Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 Aug;3(8):774-82.
doi: 10.18632/oncotarget.577.

Mutant IDH1 is required for IDH1 mutated tumor cell growth

Genglin Jin  1 , Christopher J Pirozzi, Lee H Chen, Giselle Y Lopez, Christopher G Duncan, Jie Feng, Ivan Spasojevic, Darell D Bigner, Yiping He, Hai Yan
Affiliations

Mutant IDH1 is required for IDH1 mutated tumor cell growth

Genglin Jin et al. Oncotarget. 2012 Aug.

Abstract

Frequent somatic hotspot mutations in isocitrate dehydrogenase 1 (IDH1) have been identified in gliomas, acute myeloid leukemias, chondrosarcomas, and other cancers, providing a likely avenue for targeted cancer therapy. However, whether mutant IDH1 protein is required for maintaining IDH1 mutated tumor cell growth remains unknown. Here, using a genetically engineered inducible system, we report that selective suppression of endogenous mutant IDH1 expression in HT1080, a fibrosarcoma cell line with a native IDH1(R132C) heterozygous mutation, significantly inhibits cell proliferation and decreases clonogenic potential. Our findings offer insights into changes that may contribute to the inhibition of cell proliferation and offer a strong preclinical rationale for utilizing mutant IDH1 as a valid therapeutic target.

PubMed Disclaimer

Conflict of interest statement

H. Yan receives research funding and a consulting fee from Sanofi-Aventis for research related to IDH1 mutations.

Figures

Figure 1
Figure 1. Establishment of IDH1 inducible knockdown HT1080 cell lines
A, Western blot detection of IDH1 in three different clones following treatment with 200ng/mL tetracycline for 96 hours. B, D-2HG concentration in cell lysates following treatment with 200ng/mL tetracycline for 96 hours.
Figure 2
Figure 2. Knocking down IDH1 expression inhibits HT1080 cell growth
A, 50,000 cells were seeded into 100mm cell culture dishes, with 0 or 200ng/mL tetracycline. Cells were collected and counted daily for five days. Studies were done with biological triplicates. B, 300 cells were plated into 60mm cell culture dishes. After 8-10 days, cell colonies were stained with 0.05% crystal violet. Every treatment was completed in biological triplicate. Representative cell culture dishes are shown.
Figure 3
Figure 3. Generation of cell lines with inducible knockdown of mutant or wild type IDH1
A, Strategy for generating cell lines with inducible knockdown of mutant or wild type IDH1. IDH1 or IDH1R132C full-length cDNA containing a V5 tag and lacking the 3’ non-coding sequence was infected into HT1080 cells and selected for to generate stable cell lines that exogenously express IDH1 or IDH1R132C. A tetracycline-inducible knockdown vector containing a shRNA sequence specific for the IDH1 3’ non-coding sequence was used to infect the stable cell lines that exogenously express IDH1 or IDH1R132C. Following treatment with tetracycline, the endogenous pools of wild type and mutant IDH1 were ablated, while expression of exogenous IDH1 or IDH1R132C remained intact; thereby allowing for the study of the effects of wild type or mutant IDH1 knockdown. B, Cell lines with knockdown of either wild type IDH1 or IDH1R132C as identified by Western blot following a 96 hour treatment with tetracycline. C, D-2HG concentration in cell lysates following 200ng/mL tetracycline treatment for 96 hours.
Figure 4
Figure 4. Knocking down mutant IDH1R132C inhibits cell growth in vitro
A, 50,000 cells with exogenous wild type IDH1 expression and inducible knockdown of endogenous IDH1 were seeded in 100mm cell culture dishes in triplicate and treated with 0 or 200ng/mL tetracycline. Cells were collected and counted daily for 5 days. Error bars represent one standard deviation. B, 50,000 cells with exogenous IDH1R132C expression and inducible knockdown of endogenous IDH1 were seeded in 100mm cell culture dishes in triplicate and treated with 0 or 200ng/mL tetracycline. Cells were collected and counted daily for 6 days. C, 300 cells were plated in 60mm cell culture dishes in triplicate. After 8-10 days treatment, cell colonies were stained with 0.05% crystal violet and counted. D, Indicated cell lines were treated with 0 or 200ng/mL for four days in triplicate and fixed. Cell cycle analysis was completed via FACS.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Parsons DW, Jones S, Zhang X, Lin JC, Leary RJ, Angenendt P, Mankoo P, Carter H, Siu IM, Gallia GL, Olivi A, McLendon R, Rasheed BA, Keir S, Nikolskaya T, Nikolsky Y, Busam DA, Tekleab H, Diaz LA, Jr., Hartigan J, Smith DR, Strausberg RL, Marie SK, Shinjo SM, Yan H, Riggins GJ, Bigner DD, Karchin R, Papadopoulos N, Parmigiani G, Vogelstein B, Velculescu VE, Kinzler KW. An integrated genomic analysis of human glioblastoma multiforme. Science. 2008;321:1807–1812. - PMC - PubMed
    1. Balss J, Meyer J, Mueller W, Korshunov A, Hartmann C, von Deimling A. Analysis of the IDH1 codon 132 mutation in brain tumors. Acta Neuropathol. 2008;116:597–602. - PubMed
    1. Yan H, Parsons DW, Jin G, McLendon R, Rasheed BA, Yuan W, Kos I, Batinic-Haberle I, Jones S, Riggins GJ, Friedman H, Friedman A, Reardon D, Herndon J, Kinzler KW, Velculescu VE, Vogelstein B, Bigner DD. IDH1 and IDH2 mutations in gliomas. N Engl J Med. 2009;360:765–773. - PMC - PubMed
    1. Bleeker FE, Lamba S, Leenstra S, Troost D, Hulsebos T, Vandertop WP, Frattini M, Molinari F, Knowles M, Cerrato A, Rodolfo M, Scarpa A, Felicioni L, Buttitta F, Malatesta S, Marchetti A, Bardelli A. IDH1 mutations at residue p.R132 (IDH1(R132)) occur frequently in high-grade gliomas but not in other solid tumors. Hum Mutat. 2009;30:7–11. - PubMed
    1. Hartmann C, Meyer J, Balss J, Capper D, Mueller W, Christians A, Felsberg J, Wolter M, Mawrin C, Wick W, Weller M, Herold-Mende C, Unterberg A, Jeuken JW, Wesseling P, Reifenberger G, von Deimling A. Type and frequency of IDH1 and IDH2 mutations are related to astrocytic and oligodendroglial differentiation and age: a study of 1,010 diffuse gliomas. Acta Neuropathol. 2009;118:469–474. - PubMed

Publication types