Electron-cytochemical localization of alkaline phosphatase to G cells of Necturus maculosus antrum

Summary Electron-cytochemical localization of alkaline phosphatase activity was performed on G cells of Necturus maculosus antral mucosa. Alkaline phosphatase activity was localized to the nuclear membrane, the Golgi/endoplasmic reticulum, and the limiting membranes of G cell peptide-secretion vesicles. There was no specific localization of alkaline phosphatase activity to the plasma membrane. Treatment of the tissues with levamisole (an alkaline phosphatase inhibitor) did not markedly reduce the specific alkaline phosphatase activity. Specific lead deposition was reduced by removal of the substrate from the reaction mixture. The results from this study on N. maculosus G cells demonstrate that alkaline phosphatase activity can be found in a non-mammalian gastric endocrine cell and that specific activity was localized primarily to those intracellular structures involved with protein biosynthesis.     

Prolonged agonist stimulation does not alter the protein composition of membrane domains in spite of dramatic changes induced in a specific signaling cascade

Protein composition of membrane domains prepared by three different procedures (mechanical homogenization, alkaline treatment with 1 M Na₂CO₃[pH 11.0], or extraction with nonionic detergent Triton X-100), and isolated from the bulk of plasma membranes by flotation on equilibrium sucrose density gradients, was analyzed by two-dimensional (2D) electrophoresis and compared in preparations from control (quiescent) and agonist-stimulated human embryonic kidney cells (HEK)293 or S49 cells. HEK293 cells (clone e2m11) stably expressing high levels of thyrotropin-releasing hormone receptor and G₁₁α protein were stimulated by thyrotropin-releasing hormone and S49 lymphoma cells by the β-adrenergic receptor agonist isoprenaline. Whereas sustained exposure (16h) of both cell lines to the appropriate hormones led to substantial cellular redistribution and downregulation of the cognate G proteins (G_qα/G₁₁α and G_sα, respectively), the distribution and levels of nonstimulated G_i proteins remained unchanged. The 2D electrophoretic analysis of membrane domains distinguished approx 150–170 major proteins in these structures and none of these proteins was significantly altered by prolonged agonist stimulation. Furthermore, specific immunochemical determination of a number of plasma membrane markers, including transmembrane and glycosyl-phosphatidylinositol-anchored peripheral proteins, confirmed that their detergent-extractability/solubility was not influenced by hormone treatment. Collectively, our present data indicate that sustained hormone stimulation of target cells does not alter the basic protein composition of membrane domain/raft compartments of the plasma membrane in spite of marked changes proceeding in a given signaling cascade.     

Progressive Increase In Polyamine Levels In 9l Cells in vitro During the Cell Cycle: Comparison Between Cells Isolated By Centrifugal Elutriation and Cells Grown In Synchrony

Centrifugal elutriation was used to separate 9L rat brain tumour cells into fractions enriched in the G₁, S, or G₂/M phases of the cell cycle. Cells enriched in early G₁, phase were recultured, grown in synchrony, and harvested periodically for analysis of their DNA distribution and polyamine content. Mathematical analysis of the DNA distributions indicated that excellent synchrony was obtained with low dissersion throughout the cell cycle. Polyamine accumulation began at the time of seeding, and intracellular levels of putrescine, spermidine, and spermine increased continuously during the cell cycle. In cells in the G₂/M phase of the cell cycle, putrescine and spermidine levels were twice as high as in cells in the G₁, phase. DNA distribution and polyamine levels were also analysed in cells taken directly from the various elutriation fractions enriched in G₁, S, or G₂/M. Because we did not obtain pure S or G₂/M populations by elutriation or by harvesting synchronized cells, a mathematical procedure—which assumed that the measured polyamine levels for any population were linearly related to the fraction of cells in the G₁, S, and G₂/M phases times the polyamine levels in these phases and that polyamine levels did not vary within these phases—was used to estimate ‘true’ phase-specific polyamine levels (levels to be expected if perfect synchrony were achieved). Estimated ‘true’ phase-specific polyamine levels calculated from the data obtained from cells either sorted by elutriation or obtained from synchronously growing cultures were very similar.     

Invading cancer cells are predominantly in G0/G1 resulting in chemoresistance demonstrated by real-time FUCCI imaging

Invasive cancer cells are a critical target in order to prevent metastasis. In the present report, we demonstrate real-time visualization of cell cycle kinetics of invading cancer cells in 3-dimensional (3D) Gelfoam® histoculture, which is in vivo-like. A fluorescence ubiquitination cell cycle indicator (FUCCI) whereby G₀/G₁ cells express a red fluorescent protein and S/G₂/M cells express a green fluorescent protein was used to determine the cell cycle position of invading and non-invading cells. With FUCCI 3D confocal imaging, we observed that cancer cells in G₀/G₁ phase in Gelfoam® histoculture migrated more rapidly and further than cancer cells in S/G₂/M phases. Cancer cells ceased migrating when they entered S/G₂/M phases and restarted migrating after cell division when the cells re-entered G₀/G₁. Migrating cancer cells also were resistant to cytotoxic chemotherapy, since they were preponderantly in G₀/G₁, where cytotoxic chemotherapy is not effective. The results of the present report suggest that novel therapy targeting G₀/G₁ cancer cells should be developed to prevent metastasis.     

The differentiation of T-lymphocytes. III. The behaviour of subpopulations of mouse thymus cells in short-term cell culture.

Seven human cultured lymphoblastoid cell lines (CLL) were divided into two major groups based on studies of their cell cycle characteristics and surface Ig. CLL I (lines CL, MW, HH and TM) had generation times ranging from 25–40 hr, S phase times of 10–12 hr, G₂ + M times of 6–8 hr, and demonstrated sharp differences between the percentage of SIg(+) cells in different phases of the cell cycle. Line TM was particularly discordant with the highest percentage of SIg(+) cells in G₂ + M. CLL II (lines PS, JR and HT) demonstrated generation times ranging from 18–21 hr, S phase times of 7–10 hr and G₂ + M phase times of 2 hr. In this second group, two of the three CLLs had no differences between cells taken from different points of the cell cycle. DNA synthesis and cell density could not be correlated with either of the above major parameters, i.e. cell cycle times or SIg expression. The results suggest that human CLLs fall into subgroups in which specific patterns of cellular and immune functions may predominate.     

The comparative cell cycle and metabolic effects of chemical treatments on root tip meristems. III. Chlorsulfuron

Intact and excised cultured pea roots (Pisum sativum L. cv Alaska) were treated with chlorsulfuron at concentrations ranging from 2.8 ×10^–4 M to 2.8×10^–6 M. At all concentrations this chemical was demonstrated to inhibit the progression of cells from G₂ to mitosis (M) and secondarily from G₁ to DNA synthesis (S). The S and M phases were not directly affected, but the transition steps into those phases were inhibited. Total protein synthesis was unaffected by treatment of intact roots with 2.8×10^–6 M chlorsulfuron. RNA synthesis was inhibited by 43% over a 24-h treatment period. It is hypothesized that chlorsulfuron inhibits cell cycle progression by blocking the G₂ and G₁ transition points through inhibition of cell cycle specific RNA synthesis.     

Effect of cell cycle on the regulation of the cell surface and secreted forms of type I and type II human tumor necrosis factor receptors

The cell cycle has been shown to regulate the biological effects of human tumor necrosis factor (TNF), but to what extent that regulation is due to the modulation of TNF receptors is not clear. In the present report we investigated the effect of the cell cycle on the expression of surface and soluble TNF receptors in human histiocytic lymphoma U-937. Exposure to hydroxyurea, thymidine, etoposide, bisbensimide, and democolcine lead to accumulation of cells primarily in G₁/S, S, S/G₂/M, G₂/M, and M stages of the cell cycle, respectively. Whilie no significant change in TNF receptors occurred in cells arrested in G₁/S or S/G₂ stages, about a 50% decrease was observed in cells at M phase of the cycle. Scatchard analysis showed a reduction in receptor number rather than affinity. In contrast, cells arrested at S phase (thymidine) showed an 80% increase in receptor number. The decrease in the TNF receptors was not due to changes in cell size or protein synthesis. The increase in receptors, however, correlated with an increase in total protein synthesis (to 3.8-fold of the control levels). A proportional change was observed in the p60 and p80 forms of the TNF receptors. A decrease in the surface receptors in cells arrested in M phase correlated with an increase in the amount of soluble receptors. The cellular response to TNF increased to 8- and 2-fold in cells arrested in G₁ and S phase, respectively; but cells at G2/M phase showed about 6-fold decrease in response. In conclusion, our results demonstrate that the cell cycle plays an important role in regulation of cell-surface and soluble TNF receptors and also in the modulation of cellular response. © 1995 Wiley-Liss, Inc.     

Dynamics of cell cycle phase perturbations by trabectedin (ET-743) in nucleotide excision repair (NER)-deficient and NER-proficient cells, unravelled by a novel mathematical simulation approach

Abstract. Objectives: Trabectedin (ET‐743, Yondelis^®) is a natural marine product, with antitumour activity, currently in phase II/III clinical trials. Previous studies have shown that cells hypersensitive to ultraviolet (UV)‐rays because of nucleotide excision repair (NER) deficiency, were resistant to trabectedin. The purpose of this study was to investigate whether this resistance was associated with different drug‐induced cell cycle perturbations. Materials and Methods: An isogenic NER‐proficient cellular system (CHO‐AA8) and a NER‐deficient one (CHO‐UV‐96), lacking functional ERCC‐1, were studied. Flow cytometric assays showed progressive accumulation of cells in G₂ + M phase in NER‐proficient but not in NER‐deficient cells. Applying a computer simulation method, we realized that the dynamics of the cell cycle perturbations in all phases were complex. Results: Cells exposed to trabectedin during G₁ and G₂ + M first experienced a G₁ block, while those exposed in S phase were delayed in S and G₂ + M phases but eventually divided. In the presence of functional NER, exit from the G₁ block was faster; then, cells progressed slowly through S phase and were subsequently blocked in G₂ + M phase. This G₂ + M processing of trabectedin‐induced damage in NER‐proficient cells was unable to restore cell cycling, suggesting a difficulty in repairing the damage. Conclusions: This might be due either to important damage left unrepaired by previous G₁ repair, or that NER activity itself caused DNA damage, or both. We speculate that in UV‐96 cells repair mechanisms other than NER are activated both in G₁ and G₂ + M phases.     

The activities of thymidine metabolising enzymes during the cell cycle of a human lymphocyte cell line LAZ-007 synchronised by centrifugal elutriation

The activities throughout the cell cycle of thymidine kinase (EC 2.7.1.21), dihydrothymine dehydrogenase (EC 1.3.1.2), thymidine phosphorylase (EC 2.4.2.4) and dTMP phosphatase (EC 3.1.3.35) were measured in the Epstein-Barr virally transformed human B lymphocyte line LAZ-007. Cells were synchronised at different stages of the cell cycle using the technique of centrifugal elutriation. The degree of synchrony in each cycle-stage cell population was determined by flow microfluorimetric analysis of DNA content and by measurement of thymidine incorporation into DNA. The activity of the anabolic enzyme thymidine kinase was low in the G₁ phase cells, but increased many-fold during the S and G₂ phases, reaching a maximum after the peak of DNA synthesis, then decreasing in late G₂ + M phase. By contrast, the specific activities of the enzymes involved in thymidine and thymidylate catabolism, dihydrothymine dehydrogenase, thymidine phosphorylase and dTMP phosphatase remained essentially constant throughout the cell cycle, indicating that the fate of thymidine at different stages of the cell cycle is governed primarily by regulation of the level of the anabolic enzyme thymidine kinase and not by regulation of the levels of thymidine catabolising enzymes.     

The effects of cell population density on the plasma membrane of Acanthamoeba castellanii

Plasma membranes were isolated from both exponential and stationary phase cells and their properties compared, to determine whether alterations are sustained coincident with the transition to plateau phase growth. Polyacrylamide gel electrophoresis revealed no significant differences in macromolecular composition between the two types of membrane. However, the specific activity of alkaline phosphatase (EC 3.1.3.1), an enzyme which shows enrichments in purified plasma membrane fractions relative to homogenates, was markedly reduced in preparations from stationary as compared with exponentially growing cells. The total activity per cell did not change, but in cell fractionation experiments the stationary phase cells yielded a higher proportion of the enzyme in microsomal fractions than did exponentially growing cells. This indicates that once plateau phase is attained, a greater proportion of the membrane bearing alkaline phosphatase activity is internalized as opposed to being associated with the plasmalemma.Alkaline phosphatase is known to be present on the contractile vacuole membrane. During discharge this vacuole becomes associated with the plasmalemma, an event which presumably accounts for at least part of the alkaline phosphatase in plasma membrane preparations. Thus one interpretation of the decreased levels of alkaline phosphatase in plasma membrane fractions from stationary phase cells is that they reflect a decline in the rate of water expulsion. This in turn suggests that the plasmalemma of stationary phase cells may have undergone changes leading to a decreased rate of water influx.     

Experession and integration of genes introduced into highly synchronized plant protoplasts

Summary Chimaeric genes containing the chloramphenicol acetyltransferase (CAT) coding sequence were introduced into protoplasts of suspension-cultured tobacco cells using improved conditions of electroporation (Okada et al. 1986). CAT activity became detectable in the protoplasts within 3 h, was maximal during a period of 18–36 h after electroporation, and then declined gradually. Alpha-amanitin added to the medium abolished the transient expression of the CAT gene. The closed circular form of input DNA was as effective as the linear form for the transient expression. The suspension culture was treated with aphidicolin, and S, G₂, M and G₁ phases were identified in the highly synchronized cell cycle obtained by releasing the cells from the inhibition of DNA synthesis. When a chimacric CAT gene was introduced into M phase protoplasts prepared from the synchronized culture, the transient expression of the CAT gene was 3–4 times higher than when it was introduced into protoplasts of other cell cycle phases. The frequency of stable transformation with a chimaeric neomycin phosphotransferase II gene was studied using the same system. G-418-resistant transformants were obtained from M phase protoplasts at frequencies 2–8 times those obtained from protoplasts at other cell cycle phases. The results indicate that the absence of the nuclear membrane in mitotic cells favours delivery to the nucleus of exogenous DNA introduced into the cytoplasm.     

Alkaline phosphodiesterase I and alkaline phosphatase I in plasma membranes of herpes simplex virus type 1 transformed hamster cells

Plasma membrane extracts from Herpes simplex virus type 1 transformed hamster embryo fibroblasts were chromatographed on Lens culinaris lectin coupled to Sepharose (LcH-Sepharose) and analysed by dodecyl sulphate polyacrylamide gel electrophoresis. Coomassie blue-staining revealed two major protein bands with apparent molecular weights of 125 000 and of about 75 000–90 000. In plasma membranes isolated from these tumor cells prior labeled with [³H]fucose or [³H]glucosamine these bands contained the highest amounts of incorporated radioactivity. Separation by LeH-Sepharose-affinity chromatography as well as metabolic labeling clearly demonstrates their glycoprotein character. The 125 000 protein coincides with alkaline phosphodiesterase I activity with a K_m of 6 · 10^?4 M for TMP p-nitrophenyl ester and is competitively inhibited by UDP-N-acetylglucosamine. This enzymatic activity is also present in normal hamster embryo fibroblasts. Gel electrophoresis of the Lens culinaris lectin-binding glycoproteins from plasma membranes of normal hamster embryo fibroblasts additionally revealed a strong alkaline phosphatase activity represented by an apparent molecular weight of 150 000, while HSV₁ hamster tumor cells contain only a very weak activity of this enzyme activity. HSV-lytically infected cells, however, have unchanged levels of alkaline phosphatase activity, whereas alkaline phosphodiesterase activity increases slightly.     

Noninvasive estimation of cell cycle phase and proliferation rate of human mesenchymal stem cells by phase-shifting laser microscopy

The simultaneous determination of the cell cycle phase of individual adherent mesenchymal stem cells (MSCs) using a fluorescence microscope after staining with 4′,6-diamidine-2′-phenylindole dihydrochloride and bromodeoxyuridine and the laser phase shift by phase-shifting laser microscopy (PLM) revealed that the laser phase shift of cells in the G₂/M phase was markedly higher than that of cells in the G₀/G₁ phase. Even in the synchronous cultures to G₀/G₁ and G₂/M cell cycle phases, the laser phase shift of the cells in the G₂/M phase was markedly higher than that of the cells in the G₀/G₁ phase. The analysis of the cultures of MSCs from different donors with the addition of FGF2 at different concentrations revealed that there was a marked negative correlation between the average phase shift and mean generation time. In conclusion, it is possible to estimate noninvasively the proliferation activity of MSCs population by measuring the phase shift using PLM.     

Role of membrane gangliosides in the binding and action of bacterial toxins

Summary Gangliosides are complex glycosphingolipids that contain from one to several residues of sialic acid. They are present in the plasma membrane of vertebrate cells with their oligosaccharide chains exposed to the external environment. They have been implicated as cell surface receptors and several bacterial toxins have been shown to interact with them. Cholera toxin, which mediates its effects on cells by activating adenylate cyclase, bind with high affinity and specificity to ganglioside G_M1. Toxin-resistant cells which lack G_M1 can be sensitized to cholera toxin by treating them with G_M1. Cholera toxin specifically protects G_M1 from cell surface labeling procedures and only G_M1 is recovered when toxin-receptor complexes are isolated by immunoadsorption. These results clearly demonstrate that G_M1 is the specific and only receptor for cholera toxin. Although cholera toxin binds to G_M1 on the external side of the plasma membrane, it activates adenylate cyclase on the cytoplasmic side of the membrane by ADP-ribosylation of the regulatory component of the cyclase. G_M1 in addition to functioning as a binding site for the toxin appears to facilitate its transmembrane movement. The heat-labile enterotoxin ofE. coli is very similar to cholera toxin in both form and function and can also use G_M1 as a cell surface receptor. The potent neurotoxin, tetanus toxin, has a high affinity for gangliosides G_D1b and G_T1b and binds to neurons which contain these gangliosides. It is not yet clear whether these gangliosides are the physiological receptors for tetanus toxin. By applying the techniques that established G_M1 as the receptor for cholera toxin, the role of gangliosides as receptors for tetanus toxin as well as physiological effectors may be elucidated.     

Synthesis of glycoprotein, glycolipid, protein, and lipid in synchronized L5178Y cells

Synthesis of four macromolecular classes found in membranes—glycoprotein, glycolipid, protein, and lipid—was measured as a function of time of the cell cycle in synchronized L5178Y cells. Incorporation of leucine, choline, fucose, glucosamine, or thymidine into the cells, protein, nucleic acid, or lipid was measured by pulse-labeling for ½ hr at ½ hr intervals after release from the mitotic block. The amount of protein, lipid, glycoprotein, or glycolipid released or secreted into the medium by the L5178Y cells was also measured as a function of time of the cell cycle. Cellular protein was found to be synthesized throughout the cell cycle, with the highest synthesis occurring in the S period; synthesis was depressed in the M period. Cellular glycoprotein was synthesized at approximately the same times as protein, except that the rates of glycoprotein synthesis in the S period relative to other periods were much greater than for protein. Secreted protein was synthesized throughout the cell cycle without any general pattern, except that secretion was elevated in the late S and G₂ periods. Secreted glycoprotein was similar to secreted protein. Cellular lipid and cellular glycolipid were synthesized almost exclusively in the G₂ and M periods; there was no synthesis in the G₁ and S periods. Release or secretion of glycolipid and lipid also occurred in the G₂ and M periods.     

Activation of Src Family Members Is Not Required for the Platelet-Derived Growth Factor β Receptor To Initiate Mitogenesis

The basal activity of Src family kinases is readily detectable throughout the cell cycle and increases by two- to fivefold upon acute stimulation of cells with growth factors such as platelet-derived growth factor. Previous reports have demonstrated a requirement for Src activity for the G₁/S and G₂/M transitions. With a chimeric α-β PDGF receptor (PDGFR) expressed in fibroblasts, we have investigated the importance of the PDGF-mediated increase in Src activity at the G₀/G₁ transition for subsequent cell cycle events. A mutant PDGFR chimera that was not able to detectably associate with or activate Src was compromised in its ability to mediate tyrosine phosphorylation of receptor-associated signaling molecules and initiated a submaximal activation of Erk. In contrast to these early cell cycle events, later responses such as entry of cells into S phase and cell proliferation proceeded normally when Src activity did not increase following acute stimulation with PDGF. We conclude that the initial burst of Src activity is required for efficient tyrosine phosphorylation of receptor-associated proteins such as PLCγ, RasGAP, Shc, and SHP-2 and for maximal activation of Erk. Surprisingly, these events are not required for PDGF-dependent cell proliferation. Finally, later cell cycle events do not require that Src be activated at the G₀/G₁ transition and leave open the possibility that events such as the G₁/S transition require the basal Src activity and/or activation of Src at later times in G₁.     

Enhanced expression of a chromatin associated protein tyrosine phosphatase during G0 to S transition

The non-transmembrane protein tyrosine phosphatase, PTP-S, is located predominantly in the cell nucleus in association with chromatin. Here we have analysed the expression of PTP-S upon mitogenic stimulation and during cell division cycle. During liver regeneration after partial hepatectomy, PTP-S mRNA levels increased 16-fold after 6 h (G₁ phase) and declined thereafter. Upon stimulation of serum starved cells in culture with serum, PTP-S mRNA levels increased reaching a maximum during late G₁ phase and declined thereafter. No significant change in PTP-S RNA levels was observed in growing cells during cell cycle. PTP^-S protein levels were also found to increase upon mitogenic stimulation. Upon serum starvation for 72 h, PTP-S protein disappears from the nucleus and is seen in the cytoplasm; after 96 h of serum starvation the PTP-S protein disappears from the nucleus as well as cytoplasm. Refeeding of starved cells for 6 h results in reappearance of this protein in the nucleus. Our results suggest a role of this phosphatase during cell proliferation.     

Methylmercury Effects On Cell Cycle Kinetics

Abstract. Methylmercury (MeHg) effects on cell cycle kinetics were investigated to help identify its mechanisms of action. Flow cytometric analysis of normal human fibroblasts grown in vitro in the presence of BrdU allowed quantitation of the proportion of cells in G₁, S, G₂ and the next G₁ phase. This technique provides a rapid and easily performed method of characterizing phase lengths and transition rates for the complete cell cycle. After first exposure to MeHg the cell cycle time was lengthened due to a prolonged G₁. At 3, μm MeHg the G₁ phase length was 25% longer than the control. the G₁/S transition rate was also decreased in a dose-related manner. Confluent cells exposed to MeHg and replated with MeHg respond in the same way as cells which have not been exposed to MeHg before replating. Cells exposed for long times to MeHg lost a detectable G₁ effect, and instead showed an increase in the G₂ percentage, which was directly related to MeHg concentration and length of exposure. After 8 days at 5 μM MeHg, 45% of the population was in G₂. the G₂ accumulation was reversible up to 3 days, but at 6 days the cells remained in G₂ when the MeHg was removed. Cell counts and viability indicated that there was not a selective loss of cells from the MeHg. MeHg has multiple effects on the cell cycle which include a lengthened G₁ and decreased transition probability after short term exposure of cycling cells, and a G₂ accumulation after a longer term exposure. There were no detectable S phase effects. It appears that mitosis (the G₂ accumulation) and probably synthesis of some macromolecules in G₁ (the lengthened G₁ and lowered transition probability) are particularly susceptible to MeHg.     

Spatiotemporal changes in Ha-ras p21 expression through the hepatocyte cell cycle during liver regeneration

The protein product of the ras oncogene, Ha-ras (p21), is thought to be an important regulator of cell growth. The cytoplasmic relocalization of p21 in the cell during the cell cycle suggests a transient signaling role for this protein in association with its signal transduction function. Because of the importance of this role we examined spatial patterns in vivo of p21 expression at the protein and mRNA levels in hepatocytes during compensatory growth in rat liver following partial hepatectomy. A low level of p21 was immunolocalized on the cytoplasmic membrane of nonregenerating hepatocytes. The level of hepatic p21 increased significantly and without spatial restriction within the liver from 36 to 60 hr after partial hepatectomy (PH). p21 was localized in the cytoplasm of dividing hepatocytes and on the hepatic cytoplasmic membrane. The elevated p21 level decreased and was found mainly on hepatocyte plasma membranes by 96 hr after PH. Immunogold electron microscopy showed p21 localized over mitochondrial membranes and nuclei in nondividing regenerating hepatocytes. Approximately 50% of nonregenerating hepatocytes show nuclear localization of p21. This percentage changes with time following PH. The decrease in nuclear localization was accompanied with an increase in the low number of hepatocytes which demonstrated cytoplasmic localization in nondividing hepatocytes in regenerating liver. Flow cytometric analysis revealed a significant increase of p21 at 36 hr after PH which was 12 hr after the initial induction of ras mRNA. ras mRNA level increased 1.5-fold at 24 hr after PH and a maximum twofold induction was observed at 48 hr. Cell-cycle analysis of regenerating hepatocytes indicated a synchronized first peak of cell division 36–40 hr after PH. Dual parameter flow cytometry revealed that the level of p21 in hepatocytes in S phase and G₂/M phase of the cell cycle was significantly higher than that in G₀/G₁ phase during regeneration. These findings suggest that p21 is important for the progression of regenerating hepatocytes to S phase and then to G₂/M phase.