Sensitivity to X-irradiation in relation to cell size of CHO cells synchronized in early G1

A population of line CHO Chinese hamster cells was synchronized by mitotic selection and allowed to enter early G1, after which the largest and smallest cells in the population were sorted, irradiated, and their viability determined. Despite sizeable differences in volume, metabolic capability and cell cycle progression rates, an equivalent level of survival was obtained for the two populations, indicating that the factors responsible for the volume, metabolic and progression heterogeneity do not contribute greatly to radiation sensitivity.     

Centrifugal elutriation of human lymphoid cells: synchronization and separation of aneuploid cells into diploid and tetraploid populations

Both normal and leukemic human lymphoid cell lines were separated into populations corresponding to different positions in the cell cycle by centrifugal elutriation. Each population was analyzed for cell concentration, cell volume, [3H]thymidine incorporation, percentage S phase by autoradiography, and percent G1, S, and G2/M phases by flow cytometry. The smallest cells, collected at the lowest flow rate, were in G1 phase. Cells collected at increasing flow rates progressively increased in volume and represented distinct positions in the cell cycle transition from G1 phase, through S phase, and into G2/M phase. The purity of the G1 population varied according to cell load. One hundred percent of cells were recovered and cells collected in G1- and S-phase populations proliferated in culture with patterns characteristic of synchronized cells. An aneuploidy leukemia cell line, CEM, was separated into near-diploid and near-tetraploid populations by centrifugal elutriation. This method of cell separation provides large numbers of human lymphoid cells at different positions in the cell cycle for investigating the relationship between the cell cycle and various surface membrane and metabolic properties of cells. Aneuploid leukemia and lymphoma cells can be separated by centrifugal elutriation into populations which contain different numbers of chromosomes for comparisons of their biologic properties.     

Volume growth of daughter and parent cells during the cell cycle of Saccharomyces cerevisiae a/alpha as determined by image cytometry.

The pattern of volume growth of Saccharomyces cerevisiae a/alpha was determined by image cytometry for daughter cells and consecutive cycles of parent cells. An image analysis program was specially developed to measure separately the volume of bud and mother cell parts and to quantify the number of bud scars on each parent cell. All volumetric data and cell attributes (budding state, number of scars) were stored in such a way that separate volume distributions of cells or cell parts with any combination of properties--for instance, buds present on mothers with two scars or cells without scars (i.e., daughter cells) and without buds--could be obtained. By a new method called intersection analysis, the average volumes of daughter and parent cells at birth and at division could be determined for a steady-state population. These volumes compared well with those directly measured from cells synchronized by centrifugal elutriation. During synchronous growth of daughter cells, the pattern of volume increase appeared to be largely exponential. However, after bud emergence, larger volumes than those predicted by a continuous exponential increase were obtained, which confirms the reported decrease in buoyant density. The cycle times calculated from the steady-state population by applying the age distribution equation deviated from those directly obtained from the synchronized culture, probably because of inadequate scoring of bud scars. Therefore, for the construction of a volume-time diagram, we used volume measurements obtained from the steady-state population and cycle times obtained from the synchronized population. The diagram shows that after bud emergence, mother cell parts continue to grow at a smaller rate, increasing about 10% in volume during the budding period. Second-generation daughter cells, ie., cells born from parents left with two scars, were significantly smaller than first-generation daughter cells. Second- and third-generation parent cells showed a decreased volume growth rate and a shorter budding period than that of daughter cells.     

Metabolic rewiring in drug resistant cells exhibit higher OXPHOS and fatty acids as preferred major source to cellular energetics

Alteration in metabolic repertoire is associated with resistance phenotype. Although a common phenotype, not much efforts have been undertaken to design effective strategies to target the metabolic drift in cancerous cells with drug resistant properties. Here, we identified that drug resistant AML cell line HL-60/MX2 did not follow classical Warburg effect, instead these cells exhibited drastically low levels of aerobic glycolysis. Biochemical analysis confirmed reduced glucose consumption and lactic acid production by resistant population with no differences in glutamine consumption. Raman spectroscopy revealed increased lipid and cytochrome content in resistant cells which were also visualized as lipid droplets by Raman mapping, electron microscopy and lipid specific staining. Gene set enrichment analysis data from sensitive and resistant cell lines revealed significant enrichment of lipid metabolic pathways in HL-60/MX2 cells. Further, HL-60/MX2 possessed higher mitochondrial activity and increased OXPHOS suggesting the role of fatty acid metabolism as energy source which was confirmed by increased rate of fatty acid oxidation. Accordingly, OXPHOS inhibitor increased sensitivity of resistant cells to chemotherapeutic drug and fatty acid oxidation inhibitor Etomoxir reduced colony formation ability of resistant cells demonstrating the requirement of fatty acid metabolism and dependency on OXPHOS by resistant leukemic cells for survival and tumorigenicity.     

Synchronization of mammalian cells by selection and additional chemical block studied by DNA distribution analysis and BrdUrd-Hoechst 33258-technique

Abstract. Ehrlich ascites tumour cells growing in vitro in suspension culture were separated according to volume by the technique of velocity sedimentation in a zonal rotor with a reorienting gradient. Using DNA distribution analysis the sedimentation pattern of the cells could be analysed in detail. With appropriate conditions it was possible to separate pure G1 cells. Samples could also be obtained which were enriched in S or G2 + M cells. The main limitation of the selection in this type of rotor was the reorientation of the gradient which caused disturbances during deceleration of the rotor. The synchronous growth of selected G1 cells has been studied in detail to investigate the reasons for the rather poor synchrony of these cells. The poor synchrony was found to be caused mainly by the small volume of the selected G1 cells compared with the normal volume of G1 cells in an asynchronous population. The synchronization of these cells could be essentially improved by a short treatment with excess thymidine causing a metabolic block at the G1/S border. The duration of this treatment could be minimized using DNA distribution analysis of growing cells after releasing of the block. The durations of the cell cycle phases in synchronized cells agreed with the values calculated in asynchronous cells by DNA distribution analysis and the BrdUrd-Hoechst 33258-technique.     

The ultrastructural morphology of leukemia L 1210 ascites tumor cells transplanted with different frequency

The mouse lymphatic leukemia L 1210 ascites tumor cells were transplanted every 3rd (L1210/3) or 6th day (L 1210/6) and then examined by an electron microscope. In cytoplasm of L 1210 cells thick bundles of 8 nm filaments were observed. Lysosomes were more numerous in L 1210/6 cells. Results obtained from morphometric datas shown that the relative cell volume of L 1210/6 population was 1.59 time larger as compared with L 1210/3. The index of nuclear volume to cell volume was very similar in both populations. The relative volume of endoplasmic reticulum was more than twice larger in the L 1210/3 population. The cell surface area was larger in the L 1210/6 cells as compared with this in L 1210/3 cells, but the increase in the surface was not proportional to the increase of the cell volume. The relative surface area of endoplasmic reticulum cisternae was smaller in L 1210/6 cells than in L 1210/3 population.     

A theoretical approach to zonation in a bioartificial liver

Bioartificial livers have yet to gain clinical acceptance. In a previous study, a theoretical model was utilized to create operating region charts that graphically illustrated viable bioartificial liver configurations. On this basis a rationale for the choice of operating and design parameters for the device was created. The concept is extended here to include aspects of liver zonation for further design optimization. In vivo, liver cells display heterogeneity with respect to metabolic activity according to their position in the liver lobule. It is thought that oxygen tension is a primary modulator of this heterogeneity and on this assumption a theoretical model to describe the metabolic zonation within an in vitro bioartificial liver device has been adopted. The distribution of the metabolic zones under varying design and operating parameters is examined. In addition, plasma flow rates are calculated that give rise to an equal distribution of the metabolic zones. The results show that when a clinically relevant number of cells are contained in the BAL (10 billion), it is possible to constrain each of the three metabolic zones to approximately one-third of the cell volume. This is the case for a number of different bioreactor designs. These considerations allow bioartificial liver design to be optimized.     

Light and dark smooth muscle cells in estrogen-stimulated rat myometrium.

Smooth muscle cells of different densities to transmission of electrons (termed light and dark cells) were found in rat myometrium examined in the electron microscope following fixation by immersion in glutaraldehyde. Light cells accounted for about 4% of the total population of cells. No light cells were found in tissues fixed in situ by intraarterial perfusion with glutaraldehyde. In addition to staining differences, light cells were distinguished from most dark cells by differences in nuclear, mitochondrial, endoplasmic reticular, and surface structures. The relative number of light and dark cells after in vitro fixation was not changed in tissues relaxed with adrenaline or contracted with oxytocin. Mechanical injury resulted in increased numbers of light cells. Similarly, chemical injury with metabolic inhibitors resulted in ATP depletion, followed by increased numbers of light cells and gain in water content. We concluded that light cells were produced by mechanical or metabolic damage, leading to loss of volume control mechanisms, swelling, and leakage of protein. Light cells found after fixation in vitro in numerous prior studies represent cells damaged during isolation, and not a physiological variant among smooth muscle cells.     

Variant HeLa cells selected for their resistance to ouabain

The cardiac glycoside, ouabain, normally kills HeLa cells at concentrations of about 10⁻⁷ m or greater. By treating a population of HeLa cells with increasingly higher concentrations of the drug, a variant population was obtained of HeLa cells capable of growing in medium containing 10⁻⁴ M ouabain. Inhibition of volume regulation of cells subjected to hypotonic shock was used as a measure of inhibition of active transport of Na across the plasma membrane. In that way dose-response curves for the rapid effects of ouabain and other inhibitors of active Na transport were obtained with both the original, ouabain-sensitive (OS) and the variant, ouabain-resistant (OR) cells. Three other cardiac glycosides (digoxin, digitoxin and hellebrin) and two aglycones (digitoxigenin and strophanthidjn) were found to be equally as effective as ouabain in inhibiting volume regulation of the OS cells; the concentration which produced half-maximum inhibition, I(max/2), was about 6 × 10⁻⁷ M in each case. Similar inhibition of the OR population by ouabain was observed only when the concentration exceeded 10⁻⁴ m [I(max/2)∼2.5 × 10⁻⁴ m], and the other steroid compounds had no effect on the variant cells at the highest concentrations tested (∼2 × 10⁻⁵ m). OR and OS cells differed also in their sensitivities to the cardioactive erythrophleum alkaloid, coumingine; I(max/2) for OS and OR cells was 5 × 10⁻⁸ m and 6 × 10⁻⁷ M, respectively. These results, in addition to results of ouabain binding experiments and measurements of the rates of reversal of inhibition of volume regulation, suggest that a major reason for the differential sensitivities of the two phenotypes to these drugs is different affinities of their sodium pumps for inhibitors of active transport.     

Comparison of pinocytosis and phagocytosis in Acanthamoeba castellanii

Acanthamoeba, with high rates of phagocytosis and pinocytosis of the non-concentrative type, offers favorable experimental material for investigation of similarities and possible differences in these two modes of uptake. Phagocytosis was measured by the rate of uptake of latex beads and pinocytosis by the rate of uptake of radioactive inulin and albumin. The effects of the metabolic inhibitors NaN₃, NaCN, NaF, iodoacetate, 2,4-dinitrophenol and cold were found to be identical on both forms of endocytosis. Both endocytic processes were suppressed by inhibitors of aerobic metabolism and low temperature and were not appreciably affected by inhibitors of glycolysis. The cells recovered capacity to endocytose after exposure to all these compounds except 2,4-dinitrophenol, which was irreversibly toxic. Endocytosis and O₂ consumption were measured as a function of temperature. Below 5 °C both phagocytosis and pinocytosis ceased; between 9 and 15 °C uptake was less than 10% that at 29 °C. From 16 to 29 °C uptake was a linear function of temperature for both pinocytosis and phagocytosis. Curves for O₂ consumption and endocytosis both showed breaks at about 16 °C. Concanavalin A (ConA) inhibited both types of endocytosis more than 50% at concentrations as low as 5 μg/2 × 10⁵ cells/ml. Pinocytosis and phagocytosis were also measured simultaneously in the same cells. Increasing the rate of phagocytosis suppressed pinocytosis, but the combined volume of the two forms of uptake was essentially constant. In contrast, the estimated combined surface intake varied over a two-fold range. These data show no differences between phagocytosis and pinocytosis of the non-concentrative type, and suggest that control of the rate of endocytosis is determined by the volume of an internal compartment. The volume of this compartment, estimated by measuring the volume of latex beads that “saturate” the phagocytic mechanism, amounted to about 500 μm³/cell or roughly 15% of the cell volume.     

Modes of Growth in Mammalian Cells

The increase of cell volume as a function of time was studied throughout the generation cycle in synchronous cultures of Chinese hamster cells using a Coulter aperture and a multichannel analyzer calibrated against known cell volumes. The experimental results were compared to a mathematical model of cell volume increase which considered the effect of the distribution of individual cell generation times on the progress of the population. Several modes of volume increase, including linear and exponential, were considered. The mean volume vs. time curve was rounded at the ends of the cycle even when linear growth was assumed. The experimental results show that cell volume increased in a smooth fashion as a function of time, with no discontinuities in rate detectable at periods when cells may have been undergoing metabolic shifts as, for example, through the phases associated with DNA synthesis, G₁, S, G₂. A statistical test on the comparison of the modal cell volume vs. time data to the predictions of linear and exponential growth models accepted both hypotheses within the resolution of these experiments. However, exponential growth was favored over linear growth in one cell line. Volume dispersion was almost constant with time in both sublines which is also consistent with exponential growth. Limitations of the electronic technique of volume measurement and indications for future experiments are discussed.     

Carbon dynamics of logarithmetic and stationary phase phytoplankton as determined by track autoradiography

Track autoradiographic analysis of photosynthetic radiocarbon incorporation at the cellular level indicated that the carbon uptake rate and carbon pool size of exponentially growing (log phase) Scenedesmus cells was threefold that of stationary phase cells, while carbon turnover rates were similar. Carbon fixation was uncoupled from growth and cell division in the stationary phase cells, which were larger and contained less chlorophyll per unit volume than log phase cells. Changes in the temporal pattern of isotope incorporation were evident at the cell level prior to the cessation of division and transition to stationary phase, while bulk carbon fixation responded only the second day after cell division ceased. The carbon uptake patterns of a marine nanoplankter from a nutrient-enriched natural sample resembled that of log phase cells while the control population pattern resembled that of stationary cells. The physical, biochemical, and metabolic differences between log and stationary phase cells are potentially measurable by flow cytometry procedures currently in use and under development. The use of flow cytometry to sort cell types for analysis by track autoradiography and subsequent correlation of metabolic characteristics with flow cytometry signatures is a feasible means of investigating the heterogeneity of phytoplankton metabolic state in the marine environment.     

The relationship between the rate of entry into S phase, concentration of DNA polymerase alpha, and cell volume in human diploid fibroblast-like monokaryon cells

We have examined the kinetic relationship between the rate of entry into the S phase in human diploid fibroblast-like (HDFL) monokaryon cells and (1) the concentration of DNA polymerase alpha activity and (2) the cell volume. In the former studies, a first-order dependence between the rate of entry into the S phase and the concentration of DNA polymerase alpha activity was observed, consistent with the enzyme, or a coregulated factor, being rate limiting for this metabolic process. Examination of the nature of the dependence of the rate of entry into the S phase upon cell volume revealed a more complex relationship. The results obtained in studies with synchronized cultures are consistent with the presence of two to three rate-limiting reactants when cell volume is the independent variable. Studies with asynchronous HDFL cell cultures revealed that the smallest cells in the G1 population, presumably the early G1 cells, enter the S phase at an increasing rate as a function of cell volume up to a certain size, beyond which the cells enter at a decreasing rate similar to that observed in the studies with the synchronized cultures. Similar studies examining the relationship between cell volume and the rate of entry into S phase in three established immortal cell lines revealed positive correlation between the rate of entry into S phase and cell volume throughout the size range of the G1 population. This latter observation suggests that the factors involved in the initiation of the S phase may be present in concentrations that are not rate limiting in immortal cell lines.     

Kinetics of Cell Volume Changes of Murine Lymphoma Cells Subjected to Different Agents In Vitro

Cell volume distributions obtained with an electronic particle analyzer were used to study the changes in volume of individual cells in the absence of cell division. Cultures of murine lymphoma (strain L5178-Y) cells in suspension were used in these studies. During a division delay following ionizing radiation, individual cells increased exponentially in volume with equal rate constants; these rate constants were indistinguishable from that describing the increase in cell number of an unirradiated population. When an originally log phase population of cells was prevented from increasing in number by inhibitors of DNA synthesis, individual cells increased exponentially in volume for about one generation time with the same rate constant as observed after exposure to ionizing radiation; thereafter, only the cells defining the upper half of the volume distribution continued to increase in volume, and they apparently did so with a first order rate constant proportional to their amount of DNA exceeding that present in one diploid complement of chromosomes in G(1). Cells arrested in mitosis with colchicine increased in volume for approximately 4 hr after which they remained constant in volume for almost one generation time; eventually these cells again increased in size. Inhibitors of protein and RNA synthesis inhibited the cell volume growth of irradiated cells.     

Quantification of the human Sertoli cell population: its distribution, relation to germ cell numbers, and age-related decline

The human Sertoli cell population was characterized in 14 men by histometric analysis and by direct counts of nuclei in testicular homogenates. Testes obtained at autopsy were perfused with glutaraldehyde and embedded in Epon. Nucleolar and nuclear volumes were determined by the formula of a sphere given the diameter of the nucleoli or average diameter of nuclei measured at the height and width. Nuclear volume was also estimated by adding volumes of nuclear profiles in 0.5-micron serial sections. Sertoli cell number/g was calculated by the product of the percentage nucleoli or nuclei in the parenchyma, parenchymal volume, and histologic correction factor divided by the volume of a single nucleolus or nucleus. Also, Sertoli cell nuclei were counted directly in homogenates of fixed parenchyma. Number of Sertoli cells/g was similar (P greater than 0.05) whether determined by serial sections or in homogenates, but the estimate based on the nucleolar method was higher (P less than 0.01) and the nuclear measurement method was lower (P less than 0.01) than that for serial sections. A group of 37 men aged 20 to 48 yr had significantly (P less than 0.01) more Sertoli cells than did 34 men aged 50 to 85 yr. It is concluded that: 1) the homogenate method is valid for quantification of the Sertoli cell population, 2) Sertoli cells are evenly distributed in different regions of the testis, 3) the average human Sertoli cell supports relatively few germ cells, 4) the human Sertoli cell population declines with age, and 5) there is a significant relationship between sperm production rates and number of Sertoli cells.     

Biophysical characterization of peyer’s patch lymphocytes in the B-cell deficient CBA/N mice and in their F1 hybrids

CBA/N mice bear an X-linked immunodeficiency involving abnormalities of their B lymphocytes. We investigated the electrophoretic mobility (EPM) distribution and the electronic volume of the Peyer's patch (PP) cells of these mice in comparison with those of the immunologically normal CBA/J mice. The same studies were also carried out on PP cells from the CBA/N×DBA/2 (CND2) F1 hybrid males and females that are, respectively, B-cell defective and normal. In all types of mice, PP cells could be separated by free-flow electrophoresis into a low-mobility (LM) population corresponding mainly to B cells and high-mobility (HM) population containing mostly T cells. However, while in the control mice LM (B) cells accounted for around 60–67% of PP lymphocytes, this population did not exceed 40% in the defective animals. Furthermore, LM (B) cells, but not HM (T) cells, of CBA/N and CND2 male mice were found to display abnormal physical characteristics. Thus, they possessed a higher anodic EPM (two electrophoretic fractions faster) and a slightly larger model size (134 μm³) than normal adult B cells (122–124 μm³). Similar differences were observed with PP lymphocyte preparations enriched for B cells by nylon wool adherence. These data suggest that CBA/N mice and their defective hybrids lack a population of small B lymphocytes with low anodic EPM.     

Free amino acid mediated volume regulation of isolatedNoetia ponderosa red blood cells: Control by Ca2+ and ATP

Summary The ionic and metabolic requirements of cellular volume regulation and the free amino acid (FAA) efflux from hypoosmotically stressedNoetia ponderosa (Mollusca: Arcidae) red blood cells was studied. Deletion of Ca²⁺ from 50% ASW prevented cell volume regulation and decreased the FAA efflux. Addition of Co²⁺, Mn²⁺, or La³⁺ to 50% ASW increased volume regulation and the FAA efflux, while verapamil, a Ca²⁺ antagonist, inhibited volume regulation and the FAA efflux. Volume regulation by the blood cells has a metabolic component also since DNP or incubation of the cells at 4°C both inhibited volume regulation and the FAA efflux. Thus, the FAA permeability ofN. ponderosa blood cell membranes can be manipulated by altering seawater [Ca²⁺] or by indirectly modifying intracellular levels of ATP.Abbreviations FAA free amino acid - ASW artificial seawater - DMSO dimethylsulfoxide - DNP dinitrophenol     

Morphological changes in the skin of Rana pipiens in response to metabolic acidosis

The skin of Rana pipiens excretes H+ and this excretion is increased by metabolic acidosis. The mitochondria-rich (MR) cells of the skin have been found to mediate this H+ transport. The purpose of this study was to determine if there is a change in the MR cells of the skin during metabolic acidosis and if the isolated split epithelia of frog skin maintains its capacity to excrete H+. Metabolic acidosis was induced by injecting 120 mM NH4Cl (0.025 ml/g body wt) into the dorsal lymph sac three times a day for 2 days. The frogs were sacrificed and collagenase-split skins from the abdomen of normal and metabolic acidotic frogs were mounted between 2-ml chambers. H+ fluxes into both the mucosal and serosal media were measured and reported in units of (nmol) (cm2)-1 (min)-1. An increase in H+ flux was seen on both the mucosal and serosal sides of the acidotic split skins. The isolated epithelia were fixed, postosmicated, and dehydrated in the chamber. They were then embedded in Spurr's resin and 1-micron sections were cut and stained with Paragon multiple stain. Coded slides were used to count various cell types. Sections were randomly selected and approximately 40,000 cells were counted. Four basic cell types were noted and confirmed by TEM photomicrographs; basal (B) cells, granular (G) cells, keratinized cells, and MR cells. The ratio of G + B cells:MR cells in the normal skins was 1.0:0.021. The ratio in acidotic skins was 1.0:0.34. The average percentage of cell population of MR cells in the normal skins was 2.08 + 0.18 and in acidotic skins 3.20 + 0.36 (P less than 0.005). We conclude that the split skin maintains the capacity to acidify the mucosal fluid. Additionally, during metabolic acidosis there is an increased number of MR cells in the skin and this increase may be an adaptive mechanism of the skin to excrete excess H+ during acidosis.