Carcinogenic alterations in murine liver, lung, and uterine tumors induced by in utero exposure to ionizing radiation.

The atomic bombing of Hiroshima and Nagasaki and the nuclear accident at Chernobyl raised the question of prenatal sensitivity to ionizing radiation-induced cancer. In this study, mice were exposed to single doses of gamma-radiation (0.2-2.0 Gy) at different embryonic stages. The tumor incidence increased with dose from 15% in control mice to 35% in mice irradiated with 2.0 Gy on 18 d of prenatal life. Various oncogenic events were investigated in lymphoid, liver, lung, and uterine tumors. We observed threefold to fivefold increases in myc expression in 25% of the lymphomas, and the expression of Ha-ras and p53 genes decreased in 40% and 60% of the lung tumors by twofold to fivefold. Point mutations were tissue specific: Ha-ras codon 61 mutations were found in about 40% of the liver adenocarcinomas, Ki-ras codon 12 mutations in about 17% of lung tumors, and p53 mutations in about 15% of the lymphomas. Amplification and rearrangement of the p53, myc, and Ha-, Ki- and N-ras genes were not detected. Loss of heterozygosity on chromosome 4 at the multiple tumor suppressor 1 and 2 genes was observed in all types of malignancies. Allelic losses on chromosome 11 at the p53 locus were found in lymphoid, liver, and lung tumors, but they were absent from uterine tumors. Multiple oncogenic changes were often detected. The frequency of carcinogenic alterations was similar in spontaneous and radiation-induced lymphoid, liver, and uterine tumors. In radiation-induced lung adenocarcinomas, however, the incidences of many oncogenic changes were different from those found in their spontaneous counterparts. This suggests that different oncogenic pathways are activated during spontaneous and in utero gamma-radiation-induced murine lung carcinogenesis.

Oncogenic changes in murine lymphoid tumors induced by in utero exposure to ionizing radiation.

We have investigated the oncogenic alterations in murine lymphomas induced by in utero exposure to gamma-radiation. The expression of the myc oncogene increased in 23% of the tumors. Alterations in the expression of the ras oncogenes and in the p53 tumor suppressor gene were not characteristic. The p53 gene was mutated in a low percentage of the tumors (12%). Ras mutations were not detected. Loss of heterozygosity (LOH) at the p53 locus was found in 30% of the tumors, and LOH at the mts tumor suppressor gene was detected in 23% of lymphomas. Multiple oncogenic changes were infrequent in the investigated tumors. There were no essential differences in the frequency of carcinogenic alterations in spontaneous and gamma-radiation-induced lymphomas.

Protein phosphorylation and kinase activities in tumour cells after hyperthermia.

Phosphorylation of various proteins and the activities of specific kinases were studied in tumour cells after hyperthermia. P388 lymphoid tumour cells were treated at 40-45 degrees C for 1 h in vitro. Immediately after heat treatment, particulate and cytosol cell fractions were isolated, phosphorylated proteins separated and various kinase activities were measured. Hyperthermic treatment of the cells caused a significant decrease in protein kinase C activity while the activity of calcium-ion and phospholipid-independent protein kinases increased. Phosphorylation of cytosol proteins of 120, 80, 33, 25 and 14 kDa increased significantly after hyperthermia, and protein kinase C selectively phosphorylated the last three of these proteins. The phosphorylation of three heat shock proteins (44, 70 and 85 kDa) was not changed after hyperthermic treatment. Four tyrosine kinase activities were separated. The protein tyrosine kinase activity decreased to one-tenth of the control value after 45 degrees C for 1 h hyperthermia. The changes in kinase activities and protein phosphorylation induced by hyperthermia proved to be temperature- and time-dependent.

Effect of hyperthermia and X-irradiation on survival and occurrence of metastases in mice bearing P388 tumor.

Survival of P388 lymphoid tumor-bearing mice and the occurrence of metastasis was studied after combined modality treatment with hyperthermia and X-irradiation. P388 ascites tumor cells were treated at 42 degrees C or 43.5 degrees C for 1 hr in vitro and transplanted on B6D2F1 mice intraperitoneally (i.p.) or intramuscularly (i.m.). Hyperthermic treatment at 43.5 degrees C increased the median survival time (MST). Increased life-span (ILS) was found after i.p. transplantation (54%) and after i.m. transplantation (30%). During the life-span of tumor-bearing animals, significantly fewer metastases were observed in liver and spleen after hyperthermia and 5-10% metastasis occurred after transplantation of ascites tumor cells treated at 43.5 degrees C in vitro compared with 90% in the untreated control animals. The lower occurrence of metastasis could not be ascribed to the higher cell-killing effect of hyperthermia. When both modalities were combined the best tumor growth retardation effect was obtained when ascites tumor cells were treated at 43.5 degrees C for 1 hr before being transplanted i.m. and 1 day later locally X-irradiated with 6 Gy. In this case, 77% ILS was found demonstrating a synergistic effect of the two modalities. While X-irradiation alone did not change the occurrence of metastasis, after combined modality treatment it was as low as with hyperthermia alone (5-10%). In connection with the significantly lower occurrence of metastasis, the possible alterations of P388 tumor cell membrane and surface proteins induced by in vitro hyperthermic treatment are discussed.

Transcription of human ribosomal DNA may terminate at multiple sites.

The termination of human pre-rRNA transcription has been investigated. The most abundant possible termination site was detected 360 bp downstream from the 28S gene, in front of the first SalI box of the rDNA spacer. This site, however, is partially bypassed during transcription, and three additional termination points were detected inside the heterogeneous region of the rDNA spacer. Later sites were mapped about 930, 1030 and 1110 bp downstream from the 3' end of the 28S rRNA gene. The authors suggest that the T clusters and pyrimidine-rich regions play an important role in the termination processes. They either may influence the efficiency of the SalI boxes in terminating the synthesis of pre-rRNAs or may serve as independent signals for the fail-safe termination of readthrough transcripts. In both cases transcription of human rDNA ceases at multiple sites.

New tandem repeat region in the non-transcribed spacer of human ribosomal RNA gene.

A new repetitive DNA region was identified in the non-transcribed spacer of human rDNA, namely a long (4.6 kb) sequence motif (Xbal element) was present in two copies. The repeating unit composed of two parts. One of them consisted of unique nucleotide sequences, interrupted by some simple sequences. The other, about 3.1 kb long one assembled only from highly repeated simple sequences. The unique sequence region contained two, inverted copies of the human AluI type repetitive DNA family. The authors suggest that the XbaI elements may flank the tandem arrays of human rRNA genes as terminal repeats and they might function both as the origin of rDNA replication and/or site of homologous recombination.

Inverse correlation between growth and degrading enzyme activity of seedlings after gamma and neutron irradiation of pea seeds.

The effect of gamma-, 14 MeV neutron- and fission neutron irradiation was investigated on the growth rate and degrading enzyme activities of pea seedlings. Both dormant pea seeds and 4-day-old growing seedlings were used for the experiments. Depending on the gamma dose between 15 and 300 Gy the height of pea seedlings was found shorter, and parallel with this the endogenous RNase and peroxidase activities were higher than in the unirradiated controls. Seedlings proved to be more sensitive by about one order of magnitude than seeds. Irradiation of seeds between 5 and 10 Gy slightly enhanced the growth rate of seedlings (10 per cent) and parallel with this, the RNase activity measured was lower than that in the controls. On irradiation of seedlings with 14 MeV neutrons the growth inhibition and RNase activity enhancement was only 1.3 times more effective than in the case of irradiation of seeds. The following RBE values were obtained after irradiation of seeds, related to the biological effect of gamma rays: in growth inhibition, 6 for 14 MeV neutrons and 12 for fission neutrons, and the enhancement of two enzyme activities was 15-30 for 14 MeV neutrons and 45-58 for fission neutrons. In the case of seedling irradiation with 14 MeV neutrons the RBE was 1.0 for growth inhibition and between 3 and 6 for enhancement of enzyme activity. The isoenzyme pattern of RNase also changed: two isoenzymes became predominant after the gamma irradiation of seeds, characterized by molecular weights of 21,000 and 30,000, respectively. As a result of enhanced RNase activity, the degradation of longer polysomes to monomeric ribosomes occurred. Thus after ionizing irradiation of pea seeds and seedlings an inverse correlation was found between the growth rate of pea seedlings and the activities of degrading enzymes.

Characterization of the L1NH repeat family of Novikoff hepatoma.

Long interspersed repeated sequences of the Novikoff hepatoma rat tumour cell genome were cloned and studied. No basic differences were found when the genomic organization of the Novikoff hepatoma was compared with that of other mammalian L1 families. The nucleotide sequence of the central approximately 4 kb (1 kb = 10(3) bases) part of the Novikoff hepatoma LINE (L1NH) appeared to be more highly conserved than the sequences found at the 5' and 3' ends. Moreover, the central approximately 4 kb core fragments were not always associated with the same end sequences. Thus, the occurrence of the more-conserved and more-abundant central portion in L1NH suggest that: (1) besides reverse transcription, other DNA- and/or RNA-mediated mechanisms might be involved in the dispersal of LINE families; and that (2) L1 sequences can sometimes consist of a compound unit made up of members of different L1 subunits and sequences with different genomic copy numbers.

Supercoil induced S1 hypersensitive sites in the rat and human ribosomal RNA genes.

Rat and human ribosomal RNA gene fragments in supercoiled plasmids were examined for S1 nuclease hypersensitivity. In the transcribed portion of genes the number and distribution of S1 sites were found to be species specific. No S1 sites were detected in the promoter regions. In the nontranscribed spacer (NTS), downstream of the 3' end of 28S RNA gene, S1 sites appear to be conserved in rat and human rDNAs. A rat NTS fragment (2987 nucleotides long), containing three S1 sites was sequenced and the S1 sites in this region were localized in polypyrimidine . polypurine simple repeat sequences. Other types of simple sequences, two type 2 Alu repeats and an ID sequence were also found in the sequenced region. The possible role of simple sequences and S1 sites in transcription and in recombination events of rDNA is discussed.

Inhibition of protein synthesis in developing mouse brain after fission neutron irradiation in utero.

Previous investigations showed that when pregnant mice were exposed to a single whole-body dose of 0.5 Gy fission neutrons on Day 17 +/- 2 of gestation [H. H. Vogel, Jr. and S. Antal, Radiat. Res. 98, 52-64 (1984)] about 40% of the newborn mice died and the body and brain weights of surviving animals decreased by 30-35%. Decreases of body and brain weights were most prominent by the third week after birth, but the content of nucleic acids related to wet weight did not change in liver and brain upon irradiation [S. Antal, A. Fónagy, Z. Fülöp, E. J. Hidvégi, and H. H. Vogel, Jr. Int. J. Radiat. Biol. 46, 425-433 (1984)]. Studies presented in this paper show that after a single whole-body dose of 0.5 Gy neutron irradiation on Day 18 of pregnancy protein synthesis decreased in liver and brain of 3-week-old mice irradiated in utero. Incorporation of labeled amino acids in vivo into acid soluble nuclear proteins decreased by 15% in liver and by 40% in brain. It was significantly reduced into brain histones and certain brain nonhistone proteins (separated by two-dimensional electrophoresis). The amount of H1 and H4 brain histones decreased as well. Investigations with isolated protein synthesizing systems proved that the peptide bond formation was not impaired by irradiation. The aminoacylation of transfer-RNA, however, decreased in both liver and brain by 26-34 and 34-41%, respectively. Comparing the aminoacylation capacities in the two unirradiated organs, a much lower (about one-third) capacity was found in brain than in liver. Moreover, this low aminoacylation capacity of brain decreased further by about 40% after neutron irradiation. These results suggest that in the developing irradiated brain the reduced capacity of aminoacylation of transfer-RNA might be rate limiting for the efficiency of protein synthesis.

Decreased weight, DNA, RNA and protein content of the brain after neutron irradiation of the 18-day mouse embryo.

Pregnant mice were irradiated with 0.5 Gy fission neutrons on the eighteenth day of their gestation. The average litter size at birth was unchanged but mortality increased 5-6 fold in the first 3 days. The irradiated mice were the same weight as control mice at birth but showed a progressively increasing weight deficiency up to at least 36 days as compared to controls. Brain weight was 37, 45 and 25 per cent less in 2-, 3- and 52-week old irradiated animals, respectively, and the ratio of brain weight to body weight was 25, 27 and 13 per cent less. The concentrations of DNA, RNA and protein (mg/g wet tissue) were the same in irradiated and control mice in both brain and liver at all three ages. Total DNA, RNA and protein contents of whole brain after irradiation were 56-75 per cent of the control levels. No definite decrease was observed in liver. Histological study at 6 hours after irradiation showed nuclear pyknosis in the central nervous system from definite to very severe according to the part examined. It is concluded that damage to the central nervous system of the 18-day mouse foetus after neutron irradiation is mainly due to killing and/or inhibition of the differentiation of neuroblasts.

Interaction of GGCC sequences of DNA with anticancer dianhydrogalacticol, detected by inhibition of restriction enzyme BspI.

Interaction of DNA with 1,2-5,6-dianhydro-galactitol (DAG, NSC 132 313), a bifunctional alkylating agent used in cancer therapy was studied. Treatment of lambda phage DNA with DAG in vitro protected some of the specific cleavage sites against the restriction enzyme BspI. The extent of protection depended on the concentration and time of DAG treatment but complete protection was not observed. Since the inactivation of the enzyme by DAG was excluded experimentally, the protection can be attributed to the binding of DAG to GGCC sequences of DNA. These experiments support the finding that guanine is the target of DNA alkylation by DAG (Institóris and Tamás, 1980). DAG treatment in vitro induced single strand breaks on DNA and this effect was also found to be concentration and time dependent.

Cloning and characterization of a Novikoff hepatoma ribosomal DNA-fragment containing the initiation site of transcription.

A 10.3 kilobase EcoRI fragment of Novikoff hepatoma rat ascites tumor ribosomal gene was cloned in pBR322 vector. The cloned fragment contained part of the 18S RNA gene, and about 8 kilobases of the 5' spacer region. A restriction map was constructed by cleavage of the fragment with BamHI, HindIII, KpnI, PstI, SstI and XhoI enzymes. A putative transcription initiation site for the 45S pre-ribosomal RNA was localized by P1 nuclease protection mapping at a distance of about 130 base pairs 5' to the HindIII site on the restriction map. Both the restriction map and the position of the initiation site of transcription were almost identical to those of the corresponding rat ribosomal DNA fragments.

Effect of CH-1243, a pyrido (1,2-a) pyrimidine derivative on the elevated activity of lysosomal enzymes of rabbit aorta and liver in experimental atherosclerosis.

The effect of CH-123 (3-carbethoxy-6-methyl-1-9-(carboxy-methyl)-1-4-oxo-6,7,8,9-tetrahydro-4H-pyrid o(1,2a)pyrimidine) was investigated on the activity of 4 lysosomal enzymes: beta-glucuronidase, beta-galactosidase, N-acetyl-beta-glucosaminidase and acid phosphatase obtained from aortic smooth muscle and liver cells of rabbits. Animals were fed on a 2% cholesterol diet for 4 weeks and used an experimental atherosclerotic group. In drug-treated groups, after 4 weeks of cholesterol feeding the diet was changed to regular food and the animals were treated daily either with 50 mg/kg CH-123 or with 250 mg/kg Clofibrate. The postnuclear supernatant of homogenates of liver and aortic cells was isolated, lysosomes were fractionated by sucrose density gradient centrifugation, and the activity of enzymes was measured. In cholesterol-fed animals the enzyme activities of aorta and liver was 3-5 times higher than in the control, i.e. in the group of rabbits fed regular food. On Clofibrate treatment the enzyme activities were 2-3 times higher, but on treatment with CH-123, they were only 1.2-1.8 times above the control. Experiments suggest that CH-123 treatment suppresses the elevated lysosomal marker enzyme activities in aortic and liver cells of atherosclerotic animals.

The effect of dibromo-dulcitol and dianhydro-dulcitol (galactitol) on RNA synthesis in ascites tumor cells.

The mechanism of action on RNA synthesis of anticancer dibromo-dulcitol (DBD, NSC-104800) and dianhydro-dulcitol (DAD, or elsewhere dianhydrogalactitol, DAG, NSC-132313) was investigated. Rats, bearing Yoshida or Novikoff hepatoma ascites tumor cells sensitive to these drugs were treated with doses equivalent to half the LD50 value. Nucleolar RNA (noRNA) and nuclear RNA (nRNA) were pulse labelled with 3H-uridine, isolated and fractionated on sucrose density gradient. After 18 h treatment with either drug and after 3 h with DAD noRNA synthesis increased and the rate of ribosomal RNA (rRNA) precursor processing was enhanced. Investigation of low-molecular weight nRNAs (LMW nRNAs) (separated by polyacrylamide gel electrophoresis) showed increased synthesis and/or accumulation of RNA species (5S RNA, uridylic acid rich RNAs) related to rRNA synthesis. The tritium labelled drugs were bound to distinct fractions of nRNA, separated by sucrose density gradient ultracentrifugation, both in vivo and in vitro. This fact may be explained by the formation of intra-, or intermolecular crosslinking of pre-messenger RNA. The enhanced RNA synthesis might be interpreted as an alteration in the functions of nuclear proteins, involved in the regulation of gene transcription and processing of RNA precursors.

Effect of altered membrane lipid composition and procaine on hyperthermic killing of ascites tumor cells.

The influence of membrane fluidity on hyperthermic cell killing has been investigated in ascites tumor cells. Membrane lipid composition of P388 ascites tumor cells were modified by feeding host animals with diets containing either unsaturated fatty acids (UFA) or saturated fatty acids (SFA). Both kinds of ascites were heat treated in vitro at 37, 42 or 43.5 degrees C for 30 or 60 min. The cell killing effect of hyperthermia was tested by transplantation of cells into recipient mice and survival examined. While at 37 and 42 degrees C for 1 h, there was no difference in cell killing of the two types of ascites, elevating the temperature to 43.5 degrees C the survival was significantly longer on transplantation of ascites of UFA diet. This effect was potentiated by membrane-fluidizing drugs. On the addition of 1 mM procaine during 1 h treatment at 43.5 degrees C, the ascites of SFA diet killed 80% of mice, while the ascites of UFA diet left all the mice alive at least for 3 months. Scanning electron-microscopic observations of the treated cells was performed in parallel and showed close correspondence with the results of survival studies. In conclusion, the increase of membrane fluidity by incorporating more of UFA or by the addition of membrane-fluidizing drugs or especially by the combination of both, the sensitivity of cells to heat enhanced. These experiments support the hypothesis that membranes are a target for hyperthermia.

Effect of whole-body irradiation on the synethesis of ribonucleic acid associated with nuclear ribonucleoprotein particles of rat liver.

Synthesis of the RNA of rat liver nuclear RNP particles ("informofers") was studied within 12 hrs after 1930 rad whole-body gamma-irradiation. 14C-orotic acid was administratered intravenously and nuclear RNP particles were extracted by 0.1 M and by 0.3 M salt solutions at pH 8.0. Radioactivity of the RNP particles 1.5--2.0 times higher than that of the unirradiated controls up to 6 hrs after irradiation, exhibiting a maximum at the first hour. The labelling of the 0.3 M RNP particles was higher and chased less rapidly than that of the 0.1 M RNP particles. The RNA to protein ratio and the protein composition of the RNP the RNP particles did not change after irradiation as examined by CsCl density gradient centrifugation and by acrylamide gel electrophoresis, respectively. In the cytoplasm enhanced synthesis of rapidly labelled RNA sedimenting with 40 S cytoplasmic RNP paricles could also be demonstrated after irradiation. It is concluded that the synthesis of messenger-type RNA associated with liver nuclear RNP particles increases and more RNA is transferred from the nucleus to the cytoplasm after whole-body irradiation.