VPA induces hyperacetylation, triggers viral expression, and is proapoptotic for primary cells freshly isolated from HTLV-1–infected patients with tropical spastic paraparesis. (A) Histone H3 is hyperacetylated in the presence of VPA. Peripheral blood mononuclear cells were isolated from 3 HTLV-infected, untreated HAM/TSP patients (1-3) and 3 noninfected (NI) healthy controls (4-6) and directly cultivated (ie, without cryopreservation) for 48 hours in the absence (−) or the presence (+) of 2 mM VPA. Cell lysate protein (10 μg) was analyzed by Western blot using an antibody specific for the acetylated forms of histone H3 (Ac H3) or, as a control for normalization of the protein levels, an antiactin antiserum (actin). After incubation with the appropriate alkaline phosphatase–linked conjugates, the blots were revealed by chemiluminescence and autoradiography (top 2 panels). After quantification of the luminescence signals with a luminometer and subtraction of the background, a ratio between the mean intensities generated by each antibody was calculated (Ac H3/actin ratio). (B) VPA activates expression of the viral core protein p19 in the supernatant of these primary T-lymphocyte cultures. The culture supernatants in the absence (−) or the presence (+) of VPA were collected, and expression of the viral p19 core protein was quantified using a Zeptometrix ELISA. Absolute concentrations of p19 (in pg per mL) were determined by normalization of the absorbance values with a standard curve. (C) The CD4⁺ T lymphocytes undergo increased apoptosis in the presence of VPA compared with the controls. After culture, cells were labeled with a CD4-specific antibody conjugated to allophycocyanin (APC). Apoptotic cells were identified after staining with fluorescein-isothiocyanate (FITC)–labeled annexin V (annexin) and propidium iodide (PI). Ten thousand events per thrice-labeled sample were collected by flow cytometry and analyzed with the Cellquest software. The dot plots illustrate annexin/PI (upper panels) and CD4/annexin (bottom panels) labeling of cells isolated from a HAM/TSP patient and cultivated in the absence (control) or the presence (+VPA) of VPA. The percentages of CD4⁺ annexin⁻ and CD4⁺ annexin⁺ are indicated in the upper quadrants. (D) The percentages of apoptotic CD4⁺ cells were measured in samples from the 3 HAM/TSP patients (1-3) and 3 noninfected controls (4-6) cultivated without (−) or with (+) 2 mM VPA. Note that all experiments illustrated in this figure were performed in parallel on freshly isolated noncryopreserved cells.