Cytotoxicity and anticancer studies of Bacillus cereus and Bacillus pumilus metabolites targeting human cancer cells

The metabolites of bacteria Bacillus cereus and Bacillus pumilus isolated from soil samples in Shimoga region, Karnataka (India) were tested for cytotoxicity and anticancer properties. The various solvent extract fractions obtained from the metabolites of the two bacteria were tested for their cytotoxicity against normal human liver cell lines and 2 cancer cell lines by (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole) assay. The two fractions obtained from B. cereus showed high cytotoxicity. These two fractions were further screened for anticancer activity by nuclear staining studies and DNA fragmentation analysis. Both the fractions demonstrated significant activity by membrane blebbing during nuclear staining and caused the damage the DNA patterns during DNA fragmentation analysis. On the other hand, the metabolites of B. pumilus revealed toxic effect against cancer cells as well as normal ones.     

Heterogeneity in White Blood Cells Has Potential to Confound DNA Methylation Measurements

Epigenetic studies are commonly conducted on DNA from tissue samples. However, tissues are ensembles of cells that may each have their own epigenetic profile, and therefore inter-individual cellular heterogeneity may compromise these studies. Here, we explore the potential for such confounding on DNA methylation measurement outcomes when using DNA from whole blood. DNA methylation was measured using pyrosequencing-based methodology in whole blood (n = 50–179) and in two white blood cell fractions (n = 20), isolated using density gradient centrifugation, in four CGIs (CpG Islands) located in genes HHEX (10 CpG sites assayed), KCNJ11 (8 CpGs), KCNQ1 (4 CpGs) and PM20D1 (7 CpGs). Cellular heterogeneity (variation in proportional white blood cell counts of neutrophils, lymphocytes, monocytes, eosinophils and basophils, counted by an automated cell counter) explained up to 40% (p<0.0001) of the inter-individual variation in whole blood DNA methylation levels in the HHEX CGI, but not a significant proportion of the variation in the other three CGIs tested. DNA methylation levels in the two cell fractions, polymorphonuclear and mononuclear cells, differed significantly in the HHEX CGI; specifically the average absolute difference ranged between 3.4–15.7 percentage points per CpG site. In the other three CGIs tested, methylation levels in the two fractions did not differ significantly, and/or the difference was more moderate. In the examined CGIs, methylation levels were highly correlated between cell fractions. In summary, our analysis detects region-specific differential DNA methylation between white blood cell subtypes, which can confound the outcome of whole blood DNA methylation measurements. Finally, by demonstrating the high correlation between methylation levels in cell fractions, our results suggest a possibility to use a proportional number of a single white blood cell type to correct for this confounding effect in analyses.     

Effect of enzyme-assisted extract of Sargassum coreanum on induction of apoptosis in HL-60 tumor cells

In order to effectively prepare useful components from Sargassum coreanum, enzyme-assisted extraction was adapted to extraction processing of the seaweed. Our previous studies have shown that Neutrase extract of S. coreanum exhibited the strongest antioxidant activities among ten enzymatic extracts. It is reported that many antioxidant substances have anticancer or anticarcinogenic properties. Thus, Neutrase extract, which possessed a strong antioxidant activity in the previous study, was separated into four different molecular weight fractions (<5, 5–10, 10–30, and >30?kDa) which were screened for inhibitory activities against cancer cell growth. The >30-kDa fraction inhibited cell growth more than the other fractions. We separated crude polysaccharide (CPS) from the >30-kDa fraction to show that CPS increased DNA fragmentation, apoptotic body, and apoptotic cells with hypodiploid DNA contents in HL-60 cells. This indicates that CPS suppressed the growth of cells through apoptosis. The CPS gradually increased the expression of pro-apoptotic Bax and led to the activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase. The CPS was 40.85% fucose, 27.24% galactose, and 12.99% glucose, as analyzed by bio-liquid chromatography. These findings suggest that apoptosis effect can be mediated by CPS in HL-60 cells.     

DNA replication in Escherichia coli: physical and kinetic studies of the replication point

DNA replication in Escherichia coli was followed using analytical composite agarose-acrylamide gel electrophoresis of native and denatured DNA samples from cells that had been pulse-labeled in vivo. The results obtained with samples of native DNA, combined with the different sensitivities of the various electrophoretic fractions of native DNA to enzymatic attack, led us to conclude that a significant amount of single-strandedness exists in the region of the replication site. Data obtained from kinetic analyses of the labeling of both the high molecular weight DNA and the fragments formed on denaturation of native DNA samples were consistent with the Okazaki model (Okazaki et al., 1968a,b) of DNA replication. Furthermore, the data indicated that eight to ten precursor fragments are present per fork during bacterial growth at both 20 and 37 °C, under the conditions used in this study.     

Chromatin-bound PCNA as S-phase marker in mononuclear blood cells of patients with acute lymphoblastic leukaemia or multiple myeloma

Objectives: Proliferating cell nuclear antigen (PCNA) has often been used as a marker to aid assessment of tumour growth fraction. This paper addresses the question of whether it can be used as an S‐phase marker, when the non‐chromatin‐bound form of the protein is removed by pepsin treatment. Materials and methods: Cytofluorometric measurements were carried out after immunofluorescence staining of PCNA and counterstaining of DNA. S‐phase fraction was determined with the help of windows on PCNA versus DNA scattergrams, or mathematically from DNA histograms. Results: S‐phase fractions obtained using the two methods correlated well, but did not always agree, exact discrepancies depending on the mathematical model used for histogram analysis. Conclusions: Determination of S‐phase fractions with the help of PCNA immunofluorescence staining is possible, and probably more reliable than calculation of S‐fractions from DNA histograms. It thus offers an alternative to assays involving BrdU labelling in vivo.     

In vitro cytotoxic potential of Solanum nigrum against human cancer cell lines

Plants have natural products which use to possess antiproliferative potential against many cancers. In the present study, six isolated fractions (ethyl acetate, petroleum ether, chloroform, n-butanol, ethanol and aqueous) from Solanum nigrum were evaluated for their cytotoxic effect on different cell lines. Hepatic carcinoma cell line (HepG2), cervical cancer cell line (HeLa) and baby hamster kidney (BHK) used as normal non-cancerous cells were evaluated for cytotoxicity against isolated fractions. Cell viability assay was performed to evaluate the cytotoxicity of all fractions on different cell lines followed by the lactate dehydrogenase and vascular endothelial growth factor assays of most active fraction among all screened for cytotoxic analysis. HPLC analysis of most active fractions against cytotoxicity was performed to check the biological activity of compounds. Results displayed the potent cytotoxic activity of ethyl acetate fraction of S. nigrum against HepG2 cells with IC₅₀ value of 7.89 μg/ml. Other fractions exhibited potent anticancer activity against HepG2 cells followed by HeLa cells. Fractions in our study showed no cytotoxicity in BHK cells. Cytotoxic activity observed in our current study exposed high antiproliferative potential and activity of ethyl acetate fraction against HepG2 cells. The results demonstrated that S. nigrum fractions exhibited anticancer activity against hepatic and cervical cancer cell lines with non-toxic effect in normal cells. These results reveal significant potential of S. nigrum for the therapeutic of cancers across the globe in future.     

DNA double-strand breaks activate ATM independent of mitochondrial dysfunction in A549 cells

Excessive nuclear or mitochondrial DNA damage can lead to mitochondrial dysfunction, decreased energy production, and increased generation of reactive oxygen species (ROS). Although numerous cell signaling pathways are activated when cells are injured, the ataxia telangiectasia mutant (ATM) protein has emerged as a major regulator of the response to both mitochondrial dysfunction and nuclear DNA double-strand breaks (DSBs). Because mitochondrial dysfunction is often a response to excessive DNA damage, it has been difficult to determine whether nuclear and/or mitochondrial DNA DSBs activate ATM independent of mitochondrial dysfunction. In this study, mitochondrial and nuclear DNA DSBs were generated in the A549 human lung adenocarcinoma cell line by infecting with retroviruses expressing the restriction endonuclease PstI fused to a mitochondrial targeting sequence (MTS) or nuclear localization sequence (NLS) and a hemagglutinin antigen epitope tag (HA). Expression of MTS-PstI-HA or NLS-PstI-HA activated the DNA damage response defined by phosphorylation of ATM, the tumor suppressor protein p53 (TP53), KRAB-associated protein (KAP)-1, and structural maintenance of chromosomes (SMC)-1. Phosphorylated ATM and SMC1 were detected in nuclear fractions, whereas phosphorylated TP53 and KAP1 were detected in both mitochondrial and nuclear fractions. PstI also enhanced expression of the cyclin-dependent kinase inhibitor p21 and inhibited cell growth. This response to DNA damage occurred in the absence of detectable mitochondrial dysfunction and excess production of ROS. These findings reveal that DNA DSBs are sufficient to activate ATM independent of mitochondrial dysfunction and suggest that the activated form of ATM and some of its substrates are restricted to the nuclear compartment, regardless of the site of DNA damage.     

A CYTOPLASMIC CHARACTER IN NEUROSPORA CRASSA : The Role of Nuclei and Mitochondria

Nuclear fractions isolated from mutants of Neurospora produced no effect when microinjected into mutants with complementary biochemical requirements. DNA isolated from the nuclear fractions similarly injected also had no effect. Mitochondrial fractions isolated from an abnormal inositolless strain (abn-1) produced drastic changes in the rate of growth, morphology, reproductive characteristics, and cytochrome spectra of normal inositolless strains when single hyphal compartments were microinjected and isolated, whereas the mitochondrial fractions of the wild type produced no effect. These results provide evidence for the transmission of biochemical and biological characters when mitochondria are transferred to new nucleocytoplasmic environments.     

Incorporation of phosphorus into phased cells ofCandida utilis using32P and33P

Changes in phosphorus metabolism were studied by examining the incorporation of³²P and³³P into cells ofCandida utilis growing in phased culture during a 6 h cell cycle and a post-cycle period of 6 h. Three different chemically defined media were used; these were phosphorus, nitrogen and carbon limited. The patterns of incorporation of phosphorus into RNA, DNA, lipid and cold water extractable phosphate fractions showed a non-uniform behaviour during both cell cycle and post-cycle periods. The patterns were different in all three types of media. The results showed that a cell can grow and develop at a fixed growth rate in different ways: so that the pattern of behaviour during a cell cycle is not stereotyped for a given doubling time, but largely depends upon the nutrient environment in which the cell exists.     

Effects of aloe extracts on human normal and tumor cells in vitro

Fractions of leaf extracts from 2 local types, labeledAloe vera (subsequently identified asAloe barbadensis Mill, andA. saponaria Haw.), were prepared by differential centrifugation and tested by in vitro assays for the presence of lectinlike activities and for effects on the attachment and growth of human normal and tumor cells. Fractions of extracts of fresh leaves and commercially “stabilized”Aloe vera gel had high levels of lectin-like substances measured by immunodiffusion and hemagglutination assays. Substances in fluid fractions from both fresh leaf sources were found to markedly promote attachment and growth of human normal, but not tumor, cells and to enhance healing of wounded cell monolayers. In contrast, fractions of “stabilized”Aloe vera gel were equally cytotoxic for human normal and tumor cells in vitro. Results from cell assays suggested that the observed growth promotion and wound healing effects of aloe substances in vitro may be analogous to what has been observed in vivo during healing of wounds and burns.     

A fraction from extracts of demineralized adult bone stimulates the conversion of mesenchymal cells into chondrocytes

Demineralized adult bone contains factors which stimulate nonskeletal mesenchymal cells to undergo a developmental progression resulting in de novo endochondral ossification. In this study, isolated embryonic stage 24 chick limb bud mesenchymal cells maintained in culture were utilized as an in vitro assay system for detection of specific bioactive components solubilized from adult chicken bone matrix. Guanidinium chloride extracts (4 M) of demineralized-defatted bone were fractionated and tested in limb mesenchymal cell cultures for possible effects upon growth and chondrogenesis. Two low-molecular-weight fractions were found to be active in these cultures. A cold water-insoluble, but warm Trisbuffered saline-soluble fraction provoked a dose-dependent increase in the amount of cartilage formed after 7 days of continuous exposure as evidenced by an increased number of chondrocytes observed in living cultures, elevated cell-layer-associated ³⁵S incorporation per microgram DNA, and greater numbers of toluidine blue-staining foci (i.e., cartilage nodules). Growth inhibitory substances were detected in a low-molecular-weight, water-soluble fraction; 7 days of continuous exposure to this material resulted in less cartilage formation and reduced cell numbers (accumulated DNA) on each plate. These observations demonstrate the usefulness of stage 24 chick limb bud cell cultures for identifying bioactive factors extracted from adult bone matrix. In addition, the action of these factors on mesenchymal cells may now be studied in a cell culture system.     

Persistence and Growth of Fecal Bacteroidales Assessed by Bromodeoxyuridine Immunocapture

Extraintestinal growth of fecal bacteria can impair accurate assessment of watershed health. Anaerobic fecal bacteria belonging to the order Bacteroidales are attractive candidates for fecal source tracking because they have host-specific distributions and do not grow well in the presence of high oxygen concentrations. Growth of general and human-specific fecal Bacteroidales marker organisms in environmental samples (sewage) and persistence of the corresponding genetic markers were investigated using bromodeoxyuridine (BrdU) DNA labeling and immunocapture, followed by PCR detection. Background amplification of unlabeled controls occasionally occurred when a high number of PCR cycles was used. By using fluorescent detection of PCR products obtained after 15 cycles, which was determined to be quantitative, we enriched for BrdU-labeled DNA and did not detect unlabeled DNA. By using pure cultures of Bacteroides vulgatus, the ability of Bacteroidales bacteria to take up and incorporate BrdU into nascent DNA was confirmed. Fecal Bacteroidales organisms took up and incorporated BrdU into DNA during growth. In sewage incubated aerobically at the in situ temperature, Bacteroidales genetic marker sequences persisted for at least 24 h and Bacteroidales fecal bacteria grew for up to 24 h as well. Detection by PCR using a low, quantitative cycle number decreased the sensitivity of the assay such that we were unable to detect fecal Bacteroidales human-specific marker sequences in unlabeled or BrdU-labeled fractions, even when fluorescent detection was used. Using 30 PCR cycles with unlabeled fractions, human-specific Bacteroidales sequences were detected, and they persisted for up to 24 h in sewage. These data support the utility of BrdU labeling and immunocapture followed by length heterogeneity PCR or fluorescent detection using low numbers of PCR cycles. However, this method may not be sensitive enough to identify cells that are present at low densities in aquatic environments.     

Methylation of lysine residues of histone fractions in synchronized mommalian cells

Synchronized cultures of mammalian cells were labeled with ¹⁴C-methyl methionine. Labeled methionine methyl groups were incorporated into certain histone fractions, forming methyl lysine. Incorporation of labeled methyl group into histone fractions as ¹⁴C-methyl lysine was followed through the cell cycle from late G₁ into early M. The ¹⁴C-methyl lysine contents of fractions F2a and F3 began to rise in S and reached maxima after termination of DNA and histone synthesis, coincident with the beginning of mitosis, and began to fall by mid-M. The ¹⁴C-methyl lysine content of fraction F2b rose to a maximum early in S, coincident with initiation of DNA synthesis, and rapidly decreased to its original unmethylated level by late S. Fraction F1 remained unmethylated during the period G₁-M. Evidence is presented to demonstrate differential methylation of histone fractions and to substantiate differential temporal coupling of the methylation of specific histone fractions with histone and DNA biosynthesis.     

Bacillus subtilis Cell Cycle as Studied by Fluorescence Microscopy: Constancy of Cell Length at Initiation of DNA Replication and Evidence for Active Nucleoid Partitioning

Fluorescence microscopic methods have been used to characterize the cell cycle of Bacillus subtilis at four different growth rates. The data obtained have been used to derive models for cell cycle progression. Like that of Escherichia coli, the period required by B. subtilis for chromosome replication at 37°C was found to be fairly constant (although a little longer, at about 55 min), as was the cell mass at initiation of DNA replication. The cell cycle of B. subtilis differed from that of E. coli in that changes in growth rate affected the average cell length but not the width and also in the relative variability of period between termination of DNA replication and septation. Overall movement of the nucleoid was found to occur smoothly, as in E. coli, but other aspects of nucleoid behavior were consistent with an underlying active partitioning machinery. The models for cell cycle progression in B. subtilis should facilitate the interpretation of data obtained from the recently introduced cytological methods for imaging the assembly and movement of proteins involved in cell cycle dynamics.     

Optimization of chemical induction conditions for human herpesvirus 8 (HHV-8) reactivation with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) from latently-infected BC-3 cells

Human herpesvirus 8 (HHV-8) persists as episomal DNA in latently-infected cells and can establish two alternative life cycles, latent or lytic. 12-O-tetradecanoyl-phorbol-13-acetate (TPA) is a known inducer of HHV-8 in several human primary effusion lymphoma cell lines and has been widely used for HHV-8 reactivation; however, induction conditions have differed, resulting in varying levels of virus expression. We have used HHV-8 latently-infected BC-3 cells as a model to determine critical parameters for optimizing virus reactivation by TPA. We found that cell growth properties and drug treatment conditions were important for maximum reactivation of HHV-8. Addition of TPA to cells in the early log phase of a sigmoidal growth curve, which was tightly associated with high percentage of the cells in early S phase and with lower histone deacetylase activity in the cells, provided the optimum cell conditions for latent virus to switch to lytic replication. Furthermore, increasing TPA concentration (up to 320 ng per ml) at 48 h exposure time resulted in increased virus production. The results demonstrate the use of a step-wise strategy with chemical induction that may facilitate broad detection of latent DNA viruses and novel virus discovery.     

Alkali-labile colicinogenic factor E1 DNA molecules formed in the presence of N-methyl-N'-nitro-N-nitrosoguanidine.

Plasmid ColE1 DNA of E. coli was used as a target DNA molecule to analyse the structural modification of DNA by N-methyl-N′-nitro-N-nitrosoguanidine. When the low concentration of this drug was used, both cell growth and overall DNA synthesis were neither stimulated nor inhibited, and plasmid DNA molecules were isolated as closed circles after replication. These molecules were stable for the ribonuclease treatment, but became susceptible to the alkaline hydrolysis. Such alkali-labile sites of ColE1 DNA were found in the parental strands and randomly distributed from the restriction endonuclease EcoR1 cleavage site.     

Specific Levels of DNA Methylation in Various Tissues, Cell Lines, and Cell Types of Daucus carota

The level of DNA methylation in Daucus carota was found to be tissue specific, but no simple correlation between developmental stage or age of tissue and the level of DNA methylation was found. Among three different suspension culture lines from the same variety grown under identical conditions, large differences in the level of DNA methylation were observed. The highest and lowest levels were found in two embryogenic cell lines originating from the same clone. Suspension cells from one of the embryogenic cell lines were fractionated into three morphologically defined cell types using Percoll gradient density centrifugation, and the uniformity of these fractions was evaluated by image analysis. The three cell types showed different levels of DNA methylation. The lowest level was found in the fraction containing the precursor cells of somatic embryos.     

Butyrate inhibits and Escherichia coli-derived mitogen(s) stimulate DNA synthesis in human hepatocytes in vitro

Bacterial constituents and products of the bacterial metabolism pass from the gut lumen to the portal vein and may influence the homeostasis of the liver. Our aim is to examine whether DNA synthesis of human hepatocyte cell lines is affected by constituents of Escherichia coli species as well as by intracolonic products of bacterial fermentation that reach the liver via the portal vein. Supernatant solutions and bacterial cell fractions (containing either whole dead bacteria, cell walls, cytosol or non-soluble intracellular components) of E. coli K12 and of E. coli species from rat fecal flora were separated by multi-step centrifugation, French press, and microfiltration. The supernatant solution and the cell fractions were incubated with a human hepatoma cell line (Hep-G2) and with a cell line derived from non-malignant human liver cells (Chang cells) for 24 h. The cells were labeled with tritiated thymidine before processing to autoradiography. DNA synthesis was estimated by the labeling index (LI%). DNA synthesis was also estimated following incubation of Hep-G2 cells with short chain fatty acids (acetic, propionic, butyric and succinic acid), acetaldehyde, and ammonium chloride. Epidermal growth factor and a water extract of Helicobacter pylori were used as references. The fractions of E. coli from rat fecal flora containing cytosol and non-soluble intracellular components significantly increased the labeling index in both Hep-G2 and Chang cells (p < 0.05). In addition, the supernatant solution significantly increased the LI in Chang cells (p < 0.05). Epidermal growth factor increased the LI of Hep-G2 cells dose-dependently (p < 0.05). Butyric acid reduced DNA synthesis at 10(-4) M (p < 0.05). The highest doses of acetaldehyde were cytotoxic and reduced the LI. Escherichia coli species contain mitogenic factors to human hepatocytes. The mitogen(s) are present in the supernatant solution, in the cytosol and in non-soluble intracellular components. Butyrate, which is a product of bacterial fermentation of colonic substrates inhibit DNA synthesis in the hepatocyte cell lines. Our findings suggest that soluble mitogen(s) that diffuse from the microorganism to the outer environment, intracellular bacterial constituents, and products of the bacterial metabolism that reach the liver via the portal vein may influence the cell kinetic steady-state of hepatic cells.     

Inositol Metabolism in Plants. III. Conversion of Myo-inositol-2-3H to Cell Wall Polysaccharides in Sycamore (Acer pseudoplatanus L.) Cell Culture

Prolonged growth of cell cultures of sycamore (Acer pseudoplatanus L.) on agar medium containing myo-inositol-2-³H resulted in incorporation of label predominately into uronosyl and pentosyl units of cell wall polysaccharides. Procedures normally used to distinguish between pectic substance and hemicellulose yielded carbohydrate-rich fractions with solubility characteristics ranging from pectic substance to hemicellulose yet the uronic acid and pentose composition of these fractions was decidedly pectic. Galacturonic acid was the only uronic acid present in each fraction. Subfractionation of alkali-soluble (hemicellulosic) polysaccharide by neutralization followed by ethanol precipitation gave 3 fractions, a water-insoluble, an ethanol-insoluble, and an ethanol-soluble fraction, each progressively poorer in galacturonic acid units and progressively richer in arabinose units; all relatively poor in xylose units.