HMC-1.1 and HMC-1.2 human mast cell lines were cultured in Iscove's Modified Dulbecco's Medium (Lonza, Mississauga, ON, Canada) containing 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin. Cells were maintained at 1  ×  10⁵ cells/ml at 37°C and 5% CO₂. The LAD2 cell line was cultured in StemPro-34 SFM media (Life Technologies, Burlington, ON, Canada) supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 50 mg/ml streptomycin, and 100 ng/ml recombinant human SCF (Peprotech, Rocky Hill, NJ). Cells were maintained at 1  ×  10⁵ cells/ml at 37°C and 5% CO₂ and periodically tested for expression of KIT and FcεRI by flow cytometry. The cells were used within 8 weeks of thawing from cryopreservation.

HMC-1.1, HMC-1.2 and LAD2 cells were seeded at 1.5  ×  10⁴ cells/ml, 2  ×  10⁴ cells/ml and 5  ×  10⁴ cells/ml respectively in fresh media, treated with 100 µM NaBu (Sigma-Aldrich, Oakville, ON, Canada) and counted every 2 days with a hemocytometer using trypan blue exclusion dye. Cells counts and viability were plotted.

HMC-1.1, HMC-1.2 and LAD2 cells were seeded at 5  ×  10⁴ cells/well in a 96-well plate. Following treatment with NaBu (100 µM unless otherwise indicated), 50 µl of (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) or XTT reagent (Roche, Laval, QC, Canada) was added to each well and incubated for 4 h at 37°C and 5% CO₂. Absorbance at A₄₀₅ was measured (Reference A₆₉₀) on a Varioskan plate reader (ThermoFisher Scientific, Mississauga, ON, Canada) and expressed as percent of control (untreated cells).

HMC-1.1, HMC-1.2, and LAD2 cells were suspended at 5  ×  10⁵ cells/ml in fresh media and treated with NaBu (100 µM). Subsequently, 2  ×  10⁵ cells were fixed in ice cold 70% ethanol for 2 h then resuspended in PBS. Cells were then permeabilized, treated with RNase and stained for 30 min with propidium iodide (Sigma-Aldrich) in 0.1% Triton X-100. Fluorescence of stained cells was analyzed on a FACSArray (BD Biosciences, Mississauga, ON, Canada) and cell cycle analyzed using FlowJo software (Tree Star, Ashland, OR). Results are presented as percent of cells in G2/M phase of the cell cycle.

HMC-1.1, HMC-1.2 and LAD2 were treated with NaBu (0–1000 µM) for 3 days at 37°C and 5% CO₂. Total RNA was isolated from each treatment using Trizol Reagent (Sigma-Aldrich) based on manufacturer's protocol and 1 µg of total cellular RNA was reverse transcribed into cDNA using M-MLV Reverse Transcriptase (Life Technologies) and oligo-dT primers (Integrated DNA Technologies, Toronto, ON, Canada). Primer and probe sets for target genes (Table 1) were designed using the Primer Express software (Life Technologies) and expression was analyzed on a StepOne system (Life Technologies) over 40 amplification cycles (15 s for 95°C, 45 s for 60°C). The equivalent of 20 ng of RNA was used in each reaction and performed as duplex reactions; target and GAPDH internal control amplified in the same sample. Data was obtained from three independent experiments and expressed as relative to GAPDH expression.

For analysis of KIT expression, HMC-1.1, HMC-1.2 and LAD2 cells were treated with NaBu (0–1000 µM) for 3 days at 37°C and 5% CO₂, then washed in 0.1% BSA-PBS, resuspended at 2  ×  10⁵ cells/ml in the same buffer, and subsequently incubated for 1 h with either PE-conjugated anti-CD117 antibody (eBioscience, San Diego, CA) or mouse IgG isotype control (eBioscience, San Diego, CA) antibody at 4°C in the dark. Cells were washed twice then resuspended in 0.1% BSA-PBS and measured on a FACSArray Flow Cytometer, and data analysis was performed using FlowJo software. Results are reported as Mean Fluorescent Intensity (MFI).

For LIVE/DEAD analysis and tryptase content, untreated cells or those treated with NaBu as indicated in the figure legends were prepared for flow cytometry using round-bottom 96-well plates (Sarstedt, Montréal, QC, Canada). To measure cell viability, cells were resuspended in 100 µl PBS and stained with 5 µl of 1:40 diluted LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit (Molecular Probes, Waltham, MA United States) for 30 min at 4°C. All cells were fixed using 10% formalin (Sigma-Aldrich) for at least 20 min at room temperature and were resuspended in 2% FBS + 1 mM ethylenediaminetetraacetic acid (EDTA) in PBS prior to analysis. For cells used for intracellular tryptase detection, fixed cells (stained with or without LIVE/DEAD Fixable Near-IR Dead Cell Stain or anti-human CD117-PE antibody) were blocked using 2% FBS + 1 mM EDTA in PBS for at least 16 h at 4°C followed by permeabilization with 0.1% saponin in PBS for 10 min at 4°C. The permeabilized cells were first incubated with 1.75 µl mouse anti-tryptase antibody (clone: G3; Millipore, Etobicoke, ON, Canada) in 0.1% saponin in PBS for 20 min at 4°C followed by staining with 2 µl Alexa Fluor 488-conjugated F(ab')2-goat anti-mouse IgG secondary antibody (Invitrogen, Waltham, MA, United States) for 20 min at 4°C. The cells were resuspended in 2% FBS + 1 mM EDTA in PBS prior to analysis. Compensation controls were prepared as follows: one drop of ArC (Amine Reactive Compensation Beads; Molecular Probes) and 5 µl 1:40 diluted LIVE/DEAD Fixable Near-IR Dead Cell Stain, one drop of ArC Total Antibody Compensation Beads and 2 µl Alexa Fluor 488-conjugated F(ab')2-goat anti-mouse IgG were incubated at room temperature for 30 min protected from light, washed with 1.5 ml PBS, centrifuged at 250  ×  g for 5 min, then resuspended with 250 µl flow buffer to which one drop of the respective negative beads were added. Alternatively, LIVE/DEAD-stained dead cells (positive control) were prepared by initially fixing cells with 10% formalin at 37°C for 15 min followed by staining with 5 µl of 1:40 diluted LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit for 30 min at 4°C and then a final fixation step (10% formalin, 20 min at room temperature). All samples for flow cytometry were analyzed using a LSRFortessa flow cytometer equipped with 405 nm, 488 nm, and 633 nm lasers and a High Throughput Sampler (BD Biosciences) using the following voltages: FSC: 210, SSC: 240, AlexaFluor488: 320, PE: 400, and APC-Cy7: 430. All data analysis was performed using FlowJo v10.2 software (FlowJo LLC, Ashland, OR, United States). The gating procedure involved debris exclusion using SSC-A vs. FSC-A followed by doublet discrimination using FSC-H vs. FSC-A. The percentage of LIVE/DEADLo cells in the single cell populations were used to measure viability. The average number of events analyzed for cell viability were: 0–0.25 mM, 17,196; 0.5 mM, 17,288; 1.0 mM, 16,034; 2.0 mM, 9,953 and the average number of events analyzed for intracellular tryptase expression were: 0–0.1 mM, 45,456; 0.5 mM, 45,654; 1.0 mM, 42,888.

HMC-1.1 and HMC-1.2 cells were either left untreated or were treated with the indicated concentrations of NaBu, trichostatin A (TSA; Cayman Chemical, Ann Arbor, MI, United States), suberoylanilide hydroxamic acid (SAHA; Cayman Chemical), or DMSO (0.0004% final) for four days. The cells were collected in the culture media, pelleted at 200  ×  g for 5 min, washed with HEPES buffer (10 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 5.6 mM glucose, 5.6 mM Na₂HPO₄, 1.8 mM CaCl₂, and 1.3 mM MgSO₄ at a pH of 7.4), and pelleted again at 200  ×  g for 5 min. The cells were resuspended in HEPES buffer to a concentration of 1  ×  10⁶ cells/ml and 0.4–1  ×  10⁵ cells were lysed using 0.05% Triton X-100 for 20 min on ice. In a black microplate, 30 µl cell lysate and 30 µl HEPES buffer or 60 µl of histamine dihydrochloride standards in HEPES buffer (7.8–500 ng/ml; Sigma-Aldrich) were added to duplicate wells, combined with 12 µl 1M sodium hydroxide and 2 µl 10 mg/ml o-phthalaldehyde (Sigma-Aldrich) in methanol, and incubated for 4 min at room temperature. Subsequently, 6 µl 3M hydrochloric acid was used to stop the reaction and the fluorescence intensity was measured using 360 nm (excitation) and 450 nm (emission) settings with a BioTek Synergy H1M plate reader (Winooski, VT, United States). Lower limit of detection for this assay is approximately 5–7 ng/ml (34, 35).

Untreated or NaBu-treated cells were fixed overnight at 4°C using 2% paraformaldehyde +2.5% glutaraldehyde in PBS, pH 7.4 (EM fixation buffer). After repeated washes, the cells were treated with 1% osmium tetroxide in PBS at room temperature for one hour. The cells were then sequentially dehydrated at room temperature with ethanol (30%, 50%, 70%, 90%, and 100%), 10 min per step. The cells were infiltrated with pure LR white in 100% ethanol (1:1 ratio) overnight at room temperature, followed by infiltrated with fresh LR white for one hour, and then embedded with fresh LR white and polymerized at 55°C for 24 h. The cells embedded in resin were sectioned using a microtome generating ultra-thin sections (∼100 nm) on bared copper TEM grids. Thin sections were coated with 3 nm of carbon using a Precision Etching and Coating System (model 682; Gatan, Pleasanton, CA, United States). All bright field TEM images were obtained at 100 kV in H7700 TEM (Hitachi High-Technologies, Tokyo, Japan).

Experiments were conducted at least three independent times, using three independent cultures of cells and three independent stocks of chemicals. All values are presented as mean ± standard error of the mean (SEM). Data were analyzed using Student's t-test. Differences were considered statistically significant as follows: not significant, p > 0.05; *0.05 ≥ p > 0.01; **0.01 ≥ p > 0.001; ***p ≥ 0.001.

Human mast cell lines HMC-1.1 and HMC-1.2 were cultured with 100 µM NaBu and cell proliferation was measured using a standard trypan blue cell counting protocol (Figures 1A–E). NaBu significantly inhibited HMC-1.1 proliferation after six days in culture and by eight days, very few cells were present (less than 10% of those in the untreated group; Figure 1A). NaBu decreased cell viability by approximately 20% and this effect remained consistent for up to eight days (Figure 1B). An examination of cellular metabolic activity showed that NaBu decreased overall metabolic activity of HMC-1.1 after 4 days (Figure 1C) and in a concentration-dependant manner (Figure 1D). NaBu decreased HMC-1.1 metabolic activity by approximately 25% by day four and up to 40% by day eight (Figure 1C). Furthermore, this decrease in metabolic activity was dependent on NaBu concentration and had a calculated IC₅₀ of approximately 602 µM (Figure 1D).

NaBu inhibited cell proliferation and viability of HMC-1.1 cells more potently than HMC-1.2 cells. NaBu inhibited proliferation of HMC-1.2 cells by 50% by day six (Figure 1E) and 70% by day eight, but viability of the HMC-1.2 cells decreased by only 5% by day eight (Figure 1F). Again, metabolic analysis showed that NaBu (100 µM) decreased metabolic activity by 40% by day eight (Figure 1G), and this effect was concentration-dependent (Figure 1H). Therefore, NaBu had similar effects on metabolic activity in both HMC-1.1 and HMC-1.2 cells.

NaBu is sometimes used for cell cycle synchronization (36, 37) because it regulates a number of genes related to the cell cycle and cell division such as H3 histone, C-Ha-ras, ornithine decarboxylase (36), cyclins and the Fas pathway (38). Cell division requires duplication of DNA, and this double diploid state can be quantified by incubating fixed and permeabilized cells with the stoichiometric dye, propidium iodide. Using this approach to measure cell cycle progression of NaBu-treated cells, our data showed that NaBu treatment reduced the number of HMC-1.1 cells that had progressed into the G2 and M phases of the cell cycle, thus reducing the number of cells undergoing cell division (Figures 2A,B). Furthermore, this effect increased with the concentration of NaBu such that 10 µM of NaBu decreased the number of HMC-1.1 cells in the G2 and M phases by 50% and 100 µM almost completely blocked progression of the cells into the G2/M phase (Figure 2C).

NaBu also inhibited the cell cycle progression of HMC-1.2 but this effect was not as pronounced as the effect observed with HMC-1.1 cells. As shown in Figure 2D, only the highest concentration tested of NaBu (100 µM) led to a significant decrease in the percentage of cells in the G2/M, which corresponded to approximately 50% fewer cells continuing through the cell cycle (Figure 2D).

Due to its ability to regulate the expression of several growth receptors (39, 40) and differentiation factors (41), NaBu is sometimes used to differentiate transformed cell lines into more mature phenotypes. We hypothesized that NaBu might inhibit the proliferation and division of HMC-1 cells by modulating the expression of C-KIT; which encodes a critical receptor tyrosine kinase in mast cell growth, differentiation, and function (25). Treatment with 100 µM NaBu inhibited expression of C-KIT mRNA in HMC-1.1 cells by greater than 70% (Figure 3A) and this effect required at least three days of culture with NaBu (Figure 3B).

Since changes in mRNA expression do not always correlate with changes in protein expression (42), particularly in HMC-1 cell lines (43), flow cytometry was used to measure the surface expression of the KIT receptor during NaBu treatment. NaBu (0–100 µM) treatment decreased the expression of KIT protein (Figure 3C) on the surface of HMC-1.1 cells by 50%, and this effect was observed after three days of treatment (Figure 3D).

Similar to its effect on HMC-1.1 cells, NaBu downregulated the expression of C-KIT mRNA by HMC-1.2 cells in a concentration- and time-dependent manner (Figures 3E,F). NaBu inhibited the expression of the KIT receptor on the surface of HMC-1.2 cells (Figure 3G) by 30%, and this occurred after three days of treatment (Figure 3H). However, compared to HMC-1.1, the effect of NaBu on HMC-1.2 KIT expression was less pronounced, requiring higher concentrations and longer treatment times for similar levels of inhibition.

LAD2 is a mast cell line that is considered to have a more mature phenotype than HMC-1 cells since LAD2 cells have a functional FcεR1 receptor and the ability to degranulate in response to cross-linked IgE (28). LAD2 do not harbour the V560G or D816V mutations in their KIT receptor and therefore require SCF for survival and proliferation. We next determined whether NaBu had effects on LAD2 cell proliferation and viability.

Similar to HMC-1 cells, NaBu inhibited the proliferation of LAD2 cells by as much as 50% by day eight of treatment (Figure 4A). However, NaBu had little effect on LAD2 cell viability, and 92% of the NaBu-treated cells remained viable on day 4 (Figure 4B). An analysis of metabolic rate showed that NaBu decreased the metabolic activity of LAD2 cells by almost 25% by day six of treatment (Figure 4C), similar to the effect of NaBu on HMC-1.1 and HMC-1.2 cells observed in Figure 1.

Since NaBu arrested the cell cycle in HMC-1 cells, we next determined if NaBu had the same effect on the cell cycle in LAD2 cells. Interestingly, NaBu had no significant effect on LAD2 cell cycle progression to the G2/M phase. However, in both the NaBu-treated and untreated groups (Figure 4D), the majority (more than 70%) of the cells remained in the G1/G0 phase of the cell cycle, perhaps reflecting the slow proliferation and long doubling time (approximately 10 days) of the LAD cell line (28).

Since NaBu had significant effects on C-KIT expression by HMC-1.1 and HMC1.2 cells, we next determined the effect of NaBu on C-KIT expression in LAD2 cells. NaBu inhibited the expression of C-KIT mRNA in LAD2 cells in both a concentration- and time-dependent manner. NaBu treatment decreased the expression of C-KIT compared to untreated (Figure 4E), and by three days of treatment C-KIT expression decreased by approximately 50% (Figure 4F).

NaBu also decreased the surface expression of KIT protein on LAD2 cells in both a concentration- and time-dependent manner. NaBu treatment reduced the expression of KIT on the surface of LAD2 cells by 10% (10 µM NaBu) and 17% (1000 µM NaBu; Figure 4G) compared to untreated cells. The inhibitory effect of NaBu on KIT surface expression increased with treatment time and reduced KIT expression by 17% by day 4 (Figure 4H).

Since NaBu decreased the proliferation and metabolic activity of HMC-1 cells, we next determined whether NaBu could modify some of the hallmarks of mast cell differentiation, such as tryptase and histamine expression. NaBu has been used to differentiate HL-60 into eosinophil-like cells, but usually at quite high concentrations of 500 µM or higher (44). Therefore, for the next set of experiments, HMC-1.1 and HMC-1.2 were treated with higher concentrations of NaBu (100 µM, 500 µM and 1.0 mM) to determine whether higher concentrations would have more potent effects on HMC-1 differentiation. Although 500 µM NaBu had no significant effect on HMC-1.1 viability, it significantly decreased HMC-1.2 cell viability by day eight (Supplementary Figures S1A,B). Similarly, flow cytometric analysis of cell viability indicated that 500 µM and 1.0 µM decreased viability of HMC-1.2 cells by day eight.

A preliminary analysis of tryptase content in HMC-1 sublines indicated that HMC-1.1 contain much higher amounts of tryptase than HMC-1.2 (Figures 5A,B). NaBu did not increase tryptase in HMC-1.1 cells (Figure 5A). However, NaBu treatment moderately increased tryptase content of HM-1.2 cells (Figures 5B, 5C) when they were treated with 1.0 mM NaBu.

Similar to the tryptase data, HMC-1.1 contained approximately two times more histamine per cell than HMC-1.2 (Figure 5D). NaBu treatment increased histamine content of HMC-1.2 cells in a concentration-dependent manner, reaching maximum induction at 500 µM NaBu (Figure 5D). NaBu did not increase histamine content of HMC-1.1, and although there was a trend of increased histamine content of HMC-1.1 cells treated with 1.0 mM NaBu, this was not significant when compared to the untreated cells. Lastly, two other HDACi, trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA), were tested for their ability to modify histamine content in HMC-1 cells (Figure 5E). TSA increased histamine in HMC-1.2 cells but had no effect on HMC-1.1. SAHA had no significant effect on either HMC-1.1 or HMC-1.2 compared to untreated cells.

Since NaBu caused an increase of histamine and tryptase content, we determined whether this was associated with ultrastructural changes with secretory granules in the cytoplasm. Figure 6 shows the intracellular ultrastructure of HMC-1.1 and HMC-1.2 untreated or treated with NaBu. We identified and classified granules into five types according to their ultrastructure (I, II, III, IV and V). The majority of granules in the cytoplasm of untreated HMC-1.1 have electron-dense core surrounded by sparse particulates (type I, Figures 6A–D). With NaBu treatment the electron-dense core vanished and the granules became more electron-lucent (type II, Figures 6 B&E, C&F). Interestingly, extracellular vesicles with electron-dense core were observed after 1.0 mM NaBu treatment (blue arrows in Figure 6F). Whereas, granules in untreated HMC-1.2 are uniformly filled with 60–80 nm particulates (type III, Figures 6G–J), which making the membrane of granules is hardly to tell apart from cytosol at lower magnification. After treatment with 0.5 mM NaBu, large size particulates (100–250 nm) increased but still sparsely distributed within compartments (type IV, Figures 6H–K) and some vesicles appeared in the granules. Multivesicular body (MVB) were also observed (black arrow, Figure 6H). While with 1.0 mM NaBu treatment, scroll-like or multilamellar vesicles (MLV) are shown in the majority of granules (type V, Figures 6I–L).