Saturday, June 10, 2023

Targeting Poly(ADP)ribose polymerase in BCR/ABL1-positive cells – Scientific Reports

Cells and cell culture

KOPN30, BV173, and K562 are BCR/ABL1-positive leukemia cell lines. All leukemia cell lines, as well as Ba/F3 cells, were maintained in RPMI-1640 medium supplemented with 15% fetal bovine serum (FBS) and penicillin–streptomycin (100 U/mL) at 37 °C in an atmosphere containing 5% CO2. KOPN30 cells were obtained from the University of Yamanashi School of Medicine (Yamanashi, Japan). BV173 and Ba/F3 cells were obtained from DSMZ (Braunschweig, Germany). K562 cells were obtained from the JCRB cell Bank (Osaka, Japan). Rat-1 cells were obtained from RIKEN cell bank (Tsukuba, Japan). All cell lines were tested for mycoplasma contamination. Rat-1 cells and the fibroblast line MRC5SV harboring a single integrated copy of DR-GFP (DR-GFP MRC5SV) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% FBS and penicillin–streptomycin (100 units/mL) at 37 °C in an atmosphere containing 5% CO2.

DR-GFP assay

The BCR/ABL1-expressing plasmid was constructed by subcloning BCR/ABL1into the MSCV plasmid. The DR-GFP assay was performed as previously described9. Briefly, mock or BCR/ABL1-expressing plasmids were transiently transfected into single-copy DR-GFP-integrated MRC5SV cells using X-tremeGENE 9 (Roche, Basel, Switzerland). On the next day, cells were transfected with the I-SceI expression vector pCBAS. GFP expression was monitored by flow cytometry 48 h after transfecting cells with pCBAS.

Cell death analyses

The percentage of apoptotic cells was measured by flow cytometry after staining with a combination of Annexin V (Abcam, Cambridge, MA) and propidium iodide10. Growth-inhibitory effects were assessed using a Cell Counting Kit (Dojindo, Kumamoto, Japan). A combination index, used to assess synergistic effects, was calculated using CompuSyn software11.

Rat-1 cell transformation assay

A BCR/ABL1-mediated transformation assay using Rat-1 cells was performed as previously described12.Colony number was counted on day 21. Colony-forming activity was also measured using a CytoSelect™ 96 well Cell Transformation Assay Kit (Cell Biolabs, San Diego, CA).

Isolation of adult long-term repopulating hematopoietic stem cells (LTR-HSCs)

HSCs (Linc-kit+Sca1+CD105+ cell) were harvested from 6 to 10-week-old C57BL/6 mice 4 days after intraperitoneal injection of 5-fluorouracil (5-FU; 150 mg/kg). Cells were isolated using CD105 MultiSort Kit (PE), mouse (Miltenyi Biotech, Bergisch Gladbach, Germany). Next, cells (2 × 105 cells/4 mL MethoCult M3434 (Stem Cell Technology, Vancouver, Canada)/Iscove’s MDM supplemented with 2% fetal bovine serum (FBS)) were placed in a Petri dish and incubated for 12 days. Next, cells were harvested, and 2 × 105 cells were reincubated under the same conditions.

Detection of leukemic stem cells using flow cytometry

Leukemic stem cells were labeled with ALDEFLUOR reagent (STEMCELL Technologies, Vancouver, Canada). Cells were stained with allophycocyanin (APC)-conjugated anti-CD38, APC-cyanin-7 (APC-Cy-7)-conjugated anti-CD34, and Brilliant Violet421 (BV421)-conjugated anti-CD133 antibodies (all from BD Biosciences, Franklin Lakes, NJ) for 30 min at 4 °C. Cells were then washed and resuspended in AKDEFLUOR assay buffer and 7-Actinomycin D (7-AAD). Next, cells were analyzed by flow cytometry using a BD Fortessa flow cytometer (BD Biosciences).

Breakage-fusion-bridge (BFB) formation assay

Detection of nucleoplasmic bridges was used to assess BFB frequency; the assay was optimized for mouse cells as described previously13. Briefly, mouse HSCs were isolated using immunomagnetic columns as described above (Miltenyi Biotech) and then cultured in αMEM supplemented with 20% FCS, 50 ng/mL mouse SCF, 50 ng/mL mouse FLT3 ligand, 50 ng/mL human IL-6, and 50 ng/mL human TPO. Next, cells were exposed to 2 Gy X-ray irradiation and cultured for 48 h, followed by addition of cytochalasin-D (0.6 μg/mL) for 24 h. Then, cells were released from cytochalasin-D treatment for 2 h and exposed to cold hypotonic (0.075 M KCl) solution. Finally, cells were fixed in Carnoy fluid, dropped onto slides, stained with DAPI, and examined using a fluorescent microscope at a magnification of × 400.

Immunoprecipitation and western blotting

Cells were lysed with RIPA buffer [50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 0.1% (v/v) sodium dodecyl sulfate, 1% (v/v) Nonidet-P40, and 0.04% (v/v) sodium deoxycholate] and immunoprecipitated with an anti-TBK1 antibody (Cell Signaling Technology, Danvers, MA). Precipitates were blotted using an antiphospho-TBK1 antibody (Cell Signaling Technology). After washing, primary antibodies were detected with horseradish peroxidase (HRP)-conjugated antirabbit or antimouse secondary antibodies and an ECL kit (GE Healthcare, Chicago, IL). Images of uncropped blots are provided in Supplemental data 13.

Gene expression analysis

Libraries for RNA sequencing were prepared using the NEBNext Ultra RNA Library Prep kit from Illumina (New England BioLabs) and sequenced using an Illumina NovaSeq 6000 platform in a 100–150 bp paired-end mode. Sequence reads were aligned to GRCh37 using STAR 2.7.8a14. Count data were calculated using featureCounts and normalized using edgeR15. The results of RNA sequencing were further analyzed by gene set enrichment analysis (GSEA; version 4.1.0)16.

Immunofluorescence microscopy

Cells were stained with pico488 DNA quantification regent (Lumiprobe Life ACience Solutions, Wan Chai, Hong Kong) at 37 ℃ for 2 h. After washing with PBS, cells were fixed at room temperature for 10 min in 4% formaldehyde/PBS and permeabilized at room temperature for 20 min with 0.25% Triton/PBS. Blocking was performed at room temperature for 30 min with 5% BSA and 0.1% Triton/PBS. Cells were incubated with an anti-cGAS antibody (Santa Cruz Biotechnology, Heidelberg, Germany) in blocking buffer at 4 ℃ for 1 h. After washing with PBS, cells were incubated with Goat anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 647 (Invitrogen, Massachusetts, USA). Plates were imaged using TCS SP8 (Leica, Wetzlar, Germany), and images were analyzed using ImageJ/Fiji software.

Quantitative real-time PCR (qPCR)

Total RNA was extracted using the RNeasy Mini Kit (QIAGEN, Hilden, Germany) and reverse transcribed to cDNA using the SuperScript III First-Strand Synthesis System (Invitrogen). Next, qPCR was performed using the LightCycler 480 SYBR Green I Master (Roche). The primers used for qPCR have been reported previously17.


BCR/ABL1 heterozygous transgenic mice (designated as BATg/−), originally on a DBA2 × C57BL/6 background18, and Parp1 heterozygous knockout mice (designated as Parp1+/−) on a 129Svj × C57BL/6 background19 were backcrossed for more than 15 generations to bring them close to a C57BL/6 background. Next, the two mouse strains were crossed with each other. BCR/ABL1 heterozygous transgenic mice harboring the Parp1 wild-type allele (designated as Parp1+/+BATg/−) or the Parp1 homozygous knockout allele (designated as Parp1−/−BATg/−), or Parp1 homozygous knockout mice (designated as Parp1−/−), were used for the study. All mice were bred in a specific pathogen-free unit sited in the vivarium of Tokyo Medical and Dental University. Animal care and use complied with ARRIVE guidelines and were approved by the Tokyo Medical and Dental University animal care and use committee (Protocol Number 0130266A). All methods were performed in accordance with relevant institutional guidelines and regulations.

Hematopoietic stem cell (HSC) transplantation and transduction of BCR/ABL1

The BCR/ABL1-expressing plasmid was constructed by subcloning BCR/ABL1 into the MSCV-IRES-GFP plasmid. Plat-E cells20, an ecotropic packaging cell line, were transfected with MSCV-BCR/ABL1-IRES-GFP using polyethyleneimine. Supernatants containing high titers of retrovirus were collected at 48 and 72 h and concentrated using a Retro-X Concentrator (TAKARA-Clontech, Ohtsu, Japan). LTR-HSCs were cultured overnight in αMEM supplemented with 20% FBS plus 50 ng/mL each of mouse stem cell factor (SCF), human IL-6, human FLT3 ligand, and human thrombopoietin (TPO). On Day 2, cells were placed in 24-well dishes coated with RetroNectin (TAKARA-Clontech, Shiga, Japan) and infected with concentrated retrovirus particles. At 60 h postinfection, retrovirus-infected LTR-HSCs were transplanted into mice that had received (6 h earlier) myeloablative conditioning with 9.5 Gy total body irradiation. Mice were allowed access (ad libitum) to water containing 1 mg/mL neomycin trisulfate salt hydrate and 100 U/mL polymyxin B sulfate salt.

Statistical analysis

P-values for the DR-GFP, apoptosis, cell survival, transformation, leukemic stem cell detection, and qPCR assays were calculated using a t test. Survival curves were constructed using the Kaplan–Meier method and analyzed using the log-rank test. All statistical tests were two-sided, and a p-value of < 0.05 was considered significant.

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