Biomedical subjects

W Helliger

Publications and source records attributed to W Helliger.

29 records · Page 2Linked to original sources

PubMed1988

Separation of Friend erythroleukaemic cell histones and high-mobility-group proteins by reversed-phase high-performance liquid chromatography.

A procedure for the rapid separation of histones and high-mobility-group (HMG) proteins from Friend erythroleukaemic cells (line F4N) by reversed-phase high-performance liquid chromatography is reported. By using a Nucleosil 300-5 C4 column and a multistep water-acetonitrile gradient containing 0.1% trifluoroacetic acid, the HMG-1 and HMG-2 proteins, several H1 subfractions including H1(0), H4, H2B, two H2A variants and two H3 subfractions were separated. Under changed conditions, by applying a varied acetonitrile gradient system, even two H2B variants were fractionated. The methods described seem to be a real alternative to the time-consuming polyacrylamide gel electrophoresis.

Cell Line↗

PubMed1988

A new h.p.l.c. isolation procedure for chicken and goose erythrocyte histones.

Total chicken erythrocyte histones were separated by reversed-phase h.p.l.c. using a multi-step acetonitrile gradient in a very short time (35 min). The proteins were eluted in the following order: H1, H5, H2B, H2A.2, H4, H2A.1 and H3.2. Applying a special gradient system adapted for the separation of very-lysine-rich histones, chicken erythrocyte H5 was resolved into two subfractions. Their electrophoretic mobilities were identical in both SDS and acetic acid/urea/Triton polyacrylamide-gel electrophoresis, but different in free-flow electrophoresis. Amino-acid-sequence analyses revealed that the two components only differ with respect to position 15, one having glutamine in that position and the other arginine. A separation of histones prepared from goose erythrocytes disclosed no H5 subfractionation. Furthermore, histones obtained from anaemic-chicken blood were analysed by the above-mentioned h.p.l.c. conditions. An alteration in the relation of H1 to H5 was detected, but no further differences in the number and quantity of the histones and histone variants were observed as compared with the corresponding proteins processed from normal-chicken blood.

Anemia↗

PubMed1988

Differential sensitivity of histone acetylation in nitrogen-mustard sensitive and resistant cells. Relation to drug uptake, formation and repair of DNA-interstrand cross-links.

Cultivation of Ehrlich-ascites tumor cells in the presence of N-mustard leads to a selection of cells with a defective choline carrier. As N-mustard employs the choline carrier for transport, this results in reduced drug uptake and in a decrease in drug sensitivity which is specific for N-mustard. Walker carcinoma cells with a stable pleiotropic resistance to a variety of alkylating agents and adriamycin exhibit no evidence for an impaired drug transport and show the same frequency of DNA-interstrand cross-links as the sensitive parental line. Both sensitive and resistant Walker cells exhibit equal capacities for repair of N-mustard induced DNA-interstrand cross-links. The inhibition of histone acetylation by N-mustard, however, was found to be significantly lower in the resistant Walker or Ehrlich cells compared to sensitive counterparts. Although the difference between N-mustard concentrations leading to half maximal inhibition of histone acetylation in sensitive and resistant cells is considerably smaller than the difference between N-mustard doses required for half maximal inhibition of cell proliferation the data suggest that--besides DNA-DNA cross-linking--the inhibition of histone acetylation has to be considered as an important alternative mechanism responsible for the cytotoxic activity of alkylating agents. Inhibition of histone acetylation is not due an accelerated deacetylation and is predominantly expressed in chromatin fractions soluble in 0.1 M NaCl after digestion with micrococcal nuclease.

Acetylation↗

PubMed1986

Histone separation by high-performance liquid chromatography on C4 reverse-phase columns.

Previous work in our laboratory (Lindner, H., Helliger, W., and Puschendorf, B. (1986) J. Chromatogr. 357, 301-310) described a rapid separation of H1 and core histones by reverse-phase high-performance liquid chromatography using a Bio-Rad Hi-Pore butyl (C4) silica-based column. Despite the short elution time, a high resolution of the different histone fractions, except H4 and H2A (MHP), could be obtained. In this report we present a method for the separation of H4 and H2A (MHP) as well, while maintaining a similar analysis time. By varying the gradient, trifluoroacetic acid concentration (0.05%), and flow rate (1.3 ml/min) the histones were eluted from the C4 column in the following order: H1 (MHP), H1 (LHP), H2B, H2A (LHP), H4, H2A (MHP), H3 (LHP), and H3 (MHP). LHP and MHP refer to less and more hydrophobic histone variants. The identification of the individual protein fractions was performed by comparison the retention times with pure histone markers as well as by gel electrophoresis.

Animals↗

PubMed1986

Alkylating antitumor agents reduce histone acetyl-transferase activity.

N-Mustard depresses the acetylation of histones in Ehrlich ascites and Walker carcinoma cells. It is demonstrated that this effect is not caused by an accelerated deacetylation but is due to an inhibition of the acetyl-transferase reaction. Employing 4-sulphonatoethylthio-cyclophosphamide it is demonstrated that the alkylating agent affects predominantly the acetylation of a chromatin fraction which is soluble in 0.1M NaCl after digestion with micrococcal nuclease. After removal of the alkylating agent, the recovery of histone acetylation is relatively slow and--in contrast to the repair of DNA cross-links--characterized by a 4-hr lag period. The reduction of histone acetylation by N-mustard is much less expressed in cells which are resistant to the drug than in the sensitive parental lines. This is in contrast to DNA-interstrand cross-links in Walker cells where both N-mustard sensitive and resistant cells inhibit the same cross-link frequency and identical repair rates. Based on these data it is concluded that the inhibition of histone acetylation may be an important part of the mechanism by which alkylating agents inhibit tumor growth.

Acetylation↗

PubMed1986

[Tumor biochemistry as basis for advances in tumor chemotherapy].

Oncogenes, onc-gene products and the concept of autonomous self-stimulation of tumor growth by autocrine production of growth factors are discussed with regard to the development of new antitumor agents. The significance of the plasma membrane as an attractive target in tumor chemotherapy is emphasized. Membrane bound enzymes and related reactions which are involved in growth factor dependent regulation of cell proliferation offer new targets for antitumor chemotherapy. Examples demonstrating the antiproliferative effects of phospholipase-C and calmodulin inhibitors are presented. A synergistic effect of these drugs with alkylating agents is observed. Studies devoted to the improvement of alkylating antitumor agents have also led to the plasma-membrane as a particularly suited target. The same may be true for other antitumor agents like anthracyclines and platinum complexes for which evidence exists implicating the involvement of cellular membranes in the mechanism of cytotoxic action.

Alkylating Agents↗

PubMed1984

Increase in histone acetylation and transitions in histone variants during Friend cell differentiation.

Histone acetylation of Murine Erythroleukemia Cells (MELC) has been re-examined. It is demonstrated that sodium butyrate causes hyperacetylation of core histones in inducible as well as non-inducible MELC strains. This indicates that histone hyperacetylation per se is not sufficient to activate genes. However, [3H]acetate incorporation into core histones of the inducible MELC line F4N increases after induction of differentiation with dimethylsulfoxide (DMSO), in contrast to the non-inducible variant F4+. Thus histone acetylation may play a role as an auxiliary mechanism for gene activation (and inactivation). In addition, the appearance of a histone H3 variant during differentiation of MELC is reported.

Acetylation↗

PubMed1984

Depression of histone acetylation by alkylating antitumor agents: significance for antitumor activity and possible biological consequences.

Treatment of Ehrlich ascites tumor cells with the alkylating antitumor agents triaziquonum, N-mustard and cyclophosphamide leads to a reduction in the posttranslational incorporation of 3H-acetate into histones and the extent of histone acetylation in Ehrlich ascites tumor cells. All core histones are affected. The depression of histone acetylation is not the result of a decrease in acetyl-CoA. Evidence is presented for an activation of histone deacetylase by alkylating agents. A reduction of histone deacetylation is observed after exposure to all concentrations of alkylating agents which inhibit cell proliferation. In order to evaluate the biological consequences of a reduction of histone acetylation, the extent of acetylation was modulated by either chemical acetylation or treatment with butyrate. In all cases an increase in histone acetylation leads to an enhancement of the rate of transcription. In accord with previous reports from our laboratory (1), it is concluded that the reduction of histone acetylation affects RNA synthesis. It is emphasized, however, that besides a regulation of transcription, histone acetylation may be involved in other cell functions. Thus, the complete biological consequences of the reduction of histone acetylation remain to be elucidated. In view of the antitumor activity of the alkylating agents it seems noteworthy that hepatoma AS30D cells are characterized by a remarkably higher extent of histone H4-acetylation compared to normal, adult, fetal, or regenerating liver.

Alkylation↗

PubMed1984

Depression of histone acetylation by alkylating antitumor agents in murine cells.

Treatment of Ehrlich ascites tumor cells with the alkylating agent triaziquone [2,3,5-tris(ethyleneimino)benzoquinone-1,4] and nitrogen mustard leads to a reduction of the posttranslational acetylation of histones. Acetylation of all core histones is affected. The reduction of labeling of acetylated sites is accompanied by a dose-dependent decrease in the extent of acetylation as indicated by the level of acetylation of H4. The depression of histone acetylation is expressed at all concentrations of the alkylating agents which cause significant inhibition of tumor cell proliferation. It could be excluded that the observed effects are caused by an impairment of acetyl coenzyme A synthesis.

Acetates↗

PubMed1983

Inhibition of tumor growth by an alkylation of the plasma membrane.

The effect of nitrogen mustard (2-chloro-N-2-chloroethyl-N-methylethanamine), Trenimon (2,3,5-trisethyleneiminobenzoquinone-1,4), chlorambucil (4-[p-(bis[2-chloroethyl]amino)-phenyl]butyric acid) and phosphamide mustard (N,N-bis(2-chloroethyl)-diamidophosphoric acid) on Na+/K+-ATPase, membrane fluidity and cell multiplication was studied. With the exception of chlorambucil which does not affect Na+/K+-ATPase all concentrations of the other alkylating agents which inhibit cell multiplication of Ehrlich ascites tumor cells depress the activity of the Na+/K+-ATPase. All alkylating agents--including chlorambucil--caused an increase in the apparent degree of fluorescence polarization after labelling of the plasma membrane with 1,6-diphenyl-1,3,5-hexatriene (DPH). This effect is interpreted as a decrease in membrane fluidity caused by the alkylating drugs. The decrease in membrane fluidity is due to a direct interaction of the alkylating agent with the plasma membrane and is expressed at all concentrations of the drug which inhibit cell proliferation. No effect on membrane fluidity is observed after treatment of cells resistant to nitrogen mustard. The biological consequence of a decrease in membrane fluidity was investigated by growing Friend erythroleukemia cells in the presence of 10 mM cholesterol hemisuccinate. This procedure raises the microviscosity of the plasma membrane and depresses cell proliferation.

Alkylating Agents↗

PubMedDate not supplied

Application of high-performance capillary electrophoresis to the analysis of H1 histones.

High-performance capillary electrophoresis for the separation of rat testis H1 histone variants and their phosphorylated modifications is described. The influence of buffer pH, hydroxypropylmethyl cellulose, and buffer concentration has been investigated. Under optimized conditions (500 mM phosphate buffer, pH 2, 0.03% hydroxypropylmethyl cellulose) using an uncoated capillary, eight H1 histone subfractions, including two H1(0) histones and H1t and their phosphorylated modifications, are resolved. Application of capillary electrophoresis to the separation of H1 histones provides an important new alternative to high-performance liquid chromatography (HPLC) and traditional gel electrophoresis.

Buffers↗