The R-banding pattern of the chinese hamster Don cell line

Chinese hamster cells (Don line) were treated in vivo with 5-BrdU and 332580Hoechst fluorochrome for obtaining the partial inhibition of condensation that causes the R-banding pattern. Untreated chromosomes were stained by a standard G-banding method. Statistical measurements show significant differences in the band numbers between the two treatments. The Don cell line in the authors' laboratory presents some karyotypical differences from Don cell lines studied by other authors.     

Sensitivity of two Chinese hamster cell lines to SCE induction by a variety of chemical mutagens

SCE induction in Chinese hamster Don (lung) cells was compared with that in CHO (ovary) cells exposed under identical conditions to 14 known mutagens. Test protocols used for comparison were selected following a study of Don and CHO cell responses to aflatoxin B1 and benzo[a]pyrene. In the absence of added metabolizing enzymes 9-aminoacridine, 4-nitroquinoline 1-oxide, N-methyl-N-nitrosourea, dimethylcarbamoyl chloride, beta-propiolactone, daunomycin, aflatoxin B1 and 2-aminoanthracene were directly active in both cell lines; every substance positive in CHO cells was also positive in Don cells. However, the latter detected cyclophosphamide, hydrazine sulphate, benz[c]acridine, 3-methylcholanthrene and benzo[a]pyrene without addition of S9. CHO cells did not respond equivalently to these mutagens, either in the presence or absence of S9. Other differences between the cell lines depended on chemical exposure time, S9 pre-incubation or co-incubation conditions. For example, the ability of CHO cells to detect SCEs due to 2-aminoanthracene was acutely dependent on exposure time. In addition, Don cells exhibited lower background SCE values which were less variable than those of CHO cells under the same culture conditions. Although incapable of detecting 4-dimethylaminoazobenzene (butter yellow) and not as sensitive to cyclophosphamide as certain cell lines of liver origin, the pseudodiploid Don cell line possesses other desirable characteristics required for in vitro SCE assays, particularly with regard to intrinsic metabolic activation of polycyclic aromatic hydrocarbons and related substances.     

Evolution of digestibility by Hind III: an analysis by light and electron microscopy

Digestion of Chinese hamster metaphase chromosomes from the Don cell line by Hind III restriction endonuclease followed by Giemsa staining were analysed by light and electron microscopy. The evolution of digestibility was studied and four digestion stages were characterized by different levels of chromosome structure. Three different condensation stages were established according to morphological criteria of length, width and separation among chromatids. It was observed that there are statistically significant differences in the digestion progress at the three condensation stages previously defined.     

Evolution of digestibility by Hind III: an analysis by light and electron microscopy

Digestion of Chinese hamster metaphase chromosomes from the Don cell line by Hind III restriction endonuclease followed by Giemsa staining were analysed by light and electron microscopy. The evolution of digestibility was studied and four digestion stages were characterized by different levels of chromosome structure. Three different condensation stages were established according to morphological criteria of length, width and separation among chromatids. It was observed that there are statistically significant differences in the digestion progress at the three condensation stages previously defined.     

The chinese hamster Don cell line differs from normal diploidy by one chromosome band

G-banded and C-banded prometaphase and metaphase karyotypes of the Chinese hamster Don cell line from three different laboratories were compared. All the cell lines were pseudodiploid. Each consisted of two distinct cell populations designated S1 and S2. The chromosome complements of each of these populations differed from the diploid by only one additional chromosome band on a subterminal 1p in S1 and on a median 1q in S2. The sites of these extra bands were neither constricted nor heterochromatic. All other different karyotypes, including those from seven distinct subpopulations, had basic patterns of chromosome complements that were best represented by S1 or S2, respectively the major or minor cell type of the cell line. Moreover, nearly 80% of the metaphases analyzed had a modal chromosome number of 22, and about 60% had the same chromosome composition of a cell type or subtype. Our data also suggest that Don cells were probably pseudodiploid by 1964 or before.     

Concanavalin A induces endoreduplication in cultured mammalian cells.

Concanavalin A (Con A) induced endoreduplication in an established cell line, Don, of the Chinese hamster. The inducibility of Con A was inhibited by α-methyl-D-mannoside. When a secondary culture of kidney cells (CHK), which showed the contact-inhibition of growth, was used, there was an increase in spontaneous endoreduplication. CHK cells or some of them were more sensitive to Con A than Don cells, in which few spontaneous endoreduplications were observed. Mitotic shake-off after Con A treatment led to the higher ratio of endoreduplicated cells to normal mitoses, suggesting that endoreduplicating cells do not “round-up” and probably do not condense chromosomes through the cell cycle until M is reached.     

Modulation between aerobic and anaerobic metabolism in the mutant cell line CdtR-Q

It has recently been shown that the cell line Don Q obtained by mutagenesis of wild type Chinese hamster lung fibroblasts (Don wt), presents a point mutation in the gene coding for UDP-glucose pyrophosphorylase. The persistent low level of UDP-glucose makes Don Q clone resistant to Clostridium difficile toxin B. Starting from the observation that Don Q cells exhibit many large hydrophobic cytoplasmic inclusions, that we have found to be made of neutral lipids, the aerobic metabolism of the two cell lines has been examined. The specific activity of cytochrome oxidase in Don Q cells is more than 5 times lower than that found in Don wt. Also, the activity of Complexes II + III, expressed by the activity of succinate-cytochrome c oxido-reductase, has been found to be lower in Don Q compared to wt cells. On the other hand, NADH-cytochrome c oxido-reductase activity, insensitive to rotenone, is more than doubled in Don Q. In these cells the activity of lactate dehydrogenase is very high, being able to oxidise more than 3,000 nmoles of NADH/min/mg of protein. The results obtained indicate that Don Q cells, in addition to a decreased ability to synthesise UDP-glucose, have an impairment in the respiratory chain. Such an impairment could be correlated to the increased capacity to generate a higher amount of reducing equivalents through the glycolytic activity, which can then be utilised for the synthesis of fatty acids stored in lipid droplets.     

The isolation and characterization of asparagine-requiring mutants of Chinese hamster cells

Nine asparagine-requiring mutants were isolated in culture from the Don line of Chinese hamster cells. Investigation of the asparagine requirements of the mutants, the effect of asparagine deprivation on macromolecular synthesis, and the rates of reversion to asparagine independence indicated that there were differences between the mutant clones. Biochemical analysis revealed that the defect in the mutants was due to a deficiency of the enzyme asparagine synthetase, and that the enzyme activity in the mutants and Asn⁺ revertants obtained from them was not influenced by the concentration of asparagine in the growth medium. Complementation analysis by Sendai virusmediated cell fusion indicated that the lesion behaved as a recessive trait, and was probably located in the same gene in all the mutant clones.     

Telomere erosion-induced mitotic catastrophe in continuously grown chinese hamster don cells

We have previously reported that telomere erosion is the earliest chromatin modification in cells entering the apoptotic pathway. The purpose of this investigation was to determine whether loss of telomeric DNA was involved in inducing mitotic catastrophe and death in Chinese hamster Don cells. Don, a male Chinese hamster-derived cell line which requires daily subculturing to remain diploid, was grown without subculturing for 1-4 days at 37 degrees C and analyzed cytologically. Our results indicated that (1) the frequency of metaphase chromosomes with structural anomalies was significantly higher in 3-day continuously grown cells than in 1-day control cells (8.2% vs 5.7%; P < 0.01), (2) the mitotic index was considerably lower in 3-day continuously grown cells (0.13%) than in control cells (3.64%), (3) cells grown for 3 days continuously showed a higher incidence (7.6%) of endoreduplicated metaphase chromosomes than did control cells (4.9%), (4) 4-day continuously grown Don cells showed significantly smaller amounts of telomeric DNA in interphase nuclei than did control cells, and (5) apoptotic cells were more frequent in 4-day cell cultures (40.6%) than in control cells (4.3%). These results support our earlier observations and contribute additional support for our hypothesis that telomere reduction is the cause of mitotic catastrophe and that cell death in continuously grown Don cells occurs because of the loss of telomeric DNA.     

Clastogenicity of low pH to various cultured mammalian cells.

It has been reported that low pH itself can be clastogenic to Chinese hamster ovary cells or mouse lymphoma L5178Y cells. On the other hand, there was no indication that low pH is clastogenic to rat or human lymphocytes. Therefore, in order to evaluate the generality of clastogenicity of low pH conditions, chromosomal aberration tests were carried out on Chinese hamster cell line cells (CHO-K1, CHL, Don and V79 379A) and human cells (HeLa and peripheral lymphocytes used as whole-blood cultures). The cytotoxicity of low pH to each cell line was also evaluated by counting surviving cells. The treatment medium used was Eagle's MEM containing 15 mM MES or Bis-Tris as an organic buffer to maintain the acidity of the medium for the 6-h or 24-h treatment period, and pH adjustment was done with NaOH or HCl. Chromosomal aberrations were induced at pH 6.5 or below in CHO or CHL cells, and the maximum frequency was 24.7% at pH 6.0 or 34% at pH 6.3, respectively. About 5-10% of Don or HeLa cells had aberrations over the range of pH 6.6-6.0 or pH 6.6-6.3, respectively. In V79 379A cells or human lymphocytes, however, aberrant cells amounted to about 8% at near pH 6.0, where cell survival was low (less than 20%). About 90% of aberrations induced in each cell line examined were chromatid-type gaps and breaks. When CHO or CHL cells were treated with acidic medium for 6 h plus 18 h recovery in fresh medium, about 20% of cells had aberrations including chromatid exchanges at pH 5.5 or pH 5.7, respectively. These results indicate that clastogenicity of low pH is a general finding, although the extent of it varies with cell type, and that the clastogenicity is associated with varying extents of cytotoxicity. The mechanisms of clastogenesis at low pH are not known, but might involve inhibition of DNA or protein synthesis or DNA-repair enzymes.     

In vitro stimulation of rat liver cells by homologous partial hepatectomy serum

Summary A low passage rat liver cell line demonstrated in vitro growth stimulation when cultured in the presence of serum of homologous, partially hepatectomized rats. After 4-day incubation a 3.25-fold increase in the cell population was observed in cultures supplemented with posthepatectomy serum at a dilution of 1∶10. No response was observed with sham-operated animal serum. Continous cultures of Chang human liver and Don hamster lung cells were not responsive to the posthepatectomy serum. The limitations of tetraphenylboron as a dispersing agent for primary rat liver cells are discussed. Supported by Grant 67-7 from the Illinois Division of the American Cancer Society.     

Coordination of Cytokinesis and Cell Separation by Endosomal Targeting of a Cdc42-specific Guanine Nucleotide Exchange Factor in Ustilago maydis

The small GTPase Cdc42 is a key regulator of cell polarity and cytoskeletal organization in most eukaryotic cells. In Ustilago maydis, Cdc42 and the guanine nucleotide exchange factor (GEF) Don1 regulate cytokinesis and cell separation. Don1 belongs to the FGD1 family of Cdc42-specific GEFs that are characterized by a C-terminal lipid-binding FYVE domain. Although the FGD1/frabin family of Rho-GEFs is evolutionary conserved from fungi to mammals the role of the FYVE domain for its biological function is unknown. Here, we show that the FYVE domain is specific for phosphatidylinositol-3-phosphate (PtdIns(3)P) and targets Don1 to endosomal vesicles. During cytokinesis asymmetric accumulation of Don1-containing vesicles occurs at the site of septation. We could show that FYVE-dependent localization is critical for the function of Don1 at normal expression levels but can be compensated for by overexpression of Don1 lacking a functional FYVE domain. Our results demonstrate that endosomal compartmentalization of a Cdc42-specific exchange factor is involved in the coordination of cytokinesis and cell separation.     

Cytological analysis of hybrids among triticales and trigopiros

WE STUDIED THREE DIFFERENT TRICEPIROS: (Don Santiago x Don Noé), (Cumé x Horovitz) and (Cumé x Don Noé). The tricepiro (Don Santiago x Don Noé) was obtained by crossing the triticale Don Santiago INTA (AABBRR, 2n = 6x = 42) with the trigopiro Don Noé INTA (AABBDDJJ, 2n = 8x = 56). The number of chromosomes for the F(1) was 2n = 49, the most frequent meiotic configuration being 14 bivalents and 21 univalents. The univalents were situated in the periphery of the equatorial plane, whereas the bivalents were located in the central zone. The chromatids in some of the univalents split when bivalents underwent reductional division in anaphase I. There were few laggard chromosomes or chromatids at this phase. The number of chromosomes (2n = 48-58) was high and variable, and the number of bivalents per cell (18-23) also high in F (3) individuals. In all F (8) tricepiros (Don Santiago x Don Noé), F (12) tricepiros (Cumé x Horovitz) and F (12) tricepiros (Cumé x Don Noé), the number of chromosomes (2n = 42) was the same, these retaining the rye genome, as demonstrated by GISH and FISH. These new synthesized allopolyploids constitute interesting models for investigating the evolutionary changes responsible for diploidization, and the chromosomal and genomic re-ordering that cannot be revealed in natural allopolyploids.     

Cell cycle variation associated with feeder effects in cultures of Chinese hamster fibroblasts

Sub-confluent monolayer cultures of an established line of Chinese hamster fibroblast (Don) are shown to exhibit a density-dependent stimulation of growth. Evidence is presented that both long and short range ‘feeder effects’ are involved. Using the technique of autoradiography, cell cycle parameters have been studied in sub-confluent cultures seeded at different densities to identify the source of this density-dependent variation in growth rate. The durations of S phase, G2, and mitosis are constant as indicated by “percentage labelled mitoses” curves. A simple procedure has been developed for measurement of the fraction of a cell population in the G1 state, and this fraction is shown to be inversely related to the density of the culture. It is concluded that regulation of cell growth associated with feeder effects in cultured Don cells occurs within the G1 state. The data obtained from “percentage labelled mitoses” curves are shown to be highly consistent with the predictions of the Transition Probability model for cell cycle regulation.     

Effect of precursor feeding on alkaloid accumulation by a tryptophan decarboxylase over-expressing transgenic cell line T22 of Catharanthus roseus

To obtain more insight into the regulation of terpenoid indole alkaloid (TIA) biosynthesis in Catharanthus roseus (L.) G. Don cell cultures and particularly to identify possible rate limiting steps, a transgenic cell line over-expressing tryptophan decarboxylase (Tdc), and thus having a high level of tryptamine, was fed with various amounts of precursors (tryptophan, tryptamine, loganin and secologanin) in different time schedules and analyzed for TIA production. When these precursors were added to this culture it was found that the optimal time for supplying the precursors was at inoculation of the cells into the production medium. Alkaloid accumulation by line T22 was enhanced by addition of loganin or secologanin; however, the secologanin feeding was less effective. Tryptamine or tryptophan alone had no effect on TIA accumulation. The over-expression of Tdc causes this cell line to produce quite large quantities of alkaloids after feeding loganin or secologanin. However, in combination with tryptophan or tryptamine, feeding of these precursors resulted in an even further increase of alkaloid accumulation and under optimal conditions line T22 accumulated around 1200 micromol l(-1) of TIAs whereas the control cultures accumulated less than 10 micromol l(-1) TIAs.     

营养和环境因子对长春花激素自养型细胞生长和阿玛碱生成的影响

在摇瓶中用液体培养基培养长春花（Catharanthusroseus（L．）G．Don）激素自养型细胞系C20hi,比较了不同初始糖浓度、接种量、初始pH值、光照时间、光质和摇床转速对该培养细胞生长、阿玛碱积累和释放的影响。结果表明,此细胞系具有较强的环境耐受性;一定范围内初始糖浓度增加,有利于细胞生长和生物碱生成;最适的接种量是60gFW／L;一定范围内改变培养基pH值,对生物碱生成没有显著影响;照光后生物碱生成量下降,红光较蓝光更有利于生物碱生成;最适的摇床转速是120r／min。     

Dual function of the germinal centre kinase Don3 during mitosis and cytokinesis in Ustilago maydis

Septum formation is a crucial step of cytokinesis in fungi. In the basidiomycete Ustilago maydis, the germinal centre kinase Don3 triggers initiation of a secondary septum necessary for cell separation after cytokinesis. Here we show that oligomerization of Don3 via a putative coiled-coil domain is critical for secondary septum formation. Within the Don3 sequence we detected a characteristic C-terminal sequence motif (T-motif), which determines the subcellular localization of Don3 but is not required for regulation of cell separation. This motif defines a novel family of fungal protein kinases including Sid1p, an essential component of the septation initiation network (SIN) in Schizosaccharomyces pombe. Using the yeast two-hybrid system we isolated the Don3-interacting protein Dip1, which is similar to S. pombe Cdc14p, another member of the SIN. Remarkably, deletion of dip1 did not interfere with cytokinesis in U. maydis, but both dip1 and don3 mutants were affected in nuclear envelope breakdown (NEBD) during mitosis. This phenotype has already been described for mutants, which lack the small GTPase Ras3, the U. maydis homologue of the SIN component Spg1p. We propose that the Don3 kinase exerts a dual function in the regulation of cell separation and NEBD.     

Families of replicating units in cultured hamster fibroblasts

An examination of the patterns of DNA replication in pseudodiploid Don C and diploid Don cell lines in culture has been made. Pulse-chase labelling experiments with ³H-thymidine in both synchronized and log-phase cells indicate that the newly replicated DNA can be divided into two and three large temporally distinct fractions in Don C and Don cells, respectively. This is shown radiochemically by fluctuations in the incorporation of ³H-thymidine into the DNA of synchronized cells and autoradiographically by fluctuations in counts of labelled metaphases and grain over mitotic figures. Pulse-chase experiments and fluorometric determinations indicate that the periodic incorporation of ³H-thymidine can be accounted for by discontinuous synthesis and turnover of DNA during the cell cycle.A survey of the literature reveals that fluctuations in DNA synthetic activity during the S phase are to be found in a large number of published graphs of cell population kinetics. The phenomenon is observable in both diploid and non-diploid cells. A change in the timing of DNA replicon synthesis during the S phase according to the developmental stage and age of the cell is proposed.     

长春花激素完全适应型细胞的生长和阿玛碱合成特性

从长春花激素依赖型细胞系(C20D)筛选出一种激素完全适应型的细胞系(C20hi),考察了两种细胞生长、阿玛碱合成和与吲哚生物碱生物合成相关的酶的活性,结果表明:在生长培养基上二者生长无显著差异,而C20hi细胞平均阿玛碱含量是C20D的31.9倍,在生产培养基上C20hi细胞生长较C20D快,C20hi平均阿玛碱含量是C20D的18.4倍.通过比较生产和生长培养基中C20hi细胞的色氨酸脱羧酶、异胡豆苷合酶和牛儿醇-10-脱氢酶活性,发现在生产培养基中培养细胞的3种酶活性均显著高于生长培养基,但与阿玛碱积累没有密切关系.研究结果还表明,通过5年的继代培养,激素完全适应型细胞系C20hi的阿玛碱含量是比较稳定的.     

长春花激素完全适应型细胞的生长和阿玛碱合成特性

从长春花激素依赖型细胞系（C20D）筛选出一种激素完全适应型的细胞系（C20hi),考察了两种细胞生长、阿玛碱合成和引吲哚生物碱生物合成相关的酶的活性,结果表明：在生长培养基上二生长无显差异,而C20hi细胞平均阿玛碱含量是C20D的31.9倍,在生产培养基上C20hi细胞生长较C20D快,C20hi平均阿玛碱含量是C20D的18.4倍。通过比较生产和生长培养基中C20hi细胞的色氨酸脱羧酶、异胡豆苷合酶和long牛儿醇-10-脱氢酶活性,发明,通过5年的继代培养,激素完全适应型细胞系C20hi的阿玛碱含量是比较稳定的。