Flow cytometric analysis of bromodeoxyuridine-substituted cells stained with 33258 Hoechst.

This paper describes a flow-cytometric application of the quenching of fluorescence from 33258 Hoechst stained Chinese hamster ovary-line cells due to the incorporation of 5-bromo-deoxyuridine (BrdU) into the cellular deoxyribonucleic acid. Cells were grown for 24 hr in medium containing BrdU in concentrations ranging from 1 x 10(-8) to 1 x 10(-4) M. For each concentration we measured the average fluorescence as determined by flow cytometry, the extent of BrdU substitution and the effect of the BrdU on cell growth. We determined that a BrdU concentration of 1 x 10(-5) M resulted in sufficient substitution to quench the fluorescence from 33258 Hoechst by a factor of 4, allowing discrimination between cycling and noncycling cells. The extent of BrdU substitution after growth for 24 hr in this concentration of BrdU was 64%. These data indicate the feasibility of detecting deoxyribonucleic acid synthesis in whole cells using the 33258 Hoechst-BrdU methodology.     

Microfluorometric analysis of cellular DNA following incorporation of bromodeoxyuridine.

Bromodeoxyuridine (BrdU) incorporation into cellular DNA has a differential effect on the cell-associated fluorescence of several DNA-specific dyes. After cells were treated with BrdU, flow microfluorometry was used to study the relative increase or decrease influorescence of stained cells. Bromodeoxyuridine incorporation into CHO cells increased the fluorescence of mithramycin-, olivomycin-, or chromomycin-stained cells, decreased that of propidium iodide-stained cells, and had little, if any, effect on the fluorescence of acriflavine Feulgen-stained cells. Changes in relative fluorescence of cell associated dyes are due to changes in the amounts of dye bound to cells with BrdU-substituted DNA. Colorimetric and absorbance measurement of DNA content showed that BrdU does not alter the diploid DNA content of CHO cells; however, BrdU induces perturbations in the distribution of cells about the cell cycle which cause an increase in average DNA content.     

Analysis and sorting of living cells according to deoxyribonucleic acid content.

The methods for measuring the deoxyribonucleic acid content of individual mammalian cells and sorting them on the basis of this parameter have until now required fixation or other treatment which renders the cells nonviable. Using a class of bis-benzimidazole dyes, Hoechst 33258 and 33342 and a multiparameter computer-controlled cell sorter, we have been able to stain and separate living cells in the G1, S, and G2+M phases of the cell cycle and to continue their growth in tissue culture with high retention of viability (greater than 90%) and no increase in heteroploidy. The quenching of the fluorescence of the bound dye by 5-bromodeoxyuridine incorporated into cellular deoxyribonucleic acid is being used with the flow system to detect and isolate mutants in deoxyribonucleic acid metabolism spectroscopically.     

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.     

Circadian Rhythms of Nucleic Acid Metabolism in Neurospora crassa

Wild-type, band, and fluffy strains of Neurospora crassa exhibit circadian rhythms of ribonucleic acid and deoxyribonucleic acid content in the growth-front hyphae of cultures grown on a solid medium. There is also a rhythm of (3)H-uridine incorporation into the nucleic acids of the band strain. Maximum incorporation precedes the peaks of nucleic acid content which occur during conidiation. As cultures age, ribonucleic acid content decreases rapidly and deoxyribonucleic acid content decreases gradually in standing, shake, and bubble cultures. A reduction of ribonuclease activity with age is also noted in standing and shake cultures. The nucleic acid content, nuclease activity, and changes associated with age vary with the culture conditions.     

Automated analysis of antigen-stimulated lymphocytes.

Human peripheral blood lymphocytes were stimulated with phytohemagglutinin and tuberculin-purified protein derivative to determine if flow microfluorometric techniques had the sensitivity to detect high and low levels of blastogenic response. The deoxyribonucleic acid content of the lymphocytes was analyzed after culture. Blastogenic response was found to be measurable both sensitively and reproducibly. Tritiated thymidine incorporation measurements were also made. Preliminary experiments using inactivated Herpesvirion (type I) as a stimulant showed blastogenic response detectable by flow microfluorometric deoxyribonucleic acid measurements. Refinements of this technique may prove to be useful in the study of lymphocyte response to viral antigens in patient populations.     

Inhibition of deoxyribonucleic acid replication in Bacillus brevis by ribonucleic acid polymerase inhibitors.

The incorporation of [3H]thymidine into deoxyribonucleic acid by exponentially growing cells of Bacillus brevis was inhibited by streptolydigin and rifampin in the same concentration range in which these drugs inhibit ribonucleic acid synthesis. Complete inhibition occurred within one-third generation time after drug addition, suggesting an effect on deoxyribonucleic acid chain elongation.     

Alterations of deoxyribonucleoside triphosphate pools in Escherichia coli: effects on deoxyribonucleic acid replication and evidence for compartmentation.

Inhibition of ribonucleic acid synthesis in Escherichia coli 15 TAU bar with rifampin or streptolydigin leads to large increases in the sizes of cellular ribonucleoside and deoxyribonucleoside triphosphate pools. Inhibition of protein synthesis leads to increases in the sizes of all nucleoside triphosphate pools except the guanosine triphosphate and deoxyguanosine triphosphate pools; a decrease in the size of the latter pool may be responsible for the slowing of deoxyribonucleic acid replication fork movement observed in this strain in the absence of protein synthesis. Analysis of the kinetics of incorporation of labeled precursors into deoxyribonucleic acid and into cellular pools suggests that functional compartmentation of nucleotide pools exists, allowing the incorporation of exogenously supplied precursors into deoxyribonucleic acid without prior equilibration with the cellular pools.     

Inhibition of Friend erythroleukemic cell differentiation by bromodeoxyuridine: correlation with the amount of bromodeoxyuridine in DNA

These studies were undertaken to examine the relationship between the inhibition by 5-bromodeoxyuridine (BrdU) of erythroid differentiation in Friend erythroleukemia cells and the incorporation of BrdU into DNA. Experiments were carried out in which the incorporation of BrdU into DNA and the concentration of BrdU to which the cells were exposed were varied independently of each other. In addition, the ability of deoxycytidine (dC) to reverse the effects of BrdU on hemoglobin production and to reduce the amount of BrdU in DNA was analyzed. Under all the conditions tested, the effects of BrdU were correlated with the amount of BrdU incorporated into nuclear DNA. These results differ from those of recent studies on the inhibition of pigmentation and the induction of mutations by BrdU in Syrian hamster melanoma cells. The results suggest that BrdU may be producing its biological effects by a variety of different mechanisms.     

Inhibition of Nucleic Acid and Protein Synthesis in Mouse Spleen Cells In Vitro by Azathioprine

The effect of azathioprine on macromolecular biosynthesis was studied in mouse spleen cells cultured in vitro. The rate of incorporation of (3)H-thymidine, (3)H-uridine, and (14)C-leucine into acid-insoluble material was used to measure deoxyribonucleic acid, ribonucleic acid, and protein synthesis. Results indicate that azathioprine inhibited nucleic acid and protein synthesis at levels which did not decrease cell viability.     

Photosensitizing dyes and fluorochromes as substitutes for 33258 Hoechst in the fluorescence-plus-Giemsa (FPG) chromosome technique

Using Allium cepa chromosomes after 5-bromo, 2'-deoxyuridine (BrdU) incorporation, we studied several acid and basic dyes and fluorochromes for their potential as substitutes for 33258 Hoechst in the fluorescence-plus-Giemsa (FPG) technique. All of the dyes and fluorochromes investigated showed a photosensitizing capacity which was slightly lower than 33258 Hoechst in the cases of daunomycin, phloxin, fluorescein, thioflavine T and nuclear fast red, and somewhat higher in the case of eosin Y. Observation and cytophotometric analysis of differentially Giemsa-stained sister chromatids when eosin Y was used as the photosensitizing agent revealed the unsubstituted chromatid to be reddish violet in colour (absorption maximum, 550 nm), while the BrdU-substituted chromatid was blue or pale violet blue (absorption maximum, 580 nm). These results indicate that eosin Y is a useful photosensitizing dye which could be used as a substitute for 33258 Hoechst in the FPG staining technique.     

DNA OF BROMODEOXYURIDINE–TREATED NEUROBLASTOMA CELLS

Abstract— Cells of an adrenergic clone M1 of the mouse neuroblastoma C 1300 show morphological differentiation when treated with BrdU (10⁻⁵ m ). The involvement in this phenomenon of an action of BrdU at the membrane level has been suggested (see References). Taking into account the alterations observed after addition of BrdU in culture cell lines it was interesting to investigate the incorporation of BrdU into DNA of neuroblastoma cells. Our results showed an active BrdU incorporation into the DNA of treated Ml cells (an average of 25% of all thymidine residues for the 10⁻⁵ m -BrdU concentration present in our experimental conditions). Thymidine substitution by BrdU did not occur randomly, indicating the existence of selective parts in the genome sensitive toward BrdU. In mitomycin–treated cells (at high doses) BrdU incorporation occurred significantly at a very low level when related to the total thymidines (2%). Our findings suggest that since DNA synthesis is not inhibited BrdU may act by being incorporated into DNA and thus altering gene expression in addition to a site of action on the cell membranes.     

Evaluation of intranuclear BrdU detection procedures for use in multicolor flow cytometry.

BACKGROUND: Measurement of cell proliferation via BrdU incorporation in combination with multicolor cell surface staining would facilitate studies on cell subsets that require multiple markers for their identification. However, the extent to which the often harsh cell preparation procedures required affect the staining quality of more recently developed fluorescent dyes has not been assessed. METHODS: Three cell preparation protocols for BrdU measurement were compared for their ability to maintain fluorescent surface staining and scatter parameters of in vivo BrdU-labeled cells by flow cytometry. A 10-color fluorescent panel was developed to test the quality of surface staining, following cell treatment and the ability to perform BrdU measurements on even small B lymphocyte subsets. RESULTS: All cell preparation procedures affected the quality of fluorescent and/or scatter parameters to varying degrees. Paraformaldehyde/saponin-based procedures preserved sufficient fluorescent surface staining to determine BrdU incorporation rates among all splenic B cell subsets, including B-1a cells, which constitute roughly 0.5% of cells. Turnover rates of B-1a cells were similar to immature B cells and higher than those of the other mature B cell subsets. CONCLUSION: Paraformaldehyde/saponin-based cell preparation procedures facilitate detailed cell turnover studies on small cell subsets in vivo, revealing new functional information on rare cell populations.     

Hammerhead ribozymes targeted against cyclin E and E2F1 cooperate to down-regulate coronary smooth muscle cell proliferation

BACKGROUND: Anti-proliferative drugs released from endo-vascular stents have substantially contributed to reduce in-stent restenosis rates in coronary arteries bearing single primary lesions by down-regulating coronary smooth muscle cell (CSMC) growth. However, the considerably lower drug efficacy shown in treatment of more complex coronary lesions suggests that alternative anti-proliferative approaches can be beneficial. Thus, we explored the use of hammerhead ribozymes as tools to knock down cyclin E and E2F1, two potent activators of cell proliferation which cooperate to promote the G1 to S phase transition. METHODS: Two ribozymes, one directed against cyclin E and the other against E2F1 mRNAs, were delivered by liposomes to cultured human CSMCs. The influences on cell proliferation were measured evaluating BrdU incorporation into newly synthesised DNA. The effects on cell cycle phase distribution were determined by BrdU and 7-aminoactinomycin D incorporation into DNA. RESULTS: Both ribozymes exhibited a sequence-specific and dose-dependent reduction in BrdU incorporation, which, at a concentration of 280 nM, persisted up to 4 days after transfection of CSMCs. A combined administration of the two ribozymes (210+210 nM) resulted in a more pronounced decrease in BrdU incorporation compared to the administration of an equimolar amount (420 nM) of each of them. Finally, both ribozymes induced a significant (P<0.05) reduction in S phase cells with a concomitant increase of G1/G0 and G2-M phase cells, compared to controls. CONCLUSIONS: The ribozymes selected represent potent tools to prevent CSMC proliferation, especially when administered together, and thus are ideal candidates for in vivo application.     

Fluorescent DNA probes for flow cytometry. Considerations and prospects.

Techniques employing base specific deoxyribonucleic acid (DNA)-binding fluorochromes and flow cytometry (FCM) are potentially useful for obtaining information of the compositional features of chromatin or chromosomes of mammalian cells. Fluorescent compounds which form complexes preferentially at the A-T rich regions (i.e., DNA-reactive Hoechst dyes) or the G-C rich regions (i.e., mithramycin, chromomycin, olivomycin) in DNA are available and compatible with current FCM technology as are other compounds (i.e., ethidium bromide, propidium iodide) which show little or no base specificity and bind by intercalation in the double stranded regions of helical DNA. Energy transfer between appropriate DNA-bound dyes is a reflection of the quantity and proximity of regions containing the respective base pair segments. Since extrinsic fluorescent probes provide only a measure of available binding sites or regions unobstructed by chromatin-associated or chromosomal-associated proteins, interpretations of fluorescence measurements need to be substantiated by adequate control measures.     

Quantitation of lymphocyte response to antigen by flow cytofluorometry.

The response of human peripheral blood lymphocytes to antigenic stimulation has been studied in vitro using flow cytofluorometry and an acridine orange (AO) staining technique for cellular deoxyribonucleic acid and ribonucleic acid (RNA). Antigen-stimulated "pyroninophillic" immunoblasts, identified by an increase in cellular content of RNA (red fluorescence with AO), were quantitated in triplicate cultures incubated up to 7 days with and without bacterial antigen. These results were similar to 14C-thymidine incorporation into identical cultures incubated in parallel. Cytofluorometric analysis showed a peak in percentage of immunoblasts after 6 days in culture, while maximum thymidine incorporation was seen on day 7. Cells from patients with depressed immune response secondary to cancer showed lower than normal antigen response by cytofluorometry. Kinetic studies revealed both a lower percentage of immunoblasts when compared to normal and a lower average per cell RNA content of the stimulated cells. AO cytofluorometry is suggested as a convenient method of simultaneously assessing lymphocyte proliferative and nonproliferative response to antigen.     

Quantitation of cellular deoxyribonucleic acid by flow microfluorometry.

This report characterizes for the first time an easy, reproducible means of standardizing the relative fluorescent units normally reported for flow microfluorometry. Absolute values for deoxyribonucleic acid/cell are obtained by using nucleated red blood cells as references. Cell were selected and characterized for the quantitative analysis of deoxyribonucleic acid per cell over a range from 2 pg/cell to 93 pg/cell using literature values for species having nucleated erythrocytes. Fluorescence staining by either acridine-orange (green wavelength) or propidium iodide (red wavelength) gave linear curves over the entire range investigated only when "gain controls" and current are optimized. The range was equivalent to mammalian cell values from 1 N (=3.5 pg deoxyribonucleic acid/cell) to 28 N (=91 pg deoxyribonucleic acid/cell). The standard curves obtained with nonmammalian erythrocytes were compared to mammalian free-cell preparations of bovine thymus and liver cells which fell at 6.8 and 6.9 pg deoxyribonucleic acid/cell, respectively. The routine use of these easily obtainable red blood cells will allow ready comparisons on the basis of absolute values for deoxyribonucleic acid per cell for work between experiments, work between staining procedures and dye types and work between laboratories.     

G(1)/S but not G(0)/G(1)cell fraction is related to 5-fluorouracil cytotoxicity

BACKGROUND: Bromodeoxyuridine (BrdU) cell cycle analysis using flow cytometry is of clinical interest for making treatment decisions or for predicting response and survival, through proliferation rate (labeling index or S-phase fraction) assessment or T(pot) calculation. Thymidylate synthase expression was tested in vitro, in vivo, and clinically as a prognostic factor for 5-fluorouracil (5FU) sensitivity. However, results were still controversial. Moreover, we had reported that 5FU sensitivity was related to the labeling index of untreated cell cultures. METHODS: We used six human cancer cell lines that exhibited a wide range of 5FU sensitivity. Cell cycle analysis was performed using flow cytometry monovariate propidium iodide (PI) analysis and bivariate distributions of BrdU incorporation versus DNA content. 5FU sensitivity was assayed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) colorimetric assay. RESULTS: In all cell lines, 5FU exposure resulted in a statistically significant G(1)/S accumulation. No statistically significant relationship was seen between G(0)/G(1) delay determined by monovariate analysis and 5FU sensitivity. However, 5FU sensitivity was statistically correlated to the labeling index and G(1)/S subpopulation assessed with bivariate analysis using BrdU incorporation versus DNA content. CONCLUSIONS: Cellular proliferation parameters using BrdU incorporation are more informative than PI for in vitro 5FU sensitivity. Because BrdU incorporation could be assessed clinically, it could also be informative for 5FU clinical response prediction.     

9HODE stimulates cell proliferation and extracellular matrix synthesis in human mesangial cells via PPARgamma

Plasma oxidized low-density lipoprotein (OX-LDL) levels are elevated in patients with renal diseases, including diabetic nephropathy. We examined effects of OX-LDL on cell proliferation and extracellular matrix (ECM) production by using normal human mesangial cells. Furthermore, we examined possible involvement of peroxisome proliferator-activated receptor gamma (PPARgamma). Mesangial cell proliferation with OX-LDL, 9-hydroxy-10,12-octadecadienoic acid (9HODE), and 13-hydroxy-9,11-octadecadienoic acid (13HODE), the major components of OX-LDL, were determined by 5-bromo-2'-deoxyuridine (BrdU) or 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) incorporation. The effect of OX-LDL on mesangial cell proliferation with PD98059 pretreatment was determined by BrdU incorporation. Type IV collagen, fibronectin, and PPARgamma expression with OX-LDL or 9HODE or 13HODE was determined by Western blotting. Type IV collagen expression with antisense oligonucleotide against PPARgamma pretreatment was also determined by Western blotting. The effect of PD98059 pretreatment on PPARgamma expression was determined by Western blotting. In mesangial cells exposed to isolated OX-LDL from human plasma, BrdU incorporation was increased, and this increase was deleted by PD98059. Type IV collagen expression was significantly increased by OX-LDL. 9HODE and 13HODE increased BrdU and MTT incorporation into mesangial cells and also increased expressions of Type IV collagen and fibronection, the major components of ECM. PPARgamma expression in mesangial cells was stimulated by 9HODE. The reduction of PPARgamma synthesis by pretreatment of antisense oligonucleotide against PPARgamma remarkably attenuated Type IV collagen synthesis induced by 9HODE. PPARgamma expression induced by 9HODE was also reduced by PD98059 pretreatment. These findings demonstrate that 9HODE, the major component of OX-LDL, stimulates cell proliferation and ECM production of human mesangial cells. In addition, the stimulatory effects are, at least in part, mediated by PPARgamma, which may exist in downstream of ERK1/2 pathway.     

Effects of Phenethyl Alcohol on Phospholipid Metabolism in Escherichia coli

The incorporation of labeled precursors into the deoxyribonucleic acid, ribonucleic acid (RNA), proteins, and phospholipids of Escherichia coli cultured in the presence of phenethyl alcohol (PEA) was determined. PEA inhibited the uptake of labeled uracil to the same extent in cells exhibiting relaxed and stringent control of RNA synthesis. This indicates that PEA does not primarily affect amino acid synthesis or activation. Uptake of labeled acetate into the phospholipid fraction was more sensitive to inhibition by low concentrations of PEA than was the uptake of labeled precursors into the macromolecules. Thymine starvation or the addition of nalidixic acid (10 mug/ml) had no effect on acetate incorporation. Chloramphenicol (25 mug/ml) was a much less effective inhibitor of acetate incorporation than was PEA. The distribution of labeled acetate incorporated into phospholipids was markedly affected by the presence of PEA. The uptake of acetate into phosphatidylethanolamine and phosphatidylglycerol was inhibited, whereas the uptake of acetate into the cardiolipin fraction was unaffected. Since acetate incorporation into phospholipid was quite sensitive to PEA, we suggest that the PEA-sensitive component required for the initiation of replication may be a phospholipid(s).