Prolongation of cell cycle transit time and the presence of non-cycling cells in human lymphoblastoid cells cultured under adverse conditions

Abstract. The growth characteristics of B lymphocytes infected with Epstein-Barr virus (lymphoblastoid cells) have been investigated by flow cytometric analysis of DNA content and by estimation of cell culture doubling times. It was found that the manipulative procedures involved in the cell cycle analysis resulted in a slowing of the growth rate. This slowing of growth was brought about by the prolongation of cell cycle transit times and by the entry of cells into a short-lived non-cycling pool. The entry of a proportion of the cells into the non-cycling pool may be the normal response of lymphoblastoid cells to non-optimal conditions. The non-cycling cells survived in culture with a T _1/2 of approximately 30–60 hr and continued to secrete immunoglobulin. Their surface transferrin receptors were considerably reduced, which suggests that the failure to divide may have resulted from a failure of growth factor receptors to reach a threshold value following mitosis.     

Induction of proliferation in vitro of resting human natural killer cells: IL 2 induces into cell cycle most peripheral blood NK cells, but only a minor subset of low density T cells

We report that resting human peripheral blood natural killer (NK) cells proliferate in response to recombinant interleukin 2 (rIL 2), and addition of irradiated lymphoblastoid B cells significantly increase their proliferative response. Interaction of IL 2 with the Tac IL 2 receptor expressed on activated NK cells is necessary to maintain continued growth of these cells. Experiments in which NK cell mitosis is prevented by colchicine show that the majority of peripheral blood NK cells are induced into the first cell cycle over a 6-day culture period in the presence of rIL 2. The addition of the irradiated lymphoblastoid B cell line, Daudi, to colchicine blocked cultures does not increase the proportion of cells entering cell cycle in response to rIL 2 alone. In limiting dilution analysis, only 1/1700 B73.1+ cells grow clonally in response to rIL 2. The frequency of clonal growth of NK cells in response to irradiated Daudi cells alone is minimal, whereas the addition of irradiated Daudi cells to rIL 2 stimulated cultures resulted in a 10-fold increase in clonal frequency compared with the cultures in rIL 2 alone. Therefore, Daudi cells may act by maintaining continuous proliferation of the NK cells originally responsive to IL 2. Unlike NK cells, only a minimal proportion of peripheral blood T cells proliferate in response to IL 2. These IL 2 responsive T cells are characterized by a lower bouyant density than the majority of peripheral blood T cells. These results indicate physiologic differences between peripheral blood resting NK and T cells in their ability to be induced to cycle. IL 2 is a growth factor for both cell types, but although the presence of the growth factor is sufficient for quiescent NK cells to be induced into cycle, T cells require antigenic or other mitogenic stimuli to respond to IL 2. The small proportion of light density IL 2 responsive T cells might represent in vivo activated T cells.     

The need for dynamic methods for measuring cell cycle perturbations: a study in radiation-treated lymphoblastoid cell lines of varying p53 status

Reports on the p53-related cell cycle and apoptotic responses of EBV-transformed lymphoblastoid cell lines to DNA damage have led to some confusion. This may be due to differences in the nature of the specific p53 mutations under examination, but it can also be partly attributed to methodological and analytical problems (e.g. the inappropriate use of static DNA histograms for cell cycle analysis). Taking seven lymphoblastoid cell lines derived from both normal individuals and Li-Fraumeni Syndrome/Li-Fraumeni-Like (LFS/LFL) patients of differing p53 status, we completed a detailed study of radiation-induced cell cycle perturbations. Using BrdUrd pulse labelling and flow cytometry it was found that, regardless of p53 status, the cells did not arrest in G₁ despite all of the lines showing p53 upregulation 3 hours postirradiation. The irradiated cells did, however, show a general slowing both in S-phase entry from G₁ and in movement through S-phase. These facts would not have been apparent from the analysis of static DNA histograms. The problems with the use of static methods to assess changes in the dynamics of cell cycle progression apply not only to studies involving EBV-transformed cell lines, but also to a wide range of investigations into the molecular control of cell proliferation.     

Sensitivity of flow cytometric data to variations in cell cycle parameters

Abstract We investigated to what extent flow cytometric DNA histograms are informative of cell cycle parameters. We created a computer program to simulate cell cycle progression in a generic and flexible way. Various scenarios, characterized by different models and distributions of cell cycle phase transit times, have been analysed in order to obtain the percentages of cells in the different cell cycle phases during exponential growth and their time course after mitotic block.
Cell percentages during exponential growth were insensitive to intercell variability in phase transit times and thus can be employed to estimate the relative mean phase transit times, even in the presence of non-cycling cells. However, this information is ambiguous if re-entry of such cells into the cycling status is permitted. The stathmokinetic outline gives the mean phase transit times, but also provides information about the spread, but not the form, of the phase transit time distributions, being particularly sensitive to the spread of G1 phase duration. The stathmokinetic outline also helps distinguish between scenarios considering only cycling cells, those forecasting a fraction of definitively non-cycling cells and those admitting a Go status with first-order output kinetics.     

Sensitivity of flow cytometric data to variations in cell cycle parameters

We investigated to what extent flow cytometric DNA histograms are informative of cell cycle parameters. We created a computer program to simulate cell cycle progression in a generic and flexible way. Various scenarios, characterized by different models and distributions of cell cycle phase transit times, have been analysed in order to obtain the percentages of cells in the different cell cycle phases during exponential growth and their time course after mitotic block. Cell percentages during exponential growth were insensitive to intercell variability in phase transit times and thus can be employed to estimate the relative mean phase transit times, even in the presence of non-cycling cells. However, this information is ambiguous if re-entry of such cells into the cycling status is permitted. The stathmokinetic outline gives the mean phase transit times, but also provides information about the spread, but not the form, of the phase transit time distributions, being particularly sensitive to the spread of G1 phase duration. The stathmokinetic outline also helps distinguish between scenarios considering only cycling cells, those forecasting a fraction of definitively non-cycling cells and those admitting a G0 status with first-order output kinetics.     

Fibronectin promotes cell cycle entry in smooth muscle cells in primary culture

Smooth muscle cell proliferation after arterial injury is regulated by growth factors and components of the extracellular matrix. We have previously demonstrated that fibronectin promotes a phenotypic modulation of freshly isolated rat smooth muscle cells from a contractile to a synthetic phenotype in primary culture and supports the ability of the cells to respond to growth factors. Here, we analyzed if fibronectin promotes cell cycle entry in freshly isolated rat aortic smooth muscle cells during primary culture. Cell cycle analysis showed that cells seeded on fibronectin remained in the G(0)/G(1) phase of the cell cycle during the first 6 days of culture. During this period, there was an increased expression of cyclin D1 and p27(KIP1) in the absence of exogenous growth factors. Addition of serum was followed by enhanced cyclin D1 expression, decreased p27(KIP1) levels, hyperphosphorylation of Rb protein, induction of cyclin A and cyclin D3 expression, and cell cycle progression into S phase. The results indicate that fibronectin initiates cell cycle entry in freshly isolated smooth muscle cells by promoting the induction of cyclin D1 and thereby facilitates further cell cycle progression together with growth factors.     

Regulation of K562 cell transferrin receptors by exogenous iron

Single-cell analysis of K562 human erythroleukemia cells by flow cytometry was used to demonstrate the specific role of iron in regulating transferrin receptors (TfRs) and to establish that TfR expression does not necessarily correlate with growth rate. Exogenous iron concentration in culture was manipulated by supplementing the medium with sera having different iron concentrations over the range 0.6 to 5.4 micrograms/ml, by the addition of iron in the form of FeCl3, iron-saturated serum, or diferric transferrin, and by the addition of the iron chelator Desferal (desferrioxamine). TfR expression was negatively correlated with exogenous iron content: any treatment that reduced exogenous iron supply by at least 15% resulted in as much as a 1.8-fold increase in external receptors, detected as binding by both transferrin and monoclonal anti-TfR antibodies, and a 1.5-fold increase in the pool of internal receptors, as detected by anti-TfR antibody binding. None of these treatments altered growth rate, total cellular protein content, protein synthetic rate, cell cycle distribution or cell size. The rapid (12 hr) and reversible induction of internal and external receptors by Desferal was inhibited by cycloheximide and therefore may have resulted from de novo synthesis and not just mobilization of internal receptor pool to the cell surface. The correlation between growth rate and TfR expression previously observed in these and other cells must be secondary to cellular mechanisms that maintain intracellular iron pools by regulating synthesis, recycling, and cell surface expression of TfRs.     

Cell cycle effects of an autologous growth promoter from human leukemia cells

The mechanism of action of an autologous growth promoter, produced by HL-60 cells grown in serum-free defined medium, was investigated. Determinations of cell cycle phase distribution, which showed an approx. 16% increase in S and G2M with a reciprocal decrease in G1 cells, indicated a mechanism causing decreased time in G1, resulting in a more rapid entry of cells into G2M. Adsorption of the culture supernatant with HL-60 cells resulted in a decreased promoter activity, suggesting an autologous receptor mechanism for the promoter.     

Inhibition of transferrin receptor expression by interferon-alpha in human lymphoblastoid cells and mitogen-induced lymphocytes

125I-Transferrin binding to lymphoblastoid K562 and Daudi cells markedly increased after exposure of the cells to culture conditions that stimulated proliferation. Treatment of these cells with interferon-alpha (IFN-alpha) resulted in concurrent inhibition of cell growth and of the rise in transferrin binding. Scatchard analyses revealed that IFN reduced the number of transferrin receptors without altering the binding constant. When 125I-transferrin binding was measured using permeabilized cells, the IFN-induced reduction of binding was comparable to that observed with intact cells, indicating that IFN diminished the total number of cellular transferrin receptors. We also found that addition of IFN-alpha to phytohemagglutinin-stimulated human lymphocytes inhibited the mitogen-induced enhancement of [3H]thymidine incorporation as well as surface binding of 125I-transferrin. Our findings suggest that the decrease in transferrin receptor expression on IFN-alpha-treated cells may be one of the mechanisms responsible for the antiproliferative action of IFN.     

Proliferative growth of neonatal cerebellar cells in culture: Regulation by male and by maternal serum in late gestation

Neonatal cerebellar cells were utilized as a model system to examine the effect of 20 day pregnant rat serum on proliferative growth in the CNS. Cells were prepared by mechanical dissociation and cultured as mixed cells or populations enriched in astrocytes or oligodendrocytes. Cell proliferation was estimated by measurement of DNA, protein, and/or mitochondrial reductase activity (MTT). When mixed cells were incubated with 10% male rat serum, both total DNA and protein content increased after 6 days of culture. By contrast, neither of these parameters were altered in cultures incubated with 10% pregnant serum. When cells were incubated with either male or pregnant sera, changes in MTT activity paralleled changes in protein content. Graded concentrations of pregnant serum (5–20%) added to mixed cell cultures produced consistently lower MTT values when compared with identical concentrations of male serum. In addition, MTT activity was diminished in both astrocytes and oligodendrocytes incubated with graded concentrations of pregnant sera when compared with similar concentrations of non-pregnant sera. When potential effects of these different sera on the cell cycle were examined, an increase in the number of cells in the S and G2/M phase was similar, and DNA doubling began to increase at 96 hrs in the presence of either male or 20 day pregnant sera. Thus the inhibition of cell growth by pregnant serum was not likely a result of either cytotoxicity or a delay of entry of cells into the cell cycle. To examine whether this inhibition of cell growth may reflect the effect of pregnant serum on endogenous growth factor production, we tested the production of IGF-II by cerebellar cells. Production of an endogenous source of IGF-II was apparent using an RNAse protection assay and was noted using Slot Blot analysis of mRNA extracted at sequential times during cell incubation. Mixed cell cultures also secreted immunologically defined IGF-II. These observations are consistent with the previous demonstration that the fraction of pregnant serum which bound IGF-II also inhibited cell growth. The inhibitory effect of pregnant serum was diminished by preincubating aliquots of sera with graded concentrations of IGF-I prior to adding sera to tissue culture medium. Pregnant serum inhibition was also diminished by prolonging incubation times beyond 6 days. The blunting of pregnant serum inhibition may have been consequent to either a continuing production of endogenous growth factors or to the potential emergence of resistant cells due to prolonged tissue culture incubation. Since cells studied in a primary culture of limited duration may more accurately reflect the physiologic properties of this tissue, the model presented herein could provide a new approach to study brain development.West Side Medical CenterNorthwestern University medical School     

Growth kinetics of the granulosa cell population in ovarian follicles: an approach by mathematical modelling

This paper describes, from a mathematical viewpoint, the cellular changes in the granulosa of ovarian follicles during their terminal development. A dynamic model takes into account the processes of (1) cell division, (2) exit from the cell cycle towards differentiation, and (3) apoptotic cell death. Proliferative cells leave the cycle in an irreversible way. The risk of entering apoptosis applies to non-cycling cells. Changes in the cell numbers and in the growth fraction are derived from differential equations. The transitions between the different cell states are ruled by time-dependent rates. Numerical applications of the model concern ovulating and degenerating ovarian follicles in the ewe. The main feature of the ovulating case is the progressive exhaustion of the proliferating compartment for the benefit of the non-cycling cells. From an initial mainly proliferative state the granulosa progressively switches to a highly differentiated state, so that the growth fraction continuously decreases. In the atretic cases, the pattern of changes in the total viable cell number is influenced by the follicular age at the onset of the apoptotic process and by the intensity of the cell death rate. As apoptosis affects the non-cycling cells, the growth fraction is no longer strictly decreasing. The sensitivity of the model to the parameters is studied in a more general framework than the granulosa cell population.     

Quantification of entry into and exit from the cell cycle in human lymphocyte cultures

A mathematical model is presented for the analysis of transition between cycling and non-cycling compartments by cells responding to a growth stimulus. The cellular age distribution as a function of time is derived from sequential [3H]thymidine pulse labeling indices. Rates of entry into and exit from the cycling compartment are determined on the basis of labeling indices obtained after instantaneous and long duration [3H]thymidine pulses. Analysis of an experiment involving sequential measurements over the whole lifespan of a human lymphocyte culture stimulated by phytohemagglutinin is presented as an example of the application of this method.     

Enhanced growth-dependent expression of TGF beta 1 and hsp70 genes in aortic smooth muscle cells from spontaneously hypertensive rats.

Enhanced proliferation of vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) as compared with Wistar-Kyoto rats (WKY) persists in long-term culture and is characterized by an accelerated entry of these cells into the synthetic S phase of the cell cycle and a higher specific growth rate, particularly evident at high cell density. In the present study, we investigated by Northern blot experiments the expression of genes putatively involved in the regulation of VSMC growth. One of them is the transforming growth factor beta 1 gene (TGF beta 1), a bifunctional modulator of cell growth whose action is dependent on cell density. The accumulation of TGF beta 1 mRNA was enhanced in growing SHR cells at every density studied as early as 24 h after inoculation with a further increase at later times. Protooncogenes c-fos and c-myc, which have been implicated in G1/S phase transition, have also been investigated in VSMC by Northern blot analysis. At low cell density, calf serum stimulated c-fos and c-myc mRNA expression was comparable in WKY and SHR cells whereas at high cell density, c-fos induction was higher in VSMC from SHR. SHR VSMC respond more to mitogenic stimulation and to environmental (e.g., heat) stress, particularly when growing near saturation density. hsp70 constitutes a gene family responsive to environmental stimuli (heat) and to mitogenic stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of Phosphatidylinositol 3-Kinase Is Sufficient for Cell Cycle Entry and Promotes Cellular Changes Characteristic of Oncogenic Transformation

Using a new inducible form of phosphatidylinositol 3-kinase (PI 3-kinase) we have found that PI 3-kinase activation has the following effects on cell growth and proliferation. (i) Activation of PI 3-kinase was sufficient to promote entry into S phase of the cell cycle within several hours. This was shown by activation of cyclin-dependent kinase 4 (Cdk4) and Cdk2 and by the induction of DNA synthesis. (ii) PI 3-kinase activation alone was not, however, sufficient to provide for progression through the entire cell cycle. Instead, prolonged activation of PI 3-kinase in the absence of serum stimulation resulted in apoptosis. It is possible that the cells undergo apoptosis because the PI 3-kinase-induced entry into the cell cycle is abnormal. For example, we found that the cyclin E-Cdk2 complex, which normally disappears after entry into S phase of the cell cycle, fails to be downregulated following induction by PI 3-kinase. (iii) Finally, we found that prolonged activation of PI 3-kinase in the presence of serum resulted in cellular changes that resemble those associated with oncogenic transformation. The cells reached high densities, were irregular and refractile in appearance, and formed colonies in soft agar. In contrast, neither PI 3-kinase nor serum stimulation alone could induce these changes. Our results suggest that activation of PI 3-kinase promotes anchorage-independent cell growth and entry into the cell cycle but does not abrogate the growth factor requirement for cell proliferation.     

Impaired induction of CD27 and CD28 predicts naive CD4 T cell proliferation defects in HIV disease

Many immunological defects have been described in HIV disease, including a diminished capacity of naive CD4+ T cells to expand after TCR stimulation. The mechanisms underlying impaired naive CD4+ T cell expansion in HIV disease are not well described. Using a rigorous phenotypic definition of naive T cells, we found that cell cycle entry after TCR engagement was restricted to cells that increased surface expression of costimulatory molecules CD27 and CD28. Induction of these receptors, however, was not sufficient to result in cell cycle entry among the CD4+CD31- naive T cell subset. Analyses of cells from HIV-infected persons indicated that naive CD4+CD31+ T cells from these subjects were impaired in their ability to enter the cell cycle after stimulation and this impairment was predicted by the relatively poor induction of costimulatory molecules on these cells. Thus, failure to increase surface expression of costimulatory molecules may contribute to the naive T cell expansion failure that characterizes HIV infection.     

Cdh1-APC/C, cyclin B-Cdc2, and Alzheimer's disease pathology

The anaphase-promoting complex/cyclosome (APC/C) is a key E3 ubiquitin ligase complex that functions in regulating cell cycle transitions in proliferating cells and has, as revealed recently, novel roles in postmitotic neurons. Regulated by its activator Cdh1 (or Hct1), whose level is high in postmitotic neurons, APC/C seems to have multiple functions at different cellular locations, modulating diverse processes such as synaptic development and axonal growth. These processes do not, however, appear to be directly connected to cell cycle regulation. It is now shown that Cdh1-APC/C activity may also have a basic role in suppressing cyclin B levels, thus preventing terminally differentiated neurons from aberrantly re-entering the cell cycle. The result of an aberrant cyclin B-induced S-phase entry, at least for some of these neurons, would be death via apoptosis. Cdh1 thus play an active role in maintaining the terminally differentiated, non-cycling state of postmitotic neurons--a function that could become impaired in Alzheimer's and other neurodegenerative diseases.     

Single-cell analysis of the relationship among transferrin receptors, proliferation, and cell cycle phase in K562 cells

Multiparameter single-cell analysis by flow cytometry was used to distinguish between size-related changes in K562 cell transferrin receptor (TfR) expression and changes in membrane receptor density throughout the cell cycle and over time in culture. Light-scatter pulse-width time-of-flight, a direct and readily calibrated measure of cell diameter, was used to calculate receptor density as the average number of receptors per unit cell surface area. Cell surface TfRs were unimodally distributed over the cell population and were present throughout the cell cycle. The number of receptors increased as cells progressed through the cell cycle, but cell cycle phase was also correlated with cell volume. However, when size heterogeneity was factored out by reanalysis of listmode data, there was a clear cell-cycle effect: among cells of the same size, both the number of receptors per cell and the receptor density increased from G1 to S to G2/M. TfR expression was also followed over time in culture after dilution into fresh medium. A decrease in growth rate after four days was preceded by one to two days by a decrease in both number of TfRs per cell and mean receptor density, indicating that decreased TfR expression represented true "down-regulation" and not just decreased cell size or an increase in the proportion of smaller G1 cells. This type of analysis is generally applicable for resolving the effects of cell size heterogeneity and cell cycle on membrane protein distribution and for other studies of ligand-receptor interaction.     

Effect of serum step-down on protein metabolism and proliferation kinetics of NHIK 3025 cells

Human NHIK 3025 cells growing exponentially in 30% or 3% serum had population doubling times of 19.1 and 27.6 hours, respectively. These values were equal to the calculated protein doubling times (17.6 and 26.5 hours, respectively), showing that the cells were in balanced growth at both serum concentrations. Stepdown from 30% to 3% serum reduced the rate of protein synthesis within 1–2 hours, from 5.7% hour to 4.3% hour, while the rate of protein degradation was unchanged (1.7%/hour). In cells synchronized by mitotic selection from an exponentially growing population, the median cell cycle durations in 30% and 3% serum were 17.2 and 23.6 hours, respectively, which were also in good agreement with the protein doubling times. The median G₁ durations were 7.1 and 9.6 hours, respectively. Thus the duration of G₁ relative to the total cell cycle duration was the same in the two cases. Complete removal of serum for a period of 3 hours resulted in a 3-hour prolongation of the cell cycle regardless of the time after mitotic selection at which the serum was removed. For synchronized cells, the rate of entry into both the S phase and into the subsequent cell cycle were reduced in 3% serum as compared to 30% serum, the former rate being significantly greater than the latter at both serum concentrations. Our results thus indicate that these cells are continuously dependent upon serum throughout the entire cell cycle.     

On the existence of non-cycling germinative cells in human epidermis in vivo and cell cycle aspects of psoriasis

Abstract. This report deals with the controversies of whether all germinative epidermal cells in human epidermis are in the cycling state and whether stimulated hyperproliferation of psoriatic epidermis is due to a shortening of the cell cycle time or to a recruitment of non-cycling germinative epidermal cells. Experiments were performed on human subjects in vivo . Continuous infusion of [³H]thymidine for 8½ days indicated that 40% of germinative epidermal cells reside in the non-cycling state. Proliferative stimulation by tape stripping indicated recruitment of non-cycling (G₀) germinative epidermal cells in both normal and psoriatic skin, and a prolongation (rather than a shortening) of cell cycle traverse in activated psoriatic epidermal cells.