Secondary structure and methylation of DNA in reinoculated tumors and cultures of embryonic cells transformed by oncogenic viruses]

Using the kinetic formaldehyde method the concentration of secondary structure defects (SSD) in the DNAs of ascite leukosis L 1210 cells and cultures of hamster embryonic cells transformed by virus SV-40 and polyoma was evaluated. It was found that this concentration was considerably higher than in the DNAs from normal liver and primary culture of mouse embryonic cells. The occurrence of the defects in malignant cell DNAs is not due to enzymatic degradation of DNA. Using thin-layer chromatography the content of m5C in the DNAs from 17 sources (transformed cell cultures, experimental tumours and liver cells of mouses with Ehrlich ascite carcinoma) were determined. The methylation level for all these DNAs was higher than for normal mouse and rat liver DNAs. No correlation between the SSD concentration and m5C content in the DNAs studied was observed.     

Thermoinduced structural changes in the membranes of animal organs following administration of antioxidants and during growth of malignancies

By means of weak-bound spin probes--stable iminoxyl free radicals differing in the level of hydrophobity there were studied thermoinduced structural transitions in the membranes of cell organells of animal liver after intraperitoneal injection of antioxidants and in the course of malignant growth, and in the nuclear membranes of Ehrlich ascite carcinoma as well. It has been found that during the growth of Ehrlich ascite carcinoma changes in rotationary mobility of probes in cell nuclei isolated from the liver of tumour-carrying animal are similar to the changes observed after antiocidants injection. A different pattern is observed in tumour cells. The membranes of ascite cell nuclei are characterized by a weak dependence of tau c on temperature for both probes. Within the temperature range studied no characteristic structural transitions proceeding in the nuclei of intact animals are observed.     

Detection of left-helical segments in eukaryotic DNA]

The method of DNA binding to nitrocellulose filters was applied to DNA isolated from mouse liver and Ehrlich ascite carcinoma (EAC), calf thymus, and lymphocytes from patients with chronic lymphoid leukemia. In those and phage PM2 DNA the increase in the DNA binding to the filters with a rise in NaCl concentration from 0.5 up to 4.5 M was sigmoidal being suggestive of a conformational transition. No such activity was found in the case of phage lambda or single-stranded DNA. The binding decreased dramatically after mild cleavage of DNA with DNAase I or treatment with phospholipase C or Eco RI and Hin PI restrictases. Incubation of DNA with ethidium bromide led to decrease in the amount of bound DNA. This effect was enhanced with a rise in the dye concentration. The isotherms of ethidium bromide binding to eukaryotic DNA obtained in Scatchard plots by optic titration had a component with a positive slope at low values of r. Bivalent ions (Mg2+, Zn2+) shifting the equilibrium towards the Z-form increased the proportion of macromolecules retained on the filters at NaCl concentrations of 1-3 M. Local changes in the helix conformation were studied with the help of chemical probes: diethylpyrocarbonate (guanine Z-DNA) and osmium-pyridine reagent (pyrimidines of boundary B-Z sites). These probes incorporation into samples of liver DNA, EAC, and lymphocytes resulted in chemical modification of all these samples. Modification of DNA by osmium-pyridine reagent led to inhibition of subsequent restriction by Eco RI restrictase. The data obtained are suggestive of the presence of Z-regions in the B-helix of eukaryotic DNA. A topological model of Z-site stabilization in small superhelical loops of DNA fixed by protein or lipoprotein molecules is proposed.     

Use of liposomes for incorporation of alkylating derivatives of mono- and oligonucleotides into mammalian cells]

It was shown that within the liposomes mono- and oligonucleotides and their alkylating derivatives penetrate the cells of Ehrlich ascite carcinoma and peritoneal exudate of the mice. Inside the cells the alkylating reagents are mainly utilized for modification of proteins (42--76%), RNA (5--16%) and DNA (3--9%). Presumably DNA modification is largely dependent on the penetration of the reagents into the nuclei. No significant differences in alkylation of the cell components by oligoadenylate derivatives, capable of complementary interactions with nucleic acids and mononucleotide derivatives, incapable of such interactions, were observed.     

Regulation of lipid oxidation and structural state of the nuclear membrane after the irradiation of tumor-bearing animals

Changes of antioxidative activity (AOA), lipid composition and microviscosity of different membrane regions in tumor cell nuclei and in the liver of tumor-host with Ehrlich ascite carcinoma (EAC) after irradiation were studied. On the basis of the obtained data the analysis of the control system of lipid oxidation in the membrane was carried out. This control system involves a relationship between AOA changes, lipid composition, their oxidative ability and the nuclear membrane structure. It was shown that after irradiation the control system in the nuclei of tumor cells had the same state as before irradiation and was different from the normal one. The control system in the nuclei of tumor-host liver after irradiation starts to work in a regime which is characteristic of irradiated cells. It was shown that the principle difference in the control system functioning in tumor and tumor-host nuclei disappeared after irradiation.     

Purification and characterization of thymidylate synthetase in the liver of the mouse bearing Ehrlich ascites tumor

The activity of thymidylate synthetase in the liver of the ddY strain male mouse increased transitorily according to the increase in tumor cell number at maximum 7-9 days after ip transplantation of Ehrlich ascites tumor. The enzyme was able to be purified from the tumor host mouse liver or from the normal mouse liver in the same manner as from tumor cells using Affi-Gel blue and methotrexate-Sepharose 4B affinity column chromatography. The three enzyme preparations obtained were purified at 27,000-38,000-, and 8,000-fold, and yielded total activities of 11, 3, and 16% of these homogenates, respectively. These preparations were similar in molecular weight to the whole enzyme (67,000) and its subunit (34,000), optimum pH, and Km values either for deoxyuridine 5'-monophosphate or tetrahydrofolate in the presence of formaldehyde. Furthermore, the amount of 5-fluoro-2'-deoxyuridine 5'-monophosphate forming the ternary complex with the enzyme and tetrahydrofolate paralleled the enzyme activities in the cytosol fractions of the three tissues. The characteristics of the tumor host liver enzyme were similar to those of the proliferating tissues, the Ehrlich ascites tumor.     

DNA ligase from mouse Ehrlich ascites tumor cells

The molecular (Mr = 120,000; s20, w = 5S) and catalytic properties (Km (ATP) = 3 microM; Km (nicked DNA) = 0.2 microM; Km (Mg2+) = 3 mM) of DNA ligase from mouse Ehrlich ascites tumor cells are similar to those of the enzymes from calf thymus and rodent liver. The activity level of DNA ligase from the tumor cells is about 10-fold higher than that from mouse liver. Immunochemical titration of DNA ligase with antibodies against the calf thymus enzyme showed that the higher level of DNA ligase activity in the tumor cells is due to an increase in enzyme quantity and not to elevation of the catalytic efficiency of the enzyme molecule. These results suggest that there is little apparent difference between the qualities of DNA ligases from the tumor cells and normal tissues of rodents and calf.     

Changes in the ultraviolet fluorescence and the size of Ehrlich ascites carcinoma cells in the 1st hours after X-ray irradiation

Using the Ehrlich ascite carcinoma cells, a decrease in ultraviolet fluorescence (UVF) was demonstrated one hour following a 10 Gy X-irradiation, and an increase in UVF 5 hours following the same irradiation. The same changes were demonstrated for cell turbidity. Association between the optical changes and those in cell radius and membrane protein state is discussed.     

Trichloroethylene-Induced Gene Expression and DNA Methylation Changes in B6C3F1 Mouse Liver

Trichloroethylene (TCE), widely used as an organic solvent in the industry, is a common contaminant in air, soil, and water. Chronic TCE exposure induced hepatocellular carcinoma in mice, and occupational exposure in humans was suggested to be associated with liver cancer. To understand the role of non-genotoxic mechanism(s) for TCE action, we examined the gene expression and DNA methylation changes in the liver of B6C3F1 mice orally administered with TCE (0, 100, 500 and 1000 mg/kg b.w. per day) for 5 days. After 5 days TCE treatment at a dose level of 1000 mg/kg b.w., a total of 431 differentially expressed genes were identified in mouse liver by microarray, of which 291 were up-regulated and 140 down-regulated. The expression changed genes were involved in key signal pathways including PPAR, proliferation, apoptosis and homologous recombination. Notably, the expression level of a number of vital genes involved in the regulation of DNA methylation, such as Utrf1, Tet2, DNMT1, DNMT3a and DNMT3b, were dysregulated. Although global DNA methylation change was not detected in the liver of mice exposed to TCE, the promoter regions of Cdkn1a and Ihh were found to be hypo- and hypermethylated respectively, which correlated negatively with their mRNA expression changes. Furthermore, the gene expression and DNA methylation changes induced by TCE were dose dependent. The overall data indicate that TCE exposure leads to aberrant DNA methylation changes, which might alter the expression of genes involved in the TCE-induced liver tumorgenesis.     

鼠肝细胞癌变中DNA甲基化作用的研究

Activity of DNA methylase and DNA methylation level were measured from normal mouse liver, mouse liver charged with H22a ascitic hepatoma and H22a ascitic hepatoma cell by measuring incorporation of H3-methyl. S-Adenosyl-3H-methyl-methionine (3H-SAM) was used as methyl donor. DNA methylation level of different cells were measured by HP-LC. DNA methylase activity and DNA methylation level of H22a ascitic hepatoma, mouse liver charged with H22a ascitic hepatoma are lower than normal mouse liver. Treatments of antitumor drugs lead to a rising of DNA methylase activity of tumor cell, however, the DNA methylation level of tumor cell has not rised after such treatments.     

Developmental expression of antioxidant enzymes in guinea pig lung and liver

Antioxidant enzyme activities, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and total glutathione concentration were determined in guinea pig lung and liver over the final period of gestation (days 50-68) and at several ages post-partum. Pulmonary antioxidant capacity increased markedly over the final days of gestation, individual changes ranging from 29% (glutathione) to 198% (GSH-Px). Liver antioxidant capacity was always 4-fold to 10-fold greater than that of the lung and exhibited very similar developmental profiles to those observed in the lung. From day 60 gestation to term (68 days), activity of the liver antioxidants increased, ranging from 246% (CAT) to 610% (glutathione). A number of antioxidants in both lung and liver exhibited either immediate pre- or post-birth decreases in activity. These falls could not be attributed to the way in which the results were expressed: i.e. they were similar, expressed per unit DNA, per unit protein, or per g wet wt. Following birth, liver antioxidant capacity increased such that the highest enzyme activities or glutathione concentration were recorded at 66 days post-partum. In lung, only Mn-SOD and glutathione exhibited higher levels at 66 days postpartum than at birth. In combination, these results of pulmonary and hepatic antioxidant enzyme activity indicate that the lung is not unique in acquiring increased antioxidant protection in the final period of gestation. They also suggest that a tissue's antioxidant requirement is dictated more by metabolic rate (hence free radical production) than incident partial pressure of oxygen.     

Adenosine deaminase activity in hepatomas of varying degrees of malignancy]

In highly malignant Gelshtein 22A hepatcma and ascites Ehrlich carcinoma adenosine deaminase activity was found to be reduced 3-fold as compared with that of the normal mouse liver. In less malignant hepatomas adenosine deaminase activity drops only by 20%. A certain reduction of adenosine deaminase activity was also noted in the liver to tumour-bearing mice.     

Titration of SH-groups of histone H3 in a DNP preparation of normal and neoplastic animal cells with a mercury-containing spin marker and with a DTNB preparation]

DNP samples isolated from the cells of calf thymus and Ehrlich ascite carcinoma of mice were examined. SH-groups of histone H3 of chromatin from these cells were titrated with mercury-containing spin label and with DTNB under joint action of different salt and sarcosyl concentrations on DNP. The results revealed differences in accessibility and titration of histone H3 SH-groups in DNP of normal and tumor cells with DTNB, as well as in molecular dynamics of the mercury-containing spin label introduced to these SH-groups.     

Antitumour and antioxidant activity of some Red Sea seaweeds in Ehrlich ascites carcinoma in vivo

The antitumour activities of extracts from the Red Sea seaweeds Jania rubens, Sargassum subrepandum, and Ulva lactuca were investigated in an in vivo mice model based on intramuscular injection of Ehrlich ascites tumour cells. In parallel, antioxidant activities were measured. Tumour marker levels, liver biochemical parameters, and hepatic oxidant/antioxidant status were measured to prove the anticancer and antioxidant nature of the algal extracts. Significant decreases in carcinoembryonic antigen (CEA) and a-fetoprotein (AFP) levels, activities of liver enzymes, levels of nitric oxide (NO) and malondialdehyde (MDA), and an increase in total antioxidant capacity (TAC) were recorded in groups treated with the algal extracts. Jania rubens was selected for phytochemical screening of its phytoconstituents. In addition, carotenoids, halides, minerals, lipoidal matters, proteins, and carbohydrates were studied. Furthermore, 7-oxo-cholest-5(6)-en-3-ol (1) and cholesterol (2) were isolated from the dichloromethane fraction.     

Proteinase activity of nuclei from various tissues towards endogenous histones]

The proteinase activities of nuclei isolated from tissues differing in their mitotic activities (brain, thymus, liver, ascite lymphoma) towards the histones and non-histone acid -- extractable proteins were studied. The sensitivity of different histone fractions to nuclear proteinase depends on temperature and time of nuclei incubation under conditions providing for complete dissociation of chromatin proteins from DNA (2 M NaCl--5 M urea). The proteinase activity in the brain and thymus nuclei is revealed only under prolonged (43 hrs) incubation of the nuclei at 25 degrees C, which is accompanied by partial proteolysis of histone H1. Histone H4 from brain nuclei and histone H2a from thymus nuclei are preferably degraded. In the nuclei isolated from the mice ascite cell lymphoma NK/ly and from rat liver the enzyme activity is revealed mainly towards the arginine-enriched histones H3 and H4. The proteolysis of the arginine-enriched histones in tumour cell nuclei is more complete. A high sensitivity to proteolysis was observed for non-histone acid-extractable proteins with low electrophoretic mobility, which were found in brain and tumour cell nuclei.     

Initiation of DNA synthesis in the liver and other tissues of adult mice by a growth factor (EACF) isolated from acellular fluid of the Ehrlich ascites carcinoma

The mitogenic effect of a new growth factor that we recently isolated from the acellular ascitic fluid of the Ehrlich ascites carcinoma grown in vivo was examined. We have called this factor EACF (Ehrlich ascites carcinoma factor). EACF caused initiation of DNA synthesis in the liver, submandibular gland, exorbital lacrimal gland and epithelium of the tongue of adult mice after i.p. injection at a protein concentration of 3 micrograms per 25 g of body weight. For all tissues examined, except the tongue, EACF initiated DNA synthesis at about 48 to 60 h after injection, with the maximum effect at approx. 85 h, and the stimulatory effect lasting approx. 60 h. The initiation of DNA synthesis in liver, which is normally characterized by only an occasional cell passing through the S phase, by EACF is of particular interest. The initiation of DNA synthesis in the liver was not prevented by hypophysectomy. Evidence also indicates that a similar heat-labile growth promoting factor(s) is present in calf serum.     

Changes in Liver Cell DNA Methylation Status in Diabetic Mice Affect Its FT-IR Characteristics

Background

Lower levels of cytosine methylation have been found in the liver cell DNA from non-obese diabetic (NOD) mice under hyperglycemic conditions. Because the Fourier transform-infrared (FT-IR) profiles of dry DNA samples are differently affected by DNA base composition, single-stranded form and histone binding, it is expected that the methylation status in the DNA could also affect its FT-IR profile.

Methodology/Principal Findings

The DNA FT-IR signatures obtained from the liver cell nuclei of hyperglycemic and normoglycemic NOD mice of the same age were compared. Dried DNA samples were examined in an IR microspectroscope equipped with an all-reflecting objective (ARO) and adequate software.

Conclusions/Significance

Changes in DNA cytosine methylation levels induced by hyperglycemia in mouse liver cells produced changes in the respective DNA FT-IR profiles, revealing modifications to the vibrational intensities and frequencies of several chemical markers, including ν_as –CH₃ stretching vibrations in the 5-methylcytosine methyl group. A smaller band area reflecting lower energy absorbed in the DNA was found in the hyperglycemic mice and assumed to be related to the lower levels of –CH₃ groups. Other spectral differences were found at 1700–1500 cm⁻¹ and in the fingerprint region, and a slight change in the DNA conformation at the lower DNA methylation levels was suggested for the hyperglycemic mice. The changes that affect cytosine methylation levels certainly affect the DNA-protein interactions and, consequently, gene expression in liver cells from the hyperglycemic NOD mice.     

Effect of hydrocortisone on methylation and molecular population of DNA in rat liver]

The content of 5-methyl cytosine in rat liver DNA increases 1,7-fold 8 hours after intraperitoneal injection of hydrocortisone (5 mg per 100 g animal weight). The content of GC, physicochemical parameters (Tm, delta T, etc.) and DNA renaturation pattern did not show any changes. No changes were observed in the pattern of H3-thymidine incorporation into rat liver DNA: after hydrocortisone injection the radioactivity was found to be equally distributed in all isolated sequences of DNA, differing in the degree of reiteration (specific radioactivities of these DNA, fractions are very similar). Thus, the molecular population of DNA in liver cells remains unchanged, which suggests that the hormone-induced increase in the 5-methyl cytosine content is due to a change in the DNA methylation level. The methyation level of unique sequences (COt greater than 600), i. e. that of structural genes, does not undergo any essential changes. The reversible methylation of DNA regulated by hormones seems to be one of the mechanisms controlling gene activity.