Intrinsic Fluorescence Properties and Structural Analysis of p13suc1 from Schizosaccharomyces pombe

Abstract p13suc1 acts in the fission yeast cell division cycle as a component of p34cdc2. In the present work, structural information contained in the intrinsic fluorescence of p13suc1 has been extracted by steady-state and time-resolved fluorescence techniques. In its native form, the steady-state emission spectrum of p13suc1 is centered at 336 nm. Upon denaturation by guanidine HCl (4.0 M), the emission spectrum is shifted to 355-360 nm and the fluorescence intensity decreases 70%. The same changes are not obtained with p13suc1 at 56°C or after incubation at 100°C, and the protein appears to be substantially temperature-stable. The fluorescence decay of p13suc1 is best described by three discrete lifetimes of 0.6 ns (τ1), 2.9 ns (τ2), and 6.1 ns (τ3), with amplitudes that are dependent on the native or unfolded state of the protein. Under native conditions, the two predominant decay-associated spectra, DAS-τ2 (λmax = 332 nm) and DAS-τ3 (λmax = 340 nm), derive from two different excitation DAS. Moreover distinct quenching mechanisms and collisional accessibilities (kq(τ2)≫kq(τ3)) are resolved for each lifetime. An interpretation in terms of specific tryptophan residue (or protein conformer)-lifetime assignments is presented. The decay of the fluorescence anisotropy of native p13suc1 is best described by a double exponential decay. The longer correlation time recovered (9 ns ≤ Φ2 ≤ 15ns) can be associated with the rotational motion of the protein as a whole and a Stokes radius of 21.2 A has been calculated for p13suc1. Anisotropy measurements obtained as a function of temperature indicate that, in solution, the protein exists exclusively as a prolate monomer. In 1 mM zinc, changes of the anisotropy decay parameters are compatible with subunits oligomerization

Histone post-translational modifications by HPLC-ESI-MS after HT29 cell treatment with histone deacetylase inhibitors

The goal of the present work is to establish a correlation between the degree of histone post-translational modifications and the effects caused by treatment of HT29 colon cancer cells with class I-selective (MS-275 and MC1855), class II-selective (MC1568), and non-selective (suberoylanilide hydroxamic acid (SAHA) histone deacetylase inhibitors (HDACi). This correlation could afford a mean to better understand the mechanism of action of new, more potent, and selective HDACi directly on the cells. To this end, LC coupled to MS was applied in studies of time and concentration-dependent treatment with HDACi in HT29 cells. The results were correlated to their potency of histone deacetylase inhibition and to their effects on the cell cycle. The results indicate that the four tested inhibitors show a different pattern of time- and concentration-dependent modification after treatment of HT29 cells. At the selected concentrations, they cause different histone hyperacetylation and different cell cycle effects. In particular, SAHA (non-selective HDACi) affected hyperacetylation of all histones and caused massive cell death. MC1855 (class I-selective HDACi, hydroxamate) proved to be more potent and less toxic (cell arrest in G2/M phase) than SAHA. MS-275 (class I-selective HDACi, benzamide) exhibited a higher degree of hyperacetylation of H4 and a lower degree of H2A, H2B, and H3 acetylation, causing a cell arrest in G0/G1 phase. On the contrary, MC1568 (class II-selective HDACi) produced only a modest hyperacetylation of H4, was ineffective on the other histones, and showed no effect on cell cycle in HT29 cells

IDENTIFICATION OF HISTONE MODIFICATIONS INVOLVED IN HDAC1 INHIBITION BY 9-HSA

9-hydroxystearic acid (9-HSA) is a product of endogenous lipoperoxidation identified in several human cell lines; its administration to HT29, a colon adenocarcinoma cell line, causes growth arrest in G0/G1 and differentiation toward a benign phenotype. The relevant molecular events are the increased transcription of p21WAF1, TGFbeta1, alkaline phosphatase, and subunit alfa5 of integrin. The number of genes whose activity is modified is indeed rather restricted: in particular variations of the activity of genes that regulate the cell cycle, apoptosis and differentiation have been evidenced for several HDAC1 inhibitors, but the mechanisms that induce or repress the selective activation of genes, beginning from the acetylation state of chromatin or of nuclear proteins, are not yet clear. In HT29, both histones H3 and H4 appear to be hyperacetylated as a consequence of 9-HSA administration. The isolation of histones, the identification of their isoforms as well as the determination of their modifications are essential steps of our study. Reverse phase, ion exchange or hydrophylic-interaction liquid chromatography and subsequent identification of each fraction by electrophoresis or mass spectrometry have become urepleaceble approaches for the analysis of protein modification sites. This work proposes an experimental approach for studying the different isoforms through the injection of the mixture of histones, extracted and solubilized in water, in a liquid chromatograph Jasco PU-1585 equipped with a Rheodyne Model 7725i injector, connected to a UV (JascoUV-1575) detector and to a LCQDuo (Thermo Finnigan) mass spectrometer, that is in turn interfaced to an electrospray ionization source (ESI), and equipped with an ion trap analyzer. The analysis of the peaks so obtained has been carried out by employing deconvolution programs that allow the determination of the MW of proteins and characterize and quantify also the acetylated and methylated isoforms

Cell cycle arrest induced by sulforaphane in human colon carcinoma cells HT29 is associated with the hyperacetylation of histone H4.

Introduction. Sulforaphane (SFN), a dietary isothiocyanate isolated from Brassicaceae, has been shown to prevent different cancers in laboratory animals [1]. In particular, SFN protects against colon carcinogenesis in vivo and causes a G0/G1 growth arrest and apoptosis in human colon cancer cells, by inducing p21Cip1/Waf1[2]. SFN also acts as an inhibitor of histone deacetylases (HDACs) in human embryonic kidney 293 cells and HCT116 colon cancer cells [3]. The objective of this study is to evaluate the ability of the HDAC inhibitor SFN to induce cytotoxic and cytostatic effects in HT29 colon cancer cell line. Materials and methods. Cell viability was evaluated by measuring MTT reduction. The cells were plated in 6-well dishes at equal density, grown for 24 hours, and then treated with 5 M SFN. 3H-thymidine (1 μCi/mL) was added for the last 6 hours of the incubation. The cells were washed in ice-cold PBS and 5 % trichloroacetic acid was added for 30 minutes at 4 °C, and then incubated with 0.5 N NaOH for 1 hour at 50 °C. Cell lysates were assayed for protein content by Bio Rad assay kit and measured for incorporated radioactivity. Counts were normalized for total cellular protein. To test SFN effects on HDAC activity, the total histone acetylation level was measured by pulse labelling experiments with 3H-acetate and the acetylation status of the histone classes were assayed by Western Blot and HPLC/MS. Results. SFN negatively affects HT29 growth in a dose- and time-dependent manner with a 24h IC50 equal to 22.3 M 2.4. At concentrations as low as 5 M, a significant inhibition of cell proliferation is observed, accompanied by a 47% decrease of the mitotic index, with respect to the control. Histones extracted from SFN treated HT29 cells show a 63% increase in the acetylation status; in particular SFN markedly prolonges the half- life of the acetyl groups on histone H4