Detection of surface differences between two closely related cell populations by partitioning isotopically labeled mixed cell populations in two-polymer aqueous phases. II. A correction

The partition behavior of cells in dextran-poly(ethylene glycol) aqueous phases (i.e., the cells' relative affinity for the top or bottom phase or their adsorption at the interface) is greatly dependent on the polymer concentrations and ionic composition and concentration. Appropriate selection of phase system composition permits detection of differences in either charge-associated or lipid-related surface properties. We have now developed a method that can reveal differences by partitioning that fall within experimental error if one were to compare countercurrent distribution (CCD) curves of two closely related cell populations run separately. One cell population is isotopically labeled in vitro (e.g., with 51Cr-chromate) and is mixed with an excess of the unlabeled cell population with which it is to be compared. The mixture is subjected to CCD and the relative specific radio-activities are determined through the distribution. As control we also examine a mixture of labeled cells and unlabeled cells of the same population. The feasibility of this method was established by use of cell mixtures the relative partition coefficients of which were known. The procedure was then used to test for human erythrocyte subpopulations. 51Cr-chromate-labeled human young or old red blood cells were mixed with unfractionated erythrocytes and subjected to CCD in a phase system reflecting charge-associated properties. It was found that older cells had a high, young cells (probably only reticulocytes) a low partition coefficient. Because of the small differences involved these results were not previously obtained.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of surface differences between two closely related cell populations by partitioning isotopically labeled mixed cell populations in two-polymer aqueous phases. I. Human red blood cell subpopulations

The partition behavior of cells in dextran-poly(ethylene glycol) aqueous phases (i.e., the cells' relative affinity for the top or bottom phase or their adsorption at the interface) is greatly dependent on the polymer concentrations and ionic composition and concentration. Appropriate selection of phase system composition permits detection of differences in either charge-associated or lipid-related surface properties. We have now developed a method that can reveal differences by partitioning that fall within experimental error if one were to compare countercurrent distribution (CCD) curves of two closely related cell populations run separately. One cell population is isotopically labeled in vitro (e.g., with 51Cr-chromate) and is mixed with an excess of the unlabeled cell population with which it is to be compared. The mixture is subjected to CCD and the relative specific radio-activities are determined through the distribution. As control we also examine a mixture of labeled cells and unlabeled cells of the same population. The feasibility of this method was established by use of cell mixtures the relative partition coefficients of which were known. The procedure was then used to test for human erythrocyte subpopulations. 51Cr-chromate-labeled human young or old red blood cells were mixed with unfractionated erythrocytes and subjected to CCD in a phase system reflecting charge-associated properties. It was found that older cells had a high, young cells (probably only reticulocytes) a low partition coefficient. Because of the small differences involved these results were not previously obtained. It was further determined, by repartitioning 51Cr-labeled cells from the left or right ends of a CCD of human red blood cells admixed to unlabeled unfractionated erythrocytes, that a subpopulation with higher partition coefficient exists (probably constituting the old red cells). These experiments serve to illustrate (a) that human red blood cells, contrary to a previous report, can be subfractionated by partitioning and (b) the usefulness of this new method in detecting smaller surface differences between closely related cell populations than was heretofore possible by partitioning alone.     

Detection of surface differences between two closely related cell populations by partitioning isotopically labeled mixed cell populations in two-polymer aqueous phases

The partition behavior of cells in dextran-poly(ethylene glycol) aqueous phases (i.e., the cells' relative affinity for the top or bottom phase or their adsorption at the interface) is greatly dependent on the polymer concentrations and ionic composition and concentration. Appropriate selection of phase system composition permits detection of differences in either charge-associated or lipid-related surface properties. We have now developed a method that can reveal differences by partitioning that fall within experimental error if one were to compare countercurrent distribution (CCD) curves of two closely related cell populations run separately. One cell population is isotopically labeled in vitro (e.g., with⁵¹Cr-chromate) and is mixed with an excess of the unlabeled cell population with which it is to be compared. The mixture is subjected to CCD and the relative specific radio-activities are determined through the distribution. As control we also examine a mixture of labeled cells and unlabeled cells of the same population. The feasibility of this method was established by use of cell mixtures the relative partition coefficients of which were known. The procedure was then used to test for human erythrocyte subpopulations⁵¹Cr-chromate-labeled human young or old red blood cells were mixed with unfractionated erythrocytes and subjected to CCD in a phase system reflecting charge-associated properties. It was found that older cells had a high, young cells (probably only reticulocytes) a low partition coefficient. Because of the small differences involved these results were not previously obtained. It was further determined, by repartitioning⁵¹Cr-labeled cells from the left or right ends of a CCD of human red blood cells admixed to unlabeled unfractionated erythrocytes, that a subpopulation with higher partition coefficient exists (probably constituting the old red cells). These experiments serve to illustrate (a) that human red blood cells, contrary to a previous report, can be subfractionated by partitioning and (b) the usefulness of this new method in detecting smaller surface differences between closely related cell populations than was heretofore possible by partitioning alone.     

Detection of surface differences between two closely related cell populations by partitioning. Erythrocytes from inbred and out-bred rats and rat strains

We have recently developed a new and powerful method capable of detecting, by purely physical means, surface differences between closely related red (or other) cell populations. The procedure consists of isotopically labeling (with [⁵¹Cr]chromate) aliquots of red blood cell populations. Such labeled cells are mixed with an excess of unlabeled red cells to which they are to be compared. The mixtures are subjected to countercurrent distribution in either a charge-sensitive or a non-charge-sensitive dextran-poly(ethylene glycol) aqueous phase system. The distribution curves are analyzed for total cells (in terms of hemoglobin absorbance) and labeled cells (in terms of cpm). Changes in the relative specific activities through the distribution curves are indicative of subtle differences in surface properties between such cell populations. Using this method we have found that erythrocytes from arbitrarily chosen (presumably hematologically normal) individuals differ. In the current work we have examined the surface properties of erythrocytes from Sprague-Dawley and from Lewis rats. This was done with a view to determining whether (a) differences of the type found between different humans can also be detected in other species and (b), if such differences do exist, to examine, by study of the highly inbred Lewis rat strain, whether the differences appear to have a genetic or an acquired basis. It was found that the surface properties of erythrocytes from Lewis and Sprague-Dawley rats differ as do erythrocytes among rats of the Sprague-Dawley strain. No difference was found between red blood cells from different rats of the inbred Lewis strain. These results indicate that the surface differences between red blood cells from different rats detected by partitioning have a genetic rather than acquired origin.     

Alterations in membrane surface properties during cell differentiation as measured by partition in aqueous two-polymer phase systems: Rat intestinal epithelial cells

Membrane surface properties of rat intestinal epithelial cells (crypt base to villus tips) were studied by cell partition in a two-polymer aqueous phase system. A higher partition generally reflects higher cell surface charge (or charge-associated properties) which is not necessarily the same as the charge determined by cell electrophoresis since the latter reflects only the charge at the plane of shear while the former gauges it deeper into the membrane [10]. Cells were prepared by the method of Weiser [22] which sequentially yields cell fractions from villus tips to crypt base. The isolated cells were subjected to countercurrent distribution in a dextran-polyethylene glycol aqueous two-phase system. Countercurrent distribution on the first fractions obtained by Weiser's method have a peak to the left and a smaller peak to the right indicating a surface membrane heterogeneity of upper villus cells; last fractions have a peak only to the right. When all fractions are pooled before countercurrent distribution two well-separated peaks are obtained with the right peak sometimes showing additional heterogeneities. Experiments combining isotope labeling of cells with countercurrent distribution lead us to conclude that the membrane charge (or charge-associated properties) of crypt base cells increases during differentiation and that the charge of the villus cells to which they give rise then diminishes during maturation. The charge of the bulk of the upper villus cells is the lowest of any in the intestinal cell population. The basis for the alteration in charge has not been established but the phenomenon of changing membrane surface charge (or charge-associated properties) as a function of cell differentiation, maturation and aging appears to be a general phenomenon having been found and traced in different cell populations [14, 16, 17, 28].     

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.     

Cytokeratin analysis of breast and leukemia tumor cell lines by flow cytometry

Using four human tumor cell lines, MCF-7 and T47-D from breast tumors, MOLT-4 and K-562 from leukemia, flow cytometric DNA analysis of pure and mixed cell population was performed using monoclonal antibodies to cytokeratin to distinguish cytokeratin-containing carcinoma cells from leukemia cells which do not contain cytokeratins. Surprisingly, on pure or mixed K-562 cells, we found positive labeling with KL1, CK8, and CK18 antibodies (results confirmed by immunocytology). This preliminary study has allowed a DNA analysis on epithelial cells of human breast tumors.     

Effect of surface modification of rat erythrocytes of different ages on their partitioning behavior in charge-sensitive two-polymer aqueous phases

Partitioning differences between cells in two-polymer aqueous phase systems originate from subtle differences between the surface properties of cells. Because of the exponential relation between the parameters affecting the partition ratio (P) and the P itself, differences in membrane components suspected of effecting the differential partitioning of closely related cell populations cannot be directly established by conventional chemical assay techniques. In order to study the chemical nature of the components responsible for the age-related changes in surface properties of rat red cells we have devised an approach which uses a combination of isotopic labeling of erythrocyte subpopulations of distinct cell age with different enzyme and/or chemical treatments followed by countercurrent distribution in charge-sensitive two-polymer aqueous phase systems. These studies show that: neuraminidase-susceptible sialic acid is not responsible for the cell age-related surface differences detected by partitioning; the component(s) responsible for the cell age-related surface differences can be extracted (from aldehyde-fixed red cells) with ethanol or cleaved with dilute sulfuric acid. Our data are consistent with the hypothesis that ganglioside-linked sialic acid is the chemical moiety responsible for the cell charge-associated surface differences among rat red blood cells of different ages.     

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.     

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.     

Membrane charge-associated heterogeneity of B-lymphocytes from human peripheral blood as reflected by cell partition in two-polymer aqueous phases

Aqueous solutions of dextran and of poly-(ethylene glycol) when mixed give rise to immiscible aqueous-aqueous two-phase systems which, when buffered and rendered isotonic, are suitable for the separation by partition of cells based on subtle differences in selected membrane surface properties. Mononuclear cells from human peripheral blood were obtained on a Hypaque-Ficoll cushion and were then separated on the basis of size on a velocity sedimentation gradient at unit gravity. Lymphocytes obtained in this manner were subjected to countercurrent distribution (CCD) in a phase system which reflects membrane surface charge-associated properties. Cells in the different cavities of the extraction train were examined by fluorescent techniques utilizing goat antihuman IgM and anti-human IgD (either separately or mixed) and for their ability to form rosettes with a sheep erythrocyte-antibody-complement (EAC) complex. Results indicate that the highest percentage of fluorescing cells and EAC rosetting cells are under the left part of the distribution. B-lymphocytes are highly heterogeneous and consist of at least two distinct sub-populations not attributable to a difference in surface immunoglobulins. Experimental variation and error preclude, at present, a statement relating to the partial separability of IgM- and IgD-bearing cells. Conversely, the differences in surface charge-associated properties of IgM- and IgD-bearing cells, if they exist, must be small.     

Partition behaviour of a cultured mouse mammary cancer cell line in aqueous two-phase polymer systems

Cell surface-associated changes in behaviour of cultured cells on partition in an aqueous two-phase polymer system were studied using FM3A cell line (a cultured mammary cancer of mouse) with respect to aging.The aqueous polymer system consisted of dextran, polyethyleneglycol and sodium phosphate, equilibrated at 6°C to separate into two phases. Enzyme treatment of cells with neuraminidase reduced cell electrophoretic mobility, as well as the cell partition ratio. Hyaluronidase produced no observable effects on partition and cell electrophoretic mobility, suggesting that the partition is related to sialic acid-associated cell surface charges. The pattern of change in relation to culture time was similar for both cell electrophoretic mobility and cell partition, showing a rise and fall of charge-associated cell surface change during cell growth, the maximum occurring at the beginning of exponential growth. This change was reflected in the pattern of countercurrent distribution of the cells in respective stages of growth. Countercurrent distribution with our two-phase system is expected to be capable of fractionating cell populations according to cell surface properties.     

Partition behaviour of a cultured mouse mammary cancer cell line in aqueous two-phase polymer systems.

Cell surface-associated changes in behaviour of cultured cells on partition in an aqueous two-phase polymer system were studied using FM3A cell line (a cultured mammary cancer of mouse) with respect to aging. The aqueous polymer system consisted of dextran, polyethyleneglycol and sodium phosphate, equilibrated at 6 degrees C to separate into two phases. Enzyme treatment of cells with neuraminidase reduced cell electrophoretic mobility, as well as the cell partition ratio. Hyaluronidase produced no observable effects on partition and cell electrophoretic mobility, suggesting that the partition is related to culture time was similar for both cell electrophoretic mobility and cell partition, showing a rise and fall of charge-associated cell surface change during cell growth, the maxium occurring at the beginning of exponential growth. This change was reflected in the pattern of countercurrent distribution of the cells in respective stages of growth. Countercurrent distribution with our two-phase system is expected to be capable of fractionating cell populations according to cell surface properties.     

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.     

Biologic Activity of a Nucleotide Conjugate Between Mitomycin C and Cytarabine Monophosphate

Abstract

A dual prodrug conjugate between the antimetabolite cytarabine monophosphate and the alkylating agent 2,7-diaminomitosene (derived from mitomycin C), cytaramycin, was synthesized and tested for antileukemic activity in sensitive and resistant tumors. The compound was active against parental L-1210, CCRF-CEM, HL-60 and K-562 leukemia cells but did not overcome resistance in sublines developed for (1) multidrug resistance (L-1210/MDR and K-562-R) or (2) for cytarabine resistance (CCRF-CEM/ARA-C and HL-60/ARA-C). Alkaline DNA elution tests demonstrate a predominance of strand breaking activity due to the cytarabine moiety, and a lesser degree of DNA crosslinking, due to the mitosene moiety. The conjugate was active in mice bearing P-388 leukemia (80% increased lifespan), but was not more effective than mitomycin C alone in mice bearing a cytarabine-resistant L-1210 cell line (38% to 31% increased lifespan). These findings suggest that mitomycin nucleotide conjugates do not overcome resistance to the parent antimetabolites.     

Investigations on the effects of cobalt-alkyne complexes on leukemia and lymphoma cells: cytotoxicity and cellular uptake

Cobalt-alkyne complexes represent a new class of antiproliferative drugs with high activity on cell lines derived from human solid tumors. These promising results encouraged us to evaluate also their effects against leukemia and lymphoma cells. For this purpose, we selected three cobalt complexes with (2-propyn-1-yl)acetylsalicylate (Co-ASS), 2-propyn-1-ol (Co-Prop) and diphenylacetylene (Co-Diph) ligands and investigated their growth inhibiting properties on the LAMA-84, K-562, SD-1 leukemia and U-937 lymphoma cell lines. The cobalt complexes showed high effects on LAMA-84 cells (IC(50)=7.7-16.8 microM) after 48 and 72 h of incubation, but were inactive (K-562, U-937) or low active (SD-1) on the other cell lines. The proliferation of SD-1 cells was reduced by Co-Prop (IC(50)=18.6 microM) and Co-Diph (IC(50)=7.5 microM) only after a 72 h exposure. The antiproliferative effects did not correlate with the accumulation of the drugs into the tumor cells. The time dependent uptake during 24 h determined by atomic absorption spectroscopy was comparably the same in sensitive LAMA-84 and insensitive K-562 cells.     

Cell interactions in microgravity: cytotoxic effects of natural killer cells in vitro

To study cell-to-cell interactions in microgravity we examined the functional activity of natural killer cells on board of the ISS. NK cells are the effector cells with direct cytotoxic activity to oncogenic, virus-infected cells and cells with modified differentiation. Ground-based experiments have shown that the examination of target cell lysis after incubation with NK cells is a simple and informative model for studying the influence of microgravity. NK cytotoxicity was measured as the value of non-degradeted labeled myeloblasts (K-562) after 24 hrs exposure with human lymphocytes in suspension. A special device was developed for space flight experiments. Human cultured lymphocytes and labeled K-562 cells were loaded into special syringes and delivered to the Russian segment of the ISS. Cosmonauts prepared co-cultured suspensions during the first day of microgravity, exposed them at 37 degrees C for 24 hrs and then separated H3-labeled cells on special filters. The results of ISS-8 mission showed that human NK cells in vitro remain lysis activity toward target cells in microgravity. The basal level of NK cytotoxicity was low and we did not found significant differences between "control" and "flight" values. Interferon production during the interaction between immune and target cells (ratio 10:1) in microgravity did not differ compared with ground-based control experiments. Ground exposure of the same lymphocyte samples with K-562 cells to 24 hrs clinorotation also did not lead to significant differences. These experiments paved the way for understanding the cell interaction mechanisms in space flight and the obtained results suggest that microgravity does not disrupt the interaction of NK cells with tumor cells.     

A new endogenous differentiating factor (myelopeptide-4) for myeloid cells

Along with known lymphokines involved in the regulation of hematopoiesis, a new differentiating factor (myelopeptide-4, MP-4) for myeloid cells was found. The peptide (Phe-Arg-Pro-Arg-Ile-Met-Thr-Pro) originally isolated from the culture medium of porcine bone marrow cell culture was examined for its ability to induce differentiation in two human myeloid leukemia cell lines, HL-60 and K-562. Agents with well-known differentiation-inducing activity, such as phorbol myristate acetate, dimethylsulfoxide and the lymphokines were used as a reference. It has been shown that MP-4 significantly influences the integral characteristics of metabolism, expression of surface antigens and morphology of these cells. It decreased the level of chromosomal DNA synthesis and, in parallel, increased the total protein synthesis in both HL-60 and K-562 cells. MP-4 induced the expression of CD14 monocyte-specific surface antigen and the appearance of mature monocytes/macrophages in HL-60 cell cultures. There was a good correlation of cell metabolic/morphological changes and the CD14 marker expression for HL-60 cells. A similar phenomenon was observed in K-562 cells treated with MP-4 when the levels of hemoglobin synthesis were detected in their cytoplasm. Thus, we consider MP-4 as a new endogenous differentiating factor for myeloid cells.     

Cell kinetics of the K-562 cell line in microculture

The cell kinetic parameters of K-562 leukemia cells were studied using microwell cultures in which growth was initiated from a single cell. Total population growth was studied by direct enumeration, 3H-thymidine labelling, and flow cytometry. Clonogenic cell growth was studied by replating and 3H-thymidine suicide. In 7-day clones of K-562 cells, durations of the total cell cycle, G1, S, G2, and M phases were 20.8 h, 3.5 h, 12.9 h, 3.3 h, and 1.1 h, respectively; the growth fraction was 0.92 and the cell loss factor was 0.084. Study of colony-forming cells by replating indicated that clonogenic cells comprised 40% of total cells. 3H-Thymidine suicide showed that cell-cycle duration for these cells was 22.5 h and that S-phase duration was 11.7 h.