Flow cytometry of erythropoiesis in culture: bivariate profiles of fetal and adult hemoglobin.
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Antimetabolites↗
Biomedical subjects
R M Böhmer
Publications and source records attributed to R M Böhmer.
Regulation of p21(cip1) expression by growth factors and the extracellular matrix reveals a role for transient ERK activity in G1 phase.
We have examined the regulation of p21(cip1) by soluble mitogens and cell anchorage as well as the relationship between the expression of p21(cip1) and activation of the ERK subfamily of MAP kinases. We find that p21(cip1) expression in G1 phase can be divided into two discrete phases: an initial induction that requires growth factors and the activation of ERK, and then a subsequent decline that is enhanced by cell anchorage in an ERK-independent manner. In contrast to the induction of cyclin D1, the induction of p21(cip1) is mediated by transient ERK activity. Comparative studies with wild-type and p21(cip1)-null fibroblasts indicate that adhesion-dependent regulation of p21(cip1) is important for proper control of cyclin E-cdk2 activity. These data lead to a model in which mitogens and anchorage act in a parallel fashion to regulate G1 phase expression of p21(cip1). They also show that (a) growth factors and growth factor/extracellular matrix cooperation can have different roles in regulating G1 phase ERK activity and (b) both transient and sustained ERK signals have functionally significant roles in controlling cell cycle progression through G1 phase.
Animals↗
Adhesion-dependent cell cycle progression linked to the expression of cyclin D1, activation of cyclin E-cdk2, and phosphorylation of the retinoblastoma protein.
Growth factors and cell anchorage jointly regulate transit through G1 in almost all cell types, but the cell cycle basis for this combined requirement remains largely uncharacterized. We show here that cell adhesion and growth factors jointly regulate the cyclin D1- and E-dependent kinases. Adhesion to substratum regulates both the induction and translation of cyclin D1 mRNA. Nonadherent cells fail to phosphorylate the retinoblastoma protein (Rb), and enforced expression of cyclin D1 rescues Rb phosphorylation and entry into S phase when G1 cells are cultured in the absence of substratum. Nonadherent cells also fail to activate the cyclin E-associated kinase, and this effect can be linked to an increased association of the cdk inhibitors, p21 and p27. These data describe a striking convergence in the cell cycle controls used by the two major signal transduction systems responsible for normal and abnormal cell growth. Taken together with our previous studies showing adhesion-dependent expression of cyclin A, they also establish the cell cycle basis for explaining the combined requirement for growth factors and the extracellular matrix in transit through the Rb checkpoint, entry into S phase, and anchorage-dependent growth.
3T3 Cells↗
Cytoskeletal integrity is required throughout the mitogen stimulation phase of the cell cycle and mediates the anchorage-dependent expression of cyclin D1.
The proliferation of many nontransformed cells depends on cell adhesion. We report here that disrupting the cytoskeleton in normal human fibroblasts causes the same cell cycle phenotype that is observed after blocking cell adhesion: suspended cells and cytochalasin D-treated monolayers fail to progress through G1 despite normal mitogen-induced expression of c-myc mRNA. Midway between G0 and the beginning of S-phase, cell cycle progression becomes independent of adhesion and the cytoskeleton. At this stage, the cells are also mitogen independent. Molecular analyses showed that Rb hyperphosphorylation and the induction of cyclin D1 occur slightly earlier than the transition to cytoskeleton independence. Moreover, these molecular events are blocked by cytochalasin D. Overall, our data indicate the following: 1) anchorage and cytoskeletal integrity are required throughout the mitogen-dependent part of G1; 2) mitogens and the cytoskeleton jointly regulate the phosphorylation of Rb; and 3) this interdependence is manifest in the regulation of cyclin D1.
Cell Adhesion↗
Cell density, negative proliferation control, and phosphorylation of retinoblastoma protein.
Cell density negative control (CDNC) of normal human fibroblast proliferation occurs after stimulation by mitogens with different signal transduction mechanism. Delayed exposure to agents that interfere with CDNC, such as double-stranded RNA and vanadate, reveals the existence of a biochemical event, involved in CDNC, that occurs 5-8 hr after the beginning of mitogenic stimulation. This is earlier than the point of "mitogenic commitment," defined by the duration of mitogen exposure required for cell cycle entry (8-18 hr). Phosphorylation of the retinoblastoma gene product (pRB) begins 8-10 hr after mitogen stimulation and is nearly complete at 18 hr, just as the first cells enter S-phase. CDNC prevents pRB phosphorylation. Interferon beta delays pRB phosphorylation by up to 20 hr but has little effect on the timing of mitogenic commitment. Thus mitogenic commitment is located in time between CDNC and pRB phosphorylation. When agents that cause a release from CDNC are applied to dense, negatively controlled cultures after 18 hr of EGF stimulation, pRB phosphorylation occurs 6-8 hr after release. This suggests that the negatively controlled cells process the mitogenic signal but accumulate at a restriction point. The relatively early timing of CDNC-related events in the prereplicative phase raises the possibility that pRB phosphorylation is a consequence rather than a prerequisite for release from cell density negative control.
Cell Count↗
Serum factor revealing two distant phases of negative proliferation control in mitogen-stimulated normal fibroblasts.
When quiescent normal skin fibroblasts are stimulated by mitogens such as epidermal growth factor (EGF), the proportion of cells entering a division cycle decreases with increasing cell density. The presence of a synthetic double-stranded RNA (poly I:C) enhances this density-related restriction. Fetal calf serum (FCS) as well as human serum (HS) and human platelet-poor plasma (HPPP) completely abrogate the inhibiting effect of cell density on EGF mitogenicity, both in the presence and absence of poly I:C. HS and HPPP are up to ten times more potent than FCS in overcoming density-related restriction of EGF mitogenicity in human skin fibroblasts, whereas the mitogenic potencies of FCS, HS and HPPP in the absence of EGF are identical. Thus the mitogenic activity of FCS, HS and HPPP and their ability to overcome the density-related restriction of EGF-induced proliferation may be due to different molecules. Addition of FCS or HS at various times after EGF exposure reveals two distinct control points within the prereplicative phase: one within the first 2 hours and the other between 10 and 20 hours after the beginning of EGF exposure. Thus, the interactions of EGF, serum, and poly I:C reveal the kinetics of a cell density-related mechanism of negative proliferation control.
Cell Count↗
Growth inhibition of mitogen-stimulated fibroblasts induced by double-stranded RNA depends on cell density.
Polyinosinic-polycytidylic acid [poly(I:C)], a synthetic double-stranded RNA, is an inhibitor of mitogen-induced proliferation of normal fibroblasts. We show that this inhibition depends strongly on cell density. While cultures with densities at or above confluence are completely inhibited by poly(I:C) in their proliferative response to epidermal growth factor (EGF), the proliferation of sparse (subconfluent) cultures is only delayed. Conditioned medium from dense fibroblasts exposed to poly(I:C) inhibits EGF stimulation of sparse cells, indicating that the inhibition is, at least in part, mediated by a factor released from the cells. Preincubation of quiescent cultures with poly(I:C) renders the cells refractory to the inhibitory effects of poly(I:C). This desensitization correlates with a decreased production of the inhibitor. Since the inhibition of mitogenic stimulation by poly(I:C) is completely overcome by antisera recognizing interferon-beta (IFN-beta) and interleukin-6 (IL-6), we tested the effect of IL-6 and IFN-beta on EGF mitogenicity. None of the available IL-6 preparations had any effect on cell cycle entry. IFN-beta caused a dose-dependent delay of cell division but did not affect the density-dependent proportion of cells entering the cell cycle in response to EGF. Thus, IFN-beta cannot be the sole mediator of the poly(I:C)-induced inhibition. In the presence of dexamethasone, poly(I:C) did not inhibit EGF mitogenis. Indeed, the combined presence of poly(I:C) and dexamethasone did more than just restore the density-dependent control levels of EGF stimulation; most cells entered the cell cycle even at extremely high cell densities. Thus, poly(I:C) in combination with dexamethasone could deactivate the cell density-dependent negative control of proliferation.
Biological Factors↗
Cell division analysis using bromodeoxyuridine-induced suppression of Hoechst 33258 fluorescence.
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Animals↗
Interaction of serum and colony-stimulating factor for survival of a factor-dependent hemopoietic progenitor cell line.
The growth in vitro of the murine myeloid cell line FDC-P1 depends on the presence of serum and a murine hemopoietic growth factor (either granulocyte/macrophage colony-stimulating factor (GM-CSF) or multipotential colony-stimulating factor (multi-CSF, IL3]. To determine the differential roles of serum and colony-stimulating factor (CSF) during the growth of FDC-P1 cultures, we investigated the kinetics of proliferation and death after withdrawal of serum or CSF, using flow cytometry to quantitate the numbers of vital and dead cells. After withdrawal of CSF, the cells died without entering a quiescent state. The life span of cultures lacking CSF increased with increasing concentrations of serum (greater than 50 h at 30% serum), and the cells kept dividing until they died. During the period of population death caused by the absence of CSF, the re-addition of CSF immediately prevented further cells from dying. After the withdrawal of serum in the presence of CSF, the cells continued to live and proliferate for weeks, but required high cell densities (much greater than 10(5)/ml), which suggests that the cells produced an active substance that can substitute for serum. Serum as well as serum-free conditioned medium from dense cultures made the survival and growth of FDC-P1 cultures independent of cell density. Without sufficient quantities of this activity, all cells of the population died within an interval that was much shorter than one cell cycle, which indicates that the factor acts throughout most of the cell cycle. The results suggest that both the CSF and the serum factor act together to permit cell survival, rather than to drive proliferation.
Animals↗
An autocrine activity capable of substituting for serum in cell cultures.
Provided that high cell densities (above 10(6)/ml) are maintained, a factor-dependent murine hemopoietic progenitor cell line (FDC-P1) will proliferate in serum-free medium. The conditioned medium (CM) from high-density FDC-P1 cells permits the serum-free survival of FDC-P1 cells even at low density, indicating the existence of a diffusible autocrine factor. The requirement of FDC-P1 for a colony-stimulating factor (either IL-3 or GM-CSF) is not abrogated by culturing the cells at high cell density or in the conditioned medium. Furthermore, the CM from FDC-P1 enhances the mitogenic stimulation of normal human skin fibroblasts (HSF) by epidermal growth factor (EGF): i.e., the lag period before entry into the cell cycle is shortened by up to 6 hr. The fibroblasts themselves secrete an activity into serum-free medium that appears to be required during mitogenic stimulation by EGF. The HSF-CM also allows FDC-P1 cells to survive and proliferate serum-free at low cell densities. Low concentrations of fetal calf serum or human plasma (0.2-2%) have the same effect as FDC-P1-CM and HSF-CM. We have tested many of the known growth factors, and none of them mimicked the autocrine serum replacing activity (ASRA). The activity in human plasma elutes from a gel-filtration column with an apparent molecular weight of 60,000. It appears as if cultured normal cells and cell lines produce molecules capable of complementing the growth factors required for the survival and proliferation of a range of cells in serum-free cultures.
Animals↗
Granulocytic colony-stimulating factor (G-CSF) does not induce differentiation of WEHI3B(D+) cells but is required for the survival of the mature progeny.
It has been reported previously that cells of the murine myelomonocytic leukaemia cell line WEHI3B(D+) can be induced to differentiate into mature monocytes by incubation with post-endotoxin serum (ES), granulocyte-colony-stimulating factor (G-CSF) or actinomycin D (AMD). We have investigated the kinetics of the differentiation process in suspension cultures, using growth curves and morphological markers to analyze proliferation and differentiation. At low cell densities (less than 10(5)/ml) neither ES nor G-CSF changed the rate of WEHI3B(D+) proliferation; nor did these reagents induce the differentiation of WEHI3B(D+). In contrast, AMD caused growth arrest and initiated cell maturation, which was followed by cell death. The time course of maturation depended on AMD concentration. The addition of either ES or G-CSF at the time of AMD exposure or at times up to 20 hr after the beginning of AMD exposure resulted in a delay of cell death for up to 3 days and in an accumulation of mature monocytes. The kinetics of AMD-induced maturation were not affected by the time of ES or G-CSF addition. We concluded that neither ES nor G-CSF induce the differentiation of WEHI3B(D+) cells directly, but that their presence permits the survival of mature cells produced from WEHI3B(D+) as a result of a short-range autocrine-induced differentiation.
Animals↗
Probability of transition into cell cycle does not depend on growth factor concentration.
When quiescent cells in monolayer culture are stimulated to proliferate with growth factor, the entry into S-phase or mitosis appears to follow first-order kinetics, with a probability to enter the cell cycle that depends on growth factor concentration (Smith and Martin, 1973). Suboptimal growth factor concentrations also lead to a decreased fraction of the cell population that responds to the stimulation (Brooks et al., 1984). Using flow cytometry, we have re-investigated this dual effect of growth factor concentration on cultures of quiescent normal human skin fibroblasts, stimulated with submaximal concentrations of fetal calf serum, epidermal growth factor, and platelet-derived growth factor. The size of the responding population decreased with decreasing concentration of growth factor, but the time course of cell division within this responding population was identical for all growth factor concentrations. This is in conflict with previous concepts and indicates that the entry into the proliferative state is based on a decision mechanism that cannot be adequately described using transition probabilities determined by mitogen concentration.
Bisbenzimidazole↗
Cell cycle position affects response of normal fibroblasts to phorbol esters.
Tumor-promoting phorbol ester derivatives are known to stimulate as well as inhibit the cell cycle traverse of many kinds of cells, but it is not known whether or how these effects are related to tumor promotion. It is shown here that the potency of the phorbol diester PMA (phorbol myristate acetate) to cause a delay of cell division depends on the cell cycle position at the beginning of exposure to the PMA. Quiescent human fibroblasts were stimulated with epidermal growth factor, and PMA was added to the cultures at different times after stimulation. The later PMA was added, the stronger was the mitotic delay it caused. This means that cells which become exposed while they are in an early stage of the cycle are less effectively delayed and reach mitosis earlier than those for which exposure begins at a later stage of the cycle. The data indicate that PMA is actually able to reverse the order in which the cells of a normally growing population divide. In organized tissues this could disturb the intercellular communication which may be linked to the relative cell cycle positions of neighboring cells.
Bisbenzimidazole↗
Nature of the tumor-localizing components of hematoporphyrin derivative.
Hematoporphyrin derivative linked by ether bonds (HE) was synthesized by unambiguous procedures in order to compare its properties to hematoporphyrin derivative (HpD). Reverse phase high performance liquid and gel filtration chromatography were used to compare the HE derivatives to HpD. The cellular uptake of HE derivatives was compared to HpD using the WEHI 3B (D+) cell line and was shown to be taken up to a degree and in a manner similar to HpD. The efficiency of HE porphyrins as photosensitizers was compared to HpD using the V79 cell line. HE porphyrins were more efficient in sensitizing the V79 cells than was HpD. The in vivo tumor localizing properties of HE porphyrins were compared to HpD in CBA mice bearing the C6 cerebral glioma, and BALB/c mice bearing the EMT6 mammary tumor. HE derivatives localized in both tumor models as effectively as HpD. We conclude that the properties of ether linked hematoporphyrin derivatives are very similar to properties of HpD.
Animals↗
Hematoporphyrin derivative and anthracyclines mutually inhibit cellular uptake and toxicity.
Hematoporphyrin derivative (HpD) is a fluorescent photosensitizer which localizes selectively in tumors and is used in photoirradiation therapy of various types of cancer. Photoirradiation is a local form of treatment. There may be advantages in combining it with systemic chemotherapy, and we therefore studied the interaction of HpD and various cytotoxic drugs [daunorubicin (DR), adriamycin (AD), mitozantrone (MI), cis-platinum (CP) and 5-fluorouracil (FU)] with respect to cellular uptake and toxicity. Cultured cells were incubated with HpD and the cytotoxic drugs, either simultaneously or sequentially. The amount of DR, AD and HpD taken up by cells was quantitated by measuring cellular fluorescence and by determining the specific cytotoxic action of the drugs (light-induced cell kill for HpD and loss of proliferative potential for the cytotoxic drugs). The presence of 100 mg/l HpD during drug incubation caused 30-, 12- and 6-fold reductions in the cytotoxicity of DR, AD and MI respectively. HpD-associated fluorescence and photosensitization were reduced upon co-incubation of cells with HpD and AD. When cells were incubated with HpD and AD sequentially, fluorescence was equal to the sum of fluorescence values achieved by treatment with HpD and AD alone, and the cytotoxicity was not reduced. This suggests that the effects were due to an interaction of HpD with the drugs outside the cells, resulting in a reduction of free drug available for cellular uptake. CP and FU did not interact with HpD. The results suggest that protocols which combine photoirradiation therapy and chemotherapy with anthracyclines or similar compounds need to take this interaction into account.
Animals↗
Flow cytometric detection of a two-step cell death induced by hyperthermia.
A human leukaemic cell line (REH) was subjected to various temperatures approximately greater than 42 degrees C for various time intervals; the cells were stained with a mixture of ethidium bromide and acridine orange, and red and green fluorescence were analysed by flow cytometry. Nontreated cells appeared as one cluster (V) in the biparameter histograms, but with time of heat treatment, two further discrete clusters (D1,D2) of cells appeared successively. They were distinguished by both the degree of red and green fluorescence. The kinetics of transit from one cluster to the other was dependent on temperature, the time lag between both steps becoming shorter with higher temperatures. It was shown previously that the same effect occurred during incubation with various cytostatic agents, and that only the D2 stage correlated with the stage of cell death monitored by the usual trypan blue exclusion test. Therefore the ethidium bromide technique seems to monitor an earlier stage of cell death. The decrease in the number of dye-excluding (V) cells during heat exposure occurred in two phases. After an initial decrease a plateau of number of dye-excluding cells was reached; the duration and level of this plateau depended on the temperature. The plateau was followed by a second phase where the remaining cells ceased to exclude the dye.
Acridine Orange↗
Flow-cytometric determination of fluorescence ratios between differently stained particles is dependent on excitation intensity.
By use of a flow cytometer, the fluorescence of cells stained with hematoporphyrin derivative and the fluorescence of plastic beads stained with different dyes were analysed as a function of the intensity of the exciting laser light. The ratios of the fluorescence values of stained and unstained cells as well as of stained cells and beads were sensitively dependent on excitation intensities. As a consequence of this finding, the normalization of cellular fluorescence by use of reference particles needs to be made on a well-defined and reproduced intensity of the exciting laser light.
Animals↗
Uptake of hematoporphyrin derivative by normal and malignant cells: effect of serum, pH, temperature, and cell size.
Normal and malignant cells were incubated with hematoporphyrin derivative (HPD) and their uptake and retention of HPD were analyzed by flow cytometry. In standard growth medium the amount of HPD taken up by cells was proportional to the added HPD concentration and reached a plateau level after 5-6 h of incubation. The uptake occurred in two steps; within seconds a large amount of HPD became loosely bound to the cells, presumably the outer membrane. This was followed by a slower uptake of HPD into the cytoplasm. The loosely bound portion could be washed from the cells by medium containing either fetal calf serum or serum albumin. At low temperatures the uptake into the cytoplasm was strongly reduced. A major determinant of HPD uptake was the concentration of serum in the medium. At any particular concentration of HPD below 200 mg/liter, increasing concentrations of fetal calf serum or bovine serum albumin resulted in a reduction in the amount of HPD taken up by cells. A further factor affecting uptake was the pH of the medium. At low pH (pH 6) the rate of HPD incorporation was much higher than at pH 7.4. Under identical conditions of incubation, HPD uptake was proportional to cell size as estimated using the low angle light scatter signal in the flow cytometer. Our data suggest that acidic pH, differences in extracellular serum concentrations of malignant tumor tissue, as well as the increased size of tumor cells may play an important role in the selective uptake of HPD by malignant tumors.
Animals↗