A small nuclear RNA, U5, can transform cells in vitro.

Low-molecular-weight RNA exhibiting transforming potential was identified in chemically induced lymphoma cells by the transformation of mink lung cells after transfection. The RNA was sequenced by the direct chemical method and was shown to be a small nuclear RNA, U5. The transforming potential of the RNA was further studied in quantitative transformation assays using 3Y1, a rat fibroblastic cell line. Transformed foci appeared with a latency of 3 to 4 weeks after transfection. U5-transformed 3Y1 cells frequently carried an amplified c-myc oncogene. In addition, U5 induced chromosome aberrations in transfected cells, indicating that the RNA acts as a clastogen. Transforming and clastogenic potentials were specifically inactivated when U5 was incubated with RNase H in the presence of a complementary oligonucleotide. We discuss a possible mechanism of U5-induced cell transformation.     

X-ray- and mitomycin C (MMC)-induced chromosome aberrations in spermiogenic germ cells and the repair capacity of mouse eggs for the X-ray and MMC damage

Chromosome aberrations induced at the first-cleavage metaphase of eggs fertilized with sperm recovered from spermiogenic cells which had been X-irradiated and treated with mitomycin C (MMC) at various stages were observed using in vitro fertilization and embryo culture technique. Furthermore, the repair capacity of the fertilized eggs for X-ray- and MMC-induced DNA damage which was induced in the spermiogenic cells and retained in the sperm until fertilization was investigated by analysis of the potentiation effects of 2 repair inhibitors, 3-aminobenzamide (3AB) and caffeine on the yield of chromosome aberrations. The frequency of chromosome aberrations observed in the eggs fertilized with sperm recovered from the early spermatid to late spermatocyte stage with X-irradiation of 4 Gy (16-20 days after X-irradiation) was markedly higher than that in the eggs fertilized with sperm recovered from spermatozoa to late spermatid stage (0-8 days after X-irradiation). The induced chromosome aberrations predominantly consisted of chromosome-type aberrations, the main type being chromosome fragment followed by chromosome exchange through all the spermiogenic stages. On the other hand, a high frequency of chromosome aberrations was not induced through all the stages with MMC treatment of 5 mg/kg. The remarkable potentiation effects of 3AB and caffeine were found in the eggs fertilized with sperm recovered from almost all the spermiogenic stages after X-irradiation. In the MMC treatment, a remarkable caffeine effect was observed occasionally in mid-early spermatids to late spermatocytes where a large amount of MMC damage could be induced. These results suggest that the large amount of DNA lesions induced in spermiogenic cells by X-rays and MMC persist as reparable damage until sperm maturation and are effectively repaired in the cytoplasm of the fertilized eggs.     

The stage of chromosome duplication in the cell cycle as revealed by X-ray breakage and 3H-thymidine labeling

By means of combined experiments of X-irradiation and ³H-thymidine labeling of the chromosomes which are in the phase of synthesis, and the subsequent analysis at metaphase on the autoradiographs of the chromosomal damage induced during interphase, it was shown that in somatic cells from a quasi-diploid Chinese hamster line cultured in vitro the chromosomes change their response to radiation from single (chromosome type aberrations) to double (chromatid type aberrations) in late G₁. These results are interpreted to indicate that the chromosome splits into two chromatids in G₁, before DNA replication. — By extending the observations at the second metaphase after irradiation, it was also seen that cells irradiated while in G₂ or late S when they reach the second post-irradiation mitosis still exhibit, beside chromosome type aberrations, many chromatid exchanges, some of which are labeled. Two hypotheses are suggested to account for this unexpected reappearance of chromatid aberrations at the second post-irradiation division. The first hypothesis is that they arise from half-chromatid aberrations. The second hypothesis, which derives from a new interpretation of the mechanisms of production of chromosome aberrations recently forwarded by Evans, is that they arise from gaps or achromatic lesions which undergo, as the cells go through the next cycle, a two-step repair process culminating in the production of aberrations.This work was supported in part by grant No. RH-00304 from the Division of Radiological Health, Bureau of State Services, Public Health Service, U.S.A.     

Cytogenetic effect of mitomycin C and cytosine arabinoside on mice of different genotypes]

Cytogenetic effect of mitomycin C (MC) and cytosine arabinoside (CA) on bone marrow cells of male mice of the strains 101/HY, C57BL/6Y C,3H/SnY and of the (C3HX101) F1 hybrids was studied. The frequencies of cells with chromosome aberrations after the treatment with MC at a 5 mg/kg dose were 54,4%; 41,8%; 40,4% and 26,8% in 101H, B6, C3H/Sn mice and in the F1 hybrids (C3HX101) respectively. The frequencies of cells with chromosome aberrations after the treatment with CA at a 500 mg/kg dose were 25,2%; 17,8%; 10,8% and the 101/H, B6, C3H/Sn mice and in the F1 hybrids (C3HX101) respectively. Both mutagens induced the greatest number of chromosome aberrations in the 101/H strain and the smallest number in the F1 hybrid (C3HX101). A positive correlation was established between the levels of induced and spontaneous chromosome lesions.     

新制癌菌素和平阳霉素诱发蚕豆染色体畸变的比较研究

本文报道了新制癌菌素(NCS)能诱发植物染色体畸变,同时观察了利用咖啡因后处理对NCS、PYM诱发染色体畸变的影响,研究了PYM切断DNA断头的性质。结果表明,NCS切割DNA产生3'-羟基末端和3'-磷酸末端;咖啡因能封闭3'-羟基末端抑制DNA的修复,从而提高诱变频率。PYM加咖啡因后处理,其染色体畸变频率与PYM单独处理无明显差异。说明PYM切断DNA所得到的产物,不是3'-羟基末端,而是3'-磷酸末端。     

The fate of U.V.-induced lesions affecting SCEs,chromosome abberrations and survival of CHO cells arrested by deprivation of arginine

CHO cells undergo proliferative arrest when incubated in medium deficient in the amino acid arginine (ADM). Cells arrested in this way can be released and resume mitotic activity after a brief lag period. The incidence of U.V.-induced sister chromatid exchanges (SCEs) induced in cells arrested in ADM was reduced when the cells were incubated in ADM after irradiation and prior to release. Periods of incubation in ADM of 24 and 48 h prior to release reduced the resulting SCE levels (relative to the SCE levels present in cells irradiated immediately prior to release) by an average of 35 and 45% respectively. A similar time-dependent decrease in the incidence of chromosome aberrations induced in CHO cells arrested in ADM was not observed. Despite the decrease in SCEs over time in ADM, the survival of ADM-arrested cells was not enhanced by a period of incubation in ADM after irradiation of 48 h. These observations are consistent with the hypothesis that the U.V.-induced lesions responsible for the induction of SCE are repaired in time in ADM-arrested CHO cells. Repair of those lesions resulting in chromosome aberrations was not detected in ADM-arrested CHO cells. This absence of repair of certain lesions was apparently reflected in the absence of any enhancement of cell survival.     

Different effects of 1-beta-D-arabinofuranosylcytosine and aphidicolin in S-phase cells--chromosome aberrations, cell-cycle delay and cytotoxicity

Some effects of a 2-h exposure to either aphidicolin (APC) or cytosine arabinoside (ara-C) on S-phase cells of the cell line JU56 have been measured. At a concentration of 1.5 X 10(-5) M of either drug, incorporation of tritiated thymidine into log-phase cultured was reduced by 97-99%. A 2-h exposure to either drug at the same concentration induced chromosome aberrations in cells in S when they subsequently reached mitosis. However, exposure to ara-C induced small numbers of aberrations per damaged cells, and most cells were undamaged. Exposure to APC induced gross chromosomal damage (pulverized chromosomes) in damaged cells. More cells were delayed, and for longer, after exposure to APC than after exposure to ara-C. The results of clonal assays were consistent with the assumption that chromosome aberrations are the proximal cause of reproductive cell death. In the case of ara-C, the results of this and a previous study are consistent with the assumption that cell death and chromosome aberrations are correlated with incorporation of ara-C into DNA in S-phase cells, but that these biological effects manifest themselves only with doses when inhibition of semi-conservative DNA synthesis is greater than 97%.     

Comparison of the levels of chromosome aberration and sister chromatid exchange induced by chemical mutagens in vitro

Dose dependencies of the induction of sister chromatid exchanges (SCEs) and chromosome aberrations were studied under in vivo exposure of mouse bone marrow cells to 5 alkylating agents. The efficacy of the induction of SCEs for all the substances was 20 to 60 times higher than that of the induction of chromosome aberrations. It was demonstrated that SCEs induced by chemical mutagens in vivo and in vitro are more sensitive tests than chromosome aberrations.     

Effects of 5-azacytidine on expression of endogenous retrovirus-related sequences in C3H 10T1/2 cells.

In a previous study (22) we found that transient exposure of C3H 10T1/2 mouse embryo fibroblasts to 5-azacytidine (5-azaC) induced several changes in growth properties. The treated cells showed progressive changes in morphology, saturation density, growth rate, and serum dependence. By passage 5, the cells had acquired the ability to grow in 0.3% agarose, and by passage 30, they had given rise to fully transformed foci that grew in agarose, agar, and liquid suspension. This progression was rapidly accelerated if the cultures derived from 5-azaC-treated cells were exposed for 48 h to the carcinogen benzo[a]pyrene. The present studies demonstrate that both type C and type A, but not type B, retrovirus-related sequences were expressed in the 5-azaC-treated cells. There was negligible expression of these sequences in the control 10T1/2 cells. The level of expression of the related RNAs tended to correlate with loss of anchorage dependence and other markers of an increase in the transformed phenotype. These changes were associated with hypomethylation of the corresponding cellular DNA sequences, as revealed by differential digestion with the restriction enzymes HpaII and MspI. These studies provide evidence that aberrations in DNA methylation and induction of expression of certain endogenous retroviruses may be one of a series of critical events during the course of multistage carcinogenesis, thus enhancing the evolution of malignant tumor cells.     

Cytogenetic effects of malathion insecticide on somatic and germ cells of mice

Male mice dermally exposed to single or multiple treatment (5 days/2 weeks) showed that the ability of malathion to induce chromosome aberrations in somatic (bone marrow) and germ cells (primary spermatocytes) was related to the type of treatment and dose used. Statistically significant increases of chromosome aberrations in bone marrow cells occurred after single treatment (500 and 2000 mg/kg body wt) when chromatid gaps were included and after multiple treatment (250 and 500 mg/kg) when they were excluded. No dose-response relationships were observed for either treatment. In germ cells, malathion induced a significant increase of univalents in both types of treatment but structural chromosome aberrations were induced only by multiple treatment. Malathion induced a significant decrease of the mitotic indices in the bone marrow.     

Structure of the interphase nucleus and formation of chromosome aberrations in Crepis capillaris]

A distribution of chromosome X-rays induced aberrations was studied in 2n and 4n root cells of Crepis capillaris at different stages of the mitotic cycle. Participation of various chromosomes in aberrations was found to be different from theoretically expected one found from the chromosome length. The chromosome A was characterized by excession of intrachromosome aberrations (terminal deletions, isolocuses, duplications and rings). The chromosome D very often took part in the formation of rings. The chromosome C was characterized by an excession of interchromosome aberrations and the lack of intrachromosome ones. The data obtained support the model of interphase nucleus proposed by Wagenaar. The frequency of exchanges of homologues in 2n and 4n cells well correlated with theoretically expected. The inhibition of mitosis increased the exchanges of homologues. In 2n and 4n cells asymmetrical translocations predominated over symmetrical ones.     

Combined mutagenicity of methyl methanesulfonate and ethyl methanesulfonate in Chinese hamster V79 cells.

The combined effects of methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS) on the induction of 6-thioguanine (6TG)-resistant mutants and chromosome aberrations were examined in Chinese hamster V79 cells. Cells were simultaneously treated with EMS at a concentration of D20 and MMS at various concentrations for 3, 6 or 9 h. In other experiments cells were simultaneously treated with MMS at a concentration of D20 and EMS at various concentrations for 3, 6 or 9 h. The mathematical analysis of the combined effects of both chemicals for cell killing (cytotoxicity) and 6TG-resistant mutations indicates that synergistic interactions were observed for both cell killing and mutations induced by MMS and EMS. The frequency of chromosome aberrations induced by simultaneous treatment with MMS at a concentration of D20 and EMS at various concentrations for 3 h was additive. However, the frequency of chromosome aberrations induced by EMS at a concentration of D20 and MMS at various concentrations for 3 h was not significantly different from those induced by MMS alone.     

Identification of a chromosome that controls malignancy in Chinese hamster cells.

A chromosome that controls malignancy in Chinese hamster cells has been identified by analysis of the Giemsa banding pattern of a malignant cell line transformed by simian virus 40 (SV40), non-malignant revertants from this line, segregants from the revertants that were again malignant and a cell line transformed by methylcholanthrene. The malignant cell line transformed by SV40 was near diploid and had gained additional material of chromosome 3. Revertants with a suppression of malignancy and malignant revertants from which they were derived. Malignancy of these cells was associated with the ability to form colonies in agar. Cells of a line transformed by methylcholanthrene were malignant, formed almost no colonies in agar and the only chromosome change from the normal diploid chromosome banding complement was the addition of a long arm of chromosome 3. The results indicate that chromosome 3 carriers gene(s) that control malignancy in Chinese hamster cells in cell lines transformed by a viral or a chemical carcinogen and that malignancy was induced in both cell types by an increase of these genes.     

High evolutionary conservation of the secondary structure and of certain nucleotide sequences of U5 RNA.

The nucleotide sequence of chicken, pheasant, duck and Tetrahymena pyriformis U5 RNAs as well as that of new mammalian variant U5 RNAs was determined and compared to that of rat and HeLa cells U5 RNAs. Primary structure conservation is about 95% between rat and human cells, 82% between mammals and birds and 57% between the Protozoan and mammals. The same model of secondary structure, a free single-stranded region flanked by two hairpins can be constructed from all RNAs and is identical to the model previously proposed for mammalian U5 RNA on an experimental basis (1). Thus, this model is confirmed and is likely to be that of an ancestor U5 RNA. The 3' region of the U5 RNA molecule constitutes domain A, and is common to U1, U2, U4 and U5 RNAs (2). The characteristic nucleotide sequences of domain A are highly conserved throughout the phylogenetic evolution of U5 RNA suggesting that they are important elements in the function of the four small RNAs. Another region of high evolutionary conservation is the top part of the 5' side hairpin whose conserved sequence is specific to U5 RNA. It might participate in the particular function of U5 RNA.     

A 6-Mb contig-based comparative gene and linkage map of the rat schwannoma tumor suppressor region at 10q32.3

Frequent genetic aberrations of malignant schwannomas induced by the alkylating agent N-ethyl-N-nitrosourea in hybrids from inbred BD rat strains include allelic imbalances of the telomeric 20 Mb of chromosome 5 (Dis-2) and of the telomeric 5 Mb of chromosome 10q32 (Dis-1) in 59 and 94% of the tumors, respectively. The Dis-1 minimal loss of heterozygosity consensus region extends from D10Rat4 to the telomere and harbors a putative tumor suppressor gene(s). We constructed a 6-Mb BAC/PAC contig containing more than 70 known genes, 18 mapped microsatellites, and further ESTs/reference RNAs. A continuous block of strongly conserved synteny with mouse chromosome 11E2 and human chromosome 17q25.3 was found. Combining the sequence information from the rat and closely related syntenic regions of different mammalian species produces nearly complete gene maps as a basis for a positional candidate approach and gives insight into mammalian genomic evolution.     

MAPK mediates RAS-induced chromosome instability

The generation of micronuclei is a reflection of DNA damage, defective mitosis, and loss of genetic material. The involvement of the MAPK pathway in mediating v-ras-induced micronuclei in NIH 3T3 cells was examined by inhibiting MAPK activation. Conversely, the MAPK pathway was constitutively activated by infecting cells with a v-mos retrovirus. Micronucleus formation was inhibited by the MAPK kinase inhibitors PD98059 and U0126, but not by wortmannin, an inhibitor of the Ras/phosphatidylinositol 3-kinase pathway. Transduction of cells with v-mos resulted in an increase in micronucleus formation, also consistent with the involvement of the MAPK pathway. Staining with the anti-centromeric CREST antibody revealed that instability induced by constitutive activation of MAPK is due predominantly to aberrant mitotic segregation, since most of the micronuclei were CREST-positive, reflective of lost chromosomes. A significant fraction of the micronuclei were CREST-negative, reflective of lost acentric chromosome fragments. Some of the instability observed was due to mitotic events, consistent with the increased formation of bi-nucleated cells, which result from perturbations of the mitotic spindle and failure to undergo cytokinesis. This chromosome instability, therefore, is a consequence of mitotic aberrations, mediated by the MAPK pathway, including centrosome amplification and formation of mitotic chromosome bridges.     

Morphological transformation and chromosome damage by amsacrine in C3H/10T1/2 clone 8 cells

Morphological transformation, cell survival, chromosomal aberrations and micronuclei were measured in C3H/101/2CL8 cells after 24 h exposure to amsacrine. A weak but dose-related increase in the percentage of dishes containing transformed foci occurred. As previously reported for alkylating agents, this effect was increased by treating 5 days instead of 1 day after plating. There was no evidence for gene mutation at the Na/K ATPase locus, although amsacrine induced micronuclei in a large percentage of cells and chromosomal aberrations, including interchange events and double minute chromosomes, in dividing cells. In would appear that transformation and chromosomal events may be related in amsacrine-treated C3H/10T1/2CL8 cells. The results strongly suggest that amsacrine has carcinogenic potential, possibly related to its chromosome-breaking properties.     

Chromosome aberrations induced by pyrolysates of carbohydrates in Chinese hamster V79 cells

The clastogenic activity of some pyrolysates of carbohydrates was examined in cultured Chinese hamster V79 cells. These pyrolysates include levoglucosan (LG-I), levoglucosenone (LG-II), furfural (FF), 5-(hydroxymethyl)-2-furfural (HMF), glyoxal (GL), methylglyoxal (MGL), 3-deoxy-D-glucosone (DG) and thiazolidine (TZ). LG-I did not induce a significant number of chromosome aberrations at doses up to 8000 micrograms/ml. In contrast, the related compound LG-II induced aberrations and reduced mitosis in a dose-dependent fashion at around 1/2000 of the LG-I doses. Both furan derivatives, FF and HMF, and both glyoxal derivatives, GL and MGL, induced a significant number of chromosome aberrations and a significant lowering of mitotic activity. Among these compounds, FF and MGL showed stronger clastogenic activity than HMF and GL, respectively. DG slightly but positively induced chromosome aberrations. TZ was one of the most potent clastogens among the compounds examined in this study, showing the highest incidence of aberrant cells with many exchanges at doses inducing a significant lowering of mitotic activity. The results of this study indicate the need for a re-evaluation of the thermal decomposition of carbohydrates as a source of genotoxic contaminants.