Periodate-methylamine degradation of ribonucleotides in rat liver extracts in comparison to extracts of cultured cells.

Periodate-methylamine degradation of ribonucleotides, which enables separation of deoxyribonucleotides in cell extracts by high-performance liquid chromatography, was tested on the acid-soluble fraction of L1210 cells, Ehrlich ascites tumor (ELD) cells, liver tissue, and liver cell nuclei. It was shown that dTTP, dCTP, and dATP could be clearly separated in L1210 and ELD extracts. In samples from liver tissue, however, dTTP coeluted with another compound, the dCTP peak often eluted very close to another peak, and only dATP separated quite satisfactorily. Furthermore, extracts from liver cell nuclei, isolated in glycerol, were not directly susceptible to periodate oxidation. This problem can be avoided by use of a different procedure for the isolation of cell nuclei.

Negatively charged RNase-susceptible molecules on the surface of ascites tumor cells.

RNase-susceptible ionogenic groups on the cell surface membranes of two leukemic and two nonleukemic strains of ascites tumor cells were studied by cell electrophoresis, DEAE-Sephadex A-25 column and paper chromatography, and indirect membrane immunofluorescence. RNase treatment of the nonleukemic ascites tumor cells (Ehrlich ascites tumor and Sarcoma 180) produced a significant reduction in their electrophoretic mobilities. When the cells were labeled with [3H]uridine then incubated with RNase, there was a marked increased in the radioactive nucleotides present in the incubation medium as compared to the results of the experiment with RNase-untreated controls. Indirect membrane immunofluorescence studies of nonleukemic ascites tumor cells suggest that the sites that react with anti-RNA antibody are distributed diffusely on their surfaces. RNase treatment of these cells markedly reduced their ability to react with the antibody. It thus appears that RNAs are present on the surface membrane of nonleukemic ascites tumor cells and that RNase digests these RNAs, removing negatively charged nucleotides from their electrophoretic surfaces. This results in a reduction in mobility. In contrast, leukemic ascites cells (L1210 and C1498) incubated with RNase showed no significant change in mobility or in the amount of nucleotides released into the incubation medium. Moreover, no fluorescence was found on the surface of cells examined by indirect membrane immunofluorescence. This suggests that leukemic ascites cells are devoid of RNAs on their surface.

Cytotoxic effects of dexamethasone restricted to noncycling, early G1-phase cells of L1210 leukemia.

The cytostatic and cytolytic effects of dexamethasone were studied as functions of cell cycle position in mouse L1210 leukemia cells. To this end, the cells were separated according to size by sedimentation at unit gravity in a specially designed sedimentation chamber. The fractions were analyzed by radioautography and flow cytophotometry. The size-distributions obtained by 1g sedimentation coincided with cell-cycle age distribution. With increasing fraction number, samples highly enriched in G1, S, and G2/M cells, respectively were obtained: the smallest cells being in early G1 and the largest in mitosis. In the presence of dexamethasone (10(-6)-10(-5) M), growth slowed down after a few cell cycles and the cells accumulated in early G1 phase. Lytic cell kill by continued exposure to the drug was confined to the fractions containing the small, early G1-phase cells. These fractions were also enriched in noncycling cells that were not labeled by prolonged exposure to 3H-thymidine. After removal of dexamethasone, the cells in S and G2/M phase completed cell cycle traverse but were retarded again in the G1 and early S phase of the next division cycle. The data suggest a memory effect for previous drug exposure. It is concluded that the cytostatic and cytolytic effects of dexamethasone are separate, though not unrelated events. Cytolysis is confined to the noncycling cells that in untreated populations can exit from the dividing compartment during a transitional phase of about 60 minutes subsequent to mitotic division. The cytostatic effects potentiate cytolysis by accumulating the cells in the early G1 phase and thus increasing the probability of their transit to the G0 compartment, sensitive for drug-mediated cytolysis.

RNA and DNA content of isolated cell nuclei measured by multiparameter flow cytometry.

Nuclei were isolated from various cell types including Chinese hamster ovary (CHO) and L1210 leukemia cell lines, primary cultures of fibroblasts, nonstimulated and stimulated human lymphocytes and mouse liver cells, by using different isolation techniques. The isolated nuclei were subsequently stained with acridine orange (AO) and their fluorescence was measured by flow cytometry. Various procedures designed to stain DNA versus RNA differentially with AO were tested, and the staining of isolated nuclei was compared with that of whole cells. Control incubations with RNase and DNase were performed to estimate in whole cells and in nuclei the contribution of DNA and RNA to the fluorescence intensity at the respective wavelength bands of maximum emission for DNA (F530) and RNA (F greater than 600). Depending on the cell type, 10-20% of total cell RNase-sensitive F greater than 600 is localized in the nuclei. The RNase-resistant portion of F greater than 600 of isolated nuclei represents the stainability of DNA. Suppression of cell proliferation in subconfluent cultures results in a decrease in both whole cell and in nuclear RNA content. Nonstimulated lymphocyte nuclei have considerably lower RNA content than nuclei from lymphocytes stimulated by pokeweed mitogen. Two subpopulations of nuclei having the same (2C) DNA content but differing in RNA content, are present in mouse liver; the cells entering S phase originate from the high RNA population.

Interstrand DNA crosslinking by 4,5'8-trimethylpsoralen plus monochromatic ultraviolet light. Studies by alkaline elution in mouse L1210 leukemia cells.

DNA crosslinking by 4,5',8-trimethylpsoralen plus monochromatic ultraviolet light of wavelength 365 nm was studied in mouse L1210 leukemia cells. DNA breaks and crosslinking were evaluated by alkaline elution of DNA from poly(vinyl chloride) filters. Trimethylpsoralen plus 365 nm light produced DNA crosslinks but not breaks. The kinetics of crosslinking were linear with respect to concentration and second-order with respect to light exposure time. The latter finding supports the proposed two photon mechanism for the formation of diadducts. In contrast to DNA crosslinking agents such as nitrogen mustard, nitrosoureas and platinums, trimethylpsoralen crosslinks were resistant to proteolytic digestion. Thus, trimethylpsoralen plus 365 nm light produced interstrand crosslinks, as proposed for a bifunctional agent binding to bases on opposite DNA strands.

Histone 2B can be modified by the attachment of ubiquitin.

Histone 2B in mouse and man can be modified by the post-translational addition of ubiquitin. In mouse L1210 cells, both H2B variants are modified, however only to the extent of 1-1.5% compared with about 11% of H2A. Analysis of cyanogen bromide peptides shows that ubiquitin is attached to the C-terminal part of the histone.

Histone 2A, a heteromorphous family of eight protein species.

The histone 2A faily of proteins is shown to consist of eight protein species. In addition to the previously described mammalian 2A variants H2A.1 and H2A.2, we describe two variants which are separable from each other and from variants 1 and 2 on both sodium dodecyl sulfate and acetic acid-urea gels. These two proteins H2A.X and H2A.Z are termed heteromorphous variants to distinguish them from the predominating form and its homeomorphous variants which require nonionic detergents for their resolution. The two heteromorphous variants are present in nucleosomal core particles isolated from mouse L1210 cells. In addition, these variants are found in normal mouse tissues, human HeLa cells, and chicken erythrocytes. On sodium dodecyl sulfate gels, one variant, H2A.X, has an apparent molecular weight approximately 1000 larger than H2A.1 and comprises approximately 11% of the total 2A in mouse L1210 cells. The second variant, H2A.Z, has an apparent molecular weight approximately 600 smaller than H2A.1 and comprises approximately 4% of the total 2A in mouse L1210 cells. The two heteromorphous variants have the same arginine/lysine ratio as H2A.1. In addition, a fraction of each of the four variants (approximately 11% in L1210 cells) is combined with ubiquitin. The molar sum of these eight H2A species approximately equals the number of moles of H4, H2B, or H3 in chromatin.

Series of 4.5S RNAs associated with poly(A)-containing RNAs of rodent cells.

Uninfected mouse kidney cells and mouse leukemia cells L1210 in cluture contained a series of 4.5S RNAs which was structurally identical to the series of 4.5S RNAs associated with genomic RNAs of murine retroviruses and poly(A)-containing RNAs from virus infected cells. Normal rat kidney cells and baby hamster kidney cells in culture also contained a series of 4.5S RNAs. The strucutre of the 4.5S RNAs from mouse, rat and hamster cells were very similar, but not identical. These 4.5S RNAs were not found in cultured cells of other vertebrates, such as human, monkey, cat, mink, rabbit and chicken cells.