed comparable levels of NADPH oxidase components p47- phox, p67-phox and gp91-phox. The differentiated vector control cell line produced O2. in response to receptor stimulation at rates that were not significantly different from parent HL60 cells. O2-. production by differentiated cell lines expressing mutated N17 Rap1a or N17 Rac2 dominant-negative proteins was inhibited, whereas O2-. production by the subline overexpressing wild-type Rap1a was increased by fourfold. O2-. production by the differentiated cell line expressing GTPase-defective V12 Rap1a was also significantly inhibited, a finding that is consistent with a requirement for cycling between guanosine diphosphate- and GTP-bound forms of Rap1a for continuous NADPH oxidase activation in intact neutrophils. A model is proposed in which Rac2 mediates assembly of the p47 and p67 oxidase components on the cytosolic face of the plasma membrane via cytoskeletal reorganization, whereas Rap1a functions downstream as the final activation switch involving direct physical interaction with the transmembrane flavocytochrome component of the NADPH oxidase.

A single point mutation in Drosophila dihydrofolate reductase confers methotrexate resistance to a transgenic CHO cell line

Genome ◽  
2003 ◽  
Vol 46 (4) ◽  
pp. 707-715 ◽  
Author(s):  
K Neumann ◽  
K M Al-Batayneh ◽  
M J Kuiper ◽  
J Parsons-Sheldrake ◽  
M G Tyshenko ◽  
...  
Keyword(s):  
Cell Line ◽  
Point Mutation ◽  
Cell Lines ◽  
Dihydrofolate Reductase ◽  
Cho Cells ◽  
Chinese Hamster ◽  
Single Point Mutation ◽  
Parental Line ◽  
Wild Type ◽  
Drosophila Cell

Sequence analysis of a cDNA encoding dihydrofolate reductase (DHFR) from a selected methotrexate-resistant Drosophila melanogaster cell line (S3MTX) revealed a substitution of Gln for Leu at position 30. Although the S3MTX cells were ~1000 fold more resistant to methotrexate (MTX), the karyotype was similar to the parental line and did not show elongated chromosomes. Furthermore, kinetic analysis of the recombinant enzyme showed a decreased affinity for MTX by the mutant DHFR. To determine if the resistance phenotype could be attributed to the mutant allele, Drosophila Dhfr cDNAs isolated from wild type and S3MTX cells were expressed in Chinese hamster ovary (CHO) cells lacking endogenous DHFR. The heterologous insect DHFRs were functional in transgenic clonal cell lines, showing ~400-fold greater MTX resistance in the cell line transfected with the mutant Dhfr than the wild type Dhfr. Resistance to other antifolates in the CHO cells was consistent with the drug sensitivities seen in the respective Drosophila cell lines. ELevated Levels of Dhfr transcript and DHFR in transgenic CHO cells bearing the mutant cDNA were not seen. Taken together, these results demonstrate that a single substitution in Drosophila DHFR alone can confer Levels of MTX resistance comparable with that observed after considerable gene amplification in mammalian cells.Key words: dihydrofolate reductase, methotrexate, drug resistance, point mutation.

scholarly journals Influence of Cystic Fibrosis Transmembrane Conductance Regulator on Gene Expression in Response to Pseudomonas aeruginosa Infection of Human Bronchial Epithelial Cells

2005 ◽  
Vol 73 (10) ◽  
pp. 6822-6830 ◽  
Author(s):  
Nina Reiniger ◽  
Jeffrey K. Ichikawa ◽  
Gerald B. Pier
Keyword(s):  
Cystic Fibrosis ◽  
Cell Line ◽  
Cell Lines ◽  
The Other ◽  
Transmembrane Conductance Regulator ◽  
Wild Type ◽  
Transmembrane Conductance ◽  
Bronchial Epithelial ◽  
Cystic Fibrosis Transmembrane ◽  
In Cells

ABSTRACT Chronic lung infection by Pseudomonas aeruginosa causes significant morbidity in cystic fibrosis patients initiated by the failure of innate immune responses. We used microarray analysis and real-time PCR to detect transcriptional changes associated with cytokine production in isogenic bronchial epithelial cell lines with either wild-type (WT) or mutant cystic fibrosis transmembrane conductance regulator (CFTR) in response to P. aeruginosa infection. The transcription of four NF-κB-regulated cytokine genes was maximal in the presence of WT CFTR: the interleukin-8 (IL-8), IL-6, CXCL1, and intracellular adhesion molecule 1 (ICAM-1) genes. Analysis of protein expression in two cell lines paired for wild-type and mutant CFTR with three P. aeruginosa strains showed IL-6 and IL-8 expressions were consistently enhanced by the presence of WT CFTR in both cell lines with all three strains of P. aeruginosa, although some strains gave small IL-8 increases in cells with mutant CFTR. CXCL1 production showed consistent enhancement in cells with WT CFTR using all three bacterial strains in one cell line, whereas in the other cell line, CXCL1 showed a significant increase in cells with either WT or mutant CFTR. ICAM-1 was unchanged at the protein level in one of the cell lines but did show mild enhancement with WT CFTR in the other cell pair. Inhibitions of NF-κB prior to infection indicated differing degrees of dependence on NF-κB for production of the cytokines, contingent on the cell line. Cytokine effectors of innate immunity to P. aeruginosa were found to be positively influenced by the presence of WT CFTR, indicating a role in resistance to P. aeruginosa infection.

scholarly journals Infection of a Single Cell Line with Distinct Strains of Human Cytomegalovirus Can Result in Large Variations in Virion Production and Facilitate Efficient Screening of Virus Protein Function

2015 ◽  
Vol 90 (5) ◽  
pp. 2523-2535 ◽  
Author(s):  
Anamaria G. Zavala ◽  
John M. O'Dowd ◽  
Elizabeth A. Fortunato
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Human Cytomegalovirus ◽  
Ataxia Telangiectasia ◽  
In Silico ◽  
Virus Protein ◽  
Wild Type ◽  
Ataxia Telangiectasia Mutated ◽  
Virion Production ◽  
Virus Strains

ABSTRACTPreviously, we reported that the absence of the ataxia telangiectasia mutated (ATM) kinase, a critical DNA damage response (DDR) signaling component for double-strand breaks, caused no change in HCMV Towne virion production. Later, others reported decreased AD169 viral titers in the absence of ATM. To address this discrepancy, human foreskin fibroblasts (HFF) and three ATM−lines (GM02530, GM05823, and GM03395) were infected with both Towne and AD169. Two additional ATM−lines (GM02052 and GM03487) were infected with Towne. Remarkably, both previous studies' results were confirmed. However, the increased number of cell lines and infections with both lab-adapted strains confirmed that ATM was not necessary to produce wild-type-level titers in fibroblasts. Instead, interactions between individual virus strains and the cellular microenvironment of the individual ATM−line determined efficiency of virion production. Surprisingly, these two commonly used lab-adapted strains produced drastically different titers in one ATM−cell line, GM05823. The differences in titer suggested a rapid method for identifying genes involved in differential virion production.In silicocomparison of the Towne and AD169 genomes determined a list of 28 probable candidates responsible for the difference. Using serial iterations of an experiment involving virion entry and input genome nuclear trafficking with a panel of related strains, we reduced this list to four (UL129, UL145, UL147, and UL148). As a proof of principle, reintroduction of UL148 largely rescued genome trafficking. Therefore, use of a battery of related strains offers an efficient method to narrow lists of candidate genes affecting various virus life cycle checkpoints.IMPORTANCEHuman cytomegalovirus (HCMV) infection of multiple cell lines lacking ataxia telangiectasia mutated (ATM) protein produced wild-type levels of infectious virus. Interactions between virus strains and the microenvironment of individual ATM−lines determined the efficiency of virion production. Infection of one ATM−cell line, GM05823, produced large titer differentials dependent on the strain used, Towne or AD169. This discrepancy resolved a disagreement in the literature of a requirement for ATM expression and HCMV reproduction. The titer differentials in GM08523 cells were due, in part, to a decreased capacity of AD169 virions to enter the cell and traffic genomes to the nucleus.In silicocomparison of the Towne, AD169, and related variant strains' genomes was coupled with serial iterations of a virus entry experiment, narrowing 28 candidate proteins responsible for the phenotype down to 4. Reintroduction of UL148 significantly rescued genome trafficking. Differential behavior of virus strains can be exploited to elucidate gene function.

Effects of AMN107, a Novel Aminopyrimidine Tyrosine Kinase Inhibitor, on Human Mast Cells Bearing Wild-Type or Mutated Codon 816 c-kit.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3528-3528 ◽  
Author(s):  
Srdan Verstovsek ◽  
Cem Akin ◽  
Giles J. Francis ◽  
Manshouri Taghi ◽  
Ly Huynh ◽  
...  
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitor ◽  
Cell Line ◽  
Cell Lines ◽  
Cellular Proliferation ◽  
Kinase Inhibitor ◽  
Wild Type ◽  
Kit Mutation

Abstract Background. Majority of adult patients with systemic mastocytosis (SM) have activating mutation in codon 816 of c-kit (CD117), a receptor on the surface of mast cells. This abnormality is responsible for the pathogenesis of the disease. Methods. We investigated the effects of a newly designed tyrosine kinase inhibitor, AMN107, by comparing its in vitro inhibitory potency on c-kit mutated mast cell lines and patient samples with that of imatinib mesylate, another tyrosine kinase inhibitor, effective in some patients with SM. Two cell lines, subclones of HMC-1 cells, were used: HMC-1560 carrying juxtamembrane domain mutation in codon 560 of c-kit, and HMC-1560, 816 carrying both codon 560 mutation and tyrosine kinase domain mutation in codon 816 of c-kit. Results. In HMC-1560 mast cell line carrying wild-type codon 816, AMN107 was as potent as imatinib in inhibiting cellular proliferation, with IC50 values of 108 and 74 nM respectively, while in HMC-1560, 816 cell line carrying 816 mutation, neither medication had an effect. AMN107 was also as effective as imatinib in inhibiting phosphorylation of c-kit tyrosine kinase in HMC-1560 cells. The inhibition of cellular proliferation was associated with induction of apoptosis in HMC-1560 cells. AMN107 in concentrations up to 1 uM had no effect on bone marrow mast cells carrying D816V c-kit mutation obtained from patients with mastocytosis. Conclusions. Our results suggest similar potency of AMN107 and imatinib in mast cells that carry wild-type codon 816, but no activity against codon 816 mutation carrying cells.

A Small Molecule Stat Inhibitor Blocks Stat3 and Stat5 Phosphorylation and Demonstrates Cytotoxicity In Acute Lymphoblastic Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2904-2904
Author(s):  
Robyn M. Dennis ◽  
Brandon Ballard ◽  
David John Tweardy ◽  
Karen Rabin
Keyword(s):  
Flow Cytometry ◽  
Western Blot ◽  
Cell Line ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Flow Cytometric Analysis ◽  
Positive Control ◽  
Wild Type ◽  
Atp Assay ◽  
Antibody Staining

Abstract Abstract 2904 Survival has improved dramatically in acute lymphoblastic leukemia (ALL), but further gains are unlikely using conventional chemotherapy alone. Several recently discovered, novel cytogenetic lesions with adverse prognostic impact, JAK2 activating mutations and CRLF2 rearrangements, occur in up to 15% of adult and pediatric ALL. These lesions are associated with activation of Jak2 and Stat5, and hold promise as targets for novel therapies affecting these signaling pathways. We performed in vitro testing of a novel small molecule Stat inhibitor, C188-9, in B-lineage ALL cell lines and patient samples with and without JAK2/CRLF2 alterations. C188-9 treatment for one hour at 10 μM inhibited Stat3 and Stat5 phosphorylation in ALL cell lines with JAK2 and CRLF2 alterations, but not in cell lines with wild-type JAK2 and CRLF2, as measured by phospho-flow cytometry (Fig. 1A). Only the cell lines with JAK2 and CRLF2 alterations demonstrated basal Stat5 phosphorylation on Western blot analysis, and this was inhibited by C188-9 treatment (Fig. 1B). C188-9 demonstrated cytotoxicity in ALL cell lines regardless of JAK2/CRLF2 status, with IC50s in the low micromolar concentration range (Fig. 1C). While C188-9 is undergoing investigation currently as a potent inhibitor of Stat3 in acute myeloid leukemia (AML), it also merits further investigation as an agent with Stat5 inhibitory activity and cytotoxicity in ALL. Figure 1. Effects of C188-9 in ALL cell lines. A. Stat3 and Stat5 phosphorylation were determined by flow cytometry in the ALL cell lines MHH-CALL-4 (JAK2/CRLF2 mutated) and Reh (JAK2/CRLF2 wild-type). In each condition, cells were incubated in serum-free media for one hour, followed by incubation with C188-9 or vehicle for one hour, stimulation with vehicle or pervanadate 125 mM for 15 minutes, fixation, permeabilization, phospho-antibody staining for phospho-Stat3 and phospho-Stat5, and flow cytometric analysis. B. Western blot for phospho-Stat5 in K562 cell line (positive control); MHHCALL-4 treated for one hour with C188-9 at 0, 5, or 10 uM; and RS4;11 (JAK2/CRLF2 wild-type ALL cell line). C. IC50 determination by ATP assay for C188-9 in the ALL cell lines MHH-CALL-4 and RS4;11. Each experiment was performed in triplicate. Figure 1. Effects of C188-9 in ALL cell lines. A. Stat3 and Stat5 phosphorylation were determined by flow cytometry in the ALL cell lines MHH-CALL-4 (JAK2/CRLF2 mutated) and Reh (JAK2/CRLF2 wild-type). In each condition, cells were incubated in serum-free media for one hour, followed by incubation with C188-9 or vehicle for one hour, stimulation with vehicle or pervanadate 125 mM for 15 minutes, fixation, permeabilization, phospho-antibody staining for phospho-Stat3 and phospho-Stat5, and flow cytometric analysis. B. Western blot for phospho-Stat5 in K562 cell line (positive control); MHHCALL-4 treated for one hour with C188-9 at 0, 5, or 10 uM; and RS4;11 (JAK2/CRLF2 wild-type ALL cell line). C. IC50 determination by ATP assay for C188-9 in the ALL cell lines MHH-CALL-4 and RS4;11. Each experiment was performed in triplicate. Disclosures: No relevant conflicts of interest to declare.

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