Binding of chlorozotocin and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea to chromatin and nucleosomal fractions of HeLa cells (original) (raw)
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Cancer research, 1979
Poly(adenosine diphosphate ribose) polymerase, a chromatin-bound enzyme, was stimulated 150 to 200% after treatment of HeLa cells with methylnitrosourea (MNU). In contrast, a slight inhibitory effect on enzyme activity was observed after treatment of cells with various concentrations of chloroethylnitrosoureas. To define precisely the differential effects of nitrosoureas on the enzyme activity, their interactions with chromatin substructure were studied. A nonrandom, in vivo alkylation of chromatin DNA by equimolar concentrations of MNU and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) was revealed by digestion of nuclei from drug-treated cells with micrococcal nuclease and DNase I. [methyl-14C]MNU interacted preferentially with the more accessible regions of chromatin, the internucleosome linkers, whereas, the [chloroethyl-14C]CCNU alkylated the nucleosomal core DNA to a greater extent. These two drugs also differed in their extent of covalent modification of histone and nonh...
Influence of hydrocortisone on the binding of nitrosoureas to nuclear chromatin subfractions
Cancer Research
The effects of steroid-induced modifications of chromatin structure on the extent and sites of chloroethylnitrosourea binding to chromatin were studied using log-phase HeLa cells. The cells were exposed to 0.1 to 2.0 /¿M hydrocortisone for 22 hr; this resulted in depressed DMA synthesis while transcriptional activity was stimulated. Hydrocortisone had no effect upon cellular or nuclear uptake of the two nitrosoureas under study, 0.6 rriM chlorozotocin or 1-(2-chloroethyl-3-cyclohexyl-1-nitrosourea). Both drugs were found to alkylate transcriptional chromatin preferentially, as demonstrated by DNase II and DNase I digestion. This alkylation was stimulated 2-fold by the same micromolar concentrations of hydrocortisone, 0.1 to 2.0 ¡IM, which stimulated transcription. The extent of nuclear RNA alkylation, determined using RNase T2 as a probe, was found to contribute less than 20% of total chromatin alkylation and was unaffected by steroid pretreatment. Instead, the in creased alkylation within these chromatin subfractions was attributed to a steroid-induced alteration of chromatin struc ture. Electron microscopic examination of HeLa nuclear mor phology revealed a hydrocortisone-induced disaggregation of nuclear membrane-associated heterochromatin resulting in a more heterogeneous, less condensed distribution of chromatin. Such data are consistent with a relaxation of the supercoiled chromatin structure, resulting in increased transcription and increased accessibility of potential target sites for nitrosourea alkylation.
European journal of biochemistry / FEBS, 1980
The reaction of L5178Y lymphoblast cell chromatin with the alkylating agent bis(2-chloroethyl)methylamine has been studied as a function of time, pH and reagent concentration. The reaction with DNA of chromatin from which the proteins were dissociated, as well as with purified calf thymus DNA, was studied in parallel. The extent of alkylation of DNA in intact chromatin was 4--5 times as much as in parallel free DNA samples; up to 4% of nucleotide base pairs were substituted. The extent of monofunctional substitution of the proteins was similar, on a weight basis, to that of DNA. Chromatographic analysis of the depurinated products showed that in chromatin, as in DNA, position N-7 of guanine is the major site of reaction. Up to 25% of the reaction products were guanines cross-linked as bis(2-guanin-7-yl-ethyl)methylamine, indicating a considerable degree of DNA-DNA cross linking. Column analysis shows that up to 40% of the nuclear proteins are cross-linked to DNA at 10 mM bis(2-chlor...
Biochemical Pharmacology, 1982
All of the clinically available nitrosourea antitumor agents produce serious treatment-limiting bone marrow toxicity. A reduction in this toxicity can be achieved by attaching the chloroethylnitrosourea cytotoxic group to C2 (chlorozotocin) or C1 (1-(2-chloroethyl)-3-(fl-D-glucopyranosyl)-l-nitrosourea, GANU) of glucose. Both glucose analogs are less myelotoxic in mice than 1-(2-chloroethyl)-3cyclohepyl-l-nitrosourea (CCNU) or 1-(4-amino-2-methylpyrimidin-5-yl)methyl-3-(2-chloroethyl)-3nitrosourea (ACNU), while retaining comparable antitumor activity against the murine L1210 leukemia. To define the nuclear mechanisms for this reduced myelotoxicity, alkylation of L1210 and murine bone marrow DNA was quantitated. With the use of the endonucleases micrococcal nuclease and DNase I, the sites of alkylation within the chromatin substructure were determined. Experiments were performed on L1210 leukemia or bone marrow cells that had been incubated in vitro for 2hr with 0.1 mM [14C]chloroethyl drug. The quantitative alkylation of DNA by GANU was 1.3-fold greater in L1210, as compared to bone marrow, cells. This ratio of DNA alkylation is comparable to the 1.3 ratio we previously reported for chlorozotocin [L. C. Panasci, D. Green and P. S. Schein, J. clin. Invest. 64, 1103Invest. 64, (1979]. In contrast, the ratio of alkylation (L1210:bone marrow DNA) for the myelotoxic ACNU was 0.66, similar to 0.59 for CCNU. Nuclease digestion experiments demonstrated that chlorozotocin and GANU preferentially alkylated internucleosomal linker regions of bone marrow chroinatin, while nucleosome core particles were the preferred targets of CCNU and ACNU. The reduced myelotoxicity of chlorozotocin and GANU may be correlated with the advantageous ratio of L1210:bone marrow DNA alkylation and preferential alkylation of internucleosomal regions of bone marrow chromatin.
Chemico-Biological Interactions, 1980
Treatment of HeLa cells with MNU results in a significant activation of the chromatin modifying enzyme, poly(ADP-Rib) polymerase, in nuclei and at the nucleosome level of chromatin. This appeared to be due to a direct effect of MNU on chromatin structural elements, initiating this study on the effect of MNU-induced carbamoylation at the subunit level of chromatin, the core nucleosome. Core nucleosomes were prepared by micrococcal nuclease digestion of HeLa nuclei isolated from cells treated with [carbonyl-14C]-MNU. Trypsin digestion of core nucleosomes containing carbamoylated proteins showed that 80% of the 14C-label was covalently associated with these proteins in a domain not sensitive to trypsin digestion. When core nucleosomes containing ADP-ribosylated histones were prepared from HeLa nuclei previously incubated with [3H] NAD, nearly 80% of the covalently bound ADP-Rib was proteolytically removed by trypsin. These data suggest that lysines distal to the NH2-termini of histones were most susceptible to carbamoylation, while amino acid residues near the NH2-termini were subject to ADP-ribosylation. DNA of carbonyl-modified nucleosomes, phosphorylated (32p) at the 5'~end, was digested 2-3 times faster with DNAase I than DNA in control nucleosomes. DNAase I generated the 10-base repeat pattern in these modified particles typical of native nucleosomes. Mapping the DNAase I cleavage sites revealed that sites 100, 90, 70, 60 and 50 nucleotides from the 5'-end
Binding of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea to L1210 cell nuclear proteins
Cancer research, 1976
The binding of 1-(2-chloroethyl)-3-(cyclohexyl)-1-nitrosourea (CCNU) to the proteins of the L1210 cell nucleus has been studied using both [cyclohexyl-14C]CCNU and [chloroethyl-14C]CCNU. Most of the bound [cyclohexyl-14C] moiety of CCNU was found to exist in a form that was stable in acid solution but labile and dialyzable in alkaline solution. A small amount of the cyclohexyl moiety was bound to histones in a stable, nondialyzable form. The drug/protein ratio for the H1 histone was about 0.01 to 0.02 mole/mole. No binding of the cyclohexyl group to acidic proteins or of the chloroethyl group to either histones or acidic proteins was observed. Thus, the interaction of CCNU with the proteins of the cell nucleus can be defined in terms of the modification of histones by the cyclohexyl moiety.
The Effect of Mechloroethamin on Reconstituted Chromatin
The Medical Journal of The Islamic Republic of Iran, 1997
In this study the effect of a methylated derivative of nitrogen mustard (mechloroethamin) on solubilized and reconstituted chromatin was investigated using UVNis spectroscopy, DNA and SDS gel electrophoresis and densitomeoic scanning techniques, The results indicated that the 210 and 260 nm absorbances of reconstituted chromatin were decreased in a dose�dependent manner after treatment with various concentrations of mechloroethamin. Alkylated chromatin showed difficulty in producing nucleosomes in comparison to the control, therefore a 50% reduction in absorbanccs was obtained. The amount of both core histone protein and DNA in the supernatant was decreased with increasing drug concentrations. It is suggested thatrnechloroethamin alters the interaction of ON A protein in the chromatin by producing cross�links between DNA and protein or protein and protein.
Effects of 1,3-bis(2-chloroethyl)-1-nitrosourea and related compounds on nuclear RNA metabolism
Cancer research, 1974
This study examines the effects of 1,3-bis(2-chloroethyl)-l-nitrosourea and certain related compounds on the synthesis and processing of nucleolar and nucleoplasmic RNA in cultured L1210 cells. Treatment with 0.25 mM 1,3-bis(2-chloroethyl)-lnitrosourea has little effect on either quantity or size distribution of RNA synthesized during the first 10 min after drug addition. By 30 min, however, synthesis of both RNA fractions is markedly inhibited. By contrast, the processing of 45 S nucleolar (ribosomal precursor) RNA is inhibited immediately. The inhibition of nucleolar RNA processing is due to an action at some step after the normal synthesis and methylation of 45 S species. The processing or exit of high-molecular-weight RNA from the nucleoplasmic fraction also is inhibited. Structure-activity studies of some nitrosoureas indicated that inhibition of RNA processing requires a substitution at the N-3 nitrogen of the urea moiety. The isocyanate decomposition products of 1,3-bis(2-chloroethyl)-lnitrosourea and of l-(2-chloroethyl)-3-cyclohexyl-lnitrosourea were found to be more effective than the parent compounds, suggesting that it is these isocyanate products that are responsible for the inhibition of RNA processing. In accord with this interpretation, l-(2-chloroethyl)-lnitrosourea, which lacks an N-3 nitrogen substituent and therefore cannot form an isocyanate product, was inactive. Since this nitrosourea is known to be highly active against experimental neoplasms, it is concluded that inhibition of RNA processing or other effects of isocyanate products are probably not directly responsible for the antitumor activity. These effects may instead be side effects, potentially related to toxicity.