A Detailed Study of the Complex Cell Cycle of the Dinoflagellate Crypthecodinium cohnii Biecheler and Evidence for Variation in Histone H1 Kinase Activity

ABSTRACT By adding the protein synthesis inhibitor, emetine (10^-4 M) to a highly synchronized population of Crypthecodinium cohnii Biecheler 1938 at different phases of its cycle, we were able to determine: 1. The existence and the lengthening of the G2-Phase (30 min) in the first cycle (cycle with swimming G1 phase). 2. The time of the second cell cycle phases (cycle in the cyst): G1, 30 min; S, 1.5 h; G2, 2 h and M, 2 h. These results, together with the estimation of the cell volume of the two and four swimming daughter cells emerging from the cysts, allowed us to state the existence of two transition points: G1/S and G2/M, which are necessary for completion of mitosis. We completed this refined approach of the cell cycle in studying the activities of the histone H1 kinase either in dividing or in non-dividing Crypthecodinium cohnii cells with either total soluble proteins or the isolated mitotic kinase complex. The H1 kinase activity of this purified complex is noticeably higher (twice as high) in the dividing cells than in the non-dividing ones. These data are discussed in the light of the basic characteristics of the dinokaryon, and also compared with recent biochemical observations on the same organism and studies on other higher eukaryotic protists and metazoa.     

RNA Accumulation in Relation to DNA and Protein Accumulation in Jerusalem Artichoke Callus Cultures

RNA accumulation during the synchronous early development ofJerusalem artichoke callus cultures follows a pattern of threestepwise increases in RNA per dividing cell during the firstdivision cycle. Little accumulation occurs in non-dividing cellsduring this time. These data are compared with data availablefor DNA replication, which occurs only in dividing cells, andfor protein accumulation which follows a similar pattern tothat of RNA accumulation in dividing cells, both in dividingcells and in some non-dividing cells.     

Cell cycle regulation by the pro-apoptotic gene Scotin

Scotin is a pro-apoptotic mammalian gene, which is induced upon DNA damage or cellular stress in a p53-dependent manner. In this report, we have used Drosophila as a model system to obtain a preliminary insight into the molecular mechanism of Scotin function, which was validated using the mammalian system. Targeted expression of Scotin in developing Drosophila induced apoptosis and developmental defects in wings and eyes. Co-expression of Scotin with the anti-apoptotic protein P35, while inhibited the apoptosis in both dividing and non-dividing cells, rescued adult wing or eye phenotypes only when Scotin was expressed in non-dividing cells. This suggests that mechanisms of Scotin-induced apoptosis in dividing and non-dividing cells may vary. Suppressor-enhancer screen using cell cycle regulators suggested that Scotin may mediate cell cycle arrest at both G1/S and G2/M phases. Over-expression of Scotin in mammalian cells resulted in mitotic arrest and subsequently apoptosis. Furthermore, a larger proportion of cells over-expressing Scotin showed sequestration of Cyclin B1 in the cytoplasm. These results suggest that one of the ways by which Scotin induces apoptosis is by causing cell-cycle arrest.     

A quantitative method for the analysis of mammalian cell proliferation in culture in terms of dividing and non-dividing cells

The application of the exponential growth equation is the standard method employed in the quantitative analyses of mammalian cell proliferation in culture. This method is based on the implicit assumption that, within a cell population under study, all division events give rise to daughter cells that always divide. When a cell population does not adhere to this assumption, use of the exponential growth equation leads to errors in the determination of both population doubling time and cell generation time. We have derived a more general growth equation that defines cell growth in terms of the dividing fraction of daughter cells. This equation can account for population growth kinetics that derive from the generation of both dividing and non-dividing cells. As such, it provides a sensitive method for detecting non-exponential division dynamics. In addition, this equation can be used to determine when it is appropriate to use the standard exponential growth equation for the estimation of doubling time and generation time.     

Prematurely condensed chromosomes of dividing and non-dividing cells in aging human cell cultures.

Chromosomes of dividing and non-dividing aging cells were examined by fusing senescent WI38 cells with mitotic HeLa cells to induce premature chromosome condensation (PCC). Exposure of the WI38 cells to ³H-thymidine 48 h prior to fusion allowed autoradiographic identification of cells that did not synthesize DNA (non-dividing cells). Ninety-six percent of the non-dividing cells, diploid or tetraploid, induced into PCC had single chromatids and were therefore blocked in the G1 phase of the cell cycle. Anomalous centromeric pairing of chromatids was noted in the remaining 4% of the non-dividing cells. Typical G2 configurations (double chromatids) were observed only among labeled (dividing) cells. The efficiency of PCC induction was independent of culture age. In addition, the efficiency of PCC induction was independent of phase in the cell cycle, as shown by comparison of observed frequencies with expected frequencies.     

Alternative mechanisms for homeostatic regulation of mammalian cell populations

A model for homeostatic regulation in mammalian tissues is analyzed. The model treats resting and active dividing cells, immature and mature non-dividing cells as separate populations. In the model, regulation is accomplished through control of the proportion of newly-formed cells that will become non-dividers. Four possible regulating substances, produced by dividing cells, non-dividing cells, mature non-dividing cells, and newly-formed cells respectively, are considered. Stability theorems are provided. System behavior in each instance depends on the relative values of the rate at which cells divide and the rate at which non-dividers die.     

Implications of a simple mathematical model to cancer cell population dynamics

Recent research in cancer progression and treatment indicates that many forms of cancer arise from the development of a small subpopulation of abnormal cancer stem cells (CSCs) that promote cancer growth and spread. Many potential treatments preferentially interact with cells at certain stages of the cell cycle by either selective killing or halting the cell cycle, such as intense, nanosecond-duration pulsed electric fields (nsPEFs). Simple mathematical models of unfed cancer cell populations at the plateau of their growth characteristics may estimate the long-term consequences of these treatments on proliferating and quiescent cell populations. Applying such a model with no transition from the quiescent to proliferating state shows that it is possible for the proliferating cell population to fall below 1 if the quiescent cell population obtains a sufficient competitive advantage with respect to nutrient consumption and/or survival rate. Introducing small, realistic transition rates did not appreciably alter short-term or long-term population behaviour, indicating that the predicted small cell population behaviour (< 1 cell) is not an artefact of the simpler model. Experimental observations of nsPEF-induced effects on the cell cycle suggest that such a model may serve as a first step in assessing the viability of a given cancer treatment in vitro prior to clinical application.     

The cell cycle,cell death,and cell morphology during retinoic acid-induced differentiation of embryonal carcinoma cells

Time-lapse films were made of PC13 embryonal carcinoma cells, synchronized by mitotic shake off, in the absence and presence of retinoic acid. Using a method based on the transition probability model, cell cycle parameters were determined during the first five generations following synchronization. In undifferentiated cells, cell cycle parameters remained identical for the first four generations, the generation time being 11–12 hr. In differentiating cells, with retinoic acid added at the beginning of the first cycle, the first two generations were the same as controls. The duration of the third generation, however, was increased to 15.7 hr while the fourth and fifth generation were approximately 20 hr, the same as in exponentially growing, fully differentiated cells. The increase in generation time of dividing cells was principally due to an increase in the length of S phase. Cell death induced by retinoic acid also occurred principally in the third and subsequent generations. Cell population growth was then significantly less than that expected from the generation time derived from cycle analysis of dividing cells. Cells lysed frequently as sister pairs suggesting susceptibility to retinoic acid toxicity determined in a generation prior to death. Morphological differentiation, as estimated by the area of substrate occupied by cells, was shown to begin in the second cell cycle after retinoic acid addition. These results demonstrate that as in the early mammalian embryo, differentiation of embryonal carcinoma cells to an endoderm-like cell is also accompanied by a decrease in growth rate but that this is preceded by acquisition of the morphology characteristic of the differentiated progeny.     

Proliferation/Quiescence: When to start? Where to stop? What to stock?

ABSTRACT: The cell cycle is a tightly controlled series of events that ultimately lead to cell division. The literature deciphering the molecular processes involved in regulating the consecutive cell cycle steps is colossal. By contrast, much less is known about non-dividing cellular states, even if they concern the vast majority of cells, from prokaryotes to multi-cellular organisms. Indeed, cells decide to enter the division cycle only if conditions are favourable. Otherwise they may enter quiescence, a reversible non-dividing cellular state. Recent studies in yeast have shed new light on the transition between proliferation and quiescence, re-questioning the notion of cell cycle commitment. They also indicate a predominant role for cellular metabolic status as a major regulator of quiescence establishment and exit. Additionally, a growing body of evidence indicates that environmental conditions, and notably the availability of various nutrients, by impinging on specific metabolic routes, directly regulate specific cellular re-organization that occurs upon proliferation/quiescence transitions.     

Mechanism of long slender (LS) to short stumpy (SS) transformation in the African trypanosomes

The transformation of the long slender to the short stumpy stages of the African trypanosomes is an essential part of the trypanosome life cycle. Four possible mechanisms which could control this event have been investigated. It has been shown that (a) the dividing long slender to non-dividing short stumpy transition is not a programmed event in the trypanosome life cycle; nor (b) would it appear to be initiated by some form of cell to cell contact inhibition of growth. In addition, evidence is presented which would suggest that (c) the transition is not started by the depletion of a critical growth nutrient from the environment during the growth of the trypanosomes. The last possibility (d) considered is that during trypanosome growth, a growth inhibitor-short stumpy inducer accumulates in the trypanosomes' environment. Evidence is presented which shows that plasma from infected animals can inhibit the incorporation of thymidine by the trypanosomes. These data are consistent with the suggestion of an exogenous growth inhibitor accumulating during the infection.     

Mechanism of Long Slender (LS) to Short Stumpy (SS) Transformation in the African Trypanosomes

The transformation of the long slender to the short stumpy stages of the African trypanosomes is an essential part of the trypanosome life cycle. Four possible mechanisms which could control this event have been investigated. It has been shown that (a) the dividing long slender to non-dividing short stumpy transition is not a programmed event in the trypanosome life cycle; nor (b) would it appear to be initiated by some form of cell to cell contact inhibition of growth. In addition, evidence is presented which would suggest that (c) the transition is not started by the depletion of a critical growth nutrient from the environment during the growth of the trypanosomes. The last possibility (d) considered is that during trypanosome growth, a growth inhibitor-short stumpy inducer accumulates in the trypanosomes'environment. Evidence is presented which shows that plasma from infected animals can inhibit the incorporation of thymidine by the trypanosomes. These data are consistent with the suggestion of an exogenous growth inhibitor accumulating during the infection.     

Mathematical description and analysis of cell cycle kinetics and the application to Ehrlich ascites tumor

The linear and nonlinear aspects of the dynamics of the cell cycle kinetics of cell populations are studied. The dynamics are represented by difference equations. The characteristics of cell population systems are analyzed by applying the model to Ehrlich ascites tumor. The model applied for the simulations of the growth of Ehrlich ascites tumor cells incorporates processes of cell division, cell death, transition of cells to resting states and clearance of dead cells. Comparison of the results obtained with the model and the experimental data suggests that the duration of the mean generation time of the proliferating EAT cells increases with aging of the tumor. An attempt is made to relate the prolongation of cell mean generation time with processes of cell death and dead cell clearance. Studying the transition of cells to the resting states, it becomes apparent that in fact transition of proliferating cells to the resting states occurs somewhere close to the end of the cell cycle and with a rate that varies with the age of the tumor. Time course behavior of the cell age, cell size, and cell DNA distribution with aging of the tumor are obtained. Variations in average size and average DNA contents are determined.     

Photosynthesis and carbon metabolism in photoautotrophic cell suspensions of Chenopodium rubrum from different phases of batch growth

Rapidly dividing photoautotrophic cell suspensions from Chenopodium rubrum L. assimilated about 85 μmol CO₂ (mg chlorophyll)⁻¹ h⁻¹. During the late stationary phase of culture growth, CO₂ fixation rate was reduced to about 60 μmol CO₂ (mg chlorophyll)⁻¹ h⁻¹. Actively dividing cells characteristically incorporated a smaller proportion of ¹⁴C into starch than cells from non-dividing stationary phases. In rapidly dividing cells, [¹⁴C]-turnover from free sugars, sugar-phosphates, organic and amino acids was substantially higher compared to non-dividing cells from stationary growth phase. Higher proportions of photosynthetically fixed carbon were channelled into proteins, lipids and structural components in actively dividing cells than in non-dividing cells. In the latter. ¹⁴C was preferentially channeled into starch, and a striking increase in starch accumulation was observed. The transfer of non-dividing, stationary growth-phase cells into fresh culture medium resulted in an increase in the maximum extractable activities of some enzymes involved in the glycolytic and dark respiratory pathways and in the citric acid cycle. In contrast, the maximum extractable activities of the chloroplastic enzymes, ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.38) and NADP⁺-glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) were highest after the cells had reached the stationary growth phase.     

Modelling and analysis of time-lags in some basic patterns of cell proliferation

 In this paper, we present a systematic approach for obtaining qualitatively and quantitatively correct mathematical models of some biological phenomena with time-lags. Features of our approach are the development of a hierarchy of related models and the estimation of parameter values, along with their non-linear biases and standard deviations, for sets of experimental data. We demonstrate our method of solving parameter estimation problems for neutral delay differential equations by analyzing some models of cell growth that incorporate a time-lag in the cell division phase. We show that these models are more consistent with certain reported data than the classic exponential growth model. Although the exponential growth model provides estimates of some of the growth characteristics, such as the population-doubling time, the time-lag growth models can additionally provide estimates of: (i) the fraction of cells that are dividing, (ii) the rate of commitment of cells to cell division, (iii) the initial distribution of cells in the cell cycle, and (iv) the degree of synchronization of cells in the (initial) cell population. Received: 15 September 1997/Revised version: 1 April 1998     

DURATION OF CELL CYCLES IN CULTURED ROOTS OF CONVOLVULUS

The durations of the cell cycle in physiologically different regions of the meristem of cultured roots of Convolvulus arvensis were determined by the metaphase-accumulation technique involving colchicine. The cell cycle in the root cap increases from 13 hr in the actively dividing initials of the first tier to 155 hr in the slowly dividing initials of tiers 2–4 to an indeterminate value for derivatives of the initials in the root cap columella. The cycle times for the cells of the central cylinder and cortex are 21 and 27 hr, respectively. The cells of the quiescent center have a cycle of an estimated 420 hr. The duration of the cell cycle in these different regions is discussed in relation to the increased duration of G₁ in slowly or non-dividing cells. The possible regulation of cell division by the synthesis of a cell-division factor in the quiescent center is also discussed.     

Regulation of cell division and malignant transformation

The problem of regulation of cell division is essentially a problem of understanding regulation of transition from the resting state of a cell to the dividing state and vice versa. In malignancy the ability to revert back to a normal resting state is impaired. A model is presented which attempts to explain the control of the above transitions through control of uptake of essential nutrients by a transport-inhibitory protein. Experimental evidence in favour of the model is given.     

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.     

Biochemical and enzymic changes during erythrocyte differentiation. The significance of the final cell division.

1. The haemoglobin content of developing erythroblasts was shown to increase rapidly when the cells completed the final cell division of erythroid development and passed from the dividing into the non-dividing cell compartment. 2. The activity of carbonic anhydrase was measured and shown to increase continually throughout erythroid differentiation. The activity increased most rapidly in the polychromatic stage. 3. Catalase activity did not increase significantly during erythroid differentiation until the reticulocyte stage. 4. The activity of four enzymes, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, adenosine deaminase and nucleoside phosphorylase, exhibited a similar pattern of change during erythroid differentiation. In the dividing cell compartment their activity was relatively high but exhibited a steep decline between the polychromatic stage and the orthochromatic stage, that is, as the cell completed its final cell division and moved from the dividing to the non-dividing compartment. After this the activity of these enzymes was stabilized at a relatively low value, and this activity persisted at such a value until the reticulocyte stage. 5. Lactate dehydrogenase activity also declined after the cell had crossed from the dividing into the non-dividing stage, but in this case the decline was less than in the case of the above four enzymes. 6. Adenylate kinase activity was relatively constant in the dividing cell compartment but exhibited a 60 percent increase when the cell passed from the dividing into the non-dividing compartment. 7. The cessation of cell division appears to coincide with a set of complex biochemical changes.