Selection of proliferating cybrid cells by dual laser flow sorting : Isolation of teratocarcinoma X neuroblastoma and teratocarcinoma X endoderm cybrids

An alternative method for the isolation of proliferating cybrid cells was developed, and was used to obtain teratocarcinoma X neuroblastoma and teratocarcinoma X endoderm cybrids. Enucleated neuroblastoma (or endoderm) cells labelled with fluorescent microspheres were fused with (HPRT-deficient) unlabelled teratocarcinoma cells. The cells in the fusion mixture were stained with the vital DNA stain Hoechst 33342 and the cybrids, containing both a fluorescent nucleus and fluorescent beads, were isolated by dual laser flow sorting. The purity of the sorted fraction, as determined by the percentage of cells showing HPRT activity, was 86 and 57% for the neuroblastoma and endoderm cybrids, respectively. After single cell sorting in wells of Terasaki microtest plates, clones of proliferating cybrids were obtained with cloning efficiencies of 33% (neuroblastoma and endoderm cybrids respectively. After single cell sorting in wells of Terasaki microtest parental cell lines were analysed by two-dimensional gel electrophoresis. A number of differences were found between the parental cell lines but all isolated colonies (sixteen neuroblastoma cybrids and eight endoderm cybrids) resembled the teratocarcinoma parent. These results therefore give no evidence for the existence of cytoplasmic factors in neuroblastoma or endoderm cells capable of inducing permanent differentiation of teratocarcinoma cells.     

Flow cytometric characterization of viable meiotic and postmeiotic cells by Hoechst 33342 in mouse spermatogenesis.

BACKGROUND: Spermatogenesis in adult is a complex stepwise process leading to terminally differentiated spermatozoa. The cellular heterogeneity of testis renders complex the studies on molecular aspects of this differentiation process. Analysis of the regulation of adult spermatogenesis would undoubtedly benefit from the development of techniques to characterize each germinal differentiation step. METHODS: Hoechst 33342 staining of mouse testicular cells allows characterization of an enriched population in germinal stem cell and spermatogonia, called side population. In this study, we examined the definition of the various germinal populations stained by Hoechst 33342, notably meiotic and postmeiotic cells. RESULTS: Preleptotene spermatocytes, spermatocyte I, spermatocyte II, and round and elongated spermatids were discriminated by Hoechst 33342 staining. In addition, we associated differentiation of spermatocyte I through leptotene to diplotene with changes in Hoechst 33342 red fluorescence pattern. CONCLUSIONS: Hoechst 33342 staining of viable germinal cells constitutes a valuable tool to study normal and impaired mouse adult spermatogenesis or to isolate viable cells from various differentiation stages for studies of molecular mechanisms regulating spermatogenesis.     

Isolation of anucleate cells using a fluorescence activated cell sorter (FACS II)

Human fibroblasts or mouse teratocarcinoma cells were enucleated by density gradient centrifugation in the presence of cytochalasin B (CB). The resulting mixed population of nucleated and anucleate cells was further purified by flow sorting, using the dye Hoechst 33342 as a fluorescent label for the nucleated cells. The purity of the anucleate cells obtained with this technique was at least 99%, as was shown by histological staining of the sorted fractions. Sorted enucleated fibroblasts were shown to have an intact cell membrane as indicated by their ability to convert fluorescein diacetate into fluorescein and to accumulate this product. They were found to attach and spread when cultured and showed protein synthesis immediately after enucleation, evidenced by the incorporation of [³H]leucine. Sorted enucleated teratocarcinoma cells also had an intact cell membrane, but they did not attach when cultured.     

Simultaneous analysis of relative DNA and glutathione content in viable cells by phase-resolved flow cytometry.

BACKGROUND: Analysis of the DNA cell cycle and glutathione content cannot be performed on viable cells, because the fluorescence emissions of the DNA-specific probe Hoechst 33342 and the glutathione-specific probe monobromobimane overlap completely. We decided to explore whether the emissions could be resolved by the singlet excited state lifetimes of the probes. METHODS: Viable cells were first incubated with Hoechst 33342 at 37 degrees C for 30 min and then with monobromobimane at room temperature for 10 min. Samples were excited with a sinusoidally modulated laser beam (10 MHz) in a flow cytometer. The Hoechst 33342 and monobromobimane lifetimes and fluorescence intensities were resolved by using phase-sensitive detectors. RESULTS: The observed singlet excited state lifetimes were 1.5 ns for Hoechst 33342 and 12 ns for monobromobimane. The glutathione (GSH) content was shown to increase as cells (GM130, HL60, U937) progressed through the cell cycle. However, after the data were corrected for differences in cell volume, it was found that the GSH concentration was constant throughout the cell cycle of the exponentially growing cells. CONCLUSIONS: Phase-resolved flow cytometry provides a means for the specific analysis of the GSH content/concentration as a function of the cell's position in the DNA cell cycle in viable cells.     

Enrichment and characterization of mouse putative epidermal stem cells

Epidermis, a continuously renewing tissue, is maintained by stem cells that proliferate and replenish worn out or damaged cells in the tissue during life. Cultured epidermal stem cells have great potential in scientific research and clinical application. However, isolating a pure and viable population of epidermal stem cells and culturing them has been challenging. In this study, putative epidermal stem cells of mouse were isolated by combining Hoechst 33342 and propidium iodide staining with fluorescence-activated cell sorting. Molecular markers expression pattern analysis showed that cytokeratin 14, integrin beta1 and p63 are expressed in the sorted putative stem cells, but not active beta-catenin, nestin and involucrin. Our results provide further supporting data that mouse putative epidermal stem cells could be successfully isolated by combining Hoechst dye staining with fluorescence-activated cell sorting and cultured in vitro. The cultured mouse putative epidermal stem cells could be used as a potent tool for studying stem cell biology and testing stem cell therapy.     

Separation of viable T and B lymphocytes using a cytochemical stain, Hoechst 33342

Data are presented that show that a histochemical stain, Hoeschst 33342, can be used to discriminate between viable B and T lymphocytes in the mouse. Quantitative analysis of the staining of cells from various lymphoid tissues with Hoechst 33342 using a Fluorescence-Activated Cell Sorter (FACS) indicates that two populations of cells can be identified. In the spleen approximately 60% of the lymphocytes can be classified as brightly stained with 1 microgram/ml of Hoechst 33342, while in the lymph node only 40% of the cells stain brightly. Thymocytes exhibit only the dull staining profile. Separation of these two populations from the spleen using the FACS and reanalyzing them for cell surface antigenic markers shows that the lymphocytes stained brightly with Hoechst 33342 are predominantly immunoglobulin positive, while the cells that stain less brightly express Thy 1.2. This indicates that a histochemical stain correlates directly with classical immunological markers on cell surfaces.     

Vital DNA staining and cell sorting by flow microfluorometry

A procedure has been investigated for sorting viable cells according to their DNA content. Cells are stained with the U.V. activated fluorochromes 4'6-diamidino-2-phenylindole (DAPI), Hoechst 33258 or Hoechst 33342, and sorted with a Fluorescence Activated Cell Sorter. Hoechst 33342 is a suitable vital stain for a variety of cell types. Hoechst 33258 and DAPI, however, are quantitative vital stains for CHO cells only. Cloning efficiency is unaffected by the sorting procedure, and these stains are not mutagenic at concentrations suitable for vital staining. Potential applications of this procedure to cell biology are discussed.     

Differentiation of EC cells in vitro by the fluorescent dye Hoechst 33342

The fluorescent dye Hoechst 33342 is able to differentiate F9 EC cells at low concentrations. This differentiation is accompanied by synthesis of large amounts of laminin, production of a well-developed cytoskeleton, disappearance of the SSEA-1 antigen, and synthesis of large amounts of fibronectin, all characteristics of the primitive endoderm. The dye immediately blocks the cells at the S/G2 phase of the cell cycle and produces a complete arrest in proliferation. This effect is not specific for the nullipotent F9 cell line, as multipotent EC cell lines like PCC3, P19, and PCC4 can also be easily differentiated into the same pathway by treatment with the Hoechst dye. In contrast, the dye has no remarkable effects on terminal differentiated, immortalized cells like NIH 3T3 or the parietal endoderm-like cell PYS-2.     

Biochemical markers of the progress of differentiation in cloned teratocarcinoma cell lines.

The progeny of single teratocarcinoma cells will give rise to several different cell types in vitro, and the latter were shown to be functionally differentiated by biochemical criteria. In all these studies, cloned lines of mouse teratocarcinoma cells were assayed during the course of differentiation for some biochemical products characteristic of the tissues formed. The carcinoembryonic protein, alpha-foetoprotein, was not synthesized by undifferentiated embryonal carcinoma (EC) cells, but was synthesized in increasing amounts during their differentiation to endoderm-type cells in suspension culture. alpha-Foetoprotein was shown to be a product of endoderm cells, but not all endoderm cells synthesized this protein. During the course of further differentiation when EC cells or aggregates were grown in tissue-culture dishes, other biochemical products appeared. In cultures containing predominantly nerve-type cells, there was a 30-fold increase in the specific activity of acetylcholinesterase, with concomitant appearance of the aldolase isoenzyme characteristic of mouse brain. In some cultures, a small amount of muscle-type cell formation was marked by the appearance of the MB isoenzyme of creatine phosphokinase. Generally, biochemical differentiation was immature.     

Effect of drug efflux blockers on vital staining of cellular DNA with Hoechst 33342

The present study shows that staining of certain live cells, e.g., adriamycin-resistant P388 cells, by Hoechst 33342 is difficult because of the presence of a rapid efflux pump, which reduces intracellular dye concentration. Coincubation of these refractory cells in the presence of efflux blockers such as phenothiazines (trifluoperazine) or Ca++ channel blockers (verapamil) enhances dye retention and thus leads to generation of normal DNA distribution histograms. Laser flow cytometric data is confirmed by fluorometric assays, which show that P388/R cells retain one-third the amount of Hoechst 33342, and coincubation with efflux blockers increases Hoechst retention to values similar to those of drug-sensitive P388 cells. DNA histograms of mouse splenocytes incubated with Hoechst 33342 alone have a bimodal distribution possibly because of the presence of subpopulations that do not retain the fluorochrome owing to rapid efflux. Coincubation with an efflux blocker results in the generation of unimodal DNA histograms from these cells. These preliminary studies suggest that reduced retention of Hoechst 33342 in certain cell types (because of rapid efflux) can be blocked by efflux blockers, thus leading to generation of typical DNA distribution histograms.     

Hoechst 33342 staining of mouse bone marrow: effects on colony-forming cells

We have systematically studied the effect on hemopoietic colony-forming cells of staining cellular DNA with the bisbenzimidazole dye, Hoechst 33342. Mouse bone marrow cells could be adequately stained in a 30-60 min incubation with a 5 microM concentration of stain. Flow-cytometric analysis of stained cells provided cell distributions with coefficients of variation for the G1 peaks of 6% or less under these conditions. We found considerable heterogeneity among hemopoietic colony-forming cells with respect to the toxicity of the dye. Toxicity in the proliferatively quiescent stem cell population was not changed when the population became proliferatively active. In the sequence of most sensitive to least sensitive, the five progenitors studied could be arranged as follows: CFU-M, a megakaryocyte colony-forming cell; CFU-E, a relatively differentiated erythroid precursor; BFU-E, a primitive erythroid precursor; CFU-GM, a granulocyte-macrophage precursor; and CFU-S, the spleen colony-forming cell or hemopoietic stem cell. A staining procedure involving a 30-min exposure to 5 microM Hoechst 33342 provided optimal staining and no loss in four of the five progenitor populations; the CFU-M population was diminished by about 50%. We conclude that Hoechst can be regarded as a vital DNA stain for most bone marrow precursor populations, including the hemopoietic stem cell.     

Hoechst 33342 induces radiosensitization in malignant glioma cells via increase in mitochondrial reactive oxygen species

Abstract

Mitochondrial DNA plays an important role in cellular sensitivity to cancer therapeutic agents. Hoechst 33342, a DNA minor groove binding ligand, has shown radiosensitizing effects in different cancer cell lines. In the present study, the possible binding of Hoechst 33342 with mitochondrial DNA, isolated from human cerebral glioma (BMG-1) cells, was investigated and consequences of this binding on excessive reactive oxygen species (ROS) generation in irradiated BMG-1 cells were studied. Alteration in the fluorescence spectroscopic characteristics of Hoechst 33342 suggested binding of Hoechst 33342 with isolated mitochondria and mitochondrial DNA. Persistent increase in level of ROS in the presence of Hoechst 33342 has been observed, which was further enhanced in irradiated cells. Investigations using inhibitors of ETC complex I suggested that mitochondrial bound Hoechst 33342 contributed to increased ROS, which was associated with alteration in ΔΨm and antioxidant machinery. These factors appeared to contribute in potentiating radiation-induced cell death in BMG-1 cells. The finding from these studies will be useful in designing better anti-cancer strategies.     

The location and synthesis of transferrin in mouse embryos and teratocarcinoma cells

In early postimplantation mouse development, transferrin synthesis appears to be a marker of visceral endoderm cell types. Transferrin was identified using immunoperoxidase staining, in the proximal (visceral) endoderm of the sixth-day egg cylinder, in some tissues at later stages, and in the visceral yolk sac (VYS) at all stages examined. Since the location of a plasma protein does not necessarily indicate its site of synthesis, the incorporation of labeled amino acids into transferrin was studied. Synthesis could be detected in egg cylinders on the seventh day of gestation onwards and in the VYS at all stages. However, although endoderm was the likely tissue source, its ability to synthesize transferrin after its isolation from the embryo was either much reduced or absent. The data are suggestive of a modulating influence by mesoderm and other cell types on transferrin synthesis in visceral endoderm cells. Three types of endoderm-like cells which are produced by teratocarcinoma embryonal carcinoma (EC) cells were analyzed for transferrin synthesis to assess possible parallels with the embryo. Embryoid bodies from PSA1 EC cells contained some outer endoderm cells which stained for transferrin and others which did not. The endoderm line PSA5E but not PYS-2 synthesized transferrin. The third type of endoderm-like cell (END cells) synthesized very little (OC15S1) or no (PC13 clone 5) transferrin. The conclusion that PSA5E, OC15 END, and some differentiated PSA1 cells have visceral endoderm-like character while PYS-2 reflects parietal endoderm phenotype is in agreement with published data.     

Isolation and characterization of the cDNAs corresponding to mRNAs abundant in undifferentiated mouse embryonal teratocarcinoma stem cells, but not in differentiated mouse parietal endoderm cells

As retinoic acid (RA) and dibutyryl cAMP (cAMP) treatment induces differentiation of mouse teratocarcinoma F9 cells into parietal endoderm cells in vitro, we initiated studies on the molecular mechanisms underlying early mammalian cell differentiation in this system. We constructed cDNA libraries on the poly(A)+RNAs extracted from the undifferentiated F9 cells, and screened for cDNA sequences expressed abundantly in F9 cells, but not in terminally differentiated mouse parietal endoderm PYS-2 cells. Six different cDNA clones were isolated and characterized. The levels of RNAs hybridizable to these clones were at most 5 to 24% in the PYS-2 cells when compared with those in the undifferentiated F9 cells. The six clones were classified into two groups on the basis of their responses to the RA and cAMP treatment. In F9 cells, the levels of RNAs hybridizable to the first group, which contained four clones, were decreased within 72 h after the addition of RA and cAMP, while those of the second group, which contained the remaining two clones, did not decrease significantly. One of the first group clones, named pF9-1, corresponded to the mouse "early transposon-like elements" and another, named pF9-4, hybridized to multi-size RNAs extracted from the undifferentiated F9 cells. The mouse genomic DNA sequences hybridizable to pF9-4 were repeated approximately 5,000 times, and comprise a new gene family, the expression of which is developmentally regulated in mouse F9 cells.     

Expression of fibroblast growth factor by F9 teratocarcinoma cells as a function of differentiation

F9 teratocarcinoma stem cells treated with retinoic acid (RA) and dibutyryl cAMP (but2 cAMP) differentiate into embryonic parietal endoderm. Using heparin-affinity chromatography, endothelial cell proliferation assays, immunoprecipitation, and Western analysis with antibodies specific for acidic and basic fibroblast growth factors (FGFs), we detected biologically active FGF in F9 cells only after differentiation. A bovine basic FGF cDNA probe hybridized to 2.2-kb mRNAs in both F9 stem and parietal endoderm cells and to a 3.8-kb mRNA in F9 stem cells. A genomic DNA probe for acidic FGF hybridized to a 5.8-6.0-kb mRNA in both F9 stem and parietal endoderm cells, and to a 6.0-6.3-kb mRNA only in parietal endoderm cells. Although these FGF mRNAs were present in the stem cells, we could find no evidence that F9 stem cells synthesized FGFs, whereas differentiated F9 cells synthesized both acidic and basic FGF-like proteins. We conclude that biologically active factors with properties characteristic of acidic and basic FGF are expressed by F9 parietal endoderm cells after differentiation. Differentiating embryonic parietal endoderm thus may serve as a source of FGF molecules in the developing blastocyst, where these factors appear to play a central role in subsequent embryogenesis.     

Measurement of cell-cycle phase-specific cell death using Hoechst 33342 and propidium iodide: preservation by ethanol fixation

We developed a rapid technique for preservation of Hoechst 33342/propidium iodide-stained cells, using ethanol as a fixative. Combined staining with these dyes makes possible analysis of cell-cycle phase-specific cell death. The technique relies on exclusion of propidium iodide from the viable cells, whereas Hoechst stains all of the cells. The bivariate histograms resulting from the flow cytometric analysis contain the equivalent of two single-parameter DNA histograms, one of the living and the other of the dead cell population. Preservation of staining involved addition of 25% ethanol in PBS after propidium iodide staining and before Hoechst staining. The separation between the living and the dead cell populations was maintained for over 3 days at 4 degrees C. This technique will be valuable for quantitative evaluation of the cell-cycle phase-specific effects of cytostatic or cytotoxic agents, particularly in situations where a lag period between staining and analysis is unavoidable.     

The induction of antigenic changes in a teratocarcinoma stem cell line (F9) by retinoic acid

Treatment of embryonal carcinoma cells F9 with retinoic acid results in the appearance of epithelioid cells resembling endoderm which synthesize basement membrane protein and plasminogen activator. Concomitant with the appearance of these properties of differentiated cells, the epithelial cells cease to express SSEA-1, an antigenic determinant characteristic of teratocarcinoma stem cells and early mouse embryos. Our evidence indicates that the phenotypic changes that accompany retinoic acid treatment of embryonal carcinoma cells are irreversible and a consequence of the differentiation of the cells into endoderm.     

Hoechst fluorescence intensity can be used to separate viable bromodeoxyuridine-labeled cells from viable non-bromodeoxyuridine-labeled cells

BACKGROUND: 5-Bromo-2'-deoxyuridine (BrdU) is a powerful compound to study the mitotic activity of a cell. Most techniques that identify BrdU-labeled cells require conditions that kill the cells. However, the fluorescence intensity of the membrane-permeable Hoechst dyes is reduced by the incorporation of BrdU into DNA, allowing the separation of viable BrdU positive (BrdU+) cells from viable BrdU negative (BrdU-) cells. METHODS: Cultures of proliferating cells were supplemented with BrdU for 48 h and other cultures of proliferating cells were maintained without BrdU. Mixtures of viable BrdU+ and viable BrdU- cells from the two proliferating cultures were stained with Hoechst 33342. The viable BrdU+ and BrdU- cells were sorted into different fractions from a mixture of BrdU+ and BrdU- cells based on Hoechst fluorescence intensity and the ability to exclude the vital dye, propidium iodide. Subsequently, samples from the original mixture, the sorted BrdU+ cell population, and the sorted BrdU- cell population were immunostained using an anti-BrdU monoclonal antibody and evaluated using flow cytometry. RESULTS: Two mixtures consisting of approximately 55% and 69% BrdU+ cells were sorted into fractions consisting of greater than 93% BrdU+ cells and 92% BrdU- cells. The separated cell populations were maintained in vitro after sorting to demonstrate their viability. CONCLUSIONS: Hoechst fluorescence intensity in combination with cell sorting is an effective tool to separate viable BrdU+ from viable BrdU- cells for further study. The separated cell populations were maintained in vitro after sorting to demonstrate their viability.     

DNA damage, cytotoxic effect and cell-cycle perturbation of Hoechst 33342 on L1210 cells in vitro

This study was designed to evaluate the effects of vital dye Hoechst 33342 (HO 33342), at concentrations used to obtain a good DNA histogram resolution, on DNA integrity, cell growth, and cell-cycle phase distribution of L1210 cells. HO 33342 exposure for 2 h, at 37 degrees C produced DNA single-strand breaks as assessed by the method of alkaline elution. DNA single-strand breaks were concentration dependent (in the range .5-5 micrograms/ml) and increased significantly when HO 33342 (0.5-1.5 micrograms/ml) was associated with exposure in a flow cytometer to U.V. laser beam illumination. HO 33342 produced a cytotoxic effect on cell growth even at the concentration of 0.5 microgram/ml--a concentration ten-fold smaller than those required to obtain a good DNA histogram resolution. HO 33342 produced a severe block of the cells in the G2-M phase of the cell cycle already evident 24 h after stain exposure and continuing up to 144 h after start of recovery. A new polyploid cell population (with a 4 c DNA content) not present in the unstained cells was already evident 24 h after dye exposure. The data shown in the present paper would imply caution in using sorted cells stained with HO 33342 dye for biological, biomedical, and pharmacological studies.     

Retinoic acid modulation of transmembrane signaling. Analysis in F9 teratocarcinoma cells

F9 embryonal mouse teratocarcinoma cells were differentiated to a primitive endoderm-like phenotype by retinoic acid and to a parietal endoderm-like phenotype by retinoic acid in combination with dibutyryl cyclic AMP. The secretion of tissue plasminogen activator (tPA) is a characteristic of the cells displaying the differentiated phenotypes. The fundamental question of whether tPA secretion is regulated acutely by G-protein-mediated transmembrane signaling was explored. Cells differentiated to primitive and parietal endoderm demonstrated a rapid tPA response to stimulation by beta-adrenergic agonist (isoproterenol). Adenylyl cyclase activity in response to isoproterenol and GTP, but not forskolin, was greater in primitive and parietal endoderm than F9 stem cells. Both primitive and parietal endoderm cells, but not F9 stem cells, displayed beta-adrenergic stimulation of cyclic AMP accumulation. Retinoic acid induced F9 stem cells to the primitive endoderm phenotype and increased beta-adrenergic receptor levels 3-fold. Gi alpha 2 levels declined, G beta-subunits increased, and Gs alpha levels were unchanged following differentiation to primitive endoderm. In parietal endoderm cells beta-adrenergic receptors increased 2-fold over F9 stem cells, Gi alpha 2 levels declined even further than in primitive endoderm, G beta-subunits increased compared to F9 stem cells, and Gs alpha levels again were unchanged. The marked potentiation of short-term stimulation of tPA secretion in the differentiated state may be best explained by the retinoic acid-induced increase in expression of beta-adrenergic receptors coupled with a decline in Gi alpha 2 levels. Short-term regulation by G-protein-linked receptors represents a novel mode for the control of tPA secretion.