Fractionation of K-562 cells on the basis of their surface properties by partitioning in two-polymer aqueous-phase systems

The K-562 cell line is a culture of human leukemia stem cells originally derived from a patient with chronic myelogenous leukemia in blast crisis. We have subjected such cells, in the log phase of growth, to countercurrent distribution in a charge-sensitive dextran-polyethylene glycol aqueous-phase system, a method that fractionates cells on the basis of subtle differences in their surface properties, and found that: (1) The cell population is heterogeneous since it is composed of cells with different partition ratios. (2) There is a correlation between increasing cell partition ratios and increasing cell electrophoretic mobilities. (3) Cells under different parts of the distribution curve have dissimilar ratios of cells in different parts of the cell cycle, a phenomenon that may, at least partially, be the basis for the subfractionation of these cells. There is a clear tendency for cells in G₀+G₁+early S to decrease and for those in late S+G₂+M to increase with increasing partition ratios. (4) Sialic acid is a major surface charge component of the cells as evidenced by a dramatic drop in their partition ratios after treatment with neuraminidase.     

Surface properties of irreversibly sickled cells differ from those of the bulk of sickle cells Studies by partitioning in aqueous phase systems

Counter-current distribution (CCD) of red blood cells (RBC) from individuaks with homozygous sickle cell (HbSS) disease in a charge-sensitive aqueous dextran-poly(ethylene glycol) phase system, which fractionates cells on the basis of surface properties, indicates that the percentage of irreversibly sickled cells (ISC) increases and the percentage of reticulocytes decreases with increasing cell partition ratios. The high partition ratios of ISC correspond to those of older RBC when RBC from normal individuals are subjected to CCD. Our results thus indicate that ISC differ in surface properties from those of the bulk of sickle RBC (including reticulocytes) in the population and that the difference is, most likely, charge-related. While the question as to whether ISC are indeed old cells has not yet been unequivocally answered, this view finds support in the fact that the independent parameters of ISC surface properties, as reflected by partition ratios, and densities correlate as they do in older RBC from normal individuals.     

Cell-cell affinity probed by countercurrent distribution in two-polymer aqueous phase systems

Aqueous solutions of dextran and of polyethylene glycol when mixed form immiscible liquid two-phase systems with a polyethylene glycol-rich top and a dextran-rich bottom. Such phases can be buffered and rendered isotonic and are suitable for the partition of cells. The partition coefficient of cells (i.e., their relative affinity for the top or bottom phase or their adsorption at the interface) depends on the polymer concentrations, on the ionic composition and concentration and, most sensitively, on their membrane surface properties. When two cell populations are mixed the partition coefficient of each is unaffected by the presence of the other population unless an interaction takes place between them. By countercurrent distribution of two cell populations, in a phase system selected such that the partition coefficient of one population is high (i.e., more cells in the top phase) and of the other low, one should be able to detect subtle interactions between cells in the two populations should they occur. Results of experiments with a model system consisting of human peripheral blood mononuclear cells and sheep red blood cells bear out the feasibility of this approach. At low ratios of sheep erythrocytes to mononuclear cells no interaction is apparent. With increasing ratios there is an increasing shift in the distribution curve of subpopulations of mononuclear cells (i.e., T-lymphocytes) as they interact with the sheep red cells. Substitution of rabbit red cells for sheep erythrocytes (at high ratios) reveals a small yet significant shift of a subpopulation of mononuclear cells as well. Distribution of human peripheral blood lymphocytes from patients with chronic lymphocytic leukemia (all B-lymphocytes) are essentially unaffected by the presence of sheep red cells. This sensitive new method, still in its infancy, holds out the hope for the detection of previously unknown cell-cell affinities and for probing suspected cell-cell interactions.     

The partitioning behavior of K-562 cells associated with different stages of culture growth

Partitioning behavior of cells in dextran-poly(ethylene glycol) aqueous phase systems is a sensitive reflection of the cells' surface properties. A decrease in partition ratio, in charge-sensitive phases, of a variety of cell lines as a function of culture growth has been reported by a number of investigators. The basis for this phenomenon remains unclear. We have now studied the surface properties of K-562 cells (a human cell line originally derived from a patient with chronic myelogenous leukemia in blast crisis) during suspension culture growth by countercurrent distribution. The mean partition ratio of viable cells remained constant during 220 h of culture (i.e., well into stationary phase). The decrease in mean partition ratio of the cell population as a whole during culture, previously observed and reported by others, is attributed to the lower partition ratio of non-viable cells which increase with time of culture.     

Synthesis and application of a poly(ethylene glycol)-antibody affinity ligand for cell separations in aqueous polymer two-phase systems

The possibility of producing biospecific affinity ligands for separating cells in two polymer aqueous phase systems on the basis of cell surface antigens was investigated. Rabbit anti-human erythrocyte IgG was reacted with cyanuric chloride-activated monomethyl poly(ethylene glycol) (PEG) fractions (molecular weights approximately 200, 1900, and 5000) at various molar ratios of PEG to protein lysine groups. The partition coefficient of the protein in a Dextran/PEG two-phase system increased with increasing degree of modification and increasing PEG molecular weight. There was a concomitant loss in ability to agglutinate human erythrocytes. The ability of the modified IgG to bind to a DEAE-cellulose column was almost eliminated by reaction with the PEG 5000, and was decreased to a lesser extent by PEG 1900. This PEG 1900-modified IgG substantially increased the partition of fresh or fixed human erythrocytes into the PEG-rich phase of a suitable phase system, while having no effect on rabbit cell partition. The partition increase could be inhibited by unmodified anti-human red cell IgG but not by nonspecific unmodified human IgG, demonstrating that the ligand effects were specific for the cell type against which the antibody was raised. A mixture of rabbit and human erythrocytes, which ordinarily have very similar partitions in the phase systems used, could be separated on a countercurrent distribution apparatus using the modified IgG. These results demonstrate the feasibility of producing immunologically specific affinity partition ligands for cell separation.     

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.     

Separation of human rosette- and nonrosette-forming lymphoid cells by countercurrent distribution in an aqueous two-phase system.

Mixtures of aqueous solutions of dextran and of poly(ethylene glycol) form immiscible two-phase systems suitable for the separation (by partition) of cells based on subtle differences in membrane surface properties. Lymphoid cells from human tonsils were subjected to countercurrent distribution in such a system. Analysis of cells from different parts of the countercurrent extraction train by the sheep red blood cell rosette test indicates a separation of rosette-forming and nonrosette-forming lymphocytes with the former having the higher partition coefficient. Since a major determinant of cell partition in these phases is a membrane-charge associated property it appears likely that the rosette-forming cells have a higher surface charge than the nonrosette-forming cells.     

Altered signal transduction secondary to surface IgM cross-linking on B-chronic lymphocytic leukemia cells. Differential activation of the phosphatidylinositol-specific phospholipase C

To further study the mechanisms by which surface Ig triggering activates the inositol phospholipid signaling pathway, we have used B cells from chronic lymphocytic leukemia patients which, as previously described, display two patterns of response upon sIg cross-linking: in one group this cross-linking induces an inositol phosphate release, an intracellular free Ca2+ concentration elevation and a subsequent cell proliferation; in a second group none of these events occur although there is an increased class II Ag expression following anti-mu stimulation as in the first group. We have been able to demonstrate that the phosphatidyl inositol specific phospholipase C (PI-PLC) can be activated in permeabilized B cells from the first group by direct stimulation, with GPT gamma S, of a guanine nucleotide binding (G) protein. In addition, since anti-mu + GTP gamma S stimulate an increased inositol phosphate production in these cells, this suggests that surface Ig cross-linking activates PI-PLC via a G protein. However, in cells from the second group no inositol phosphate is released after GTP gamma S stimulation although PI-PLC can be directly activated by high Ca2+ concentrations. This reflects in these cells, an interruption of the signaling cascade sIg/G protein/PI-PLC at the level of the G protein or at the G protein/PI-PLC coupling. In cells from both groups PMA treatment, which is known to alter phosphatidyl inositol metabolism in B cells, completely inhibits PI-PLC activation even by high Ca2+ concentrations. These studies show that the phosphatidyl inositol-dependent signaling cascade after surface Ig triggering can be altered at different levels in B cells.     

Endothelial cell subpopulations in vitro: cell volume, cell cycle, and radiosensitivity

Vascular endothelial cells (EC) are important clinical targets of radiation and other forms of free radical/oxidant stresses. In this study, we found that the extent of endothelial damage may be determined by the different cytotoxic responses of EC subpopulations. The following characteristics of EC subpopulations were examined: 1) cell volume; 2) cell cycle position; and 3) cytotoxic indexes for both acute cell survival and proliferative capacity after irradiation (137Cs, gamma, 0-10 Gy). EC cultured from bovine aortas were separated by centrifugal elutriation into subpopulations of different cell volumes. Through flow cytometry, we found that cell volume was related to the cell cycle phase distribution. The smallest EC were distributed in G1 phase and the larger cells were distributed in either early S, middle S, or late S + G2M phases. Cell cycle phase at the time of irradiation was not associated with acute cell loss. However, distribution in the cell cycle did relate to cell survival based on proliferative capacity (P less than 0.01). The order of increasing radioresistance was cells in G1 (D0 = 110 cGy), early S (135 cGy), middle S (145 cGy), and late S + G2M phases (180 cGy). These findings 1) suggest an age-related response to radiation in a nonmalignant differentiated cell type and 2) demonstrate EC subpopulations in culture.     

Cell-Cycle Dependent Appearance of Murine Leukemia-Sarcoma Virus Antigens

Normal rat kidney (NRK) cells, NRK cells infected with Rauscher murine leukemia virus, and NRK cells infected with Kirsten murine sarcoma-leukemia virus (NRK-K) were synchronized by a double thymidine block. At intervals after release from thymidine blockage, the cells were examined for the presence of viral antigens in the cytoplasm and on the cell surface by immunofluorescent microscopy by using goat anti-Rauscher murine leukemia virus and goat anti-Moloney leukemia virus (Tween-ether disrupted) sera. Detection of viral antigens in the cytoplasm was periodic during the cell cycle. Antigens were detected first during the S phase, increased during the G2 phase, and disappeared during the M and G1 phases. A similar pattern of surface immunofluorescence was observed. Infectious virus was detected in culture fluids from synchronized cells during the M phase. Surface immunofluorescence was detected in NRK-K cells with anti-Rauscher murine leukemia virus and may represent the presence of group-specific antigens on the cell surface. Control, uninfected NRK cells, which did not normally fluoresce, showed weak immunofluorescence during the S and G2 phases after synchronization. Synchronization can be used to amplify latent oncornavirus expression.     

Nucleotide sequences in the RNA of mammalian leukemia and sarcoma viruses.

The nucleotide sequences in viral RNA from purified murine sarcoma and hamster leukemia viruses (S+H+) from HTG-1 cells and Rauscher leukemia virus (RLV) from JLS-V 9 cells have been examined by polynucleotide agarose affinity chromatography. There is at least one copy of poly(A) sequences per genomic viral RNA molecule. After heat denaturation of genomic viral RNA (S+H+), there are two types of viral subunits for 34S and 28S species: one that contains poly(A) sequences and one that does not. There are no detectable poly(U) tracts in the viral RNA. However, poly(C) sequences and poly(G) tracts were detected in viral RNA, although less poly(G) than poly(C) tracts were observed. In addition, heat-denatured genomic viral RNA has a greater affinity for poly(G) agarose column than native genomic viral RNA.     

Alteration of cell cycle progression in human leukemia cell line (KOPM-28) induced by 12-o-tetradecanoylphorbol-13-acetate

Terminal cell differentiation usually results in an irreversible arrest in the G1 phase of the cell cycle and loss of cell renewal ability. Human promyelocytic leukemia HL-60 cells induced with 12-o-tetradecanoylphorbol-13-acetate (TPA) differentiate into monocytes/macrophages and accumulate in G1. We determined the effect of TPA on the growth kinetics of a human leukemia cell line (KOPM-28), which developed several of the characteristics of megakaryocytes in response to TPA, such as the surface antigen complex IIb/IIIa, platelet peroxidase and polyploidy. Cell growth was immediately and completely inhibited by TPA. Flow cytometric analysis of cellular DNA content revealed a gradual decrease in cells in G1 and an accumulation of cells in G2. These data suggest that TPA prolonged G1 and rapidly arrested the cells in G2. Synchronized cells were utilized to further analyze the rapid G2 arrest. Cells arrested with aphidicolin at the G1/S interphase were released, and the effects of TPA (added at different intervals) on cell cycle progression were examined 14 h after release. The results showed that TPA added at the end of the S phase, as well as at the G1/S interphase incompletely but distinctly arrested cells in G2. Moreover, G2 arrest was observed when TPA was added to cells released from a colcemid-induced G2/M block, suggesting that cells already in G2 were inhibited by TPA from moving through M to G1. Since some cells became multi-nucleated in the course of incubation with TPA, this G2 accumulation may have resulted at least in part from a prolongation of the phase or a transient G2 block. These changes in cell cycle progression induced by TPA may be characteristic of and/or related to megakaryocytic differentiation of hemopoietic precursor cells.     

Aging of erythrocytes results in altered red cell surface properties in the rat,but not in the human Studies by partitioning in two-polymer aqueous phase systems

Aqueous solutions of dextran and of poly(ethylene glycol) when mixed give rise to two-phase systems useful in separating cells, on the basis of their surface properties, by partitioning. Depending on whether salts with unequal or equal affinity for the two phases are chosen, phases with or without an electrostatic potential difference between the phases are obtained. At appropriate polymer concentrations the former yield cell partition coefficients (i.e., the quantity of cells in the top phase as a percentage of total cells added) based on charge-associated surface properties while the latter reflect membrane lipid-related parameters. With increasing cell age, rat erythrocytes have diminishing partition coefficients in both charged and uncharged phases. Using the elevated aspartate aminotransferase levels of younger red cells as a marker, we have now found that young mature erythrocytes of human do not have the highest partition coefficient in the red cell population as they do in rat. Experiments with isotopically labeled dog red cells yield results similar to those found with human erythrocytes. Furthermore, density-separated young and old red cells from human give overlapping countercurrent distribution curves. Finally, counter-current distribution of human red blood cells followed by pooling of cells from the left and right ends of the distribution and subjection of these cells to a redistribution gives curves that overlap with each other and with the original countercurrent distribution. This indicates that not only are human red cells not subfractionated based on possible age-related surface alterations, but also that they are not subfractionated by partitioning based on any surface parameter.These results are consistent with our previous findings that membrane sialic acid/hemoglobin absorbance is essentially constant through the extraction train after countercurrent distribution of human erythrocytes in a charged phase system; and with the recent reports of others that there is no difference in electrophoretic mobility between human young and old red cells.     

Aging of erythrocytes results in altered red cell surface properties in the rat, but not in the human. Studies by partitioning in two-polymer aqueous phase systems

Aqueous solutions of dextran and of poly(ethylene glycol) when mixed give rise to two-phase systems useful in separating cells, on the basis of their surface properties, by partitioning. Depending on whether salts with unequal or equal affinity for the two phases are chosen, phases with or without an electrostatic potential difference between the phases are obtained. At appropriate polymer concentrations the former yield cell partition coefficients (i.e., the quantity of cells in the top phase as a percentage of total cells added) based on charge-associated surface properties while the latter reflect membrane lipid-related parameters. With increasing cell age, rat erythrocytes have diminishing partition coefficients in both charged and uncharged phases. Using the elevated aspartate aminotransferase levels of younger red cells as a marker, we have not found that young mature erythrocytes of human do not have the highest partition coefficient in the red cell population as they do in rat. Experiments with isotopically labeled dog red cells yield results similar to those found with human erythrocytes. Furthermore, density-separated young and old red cells from human give overlapping countercurrent distribution curves. Finally, countercurrent distribution of human red blood cells followed by pooling of cells from the left and right ends of the distribution and subjection of these cells to a redistribution gives curves that overlap with each other and with the original countercurrent distribution. This indicates that not only are human red cells not subfractionated based on possible age-related surface alterations, but also that they are not subfractionated by partitioning based on any surface parameter. These results are consistent with our previous findings that membrane sialic acid/hemoglobin absorbance is essentially constant through the extraction train after countercurrent distribution of human erythrocytes in a charged phase system; and with the recent reports of others that there is no difference in electrophoretic mobility between human young and old red cells.     

Cell cycle-related proteins: a flow cytofluorometric study in human tumors

We used 2-parameter flow cytometry (FCM) to investigate the relationship between the cell cycle phases and 3 proteins whose expression is known to increase in proliferating cells: the surface antigen transferrin receptor (Trf-r), the "cyclin" (a proliferating cell nuclear antigen, PCNA), and the nuclear antigen recognized by the monoclonal antibody (MoAb) Ki-67. FITC-labeled antibodies against Trf-r, PCNA, and the Ki-67-reactive antigen, as well as propidium iodide-DNA distribution, were simultaneously measured on human leukemia HL-60 and K562, and breast carcinoma MCF-7 cell lines and on fresh human leukemic and glioblastoma cells. The 70% ethanol fixation for Trf-r and PCNA and the 95% acetone fixation for Ki-67 plus permeabilization (with 0.1% and 1% Triton X100, respectively, for the surface and the nuclear antigens) produced cell suspensions with negligible cell clumping, high-quality DNA profiles, and bright specific immunofluorescent staining. The investigated proteins have different relationships with the proliferative state of the cell. Trf-r is expressed mainly at the transition from G0/G1 to S-phase. PCNA expression is prominent in late G1 and through S-phase and decreases in G2-M. The Ki-67-reactive antigen is widely distributed in G1, S, and G2-M phases. Knowledge regarding the relationships between proliferation-associated antigens and cell cycle phase in normal and neoplastic cells could improve our understanding of the mechanisms underlying growth regulation and neoplastic transformation. Bivariate FCM is an easy method for obtaining these data from large numbers of cells.     

UNCOUPLING OF SILICON COMPARED WITH CARBON AND NITROGEN METABOLISMS AND THE ROLE OF THE CELL CYCLE IN CONTINUOUS CULTURES OF THALASSIOSIRA PSEUDONANA (BACILLARIOPHYCEAE) UNDER LIGHT,NITROGEN, AND PHOSPHORUS CONTROL1

The elemental composition and the cell cycle stages of the marine diatom Thalassiosira pseudonana Hasle and Heimdal were studied in continuous cultures over a range of different light‐ (E), nitrogen‐ (N), and phosphorus‐ (P) limited growth rates. In all growth conditions investigated, the decrease in the growth rate was linked with a higher relative contribution of the G2+M phase. The other phases of the cell cycle, G1 and S, showed different patterns, depending on the type of limitation. All experiments showed a highly significant increase in the amount of biogenic silica per cell and per cell surface with decreasing growth rates. At low growth rates, the G2+M elongation allowed an increase of the silicification of the cells. This pattern could be explained by the major uptake of silicon during the G2+M phase and by the independence of this process on the requirements of the other elements. This was illustrated by the elemental ratios Si/C and Si/N that increased from 2‐ to 6‐fold, depending of the type of limitation, whereas the C/N ratio decreased by 10% (E limitation) or increased by 50% (P limitation). The variations of the ratios clearly demonstrate the uncoupling of the Si metabolism compared with the C and N metabolisms. This uncoupling enabled us to explain that in any of the growth condition investigated, the silicification of the cells increased at low growth rates, whereas carbon and nitrogen cellular content are differently regulated, depending of the growth conditions.     

INTRAPOPULATION KINETICS OF THE MITOTIC CYCLE

Data obtained with time lapse cinemicrographic techniques showed that the distribution of generation times for exponentially proliferating human amnion cells in culture is skewed to the right and that reciprocals of generation times appear normally distributed. As shown for bacteria, the true age distribution is much broader than theoretical distributions which fail to take into account the dispersion of generation times. By means of the technique utilizing autoradiographic detection of tritiated thymidine in cells whose mitotic histories were recorded by time lapse cinemicrography, it was shown that the G1 distribution is similar to the generation time distribution but is more variable. In our experiments, the G2 + prophase distribution resembled the generation time and G1 distributions. The data suggested two possibilities for S: either it is relatively constant, or it is inversely related to the lengths of G1 and G2 + prophase. Since G1 is more variable than the total cycle, and G2 + prophase more variable than the computed sum of S + G2 + prophase + metaphase, it was concluded that the relationships between parts of the cycle are non-random and that compensating mechanisms apparently help regulate the lengths of successive parts of the mitotic cycle in individual cells.     

In vivo growth kinetics of P388 and L1210 leukemias

The P388 lymphocytic leukemia and the L1210 lymphoid leukemia are used as test systems for putative cytotoxic drugs. These leukemias are also used to investigate the perturbation of cell cycle progression of various chemical compounds in more detail. There is little information on the normal growth kinetics in vivo of these leukemias. In the present report we therefore present the results from growth kinetic studies of P388 and L1210 leukemic cells growing in ascites form in mice. We used 3H-TdR autoradiography, DNA flow cytometry and the stathmokinetic method. During exponential growth both leukemias showed a growth fraction of unity. Whereas no significant cell loss was observed during the early growth phase of P388 cells, cell loss was indicated by a discrepancy between potential and actual doubling times during exponential growth of L1210 cells. During the phase of growth retardation, the proportion of G1 and G2 cells increased at the expence of a reduced S phase fraction in the P388 leukemia, whereas only small changes in cell cycle distributions were seen with time after inoculation of L1210 cells. An increasing discrepancy in the reduction of the S phase fraction and the 3H-TdRLI was seen in the P388 cells with time after inoculation. Thus, a majority of P388 cells with S phase DNA content were unlabelled during the late phase of growth restriction, indicating resting cells in S phase. A good correlation was found between the 3H-TdR LI and S phase fraction throughout the life history of L1210 cells, revealing considerable differences in in vivo growth kinetics between the two leukemias. Such differences should be considered when evaluating test results.     

Changes in the distribution of a major fibroblast protein, fibronectin, during mitosis and interphase

The distribution of a major fibroblast protein, fibronectin, was studied by immunofluorescence and immunoscanning electron microscopy in cultures of human and chicken fibroblasts during different phases of the cell cycle. The main findings were: (a) In interphase cells, the intensity of surface-associated fibronectin fluorescence correlated with that of intracellular fibronectin fluorescence. (b) The intensity of the fluorescence of both surface-associated and intracellular fibronectins was not changed in cells that were synthesizing DNA. (c) Mitotic cells had reduced amounts of surface-associated but not of intracellular fibronectin. The surface fibronectin that remained on meta-, ana-, or telophase cells had a distinct punctate distribution and was also localized to strands attaching the cells to the substratum. Fibronectin strands first reappeared on the surface of flattening cytoplasmic parts of telophase cells. (d) Fibronectin was also detected in extracellular fibrillar material on the growth substratum, particularly around dividing cells. Thus, surface-associated fibrillar fibronectin was present during G(1), S, and G(2) but in cells undergoing mitosis the distribution was altered and the amount appeared to be reduced. The observations on the distribution of surface-associated fibronectin suggest that rather than being involved in growth control this fibronectin plays a structural role in interactions of cells with the environment.