br Modulation of alternative pre
Modulation of alternative pre-mRNA splicing with ceramide allows Biochemical Pharmacology 160 (2019) 134–145
cancer WAY 316606 to express pro-apoptotic isoforms of BCL-x and caspase-9 [25,26], and even to restore wt p53 levels and functions in cancer cells carrying a p53-deletion mutation . In pre-mRNA, adenosine can be methylated by a methyltransferase complex containing methyl-transferase-like 3 (METTL3) to form N6-methyladenosine (m6A) [28,29]. Emerging evidence implicates that the m6A reader protein YTHDF1 selectively binds m6A-modified pre-mRNAs, with higher affi-nity, and that this can in turn cause alternative splicing, thereby al-tering protein translation and levels thereof, thus bringing about m6A-dependent deleterious changes in protein expression in cancer cells [28,30]. Herein, we report our study results of the methylation of adenosine to generate m6A in R273H missense mutant of p53 pre-mRNA transcripts, and consequent preferential effects on mutant pro-tein expression and oncogenic GOF in association with cancer drug resistance.
2. Materials and methods
2.1. Cell lines and cell culture
Human colon cancer SW48 cells, and also cells of its corresponding heterozygous TP53 missense mutant line (SW48/TP53 (R273H/+), in-troduced by a CRISPR/Cas9 editing system), were purchased from Horizon Discovery (HD 103-008, Waterbeach, Cambridge, UK) . SW48-Dox and TP53-Dox sublines of SW48 and SW48/TP53 (R273H/+) cells were generated by culture in 10% FBS RPMI-1640 medium in-cluding 2 mM L-glutamine and 25 mM sodium bicarbonate, supple-mented with 10% FBS and 800 µg/mL geneticin (G418), with the addi-tion of 25 nM doxorubicin (Dox) for 16 weeks (∼26 passages) . SW48-5FU and TP53-5FU sublines of SW48 and SW48/TP53 (R273H/+), respectively, were cultured in medium including 1 µM 5-fluorouracil (5-FU) for 16 weeks (∼27 passages). Human WiDr (homozygous R273H p53) colon cancer cells were purchased from ATCC (Manassas, VA, USA). Cells were cultured in RPMI-1640 or ATCC-formulated EMEM containing 10% fetal bovine serum (FBS), 100 units/mL penicillin, 100 µg/mL streptomycin and 584 mg/L L-glutamine, and maintained in an incubator humidified with 95% air and 5% CO2 at 37 °C. Cell culture medium and supplementary materials were purchased from Thermo Fisher Scientific (Waltham, MA, USA) unless otherwise stated. The cell lines were ana-lyzed by short tandem repeat (STR) profiling (Gene Resources Core Fa-cility, Johns Hopkins University) and compared against publically available databases (DSMZ and ATCC) for authentication.
2.2. Cell viability assay
Cell viability was determined by quantitation of ATP, an indicator of live cells, using the CellTiter-Glo luminescent cell viability assay kit (Promega, Madison, WI, USA), as described previously [19,32]. Briefly, cells (4000 cells/well) were grown in 96-well plates with 10% FBS supplemented RPMI-1640 medium. Cells were treated with indicated agents in 5% FBS medium for 72 h. For combination treatments, cells (3 × 106/100-mm dish; 4000 cells/well in 96-well plates) were pre-treated with D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-pro-panol (PDMP; 5 µM) or neplanocin A (NPC, 20 nM), fumonisin B1 (FB1, 25 µM), Shiga toxin 1B-subunit (STxB, 100 nM), PP2 (500 nM) and FH535 (5 µM) in 5% FBS medium for 48 h. ATP quantitation for cell viability assessment was accomplished with a Synergy HT microplate reader (BioTek, Winnooski, VT, USA), following incubation with Cell-Titer-Glo reagent.
2.3. Induction and detection of apoptosis
Apoptosis was carried out using an APO-DIRECT kit (BD Biosciences, San Jose, CA, USA) and propidium iodide (PI), as described previously [32,33]. Briefly, cells were pretreated with NPC (20 nM) or vehicle in 5% FBS medium for 3 days, and then co-treated with NPC and
Dox (100 nM) for an additional 48 h to induce apoptosis. After fixing with paraformaldehyde (1% w/v) and 70% ethanol, cells (1 × 106 cells/ml) were incubated with FITC-labeled Br-dUTP (5 μg/ 100 μl) and then PI (5 μg/100 μl) in staining buffer containing RNase for apoptosis detection. Cells were then analyzed using an imaging flow cytometry system, namely an ImageStreamX Mark II with high-resolu-tion microscope (Amnis, Seattle, WA, USA). For each sample, 5000 events were counted in triplicate. Data were analyzed with the IDEAS® Software package (Amnis).
2.4. N6-Methyladenosine RNA immunoprecipitation (MeRIP) and RT-qPCR
After treatments, total RNA was extracted from cells of SW48-Dox, TP53-Dox and WiDr cell lines using SV total RNA isolation kits (Promega). MeRIP was performed using the Magna MeRIP m6A kit (Cat.
# 17-10499, EMD Millipore, Billerica, MA, USA), as described in pre-vious studies [28,34] and per kit instructions. Briefly, equal amounts of total RNA (300 µg in 1 µg/µl aliquots) were fragmented with heating in a thermocycler, extracted, and validated with 1.5% agarose gel elec-trophoresis, and then quantitated via NanoDrop spectrometry. Frag-mented RNA samples (∼100 nt, 280 µg) were incubated with anti-m6A antibody (clone 17-3-4-1; 10 µg) bound-protein A/G magnetic beads in IP buffer for 2 h at 4 °C, eluted with elution buffer, and purified in RNeasy mini-columns. For control experiments, MeRIP was carried out with mouse IgG replacing m6A antibody, under the same conditions.