Presence of spectrin in untreated friend erythroleukemic cells. Its accumulation upon treatment of the cells with dimethyl sulfoxide

Friend leukemia cells (FLC) are nucleated erythroid precursors, and are markedly stimulated towards more advanced stages of differentiation by treatment with dimethyl sulfoxide (DMSO). The presence of spectrin, an erythrocyte membrane protein, has been investigated in untreated and in DMSO-treated FLC by indirect immunofluorescence and by analysis in SDS-polyacrylamide gel electrophoresis of low-ionic-strength cell extracts immuno-precipitated with a monospecific anti-spectrin serum. Spectrin is detectable in significant amounts in the “inducible” clones prior to DMSO stimulation, and accumulates 4- to 5-fold upon addition of this compound to the cultures. Spectrin accumulation occurs rather early (24 hours after cell seeding) and reaches its peak on the third day, to decline thereafter. Semiquantitative determinations of spectrin amounts present in DMSO-stimulated 745A and A°1 cells on the third day after treatment were 2.4 × 10⁵ and 3.0 × 10⁵ molecules/cell, respectively. Spectrin is also detectable in very low amounts in an “uninducible” line of FLC, and is not accumulated upon DMSO treatment thereof, whereas treatment with hemin does cause a significant increase of spectrin-positive cells. These data indicate that spectrin is a convenient “early” marker for in vitro studies of erythropoiesis.     

Influence of irradiation and pentoxifylline on histone H3 phosphorylation in human tumour cell lines

Phosphorylation of histone H3 at Ser-10 correlates with chromatin condensation and this amino terminal modification is now recognized as a specific marker of mitosis. We have monitored the appearance of cells showing histone H3 phosphorylation in four human tumour cell lines to identify cell cycle progression after irradiation. In the human melanoma cell lines Be11 and MeWo and in the squamous cell carcinoma lines 4197 and 4451 a dose of 7 Gy of Co-gamma irradiation increases the number of cells binding anti-histone H3-P antibody 1-8-fold in a p53-independent manner. In the p53 mutant cell lines MeWo and 4451 H3-P phosphorylated cells can be detected as early as 30 min and show a maximum 1 h post-irradiation. In the cell lines Be11, 4197 and 4451 the early wave of H3 phosphorylated cells is followed by a second wave, which reaches a maximum 4.5-7 h post-irradiation and then declines. These events are attributed to damage-induced cell cycle blocks in the G1 and G2 phase of the cell cycle. Addition of the dose modifying drug pentoxifylline before irradiation increases the appearance of cells showing early and the late H3 phosphorylation. When pentoxifylline is added 12-24 h post-irradiation when the cell cycle blocks have reached their maximum the appearance of cells with phosphorylated H3 increases 3-5-fold in the p53 mutant cell lines MeWo and 4451. These observations are consistent with the function of the drug as a G2 block abrogator. The large H3 phosphorylation signal in p53 mutant cells is consistent with early entry of a cohort of G2 cells into mitosis. The smaller H3-P signal in p53 wild type cells correlates with the lower proportion of stable G2 populations in G1 blocked cells. These results indicate that pentoxifylline influences the appearance of histone H3 phosphorylated cells in a manner strongly dependent on the number of cells in G2 phase. This suggests that addition of pentoxifylline indeed abrogates the G2 block and thereby facilitates early entry into mitosis.     

Dinucleoside tetraphosphate variations in cultured tumor cells during their cell cycle and growth

Asynchronous and synchronized cultures of A549 and HTC cells were used to detect possible, cell cycle or cell density specific variations in the intracellular pools of dinucleoside tetraphosphates (Ap4X). No important variations of the nucleotide pools were observed during cell growth. When HTC cells were released from mitotic arrest, a decrease by a factor of N3 Ap4X and ATP levels was observed when the cells entered the G1 phase. This decrease is essentially due to cell doubling. When A549 cells were released from an arrest at the G1/S boundary, the nucleotide pool size increased slightly during the G2 phase just before mitosis. This result is in agreement with both earlier data from our laboratory and the observed decrease in Ap4X pool after release from mitotic-arrested HTC cells. These results suggest that the Ap4X and ATP pools are only subjected to very small variations during the cell cycle, essentially in the G2 phase and after mitosis.     

Reversible G1 arrest in the cell cycle of human lymphoid cell lines by dimethyl sulfoxide

Proliferation of human B- and T-lymphoid cell lines including Raji and Akata cells was found to be arrested at the G1 stage in the cell cycle by dimethyl sulfoxide (DMSO). The G1 arrest by DMSO occurred gradually and was completed within 96 h after addition of 1.5% DMSO concomitantly with a decrease in growth rate. Progression of G1-phase cells containing a larger amount of RNA into S-phase began 9-12 h after removal of DMSO. At 24 h, the DNA pattern of the cell cycle was similar to that of nontreated log-phase cells. The expression of six differentiation markers on the lymphoid cells was not appreciably changed by treatment with DMSO. On the other hand, the expression of transferrin receptor (one of the growth-related markers) on G1-phase cells 96 h after addition of DMSO was decreased to one-fourth that on log-phase cells and was completely restored 24 h after removal of DMSO. These results indicate that DMSO, known as an inducer of differentiation in several myeloid cell lines, acts as an agent inducing G1 arrest in the cell cycle of the lymphoid cells.     

Mitosis may be an obligatory route to terminal differentiation in the Friend erythroleukemia cell

In previous studies, it was shown that treatment of Friend erythroleukemia (FEL) cells with dimethylsulfoxide (DMSO) and the poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide (3AB) blocked the differentiation pathway just prior to commitment. These studies show that the exposure of DMSO(+3AB)-induced cells to the mitotic inhibitors colcemid or nocodazole resulted in commitment to terminal differentiation. Expression of differentiated phenotype required further incubation without the mitotic inhibitors. Microscopic examination indicated that the number of cells blocked in mitosis and those that differentiated were approximately equivalent. These observations suggest that commitment had occurred during mitosis and that expression of the differentiated state occurred after completion of mitosis. Since commitment was not inhibited by blocking DNA replication by aphidicolin or cytokinesis by cytochalasin B, mitosis may be the only phase of the cell cycle required for commitment.     

Sulfhydryl groups and the differentiation of murine erythroleukemia cells.

The importance of cysteine and sulfhydryl groups has been demonstrated in relation to the differentiation and respiration of Friend erythroleukemia cells (FLC). The respiratory rate of undifferentiated FLC was higher basally (5.06 ± 0.16 vs. 3.10 ± 0.09 nmoles 02/min/10⁶ cells) and was further 70% stimulated by addition of cysteine, whereas DMSO-induced differentiated cells were insensitive. A sulfhydryl blocking agent (PCMS) was capable of maintaining the differentiated state of FLC cultured in the absence of DMSO and this effect appeared to be reversible upon removal of the PCMS.     

Changes in intracellular cations during the cell cycle in HeLa cells

Intracellular Na+, K+, and Mg2+ concentrations have been measured during the HeLa cell cycle and compared with changes in oxygen utilization and macromolecular synthesis. Cell water content remains relatively constant at 79 +/- 1% during the cell cycle. A biphasic change in intracellular Na+ occurs with low values as cells reach peak S phase and again in early G1. The decrease in S coincides with an increase in cell volume during increased macromolecular synthesis. The fall in intracellular Na+ during mitosis/early G1 coincides with decreased energy utilization as macromolecular synthesis decreases with a continued decrease in [Na+]i in G1 corresponding to a period of increasing cell volume and an increase in protein synthesis. Intracellular Na+ is relatively high during late S/G2 when phosphate incorporation into protein and phospholipid is maximal. Intracellular K+ concentrations largely parallel intracellular Na+ levels although the intracellular K+:Na+ ratio is significantly lower as the cell volume increases during late G2/mitosis. Additions of a Na+-pump inhibitor (strophanthidin) not only caused a rise in [Na+]i and fall in [K+]i but also inhibited protein synthesis. Conversely, addition of a protein synthesis inhibitor (cycloheximide) blocked amino acid incorporation and produces a fall in intracellular Na+ levels. These findings indicate that intracellular Na+ and K+ play an important role in regulating cell hydration during the cell cycle and that changes in Na+, K+-ATPase activity, synthesis and/or utilization of high energy phosphate compounds, fluid phase turnover (endocytosis), Na+:H+ exchange (pHi), Donnan forces, and ionic adsorption may all be involved.     

Cell cycle-dependent changes in Golgi stacks, vacuoles, clathrin-coated vesicles and multivesicular bodies in meristematic cells of Arabidopsis thaliana: A quantitative and spatial analysis

Cytokinesis in plants involves both the formation of a new wall and the partitioning of organelles between the daughter cells. To characterize the cellular changes that accompany the latter process, we have quantitatively analyzed the cell cycle-dependent changes in cell architecture of shoot apical meristem cells of Arabidopsis thaliana. For this analysis, the cells were preserved by high-pressure freezing and freeze-substitution techniques, and their Golgi stacks, multivesicular bodies, vacuoles and clathrin-coated vesicles (CCVs) characterized by means of serial thin section reconstructions, stereology and electron tomography techniques. Interphase cells possess ∼35 Golgi stacks, and this number doubles during G2 immediately prior to mitosis. At the onset of cytokinesis, the stacks concentrate around the periphery of the growing cell plate, but do not orient towards the cell plate. Interphase cells contain ∼18 multivesicular bodies, most of which are located close to a Golgi stack. During late cytokinesis, the appearance of a second group of cell plate-associated multivesicular bodies coincides with the onset of CCV formation at the cell plate. During this period a 4× increase in CCVs is paralleled by a doubling in number and a 4× increase in multivesicular bodies volume. The vacuole system also undergoes major changes in organization, size, and volume, with the most notable change seen during early telophase cytokinesis. In particular, the vacuoles form sausage-like tubular compartments with a 50% reduced surface area and an 80% reduced volume compared to prometaphase cells. We postulate that this transient reduction in vacuole volume during early telophase provides a means for increasing the volume of the cytosol to accommodate the forming phragmoplast microtubule array and associated cell plate-forming structures.     

Disappearance of a 32 000 D chromatin polypeptide during early erythroid differentiation of Friend leukemia cells

The patterns of non-histone chromatin proteins have been investigated in dimethyl sulphoxide (DMSO)-stimulated Friend leukemia cells (FLC) undergoing the “early” events of erythroid differentiation. Sucrose-purified whole nuclei were lysed and proteins extracted with 6 M urea and 4 M guanidium hydrochloride. The proteins were analysed in SDS-and in SDS-urea-polyacrylamide gel electrophoresis. The disappearance of a 32 000 D chromatin protein component was observed in cells of the 745A “inducible” line harvested as early as 24 h after DMSO treatment, as compared with untreated 745A cells and to cells harvested 6 and 12 h after DMSO addition to the cultures. By contrast, a 32 000 D chromatin protein component is always present in untreated as well as in DMSO-treated cells of the “uninducible” Fw line of FLC.     

Distribution of IP25 in chromatin and its possible involvement in chromatin condensation

Friend leukaemic cells (FLC) were induced to differentiate with dimethyl sulfoxyde (DMSO), hexamethylenbis-acetamide (HMBA) and sodium butyrate (SB) and the phospholipid composition was analyzed. The phospholipid composition of differentiated cells differed from that of non differentiated cells and also varied according to inducer. The ratios of the percentage of phosphatidyl choline (PC) to that of phosphatidyl ethanolamine (PE) or sphingomyelin (SPH) increased by about 2-fold in DMSO or SB induced FLC. These ratios did not vary in HMBa induced FLC. Furthermore the fatty acid composition of PC and PE obtained from differentiated cells varied according to the inducer. Although these changes appeared to be related to the inducers, it can not be excluded that the differentiated state also contributes to these changes.     

Regulation of a group of abundant mRNA sequences during Friend cell differentiation

The changes occurring in the pattern of genes expressed at the polysomal level during induction of Friend cell differentiation with 1.5% dimethylsulfoxide (DMSO) have been examined in two ways. First, homologous and heterologous hybridization experiments between cDNA and polysomal poly(A)⁺ mRNA from differentiated and undifferentiated cells show that about 8000 mRNAs are expressed at both stages of differentiation, the major change being the accumulation of α+β-globin mRNA after DMSO treatment. The vast majority of the mRNA sequences do not change qualitatively, remaining homologous between the undifferentiated and differentiated state. However, in addition to the accumulation of α+β-globin mRNA there is a decrease, after DMSO treatment, in the concentration of abundant and semiabundant sequences found in undifferentiated cells. From control studies with Friend cell variants and fractionated cDNA probes enriched in these sequences, it is shown that the decrease in the abundance of these mRNAs is related to the process of differentiation and not an artefact of DMSO treatment. Comparison of the polysomal poly(A)⁺ mRNAs in differentiated cells to those in pluripotential embryonal carcinoma (EC) cells shows that the vast majority of the sequences are homologous and hence not erythropoiesis specific. Second, comparison of these mRNA populations by in vitro translation and analysis of the protein products on two-dimensional gels also shows that among the more abundant proteins very few qualitatively new proteins appear after differentiation and that the majority are the same as those translated in EC mRNA. There are several proteins prominent in undifferentiated cells which diminish after DMSO treatment, in agreement with the findings from the cDNA studies.     

An Analysis of Cell Proliferation in the Apical Meristem of Lateral Roots of Vicia faba L.

The relative proportions of the various proliferating and quiescentcells, cell doubling time, mean cycle time and the durationof the mitotic cycle and its various phases as measured fromthe passage of labelled cells through mitosis have been determinedfor the initial cells of the cap, epidermis, cortex and stele,for the epidermis together with the cortex and for the steleat various distances basal to the cap-quiescent centre boundaryin 1-cm long lateral roots of Vicia faba. Cell doubling timegenerally increased basally along these tissues as a resultof a gradual decrease in the size of the proliferating populationof cells. Cycle time of the fast-dividing cell population, however,was less between 750 and 800 µm basal to the cap-quiescentcentre boundary than in the corresponding initial cells largelyas a result of a decrease in the duration of G1, although changesalso took place in the durations of the other phases of themitotic cycle as cells were displaced basally along the root.From data reported in this paper as well as other results inthe literature, it appears that the proportions of quiescentcells arrested in G1 and G2 vary in the different groups ofinitial cells. Moreover, the proportion arrested in G1 appearsto decrease basally along each tissue, while that in G2 increases.     

Changes in the testis interstitium of Sprague Dawley rats from birth to sexual maturity

Changes in the rat testis interstitium from birth to adulthood were studied using Sprague Dawley rats of 1, 7, 14, 21, 28, 40, 60, and 90 days of age. Our objectives were 1) to understand the fate of the fetal Leydig cells (FLC) in the postnatal rat testis, 2) to determine the volume changes in testicular interstitial components and testicular steroidogenic capacity in vitro with age, 3) to differentially quantify FLC, adult Leydig cells (ALC), and different connective tissue cell types by number and average volume, and 4) to investigate the relationship between mesenchymal and ALC numbers during testicular development. FLC were present in rat testes from birth to 90 days, and they were the only steroidogenic cells in the testis interstitium at Days 1 and 7. Except for FLC, all other interstitial cell numbers and volumes increased from birth to 90 days. The average volume of an FLC and the absolute volume of FLC per testis were similar at all ages except at Day 21, when lower values were observed for both parameters. FLC number per testis remained constant from birth through 90 days. The observations suggested that the significance of FLC in the neonatal-prepubertal rat testis is to produce testosterone to activate the hypothalamo-hypophyseal-testicular axis for the continued development of the male reproductive system. ALC were the abundant Leydig cell type by number and absolute volume per testis from Day 14 onwards. The absolute numbers of ALC and mesenchymal cells per testis increased linearly from birth to 90 days, with a slope ratio of 2:1, respectively, indicating that the rate of production of Leydig cells is 2-fold greater than that of mesenchymal cells in the postnatal rat testis through 90 days. In addition, this study showed that the mesenchymal cells are an active cell population during testis development and that their numbers do not decrease but increase with Leydig cell differentiation and testicular growth up to sexual maturity (90 days).     

Early transport changes during erythroid differentiation of Friend leukemic cells

Early transport changes occurring during Friend erythroleukemic cell differentiation are reported. A decrease in the rate of 86Rb transport was observed beginning approximately five hours after stimulation with 1.5% dimethylsulfoxide (DMSO), a potent inducer of Friend cell differentiation. By 12 to 14 hours after DMSO addition, the transport rate had stabilized at close to 60% of control level. This decrease in the rate of 86Rb transport preceded a previously reported decrease in cell volume. Other chemical inducers of Friend cells, such as hypoxanthine and ouabain, also caused early decreases in 86Rb influx. In contrast, xanthine, which does not induce Friend cell differentiation, also did not affect 86Rb influx. The transport of two amino acid analogues, alpha-aminoisobutyric acid and 2-aminobicyclo [2,2,1]-heptane-2-carboxylic acid, which differ in their mode of uptake, was also measured following induction by DMSO. The transport rates of both compounds decreased after a 12-hour exposure to DMSO. In contrast, the uptake of 3H-colchicine, a drug which diffuses passively across the cell membrane, was not significantly affected. Studies with several variant cell lines which do not synthesize hemoglobin in response to DMSO indicate that these non-inducible cells can be divided into two classes--those that demonstrate early changes in transport very similar to the changes observed in inducible cell lines and those which exhibit only small changes in transport. Results obtained using a revertant clone have helped to distinguish between those transport changes which are associated with the induction of hemoglobin synthesis and those which are not. In addition, these early transport changes may be useful in defining the stage in the differentiation process at which a particular variant line is blocked.     

Cell cycle-specific changes in β-adrenergic receptor concentrations in C6 glioma cells

The concentration of β-adrenergic receptors in synchronized C6 cells is lowest during mitosis and G₁ (1000–2000 receptors/cell). A rapid increase in β-receptor concentration occurs in early S phase to 9000 receptors/cell, followed by a decrease during G₂ to the low concentration found at mitosis. In non-synchronized C6 cell populations, mitotic cells have one-half of the average β-receptor concentration of the population as a whole, confirming the observations made with synchronized cells.     

Terminal differentiation in cultured Friend erythroleukemia cells.

Two populations of differentiated, hemoglobin-containing cells have been identified in cultures of Friend murine erythroleukemia cells (Friend cells): terminally differentiated benzidine-positive (B+) cells that are no longer capable of proliferation and are arrested in the G1 phase of the cell cycle, and their precursors, traversing B+ cells which undergo two or three cell divisions before reaching their terminally differentiated state. Thus Friend cells in suspension culture retain a limited capacity to synthesize DNA and divide after commitment to erythroid differentiation. We identified terminally differentiated cells using autoradiography after benzidine staining. We also developed a quantitative flow microfluorometric assay to distinguish cells that are terminally differentiated from those cells committed to differentiation but still capable of proliferation.We developed a purification procedure to isolate terminally differentiated Friend cells. Their DNA content was the same as that of the undifferentiated cells in G1 by both the diphenylamine reaction and a fluorescence assay. No loss of DNA was detected during the differentiation of Friend cells. As many as 72% of the total cells in a culture induced with DMSO (88% B+) were differentiated cells arrested in G1. As a control, a DMSO-resistant line derived from 745A neither differentiated nor arrested in G1 after growth in the presence of DMSO. The results of these studies were obtained using several compounds that induce differentiation and three independently isolated clones of 745A. We also observed arrest of differentiated cells in G1 with the two other well characterized, independently derived erythroleukemia cell lines, F4-1 and T3-C1-2.     

Cell cycle parameters inAedes albopictus mosquito cells

Summary We have analyzed cell cycle parameters for theAedes albopictus C7-10 mosquito cell line, which has been systematically developed for somatic cell genetics, expression of transfected genes, and synthesis of hormone-inducible proteins. In rapidly cycling cells, we measured a generation time of 10–12 h. The duration of mitosis (M) was ≤1 h, and the DNA synthesis phase (S) required 6 h. UnlikeDrosophila melanogaster Kc cells, in which the G2 gap is substantially longer than G1, in C7-10 cells G1 and G2 each lasted approximately 2h. In these cells, the duration of both S and G2 was independent of the population doubling time, and the increase in population doubling time as cells approached confluency was due to prolongation of G1. When treated with the insect steroid hormone, 20-hydroxyecdysone, C7-10 mosquito cells complete the cycle in progress before undergoing a reversible arrest.     

Significance of the cell cycle in commitment of murine erythroleukemia cells to erythroid differentiation.

The relationship between differentiation of murine erythroleukemia cells (MEL) induced by DMSO and the cell division cycle has been analyzed. We demonstrate that incubation in the presence of DMSO increases the length of the G1 phase of the cell cycle. A method of synchronization of MEL cells by unit gravity sedimentation has been developed and characterized. Using this method, a series of synchronized cell populations covering the entire cell division cycle can be generated simultaneously. Cells synchronized by this technique were challenged with DMSO and analyzed for kinetics of commitment to the differentiation program. Our results indicate that populations of cells in G1 or G2 at the time of addition of inducer give rise to a greater proportion of committed cells than an unfractionated population, while cells in S phase result in a lower percentage of committed cells than the unfractionated population when cultured in DMSO.     

The durations and compositions of cell cycles in embryos of the leech, Helobdella triserialis

When tritiated thymidine triphosphate ([(3)H]TTP) or its immunohistochemically detectable analogue, bromodeoxyuridine triphosphate (BrdUTP), is injected into blastomeres of leech embryos it passes throughout the entire embryo and is rapidly incorporated (within 2 min after injection) into nuclei of cells synthesizing DNA (S phase). In the same embryos a DNA-specific stain can be used to identify cells in mitosis (M phase) or nonreplicative interphase (G(1) or G(2) phase) on the basis of nuclear or chromosomal morphology. Using this procedure, we have determined the lengths and compositions of the mitotic cell cycles of identifiable cells in early embryos of the leech, Helobdella triserialis, and have analysed how the cell cycles change during the first seven stages of development. The relatively short cell cycles of the early blastomeres comprise not only phases of M and S, but also postreplicative gap (G(2)) phases. The lengthening of the cell cycles that occurs as development progresses is primarily accomplished by an increase in the length of G(2) and secondarily by an increase in the length of S and,in some instances, the addition of a prereplicative gap(G(1)) phase; M phase remains relatively constant. These data suggest that the durations of the cell cycles of embryonic cells are regulated by a variety of mechanisms.     

Lipid composition of Friend leukemia cells following induction of erythroid differentiation by dimethyl sulfoxide

The effects of dimethyl sulfoxide (DMSO)-induced differentiation of Friend leukemia cells in vitro on the lipid composition of these cells have been examined. DMSO had no early effect on the incorporation of either [¹⁴C] glycerol or [³H] methyl choline chloride into the total lipids or individual phospholipids of Friend cells up to 240 min after addition of the inducer. Examination of DMSO-diferentiated Friend cell phospholipids revealed a percentage composition which was similar to control cells, with phosphatidylcholine and phosphatidylethanolamine in both uninduced and differentiated cells accounting for over 75% of the total phospholipid. Sphingomyelin levels were significantly lower in Friend cells than in normal adult mouse erythrocytes, and differentiation of murine erythroleukemia cells resulted in a further lowering of this phospholipid. In contrast, a significant increase in the level of phosphatidylethanolamine occured as a result of maturation. Fatty acid analysis of major lipid classes of differentiated Friend cells showed significant reduction in saturation, but no alteration in chain length in comparison to undifferentiated cells. A pronounced decrease in the cellular content of both free and esterified cholesterol, which resulted in a 45% decrease in the ratio of cholesterol/phospholipids, occurred in cells differentiated by the polar solvent. The findings indicate that erythrodifferentiation induced by DMSO results in a variety of changes in the lipid composition of the membranes of Friend leukemia cells.