Fig. 1
MultiCycle software-transformed histogram of propidium iodide-DNA fluorescence of THP1 cells grown for 24 h in co-culture with A549 cells. Original histogram was obtained using epics XL flow cytometer. Gating was set for the control probe (THP1 cells) and applied to all experimental samples. Cell distribution was quantified using MultiCycle software as subdiploid (‘early’ G0/G1 cells), diploid (G0/G1 peak)-pre-DNA synthesis/resting, S-phase-DNA synthesis, and G2/M-post-DNA-synthesis/mitosis phases
Cell viability was quantified after 24 h of cell growth in CS-conditioned media using mitochondrial-dependent MTT (3-[4,5-dimethylthiazolyl-2] 2,5-diphenyltetrazoliumbromide) reduction to purple formazan, with colorimetric detection (Niks and Otto 1990). Changes in absorbance in viable cells were measured at 570 nm, with 630 nm as a reference wavelength. Cell viability was estimated as a percentage of the control. Time-dependent toxicity was assessed using lactate dehydrogenase (LDH) release assay (LDH cytotoxicity kit, ScienCell, Carlsbad, CA). LDH release to the culture medium was compared to total enzyme activity in sonicated cells.
2.4 Oxidative Stress
Reactive oxygen intermediates were quantified using dichlorodihydrofluorescein diacetate (H2DCFDA; Sigma-Aldrich, St. Louis, MO) (Ubezio and Civoli 1994). Cells were loaded with 5 μM H2DCFDA for 30 min, washed, resuspended in phosphate-buffered saline, and assayed by flow cytometry. Green dichlorofluorescein (DCF) fluorescence was captured on F11 channel of flow cytometer (Epics XL, Coulter Electronics, High Wycombe, UK) and registered as histograms of fluorescence distribution.
2.5 Expression of Acetylated Histone 3 and Extracellular Activation Markers
Acetylated histone H3 levels were measured in formaldehyde (1 %)-fixed and ethanol (70 %)-refixed cells with acetylated histone H3 (AcH3)-specific monoclonal fluorescent antibodies (Acetyl-Histone H3 (Lys9) Antibody; Rabbit mAb Alexa Fluor 488 Conjugate; Cell Signalling Technology Inc., Danvers, MA), corresponding isotype control antibody, and flow cytometry (Coulter Electronics, High Wycombe, UK) detection (Ronzoni et al. 2005).
The percentages of CD14+, HLA-DR+, CD11a+, and CD11b+ THP1 cells were determined using specific monoclonal fluorescent antibodies (Beckman-Coulter, Warsaw, Poland), corresponding isotype controls, and flow cytometry detection. Cells were diluted to 105 cells per ample and 10 μl of a commercial antibody solution was added to cell suspension and allowed to bind for 30 min at room temperature in darkness. The cells were washed with phosphate buffered saline, fixed with CellFIX™ (Becton Dickinson, Oxford, UK) and run on an Epics XL flow cytometer (Coulter Electronics, High Wycombe, UK). Three thousand total events were collected per sample.
2.6 Statistical Analysis
Results were expressed as means ± SD of 6–10 assays. Statistical analysis was performed with a statistics package-Statistica 6.0 software (Statsoft, Cracow, Poland) using one-way or two-way ANOVA followed by Bonferroni post-hoc tests for selected pairs of data. A p value of less than 0.05 was considered statistically significant.
3 Results
Cytotoxicity of CS applied for 24 h to the cells was tested initially by the MTT test (Table 1). In A549 cells grown for 24 h in CS-conditioned medium very significant toxicity was detected. MTT values in this group were lower by about 78 % (p < 0.01), while no toxicity was noticed in THP1 cells. Since cytotoxicity data in the MTT test reflect not only cell damage but also alterations in cell proliferation, CS cytotoxicity to A549 cells was further characterized in time-dependent experiments. Increased LDH levels were found in the culture medium of A549 cells exposed to CS already after 1 h of cell treatment (p < 0.05). Then, LDH levels increased with incubation time to reach 36 % (p < 0.01) of total enzyme activity (sonicated cells) after 24 h of cell treatment.
Table 1
Cigarette smoke (CS) cytotoxicity (MTT test), proliferation (PI-DNA assay), oxidative stress (DCF fluorescence), expression of acetylated histone H3 (flow cytometry) and expression of extracellular markers of immune activation (flow cytometry) in control alveolar epithelial cells (A549) and in human monocyte cell line (THP1) as well as in cells grown in smoke-conditioned medium for 24 h
A549 cells | THP1 cells | |||
|---|---|---|---|---|
Control | CS | Control | CS | |
MTT (% of control) | 100 ± 16 | 22 ± 6** | 100 ± 17 | 118 ± 24 |
LDH (% of total activity) | ||||
1 h | – | 11 ± 5* | – | – |
6 h | – | 18 ± 7** | – | – |
12 h | – | 27 ± 9** | – | – |
24 h | – | 36 ± 11** | – | – |
Oxidative stress (relative units) | ||||
1 h | 100 ± 11 | 844 ± 55** | 100 ± 16 | 153 ± 66** |
6 h | 100 ± 13 | 527 ± 66** | 100 ± 17 | 187 ± 34** |
12 h | 100 ± 17 | 161 ± 41* | 100 ± 21 | 221 ± 42** |
24 h | 100 ± 19 | 62 ± 33* | 100 ± 18 | 266 ± 48** |
Cytotoxicity (% of ‘early’ G0/G1 cells) | 7 ± 3 | 47 ± 11** | 6 ± 3 | 11 ± 6* |
Proliferation (% of S-G2/M cells) | 43 ± 7 | 9 ± 3** | 27 ± 5 | 19 ± 4* |
Expression of acetylated histone H3 (relative units) | 100 ± 14 | 85 ± 17 | 100 ± 19 | 65 ± 17** |
CD14+ (% of cells) | – | – | 5 | 94 ± 11** |
HLA-DR+ (% of cells) | – | – | 5 | 47 ± 9** |
CD11a+ (% of cells) | – | – | 5 | 69 ± 10** |
CD11b+ (% of cells) | – | – | 5 | 46 ± 8** |
We also examined oxidative stress in cells grown in CS-conditioned media. In A549 cells, the stress was the highest after 1 h. At that time registered values of DCF fluorescence were more than 8 times higher (p < 0.01) than baseline reference values. In the 6th hour, oxidative stress was still very high (increased by more than 5 times; p < 0.01) and then lower values were observed, but after 12 h of cell growth in CS-conditioned media the stress was still significant (increased by 61 %; p < 0.05). After 24 h, the DCF fluorescence was lower than control values but at that time DCF fluorescence histograms became broad and bimodal (results not shown) due to significant toxicity. In THP1 cells, oxidative stress increased progressively with time attaining the highest values (about 2.5 times higher than baseline) after 24 h of cell treatment with CS.
Table 1 shows flow cytometry data of PI-DNA fluorescence reflecting both cytotoxicity of CS and changes in proliferation rates of A549 and THP1 cells grown for 24 h in CS-conditioned media. Damaged cell numbers were assessed as ‘early’ G0/G1 cells, while cell proliferation was quantified as fractions of S + G2/M cells (Fig. 1). About 47 % of A549 cells were damaged (p < 0.01) by CS, while in THP1 cells the fraction of damaged cells was about 11 % (p < 0.05). CS decreased cell proliferation particularly in A549 cells, where an almost 81 % (p < 0.01) decrease in cell growth dynamics was observed comparing to control cells. In THP1 cells, proliferation was also reduced, by about 21 % (p < 0.05).
Next we determined the expression of acetylated histone H3 (AcH3) in both cell types. CS did not alter AcH3 levels in A549 cells, while in THP1 cells AcH3 level was reduced by about 35 % (p < 0.01). It should be stressed however, that expression of AcH3 was assessed only in viable cells. Incubation of THP1 cells in a smoke-conditioned medium resulted in increased expression of antigens typical for monocyte activation. At baseline, 5 % of cells in each sample were set as antigen positive. CS treatment significantly increased CD14-positive cell numbers (to 95 % of cells; p < 0.01), HLA-DR was expressed in 47 % (p < 0.01) of cells, while corresponding values in CD11a and CD11b were 69 % (p < 0.01) and 46 % (p < 0.01), respectively.
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