ABSTRACT
UNLABELLED: Saponins are natural substances produced by a large number of plants, one of which is Tribulus terrestris L. (TT). They have been reported to possess an antitumor activity exerted by regulating various signaling pathways in the cell. Although the mechanisms of action of saponin extracts from various plants have been widely studied, limited data are available about TT. The present study aimed to analyze the impact of saponin extract from TT on cell processes in breast carcinoma cell lines. The variations in expression of a group of 32 selected genes were examined by real-time PCR after saponin treatment of MCF7 and MCF10A cell lines. Only three genes - CXCR4, CCR7 and BCL2, showed changes in their mRNA levels after the application of the herb extract. While CXCR4 expression was reduced in both cell lines, CCR7 and BCL2 levels decreased only in tumorigenic MCF7 cells, implying cell-specificity of the saponin action. Our results suggested that TT extract containing saponins was likely to affect the processes of apoptosis and metastasizing of cancer cells. Further in vivo studies will show its applicability as an anticancer therapeutic agent. KEYWORDS: saponins, Tribulus terrestris, breast cancer, CXCR4, CCR7, BCL2.
ABSTRACT
Checkpoint kinase 2 (CHEK2) is a DNA damage-activated protein kinase implicated in cell cycle checkpoint control. The significance of CHEK2 alterations for breast cancer incidence and clinical behavior is not clear. In this study we determined the mutational spectrum and the level of promoter hypermethylation of CHEK2 gene in a group of 145 Bulgarian patients with breast cancer. A special emphasis was put on the clinical impact of CHEK2 alterations for breast cancerogenesis. PCR-SSCP-sequencing analysis of the entire coding sequence of CHEK2 gene was performed to estimate the mutational profile of tumor samples. Methylation-sensitive SSCP was applied to determine the methylation status in CpG clusters implicated in CHEK2 silencing. Clinical significance of CHEK2 alterations was evaluated using standard statistical methods. Mutations in CHEK2 were identified in 9.65 % of the patients. Two novel missense substitutions Thr476Met (C >T) and Ala507Gly (C>G), and a novel silent variant Glu79Glu (A>G) were registered. However, hypermethylation was not found in any of the studied cases. Comparison with clinical characteristics showed that CHEK2 positive women have predominantly lobular type of breast carcinoma (Ñ=0.04) and PR+ status (p=0.092). CHEK2 mutations correlated significantly with ATM+ status (p=0.046). All patients with the Glu79Glu variant were progesterone receptor positive (p=0.004). A decrease in overall survival (p = 0.6301) and a threefold increased independent risk of death (HR = 3.295, 95%CI 0.850-12.778, p = 0.085) in CHEK2+patients was found. Our data indicate the significance of CHEK2 gene alterations in contrast to promoter hypermethylation in breast cancerogenesis. Specificity of CHEK2 mutational profile for the Bulgarian population was found. Though CHEK2 mutational status correlated with more favorable clinical characteristics, including positive progesterone receptor and lobular histological type, it independently increased the risk of death in these patients.
Subject(s)
Breast Neoplasms/mortality , Protein Serine-Threonine Kinases/genetics , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Checkpoint Kinase 2 , CpG Islands , DNA Methylation , Female , Humans , Mutation , Promoter Regions, Genetic , RiskABSTRACT
During the last few decades a substantial amount of evidence has accumulated proving that the abrogation of the normal p53 pathway is a critical step in the initiation and progression of tumors. Decoding the genetic mechanisms involved in carcinogenesis requires screening for consistent genetic tumor alterations, including those concerning the p53 gene. Thus, practical, efficient, and inexpensive techniques for accurate determination of p53 mutational status are needed. Polymerase chain reaction/single-strand conformation polymorphism (PCR-SSCP) analysis is considered to be a useful tool to investigate the role of the p53 gene in the development and progression of human cancers. The sensitivity of the method can be increased considerably by varying the experimental conditions. Here we demonstrate a scheme of PCR-SSCP optimization for detection of p53 gene mutations of patients with various cancers. Optimal conditions for PCRSSCP of p53 exons 4-9 are reported. Such PCR-SSCP optimization could allow an increase in the sensitivity and reproducibility of the technique and facilitates screening of large series of patients to assess the clinical significance of p53 mutations in human cancers. Using the optimized PCR-SSCP analysis we screened Bulgarian patients with invasive breast cancer for p53 gene mutations and registered a 33.33% frequency of mutations. To date, there are no data concerning the p53 status of Bulgarian breast cancer patients. Screening for p53 gene mutations enables an accurate and routine determination of the p53 status of patients with cancer and may be applied in clinical oncology to cancer diagnosis, prediction of prognosis and response to treatment.
Subject(s)
Breast Neoplasms/genetics , Genes, p53/genetics , Neoplasm Invasiveness/genetics , Polymerase Chain Reaction/methods , Polymorphism, Single-Stranded Conformational , Base Sequence , Breast Neoplasms/pathology , Breast Neoplasms/surgery , Bulgaria , Carcinoma, Ductal/genetics , Carcinoma, Ductal/pathology , DNA Primers , Female , Humans , Neoplasm Staging , Templates, Genetic , White PeopleABSTRACT
We describe an altered mobility for acetylated histone isoforms in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Isoforms of histones H3 and H4 with a higher acetylation degree have a slightly faster electrophoretic mobility. Since acetylation neutralizes the positive charge of the epsilon-amino group of lysine, without significantly changing the molecular mass of the protein, the acetylation-dependent mobility shift could be explained by the increase of the net negative charge of the SDS-histone complexes. A possible consequence of this differential mobility for the acetylation site determination by protein microsequencing from SDS gels is discussed.
Subject(s)
Electrophoresis, Polyacrylamide Gel/methods , Histones/isolation & purification , Acetylation , Animals , Blood Protein Electrophoresis/methods , Blood Protein Electrophoresis/statistics & numerical data , Chickens , Electrochemistry , Electrophoresis, Polyacrylamide Gel/statistics & numerical data , Erythrocytes/chemistry , Histones/blood , Histones/chemistry , Reproducibility of Results , Sodium Dodecyl SulfateABSTRACT
Gcn5p is the catalytic subunit of several type A histone acetyltransferases (HATs). Previous studies performed under a limited range of solution conditions have found that nucleosome core particles and nucleosomal arrays can be acetylated by Gcn5p only when it is complexed with other proteins, e.g. Gcn5-Ada, HAT-A2, and SAGA. Here we demonstrate that when assayed in buffer containing optimum concentrations of either NaCl or MgCl2, purified yeast recombinant Gcn5p (rGcn5p) efficiently acetylates both nucleosome core particles and nucleosomal arrays. Furthermore, under conditions where nucleosomal arrays are extensively folded, rGcn5p acetylates folded arrays approximately 40% faster than nucleosome core particles. Finally, rGcn5p polyacetylates the N termini of free histone H3 but only monoacetylates H3 in nucleosomes and nucleosomal arrays. These results demonstrate both that rGcn5p in and of itself is catalytically active when assayed under optimal solution conditions and that this enzyme prefers folded nucleosomal arrays as a substrate. They further suggest that the structure of the histone H3 N terminus, and concomitantly the accessibility of the H3 acetylation sites, changes upon assembly into nucleosomes and nucleosomal arrays.
Subject(s)
Nucleosomes/metabolism , Saccharomyces cerevisiae Proteins , Trans-Activators/metabolism , Acetylation , Acetyltransferases/metabolism , Animals , Cations, Divalent , Cell Cycle Proteins , Chickens , Histone Acetyltransferases , Histones/metabolism , Recombinant Proteins/metabolism , Substrate Specificity , Transcription Factors , p300-CBP Transcription FactorsABSTRACT
Multiple enzyme forms of histone deacetylase and histone acetyltransferase exist in germinating maize embryos. We analyzed the association of the different enzymes to chromatin by ion exchange chromatography of subcellular fractions from different time points of embryo germination. The vast majority of histone deacetylase HD-1A was not bound to chromatin, since it was solubilized during chromatin isolation, regardless of its phosphorylation state and the phase of embryo germination. In contrast, HD-2 was chromatin bound during the entire germination pathway. Histone deacetylase HD-1B was present in a chromatin-bound and a soluble form; the ratio between these two forms changed during germination. Both nuclear histone acetyltransferases, HAT-A1 and HAT-A2, were tightly chromatin-bound and could only be released from chromatin by salt extraction. To test whether histone acetyltransferases or deacetylases are associated with the nuclear matrix, we analyzed nuclear matrix preparations from yeast, Physarum, and maize step by step for both enzyme activities. This analysis confirmed that part of the activity is chromatin bound, but no significant enzyme activity could be found in the final nuclear matrix, regardless of the preparation protocol. This result was further substantiated by detailed analysis of histone deacetylases and acetyltransferases during cellular fractionation and nuclear matrix preparation of chicken erythrocytes. Altogether our results suggest that the participation of these enzymes in different nuclear processes may partly be regulated by a distinct location to intranuclear components.
Subject(s)
Acetyltransferases/analysis , Cell Nucleus/enzymology , Histone Deacetylases/analysis , Histones/metabolism , Saccharomyces cerevisiae Proteins , Subcellular Fractions/enzymology , Acetylation , Animals , Chickens , Chromatography, Ion Exchange , Chromatography, Liquid/methods , Erythrocytes/enzymology , Histone Acetyltransferases , Male , Nuclear Matrix/enzymology , Physarum polycephalum/enzymology , Saccharomyces cerevisiae/enzymology , Seeds/enzymology , Seeds/growth & development , Zea mays/enzymologyABSTRACT
Core histones can be modified by reversible, posttranslational acetylation of specific lysine residues within the N-terminal protein domains. The dynamic equilibrium of acetylation is maintained by two enzyme activities, histone acetyltransferase and histone deacetylase. Recent data on histone deacetylases and on anionic motifs in chromatin- or DNA-binding regulatory proteins (e.g. transcription factors, nuclear proto-oncogenes) are summarized and united into a hypothesis which attributes a key function to histone deacetylation for the binding of regulatory proteins to chromatin by a transient, specific local increase of the positive charge in the N-terminal domains of nucleosomal core histones. According to our model, the rapid deacetylation of distinct lysines in especially H2A and H2B would facilitate the association of anionic protein domains of regulatory proteins to specific nucleosomes. Therefore histone deacetylation (histone deacetylases) may represent a unique regulatory mechanism in the early steps of gene activation, in contrast to the more structural role of histone acetylation (histone acetyltransferases) for nucleosomal transitions during the actual transcription process.
Subject(s)
Chromatin/metabolism , Histone Deacetylases/metabolism , Transcription Factors/metabolism , Animals , Humans , Models, Molecular , Protein Binding/physiologyABSTRACT
Mono Q ion exchange high performance liquid chromatography (HPLC) reveals that the main histone deacetylase activity (HD1) of germinating Zea mays embryos consists of multiple enzyme forms. Chromatography of HD1 after treatment with alkaline phosphatase yields two distinct histone deacetylase forms (HD1-A, HD1-B). The same is true for chromatography after phosphatase treatment of a total cell extract. One of these enzyme forms (HD1-A) is subject to phosphorylation, which causes a change in the substrate specificity of the enzyme, as shown with HPLC-purified individual core histone species; the substrate specificity for H2A increases more than 2-fold after phosphorylation, whereas the specificity for H3 decreases to about 60%. The total histone deacetylase activity is quantitatively released from isolated nuclei after extraction with moderate ionic strength buffers; no significant residual enzyme activity could be detected in the nuclear matrix.
Subject(s)
Histone Deacetylases/metabolism , Isoenzymes/metabolism , Zea mays/enzymology , Animals , Chickens , Chromatography, High Pressure Liquid , Erythrocytes , Histone Deacetylases/isolation & purification , Histones/blood , Histones/isolation & purification , Isoenzymes/isolation & purification , Phosphorylation , Substrate SpecificityABSTRACT
DEAE-Sepharose chromatography of extracts from Zea mays meristematic cells revealed multiple histone acetyltransferase and histone deacetylase enzyme forms. An improved method for nuclear isolation allowed us to discriminate nuclear and cytoplasmic enzymes. Two nuclear histone acetyltransferases, A1 and A2, a cytoplasmic B-enzyme and two nuclear histone deacetylases, HD1 and HD2, have been identified. The histone specificity of the different enzyme forms has been studied in an in vitro system, using chicken erythrocyte histones as substrate. The cytoplasmic histone acetyltransferase B is the predominant enzyme, which acetylates mainly histone H4 and to a lesser extent H2A. The nuclear histone acetyltransferase A1 preferentially acetylates H3 and also H4, whereas enzyme A2 is specific for H3. This substrate specificity was confirmed with homologous Z. mays histones. The two histone deacetylases differ from each other with respect to ionic strength dependence, inhibition by acetate and butyrate, and substrate specificity. The strong inhibitory effect of acetate on histone deacetylases was exploited to distinguish different histone acetyltransferase forms.
Subject(s)
Acetyltransferases/metabolism , Histone Deacetylases/metabolism , Histones/metabolism , Saccharomyces cerevisiae Proteins , Zea mays/enzymology , Acetylation , Cell Differentiation , Chromatography, Ion Exchange , Histone Acetyltransferases , Isoenzymes/metabolism , Substrate Specificity , Zea mays/cytologyABSTRACT
Multiple forms of histone acetyltransferases and histone deacetylases, which have been separated and characterized in the accompanying manuscript (López-Rodas, G., Georgieva, E. I., Sendra, R., and Loidl, P. (1991) J. Biol. Chem. 266, 18745-18750), together with in vivo acetate incorporation, were studied during the germination of Zea mays embryos. Total histone acetyltransferase activity increases during germination with two maxima at 40 and 72 h after start of germination. This fluctuation is mainly due to the cytoplasmic B-enzyme which predominantly acetylates histone H4 up to the diacetylated form. The nuclear histone acetyltransferase A2, specific for H3, is low throughout germination, except at 24 h, when it transiently becomes the main activity. Both enzymes are also present in the dry embryo, whereas the second nuclear enzyme A1, specific for H3 and H4, is absent in the initial stage of differentiation. The two histone deacetylases, HD1 and HD2, exhibit entirely different patterns. Whereas HD1 activity is low in the dry embryo and increases during germination, HD2 is the predominant enzyme at the start of differentiation, but almost disappears at later stages. Analysis of the in vivo acetate incorporation reveals that H4 is present in up to tetraacetylated subspecies. The pattern of acetate incorporation into core histones closely resembles the fluctuations of histone acetyltransferase B. Based on the analysis of thymidine kinase activity a close correlation was established between histone acetyltransferase B and DNA replication, whereas the A2 enzyme is associated with transcriptional activity. Histone deacetylase HD1 obviously serves a specific function in the dry embryo and could be a prerequisite for DNA repair processes. The study confirms the idea of DNA repair processes. The study confirms the idea of multiple functions of histone acetylation and assigns distinct enzymes, involved in this modification, to certain nuclear processes.
Subject(s)
Acetyltransferases/metabolism , Histone Deacetylases/metabolism , Histones/metabolism , Protein Processing, Post-Translational , Saccharomyces cerevisiae Proteins , Zea mays/enzymology , Acetylation , Animals , Cell Differentiation , Chickens , Histone Acetyltransferases , Kinetics , Macromolecular Substances , Plant Proteins/metabolism , Substrate Specificity , Zea mays/embryologyABSTRACT
Yeast microorganisms from Candida genus are investigated for their superoxide dismutase (SOD) and catalase activity during cultivation on N-alkanes. The later caused a considerable increase of Cu/Zn SOD activity of yeast cells in comparison with glucose. A correlation between SOD and catalase activity existed. It is further observed that cells of Candida lipolytica 68-72 which contain a high level of Cu/Zn SOD were more resistant to lethality of exogenous O2-. An over-production of Cu/Zn SOD during the assimilation of N-alkanes by yeasts is also connected to their considerable resistance to increased concentrations of Cu2+ and Zn2+ ions in the nutrient medium. The results are consistent with the assumption that the enhanced resistance of yeast cells to O2- and high concentrations of Cu2+ and Zn(2+)-ions are due to the increased activity of Cu/Zn SOD and that SOD is involved in the protection of some cellular components. Polyacrylamide gel electrophoresis of Candida lipolytica cell-free extracts revealed the same chromatic bands of SOD activity under growth on glucose and N-alkanes. The type of the carbon source used from yeast cells as a single source of carbon and energy had no influence on the SOD profile of the cell.
Subject(s)
Alkanes/metabolism , Candida/enzymology , Superoxide Dismutase/physiology , Alkanes/pharmacology , Candida/drug effects , Candida/growth & development , Catalase/biosynthesis , Free Radicals , Glucose/pharmacology , Oxidation-Reduction , Superoxide Dismutase/antagonists & inhibitors , Superoxide Dismutase/biosynthesis , Superoxides/pharmacologyABSTRACT
Histones from maize embryos and seedlings have been isolated using a fast extraction procedure. Three different electrophoretic systems have been applied for the study of the heterogeneity of maize core histones. Electrophoresis in acetic acid/urea polyacrylamide gels, containing high concentrations of urea, resulted in optimum fractionation of the core histones and especially of histone H4. Sodium dodecyl sulfate-containing polyacrylamide gels were not useful for the fractionation of maize histone classes H2a and H2b, nor for the various subfractions of H3 and H4. Gels containing Triton X-100, used for the dimension in two-dimensional electrophoresis proved to be efficient for the separation of all histone classes, as well as their structural variants and chemical modifications. Maize core histones have been oxidized in an attempt to define which of the Triton X-100 resolved subfractions represent oxidation forms.
Subject(s)
Histones/analysis , Zea mays/analysis , Cell Nucleus/analysis , Electrophoresis, Gel, Two-Dimensional , Electrophoresis, Polyacrylamide Gel , Phylogeny , Zea mays/embryologyABSTRACT
Chromatin was fractionated by digestion with deoxyribonuclease II and precipitation with MgCl2. The Mg2+-soluble fraction, known to be enriched in transcribed DNA sequences, was enriched also in high mobility group proteins 1 and 2 and contained almost all other acid-soluble nonhistone proteins.