The expression of virus-specific nucleotide sequences in Rous sarcoma cells.

Poly(A)-enriched virus-specific mRNAs (v-mRNA) are revealed in the subcellular fractions (nuclei, mitochondria, polyribosomes and cytosol) from chicken and hamster Rous sarcoma cells. The qualitative and quantitative differences in v-mRNA populations from permissive and non-permissive tumors are shown. The sedimentation analysis of v-mRNAs allowed to ascertain the presence of three molecular classes of v-mRNA in nuclei and cytoplasm of chicken Rous sarcoma cells (35S, 28S and 22S, and 35S, 26-28S and 20S, respectively). While two size classes (33S and 20S) are shown in hamster Rous sarcoma nuclei, only 24S v-mRNA is revealed in the cytoplasm of its tumor tissue. Thus, the oncovirus gene expression is restricted in the non-permissive tumor cells, unlike of the permissive chicken Rous sarcoma cells, both in the level of transcription, and in the processing and transfer v-mRNAs from nucleus to cytoplasm. The virus gene function peculiarities in the tumors of birds and mammals are discussed.

Provirus DNA sequences in Rous sarcoma cell genome.

Non-producer hamster and virus-producing chicken Rous sarcoma cells contain a complete avian sarcoma virus (ASV)-provirus DNA (pro-DNA). Unlike this, in normal hamster liver DNA to ASV--specific sequences are absent. Moreover, the nuclear chicken Rous sarcoma and chick embryo cell (CEC) DNA preparations contain practically all endogenous chicken virus (ECV)-specific sequences, while the hamster tumor DNA was annealed with only more than half of the ECV-RNA sequences. Thus, the pro-DNA of permissive hosts contains so-called "sarcoma-specific", "common" and "endogenous--specific" nucleotide sequences. The "sarcoma-specific" and "common" sequences are present in the hamster sarcoma pro-DNA, only. Both the pro-DNA of permissive cells and the pro-DNA of non-permissive cells consist of moderately reiterated and unique sequences. The "common" regions of pro-DNA are localized, mainly, in the unique zone, while "sarcoma-specific" and "endogenous-specific" sequences of pro-DNA - in the moderately reiterated and unique zones. The pro-DNA organization of permissive and non-permissive hosts is discussed.

Virus-specific sequences in nuclear DNA from non-producer hamster Rous sarcoma.

Proviral DNA from non-producer Rous sarcoma in Syrian hamster contains practically all the nucleotide sequences presented in 125I-labeled RNA from Rous sarcoma virus, Carr-Zilber strain. Virus-specific sequences consist of moderately reiterated and unique DNA regions. The amount of Rous sarcoma virus-specific provirus equivalents in hamster Rous sarcoma DNA is equal to 5.2 +/- 0.01. Experiments on transfection show that proviral DNA studied possesses biological activity in respect to cell transformation and virus production. The infectivity of DNA from hamster tumor does not depend on the expression of group-specific (gs) antigen in the recipient cells.

Polyadenylated RNA molecules and polyribosomes in tumours of chemical and viral origin.

The distribution of poly(A)-enriched mRNA in nuclei, mitochondira, free and membrane-bound polyribosomes from normal C3HA mouse and Syrian hamster livers and normal chicken fibroblasts has been compared with that in corresponding subcellular fractions in a transplantable, chemically induced MD hepatoma, non-virus-producer hamster and virus-producer chicken Rous sarcomas. It has been shown that the content of poly(A)-RNA is increased in all tumour fractions except in free polyribosomes. The distribution of different classes of polysomes i.e. free, membrane-bound and mitochondrial outer-membrane-associated polysomes in tumour cells was changed in comparison to that in normal cells. It is concluded that in tumours of chemical and viral origin, the observed changes in the two components of the protein-synthesizing apparatus occur simultaneously.

Nucleic acids from subcellular fractions of N-nitrosodiethylamine-induced hepatoma in mice. II. Changes in gene expression during tumor progression.

The hybridization properties of in vivo labeled nuclear and mitochondrial ribonucleic acids of an transplantable hepatoma were studied. It was shown that during tumor progression the repression of nuclear genome revelead at its early stages was replaced by the de-repression at its later stages. The hybridizability of mitochondrial RNA with nuclear DNA was not changed.

Behavior of mitochondria from normal and tumor cells in isopycnic sucrose gradients.

Rat and mouse liver mitochondria, when centrifuged in a sucrose density gradient (25--50%, w/w), showed the presence of heavy (H) and light (L) subfractions with buoyant densities 1.185 and 1.170--1.165 g/ml, respectively. Mild treatment with digitonin or EDTA (30 mM) shifted H-subfraction of mitochondria into the lighter zone of the gradient and as a result of this the mitochondria were distributed as a homogenous band with buoyant density 1.170--1.165 g/ml. Mitochondria isolated from both MD hepatoma and Zajdela rat hepatoma were characterized by a homogenous banding with buoyant density 1.160--1.165 g/ml. Regarding to this, the content and patterns of polyribosomes bound to outer membranes of mouse tumor mitochondria were studied. Analysis of polyribosomes as well as the results of RNA polyacrylamide gel electrophoresis indicate that the content of these polyribosomes in tumor mitochondria is less than that in normal liver ones. However, the decrease of cancer cell membrane-bound polyribosomes cannot account for the differences in buoyant densities of mitochondria from normal and tumor tissues.

Studies of poly(A+)-RNA in mouse hepatoma and cortisone-stimulated rat liver.

The content of poly(A)-containing RNA in subcellar fractions has been investigated both in cortisone-treated rat liver and experimental hepatoma cells. The fractions included nuclei, cytoplasm, mitochondria, free and membrane-bound polyribosomes. 1) In both cases of genome activation (cortisone induction and hepatoma cells) an increase in poly(A) content of all subcellular fractions except free polyribosomes was observed. 2) Cortisone was found to induce elongation of poly(A) segments detected in both nuclei and cytoplasm. 3) An increase in the poly(A) block size also was found to be stimulated in nuclei and cytoplasm of hepatoma cells. 4) The observed elongation in poly(A) length occurred against the background of an increase of the population of of poly(A)-RNA's.

Presence of ribonucleotide sequences complementary to Rous sarcoma virus (RSV) RNA in chicken cells infected with RSV.

RNA--RNA molecular hybridization between [125I] RNA from Rous sarcoma virus virions and RNAs isolated from various subcellular fractions, i.e. nuclei, mitochondria, free and membrane-bound polyribosomes, from tumors induced by RSV in chickens resulted in the formation of RNAase-resistant hybrids only with the RNA of mitochondria and membrane-bound polysomes. The origin of complementary regions in the RNAs from these organelles is discussed.

Dependence of 1,2-dimethylhydrazine metabolism on its treatment schedule.

The content of cytochromes P-450 and b5 in rat liver microsomes, as well as the extent of labeling of nucleic acids and proteins of the liver and kidneys and of mucosa from different intestinal segments, was studied in rats injected daily or once a week subcutaneously with similar total doses of 1,2-dimethyl-hydrazine (SDMH) and in untreated rats. Daily SDMH administrations led to a decrease in cytochrome P-450 activity. Pretreatment of rats with unlabelled SDMH resulted in decreased labeling of DNA, RNA, proteins, and acid-soluble fractions after [3H]SDMH injection. A more pronounced effect was found after the daily treatment.

Distribution and carcinogenic action of 1,2-dimethylhydrazine (SDMH) in rats.

The radioactivity in the blood, bile, and contents from different parts of the gastro-intestinal tract was estimated for different time intervals up to 24 h after 3H-SDMH injection to rats. 65% of the radioactivity was excreted in the urine. Of the total quantity of radioactive products entering the intestine, 96% is brought with bile and only 4% from the circulation through the wall of the intestine. This latter small amount of SDMH metabolites plays a leading role in the genesis of intestinal tumours. This conclusion was proved by the observation that the intestinal tumours developed in different isolated segments of the gut where the entry of bile was excluded and by published data indicating that SDMH is excreted unchanged in the bile. It was shown that the carcinogenic effect depends upon the dose schedule of carcinogen treatment, probably, due to the changes in the SDMH metabolism. The optimal conditions for induction of intestinal tumours occur after administration of SDMH in a dose of 21 mg/kg body weight once a week. Hypothetic SDMH metabolic pathways leading to tumour production have been considered in the light of available experimental data.

Nucleic acids from subcellular fractions of N-nitrosodiethylamine-induced hepatoma in mice.

During eight successive isologous passages of hepatoma induced in male C3HA mice by N-nitrosodiethylamine, no common features of tumor progression were observed, although both the mitotic pattern and ploidy differed from generation to generation. These additional cytologic criteria allowed the biochemical examination of material least changed due to tumor progression. Tumor nDNA's were characterized by greater actinomycin D (AD)- and acridine orange (AO)-binding abilities than were normal nDNA's; this could have resulted from a higher proportion of double-stranded regions in tumor DNA. Isolated tumor deoxyribonucleoprotein had both lower template activity in an RNA polymerase system and fewer AD- and AO-binding sites, when compared with the activity and sites from normal mouse liver. RNA-DNA hybridization data with the above-mentioned findings showed that in hepatoma, part of the nuclear genome was repressed. Also, RNA "new classes" appeared and a certain proportion of nuclear genes controlling mitochondrial protein biosynthesis were derepressed in tumor mitochondria. The hybridization of mitochondrial RNA (mtRNA) and DNA revealed new classes of pulse-labeled RNA's in in vitro-incubated liver mitochondria that were absent from intact cell organelles; the hybridization properties of in vivo- and in vitro-formed hepatoma mtRNA's were similar. Competition and hybridization experiments demonstrated that in tumor mitochondria in vivo, some new classes of RNA existed. Hepatoma mitochondrial mRNA had a higher metabolic stability than did normal mRNA.

The mechanism of carcinogenic action of 1,2-dimethylhydrazine (SDMH) in rats.

The radioactivity level in blood, bile, urine and contents of parts of the gastro-intestinal tract in rats was studied after subcutaneous administration of 3-H-1,2-dimethylhydrazine (3-H-SDMH) which induces colonic tumours. The alkylation of DNA, RNA and protein in the intestinal mucosa, liver and kidneys was estimated 1 h to 28 days after 3-H-SDMH treatment from the 3-H-incorporation into these macromolecules. Administration of 3-H-1,2-diethylhydrazine (3-H-SDEH) which does not induce intestinal tumours was made as a control. Fifteen to 30 min after 3-H-SDMH treatment, marked radioactivity was found in blood, bile, urine and in contents of all regions of gastro-intestinal tract. After 3-H-SDMH administration no label occurred in the contents of localized segments of gastro-intestinal tract although it was present in blood, bile and urine. 3-H-SDMH methylated DNA, RNA and proteins of intestinal mucosa, liver and kidney to a high degree. One hour after 3-H-SDMH treatment the incorporation of label into protein of intestinal mucosa was higher than into liver and kidneys. 3-H-SDEH did not alkylate macromolecules in these organs but did so in thymus, spleen and brain, which are target organs for this carcinogen. After total hepatectomy, 3-H-SDMH did not methylate macromolecules of the intestinal mucosa. The following mechanism for the carcinogenic effect of SDMH is suggested. A carcinogenic metabolite of SDMH forms, in the liver, a conjugate with glucuronic acid. This glucuronide enters the gut both with bile and directly via the circulation. Microbial beta-glucuronidase releases the active metabolite which, in turn, alkylates tissue macromolecules.