Expression of p21Waf1/Cip1 and cyclin D1 is increased in butyrate-resistant HeLa cells.

Sodium butyrate induced cell cycle arrest in mammalian cells through an increase in p21Waf1/Cip1, although another study showed that this arrest is related to pRB signaling. We isolated variants of HeLa cells adapted to growth in 5 mm butyrate. One of these variants, clone 5.1, constitutively expressed elevated levels of p21Waf1/Cip1 when incubated in regular growth medium and in the presence of butyrate. Despite this elevated level of p21Waf1/Cip1, the cells continue to proliferate, albeit at a slower rate than parental HeLa cells. Western blot analyses showed that other cell cycle regulatory proteins were not up-regulated to compensate for the elevated expression of p21Waf1/Cip1. However, cyclin D1 was down-regulated by butyrate in HeLa cells but not in clone 5.1. We conclude that continued expression of cyclin D1 allowed clone 5.1 to grow in the presence of butyrate and elevated levels of p21Waf1/Cip1.

DNA-dependent protein kinase-independent activation of p53 in response to DNA damage.

Phosphorylation at serine 15 of the human p53 tumor suppressor protein is induced by DNA damage and correlates with accumulation of p53 and its activation as a transcription factor. The DNA-dependent protein kinase (DNA-PK) can phosphorylate serine 15 of human p53 and the homologous serine 18 of murine p53 in vitro. Contradictory reports exist about the requirement for DNA-PK in vivo for p53 activation and cell cycle arrest in response to ionizing radiation. While primary SCID (severe combined immunodeficiency) cells, that have defective DNA-PK, show normal p53 activation and cell cycle arrest, a transcriptionally inert form of p53 is induced in the SCID cell line SCGR11. In order to unambiguously define the role of the DNA-PK catalytic subunit (DNA-PKcs) in p53 activation, we examined p53 phosphorylation in mouse embryonic fibroblasts (MEFs) from DNA-PKcs-null mice. We found a similar pattern of serine 18 phosphorylation and accumulation of p53 in response to irradiation in both control and DNA-PKcs-null MEFs. The induced p53 was capable of sequence-specific DNA binding even in the absence of DNA-PKcs. Transactivation of the cyclin-dependent-kinase inhibitor p21, a downstream target of p53, and the G1 cell cycle checkpoint were also found to be normal in the DNA-PKcs -/- MEFs. Our results demonstrate that DNA-PKcs, unlike the related ATM protein, is not essential for the activation of p53 and G1 cell cycle arrest in response to ionizing radiation.

Attenuation of apoptotic DNA fragmentation by amiloride.

Amiloride is a K+-sparing diuretic that effectively inhibits the Na+/H+ transporter in the plasma membrane of most mammalian cells. We have examined the effects of amiloride on the progression of apoptosis in HL-60 cells induced by camptothecin (CAM), cycloheximide (CHX), and 20 Gy gamma irradiation. Spectrofluorometric measurements on cell populations showed an inhibition of Na+/H+ transporter activity and a corresponding decrease in intracellular pH following treatment with amiloride alone, or in combination with the apoptosis-inducing agents. Flow cytometric cell cycle analysis, in combination with DNA strand break analysis, indicated that amiloride diminished endonuclease-mediated degradation of nuclear chromatin 3 h following treatment with CAM or CHX, and prevented degradation for 3 h following gamma radiation treatment. Apoptosis-associated DNA degradation was significantly greater for all three agents in the absence of amiloride. Protection from radiation-induced apoptosis was transient, since apoptotic subpopulations were observed, but still at a decreased level, 5 h following irradiation. Amiloride was as effective as zinc, an inhibitor of Ca2+/Mg2+-dependent endonucleases, in reducing or delaying the onset of endonuclease activity. Data presented show that effects of amiloride on membrane Na+/H+ transporter activity and intracellular pH can potentially affect apoptotic signaling cascades, leading to a retardation in the rate of progression to an apoptotic cell death. Results also point to the involvement of intracellular pH and Ca2+ in the regulation of apoptotic endonuclease activity, and the need for a functional Na+/H+ exchanger for the induction of apoptosis.

Apoptosis induced with different cycle-perturbing agents produces differential changes in the fluorescence lifetime of DNA-bound ethidium bromide.

Fluorescence lifetime analysis was used in combination with conventional flow cytometric analysis to monitor changes in residual chromatin in apoptotic HL-60 cell populations following treatment with camptothecin, cycloheximide, genistein, H7, and gamma radiation. Data presented show that all of these metabolic inhibitors, which act through different signaling cascades, produce apoptotic subpopulations with decreased but different lifetimes for DNA-bound ethidium bromide (EB). Additionally, treatment with certain agents reduced the fluorescence lifetime in the apoptotic cells prior to extensive endonuclease degradation of DNA and the appearance of the typical sub-G0/G1 peak in the DNA histogram. A lifetime value of 21.15 +/- 0.12 ns was obtained for EB bound to nonapoptotic cells, while values for EB bound to the apoptotic subpopulations following treatment with the different agents were: camptothecin, 19.87 +/- 0.08 ns; cycloheximide, 19.39 +- 0.02 ns; H7, 19.77 +/- 0.03 ns; genistein, 20.04 +/- 0.04 ns; and gamma radiation, 19.67 +/- 0.03 ns. Traditional methods of analysis, including gel electrophoresis or morphology assessment, revealed no significant differences among apoptotic subpopulations induced by treatment with these agents. Our data suggest that the mode of action of the various agents induces structural changes in chromatin organization that differentially alter accessibility of DNA to endonuclease digestion. Subsequent fluorescence lifetime analysis appears sensitive to the resulting differences in the residual chromatin in apoptotic cells following DNA cleavage. Results presented indicate that lifetime analysis, used in conjunction with conventional flow cytometry, can be useful for early detection of apoptosis-induced chromatin changes and may also potentially provide new information on the effects of different apoptosis-inducing agents.

Reduction in the radiation-induced late S phase and G2 blocks in HL-60 cell populations by amiloride, an efficient inhibitor of the Na+/H+ transporter.

Recent investigations that showed that amiloride delayed or inhibited apoptosis indicated it might also attenuate cell cycle checkpoints activated by ionizing radiation. In this report, single- and dual-parameter flow cytometry were used to investigate the effects of amiloride on cell cycle progression, and the effectiveness of amiloride to attenuate the S and G2 phase checkpoint responses induced by 2.5, 5.0, and 7.5 Gy of gamma radiation. The late S-phase delay, noted at 8 h following irradiation, and a radiation-induced G2 block, which was maximum at 16 h after irradiation, were both significantly reduced in amiloride-treated samples. Attenuation of the radiation-induced late S phase and G2 blocks resulted in cell division without apparent apoptosis or necrosis over a 24-h period. Results presented indicate that amiloride reduces the radiation-induced G2 block in HL-60 cell populations almost equally well as caffeine and to a greater extent than staurosporine. Immunofluorescent detection and quantitation of cyclin B1 expression demonstrated that amiloride only significantly reduced cyclin B1 expression following 5.0 Gy, when there was a notable induction of a significant G2 delay, followed by a relatively rapid recovery in cycling potential. The results suggest that amiloride affects the radiation-triggered signaling cascades to alter the kinase activity of proteins associated with mitotic progression, particularly the cyclin B1-p34cdc2 complex. Alternatively, alterations in intracellular ion concentrations induced by amiloride may lead to changes in Ca2+-dependent signaling cascades and thereby decrease the radiation-mediated cell cycle perturbations.

Flow cytometric fluorescence lifetime analysis of DNA-binding probes.

A new dimension has been added to multiparameter flow cytometric analysis through the recent development of techniques for rapidly measuring the fluorescence lifetime of probes bound to single cells. The lifetime measurements are made by phase-sensitive detection techniques in a flow cytometer (FCM) that also analyzes fluorescence intensity and other optical properties of stained cells. These lifetime assays have potential for elucidating the microenvironment of the interaction of fluorochrome probes and subcellular target molecules. Alterations in the lifetime of DNA probes have been observed in cells in different phases of the cell cycle, in different cell types, in differentiating cells, and in apoptotic cells with damaged chromatin. Lifetime differences noted also for intercalating dyes bound to DNA and dsRNA, indicated modifications in the modes of binding and provide the potential for analyzing both corformational states and nucleic acid metabolism. Future developments in the technology will provide multiple lifetime assays and thereby allow for detection and quantitation of selected subcellular probe-complexes with different lifetime signatures. These novel assays will expand the applications for quantitative studies on the binding of various chemical agents to DNA and other molecular targets in cells, and further improve methods for rapid screening of chemotherapeutic agents or environmentally toxic compounds.

Monitoring uptake of ellipticine and its fluorescence lifetime in relation to the cell cycle phase by flow cytometry.

Ellipticine (5,11-dimethyl-6H-pyrido[4,3-b]carbazole) is a green fluorescent plant alkaloid that inhibits DNA topoisomerase II activity and possesses pharmacologic activity toward both murine and human leukemias in vivo. In this flow cytometric study, the uptake of ellipticine was monitored as a function of cell volume and cell cycle phase in viable human promyelocytic (HL-60) cells costained with the DNA fluorochrome Hoechst 33342. Uptake of ellipticine was time and dose dependent; however, drug content was quantitatively similar in all phases of the cell cycle when normalized for DNA content or similar to cell size when correlated with cell volume. The fluorescence lifetime values of ellipticine in HL-60 cells, as analyzed by novel flow cytometric analysis, reached a plateau when the intra-cellular ellipticine intensity was still rising with increasing drug concentration. Since the free drug and the different subcellular ellipticine complexes, including DNA and RNA, had different lifetime values, the changes in the lifetime values appear to reflect differing proportions of unbound drug to that bound to different cellular constituents in the cells. Further development of phase-sensitive flow cytometry will provide for multiple lifetime determinations so that quantitation of drugs bound to the different cellular components can be performed along with the simultaneous determination of total drug uptake and cell cycle position. Such analyses should provide useful information for the design of drugs with greater affinity for cytotoxic targets.

Cell cycle-dependent protein expression of mammalian homologs of yeast DNA double-strand break repair genes Rad51 and Rad52.

Recently, human and rodent homologs of yeast repair genes Rad51 and Rad52 have been identified and proposed to play roles in DNA double-strand break (DSB) repair. In this study, cell cycle-dependent expression of human and rodent RAD51 and RAD52 proteins was monitored using two approaches. First, flow cytometric measurements of DNA content and immunofluorescence were used to determine the phase-specific levels of RAD51 and RAD52 protein expression in irradiated and control populations. The expression of both proteins was lowest in G0/G1, increased in S and reached a maximum in G2/M. No difference was found in the whole-cell level of RAD51 or RAD52 protein expression between gamma-irradiated and control cell populations. Second, cell cycle-dependent protein expression was confirmed by Western analysis of populations synchronized in G0, G1 and G2 phases. Analysis of V3, a hamster equivalent of SCID, indicates that the protein level increases of RAD51 and RAD52 from G0 to G1/S/G2 do not require DNA-PK.

Association of G1/S-phase and late S-phase checkpoints with regulation of cyclin-dependent kinases in Chinese hamster ovary cells.

We investigated the time-dependent effects of 8 Gy of gamma radiation on the activities of cyclin-dependent kinases (Cdk's) and the incorporation of the thymidine analog bromodeoxyuridine (BrdU) throughout the S phase in Chinese hamster ovary (CHO) cells. The in vitro Cdk activities of immunoprecipitated cyclin E, cyclin A and Cdk2 were reduced about 30% per cell within 0.5-1 h after irradiation, but they recovered at different rates. The kinase activity of the cyclin E-Cdk2 complex recovered first and exceeded the control values by 1.5-2 h after irradiation. Cyclin A-Cdk activities began to recover at 3-4 h after irradiation, and cyclin E/A-Cdk2 activities recovered at intermediate rates. The super-recovery of cyclin E-Cdk2 coincided with the appearance of a small synchronous population of cells entering into S phase, consistent with transient G1-phase delay/recovery regulated by cyclin E-Cdk2, whereas the activities of cyclin A-Cdk's (75% cyclin A-Cdk2; 25% cyclin A-Cdc2 when inhibition was maximal) were correlated with rates of total DNA synthesis. Multivariate flow cytometry analyses of BrdU incorporation demonstrated that radiation-induced inhibition of DNA synthesis occurred predominantly within the last quarter of S phase and that the majority of the irradiated cells failed to enter G2 phase for 4-5 h. The recovery of cyclin A-Cdk activities coincided with increased levels of total DNA synthesis and BrdU incorporation into cells within the last quarter of S phase. Western blot analysis demonstrated that levels of Waf1/p21 did not increase during inhibition of cyclin A-Cdk's and DNA synthesis in the irradiated p53-mutated CHO cells; however, Cdc2 and Cdk2 exhibited increased levels of phosphotyrosine. The results (1) indicate that the transient G1-phase delay or G1/S-phase checkpoint (Lee et al., Proc. Natl. Acad. Sci. USA 94, 526-531, 1997) is mediated by inhibition of cyclin E-Cdk2 and (2) point to the existence of a radiation-induced S-phase checkpoint located about 75% into S phase involving the inhibition of cyclin A-Cdk's by a p53/Waf1-independent pathway in CHO cells.

Modulation in cell cycle and cyclin B1 expression in irradiated HeLa cells and normal human skin fibroblasts treated with staurosporine and caffeine.

The comparative effects of staurosporine or caffeine on G2-phase arrest and cyclin B1 expression in human skin fibroblasts (HSF) and transformed HeLa cells following gamma-irradiation were examined by flow cytometry. Contrary to some earlier reports with HeLa cells, the arrest in G2 after irradiation was accompanied by an increase in cyclin B1 levels in both asynchronous and synchronized HeLa cells irradiated in early S phase. Caffeine and staurosporine were equally effective in attenuating both the radiation-induced increase in cyclin B1 expression and the prolongation of G2 in synchronous and asynchronous HeLa cell populations. Staurosporine treatment was less effective in down-regulating cyclin B1 expression in asynchronous HeLa cells at earlier time points following irradiation when compared to caffeine-treated cells. In synchronized HeLa cells, down-regulation of an irradiation-induced increase in cyclin B1 expression was similar to either staurosporine or caffeine treatments, with caffeine being more effective at later time points. An increase in cyclin B1 expression was also observed in irradiated HSF cells (synchronous and asynchronous), which decreased when the cells were treated with staurosporine or caffeine. However, staurosporine was ineffective in attenuating the radiation-induced prolongation of G2 in synchronous and asynchronous HSF cells, whereas treatment of irradiated synchronous or asynchronous HSF cells with caffeine significantly reduced the prolongation of G2. These results suggest that both staurosporine and caffeine treatments act on different pathways of cell cycle control in normal and transformed cells, in terms of attenuation of G2 block and diminution of elevated levels of cyclin B1 expression, in response to radiation.

New flow cytometric technologies for the 21st century.

The envelope that defines the limits within which flow cytometry was developed is being rapidly expanded. For example: detection sensitivity has been extended to single molecules, the size range of "particle" analysis now extends from DNA fragments to plankton (1,000.+ microns), cell and chromosome sorting rates are being increased dramatically by using inactivation procedures (50,000 per second versus 2,000 per second), rapid kinetic flow cytometry enables real-time analysis of molecular assembly and cell function in the sub-second time domain, the lifetime of a fluorochrome bound to a single cell can be measured with nsec precision, and classical karyotype information (cell to cell heterogeneity) can be determined in a flow based system. These frontiers have greatly expanded the range of new and exciting flow cytometric based biomedical applications. New enabling technologies have provided the means to measure DNA cleavage by the structure-specific nuclease, human Flap Endonuclease (FEN-1), in the 300 msec time frame. Phase sensitive measurements and fluorescence lifetime are proving to be major advances for understanding molecular environments that change with, for example, the process of apoptosis. The ability to detect single fluorescent molecules has been applied to the analysis of DNA fragments obtained from enzymatic digestion of lambda DNA. This technology is being used to rapidly and very accurately size DNA fragments for the human genome project. Optical chromosome selection is a faster, better, less complex approach to chromosome sorting. This method is based on the induction of specific damage to the DNA of selected chromosomes. Lastly, the miniaturization of a single cell fractionator has made it possible to perform single cell flow cytogenetics.

Differential effects of deuterium oxide on the fluorescence lifetimes and intensities of dyes with different modes of binding to DNA.

Deuterium oxide (D2O) increases both the fluorescence lifetime and the fluorescence intensity of the intercalating dyes propidium iodide (PI) and ethidium bromide (EB) when bound to nucleic acid structures. We have used spectroscopic analysis coupled with conventional and phase-sensitive flow cytometry to compare the alterations in intensity and lifetime of various DNA-binding fluorochromes bound to DNA and Chinese hamster ovary (CHO) cells in the presence of D2O vs phosphate-buffered saline (PBS). Spectroscopic and flow cytometric studies showed a differential enhancement of intensity and lifetime based on the mode of fluorochrome-DNA interaction. The fluorescence properties of intercalating probes, such as 7-aminoactinomycin D (7.AAD) and ethidium homodimer II (EthD II) were enhanced to the greatest degree, followed by the probes TOTO and YOYO, and the non-intercalating probes Hoechst 33342 (HO) and 4,6-diamidino-2-phenylindole (DAPI). The non-intercalating probe mithramycin (MI) gave unexpected results, showing a great enhancement of fluorescence intensity and lifetime in D2O, indicating that when staining is performed in PBS, much of the MI fluorescence is quenched by the solvent environment. Apoptotic subpopulations of HL-60 cells had a shorter lifetime compared to non-apoptotic subpopulations when stained with EthD II. These results indicate that accessibility of the dye molecules to the solvent environment once bound to DNA, leads to the differential enhancement effects of D2O on fluorescence intensity and lifetime of these probes.

Alterations in the progression of cells through the cell cycle after exposure to alpha particles or gamma rays.

A G1-phase delay after exposure to alpha particles has not been report ed previously, perhaps because immortalized cell lines or cell lines from tumor cells were used in past studies. Therefore, we compared the effects of alpha particles (0.19 or 0.57 Gy) and approximately equitoxic doses of gamma rays (2 or 4 Gy) on progression of cells through the cell cycle in normal human skin fibroblasts. Cell cycle analyses were performed using flow cytometry by measuring incorporation of bromodeoxyuridine (BrdUrd) in each phase of the cell cycle up to 44 h after irradiation. We observed an alpha-particle-induced G1-phase delay in human skin fibroblasts even at the lowest dose, 0.19 Gy. At equitoxic doses, more pronounced and persistent G1-phase delays and arrests were observed in gamma-irradiated cultures in that increased fractions of the G1-phase cells remained BrdUrd- over the course of the study after gamma-ray exposure compared to cells exposed to alpha particles. In addition, G1-phase cells that became BrdUrd+ after gamma irradiation re-arrested in G1 phase, whereas BrdUrd+ G1-phase cells in alpha-particle-irradiated cultures continued cycling. In contrast, comparable percentages of cells were delayed in G2 phase after either alpha-particle or gamma irradiation. Both gamma and alpha-particle irradiation caused increases in cellular p53 and p2lCip1 shortly after the exposures, which suggests that the G1-phase delay that occurs in response to alpha-particle irradiation is dependent on p53 like the initial G1-phase delay induced by gamma rays.

Interactions of intercalating fluorochromes with DNA analyzed by conventional and fluorescence lifetime flow cytometry utilizing deuterium oxide.

Deuterium oxide (D2O) has been shown in previous studies to increase both the fluorescence lifetime and fluorescence intensity of propidium iodide (PI) and ethidium bromide (EB) when bound to nucleic acid structures. We have used spectroscopic analysis and conventional and phase-sensitive flow cytometry to compare changes in PI and EB fluorescence intensity and lifetime bound to DNA and fixed Chinese hamster ovary (CHO) cells in the presence of D2O vs. phosphate-buffered saline (PBS). Spectroscopic and flow cytometric studies showed a twofold enhancement of fluorescence intensity of PI and EB bound to fixed CHO cells in D2O and a 5 ns increase in PI and EB fluorescence lifetimes in D2O. The fluorescence lifetime of HL-60 cells stained with PI or EB was found to be 1-2 ns different from that of CHO cells, indicating that the lifetime of these fluorochromes is sensitive to chromatin configuration in different cells types. Apoptotic subpopulations of HL-60 cells had a significantly reduced fluorescence lifetime compared to nonapoptotic subpopulations. Results indicate that different chromatin states, or differences in the structures of PI and EB, lead to alterations in the fluorescence intensity and fluorescence lifetime of these intercalating probes.

A ribonucleotide reductase inhibitor, MDL 101,731, induces apoptosis and elevates TRPM-2 mRNA levels in human prostate tumor xenografts.

MDL 101,731, (E)2'-fluoromethylene-2'-deoxycytidine, is an irreversible inhibitor of ribonucleotide diphosphate reductase and causes regression of human tumors in nude mouse models. Messenger RNA levels for testosterone-repressed prostatic message-2 (TRPM-2), a transcript that increases in human tumor xenografts undergoing programmed cell death, were analyzed by in situ hybridization. Xenografts derived from a human prostate tumor cell line (PC-3) regressed following treatment with MDL 101,731 and the relative levels of TRPM-2 mRNA increased up to threefold in drug-treated animals. Apoptosis in the tumor xenografts was further indicated by in situ labeling of DNA strand breaks by incorporation of biotinylated-dUTP with terminal deoxynucleotidyl transferase. In vitro, PC-3 cells incubated with MDL 101,731 showed evidence of apoptosis based on flow cytometry and DNA laddering. These data support the hypothesis that MDL 101,731 stimulates programmed cell death in regressing PC-3 xenografts.

Host range and cell cycle activation properties of polyomavirus large T-antigen mutants defective in pRB binding.

We have examined the growth properties of polyomavirus large T-antigen mutants that are unable to bind pRB, the product of the retinoblastoma tumor suppressor gene. These mutants grow poorly on primary mouse cells yet grow well on NIH 3T3 and other established mouse cell lines. Preinfection of primary baby mouse kidney (BMK) epithelial cells with wild-type simian virus 40 renders these cells permissive to growth of pRB-binding polyomavirus mutants. Conversely, NIH 3T3 cells transfected by and expressing wild-type human pRB become nonpermissive. Primary fibroblasts from mouse embryos that carry a homozygous knockout of the RB gene are permissive, while those from normal littermates are nonpermissive. The host range of polyomavirus pRB-binding mutants is thus determined by expression or lack of expression of functional pRB by the host. These results demonstrate the importance of pRB binding by large T antigen for productive viral infection in primary cells. Failure of pRB-binding mutants to grow well in BMK cells correlates with their failure to induce progression from G0 or G1 through the S phase of the cell cycle. Time course studies show delayed synthesis and lower levels of accumulation of large T antigen, viral DNA, and VP1 in mutant compared with wild-type virus-infected BMK cells. These results support a model in which productive infection by polyomavirus in normal mouse cells is tightly coupled to the induction and progression of the cell cycle.

Inhibition of histone phosphorylation by staurosporine leads to chromosome decondensation.

In mammalian cells, hyperphosphorylation of histone H1 and phosphorylation of histone H3 correlate well with the G2 phase to metaphase condensation of chromosomes, and these phosphorylations most probably have a role in initiating and controlling the entry into mitosis. The protein kinase inhibitor staurosporine has been used to examine the role of H1 and H3 phosphorylations in controlling chromosome condensation in the mouse FM3A cell line. We present evidence that (i) staurosporine inhibits the protein kinases that phosphorylate histone H1 during mitosis, (ii) staurosporine also inhibits the histone H3-specific kinase, (iii) the inhibition of these kinase activities prevent cells from entering mitosis, and (iv) addition of staurosporine to cells already arrested at metaphase by nocodazole causes a rapid dephosphorylation of histones H1 and H3 and the decondensation of the metaphase chromosomes. The results show that the hyperphosphorylation of histone H1 and phosphorylation of histone H3 are required to maintain metaphase chromosomes in their condensed state.

TOTO and YOYO: new very bright fluorochromes for DNA content analyses by flow cytometry.

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.