Author Affiliations
- Nikki A. Evensen1,
- P. Pallavi Madhusoodhan1,
- Julia Meyer2,
- Jason Saliba1,
- Ashfiyah Chowdhury1,
- David J. Araten3,
- Jacob Nersting4,
- Teena Bhatla1,
- Tiffaney L. Vincent5,
- David Teachey5,
- Stephen P. Hunger5,
- Jun Yang6,
- Kjeld Schmiegelow4 and
- William L. Carroll1⇑
- 1Departments of Pediatrics and Pathology, Perlmutter Cancer Center, NYU-Langone Medical Center, New York, NY, USA
- 2Huntsman Cancer Institute, University of Utah Medical Center, Salt Lake City, USA
- 3Department of Medicine, Perlmutter Cancer Center, NYU-Langone Medical Center, New York NY, USA
- 4Department of Pediatrics and Adolescent Medicine, The University Hospital Rigshospitalet, Copenhagen, Denmark
- 5Department of Pediatrics and the Center for Childhood Cancer Research, Children’s Hospital of Philadelphia and The Perelman School of Medicine at The University of Pennsylvania, Philadelphia, PA, USA
- 6St. Jude Children’s Research Hospital, Memphis, TN, USA
- Correspondence:
william.carroll{at}nyumc.org
Data supplements
Evensen et al. Supplementary Appendix
Evensen et al. Supplementary appendix provided by the authors.
Files in this Data Supplement:
Disclosures and Contributions
Files in this Data Supplement:
ARTICLE FIGURES & DATA
Figures
- Figure 1.
Knockdown of MSH6 in mismatch repair (MMR) proficient cells lead to decreased sensitivity to thiopurines. (A-C, left) Western blot analysis of whole cell lysates from 697 (A), UOCB1 (B), and Reh and RS4;11 (C). (A-C, right) Apoptotic cells measured by Annexin V and 7AAD staining followed by flow cytometry after 5 days of treatment. Graphs represent 3 experiments each performed with duplicates. Bars indicate mean+Standard Deviation.
- Figure 2.
Knockdown of MSH6 leads to increased tolerance of incorporated thioguanine (TGN). (A and B) TGN incorporation into DNA was measured over time after treatment with 0.1 μg/mL of 6-thioguanine (6-TG) in 697 (A) and Reh (B) cells using liquid chromatography-tandem mass spectrometry. A representative graph from 3 independent experiments is shown. (C) Combined ratio of TGN fmole/μg of DNA in MSH6 shRNA1 knockdown (KD) cells compared to non-targeting (NT) cells from 3 experiments. Bars indicate mean+Standard Deviation.
- Figure 3.
Thiopurine treatment resulted in an S phase arrest, which was abrogated upon knockdown of MSH6. 697 (A) and UOCB1 (B) NT and MSH6 shRNA1 and 2 expressing cells were treated with indicated drug for 5 days. Cells were fixed with 70% ethanol, treated with RNAse, and then stained with propidium iodide. DNA content was analyzed by flow cytometry. Representative images from 3 individual experiments are shown. A one-way ANOVA was performed to determine statistical significance of the increase in % of cells in S phase at each time point.
- Figure 4.
Thiopurine treatment leads to activation of cell cycle regulator Chk1 and DNA repair that ultimately resulted in DNA damage and cell death. Western blot analysis of whole cell lysates from 697 (A) and UOCB1 (B) non-targeting (NT), MSH6 shRNA1, and shRNA2 cells after treatment with 6-thioguanine (0.1 μg/mL, and 0.025 μg/mL, respectively). (C) Untreated cells; numbers are hours after treatment. Blots were probed for Chk1 activation, γH2AX for DNA damage, and apoptosis marker p53. Total Chk1, actin, and total H2AX were used as loading controls. Images are representative of 3 individual experiments.
- Figure 5.
Knockdown of MSH6 did not lead to a mutator phenotype or increased mutation rate. (A) Microsatellite instability (MSI) was measured in diagnosis/relapse pairs that had relapse specific, heterozygous MSH6 deletions and in 697 non-targeting (NT) and MSH6 shRNA1 cells left untreated or treated with 0.05 μg/mL of 6-thioguanine (6-TG) for 5 days. (B) Mutation rate in the PIC-A gene was measured in 697 NT and MSH6 shRNA1 clones that were expanded for 2–3 weeks with or without 6-TG. The cells were analyzed for loss of GPI-dependent cell surface markers, including FLAER, CD48, CD52, and CD59 using flow cytometry. (Left) Individual mutation rates/cell divisions for each clone; the line represents mean+Standard Deviation. (Right) Mutation rates/cell divisions for three clones with and without 6-TG treatment.
- Figure 6.
Knockdown of MSH6 leads to decreased sensitivity to purixan in vivo. (A) Bioluminescence imaging (BLI) of mice injected with UOCB1 non-targeting (NT) or MSH6 shRNA1 cells. Six days after injection mice were imaged and then randomized to treatment. Treatment was started on day 7 and images were taken again on days 13 and 17. C: PBS control treatment; T: purixan treatment. (B) Quantification of total flux was determined by analyzing the BLI images using Living Image software. (C) Western blot to confirm knockdown of MSH6 in cells used to inject mice. Actin was used as loading control.
Vol 103 Issue 5
