No abstract

Flow cytometric (FCM) studies were performed on nuclei, ethanol-fixed CHO cells, and isolated human GM130 chromosomes stained with two new cyanine dyes, TOTO and YOYO. These fluorochromes, which are dimers of thiazole orange and oxazole yellow, respectively, have high quantum efficiencies and exhibit specificities for both DNA and RNA. Bound to dsDNA in solution, TOTO and YOYO emit at 530 and 510 nm, respectively, when excited at 488 nm and 457 nm, wavelengths available from most lasers employed in FCM. RNase-treated CHO nuclei, stained with either TOTO or YOYO, provided DNA histograms, with low coefficients of variation, that were as good as or better than those obtained with nuclei stained with propidium iodide (PI) or mithramycin (MI). In addition, by comparison on an equimolar basis, nuclei stained with YOYO fluoresced over 1,000 times more intensely than nuclei stained with MI. Fluorescence ratio analyses of nuclei stained with both YOYO and Hoechst 33258 showed that the ratio of YOYO to Hoechst fluorescence remained relatively constant for G1 and S phase cells, but decreased significantly for cells in G2/M. These results indicate that the cyanine dyes may be useful in examining specific changes in chromatin structure during G2/M phases of the cell cycle. Ethanol-fixed CHO cells stained with TOTO or YOYO did not yield reproducible DNA histograms of good quality, presumably because of the poor accessibility of DNA to these large fluorochromes. However, bivariate analyses of human GM130 chromosomes stained with TOTO or YOYO alone and excited sequentially with uv and visible wave-lengths showed resolution of many individual chromosome peaks similar to results obtained for chromosomes stained with HO and chromomycin A3. Collectively, these studies show potential advantages for the use of these new cyanine dyes in FCM studies that require the sensitive detection of DNA.

Following the recently reported trapping of biological particles by finely focused laser beams, we report on the automated micromanipulation of cells and other microscopic particles by purely optical means as well as on a newly observed interaction between particles in the trapping beam. A simple instrument is described which allows single cells to be positioned with high accuracy, transported over several millimeters, and automatically sorted on the basis of their optical properties. These operations are performed inside a small enclosed chamber without mechanical contact or significant fluid flow. Potential applications of this technique in experimental cell biology are discussed.

Mithramycin added to mammalian cells fixed in aqueous ethanol is bound to DNA and fluoresces in direct proportion to the cellular DNA content. Quantitative fluorescence measurement by means of a high-speed flow system instrument provides a rapid method for cell-cycle analysis and for the first time permits continuous monitoring of cell-cycle kinetics during ongoing experiments.

A cytochemical method was developed to differentially stain cellular DNA, RNA, and proteins with fluorochromes Hoechst 33342, pyronin Y, and fluorescein isothiocyanate, respectively. The fluorescence intensities, reflecting the DNA, RNA, and protein content of individual cells, were measured in a flow cytometer after sequential excitation by three lasers tuned to different excitation wavelengths. The method offers rapid analysis of changes in the cellular content of RNA and protein as well as in the RNA-protein, RNA-DNA, and protein-DNA ratios in relation to cell cycle position for large cell populations. An analysis of cycling cell populations (exponentially growing CHO cultures) and noncycling CHO cells arrested in the G1 phase by growth in isoleucine-free medium demonstrated the potential of the technique.

No abstract

ios Alamos National laboratory, 10s Alamos, New Mexico 87545 (H.C., 1.S.)Using flow cytometry, populations of Chinese hamster ovary cells, asynchronous and synchronized in the cycle, were measured with respect to cellular RNA-and protein-content, as well as cell light scatter properties. Heterogeneities of cell populations were expressed as coefficients of variation (c.v.) in percent of the respective mean values. Populations of cells immediately after mitosis have about 15% higher C.V. than mitotic cell populations, regardless of whether RNA, proteins, or light scatter are measured. These data indicate that cytoplasmic constituents are unequally distributed into the daughter cells during cytokinesis and that unequal cytokinesis generates intercellular metabolic variability during the cycle. An additional increase in heterogeneity, although of smaller degree, occurs during C, phase. Populations of S-phase cells measured in the selective window equivalent to 15-60 min progression through the cycle, i.e., comparable with the mitotic and postmitotic populations, are the most uniform, having 20-30% lower C.V. than the postmitotic cells. Cell progression through S does not involve any significant increase in intercellular variability with respect to RNA or protein content. In unperturbed exponentially growing cultures a critical RNA content is required for G, cells prior to their entrance into S. Thus, the cells equalize in G, with respect to RNA and protein and, during the transition from the period (compartment) of equalization (G,J to the prereplicative compartment (C,@), they exhibit minimal heterogeneity. The cell residence times in the equalization compartments are exponentially distributed, which may reflect the randomness generated by the uneven division of metabolic constituents to daughter cells during cytokinesis. The cell heterogeneities were presently estimated at two metabolic levels, transcription (RNA content) and translation (proteins). The most uniform were populations stained for RNA and the highest variability was observed after staining of proteins. This suggests that the regulatory mechanisms equalizing cells in the cell cycle may operate primarily at the level of DNA transcription.Cell populations of a given type or line are heterogeneous with respect to the cell size as well as protein or RNA content of individual cells. There is also high intercellular variation in rates of progression through the cell cycle (see reviews: Baserga, 1976;Prescott, 1976). Despite this heterogeneity, the mean values of the cell size or content of cell constituents remain constant for a given cell type, from generation to generation. The degree of heterogeneity of the populations also remains constant over generations. Thus, mechanisms do operate during the cell cycle, "equalizing" the populations, i.e., precluding the appearance of cells with extremely low or high protein or RNA-content, or cells traversing the cycle at extreme rates. In addition, there is a control over the mean values of these parameters, so tha...

We have shown that nontransformed mammalian cells arrest early in the G1 phase of the cell cycle when treated with exceedingly low concentrations of the nonspecific kinase inhibitor staurosporine, whereas transformed cels continue to progress through the cell cycle. We have now treated normal or transformed human skin fibroblasts with four other kinase inhibitors. Three of these inhibitors are highly specific: KT5720 inhibits cAMP-dependent protein kinme, KT5823 inhibits cGMP dependent protein kinase, and KT5926 Inhibits myosin light-chain kinase. The fourth inhibitor K252b has a moderate specificity for protein kinase C but also inhibits the three kinaes just mentioned. We have found that these inhibitors reversibly arrest normal human skin fibroblasts at different times in the G1 phase but do not affect the cell cycle poession of nformed cells. The times of arrest within the G1 phase can be divided Into two categories. Two of the inhibitors, KT5926 and K252b, act at an early time that is -4 h after the transition from Go to G1. The cAMP-and cGMPdependent protein kinase inhibitors rest cells at a later tue that is =6 h after the Go/G1 boundary.These data indite that there are multiple kinase-medated phosphorylitions of different substrates that are essential for the pgression of normal cells, but not transformed cells, through the G1 phase. These inhibitors provide us with a set of biochemical probes that should be invaluable in the study ofthe function of kinases during GI phase progression of normal cells.