Isocitrate dehydrogenase 1 (IDH1) mutations are common in gliomas, acute myeloid leukemia, and chondrosarcoma. The mutation ‘hotspot’ is a single arginine residue, R132. The R132H mutant of IDH1 produces the 2-hydroxyglutarate (2-HG) carcinogen from α-ketoglutarate (α-KG). The reduction of α-KG induces the accumulation of hypoxia-inducible factor-1α subunit (HIF-1α) in the cytosol, which is a predisposing factor for carcinogenesis. R132H is the most common IDH1 mutation in humans, but mutations at the R132 residue can also occur in tumor tissues of dogs. The current study reported the discovery of a novel Tyr208Cys (Y208C) mutation in canine IDH1 (cIDH1), which was isolated from 2 of 45 canine chondrosarcoma cases. As the genomic DNA isolated from chondrosarcoma tissue was mutated, but that isolated from blood was not, Y208C mutations were considered to be spontaneous somatic mutations. The isocitrate dehydrogenase activity of the Y208C mutant was attenuated compared with that of wild-type (WT) cIDH1, but the attenuation of Y208C was less intense than that of the R132H mutation. The induction of HIF-1α response element activity and cell retention of HIF-1α were not increased by Y208C overexpression.
Gliomas are intracranial tumors that are thought to develop from astrocytes or oligodendrocytes (
Although intracranial tumors in dogs, such as meningiomas and gliomas, are relatively common brain diseases (
In this study, we found the novel mutation Y208C in cIDH1 by sequencing formalin-fixed paraffin-embedded (FFPE) canine chondrosarcoma tissues. We compared the production ability of NADPH and induction of HIF-1α between the wild-type (WT) and cIDH1 mutants. Furthermore, the dimerization ability necessary to exert the enzyme activity of IDH1 was estimated
The genomic DNA of the FFPE tissue from paraffin scrolls (
With permission from the University Ethics Committee, we obtained tissue samples from the Department of Veterinary Pathology, School of Veterinary Science, Nippon Veterinary and Life Science University (approval no. 11-50, 27 May 2018). All samples were classified by veterinary pathologists according to the World Health Organization classification (
HeLa and MDCK cells were purchased from the American Type Culture Collection (ATCC). The cell lines were maintained in Dulbecco's modified Eagles medium (Wako) supplemented with 10% fetal bovine serum, penicillin, and streptomycin (Applied Biosystems) and incubated at 37°C in a 5% CO2 atmosphere.
To measure the production of NADH and NADPH, cIDH1-transfected cells (5×104) were processed using the Isocitrate Dehydrogenase Activity Colorimetric Assay Kit (BioVision) according to the manufacturer's instructions. The reaction mix was treated for 10 min, and the optical density at 450 nm was measured using an iMark microplate reader (Bio-Rad Laboratories).
HeLa cell transfection was performed in a 96-well plate at 80% confluency. The vector containing the HIF-1α response element pGL4.42[
HeLa cells were harvested in a 24-well plate at a density of 1×105 cells/well and transfected with 250 ng of HA-tagged, full-length WT, R132H, or Y208C mutant of cIDH1 in pMACS Kk.HA-C (Miltenyi Biotec). The assay was performed using the coupled enzymatic assay method according to the manufacturer's instructions (Sigma-Aldrich; Merck KGaA, catalog no. MAK054). In this method, α-KG concentration is determined by a coupled enzyme assay, which results in a colorimetric (570 nm) product that, in turn, is proportional to the amount of α-KG present in the sample.
For the CoCl2 (Wako) experiments to induce HIF-1α expression, 2×105 HeLa cells were seeded in 6-well plates for 24 h before being treated with 100 µM CoCl2 for an additional 24 h.
Immunoblotting was performed using the following primary antibodies: Rabbit polyclonal anti-HA (561, 1:1,000; MBL), anti-β-actin (PM053, 1:2,000; MBL), rabbit polyclonal anti-HIF-1α (#3716, 1:1,000; Cell Signaling Technology), and anti-Halo antibody (G9281, 1:1000; Promega). Horseradish peroxidase-conjugated secondary antibodies and EzWestLumi plus (ATTO) were used for detecting antibody-bound proteins.
We retrieved the crystal structure of the human IDH1 dimer from the Research Collaboratory for Structural Bioinformatics Protein Data Bank at
For the mammalian cell two-hybrid assay (MTH), WT and Y208C cIDH1 cDNA were cloned into the
We cloned the Halo- or HA-tagged, full-length WT, R132H or Y208C mutant into the pFN21A (Promega) or pMACS Kk.HA-C vector, respectively. The expression of the Halo- and HA-tagged constructs in the HeLa cells was induced using FuGENE HD Transfection Reagent (Promega), and the transfected cells were grown for 48 h. The cells were then lysed and pulled down using Mammalian Lysis Buffer (Promega) containing Protease Inhibitor Cocktail (Promega) for 15 min, and cellular debris was cleared by centrifugation at 12,000 × g for 10 min. In total, 50 µl of Magne Halo-Tag Beads (Promega) equilibrated with TBS containing 0.05% IGEPAL CA-630 (TBS+) was added to the supernatant. The samples were incubated for 20 min at 22°C with rotation. The supernatant was discarded, and the protein-captured beads were washed thrice with TBS+ and suspended in SDS-PAGE loading buffer. The samples were analyzed by immunoblotting using anti-Halo or anti-HA antibody and horseradish peroxidase-conjugated anti-rabbit IgG antibody (GE Healthcare). The blots were developed using EzWestLumi plus reagents.
HeLa cells in 6-well plates were transfected with either WT, R132H or Y208C of cIDH1 expression plasmids (1 µg/well). After 48 h of transfection, the cells were lysed with mammalian lysis buffer (Promega) supplemented with a protease inhibitor cocktail (Promega). Post lysis, the samples were centrifuged (15,000 × g for 15 min at 4°C) to obtain the supernatant accordingly. Total protein levels were measured using BCA (Nacalai Tesque). Equal amounts of proteins (100 µg/condition) were then incubated with glutaraldehyde at different concentrations (0, 0.04, 0.1, 0.25 or 0.5%) and incubated on ice for 30 min accordingly. To make a working solution of glutaraldehyde, commercially available 25% glutaraldehyde solution was diluted in PBS and discarded after use. To quench the reaction, sample buffer was added to obtain the following final concentrations: 250 mM Tris-HCl, pH 8.5; 2% lithium dodecyl sulfate; 100 mM DTT; 0.4 mM EDTA; 10% glycerol; and 0.2 mM bromophenol blue. Samples were then separated by SDS-PAGE and the monomer and dimer of cIDH1 was detected using anti-HA antibody accordingly.
NOX activity was evaluated by assessing the superoxide production by lucigenin-enhanced chemiluminescence (
Data are expressed as mean ± standard deviation (SD). Analysis of variance (ANOVA) with a Tukey's post-hoc test was used when multiple comparisons were required. P<0.01 was considered statistically significant.
Genomic DNA was isolated from tumor tissues and blood. PCR amplification of the coding exons of cIDH1 (
The formation of NADPH in multiple types of cIDH1-overexpressing HeLa and MDCK cells was measured using colorimetric analysis. The productivity of NADPH in the R132H mutant of cIDH1 was significantly lower than that in WT, and Y208C showed moderate productivity in both HeLa and MDCK cells (
To evaluate HIF-1α induction by overexpression of the cIDH1 mutant, a reporter assay was performed to measure under the control of a promoter containing hypoxic response element sites (
To predict the functional alteration based on the IDH1 mutation, the protein structure editing tool in the UCSF Chimera software package was used to analyze the possible structural outcomes of Y208C substitutions. Y208 is located adjacent to the binding surface of the IDH1 dimerization form. Y208 showed hydrogen bonds with amino acids belonging to the intra-strand W245, E247, R249, M254, Q257, and W267 (
NOX activity in WT or mutant cIDH1-transfected HeLa cells was assessed. The WT of cIDH1-transfected cells showed a significant increase compared with the empty vector-transfected control, but the R132H and Y208C mutant transfectants did not show (
Two cases of novel Y208C mutations in CS tissues were not detected in the genomic DNA isolated from blood; therefore, the Y208C mutation appeared to be spontaneous somatic mutations. Although the sensitivity for detection by Sanger sequencing of R132 mutation in human cases is sometimes low (
The R132H mutated IDH1 proteins lose normal catalytic activity for α-KG and produce less NADPH. Instead, the abnormal enzymatic activity produces 2-HG and consumes NADPH (
The R132H mutation of IDH1 produces 2-HG from α-KG, which reduces α-KG-dependent prolyl hydroxylases, which regulate HIF-1α levels (
In
IDH1 mutation causes a change in NADPH production, so we predicted that IDH1 mutation may affect NOX activity, which reflects the amount of reactive oxygen species (ROS). To elucidate the cause of tumorigenesis due to the Y208C mutation, we analyzed NOX activity in cIDH1-transfected cells. NOX activity was significantly higher in WT IDH1-transfected cells, and there was no significant difference between the negative control and both R132H and Y208C mutant transfected cells. This result suggests that WT IDH1 could produce more NADPH, a source of ROS, but IDH1 mutants lose the ability to produce more NADPH; therefore, mutant transfected cells could not produce ROS. Since there was no difference in the results of ROS productivity between R132H and Y208C, the mechanism of carcinogenesis of the Y208C mutation remains unclear. This study is the first experimental report to describe the relationship between canine IDH1 mutation and NOX activity. Future studies will need to elucidate the mechanism of tumorigenesis of the Y208C mutation. Furthermore, future studies will have to look for Y208C mutations in various tumors.
In conclusion, we identified for the first time Y208C spontaneous somatic mutations of canine IDH1 in chondrosarcomas and assessed the impact of these mutations on IDH1 functions. Y208C mutation attenuated the NADPH production ability but did not enhance HIF-1α retention in CoCl2-treated cells. This phenomenon was caused by the attenuation of the dimerization ability of the Y208C mutation. We hope that the precise analysis of IDH1 functional changes can help elucidate the tumorigenesis involvement of the Y208C IDH1 mutation.
Not applicable.
This work was supported by KAKENHI scientific research grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (grant nos. 18H02334 and 19K06390).
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
SK, MS, MMi, MMo, KI, MW and KO designed the study and performed the bioinformatics analysis. SK, MU, NK, MMa, YM, DA, ASE, EO and TO performed the laboratory experiments. KO and YT performed statistical analysis and wrote the manuscript. MW, YT, TO and KO supervised the study. All authors have read and approved the final version of the manuscript.
The current study was approved by the University Ethics Committee of the Department of Veterinary Pathology, School of Veterinary Science, Nippon Veterinary and Life Science University (approval no. 11-50, 27 May 2018).
Not applicable.
The authors declare that they have no competing interests.
Detection and characterization of the canine IDH1 Y208C mutation. (A) The amino acid sequence comparison of human (NP_005887.2) and canine (BBC43078.1) IDH1. A total of 401/414 residues were identical. The bold residues represent R132 and Y208, respectively. (B) Eight exons were amplified from genomic DNA isolated from canine CS tissues (left). Electropherogram results demonstrated a single PCR band amplification (right). Amplicon sizes are provided under the left panel. (C) Electropherograms of Sanger sequencing conducted on CS cases 7 and 12. (D) Photomicrographs of canine CS in case number 7, which demonstrates representative CS pathogenesis, as indicated by hematoxylin and eosin staining (scale bar, 50 µm). IDH1, isocitrate dehydrogenase 1; CS, chondrosarcoma; Ex, exon.
Measurement of the functional alterations between WT and mutant cIDH1. (A) Upper graphs indicate the production of NADPH in HeLa (left) and MDCK (right) cells transfected with WT, R132H or Y208C cIDH1, as determined through a colorimetric assay. Western blotting of the lower panel indicates the even expression of transfected cIDH1s, which was fused with HA. **P<0.01 as indicated (n=4). (B) Intracellular levels of α-KG were quantitated using an enzymatic assay and the value of empty vector transfection was 100%. *P<0.05 and **P<0.01 as indicated (n=3). (C) Luciferase assay demonstrating HIF-1α promoter activity in HeLa cells transfected with WT or mutant cIDH1. (D) Western blotting demonstrating HIF-1α retention in HeLa cells transfected with WT and mutant cIDH1 with 10 µM CoCl2. β-actin was used as the loading control. WT, wild-type; cIDH1, canine isocitrate dehydrogenase 1; HA, hemagglutinin; α-KG, ketoglutarate; emp, empty; HIF-1α, hypoxia-inducible factor-1α.
Generation of cellular ROS by NOX in cIDH1-transfected cells. Cellular NOX activity in WT and mutant cIDH1-transfected cells. Temporal ROS generation (RLU/mg protein) by NOX was detected using lucigenin chemiluminescence. Data are presented as the mean ± SD (n=3). **P<0.01 as indicated. ROS, reactive oxygen species; NOX, NADPH oxidase; cIDH1, canine isocitrate dehydrogenase 1; WT, wild-type; emp, empty.
Primer pairs to amplify canine Isocitrate dehydrogenase 1 exons.
Exon | Primer sequences |
---|---|
3 | F: 5′-GCAGCCTCAAAAGCCACACACGC-3′ |
R: 5′-TGTACTTATCTTTAAGCATCCC-3′ | |
4 | F: 5′-CGTTGTGCGCCATCACACAG-3′ |
R: 5′-CACTTAAAGGGAGTAGTCAC-3′ | |
5 | F: 5′-TGATCTTGAGTCTATACCAG-3′ |
R: 5′-TGGCTAGTTCCCTTTGTGTC-3′ | |
6 | F: 5′-GACTTTCTTCCAATCACGTG-3 |
R: 5′-TATGCCCTTAACTTTATGGG-3′ | |
7 | F: 5′-GCCTGATGCAAGACTCGATC-3′ |
R: 5′-TTCATTGATGACTACACATGC-3′ | |
8 | F: 5′-GGACCCTGCTTCCTGAGAGG-3′ |
R: 5′-GGACCCTGCTTCCTGAGAGG-3′ | |
9 | F: 5′-TCTGCTCAACAGCAAGACAG-3′ |
R: 5′-TGACTGTGCTCCTTCCACAG-3′ | |
10 | F: 5′-GTGGCCGAGCTGCCAGTGCAGGC-3′ |
R: 5′-CCTGCCACGTTCACGAGGGTG-3′ |
F, forward; R, reverse.