Anti-(U1)snRNP autoantibodies inhibit homologous pairing activity of the human recombination complex.

The co-purification of the U1snRNP particle with a high-molecular-weight human homologous pairing activity has been observed consistently. Using human autoimmune sera directed against various snRNPs, it has been found that autoantibody binding to antigenic determinants specifically associated with the U1snRNP particle inhibits the formation of paired DNA molecules by the human homologous pairing activity. Immunoprecipitation of U1snRNP with anti-(U1)RNP autoantibodies significantly reduced the homologous pairing activity in these fractions. NaDodSO4-PAGE analysis of immunoprecipitated samples has revealed their content to be mostly composed of anti-(U1)RNP precipitable material. Taken together, these results suggest that some biochemical reactions in the process of homologous pairing promoted by high-molecular-weight complex are dependent upon U1snRNP components. It is postulated that the U1snRNP may be associated with the recombination complex in human cells.

Heterochromatic proteins specifically enhance nickel-induced 8-oxo-dG formation.

7,8-Dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG) was measured as an indicator of nickel-induced oxidative base damage in the presence of H2O2. Heterochromatic proteins isolated from Chinese hamster liver cells enhanced the formation of 8-oxo-dG induced by NiCl2 and H2O2 in vitro, whereas euchromatic proteins inhibited this reaction. The inhibitory effect of euchromatic proteins on dG oxidation may be due to the oxygen radical scavenging effects of low molecular weight protein-rich fractions. Gel electrophoresis confirmed that histone H1 was present at a higher concentration in heterochromatin than in euchromatin. It is believed that the presence of nickel-protein complexes in cells is crucial for the formation of reactive oxygen species (ROS). We found that Ni2+ binds to histone H1 and core histones as determined by 63Ni autoradiography of proteins on nitrocellulose membranes. In vitro studies showed that commercially purified histone H1, and to a considerably lesser extent core histones, enhanced the NiCl2 and H2O2 catalyzed formation of 8-oxo-dG in a reaction containing free dG base. Since histone H1 is a lysine- and alanine-rich protein, the levels of 8-oxo-dG induced by NiCl2 and H2O2 were studied in the presence of these amino acids and found to be enhanced by them. These results suggest that nickel may specifically produce oxidative DNA damage in heterochromatin because of the nature of its binding to histone H1 and core histones. This selective oxidation of genetically inactive heterochromatin may explain why nickel compounds which generate oxygen radicals and oxidize DNA bases are inactive in most gene mutation assays.

Carcinogenic nickel silences gene expression by chromatin condensation and DNA methylation: a new model for epigenetic carcinogens.

A transgenic gpt+ Chinese hamster cell line (G12) was found to be susceptible to carcinogenic nickel-induced inactivation of gpt expression without mutagenesis or deletion of the transgene. Many nickel-induced 6-thioguanine-resistant variants spontaneously reverted to actively express gpt, as indicated by both reversion assays and direct enzyme measurements. Since reversion was enhanced in many of the nickel-induced variant cell lines following 24-h treatment with the demethylating agent 5-azacytidine, the involvement of DNA methylation in silencing gpt expression was suspected. This was confirmed by demonstrations of increased DNA methylation, as well as by evidence indicating condensed chromatin and heterochromatinization of the gpt integration site in 6-thioguanine-resistant cells. Upon reversion to active gpt expression, DNA methylation and condensation are lost. We propose that DNA condensation and methylation result in heterochromatinization of the gpt sequence with subsequent inheritance of the now silenced gene. This mechanism is supported by direct evidence showing that acute nickel treatment of cultured cells, and of isolated nuclei in vitro, can indeed facilitate gpt sequence-specific chromatin condensation. Epigenetic mechanisms have been implicated in the actions of some nonmutagenic carcinogens, and DNA methylation changes are now known to be important in carcinogenesis. This paper further supports the emerging theory that nickel is a human carcinogen that can alter gene expression by enhanced DNA methylation and compaction, rather than by mutagenic mechanisms.

Chromosomal changes in relation to tumorigenicity of the murine L1210 leukemia line.

A spontaneous loss of tumorigenicity was observed in long-term in vitro culture of the cell line L1210(VAII), which was derived from mouse lymphoblastic leukemia L1210(V) of DBA/2 mice. The loss of tumorigenicity required at least 12 months of in vitro culture and was accompanied by the appearance of a new karyotypically distinct population. The dominant cell variant of the in vitro population that lost tumorigenicity showed duplication of a fragment within the M2 marker chromosome. The loss of tumorigenicity could be interpreted as a dose effect on tumor growth control by gene(s) localized within this marker chromosome.

Banding karyotype analysis of murine leukemia L1210 cell lines with special regard to changes associated with a shift from in vivo to in vitro growth.

An attempt was made to establish permanent in vitro cultures of L1210 leukemia cells under close chromosome control. The investigations involved three independent adaptations. The karyotype was analyzed by the G-banding method. Chromosome analysis of the in vitro cell populations was carried out as soon as they could be analyzed, i.e. immediately after they obtained stable growth in the culture. By comparing the karyotype of the in vivo parental cell line with that of the derivative sublines in vitro, it was found that only some karyotypic variants present in the heterogeneous original population in vivo were able to produce cell lineages. In some cases rapid overgrowth of the cell populations by newly formed karyotypic variants was observed. In all independently obtained sublines changes in the karyotype involved the same chromosomes, thus suggesting that these changes were non-random for the process of cell line establishment in the system investigated. The observed changes in the number of copies of chromosomes 9 and 15 in the cells from the culture seem to indicate that the ratio of the genes localized in these chromosomes may be important for the growth in perpetuation of the cells.

G-banding patterns in mouse lymphoblastic leukemia L1210.

The karyotype of murine lymphoblastic leukemia L1210 was studied with the use of the trypsin-Giemsa technique. The line was hypodiploid with a modal number of 38 chromosomes, including three biarmed chromosomes. All cells examined contained at least one copy of each normal chromosome with the exception of chromosome #1, in which a whole copy along with parts of the second copy was found in the marker chromsome's composition. In 65% of the cells, trisomy of chromosome #15 was observed. Nearly all cells contained only one X-chromosome. The 13 marker chromosomes that frequently occurred are described, and the presumed origin of seven of these chromosomes is described in detail. Comparison of the karyotype of murine L1210 leukemia with the karyotypes of other luekemias of the mouse showed the existence of similar chromosome aberrations. Among these aberrations, the trisomy of chromosome #15 and monosomy of the X-chromosome may be of special importance. The possible role of karyotypic changes in the genesis of the malignant phenotype of the cells is discussed.

Evaluation of measurements of infectious titers of the vaccinia virus in passaged human embryonic fibroblasts.

Measurements of infectivity of the vaccinia virus on 11 cell lines derived from human embryos and passaged several times were analyzed statistically. Various cell lines showed different sensitivity and differentiated response in various passages.