Successive evaluation of multiple parameters in individual cells: The SEMPIC photometric system

Summary An automated microphotometric system has been designed for the successive assessment of a maximum number of parameters from individual cells. The hardware and software elements are described as well as the various photometric functions that can be performed. The aim is to obtain a multiparameter analysis of morphology, cytochemistry, cell surface characteristics, and metabolic activity including cell cycle characteristics for any set of cells selected from a sample. The combination of DNA synthesis rate determination in a really quantitative approach with the analysis of features of nuclear chromatin texture opens up a new field for associating morphology with functional properties.Examples of application are presented to demonstrate the applicability of the system in a heterogeneous cell population of a very limited sample size. For this purpose, human peripheral blood cells grown in diffusion chambers in preirradiated mice were investigated. It is shown that, depending on their functions, different cell types may have quite different nuclear-to-cell area ratios. Further, a crude morphological parameter, such as the degree of basophilia of Giemsa-stained cells, may be associated with the rate of DNA synthesis, thus bearing information on the replicative activity of a cell. Cell surface properties related to the leukemia-associated cALL antigen are investigated in a human-derived cell line and correlated with cell-cycle characteristics. It is concluded that different antigenic sites rather than cell-cycle dependent differences of antigen density account for the outcome of a bimodal distribution.     

The cell cycle associated change of the Ki-67 reactive nuclear antigen expression

The change of human nuclear antigen expression in proliferating cells recognized by a monoclonal antibody, Ki-67, during the cell cycle was investigated in HeLa S3 cells using a bivariate-flow-cytometric analysis. The antigen was immunocytochemically stained with FITC, and DNA was stained with propidium iodide (Pl). The expression of the antigen increased with cell-cycle progression, especially in the latter half of S-phase and reached a maximum at G2M-phase, although its content varied greatly from cell to cell. The cells in which DNA synthesis was inhibited by treatment with hydroxyurea increased markedly in the antigen expression (as compared to untreated cells). Treatment with adriamycin also elevated the antigen content. After digestion with DNase I, but not after RNase treatment, FITC fluorescence from the antigen disappeared. These results suggest that the Ki-67 antigen is bound to DNA and its expression does not depend on DNA replication. Although the biological implications of the antigen remain unresolved, the antigen may be considered to be essential for maintaining the proliferating state of cells.     

The kinetics of simian virus 40-induced progression of quiescent cells into S phase depend on four independent functions of large T antigen.

Microinjection of purified simian virus 40 large-T-antigen protein or DNA encoding T antigen into serum-starved cells stimulates them to re-enter the cell cycle and progress through G1 into the S phase. Genetic analysis of T antigen indicated that neither its Rb/p107-binding activity nor its p53-binding activity is essential to induce DNA synthesis in CV1P cells. However, T antigens bearing missense mutations that inactivate either activity induced slower progression of the cells into the S phase than did wild-type T antigen. Inactivation of both activities resulted in a T antigen essentially unable to induce DNA synthesis. Missense mutations in either the DNA-binding region of the N terminus also impaired the ability of full-length T antigen to stimulate DNA synthesis in CV1P cells. The wild-type kinetics of cell cycle progression were restored by genetic complementation after coinjection of plasmid DNAs encoding different mutant T antigens or coinjection of purified mutant T-antigen proteins, suggesting that the four mitogenic functions of T antigen are independent. The maximal rate of induction of DNA synthesis in secondary primate cells and established rodent cell lines required the same four functions of T antigen. A model to explain how four independent activities could cooperate to stimulate cell cycle progression is presented.     

A new endogenous differentiating factor (myelopeptide-4) for myeloid cells

Along with known lymphokines involved in the regulation of hematopoiesis, a new differentiating factor (myelopeptide-4, MP-4) for myeloid cells was found. The peptide (Phe-Arg-Pro-Arg-Ile-Met-Thr-Pro) originally isolated from the culture medium of porcine bone marrow cell culture was examined for its ability to induce differentiation in two human myeloid leukemia cell lines, HL-60 and K-562. Agents with well-known differentiation-inducing activity, such as phorbol myristate acetate, dimethylsulfoxide and the lymphokines were used as a reference. It has been shown that MP-4 significantly influences the integral characteristics of metabolism, expression of surface antigens and morphology of these cells. It decreased the level of chromosomal DNA synthesis and, in parallel, increased the total protein synthesis in both HL-60 and K-562 cells. MP-4 induced the expression of CD14 monocyte-specific surface antigen and the appearance of mature monocytes/macrophages in HL-60 cell cultures. There was a good correlation of cell metabolic/morphological changes and the CD14 marker expression for HL-60 cells. A similar phenomenon was observed in K-562 cells treated with MP-4 when the levels of hemoglobin synthesis were detected in their cytoplasm. Thus, we consider MP-4 as a new endogenous differentiating factor for myeloid cells.     

Correlation between nuclear morphology and rate of deoxyribonucleic acid synthesis in a normal cell line.

Chinese hamster fibroblasts were investigated for the existence of correlations between proliferative activity and nuclear morphology. As a proliferative parameter, the rate of DNA synthesis of individual cells was determined by quantitative 14C-autoradiography. In a second step the images of the Feulgen-stained nuclei were digitized for extraction of features of morphology and texture. These features were correlated with the corresponding DNA synthesis rate values. The following relationships were found: Round nuclei have higher rates of DNA synthesis than flat ones. The more chromatin is packed at the nuclear rim, possibly representing heterochromatin, the lower the rate of DNA synthesis. The DNA synthesis rate also correlates with the graininess of chromatin. Larger areas of condensed chromatin are associated with lower rate values. A fine and irregular network of chromatin, as is typical of immature cell types, is associated with a high rate of DNA synthesis. Although these results are presently confined to the cell line investigated, parallels seem to exist to other cell types, such as erythropoietic cells, which await further investigation.     

Bovine Brain Endothelial Cells Express Tight Junctions and Monoamine Oxidase Activity in Long-Term Culture

The passage of substances across the blood-brain barrier is regulated by cerebral capillaries which possess certain distinctly different morphological and enzymatic properties compared to capillaries of other organs. Investigations of the functional characteristics of brain capillaries have been facilitated by the use of cultured brain endothelial cells, but in most studies a number of characteristics of the in vivo system are lost. To provide an in vitro system for studies of brain capillary functions, we developed a method of isolating and producing a large number of bovine brain capillary endothelial cells. These cells, absolutely free of pericyte contamination, are subcultured, at the split ratio of 1:20 (20-fold increase of the cultured surface), with no apparent changes in cell morphology up to the fiftieth generation (10 passages). Retention of endothelial-specific characteristics (factor VIII-related antigen, angiotensin-converting enzyme, and nonthrombogenic surface) is shown for brain capillary-derived endothelial cells up to passage 10, even after frozen storage at passage 3. Furthermore, we showed that bovine brain capillary endothelial cells retain, up to the fiftieth generation, some of the characteristics of the blood-brain barrier: occurrence of tight junctions, paucity of pinocytotic vesicles, and monoamine oxidase activity.     

Reversible G1 arrest of a human lung epithelial cell line by staurosporine.

Staurosporine, a microbial-derived protein kinase inhibitor, reversibly blocked non-synchronized, replicating cultures of the human lung epithelial cell line EKVX in the G1 phase of cell cycle and inhibited DNA synthesis and cell replication. The mechanism of this cell-cycle arrest in EKVX cells by staurosporine was likely due to inhibition of protein kinase C (PKC) because: 1) dose-dependent inhibition of DNA synthesis occurred at levels of staurosporine that inhibit phosphorylation of PKC substrate, 2) inhibition of DNA synthesis was also seen after treatment with another PKC inhibitor H7, but not by the chemically similar HA1004, which has a relative inhibitory specificity for cAMP-dependent protein kinase, and 3) the DNA synthesis was not inhibited by specific tyrosine kinase inhibitors Genistein and Lavendustin A at concentrations that inhibit tyrosine kinase activity. Removal of staurosporine from cell culture media resulted in a rebound in PKC activity and synchronized DNA synthesis in EKVX cultures. The reversibility of the inhibition was noted even after 5 days of treatment with staurosporine, and DNA synthesis remained synchronized for at least two rounds of cell replication after removal of staurosporine. Flow cytometric analysis confirmed that more than 90% of the cell population was blocked in the G1 phase after cells were treated with staurosporine for 24 h. Agents such as staurosporine may be useful for synchronizing cell populations to study cell-cycle specific biochemical events important for the regulation of cell replication in the EKVX cell line.     

Neurons are replenished in cultures of embryonic chick optic tectum after immunomagnetic depletion

The effect of intercellular interactions on the determination and differentiation of early embryonic brain cells was tested by immunomagnetic cell separation techniques. Using the A2B5 monoclonal antibody, which in chick brain reacts with a neuron-specific surface ganglioside, we produced initially pure populations of optic tectum cells devoid of the antigen. A coincident depletion of neurofilament(+) cells (95%) and nonneuronal growth characteristics of the separated A2B5(-) cells indicated that the vast majority of neurons had been removed initially. Surprisingly, A2B5(+) cells rapidly appeared in separated A2B5(-) cell cultures. After 1 day, the percentage of A2B5(+) cells in separated cell cultures equalled those in unseparated cultures (approximately 50%). By a week in culture, A2B5(+) cells developed neuronal morphology and contained neurofilaments. A2B5(-) to (+) conversion was a regulated phenomenon in that removal of different proportions of the (+) cells resulted in different numbers of (-) to (+) conversions. New DNA synthesis was not required for the acquisition of cell surface A2B5 antigen or for differentiation of cells into definitive A2B5(+) neurons. Our results demonstrate that postmitotic embryonic brain contains cells which are capable of replacing depleted neurons in vitro.     

DNA methyltransferase inhibition in normal human fibroblasts induces a p21-dependent cell cycle withdrawal

Maintenance of methylation patterns in the mammalian genome by DNA (cytosine-5) methyltransferases (DNAMeTase) is required for normal cell and tissue function. Inhibition of DNAMeTase in cultured cells induces the expression of p21, a cyclin-dependent kinase (Cdk) inhibitor critical for cells to enter replicative senescence. We investigated the effects of DNAMeTase inhibition in normal human fibroblasts and found that it induces an irreversible growth arrest. Cells arrested by DNAMeTase inhibition became enlarged and had a flat morphology, exhibited an increased expression of collagenase and p21, and the DNA synthesis block could be overcome by the introduction of the SV40 large T antigen, all characteristics of senescent cells. In contrast, normal human fibroblasts lacking a functional p21 gene fail to undergo cell cycle arrest following DNAMeTase inhibition, indicating that p21 is an essential component of this arrest. Furthermore, DNAMeTase activity was reduced as cells approached the end of their proliferative potential. These data suggest that DNAMeTase could be an integral part of the mechanisms by which cells count the number of cell divisions completed and initiate a signaling cascade that ultimately results in the senescent phenotype.     

Cell cycle: check for asynchrony

During metazoan development cells destined for different fates become asymmetric, not just in morphology and developmental potential but also in cell-cycle timing. A recent study has now shown that differential cell-cycle timing in the first cell divisions of the Caenorhabditis elegans embryo is in part controlled by a DNA replication checkpoint.     

Timed,sequential administration of paclitaxel improves its cytotoxic effectiveness in a cell culture model

Paclitaxel (taxol) is a chemotherapeutic agent frequently used in combination with other anti-neoplastic drugs. It is most effective during the M phase of the cell-cycle and tends to cause synchronization in malignant cells lines. In this study, we investigated whether timed, sequential treatment based on the cell-cycle characteristics could be exploited to enhance the cytotoxic effect of paclitaxel. We characterized the cell-cycle properties of a rapidly multiplying cell line (Sp2, mouse myeloma cells) by propidium-iodide DNA staining such as the lengths of various cell cycle phases and population duplication time. Based on this we designed a paclitaxel treatment protocol that comprised a primary and a secondary, timed treatment. We found that the first paclitaxel treatment synchronized the cells at the G2/M phase but releasing the block by stopping the treatment allowed a large number of cells to enter the next cell-cycle by a synchronized manner. The second treatment was most effective during the time when these cells approached the next G2/M phase and was least effective when it occurred after the peak time of this next G2/M phase. Moreover, we found that after mixing Sp2 cells with another, significantly slower multiplying cell type (Jurkat human T-cell leukemia) at an initial ratio of 1:1, the ratio of the two different cell types could be influenced by timed sequential paclitaxel treatment at will. Our results demonstrate that knowledge of the cell-cycle parameters of a specific malignant cell type could improve the effectivity of the chemotherapy. Implementing timed chemotherapeutic treatments could increase the cytotoxicity on the malignant cells but also decrease the side-effects since other, non-malignant cell types will have different cell-cycle characteristic and be out of synch during the treatment.     

Cell cycle-dependent dynamic association of cyclin/Cdk complexes with human DNA replication proteins

We have previously described the isolation of a replication competent (RC) complex from calf thymus, containing DNA polymerase alpha, DNA polymerase delta and replication factor C. Here, we describe the isolation of the RC complex from nuclear extracts of synchronized HeLa cells, which contains DNA replication proteins associated with cell-cycle regulation factors like cyclin A, cyclin B1, Cdk2 and Cdk1. In addition, it contains a kinase activity and DNA polymerase activities able to switch from a distributive to a processive mode of DNA synthesis, which is dependent on proliferating cell nuclear antigen. In vivo cross-linking of proteins to DNA in synchronized HeLa cells demonstrates the association of this complex to chromatin. We show a dynamic association of cyclins/Cdks with the RC complex during the cell cycle. Indeed, cyclin A and Cdk2 associated with the complex in S phase, and cyclin B1 and Cdk1 were present exclusively in G(2)/M phase, suggesting that the activity, as well the localization, of the RC complex might be regulated by specific cyclin/Cdk complexes.     

Measurement of proliferation nuclear and membrane markers in tumor cells by flow cytometry

Nuclear and membrane markers that have been related to proliferative activity were measured by flow cytometry. The markers studied were transferrin receptor (TR), Ki-67 antigen, and epidermal growth factor receptor (EGFR). Two-color analysis for DNA via propidium iodide binding and for antigen expression via either a direct or indirect immunofluorescence assay was performed on three different cell lines and a solid human tumor model. The three cell lines tested were MCF-7 (breast), K-562 (leukemia), and A431 (a squamous cell). The solid tumor was obtained by subcutaneous injection of A431 cells into an athymic nude mouse. Our results demonstrate that TR are cell-cycle specific and can be readily measured in the cell lines. Ki-67 antigen is also cell-cycle specific in the cell lines tested, but the mean channel specific fluorescence uptake varies in the cell types. Finally, the EGFR was observed only in the A431 cell line, with most cells equally expressing this receptor. A bimodal distribution of EGFR was observed in A431 cells obtained from a solid tumor grown in an athymic nude mouse system. This suggests that cell line analysis may not always represent what might be observed under in vivo conditions. There are advantages to flow cytometry measurements of these factors which might be useful in predicting how patients should be treated and possibly the prognosis of cancer patients.     

A mathematical model of the effects of hypoxia on the cell-cycle of normal and cancer cells

The evolution of the cell-cycle is known to be influenced by environmental conditions, including lack of extracellular oxygen (hypoxia). Notably, hypoxia appears to have different effects on normal and cancer cells. Whereas both experience hypoxia-induced arrest of the G1 phase of the cell-cycle (i.e. delay in the transition through the restriction point), experimental evidence suggests that only cancer cells undergo hypoxia-induced quiescence (i.e. the transition of the cell to a latent state in which most of the cell functions, including proliferation, are suspended). Here, we extend a model for the cell-cycle due to Tyson and Novak (J. Theor. Biol. 210 (2001) 249) to account for the action of the protein p27. This protein, whose expression is upregulated under hypoxia, inhibits the activation of the cyclin dependent kinases (CDKs), thus preventing DNA synthesis and delaying the normal progression through the cell-cycle. We use a combination of numerical and analytic techniques to study our model. We show that it reproduces many features of the response to hypoxia of normal and cancer cells, as well as generating experimentally testable predictions. For example our model predicts that cancer cells can undergo quiescence by increasing their levels of p27, whereas for normal cells p27 expression decreases when the cellular growth rate increases.     

The acquisition and maintenance of cytolytic activity by CD4+ murine T-lymphocyte clones.

The class II MHC antigen-specific CTL clones described in this report lose lytic activity when grown in exogenous rIL-2, but regain lytic activity when rIL-2 is removed from the culture medium. Using this cell model, we have investigated the metabolic activities (i.e., DNA, RNA, and protein synthesis) required for CTL to acquire or down-regulate lytic activity. DNA synthesis inhibitors (hydroxyurea and cytosine-arabinoside) and irradiation did not prevent CTL from gaining lytic activity. However, when protein or RNA synthesis was inhibited, these CTL could no longer acquire lytic activity. Furthermore, evidence showed that continuous RNA and protein syntheses were essential for CTL to exert their lytic function. Studies on cell surface antigen expression of CD3, CD4, Thy-1, and LFA-1 revealed no significant difference of antigen expression between a cloned CTL in its lytic and nonlytic states. Our data suggested that the synthesis of certain proteins and their encoded mRNA are essential for CTL to exert its lytic function and these proteins are not the cell surface antigens involved in CTL-target recognition or binding. Data also indicated that a granule enzyme, serine-esterase, was not involved in the expression of lytic activity in these CTL clones.     

Control of cell-cycle-associated tetrahydrobiopterin synthesis in rat thymocytes.

The cell-cycle progression of rat thymocytes from G0 through G1 to DNA synthesis is associated with a transient synthesis of H4biopterin, the concentration of which reaches a maximum at the time of S-phase entry and then decreases. This synthesis of H4biopterin is controlled by the specific activity of GTP cyclohydrolase I, which peaks in G1/S cells. In contrast, the catalytic activity of sepiapterin reductase remains constant throughout the cell-cycle. At G0 the steady state mRNA levels specific for GTP cyclohydrolase I and sepiapterin reductase, respectively, are below the limits of detection. Both accumulate as the thymocytes progress through the cell-cycle but lack cyclic down regulation. The data indicate that the variations in H4biopterin synthesis during the cell-cycle are caused by growth regulated increase in GTP cyclohydrolase I mRNA expression, with subsequent post-translational inactivation. This latter is likely due to the degree of enzyme phosphorylation.     

Cell-cycle regulators are involved in transient cerebral ischemia induced neuronal apoptosis in female rats

Recent evidence indicates that cell-cycle regulating proteins are involved in apoptotic process in post-mitotic neurons. In this study, we examined cell-cycle regulators for G1/S cell-cycle progression after a transient focal cerebral ischemia induced by middle cerebral artery (MCA) occlusion. In the cerebral frontoparietal cortex, we observed a marked induction of Cyclin D1 (a coactivator of Cdks), and proliferating cell nuclear antigen (PCNA), together with upregulated Cdk kinase activities. This process is accompanied with multiple phosphorylation of retinoblastoma (Rb) protein at Cdk phosphorylation sites in neurons from the ischemic cortex. We further examined DNA synthesis by the incorporation of BrdU, a nucleotide analog that incorporates into newly synthesized DNA. Within 24-h of reperfusion after 60-min occlusion, substantial BrdU-positive neurons were observed in the ischemic cortex. Inhibition of Cdk4 activity during this ischemia/reperfusion is highly neuroprotective. These results suggest that ischemia/reperfusion cerebral damage induces signalings at the G1/S cell-cycle transition, and may constitute a critical step in the neuronal apoptotic pathway in ischemia/reperfusion induced neuronal damage.     

Towards a further understanding of the growth-inhibiting action of oxygen deficiency. Evaluation of the effect of antimycin on proliferating Ehrlich ascites tumour cells

The effect of 1 microM antimycin on the proliferative properties, metabolism and basic cell composition of Ehrlich ascites tumour cells cultured in the second in vitro passage was studied. Continuous drug exposure of asynchronous cells caused rapid cessation of cell growth, characterized by the cell number and DNA, RNA and protein content of cultures. Cells cease to consume oxygen and enhance their glycolytic activity. Uptake of labelled thymidine into acid-insoluble material was far below that of the controls, whereas incorporation of labelled uridine exceeded that of controls, as was also observed with other inhibitors of the respiratory chain (sodium cyanide, 2-thenoyltrifluoroacetone, or anaerobiosis). The influence of antimycin on cells at different stages of the cell cycle was tested using cells enriched in either G1, S or G2 phase by centrifugal elutriation. DNA histograms (flow cytometry) and pulse-labelling index curves gave detailed insight into cell-cycle progression of antimycin-treated cells: G1 and early S cells remained stationary; G2 cells still passed from G2 into mitosis to remain subsequently in a non-growing state in G1; S cells were either slowed or halted. Supplementation of antimycin-containing cultures with exogenous pyrimidine nucleosides stimulated reprogression of G1 cells without changing their ATP content. The results of the current experiments are interpreted as supporting the concept that growth cessation of G1 cells under respiratory insufficiency is not predominantly caused by impairment of respiratory phosphorylation but may be the consequence of a lack of precursors for DNA and RNA synthesis.     

Differential ability of fixed antigen-presenting cells to stimulate nominal antigen-reactive and alloreactive T4 lymphocytes

The capacity of paraformaldehyde-fixed human antigen-presenting cells (APC) to induce responses by autologous, freshly isolated peripheral blood T4 cells was examined and was compared with their ability to stimulate allogeneic T4 cell DNA synthesis. Fixation of glass-adherent cells (AC) with as little as 0.06% paraformaldehyde abolished leucine incorporation, whereas fixation with 0.75% paraformaldehyde caused death of greater than 98% of the AC. Control APC were able to take up and present the soluble antigens streptokinase-streptodornase (SK-SD), tetanus toxoid, or tuberculin-purified protein derivative to autologous Ia-depleted T4 cells. Fixation with greater than 0.06% paraformaldehyde eliminated such ability. When AC were incubated with antigen overnight and were then fixed, however, they were able to present nominal antigen to autologous T4 cells in a genetically restricted manner that was blocked by monoclonal antibodies directed against monomorphic determinants on class II major histocompatibility complex (MHC) molecules. Despite the ability to present nominal antigen, paraformaldehyde-fixed AC were unable to induce allogeneic T4 cell proliferation. Similar results were observed when non-T cells or spleen cells were used as stimulators. The inability of fixed APC to stimulate allogeneic T4 cell DNA synthesis was not reversed by increasing the number of fixed APC or by the addition of control AC autologous to the responding cells. Moreover, interleukins 1 and 2 either alone or in combination also failed to permit maximal T cell proliferation in response to fixed allogeneic APC. The differential effects of fixation on nominal antigen and alloantigen presentation could not be explained by the loss of membrane thymocyte stimulatory activity on fixed AC. These results indicate that antigen-bearing fixed APC are competent to stimulate proliferation by antigen-reactive T4 cells, but are deficient at inducing allogeneic T4 cell DNA synthesis. The differential sensitivity of these two Ia-restricted functions of APC to chemical denaturation (reductive methylation) by paraformaldehyde suggests that the allodeterminants and restriction elements for nominal antigen on MHC class II molecules can be functionally dissociated.