DNA Methylation Changes in Valproic Acid-Treated HeLa Cells as Assessed by Image Analysis, Immunofluorescence and Vibrational Microspectroscopy

<div><p>Valproic acid (VPA), a well-known histone deacetylase inhibitor, has been reported to affect the DNA methylation status in addition to inducing histone hyperacetylation in several cell types. In HeLa cells, VPA promotes histone acetylation and chromatin remodeling. However, DNA demethylation was not checked in this cell model for standing effects longer than those provided by histone acetylation, which is a rapid and transient phenomenon. Demonstration of VPA-induced DNA demethylation in HeLa cells would contribute to understanding the effect of VPA on an aggressive tumor cell line. In the present work, DNA demethylation in VPA-treated HeLa cells was assessed by image analysis of chromatin texture, the abundance of 5-methylcytosine (5mC) immunofluorescence signals and Fourier transform-infrared (FT-IR) microspectroscopy centered on spectral regions related to the vibration of–CH₃ groups. Image analysis indicated that increased chromatin unpacking promoted by a 4-h-treatment with 1.0 mM VPA persisted for 24 h in the absence of the drug, suggesting the occurrence of DNA demethylation that was confirmed by decreased 5mC immunofluorescence signals. FT-IR spectra of DNA samples from 1 mM or 20 mM VPA-treated cells subjected to a peak fitting analysis of the spectral window for–CH₃ stretching vibrations showed decreased vibrations and energy of these groups as a function of the decreased abundance of 5mC induced by increased VPA concentrations. Only the 20 mM-VPA treatment caused an increase in the ratio of -CH₃ bending vibrations evaluated at 1375 cm^-1 in relation to in-plane vibrations of overall cytosines evaluated at 1492 cm^-1. CH₃ stretching vibrations showed to be more sensitive than–CH₃ bending vibrations, as detected with FT-IR microspectroscopy, for studies aiming to associate vibrational spectroscopy and changes in DNA 5mC abundance.</p></div

Statistics for the FT-IR band peak related to–CH₃ stretching vibrations.

<p>Statistics for the FT-IR band peak related to–CH₃ stretching vibrations.</p

Details of the IR spectral window in the 1530–1350 cm^-1 range.

<p>Spectral profiles are shown for DNA from 1 mM VPA-treated (red line), 20 mM VPA-treated (olive line) treated and untreated (black line) cells. A, absorbances.</p

Immunofluorescence signals for DNA 5mC in VPA-treated HeLa cells.

<p>A reduction in the DNA 5mC fluorescence signals occurred with the VPA treatment (A, B). The intensity of the fluorescence signals was higher in the 20 mM-VPA-treated cells compared with the 1 mM-VPA-treated cells (A, B). Image fluorescence intensity is also shown using the ImageJ 3D plugin software (C). Arbitrary units are based on the 8-bit intensity scale (0–255). The bars equal 20 μm.</p

Optical anisotropy of DNA samples extracted from HeLa cells for FT-IR analysis as observed using polarization microscopy.

<p>Birefringence images of DNA from untreated control (A) and 1 mM VPA- (B) and 20 mM VPA-treated cells (C). An example of images obtained using a differential interference contrast (DIC) system is also shown for DNA from 1 mM VPA-treated cells (D). Blue and yellow interference colors against a red background (D) resulted from the orientation of the DNA fibers in relation to the gamma axis of Berek’s U-CBE compensator inserted into the microscope. This procedure was used to validate that the negative sign of the birefringence, typical of double-stranded B-DNA, was achieved. The outer edge of the DNA drops dried on slides is indicated (o). The bars equal 50 μm.</p

Details of the IR spectral window in the 2990–2850 cm^-1 wavenumber range.

<p>The band peak in (A) is shown as originally obtained from normalized spectra for DNA from 1 mM VPA-treated (red line), 20 mM VPA-treated (olive line) and untreated (black line) cells. The same FT-IR spectral profiles after peak fitting using Grams/AI 8.0 software, Gaussian function and low sensitivity level are shown for the untreated sample (B), the 1 mM VPA-treated cells (C), and the 20 mM VPA-treated cells (D). Original traces appear as red lines, and fitted peaks are represented as green lines (B-D). Small A, absorbances.</p

FT-IR average spectral profiles for DNA from 1 mM VPA-treated (red line), 20 mM VPA-treated (olive line), and untreated (black line) HeLa cells.

<p>Non-normalized spectra (A, B) and spectra normalized with respect to the highest band peak (C, D) are shown. (B) and (D) are spectral details from (A) and (C), respectively. The arrow in (A) indicates ~1707 cm^-1 wavenumber position. Small A, absorbances.</p

Increased inflammasome and caspase-1 activity in the thymus of infected mice.

<p>Male BALB/c mice (n = 5 mice/group for each analyses per experiment replicate) were inoculated with 5x10⁶ Pb18 yeasts contained in PBS intraperitoneally or with only PBS (control group). One hundred and twenty days after inoculation, mice were killed and the thymus was collected and processed individually for analysis. A) Increased initiator caspase-8 gene expression on 120dpi group compared to the naive group. Increased inflammatory caspase-1 gene expression on 120dpi group compared to the naive group. Increased NLRP3 inflammasome gene expression on 120dpi group compared to the naive group. B) Increased pro-caspase-1 production and increased caspase-1 activity on 120dpi group compared to the naive group. C) Increased NLRP3 inflammasome complex assembly on 120dpi compared to the naive group. Statistical analysis was carried out with Student’s t-test. **p<0.01, ****p<0.0001. Representative data from three independent experiments with similar results. Expression levels of genes were represented as a relative copy numbers by using the method of delta threshold (2^-ΔΔCt). Image J software (NIH, MD, USA) was used for the estimation of the pro-caspase-1, the active form of caspase-1 and NLRP3 inflammasome assembly, through a GAPDH ratio.</p

Prolonged <i>Paracoccidioides brasiliensis</i> infection leads to thymic atrophy and intense fungal burden.

<p>Male BALB/c mice (n = 5 mice/group for each analyses per experiment replicate) were inoculated with 5x10⁶ Pb18 yeasts contained in PBS or with only PBS (control group), intraperitoneally. One hundred and twenty days after inoculation, mice were killed and the thymus removed for analysis. A) Thymus from the 120dpi group was smaller in size and weighted less than the naive group. (B) Hematoxilin-Eosin staining showed histologic disorganization in 120dpi, and no evidence of typical cortical (C) and medullary (M) regions, while naive mice showed normal histologic distribution. In more detail below, giant cells and granuloma formation is present in 120dpi. Silver impregnation revealed massive fungal infiltration in the thymic medulla in 120dpi. Statistical analysis was carried out with Student’s t-test. **p<0.01. Results are expressed by Mean ± SEM. Images are representative of three independent experiments with similar results. The images were taken in an Olympus BX50. Magnification 40x (upper and lower panels); 100x (middle panel).</p

Recirculating mature T cells home to infected thymuses, leading to increased frequency of cytokine producing Th17 and T CD8⁺ IFN-γ⁺.

<p>Male BALB/c mice (n = 5 mice/group for each analyses per experiment replicate) were inoculated with 5x10⁶ Pb18 yeasts contained in PBS intraperitoneally or with only PBS (control group). One hundred and twenty days after inoculation, mice were killed and the thymus was collected and processed individually for analysis. A) Frequency of CD44^hiCD24^lo T cells increased among CD4⁺ and CD8⁺ subpopulations in 120dpi compared to the naive group. B) Cytokine producing T cells, Th17 and CD8⁺IFNγ⁺ was found in 120dpi, while practically absent in the naive group. Representative data from three independent experiments with similar results. At least 20000 events were analyzed with FlowJo vX.0.7 (Tree Star Inc., Ashland, OR, USA). Statistical analysis was carried out with Student’s t-test. ***p<0.001, ****p<0.0001. Representative data from three independent experiments with similar results.</p