Do individual allocyclic chromosomes in metaphase reflect their interphase domains?

Summary Individual S phase allocyclic chromosomes have been analyzed in Bloom syndrome lymphocytes, in cells with an r(9), and in hypotetraploid Ehrlich mouse ascites cells treated with 1-methyl-2-benzyl hydrazine. On the basis of the following observations, we conclude that such chromosomes more or less reflect their domains in interphase: (1) The S phase allocyclic chromosomes have the same structure as S phase prematurely condensed chromatin (PCC) in fused cells; in other words they form limited areas of chromatin dots; (2) the allocyclic chromosome is the only chromosome in a metaphase plate which synthesizes DNA simultanneously with interphase nuclei; (3) the size of the allocyclic chromosomes is related to the size of the corresponding metaphase chromosome; and (4) the S phase allocyclic chromosomes resemble closely the chromosome domains in interphase made visible with biotinylated human DNA. A variety of evidence shows that most allocyclic chromosomes are simply left behind in their cycle, which presumably is caused by a deletion or inactivation of a hypothetical coiling center situated on each chromosome arm.     

Interstrand crosslink-induced radials form between non-homologous chromosomes, but are absent in sex chromosomes

Fanconi anemia (FA) and cells lacking functional BRCA1 and BRCA2 proteins are hypersensitive to interstrand crosslinking (ICL) agents and show increased numbers of chromosomal breaks and radials. Although radial formation has been used to diagnose FA for more than 30 years, there has been little analysis of these characteristic formations. In this study, radials were analyzed from FA-A and FA-G fibroblasts as well as normal and retrovirally-corrected FA-A fibroblasts treated with high doses of ICLs. Radials were found to only involve non-homologous chromosome interactions and to be distributed nearly randomly along the length of chromosomes. Sites on chromosomes that did show increased frequency of radial involvement did not correlate with known fragile sites or pericentric regions. Hybrid radials were observed between mouse and human chromosomes in human-mouse hybrid cells produced by microcell-mediated chromosome transfer of mouse chromosomes into human FA-A fibroblasts. Both X and Y chromosomes were notably not involved in radials. These observations suggest that ICL repair may involve short stretches of homology, resulting in aberrant radial formation in the absence of FA proteins.     

Chromosomal assignments of the genes for neuroendocrine convertase PC1 (NEC1) to human 5q15-21, neuroendocrine convertase PC2 (NEC2) to human 20p11.1-11.2, and furin (mouse 7[D1-E2] region).

The chromosomal localization of the genes coding for the pro-protein and pro-hormone convertases PC1, PC2, and Furin has been achieved by in situ hybridization. The genes for PC1 and PC2 were located on human chromosomes 5q15-21 and 20p11.1-11.2, respectively. The gene for Furin was assigned to the mouse chromosome 7D1-7E2 region. These data complete the chromosomal localization of these three convertases in both human and mouse. The results confirm the regional correspondence of the human chromosomes 15 and mouse chromosomes 7, as well as between human chromosome 20 and mouse chromosome 2. Furthermore, the identification of the NEC1 locus on human chromosome 5 and mouse chromosome 13 suggests a conservation of synthenic regions between these regions of the human and mouse genomes.     

contribution to the study of nuclear total proteins and DNA during the mitotic cycle in fibroblasts cultivated in vitro and in Ehrlich ascites cells

Summary The dry weight and total protein content of nuclei has been measured by interferometry in living or fixed cells cultivated in vitro (freshly prepared chick, mouse or rat embryo fibroblasts) and in fixed Ehrlich ascites tumor cells of the mouse growing in vivo. The DNA content was estimated by cytophotometry after Feulgen reaction in the same nuclei. The dry weight of nucleoli in fibroblasts and the dry weight and DNA content of chromosomes in dividing fibroblasts and Ehrlich tumor cells have also been measured.During the interphase in fibroblasts, the dry weight of the living nucleus and the nuclear total protein content as measured in fixed cells doubles during the preparation for mitosis, as the DNA content does. In chick and mammal fibroblasts and within the limits of accuracy of our measurements, the synthesis curves for nuclear proteins and DNA do not seem to be necessarily identical.In our fibroblasts, the nucleolar total dry weight per nucleus doubles during the interphase (nucleolar preparation for mitosis); it increases in proportion to the nuclear total protein content, even in polyploid nuclei.During the mitosis, the chromosomes contain all the DNA of the nucleus but some nuclear proteins (non chromosomal proteins) seem to move into the cytoplasm during the mitosis and return into the nucleus at the post-telophase.According to our observations, Ehrlich ascites mouse tumor cells are near-tetraploid as far as the number of chromosomes, nuclear total protein content and DNA content are concerned. During the preparation for mitosis, these amounts double but no necessary close time relation seems to link these premitotic syntheses. Prom this point of view, our results show no clear-cut differences between these tumor cells and the fibroblasts. Except the polyploidy, the behaviour of nuclear proteins and DNA during mitosis in the tumor cells is the same as that observed in our fibroblasts.The effects of various antimitotic agents on rat fibroblasts cultivated in vitro have also been studied with our cytochemical methods. Our measurements of nuclear protein, DNA and nucleolar material content have been made in cells in which mitosis was prevented by alkylating agents, beryllium sulphate, RNase or neutral DNase. The effects of colchicine on these cellular parameters have also been studied.     

Electrophoretic mobility of mouse cells and homologous isolated nuclei

The electrophoretic mobilities of Ehrlich ascites, sarcoma 37 ascites, mouse liver cells and their isolated nuclei were measured under similar environmental conditions. No differences in mobility were detected between cells and homologous nuclei from the same cell population and it was concluded that their surface charge densities were probably the same. The effect of neuraminidase on Ehrlich ascites and liver cells and nuclei was also determined; neuraminidase reduced the mobility of Ehrlich ascites cell nuclei as well as cells. The reduction in mobility of cells and nuclei prepared by a sucrose method was the same; however, the reduction in mobility of citric acid prepared nuclei was less than that of citric acid treated cells. The reduction in mobility of both liver cells and nuclei was small or insignificant. It is suggested that although cells and nuclei have similar electrophoretic mobilities, possibly different groups contribute to their surface charge.     

Flow cytometry isolation and improved visualization of sorted mouse chromosomes. Purification of chromosomes X and ISO-1 from cell lines with Robertsonian translocations

While analysis and sorting of human chromosomes by flow cytometry has been widely used, isolation of a pure mouse chromosome remains very difficult, since most murine chromosomes are quite similar in size. To overcome this problem, we have analysed mouse cell lines having either Robertsonian translocations or isochromosomes. The resulting metacentric chromosomes are very different in size and in morphology from normal mouse acrocentric chromosomes. These characteristics have been analysed by computer-monitored flow cytometry, facilitated by improvements in the chromosome extraction procedure. Signals characteristic of the iso-lq chromosome in cell line PCC4 azaR1, and of the normal X chromosome in the mouse strain 22CD have thus been obtained. These chromosomes have been sorted and can be easily recognized by fluorescence microscopy when collected onto serum-albumin-coated microscope slides. The technical modifications made, coupled with the existence of a great diversity of metacentric chromosomes resulting from Robertsonian translocations, should allow the purification of a number of different mouse chromosomes.     

Uridine kinase: altered subunit size or enzyme expression as a function of cell type, growth stimulation, or mutagenesis

Using antibody prepared against pure uridine kinase from Ehrlich ascites cells, we have measured the expression of enzyme protein by the Western blot technique. Variations were observed in the Mr of the enzyme subunit for uridine kinase from different species: 32,000 (mouse Ehrlich ascites cells), 30,000 (normal human lymphocytes), 28,000 (mouse tissues), 27,500 (rat tissues). For different normal tissues from the same species, there was no significant variation in the subunit size. Transformed human and mouse cell lines, selected for a deficiency of uridine kinase activity in the presence of inhibitors activated by this enzyme, expressed two cross-reacting proteins, one with a normal (30,000) and one with a smaller (21,000) subunit molecular weight than was found in the parental cell line (human lymphoma), or only a smaller protein of Mr 25,000 (mouse lymphoma). Our results show that selection protocols using metabolite inhibitors do not always repress the expression of the enzyme but instead may lead to selection of those cells that have a mutation in the uridine kinase gene, resulting in the expression of an inactive enzyme. The expression of uridine kinase protein changes when cells are stimulated to divide. For both mouse fibroblasts and human lymphocytes, expression of uridine kinase protein as well as activity clearly increased after cells were stimulated to grow. In fibroblasts, increases are seen by 3 hr after stimulation, and plateau after 9 hr at a sevenfold increase. In lymphocytes, no change is seen until 12 hr after stimulation, and a plateau is not reached until 72 hr, with a total increase of approximately 50-fold. There has been considerable interest in the possibility of uridine kinase isozymes. Except for cells that have been mutagenized, the present results show that, as judged by subunit molecular weight, there appears to be only one enzyme form in normal and neoplastic cells or in cells in which uridine kinase activity is induced.     

Telomere loss in somatic cells of Drosophila causes cell cycle arrest and apoptosis

Checkpoint mechanisms that respond to DNA damage in the mitotic cell cycle are necessary to maintain the fidelity of chromosome transmission. These mechanisms must be able to distinguish the normal telomeres of linear chromosomes from double-strand break damage. However, on several occasions, Drosophila chromosomes that lack their normal telomeric DNA have been recovered, raising the issue of whether Drosophila is able to distinguish telomeric termini from nontelomeric breaks. We used site-specific recombination on a dispensable chromosome to induce the formation of a dicentric chromosome and an acentric, telomere-bearing, chromosome fragment in somatic cells of Drosophila melanogaster. The acentric fragment is lost when cells divide and the dicentric breaks, transmitting a chromosome that has lost a telomere to each daughter cell. In the eye imaginal disc, cells with a newly broken chromosome initially experience mitotic arrest and then undergo apoptosis when cells are induced to divide as the eye differentiates. Therefore, Drosophila cells can detect and respond to a single broken chromosome. It follows that transmissible chromosomes lacking normal telomeric DNA nonetheless must possess functional telomeres. We conclude that Drosophila telomeres can be established and maintained by a mechanism that does not rely on the terminal DNA sequence.     

Chromosomal alterations in cell lines derived from mouse rhabdomyosarcomas induced by crystalline nickel sulfide

Summary Prior studies have shown a preferential decondensation (or fragmentation) of the heterochromatic long arm of the X chromosome of Chinese hamster ovary cells when treated with carcinogenic crystalline NiS particles (crNiS). In this report, we show that the heterochromatic regions of mouse chromosomes are also more frequently involved in aberrations than euchromatic regions, although the heterochromatin in mouse cells is restricted to centromeric regions. We also present the karyotypic analyses of four cell lines derived from tumors induced by leg muscle injections of crystalline nickel sulfide which have been analyzed to determine whether heterochromatic chromosomal regions are preferentially altered in the transformed genotypes. Common to all cell lines was the presence of minichromosomes, which are acrocentric chromosomes smaller than chromosome 19, normally the smallest chromosome of the mouse karyotype. The minichromosomes were present in a majority of cells of each line although the morphology of this extra chromosome varied significantly among the cell lines. C-banding revealed the presence of centromeric DNA and thus these minichromosomes may be the result of chromosome breaks at or near the centromere. In three of the four lines a marker chromosome could be identified as a rearrangement between two chromosomes. In the fourth cell line a rearranged chromosome was present in only 15% of the cells and was not studied in detail. One of the three major marker chromosomes resulted from a centromeric fusion of chromosome 4 while another appeared to be an interchange involving the centromere of chromosome 2 and possibly the telomeric region of chromosome 17. The third marker chromosome involves a rearrangement between chromosome 4 near the telomeric region and what appears to be the centromeric region of chromosome 19. Thus, in these three major marker chromosomes centromeric heterochromatic DNA is clearly implicated in two of the rearrangements and less clearly in the third. The involvement of centromeric DNA in the formation of even two of four markers is consistent with the previously observed preference in the site of action of crNiS for heterochromatic DNA during the early stages of carcinogenesis.     

The role of extracellular calcium ions in HVJ (Sendai virus)-induced cell fusion

The biochemical and biophysical roles of extracellular calcium ions in HVJ (Sendai virus)-induced cell fusion were studied. (1) Various kinds of cell, such as Ehrlich ascites tumor cells, mouse melanoma cells (B16-CW1 cells) and human epidermoid carcinoma cells (KB cells), could fuse in Ca2+-free medium containing a cheletor, glycoletherdiaminetetraacetic acid, in the same way as in Ca2+-containing medium. (2) The ATP content in Ehrlich ascites tumor cells decreased rapidly when the cells were treated with the virus in Ca2+-free medium but not in Ca2+-containing medium. (3) Intracellular adenine nucleotides leaked out into the reaction medium when the cells were treated with the virus in Ca2+-free medium but not in Ca2+-containing medium. (4) On addition of the virus, O2 consumption of Ehrlich ascites tumor cells decreased in Ca2+-free medium, but not in Ca2+-containing medium. (5) HVJ (Sendai virus) did not affect production of lactate by Ehrlich ascites tumor cells in both Ca2+-free medium and Ca2+-containing medium. These observations suggest that the role of extracellular Ca2+ in virus-induced cell fusion is to maintain the ATP and other intracellular metabolite contents at normal levels instead of triggering the fusion reaction itself.     

Analysis of radiation injuries of DNA from liver and transplanted tumors in vitro

A comparative study of some radiation effects on DNAs from mouse liver, Ehrlich ascites tumor and hepatoma 22A was made after X-irradiation of the 0.02% aerated solutions in 0.1 M NaCl. The electrophoretic and spectrophotometric methods were used to measure the radiation-chemical yields of single- and double-strand breaks, DNA secondary structure defects, a malonaldehyde analogue and pyrimidine hydroperoxides. DNA from tumors was found to be more affected by radiation than DNA from normal liver: this may be due to a higher degree of base damage (the yield of hydroperoxides) and to transformation of original secondary structure defects into single-strand breaks at low irradiation doses.     

The inhibitory effect of l-cysteine and its derivatives on glycolysis in Ehrlich ascites tumour cells

(1) l-Cysteine inhibits aerobic glycolysis and restores the Pasteur effect in Ehrlich ascites tumour cells or in their supernatants, while d-cysteine has no effect on this process. (2) Other compounds which have configuration l at the α-carbon and a thiol group in the β-position (penicillamine) or restore them in vivo (3-mercaptopyruvate, cystine or l-serine together with l-homocysteine) also show inhibitory properties. (3) dl-Homocysteine with a free thiol group in the γ-position, reduced glutathione, methionine and products of cysteine oxidation (cysteic acid, taurine) do not inhibit tumour aerobic glycolysis. (4) Glycolysis of normal tissue supernatants (mouse liver and muscle) is not sensitive to the inhibitory effect of cysteine. (5) Metabolic studies showing a cysteine-induced decrease in ATP content, coupled with cross-over of the pyruvate and 2-phosphoenolpyruvate concentrations in Ehrlich ascites tumour cells, indicate that tumour pyruvate kinase is an enzyme sensitive to cysteine inhibition. (6) Enzymatic studies carried out both after preincubation of Ehrlich ascites tumour cells with cysteine or during direct action of this substance on tumour and normal tissue supernatants indicate the presence of a cysteine-sensitive isoenzyme besides the normal cysteine-insensitive pyruvate kinase in tumour material.     

Heterogeneity of alkaline ribonuclease in the mouse and Ehrlich ascites cells.

The specific activity of alkaline RNase II was l00 to 1800 times higher in mouse pancreas than in mouse liver, serum, ascites fluid, and Ehrlich ascites cell grown intraperitoneally. Ehrlich ascites cells grown in cell culture medium had a much lower alkaline RNase II activity than cells grown intraperitoneally. Chromatography on CM-52 cellulose of acid- and heat-treated preparations showned a considerable heterogeneity of the mouse enzymes. Depending on the source of the extract, two to six forms fo alkaline RNase were eluted. Pancreatic extract contained two RNase forms. These also seemed to be present as minor components in preparations from other sources except Ehrlich ascites cells grown in vitro. Ehrlich ascites cells grown in vivo contained forms of the RNase which were not present in other extracts. Possible reasons for this heterogeneity were investigated. In addition to their stability to acid and heat the different RNase forms were similar in that they were much more active at alkaline pH than at acidic pH, they did not require divalent metal ions for activity, and they degraded RNA 'endonucleolytically.' Also, native DNA, denatured DNA, and poly A were poor substrates compared with RNA. Some differences seemed to exist, however, with respect to their abilities to degrade poly U and poly C and their sensitivities to the endogenous RNase inhibitor.     

用~（31）P磁共振谱对艾氏腹水癌和肉瘤S－180细胞代谢的研究

艾氏腹水癌细胞和肉瘤Ｓ－１８０细胞是抗肿瘤药物筛选常用细胞株．实验采用取自小鼠腹腔的第７～８天的艾氏腹水癌和Ｓ－１８０细胞,用３１Ｐ磁共振谱测得了细胞内小分子含磷代谢成分;计算了细胞内ｐＨ值;还用３１Ｐ谱探讨了作用机制不同的三种抗代谢物：碘乙酸、２,４－二硝基苯酚及棉酚对艾氏腹水癌细胞代谢的影响     

The phosphorylation of high mobility group proteins 14 and 17 and their distribution in chromatin

The phosphorylation of the high mobility group (HMG) proteins has been investigated in mouse Ehrlich ascites, L1210 and P388 leukemia cells, human colon carcinoma cells (HT-29), and Chinese hamster ovary cells. HMG 14 and 17, but not HMB 1 and 2, were phosphorylated in the nuclei of all cell lines with a serine being the site of modification for both proteins in Ehrlich ascites cells. Phosphorylation of HMG 14 and 17 was greatly reduced in cultured cells at plateau phase in comparison to log phase cells, suggesting that modification of HMG 14 and 17 is growth-associated. However, phosphorylation was not linked to DNA synthesis, since incorporation of 32P did not vary through G1 and S phase in synchronized Chinese hamster ovary cells. Treatment of HT-29 or Ehrlich ascites cells with sodium butyrate reduced HMG phosphorylation by 30 and 70%, respectively. The distribution of the phosphorylated HMG proteins in chromatin was examined using micrococcal nuclease and DNase I. 32P-HMG 14 and 17 were preferentially associated with micrococcal nuclease-sensitive regions as demonstrated by the release of a substantial fraction of the phosphorylated forms of these proteins under conditions which solubilized less than 3% of the DNA. Short digestions with DNase I did not show a marked release of 32P-HMG 14 or 17.     

Immunospecificity of non-histone chromosomal proteins in 89Sr-induced osteogenic sarcoma (mouse).

Antibodies against non-histone chromosomal proteins for 89Sr-induced osteogenic sarcoma (mouse) were prepared by immunization of rabbits. The immunoreactivity of this antigen was then compared with those of non-histone chromosomal proteins from Ehrlich ascites tumor, normal mouse liver, and calf thymus by the method of quantitative microcomplement fixation. The non-histone chromosomal proteins of 98Sr-induced osteogenic sarcoma, fractionated by hydroxylapatite chromatography, exhibited significant affinity for the antibodies. Similar proteins from Ehrlich ascites tumor, normal mouse liver, or calf thymus were virtually inactive, indicating the tissue-specificity of 89Sr-induced osteogenic sarcoma proteins.     

Elevated polyamine levels in erythrocytes of SLC:ICR mice inoculated with ehrlich ascites carcinoma cells

Ehrlich ascites carcinoma cells (4×10⁵ cells/mouse) were inoculated intraperitoneally in 7-week-old SLC:ICR mice, and polyamine levels in peripheral erythrocytes and in ascites cells were determined periodically. Polyamine levels in peripheral erythrocytes increased linearly until 10 days after cell inoculation, while ascites cells showed exponential growth.The effect of carbazilquinone on cellular growth and polyamine levels in erythrocytes was also studied. When 1 or 2mg/kg of carbazilquinone was injected intraperitoneally on day 4 or on day 7, cellular growth was suppressed and the survival time of the mice was lengthened. The polyamine levels in erythrocytes were also markedly decreased 3 days after the carbazilquinone injection.These results suggest that the polyamine levels in peripheral erythrocytes are closely related to the cellular growth of Ehrlich ascites carcinoma cells.     

Inhibiting effect of vitamins C and B12 on the mitotic activity of ascites tumors.

The mitotic activity of the transplantable mouse tumors, Sarcoma 37, Krebs-2, and Ehrlich carcinomas, in the ascites form, were inhibited after treatment with a mixture of vitamins C and B12 with no apparent toxic side effects. These vitamins when administered alone, at the same dosage, did not seem to have any apparent effect on mitosis or the morphology of the cells studied. Microscopic examination of the stained ascites fluid taken from the mice treated with the vitamin mixture showed few tumor cells, and these in various stages of disintegration. Also, an increase in lymphocytes, monocytes and neutrophils were noticed; however, later in the experiment, no tumor cells could be found and monocytes and macrophages were abundant.     

Effect of electrostatic fields on the chromosomes of Ehrlich ascites tumor cells exposed in vivo

An effect of electrostatic fields on the chromosomes of Ehrlich ascites tumor cells exposed in vivo has been demonstrated. Cells exposed to horizontal electrostatic fields for two weeks had almost a threefold increase in the percentage of abnormal chromosomes when compared to control cells or cells exposed to vertical electrostatic fields for the same period. Extended exposure times of 4--15 weeks resulted in the disappearance of the aberrant chromosomes. It is suggested that the effected cells were incapable of cellular replication resulting eventually in their disappearance via cell death.