The morphology of erythroid cells separated by density gradient centrifugation through ficoll

Summary The regenerating blood of geese injected with phenylhydrazine was subjected to large scale, zonal centrifugation through density gradients of Ficoll. In this way, erythroid cells were fractionated according to their respective stages of development. Highly enriched fractions were obtained, containing cells that were well preserved as assessed by both light and electron microscopy. The separated cells exhibited ribosome density and nucleic acid and protein staining patterns typically associated with erythrocyte differentiation. Morphometric analysis of nuclei indicated that despite an apparent net increase in the amount of compact chromatin during development, comparatively little difference existed between the volumes of condensed chromatin present in immature and mature cells. Instead, there was a three fold decrease in nuclear volume between young erythroblasts and reticulocytes, coupled with a concomitant decrease in the volume occupied by dispersed chromatin, RNP and nucleoli. These observations are discussed in relation to molecular changes associated with nuclear differentiation in erythroid cells.Supported by grants from the National Research Council of CanadaWe thank Dr. G. Setterfield for assistance with the EM data and we are grateful to the N.R.C. for use of centrifuges and the zonal rotor     

Osmotic properties of ehrlich ascites tumor cells during the cell cycle

Ehrlich ascites tumor cells were grown and maintained in continuous spinner culture. The population of dividing cells was synchronized by a double thymidine block technique. Cell cycle phases were determined graphically by plotting mitotic index, cell number, and DNA synthesis against time. Changes in the osmotic properties of Ehrlich ascites tumor cells during the cell cycle are described. Permeability to water is highest at the initiation of S and progressively decreases to its lowest value just after mitosis. Heats of activation for water permeability vary during the cell cycle, ranging from 9–14 kcal/mole. Results may imply changes in the state of water in the membrane during the cycle. The volume of osmotically active cell water is highest during S and early G2 and decreases during the mitotic phase, as cells undergo division. Total water content remains stable at 82% (w/w) during the cycle. Total concentration of the three major ions (Na, K, Cl), expressed as mEq/liter total cell volume, does not change. The fraction of total cell water which is osmotically active (Ponder's R) decreased gradually from 0.75 at S to about 0.56 following mitosis. Findings suggest that a fraction of the total water within the cell exists in a “bound” form and is, therefore, incapable of being shifted under the driving force of osmotic pressure. This fraction of bound water increases during the cell cycle. Possible alterations in membrane fluidity and the state of water in the cell are discussed.     

Electrolyte and non-electrolyte distribution in the ehrlich ascites tumor cells during the cell cycle

In a previous study, evidence was presented for changes in the state of water and osmotically active solutes during the cell cycle. Total water was constant at 82% (w/w), while the fraction of water that was osmotically active decreased from a maximum during S to a minimum at mitosis. Total Na⁺, K⁺, and C1^? in milliequivalents per liter of cell water remained constant. Therefore, electrolytes are sequestered in the osmotically inactive water. Evidence is now presented that Na⁺ exists primarily as one compartment, with a second, slower compartment appearing during S and disappearing during G2. Na⁺ is completely exchangeable during the entire cell cycle. The distribution of other penetrating solutes was also investigated. When placed in hyperosmotic ethylene glycol solutions, cells first shrink, then swell to their original volumes. ¹⁴C-ethylene glycol distributes in 89% of cell water throughout the cell cycle. However, ¹⁴C-urea distributes in anywhere from 86–100% of the cell water, depending on the stage in the cell cycle. Both solutes are at chemical equilibrium in water in which they are distributed, but they differ in their effects on cell volume. The final volume at which cells equilibrate in urea varies with the concentration of urea in the environment and with time into the cell cycle. Results suggest a loss of osmotically active particles or decreased osmotic activity of urea.     

Anticlastogenicity of chlorophyllin in the different cell cycle phases in cultured mammalian cells

Chlorophyllin (Chln), a sodium-copper salt derivative of chlorophyll, like chlorophyll-a and -b found in green plants, has been studied for its protective action against the carcinogenic effects of various physical and chemical agents and in relation to the mutagenic and clastogenic activities of genotoxic agents. The aim of the present study was to evaluate chlorophyllin in different phases of the cell cycle for clastogenicity and anticlastogenicity, the latter in reversing DNA damage induced by ethyl methane sulfonate (EMS). The test for chromosomal aberrations was performed in cultured mammalian cells (CHO-K1). The three Chln concentrations tested (6.25, 12.5 and 25 microg/ml) were not clastogenic and damage induced by EMS (1240 microg/ml) was reduced in cells treated with Chln as well during S (25-48%) and G2/S (70-80%). The results demonstrate a greater protective effectiveness of Chln against EMS during G2/S.     

DNA repair in UV-irradiated heteroploid cells at different phases of the cell cycle

The variation of DNA repair activity during the cell cycle was studied by analysing the UV-stimulated DNA synthesis in cells synchronized in mitosis. This activity was detected both by autoradiography and by directly measuring the incorporation of tritiated thymidine in cells irradiated and incubated in the presence of hydroxyurea. Cells in all phases were found to be able to perform repair. However the activity appeared to be considerably lower in mitotic cells than in cell in other phases. Increasing values of repair capacity were observed in G1 cells, in mixed G2, S and M cells and in asynchronous cells. The relationship between these findings and data on survival rates in the same synchronized cells is discussed.     

Enucleation of cells by density gradient centrifugation

HEp-2 cells can be enucleated by ultracentrifugation in a colloidal silica (PTL) density gradient, containing cytochalasin B. Under optimal conditions, more than 70% of the cells are enucleated. Purification up to 97% is carried out by centrifugation at low speed through a second, preformed PTL density gradient. The enucleated cells show a high viability, as tested by [³H]leucine incorporation. The method described was developed for enucleation of high quantities of cells and has the advantage that it can be used for cell types which do not adhere firmly enough to a carrier to be centrifuged as a monolayer.     

Isolation of plasma membranes from corn roots by sucrose density gradient centrifugation: an anomalous effect of ficoll

An investigation was conducted into the isolation of plasma membrane vesicles from primary roots of corn (Zea mays L., WF9 × M14) by sucrose density gradient centrifugation. Identification of plasma membranes in cell fractions was by specific staining with the periodic-chromic-phosphotungstic acid procedure. Plasma membrane vesicles were rich in K⁺-stimulated ATPase activity at pH 6.5, and equilibrated in linear gradients of sucrose at a peak density of about 1.165 g/cc. It was necessary to remove mitochondria (equilibrium density of 1.18 g/cc) from the homogenate before density gradient centrifugation to minimize mitochondrial contamination of the plasma membrane fraction. Endoplasmic reticulum (NADH-cytochrome c reductase) and Golgi apparatus (latent IDPase) had equilibrium densities in sucrose of about 1.10 g/cc and 1.12 to 1.15 g/cc, respectively. A correlation (r = 0.975) was observed between K⁺-stimulated ATPase activity at pH 6.5 and the content of plasma membranes in various cell fractions. ATPase activity at pH 9 and cytochrome c oxidase activity were also correlated.     

Heterogeneity in tumor cell energetic metabolome at different cell cycle phases of human colon cancer cell lines

In this present study, the efficacy of metabolomics as a tool for tumor cell energetics for in vitro cell cultures was demonstrated with full competence for the first time by elucidating the anabolic and energy-yielding segments of glycolysis and glutaminolysis, which constitute a part of energy metabolism in tumor cells. By synchronizing colon cancer cells SW480 and SW620 in culture, the metabolome specific to cell cycle phases was analyzed using nuclear magnetic resonance spectroscopy. At the G₁/S transition of the cell cycle (i.e. transition from cell growth to duplication of genetic material), the majority of the energy production was realized by glycolysis through a high channeling of glucose carbons towards lactate. During the late S phase, the majority of energy was produced by glutaminolysis through a high channeling of glutamine carbons towards lactate, while the glucose carbons were channeled towards bio-synthetic pathways. These results indicate that the metabolism of proliferating cells is heterogeneous throughout the cell cycle and can be better interpreted on the basis of different cell cycle phases. These findings could be exploited for the development of a tool for tumor diagnosis as well as for targeting tumors.     

Phosphorus incorporation in the ehrlich ascites tumor cell

Data from isotopic uptake experiments were used to measure the kinetics of labelling of cellular phosphate, ATP and ADP in the Ehrlich ascites tumor cell. The results show that steady state phosphate exchange flux was 0.333 ± 0.052 (S.E.) μmoles per 10⁷ cells per hour at 37°, and that the specific activity of phosphate was the same as P_γ ATP. Metabolic inhibition reduced the phosphate flux by 30–50%. A model, based on oxidative phosphorylation and the adenylate kinase reaction is used to interpret the labelling sequence of P_β ATP and P_β ADP, and its dependence on P_γ ATP.     

Spontaneous apoptosis of melanotic and amelanotic melanoma cells in different phases of cell cycle: relation to tumor growth

Since the spontaneous alteration of native melanotic (Ma) into amelanotic (Ab) transplantable melanoma line it has been observed that this alteration is accompanied by the acceleration of growth of Ab line. The aim of the present study was to check and estimate spontaneous apoptosis of cells from cell cycle phases. Cytometric cell cycle analysis was performed by staining cells with propidium iodide (PI). Apoptosis estimated by the TUNEL method, alterations in the plasma membrane structure (annexin V staining), changes in the mitochondrial transmembrane potential--delta psi m (JC-1 staining) showed that amelanotic melanoma cells have decreased ability to undergo spontaneous apoptosis. The obtained results showing that in the native melanotic line about 30% of cells are in S+G2/M phases and that 33% of these cells undergo apoptosis could lead to the conclusion that the slower growth of this melanoma line is the result of lower proliferation activity and higher rate of apoptosis of these tumor cells. The number of cells in S+G2/M phases in amelanotic melanoma line increases up to 40% and only 7% of them undergo apoptosis. This observation seems to suggest that the expansive growth of this melanoma line depends mainly on the decreased ability to undergo spontaneous apoptosis, especially in case of cells from S+G2/M phases. Moreover, the obtained results indicate that alteration of melanotic line into amelanotic one, accompanied by differences in many biological features also concerns basic cell processes such as cell cycle and cell death.     

Separation of live cells in different phases of the cell cycle for gene expression analysis

BACKGROUND: Homogeneity of cell populations is a basic requirement for gene expression analyses of the cell cycle, such as those based on microarrays. The most common approach to obtain specific populations is the use of synchronization methods that increase the number of cells representing a certain cell cycle stage. On the one hand, conventional synchronization usually causes undesirable effects. On the other hand, cell separation methods may imply loss of RNA quality, another limiting factor for expression profiling. We describe a new strategy to specifically separate live cells in different phases of the cell cycle (G(1) and G(2)/M) to obtain good quality RNA for gene expression analyses. METHODS: The experimental design included sorting G(1) and G(2)/M cells with the vital fluorochrome Hoechst 33342, followed by RNA isolation from the sorted cells. RESULTS: Sorted living G(1) and G(2)/M cells, analyzed by immunocytochemistry and laser scanning cytometry, showed strong enrichment. The quality and specificity of the isolated RNA were demonstrated by northern blot. CONCLUSIONS: This new approach has many potential applications, such as expression profiling of specific cell populations after eliminating the irrelevant data produced by cells in other stages of the cycle.     

Susceptibility of Hep3B cells in different phases of cell cycle to tBid

tBid is a pro-apoptotic molecule. Apoptosis inducers usually act in a cell cycle-specific fashion. The aim of this study was to elucidate whether effect of tBid on hepatocellular carcinoma (HCC) Hep3B cells was cell cycle phase specific. We synchronized Hep3B cells at G0/G1, S or G2/M phases by chemicals or flow sorting and tested the susceptibility of the cells to recombinant tBid. Cell viability was measured by MTT assay and apoptosis by TUNEL. The results revealed that tBid primarily targeted the cells at G0/G1 phase of cell cycle, and it also increased the cells at the G2/M phase. 5-Fluorouracil (5-FU), on the other hand, arrested Hep3B cells at the G0/G1 phase, but significantly reduced cells at G2/M phase. The levels of cell cycle-related proteins and caspases were altered in line with the change in the cell cycle. The combination of tBid with 5-FU caused more cells to be apoptotic than either agent alone. Therefore, the complementary effect of tBid and 5-FU on different phases of the cell cycle may explain their synergistric effect on Hep3B cells. The elucidation of the phase-specific effect of tBid points to a possible therapeutic option that combines different phase specific agents to overcome resistance of HCC.     

Purification of cell wall fragments by sucrose gradient centrifugation

Summary A procedure for purification of cell wall fragments was developed. The method utilizes sucrose density gradients to efficiently remove soluble enzyme and membrane contaminants from the cell wall. Purification at each stage was monitored biochemically by the removal of cytoplasmic associated markers and ultrastructurally by thorough electron microscopic examination of the isolated cell wall fractions. Cell walls purified by the procedure were compared to those purified by the more conventional multiple washing procedure.     

Separation of hormone-sensitive yeast cells by density gradient centrifugation

Cells in cultures of haploid strains of Saccharomyces cerevisiaein stationary phase were separated into interface fraction andpellet fraction by density gradient centrifugation. Cells inpellet fraction expanded in response to yeast sexual hormoneand animal sex hormones, whereas cells in interface fractiondid not. ¹Present address: Department of Biology, Faculty of Science,Osaka City University, Sumiyoshi-ku, Osaka, Japan (Received July 16, 1970; )