Verrell M. Randolph

Induction of histone acetylation and inhibition of growth of mouse erythroleukemia cells by S-allylmercaptocysteine

Growth-inhibitory effects on DS19 mouse erythroleukemia cells were seen in the micromolar concentration range with allicin and S-allylmercaptocysteine and in the millimolar range with allyl butyrate, allyl phenyl sulfone, and S-allyl cysteine. Increased acetylation of histones was induced by incubation of cells with the allyl compounds at concentrations similar to those that resulted in the inhibition of cell proliferation. The induction of histone acetylation by S-allylmercaptocysteine was also observed in Caco-2 human colon cancer cells and T47D human breast cancer cells. In contrast to the effect on histone acetylation, there was a decrease in the incorporation of phosphate into histones when DS19 cells were incubated with 25 μM S-allylmercaptocysteine. Histone deacetylase activity was inhibited by allyl butyrate, but there was little or no effect with the allyl sulfur compounds examined in this study. A similar degree of downregulation of histone deacetylase and histone acetyltransferase was observed when DS19 cells were incubated with S-allylmercaptocysteine or allyl isothiocyanate. The induction of histone acetylation by S-allylmercaptocysteine was not blocked by a proteasome inhibitor. The mechanism by which S-allylmercaptocysteine induces histone acetylation remains to be characterized. It may be related in part to metabolism to allyl mercaptan, which is a more effective inhibitor of histone deacetylase.

Induction of histone acetylation in mouse erythroleukemia cells by some organosulfur compounds including allyl isothiocyanate

In previous studies we observed that some allyl sulfides can cause increased acetylation of histones and differentiation in DS19 mouse erythroleukemia cells. In the present work we observed increased acetylation of histones with allyl isothiocyanate and butanethiol but not with butyl sulfide or butyl disulfide. Increased acetylation of histones was established by change in electrophoretic mobility, incorporation of [3H]acetate or immunoblotting. Histone deacetylase in nuclei of DS19 cells was inhibited 74% by 0.5 mM allyl mercaptan and 43% by 0.5 mM butanethiol but was not significantly affected by 0.5 mM allyl isothiocyanate. There was some degree of reversibility in the effect of allyl isothiocyanate when the cells were incubated for 15 hr in fresh medium. The data suggested that allyl isothiocyanate may stimulate histone acetylation rather than inhibit histone deacetylation. Addition of allyl isothiocyanate, however, had very little or no additional effect on the induction of histone acetylation caused by trichostatin A. Histone acetyltransferase activity determined in cell homogenates was not increased by preincubation of cells with allyl isothiocyanate or inclusion of allyl isothiocyanate in the assay medium. It was concluded that treatment of mouse erythroleukemia cells with allyl isothiocyanate can cause increased acetylation of histones but the mechanism for this effect requires further elucidation.

Recovery of Native Proteins from Preparative Electrophoresis Gel Slices by Reverse Polarity ElutioN

A technique for high yield recovery of native, biologically active proteins from preparative polyacrylamide gel slices by reverse polarity elution is described. No apparatus other than the standard slab gel electrophoresis system is required. Several proteins have been recovered in biologically active form at a 90% yield, in quantities ranging from 0.4 mg to 4.2 mg. The method is effective with both small (9,000 dalton) and large (186,000 dalton) polypeptides. Both simple and complex proteins are recovered intact. For example, the copper-zinc and manganese superoxide dismutases from crude soybean extracts are active upon recovery. Similarly, the vitamin D-dependent calcium binding proteins from rat kidney and intestine are isolated by this method in homogeneous, active form.

Oxidation of one-carbon compounds to formate and carbon dioxide in rat liver mitochondria.

Abstract In rat liver mitochondria, swollen with phosphate and supplemented with NAD+, the oxidation of the methyl carbon of sarcosine to formate is enhanced by the addition of NADP+. No carbon dioxide is formed. Formaldehyde and serine, which are the only oxidation products of the methyl group in the absence of the pyridine nucleotides, are decreased by an amount equal to the formate produced. Carbon dioxide, as well as formate, is produced when the mitochondria are treated with EDTA, even without the addition of the pyridine nucleotides. When the mitochondria are exposed to pyrophosphate without added NAD+ and/or NADP+, all of the oxidized sarcosine-methyl can be recovered as formate, [3-C]serine, and carbon dioxide. Formaldehyde accumulates only if the system is supplemented with Mg2+. In the presence of NADP+ or the combined pyridine nucleotides, serine accumulation is depressed by an amount equal to the increase in carbon dioxide production. Both carbons of glycine and the 3-C of serine can also be oxidized to carbon dioxide in the pyrophosphate-treated mitochondria. The oxidation of the methyl carbon of S-adenosylmethionine to formaldehyde, [3-C]serine, formate, and carbon dioxide requires a whole homogenate supplemented with glycine. Neither exogenous formaldehyde nor formate is oxidized to carbon dioxide in any of the mitochondrial systems capable of converting sarcosine-methyl to carbon dioxide. Under conditions in which [N5,N10-14C-methylene]- and [N10-14C-formyl]tetrahydrofolate can be isolated as intermediate products of [14CH3]sarcosine, exogenous [N5,N10-14C-methylene]tetrahydrofolate can also be converted to [3-14C]serine, [14C]formate, and [14C]carbon dioxide.

Pseudo-inhibitors of neutrophil superoxide production: Evidence that soybean-derived polypeptides are superoxide dismutases

We have previously reported the purification of polypeptides from soybean which are potent inhibitors of superoxide production by human neutrophils. We now report that neither oxygen uptake nor hydrogen peroxide production by stimulated neutrophils is affected by these inhibitors. Furthermore, the E-1 and E-3 polypeptides inhibit ferricytochrome c reduction by a xanthine oxidase superoxide generation system. The inhibitory activity of E-3 in the model system is blocked by 1 mM KCN while E-1 is only slightly cyanide sensitive. Atomic absorption analysis of E-1 and E-3 polypeptides reveal copper in the latter and manganese in the former. Thus, E-3 is a copper-containing superoxide dismutase while E-1 appears to be a manganese-containing superoxide dismutase.

N-methyl oxidation in liver mitochondria of triiodothyronine-treated and thyroidectomized rats

Abstract The levels of sarcosine dehydrogenase and acid-nonextractable flavin in the inner matrix of mitochondria of rat liver are decreased in animals treated with triiodothyronine and are elevated in the mitochondria obtained from thyroidectomized animals. Administration of triiodothyronine does not affect the electron-transfer flavoprotein associated with the sarcosine dehydrogenase or the relative amounts of soluble and membrane-bound proteins of the mitochondria. In phosphate-washed mitochondria from either the controls or the triiodothyronine-treated rats, the O 2 uptake equals the total of the [ 14 C]formaldehyde and [β- 14 C]serine isolated as reaction products of the sarcosine-[ 14 C]methyl group. In contrast to its restraint of sarcosine or choline oxidation in preparations capable of oxidative phosphorylation, ADP does not inhibit the oxidation of these substrates in mitochondria of rats given triiodothyronine.

DNA in cytosol fractions obtained by differential centrifugation of homogenates of rapidly growing liver tumors

Preliminary work revealed that the cytosol fractions obtained by differential centrifugation of homogenates of the rapidly growing Morris hepatomas 7777 and 7288CTC contained substantially more DNA than could be detected with the more slowly growing hepatoma 5123C or the liver of tumor-bearing rats. The amount of DNA in the cytosol fractions of the rapidly growing hepatomas was decreased by concentrations of cations which are known to promote precipitation of solubilized chromatin. Data on the incorporation of [3H]thymidine into DNA of nuclear and cytosol fractions and the time-dependent effects of hydroxyurea were compatible with initial nuclear synthesis and later release of the DNA seen in the cytosol fractions. The amount of this DNA was increased in larger tumors and was less in isolated cells than in solid tumors. Postmortem changes in the liver and hepatoma 5123C, but not in hepatomas 7777 and 7288CTC, resulted in a large increase in DNA appearing in the cytosol fraction. The results indicated that the measurement of DNA in cytosol fractions may serve as a guide to the existence of necrosis in tumors, but is not always proportional to the degree of necrosis.