DNA topoisomerase I and II activities during cell proliferation and the cell cycle in cultured mouse embryo fibroblast (C3H 10T1/2) cells

We have used C3H 10T1/2 cells to examine the regulation of topoisomerase activities during cell proliferation and the cell cycle. The specific activity of topoisomerase I was about 4-fold greater in proliferating (log phase) cells than in non-proliferating (confluent) cells. In synchronized cells, the bulk of the increased activity occurred during or just prior to S phase, depending upon the method of synchronization. A smaller increase in activity also occurred during G1 phase. The increase in activity during S phase was not altered by a hydroxyurea block at the G1/S phase boundary indicating that it is not directly coupled to DNA synthesis and is not the result of topoisomerase I gene dosage. The increase was inhibited by blocking cells at mid-G1 phase using isoleucine deprivation. Thus, the increase in activity during S phase is dependent on events occurring during mid- to late G1 phase. In contrast to the changes in topoisomerase I levels, the specific activity of topoisomerase II showed no detectable difference in proliferating vs non-proliferating cells. In addition, no detectable difference in topoisomerase II specific activity was seen in G1, S and M phases of the cell cycle. The differences in the activity profiles of the topoisomerases I and II during the cell cycle suggest that the two activities are regulated independently and may be required for different functions.     

Effect of cyclophosphamide on the proliferation of L 1210 ascites tumour cells and of jejunal crypt cells in the mouse

Cyclophosphamide (CY) does not act in a cell-cycle specific manner, i.e. exclusively on proliferating cells. It also kills non-proliferating cells, as shown by application of CY to L 1210 ascites tumour-bearing mice during plateau phase growth of the tumour. Moreover, treatment with CY of L 1210 ascites tumour cells, double-labelled with [3H] and [14C]-thymidine, suggests that CY is not cell cycle phase dependent, but kills cells out of all cycle phases. There is also an extensive cytocidal effect of CY (300 mg/kg) on the jejunal crypt cells of the mouse, which is even more pronounced than that of cisplatinum (DDP, 13 mg/kg). However, rapid regeneration of crypt cells occurs after treatment with the drugs.     

Cultured human tumour cells may be arrested in all stages of the cycle during stationary phase: demonstration of quiescent cells in G1, S and G2 phase

Six human colon carcinoma cell lines were induced to enter stationary phase of growth by nutrient deprivation and cell crowding. Growth kinetics parameters (cell number, flow cytometric analysis of DNA distribution, and labelling and mitotic indices) were measured sequentially for all lines during the various stages of in vitro growth. Our results demonstrated that a substantial fraction of cells (9-18%) were located in G2 phase when they changed from an exponential to a stationary mode of growth. Moreover, a large number of cells in stationary phase of growth had an S-phase DNA content, as determined by flow cytometry, but failed to incorporate radioactive DNA precursors (up to 15-fold difference). To substantiate these findings, cells in stationary phase of growth were induced to enter exponential growth by re-seeding in fresh medium at a lower density. Subsequently observed changes in DNA-compartment distribution, and in labelling and mitotic indices were those expected from cells that had been arrested at different stages of the cycle during their previous stationary phase. Thus, the non-proliferating quiescent state (Q), traditionally located 'somewhere' in G1 phase, appears to be composed also of cells that can be arrested at other stages of the cycle (Qs and QG2). Although the proportion of such cells is rather small, their contribution to the growth kinetics behaviour of human in vivo tumours will become apparent following 'recruiting' or 'synchronizing' clinical manoeuvres and will prevent the formation of a clear-cut wave of synchronized cells.     

Effect of Cyclophosphamide On the Proliferation of L 1210 Ascites Tumour Cells and of Jejunal Crypt Cells In the Mouse

Abstract. Cyclophosphamide (CY) does not act in a cell-cycle specific manner. i.e. exclusively on proliferating cells. It also kills non-proliferating cells, as shown by application of CY to L 1210 ascites tumour-bearing mice during plateau phase growth of the tumour. Moreover, treatment with CY of L 1210 ascites tumour cells, double-labelled with [³Hl and [¹⁴C]-thymidine, suggests that CY is not cell cycle phase dependent, but kills cells out of all cycle phases.
There is also an extensive cytocidal effect of CY (300 mg/kg) on the jejunal crypt cells of the mouse, which is even more pronounced than that of cisplatinum (DDP, 13 mg/kg). However, rapid regeneration of crypt cells occurs after treatment with the drugs.     

Kinetic analysis of cell size and DNA content distributions during tumor cell proliferation: Ehrlich ascites tumor study.

In order to study the growth dynamics of proliferating and non-proliferating cells utilizing discrete-time state equations, the cell cycle was divided into a finite number of age compartments. In analysing tumor growth, the kinetic parameters associated with a retardation in the growth rate of tumors were characterized by computer simulation in which the simulated results of the growth curve, the growth fraction, and the mean generation time were adjusted to fit the experimental data. The cell age distibution during the period of growth was obtained and by a linear transformation of the state transition matrices, was employed to specify the cell size and DNA content distributions. In an application of the model, the time-course behavior of cell cycle parameters of Ehrlich ascites tumor is illustrated, and the parameters important for the transition of cells in the proliferating compartment to the non-proliferating compartment are discussed, particularly in relation to the G1-G0 and G2-G0 transitions of non-cycling cells as revealed by the variation of cell size distribution.     

Extracts of proliferating and non-proliferating human cells display different base excision pathways and repair fidelity

Base excision repair (BER) of damaged or inappropriate bases in DNA has been reported to take place by single nucleotide insertion or through incorporation of several nucleotides, termed short-patch and long-patch repair, respectively. We found that extracts from proliferating and non-proliferating cells both had capacity for single- and two-nucleotide insertion BER activity. However, patch size longer than two nucleotides was only detected in extracts from proliferating cells. Relative to extracts from proliferating cells, extracts from non-proliferating cells had approximately two-fold higher concentration of POLβ, which contributed to most of two-nucleotide insertion BER. In contrast, two-nucleotide insertion in extracts from proliferating cells was not dependent on POLβ. BER fidelity was two- to three-fold lower in extracts from the non-proliferating compared with extracts of proliferating cells. Furthermore, although one-nucleotide deletion was the predominant type of repair error in both extracts, the pattern of repair errors was somewhat different. These results establish two-nucleotide patch BER as a distinct POLβ-dependent mechanism in non-proliferating cells and demonstrate that BER fidelity is lower in extracts from non-proliferating as compared with proliferating cells.     

Identification of 'Non-Proliferating' B16 Melanoma Cells Using Monoclonal Antibody (Ad203) Against the M1 Subunit of Ribonucleotide Reductase

Abstract. Ribonucleotide reductase catalyses a critical reaction in DNA synthesis. Its M1 subunit is present during all proliferative phases of the cell cycle, but apparently not in the quiescent phase Go. We have used a monoclonal antibody (AD203) directed against the M1 subunit to distinguish immunocytochemically proliferating from non-proliferating cultured B16 mouse melanoma cells during exponential growth. the presumption that AD203 unstained cells constituted a non-proliferating fraction was tested by simultaneously counting the cells that failed to incorporate bromodeoxyuridine (BrdU) into DNA during a prolonged BrdU exposure. the proportion of cells which did not incorporate BrdU was found to correlate closely with the proportion not staining with AD203 and therefore presumably lacking the M1 subunit. the respective morphological features of AD203 stained and unstained cells were found not to differ significantly.     

Development of a structured model for batch cultures of lactic acid bacteria

A combined stochastic-deterministic model able to predict the growth curve of microorganisms, from inoculation to death, is presented. The proposed model is based on the assumption that microorganisms can experience two different physiological states: non-proliferating and proliferating. The former being the physiological state of the cells right after their inoculation into the new extracellular environment; the latter the state of microorganisms after adaptation to the new medium. To validate the model, a Lactobacillus bulgaricus strain was tested in a medium at pH 4.6 at two different temperatures (42°C and 35°C). Curves representing the bacterial growth cycle were satisfactorily fitted by means of the proposed model. Moreover, due to the mechanistic structure of the proposed model, valuable quantitative information on the following was obtained: rate of conversion of non-proliferating cells into proliferating cells, growth and death rate of proliferating cells, and rate of nutrient consumption.     

Cultured Human Tumour Cells May Be Arrested In All Stages of the Cycle During Stationary Phase: Demonstration of Quiescent Cells In G1, S and G2 Phase

Six human colon carcinoma cell lines were induced to enter stationary phase of growth by nutrient deprivation and cell crowding. Growth kinetics parameters (cell number, flow cytometric analysis of DNA distribution, and labelling and mitotic indices) were measured sequentially for all lines during the various stages of in vitro growth. Our results demonstrated that a substantial fraction of cells (9–18%) were located in G₂, phase when they changed from an exponential to a stationary mode of growth. Moreover, a large number of cells in stationary phase of growth had an S-phase DNA content, as determined by flow cytometry, but failed to incorporate radioactive DNA precursors (up to 15-fold difference). to substantiate these findings. cells in stationary phase of growth were induced to enter exponential growth by re-seeding in fresh medium at a lower density. Subsequently observed changes in DNA-compartment distribution, and in labelling and mitotic indices were those expected from cells that had been arrested at different stages of the cycle during their previous stationary phase. Thus, the non-proliferating quiescent state (Q), traditionally located ‘somewhere’ in G₁, phase, appears to be composed also of cells that can be arrested at other stages of the cycle (Q_s, and Q_G). Although the proportion of such cells is rather small, their contribution to the growth kinetics behaviour of human in vivo tumours will become apparent following ‘recruiting’ or ‘synchronizing’ clinical manoeuvres and will prevent the formation of a clear-cut wave of synchronized cells.     

Epidermal growth factor stimulates proliferation and DNA synthesis in ciliate cells

We studied the effect of murine epidermal growth factor on cell proliferation and DNA synthesis in macronuclei of ciliate Tetrahymena pyriformis G1. Mitogenic effect of epidermal growth factor on proliferation-induced tetrahymena cells has been revealed. This effect is due to the induced progression of cells at G1 and, consequently, their earlier entering DNA synthesis phase of the first cell cycle. Epidermal growth factor had no mitogenic effect on the resting cells from stationary culture (G0 phase) whose development is independent of the growth factors in the medium.     

KINETIC ANALYSIS OF CELL SIZE AND DNA CONTENT DISTRIBUTIONS DURING TUMOR CELL PROLIFERATION: EHRLICH ASCITES TUMOR STUDY

In order to study the growth dynamics of proliferating and non-proliferating cells utilizing discrete-time state equations, the cell cycle was divided into a finite number of age compartments. In analysing tumor growth, the kinetic parameters associated with a retardation in the growth rate of tumors were characterized by computer simulation in which the simulated results of the growth curve, the growth fraction, and the mean generation time were adjusted to fit the experimental data. The cell age distribution during the period of growth was obtained and by a linear transformation of the state transition matrices, was employed to specify the cell size and DNA content distributions. In an application of the model, the time-course behavior of cell cycle parameters of Ehrlich ascites tumor is illustrated, and the parameters important for the transition of cells in the proliferating compartment to the non-proliferating compartment are discussed, particularly in relation to the G₁-G₀ and G₂-G₀ transitions of non-cycling cells as revealed by the variation of cell size distribution.     

Correlation between histone phosphorylation and tumor ageing in Ehrlich ascites tumor cells

The histone fraction F1 can be divided into subfractions by gel electrophoresis. The microheterogeneity of F1 histone has been investigated in EAT cells in mice between 3 and 16 days after inoculation. The cell number per mouse increases during the first 8 days (proliferation phase); thereafter it remains constant (non-proliferating phase). We could demonstrate that the number of F1 subfractions is reduced from 5 in proliferating cells to 3 in non-proliferating ones. In short term experiments using [³²P]phosphate the label was only found in F1 histone from proliferating cells but not in that from resting cells. However, F2a1 histone, which is the other phosphorylated histone in interphase cells, was labelled in young and old cell populations. When ³²P-labelled F1 histone was treated with alkaline phosphatase not only was the label split off but also the number of subfractions was reduced from 5 to 3. These results lend additional evidence to the hypothesis that at least some of the F1 subfractions are derived from the same protein by phosphorylation.     

The modulation of serine metabolism in hepatoma 3924A during different phases of cellular proliferation in culture.

The activities of 3-phosphoglycerate dehydrogenase and serine hydroxymethyltransferase increased markedly during the transition of hepatoma cells from a resting non-proliferating culture into the proliferating growth phase. Activities declined as cells reached confluency and entered the plateau growth phase. This pattern was paralleled by changes in [14C]serine incorporation into nucleic acids. The experiments support the hypothesis that the biosynthesis of serine is metabolically coupled to its utilization for nucleotide precursor formation in cancer cells.     

Effect of antioxidants on the proliferation of human diploid cells at various phases of the culture growth

Some antioxidants (2-ethyl-6-methyl-3-hydroxypyridine chlorhydrate and some of its derivatives and 4-methyl-2,6-di-tert-butylphenol) have been shown to stimulate proliferation of young and old diploid cell at all phases of culture growth (lag and stationary phases). It is supposed that the mechanism of this effect may depend on stimulation of dreaming cell to division.     

Relation between the damaging effect of surface-active compounds on Escherichia coli cells and the phase of culture growth

The techniques of cell electrophoresis and electro-orientation spectroscopy were used to study the effect of sodium dodecyl sulfate (SDS) and cetyl trimethyl ammonium bromide (CTAB) on Escherichia coli K-12 cells from the culture at the exponential and stationary growth phases. SDS (2 x 10(-4) M) considerably damaged cells at the exponential phase, particularly at pH less than 6.0, whereas cells at the stationary phase were damaged to a less degree and only at pH less than 5.3 or after their treatment with Trilon B. The damaging effect of SDS decreased in an isotonic medium (0.25 M sucrose) as compared to a hypotonic medium (distilled water). CTAB also damaged cells at the exponential phase more than those at the stationary phase, and its damaging action decreased with pH. Mg2+, Ca2+, and Sr2+ cations diminished the degree of cell damage with CTAB, but did not exert any noticeable protection in the case of SDS. The different sensitivity of cells at the exponential and stationary growth phases may be associated with changes in their surface electric charge and with the existence of hydrophobic regions on the cell surface. The higher electric charge of cells at the stationary growth phase is presumed to stem from a rise in the amount of surface lipopolysaccharides which bear a negative electric charge.     

Distribution of acid hydrolases in subcellular fractions of proliferating vs non-proliferating fibroblasts

We have employed colloidal silica (Percoll) density-gradient subcellular fractionation technique to examine the distribution of lysosomal hydrolases between intermediate vesicles (primary lysosomes) and secondary lysosomes in contact-inhibited non-proliferating vs proliferating chicken embryo fibroblasts. We find that the activities of lysosomal specific enzymes from both phases of growth are distributed within two peaks; however, the relative amounts differ markedly. In normal, non-proliferating cells approx. 60% of the total activities of cathepsin B, beta-mannosidase, alpha-fucosidase, beta-galactosidase and hexosaminidase is recovered in the heavier density fraction corresponding to secondary lysosomes, while less than 9% of the enzyme activities are recovered in the light-density peak. With transformed cells, between 16 and 22% of activity for these enzymes are recovered in the lighter density intermediate vesicle fraction, when less than 40% of the enzyme activities recovered in the heavy density fraction. beta-Glucuronidase distribution was different from that of the above enzymes. First, a more even distribution between the two lysosomal fractions was found with non-proliferating normal cells (33% in heavy-density fraction and 21% in light-density fraction), whereas more than 40% of the total enzyme activity was recovered in the lighter density fraction from transformed cells. Also, the amount of cathepsin B contained in the vesicle fractions is increased severalfold relative to that of contact-inhibited normal cells. However, the apparent differences in enzyme distribution between confluent normal and transformed cells are not found when vesicles are prepared from subconfluent, actively proliferating cultures. We have also compared the Percoll density gradient patterns of membrane vesicles from proliferating and non-proliferating human fibroblasts, since most earlier studies utilized this system. Again, we find that the majority of beta-hexosaminidase activity (41%) of contact-inhibited, confluent cells is recovered in the heavier density fraction with less than 15% in the lighter density fraction. Also, the distribution of beta-hexosaminidase between the heavy density and light density vesicle fractions is altered in homogenates from exponentially growing cells, being 22% and 26% respectively. We conclude that the distribution of lysosomal hydrolases between the two vesicle populations is growth-phase dependent and is markedly heterogeneous in proliferating cells.     

Amiloride and glucose effects on the intracellular pH of Yoshida rat ascites hepatoma AH-130 cells grown in vivo

The equilibrium distribution of 5,5-dimethyloxazolidine-2,4-dione (DMO) between intra- and extracellular volume was used to estimate the intracellular pH in Yoshida rat ascites hepatoma AH-130 cells under different growth conditions (log, midlog and stationary). The cells were suspended in a Krebs-Ringer 25 mM phosphate buffer and the effects of variation of external pH, of glucose and amiloride addition on intracellular pH were measured. Proliferating cells had higher intracellular pH than stationary phase cells and this difference was inhibited by amiloride. On addition of glucose the fall in external pH was similar in all conditions and corresponded to lactate production. However, the intracellular pH decreased only in proliferating cells. Stationary phase cells showed an amiloride-sensitive cytoplasmic alkalinization with glucose. Glucose addition also caused prompt recovery to a normal polysomal pattern in these cells that might suggest increased efficiency of the initiation step of protein synthesis under these conditions. The data thus suggest that the increased intracellular pH of proliferating and of glucose-treated stationary phase cells is linked to the rate of protein synthesis and is mediated by the amiloride-sensitive Na+/H+ exchange system. This could lead to increased intracellular Na+ concentration under these conditions and to initiation of growth.     

Differential human interferon alpha receptor expression on proliferating and non-proliferating cells

The expression of interferon-alpha (IFN-alpha) receptors was studied on a variety of human cells, using monoiodinated IFN-alpha 2 probes. Steady-state binding at 4 degrees C revealed a single class of non-interacting IFN receptor on peripheral blood lymphocytes, and tonsillar B lymphocytes, which are both known to be G0/G1 resting cell populations. The binding affinity of this class of receptor was found to be on the order of 5 X 10(-10) M, expressed as an apparent dissociation constant (Kd). However, cells proliferating either in culture or in vivo were found to express a heterogeneity in IFN-alpha 2 binding. Such binding could be objectively resolved (by a version of the LIGAND program of P. Munson) into a two-site receptor model. Hill plots of binding to proliferating cells indicated a negative cooperativity in the interaction of IFN and receptor. The high-affinity component, expressed on proliferating cells, typically exhibits a Kd of (1-10) X 10(-11) M, while the lower-affinity component indicates a Kd of (1-10) X 10(-9) M. Furthermore, the low-affinity component is apparently expressed on the order of 10-200 times the copy number, per cell, of the high-affinity site. Affinity-labeling experiments revealed that, in addition to the 140-160-kDa IFN-binding complex reported by others, both the proliferating and non-proliferating cell populations possess a novel IFN-binding component of 60 kDa.