PROPERTIES OF G0 CELLS: VARIATIONS IN THE PROLIFERATIVE RESPONSE FOLLOWING ISOPRENALINE

The proliferative behaviour induced in the acinar cells of the rat submaxillary gland in response to isoprenaline has been used to examine the transit time of cells from a quiescent (G₀) state into the S phase. Cumulative ³H-TdR labelling index curves were constructed to determine the mean time interval (G_is time) between stimulation with isoprenaline and entry into the S phase. Data were collected for the proliferative wave induced by three sequential injections of isoprenaline, and the effects of varying the interval between the second and third injections of isoprenaline, and of changing the dose of the drug, were examined. Intervals of 28, 52 and 76 hr between isoprenaline injections resulted in mean G_is times of 16-2, 20-9 and 25-6 hr respectively. It was concluded that the G_is time depended on the recent history of cells with respect to stimulation, but not division. The results are considered in terms of two models, in one of which the time to leave G₀ is variable, whilst in the other the cells leave G₀ immediately the stimulus is applied.     

Characteristics of the isoprenaline stimulated proliferative response of rat submaxillary gland.

A simple stochastic model has been developed to determine the cell cycle kinetics of the isoprenaline stimulated proliferative response in rat acinar cells. The response was measured experimentally, using 3H-TdR labelling of interphase cells and cumulative collections of mitotic cells with vincristine. The rise and fall of the fraction of labelled interphase cells and of metaphase cells is expressed by the product of the proliferative fraction and a difference of probability distributions. The probability statements of the model were formulated and then compared by an iterative fitting procedure to experimental data to obtain estimates of the model parameters. The model when fitted to the combined fraction labelled interphase (FLIW) and fraction metaphase (FMWa) waves gave a mean Gis transit time of 21-2 hr, mean Gis +S transit time of 27-0 hr, and mean Gis + S + G2 transit time of 35-8 hr for a single injection of isoprenaline, where Gis is the initiation to S phase time. When successive injections of isoprenaline were given at intervals of 24 and 28 hr the corresponding values after the third injection were 12-4 hr, 20-8 hr and 25-7 hr respectively. The variance of the Gis phase dropped from 18-1 to 1-3 while the other variances remained unchanged. The estimated proliferative fraction was 0-24 after a single injection of isoprenaline, and 0.31 after three injections of the drug. Independently determined values of the proliferative fraction, obtained from repeated 3H-TdR injections, were 0-21 and 0-36 respectively.     

CHARACTERISTICS OF THE ISOPRENALINE STIMULATED PROLIFERATIVE RESPONSE OF RAT SUBMAXILLARY GLAND

A simple stochastic model has been developed to determine the cell cycle kinetics of the isoprenaline stimulated proliferative response in rat acinar cells. The response was measured experimentally, using ³H-TdR labelling of interphase cells and cumulative collections of mitotic cells with vincristine. The rise and fall of the fraction of labelled interphase cells and of metaphase cells is expressed by the product of the proliferative fraction and a difference of probability distributions. The probability statements of the model were formulated and then compared by an iterative fitting procedure to experimental data to obtain estimates of the model parameters. The model when fitted to the combined fraction labelled interphase (FLIW) and fraction metaphase (FMW,) waves gave a mean G_is transit time of 21-2 hr, mean G_is+ S transit time of 270 hr, and mean G_is+ S + G₂ transit time of 35-8 hr for a single injection of isoprenaline, where G_is is the initiation to S phase time. When successive injections of isoprenaline were given at intervals of 24 and 28 hr the corresponding values after the third injection were 12-4 hr, 20-8 hr and 25-7 hr respectively. The variance of the G_is phase dropped from 18-1 to 1–3 while the other variances remained unchanged. The estimated proliferative fraction was 0–24 after a single injection of isoprenaline, and 0–31 after three injections of the drug. Independently determined values of the proliferative fraction, obtained from repeated ³H-TdR injections, were 0–21 and 0–36 respectively.     

CELL KINETICS IN THE SEBACEOUS GLANDS OF THE MOUSE. I. THE GLANDS IN RESTING SKIN

Cell proliferation, differentiation and migration have been studied in the sebaceous glands of DBA-2 mice in the resting (telogen) phase of hair growth. Cells labelled by a single injection of tritiated thymidine start to leave the glands of adult male mice 5 days later. About 80% of the proliferative cells in the basal layer have a cell cycle time of 40 hr or less. In 18% of the proliferative cells G₁ is at least 4 days long and 16% have a G₂ phase longer than 17 hr. The S phase is about 7.5 hr long and cells spend at least 21 hr in the basal layer before migrating into the differentiating cell region. The glands of mature female and immature mice are smaller than those of the mature male. They have fewer, smaller cells and a much lower labelling index.     

OBSERVATIONS ON HUMAN MONOCYTE KINETICS AFTER PULSE LABELING

Monocyte kinetics were studied in seven hematologically normal individuals using in vivo pulse labeling with tritiated thymidine. Although occasional labeled cells appear in the peripheral blood within 4 or 5 hr of the administration of label, a significant outflow from the marrow begins 13–26 hr later. This interval is occupied by the G₂ and M phases of the mitotic cycle since mitotic cells are not observed in the peripheral blood. The duration of the DNA synthesis phase of monocytes is measured at 34 hr ≈ 1.8 hr. Cells do not enter this phase while circulating since exposure of circulating cells to tritiated thymidine does not result in any uptake. If monocytes are not 'end'cells which have completed their mitotic activity before leaving the marrow they must at least be inhibited from further proliferative activity until they are permanently sequestered in other tissues.
The generation time is probably not less than 40 hr and data derived from the mean grain counts of labeled cells suggest that it is often more than 70 hr. The total daily output of monocytes in man is 9.4 × 10⁸ cells per 24 hr ≈ 3.3 × 10⁸.
Cells leave the bloodstream with a half-time of about 71 hr thereby proving themselves to be considerably more durable than neutrophils which have a half-life in the neighborhood of 6 hr.     

Analysis of the effect of hydroxyurea on stem cell (CFU-s) kinetics

Abstract. Hydroxyurea induces profound changes in the pluripotential haemopoietic stem cell (CFU-s) kinetics. The main feature of these changes is a synchronous entry of resting Go CFU-s into the cell cycle. The analysis of the passage of the CFU-s cohort through the cell cycle has been largely based on the examination of the fraction of CFU-s which synthesize DNA in the S phase of the cell cycle. This analysis has, however, been hampered by the fact that both the sensitivity of the S phase CFU-s to hydroxyurea and their sensitivity in the [³H] thymidine suicide technique vary as the cells pass through the S phase. Methods which overcome these difficulties have been used in the experiments presented in this paper.
It was demonstrated that hydroxyurea kills only about 80% of the S phase CFU-s. The sensitivity to hydroxyurea gradually decreases as the cells approach the middle part of the S phase and increases again as the cells enter the late portions of the S phase.
The degree of CFU-s synchrony at the point of entry into and exit from, the S phase has been established. Mathematical analysis of the available data suggests that CFU-s pass through the S phase with a mean transit time of 4–79 hr (standard deviation, 1.45 hr).
Hydroxyurea, administered in vivo , blocks CFU-s in the late G1 phase. The duration of this G1-S block, induced by a dose of 1000 mg of hydroxyurea per kg body weight, is approximately 2 hr. The CFU-s in the middle of the S phase, which survive hydroxyurea administration, are also blocked in their passage through the S phase. These cells, however, seem to finish the S phase with a delay of approximately 2 hr.     

Cytochalasin D inhibits the progression from the Go to S phase at the mid-prereplicative stage in GC-7 cells stimulated with serum

When the growth of serum-arrested GC-7 cells, a clone from African green monkey kidney, was induced by the addition of 10% calf serum, they began to enter S phase after 15-16 h. When stimulated cells were cultured in the presence of 0.6 micrograms/ml of cytochalasin D, the entrance into S phase was inhibited. Treatment of cells with cytochalasin D during the period earlier than 8 h or later than 11 h after the serum stimulation showed no or little inhibitory effect on the entrance of cells into S phase. Inhibition of the entrance into S phase was observed only when stimulated cells were treated with cytochalasin D during the periods including 9-10 h after stimulation. A rapid increase in protein synthesis occurred 9-12 h after the serum stimulation and was inhibited in the presence of cytochalasin D. These and other results suggested that in the course of the prereplicative process from Go through S phase only the stage around 9-10 h after the start of the cell cycle was sensitive to cytochalasin D and that the block of the cycle was correlated with the inhibition of protein synthesis at this stage.     

Macrophage-mediated cytostatic activity blocks lymphoblast cell cycle progression independently in both G1 phase and S phase

Recent work has shown that macrophage-mediated cytostatic activity inhibits cell cycle traverse in G1 and/or S phase of the cell cycle without affecting late S, G2, or M phases. The present report is directed at distinguishing between such cytostatic effects on G1 phase or S phase using the accumulation of DNA polymerase alpha as a marker of G1 to S phase transition. Quiescent lymphocytes stimulated with concanavalin A undergo a semisynchronous progression from G0 to G1 to S phase with a dramatic increase in DNA polymerase alpha activity between 20 and 30 hr after stimulation. This increase in enzyme activity was inhibited, as was the accumulation of DNA, when such cells were cocultured with activated murine peritoneal macrophages during this time interval. However, if mitogen-stimulated lymphocytes were enriched for S-phase cells by centrifugal elutriation and cocultured with activated macrophages for 4-6 hr, DNA synthesis was inhibited but the already elevated DNA-polymerase activity was unaffected. Similar results were obtained when a virally transformed lymphoma cell line was substituted as the target cell in this assay. These results show that both G1 and S phase of the cycle are inhibited and suggest that inhibition of progression through the different phases may be accomplished by at least two distinct mechanisms.     

Cell cycle kinetics of aerated, hypoxic and re-aerated cells in vitro using flow cytometric determination of cellular DNA and incorporated bromodeoxyuridine

The effects of extreme hypoxia on cell cycle progression were studied by simultaneous determination of DNA and bromodeoxyuridine (BrdU) contents of individual cells. V79-379A cells were pulse-labelled with BrdU (1 microM, 20 min, 37 degrees C) and then incubated for up to 12 hr in BrdU-free medium under either aerated or extremely hypoxic conditions. After the incubation interval (0-12 hr), the cells were trypsinized and fixed in 50% EtOH. Propidium iodide and a fluorescein-labelled monoclonal antibody to BrdU were then used to quantify DNA content and incorporated BrdU, respectively. Measurements in individual cells were made by simultaneous detection of green and red fluorescence upon excitation at 488 nm using flow cytometry. Bivariate analysis revealed progression of BrdU-labelled cells in aerated cultures out of S phase, into G2 and cell division, with halving of mean fluorescence, and back into S phase by approximately 9 hr after the BrdU pulse. Hypoxia immediately arrested cells in all phases of the cell cycle. Both the DNA distribution and the bivariate profile of cells that were fixed from 2 to 12 hr after induction of hypoxia were identical to the 0 hr controls. The percent of cells with green fluorescence in a mid-S phase window remained 100% and the mean fluorescence of these cells remained at control (0 hr) levels. This indicates that, under hypoxic conditions, cells were moving neither into nor out of S phase. Cultures that had been hypoxic for 12 hr exhibited an increasing rate of BrdU uptake with time after re-aeration. Re-aerated cells were able to complete or initiate DNA synthesis, but their rates of progression through the cell cycle were markedly reduced. A large fraction of cells appeared unable to divide up to 12 hr following release from hypoxia.     

Cell Cycle Kinetics of Aerated, Hypoxic and Re-Aerated Cells In Vitro Using Flow Cytometric Determination of Cellular Dna and Incorporated Bromodeoxyuridine

Abstract. The effects of extreme hypoxia on cell cycle progression were studied by simultaneous determination of DNA and bromodeoxyuridine (BrdU) contents of individual cells. V79-379A cells were pulse-labelled with BrdU (1 μM, 20 min, 37°C) and then incubated for up to 12 hr in BrdU-free medium under either aerated or extremely hypoxic conditions. After the incubation interval (0-12 hr), the cells were trypsinized and fixed in 50% EtOH. Propidium iodide and a fluorescein-labelled monoclonal antibody to BrdU were then used to quantify DNA content and incorporated BrdU, respectively. Measurements in individual cells were made by simultaneous detection of green and red fluorescence upon excitation at 488 nm using flow cytometry. Bivariate analysis revealed progression of BrdU-labelled cells in aerated cultures out of S phase, into G₂ and cell division, with halving of mean fluorescence, and back into S phase by approximately 9 hr after the BrdU pulse. Hypoxia immediately arrested cells in all phases of the cell cycle. Both the DNA distribution and the bivariate profile of cells that were fixed from 2 to 12 hr after induction of hypoxia were identical to the 0 hr controls. the percent of cells with green fluorescence in a mid-S phase window remained 100% and the mean fluorescence of these cells remained at control (0 hr) levels. This indicates that, under hypoxic conditions, cells were moving neither into nor out of S phase. Cultures that had been hypoxic for 12 hr exhibited an increasing rate of BrdU uptake with time after re-aeration. Re-aerated cells were able to complete or initiate DNA synthesis, but their rates of progression through the cell cycle were markedly reduced. A large fraction of cells appeared unable to divide up to 12 hr following release from hypoxia.     

Isoprenaline-induced cell proliferation in mouse salivary glands: the effect of castration

The proliferative response to isoprenaline in the submaxillary and parotid glands of the Balb/c mouse has been studied in the intact male and female, and also in the male castrated one month prior to stimulation. The hyperplastic response of the acinar cells has been monitored by serial measurements of the flash tritiated thymidine labelling index and the mitotic index. Castration caused the atrophy of the granular ducts in the submaxillary gland, and therefore an increased predominance of the acini. At one month after castration the acini occupied an area almost 1.5-fold greater than that of the granular ducts, but this was not as great as in the intact female gland where acini occupied twice the area of the granular ducts. Hyperplasia was induced by a single injection of isoprenaline (0.3 mM/kg body weight). The response of the submaxillary gland in the intact male and intact female was very similar, DNA synthesis commencing 21-24 h after stimulation and mitotic activity first noted after 33-36 h. On the other hand, in the submaxillary gland of the castrated male, DNA synthesis began after only 18-21 h and mitotic activity after only 27-30 h. A metaphase arrest experiment with vincristine confirmed the more prompt response in the castrated animals; between 33-36 h after isoprenaline injection, the rate of entry of cells into mitosis was 4 cells/100 cells/h in the castrated group but only 0.4 cells/100 cells/h in the intact males. Thus castration appears to bestow a unique state of responsiveness upon the submaxillary gland to isoprenaline stimulation. The mechanisms underlying this change are not yet understood, for it is paradoxical that atrophy of a structural component rich in specific protein growth factors can alter the format of isoprenaline-induced hyperplasia in acinar cells that produce secretory glycoproteins.     

微管解聚对生长因子在DNA合成中的作用

PPP (platelet-poor plasma) alone can not stimulate DNA synthesis in Go C3H/10T1/2 cells.50 ng/ml of EGF promoted partial Go cells to enter S phase. However, there was an apparent synergic effect of simultaneous treatment with 50 ng/ml EGF and 5%PPP, their synergic effect to stimulate DNA synthesis in Go cells was the same as 10% calf serum. Taxol can resist the depolymerization of microtubules. After treatment with taxol (10 mumol/L), the progression from Go to S phase in C 3 H 10 T 1/2 cells was inhibited. This inhibition was especially exhibited at early stage of transition from Go to S phase. The result indicated that Go cells can not enter S phase without the depolymerization of microtubules. It showed that DNA synthesis was stimulated by the simultaneous treatment with colcemid (0.1 microgram/ml) and growth factors (50 ng/ml EGF + 5% PPP or 10% Calf serum). But without the stimulation of growth factors, the unique effect of depolymerization of microtubules can not stimulate DNA synthesis. The results present evidence indicating that the depolymerization of microtubules has the potency to elevate DNA synthesis in Go cells stimulated by growth factors. This potency was also appeared at early stage of progression from Go to S phase. We suggest that the depolymerization of cytoplasmic microtubules and synergic effect of growth factors are involved in account for the transition from Go to S phase in C 3 H 10 T 1/2 cells.     

Flow cytometric analysis of the recruitment of G0 cells in human epidermis in vivo following tape stripping

Tape stripping of human skin elicits a proliferative response of a synchronously-dividing group of cells. The progress of this cohort of cells has been monitored using two windows in the cell cycle, one located in mid-S phase and the other centred around G2 + M. The cellular DNA is measured with flow cytometry, the windows are defined by two ranges in the DNA histogram. The cohort can be described as the recruitment of cells from a pre-existing G0 compartment which consists of 76% of all proliferative cells. The duration of the S phase is calculated to be 10.2 hr and G2 + M phase 5.1 hr. The cell cycle time of 39 hr for normal human keratinocytes derived from these figures is in line with recent values obtained by different techniques.     

Thrombin, epidermal growth factor, and phorbol myristate acetate stimulate tubulin polymerization in quiescent cells: a potential link to mitogenesis.

Previous studies suggest that alterations in the microtubule (MT)-tubulin equilibrium during G0/G1 affect mitogenesis. To determine the effect of growth factors on the MT-tubulin equilibrium, we developed a radioactive monoclonal antibody binding assay (Ball et al.: J. Cell. Biol. 103:1033-1041, 1986). With this assay, 3H-Ab 1-1.1 binding to cytoskeletons in confluent populations of cultured cells is proportional to the number of tubulin subunits polymerized into MTs. We now show that purified alpha-thrombin increases 3H-Ab 1-1.1 binding to cytoskeletons of serum-arrested mouse embryo (ME) fibroblasts from 1.5- to 3-fold. This stimulation is dose-dependent and correlates with concentrations of thrombin required for initiation of DNA synthesis. Other mitogenic factors, epidermal growth factor (EGF) and phorbol 12-myristate 13-acetate (PMA), also stimulate MT polymerization. Addition of colchicine (0.3 microM) eight hours after growth factor addition, blocks stimulation of 3H-thymidine incorporation by thrombin, EGF, or PMA, suggesting that tubulin polymerization or subsequent events triggered by MT polymerization are required for cells to enter a proliferative cycle. Consistent with models for autoregulation of tubulin synthesis, thrombin, EGF, and PMA all increase tubulin synthesis 9 to 15 hr after growth factor addition, raising the possibility that the decrease in free tubulin and subsequent stimulation of tubulin synthesis is linked to progression of cells into a proliferative cycle. Colchicine addition to these cells also stimulates DNA synthesis, but colchicine-stimulated cells enter S phase 6 to 8 hr later than those stimulated by growth factors. This delayed stimulation may be related to the time required for degradation of tubulin-colchicine complexes below a critical level. These data suggest that regulation of cell proliferation may be linked to increased MT polymerization and the resulting decrease in free tubulin pools.     

Cytokinetic Analysis of the Expanding Kupffer-Cell Population Inrat Liver

Abstract. Zymosan stimulation in rats provides a useful model for studying the expansion of the Kupffer-cell population in liver, which represents the major population of tissue macrophages. This study, using tritiated-thymidine-labelling experiments, demonstrates that during population expansion at least 90% of the resident macrophages (Kupffer cells) develop proliferative activity. the mean duration of the cell cycle is estimated to be 52 hr, with an S phase of 7 hr. We have calculated that about 75% of population expansion results from local Kupffer-cell replication, whereas the remaining growth results from extrahepatic recruitment of macrophage precursors. These findings conflict with a concept of the mononuclear phagocyte system, which states that resident macrophages are (monocyte-derived) non-dividing end-cells.     

Cytokinetic analysis of the expanding Kupffer-cell population in rat liver

Zymosan stimulation in rats provides a useful model for studying the expansion of the Kupffer-cell population in liver, which represents the major population of tissue macrophages. This study, using tritiated-thymidine-labelling experiments, demonstrates that during population expansion at least 90% of the resident macrophages (Kupffer cells) develop proliferative activity. The mean duration of the cell cycle is estimated to be 52 hr, with an S phase of 7 hr. We have calculated that about 75% of population expansion results from local Kupffer-cell replication, whereas the remaining growth results from extrahepatic recruitment of macrophage precursors. These findings conflict with a concept of the mononuclear phagocyte system, which states that resident macrophages are (monocyte-derived) non-dividing end-cells.     

Quantitative analysis of the proliferative activity induced in murine thymocytes by concanavalin A.

A quantitative analysis of the proliferative response induced in murine thymocytes by concanavalin A (Con A) is described. Exogenous 3H-thymidine labels 35 to 40% of the newly incorporated TMP residues under optimal conditions. The density label 5-bromo-2-deoxuridine (BrUdR) does not affect DNA metabolism in this system. With this nucleoside, it is shown that newly synthesized DNA is the result of semi-conservative replication, not repair. Double labeling of DNA provides a monitor for cells traversing the cell cycle (S phase to subsequent S phase). The average cycle time is 12.5 hr, and the shortest cell cycle time is 10 hr. The growing fraction of active cells is about two-thirds. The data show that different subpopulations of thymocytes begin proliferating after various times in culture. Once effectively stimulated by Con A, some of the cells can traverse the cell cycle at least twice more after the mitogen is removed.     

Response of T cells in vivo induced by repeated superantigen treatments at different time intervals

We have investigated the response of T cells to staphylococcal enterotoxin A （SEA） injections in vivo. We found that a single injection of SEA with an optimal dose of 10μg increased the expression of both CD4 and CD8 significantly. There was expansion of SEA-reactive T cells in vivo after SEA re-injection and the time interval between injections strongly influenced the responsiveness of CD4^＋ and CD8^＋ T cells. Anergy of T cells was observed after three SEA treatments. The time interval between injections mainly affected the unresponsiveness of CD4^＋ T cells, not CD8^＋ T cells. Marked deletion followed by anergy of CD4^＋ T cells was induced at short intervals, and anergy without obvious deletion of CD4^＋ T cells was induced at long intervals. We also found that the anergic state was reversible in vivo. Repeated SEA stimulation led to down-regulation of interleukin （IL）-2, and high levels of IL-10. This study showed that both CD4^＋ and CD8^＋ SEA-primed T cells were responsive to SEA rechallenge in vivo, and a third injection was needed to induce the anergy of T cells.     

BrdU-Hoechst flow cytometry reveals regulation of human lymphocyte growth by donor-age-related growth fraction and transition rate

An improved BrdU-Hoechst flow assay was applied to cell kinetic studies of human lymphocyte cultures during a 24-96 hr interval after PHA stimulation. The assay shows that the duration of the initial lag phase and the proportions of noncycling cells increase as a function of donor age, whereas the rates of transition from each cell cycle compartment to the next decrease. Cell cycle arrest occurs in the first S and G2 phase after stimulation of lymphocytes from a 75-year-old donor but not from younger donors. The data are consistent with several models of cell cycle kinetics, so long as these models are modified to include a fraction of noncycling cells in each cell cycle compartment.