Relationship between DNA damage response, initiated by camptothecin or oxidative stress, and DNA replication, analyzed by quantitative 3D image analysis.

A method of quantitative analysis of spatial (3D) relationship between discrete nuclear events detected by confocal microscopy is described and applied in analysis of a dependence between sites of DNA damage signaling (γH2AX foci) and DNA replication (EdU incorporation) in cells subjected to treatments with camptothecin (Cpt) or hydrogen peroxide (H2O2). Cpt induces γH2AX foci, likely reporting formation of DNA double-strand breaks (DSBs), almost exclusively at sites of DNA replication. This finding is consistent with the known mechanism of induction of DSBs by DNA topoisomerase I (topo1) inhibitors at the sites of collisions of the moving replication forks with topo1-DNA "cleavable complexes" stabilized by Cpt. Whereas an increased level of H2AX histone phosphorylation is seen in S-phase of cells subjected to H2O2, only a minor proportion of γH2AX foci coincide with DNA replication sites. Thus, the increased level of H2AX phosphorylation induced by H2O2 is not a direct consequence of formation of DNA lesions at the sites of moving DNA replication forks. These data suggest that oxidative stress induced by H2O2 and formation of the primary H2O2-induced lesions (8-oxo-7,8-dihydroguanosine) inhibits replication globally and triggers formation of γH2AX at various distances from replication forks. Quantitative analysis of a frequency of DNA replication sites and γH2AX foci suggests also that stalling of replicating forks by Cpt leads to activation of new DNA replication origins. © 2013 International Society for Advancement of Cytometry.

Leukemia treatment in severe combined immunodeficiency mice by antisense oligodeoxynucleotides targeting cooperating oncogenes.

Transformation of hematopoietic cells by the p210bcr/abl tyrosine kinase appears to require the expression of a functional MYC protein, suggesting that simultaneous targeting of BCR-ABL and c-myc might be a rational strategy for attempting treatment of Phil-adelphia leukemia. To test this hypothesis, severe combined immunodeficiency mice injected with Philadelphia leukemic cells were treated systemically with equal doses of bcr-abl or c-myc antisense oligodeoxynucleotides (ODNs) or with both ODNs in combination. Compared with the mice treated with individual agents, the disease process was much slower in the group treated with both ODNs, as revealed by flow cytometry, clonogenic assay, and reverse transcriptase-polymerase chain reaction analysis to detect leukemic cells in mouse tissue cell suspensions, and by enumeration of liver metastases. The retardation of the disease process was positively correlated with a markedly increased survival of leukemic mice treated with both ODNs. These data demonstrate the therapeutic potential of targeting multiple cooperating oncogenes.

Different sensitivity of DNA in situ in interphase and metaphase chromatin to heat denaturation.

Heat denaturation of DNA in situ, in unbroken cells, was studied in relation to the cell cycle. DNA in metaphase cells denatured at lower temperatures (8 degrees-10 degrees C lower) than DNA in interphase cells. Among interphase cells, small differences between G1, S, and G2 cells were observed at temperatures above 90 degrees C. The difference between metaphase and interphase cells increased after short pretreatment with formaldehyde, decreased when cells were heated in the presence of 1 mM MgCl2, and was abolished by cell pretreatment with 0.5 N HCl. The results suggest that acid-soluble constituents of chromatin confer local stability to DNA and that the degree of stabilization is lower in metaphase chromosomes than in interphase nuclei. These in situ results remain in contrast to the published data showing no difference in DNA denaturation in chromatin isolated from interphase and metaphase cells. It is likely that factors exist which influence the stability of DNA in situ are associated with the super-structural organization of chromatin in intact nuclei and which are lost during chromatin isolation and solubilization. Since DNA denaturation is assayed after cell cooling, there is also a possibility that the extent of denatured DNA may be influenced by some factors that control strand separation and DNA reassociation. The different stainability of interphase vs. metaphase cells, based on the difference in stability of DNA, offers a method for determining mitotic indices by flow cytofluorometry, and a possible new parameter for sorting cells in metaphase.

DNA denaturation in situ. Effect of divalent cations and alcohols.

Heat denaturation profiles of rat thymus DNA, in intact cells, reveal the presence of two main DNA fractions differing in sensitivities to heat. The thermosensitive DNA fraction shows certain properties similar to those of free DNA: its stability to heat is decreased by alcohols and is increased in the presence of the divalent cations Ca2+, Mn2+, or Mg2+ at concentrations of 0.1-1.0 mM. Unlike free DNA, however, this fraction denatures over a wide range of temperature, and is heterogeneous, consisting of at least two subfractions with different melting points. The thermoresistant DNA fraction shows lowered stability to heat in the presence of Ca2+, Mn2+, or Mg2+ and increased stability in the presence of alcohols. It denatures within a relatively narrow range of temperature, consists of at least three subfractions, and, most likely, represents DNA masked by histones. The effect of Ca2+, Mn2+, or Mg2+ in lowering the melting point of the thermoresistant DNA fraction is seen at cation concentrations comparable to those required to maintain gross chromatin structure in cell nuclei or to support superhelical DNA conformation in isolated chromatin (0.5-1.0 mM). It is probable that factors involved in the maintenance of gross chromatin organization in situ and/or related to DNA superhelicity also have a role in modulating DNA-histone interactions, and that DNA-protein interactions as revealed by conventional methods using isolated chromatin may be different from those revealed when gross chromatin morphology remains intact.

Normal and perturbed Chinese hamster ovary cells: correlation of DNA, RNA, and protein content by flow cytometry.

Quantitative, correlated determinations of DNA, RNA, and protein, as well as RNA to DNA and RNA to protein ratios, were performed on three-color stained cells using a multiwavelength-excitation flow cytometer. DNA-bound Hoechst 33342 (blue), protein-fluorescein isothiocyanate (green), and RNA-bound pyronin Y (red) fluorescence measurements were correlated as each stained cell intersected three spatially separated laser beams. The analytical scheme provided sensitive and accurate fluorescence determinations by minimizing the effects of overlap in the spectral characteristics of the three dyes. Computer analysis was used to generate two-parameter contour density profiles as well as to obtain numerical data for subpopulations delineated on the basis of cellular DNA content. Such determinations allowed for analysis of RNA to DNA and RNA to protein ratios for cells within particular regions of the cell cycle. The technique was used to study the interrelationship of DNA, RNA, and protein contents in exponentially growing Chinese hamster ovary cells as well as in cell populations progressing the cell cycle after release from arrest in G1 phase. The sensitivity of the method for early detection of conditions of unbalanced growth is demonstrated in the comparison of the differential effects of the cycle-perturbing agent, adriamycin, on cells treated either during exponential growth or while reversibly arrested in G1 phase.

Quantitative studies on enzymes in structures in striated muscles by labeled inhibitor methods. I. The number of acetylcholinesterase molecules and of other DFP-reactive sites at motor endplates, measured by radioautography.

Di-isopropylfluorophosphate (DFP) labeled with phosphorus-32 was applied to fragments of the diaphragm and sternomastoid muscles of the mouse, in conditions in which it saturated all available sites at the motor endplates. After adequate washing and exchange with unlabeled DFP, single endplates were obtained by microdissection and their radioactivity was found by beta track radioautography. The number of sites phosphorylated by DFP-(32)P per endplate was relatively constant for each muscle: in the sternomastoid, about 9 x 10(7) sites per endplate, in the diaphragm, about 3 x 10(7). Reaction with DFP-(32)P was abolished by prior treatment with unlabeled DFP. Labeling was unaffected by prior fixation in formaldehyde, but was inversely proportional to the time of incubation in the Koelle staining medium, when this preceded labeling. The contribution of acetylcholinesterase (AChase) to this total number of DFP-reactive sites was determined by three methods. The first involved reactivation of the phosphorylated AChase by pyridine-2-aldoxime methiodide (2-PAM), in conditions in which the reactivation of other enzymes would be insignificant. The other two methods involved protection of the active centers of AChase from phosphorylation by labeled DFP by use of 284C51, an inhibitor highly specific for this enzyme, or by use of eserine. Each of these methods indicated that about 35% of the DFP-reactive sites at endplates of the sternomastoid and diaphragm are AChase. The mean number of AChase molecules was thus found to be 3.1 x 10(7) and 1.1 x 10(7)per endplate in sternomastoid and diaphragm, respectively. No significant reaction of labeled DFP with muscle and nerve was observed. Mast cells in the muscle had a concentration of DFP-reactive sites far higher than the endplates.

Relation of mammalian sperm chromatin heterogeneity to fertility.

Flow cytometry of heated sperm nuclei revealed a significant decrease in resistance to in situ denaturation of spermatozoal DNA in samples from bulls, mice, and humans of low or questionable fertility when compared with others of high fertility. Since thermal denaturation of DNA in situ depends on chromatin structure, it is assumed that changes in sperm chromatin conformation may be related to the diminished fertility. Flow cytometry of heated sperm nuclei may provide a new and independent determinant of male fertility.

Correlated measurements of DNA, RNA, and protein in individual cells by flow cytometry.

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.

Autoradiography with tritiated methotrexate and the cellular distribution of folate reductase.

Bound methotrexate has been revealed by an autoradiographic procedure, presumed to introduce a method for cytochemical study of folate reductase. Preferential localization is seen in kidney proximal tubules, intestinal epithelium, and nuclei of parenchymal liver cells in mice. The extremely firm binding and prolonged retention of this drug should render it suitable as an inert label for the autoradiographic study of cell migrations and lifetimes.

Increased sensitivity of lymphocytes from people over 65 to cell cycle arrest and chromosomal damage.

Flow cytometry revealed that, in the presence of tritiated thymidine, a greater percentage of phytohemagglutinin-stimulated lymphocytes from old human donors were arrested in the G2 or M phase than were cells from young donors. Furthermore, lymphocytes from old donors showed significantly more chromosomal damage than did lymphocytes from young donors. Lymphocyte cultures from old or young donors not exposed to tritiated thymidine had the same percentage of cycling lymphocytes in G2 or M, although the number of lymphocytes stimulated by phytohemagglutinin to enter the cell cycle was significantly lower in cultures from old donors. Thus, the impaired incorporation of tritiated thymidine by phytohemagglutinin-exposed lymphocytes from old humans reflects both an impaired proliferative response to phytohemagglutinin and an increased sensitivity to the radiobiological effects of tritiated thymidine.

BubR1 is involved in regulation of DNA damage responses.

Defective mitotic spindles or an impaired spindle-kinetochore interaction activates the spindle checkpoint. We have previously shown that BubR1 haplo-insufficiency results in enhanced genomic instability and tumorigenesis in mice. Here we report that BubR1 deficiency also leads to a compromised response to DNA damage. Following treatment with doxorubicin, BubR1(+/-) murine fibroblast cells (MEF) were defective in undergoing G(2)/M arrest. Thus, whereas in the presence of DNA damage BubR1(+/+) MEF cells remained arrested in mitosis, BubR1(+/-) MEFs rapidly exited from mitosis and divided. The impaired mitotic arrest of BubR1(+/-) MEFs was associated with low levels of phospho-histone H2AX, p53, and p21 after DNA damage caused by treatment with both doxorubicin and ultraviolet light (UV). The impaired expression of p53 and p21 was also confirmed in human cell lines with BubR1 knockdown via RNA interference. Affinity pull-down coupled with mass spectrometry identified Poly(ADP-ribose) polymerase 1 (PARP-1) as one of the proteins interacting with BubR1. Reciprocal co-immunoprecipitation analysis confirmed the physical interaction between BubR1 and PARP-1. Our further study revealed that the ability of retaining intact PARP-1 or its cleavage product p89 was compromised in BubR1(+/-) MEFs upon treatment with doxorubicin or UV. Given that PARP-1 mediates DNA damage responses and regulates the activity of p53, our studies suggest that there exists a cross-talk between the spindle checkpoint and the DNA damage checkpoint and that BubR1 may play an important role in mediating the cross-talk.

Constitutive histone H2AX phosphorylation and ATM activation are strongly amplified during mitogenic stimulation of lymphocytes.

OBJECTIVES: We recently postulated that constitutive activation of Ataxia Telangiectasia, Mutated (CAA) and constitutive histone H2AX phosphorylation (CHP) seen in cells not treated with genotoxic agents are the events triggered by DNA damage caused by endogenous reactive oxygen species (ROS), the product of mitochondrial oxidative metabolism. The aim of this study was to seek further evidence in support of this postulate, namely to test whether the levels of CAA and CHP correlate with cells metabolic activity. MATERIALS & METHODS: Peripheral blood lymphocytes are non-cycling (G(0)) cells characterized by minimal rate of oxidative metabolism. A dramatic rise in transcriptional and translational activity, an increase in number of mitochondria, and induction of DNA replication, occur during their mitogenic stimulation. This classic model of cell activation was chosen to study a possible correlation between CAA and CHP versus metabolic activity and generation of ROS. RESULTS: The levels of CAA and CHP in lymphocytes were increased many-fold during their stimulation. This increase was paralleled by the rise in extent of endogenously generated ROS. The growth of stimulated lymphocytes in the presence glucose antimetabolite 2-deoxy-D-glucose led to markedly lowered translational activity, decreased ROS generation and correspondingly attenuated CHA and CAA. CONCLUSIONS: The present data are consistent with our postulate that CHP and CAA report DNA damage by endogenous oxidants whose level correlates with metabolic activity. Because cumulative DNA damage by ROS generated via oxidative metabolism is considered the key mechanism responsible for cell ageing and senescence the data imply that these processes are delayed in G(0) quiescent lymphocytes or stem cells as compared with proliferating cells.

Human keratinocyte culture. Identification and staging of epidermal cell subpopulations.

Stratification of human epidermal cells into multilayered sheets composed of basal and suprabasal layers (resembling the stratum germinativum and stratum spinosum of the epidermis) was studied in a dermal component-free culture system. Although no stratum corneum developed in vitro, this culture system provided a method to study early events in human keratinocyte differentiation. Multiparameter flow cytometric analysis of acridine orange-stained epidermal cells from these cultures revealed three distinct subpopulations differing in cell size, RNA content, and cell cycle kinetics. The first subpopulation was composed of small basal keratinocytes with low RNA content and a long generation time. The second subpopulation consisted of larger keratinocytes, having higher RNA content and a significantly shorter generation time. Finally, the third subpopulation contained the largest cells, which did not divide, and represent the more terminally differentiated keratinocytes. This in vitro approach provides discriminating cytochemical parameters by which the maturity of the epidermal cell sheets can be assessed prior to grafting onto human burn patients.

Onconase, an anti-tumor ribonuclease suppresses intracellular oxidative stress.

Onconase (ONC), an antitumor ribonuclease from oocytes of a frog Rana pipiens, capable of inducing apoptosis in many cell lines is synergistic with several other anticancer drugs. Since cytotoxic effects of numerous drugs are modulated by reactive oxygen intermediates (ROI), we have studied effects of ONC on the intracellular level of oxidants in several normal cell types as well as tumor cell lines. It is demonstrated for the first time that ONC substantially decreases the content of ROI in all cell lines studied. This effect depends on the ribonucleolytic activity of the enzyme and is due to both, decreased rate of ROI generation and accelerated rate of their degradation. Onconase decreases the mitochondrial transmembrane potential and consequently, generation of ATP. Simultaneously the enzyme decreases the expression of an antiapoptotic protein Bcl-2, and upregulates the proapoptotic Bax protein. These finding are consistent with the enzyme propensity to induce apoptosis. The observed antioxidant activity of ONC may be an important element of its cytotoxicity towards cancer cells. The enzyme seems to exert its biological activities by interfering with the redox system of cellular regulation.

Binding of anti-Z-DNA antibodies in quiescent and activated lymphocytes: relationship to cell cycle progression and chromatin changes.

Although regions of DNA reacting with anti-Z-DNA antibodies have been identified in the polytene chromosomes of Drosophila spp. and the metaphase chromosomes from a number of different mammalian species, the biological role of this DNA is unknown. Flow cytometry was used in the present studies to quantitate the binding of anti-Z-DNA antibodies in quiescent and activated human peripheral blood lymphocytes; the antibody binding was then correlated with cell cycle phase. The data show that quiescent (G0 or G1Q) lymphocytes are heterogeneous with respect to their reaction with anti-Z-DNA antibodies. The transition from quiescence (G1Q) into the cell cycle (G1), which involves decondensation of chromatin, did not result in any significant change in binding of these antibodies. In contrast, progression of cells from G1 through S and G2 is correlated with a 27% decrease in anti-Z-DNA antibody reactivity relative to total DNA content. No significant change was observed during the transition from G2 to mitosis (M).

Extent of constitutive histone H2AX phosphorylation on Ser-139 varies in cells with different TP53 status.

In response to DNA damage by genotoxic agents, histone H2AX is phosphorylated on Ser-139. However, during the cell cycle, predominantly in S and G(2)M phase, histone H2AX is also phosphorylated in untreated normal and tumour cells. This constitutive H2AX phosphorylation is markedly reduced by exposure of cells to the reactive oxygen species scavenger N-acetyl-L-cysteine. Therefore, it appears likely that constitutive H2AX phosphorylation reflects the ongoing oxidative DNA damage induced by the reactive oxygen species during progression through the cell cycle. Because the tumour suppressor p53 (tumour protein p53) is known to induce transcription of genes associated with cell response to oxidative stress, we have compared the intensity of constitutive H2AX phosphorylation, and the effect of N-acetyl-L-cysteine on it, in cells with different tumour protein p53 status. These were human lymphoblastoid cell lines derived from WIL2 cells: TK6, a p53 wt line, NH32, a tumour protein p53 knock-out derived from TK6, and WTK1, a WIL2-derived line that expresses a homozygous mutant of tumour protein p53. Also tested were the tumour protein p53-null promyelocytic HL-60 cells. The degree of constitutive H2AX phosphorylation was distinctly lower in NH32, WTK1 and HL-60 compared to TK6 cells in all phases of the cell cycle. Also, the degree of attenuation of constitutive H2AX phosphorylation by N-acetyl-L-cysteine was less pronounced in NH32, WTK1, and HL-60, compared to TK6 cells. However, the level of reactive oxygen species detected by the cells' ability to oxidize carboxyl-dichlorodihydrofluorescein diacetate was not significantly different in the cell lines studied, which would suggest that regardless of tumour protein p53 status, the level of oxidative DNA damage was similar. The observed higher level of constitutive H2AX phosphorylation in cells harbouring wt tumour protein p53 may thus indicate that tumour protein p53 plays a role in facilitating histone H2AX phosphorylation, an important step in the mobilization of the DNA repair machinery at the site of DNA double-strand breaks.

Synchronization in the cell cycle by inhibitors of DNA replication induces histone H2AX phosphorylation: an indication of DNA damage.

Several methods to synchronize cultured cells in the cell cycle are based on temporary inhibition of DNA replication. Previously it has been reported that cells synchronized this way exhibited significant growth imbalance and unscheduled expression of cyclins A and B1. We have now observed that HL-60 cells exposed to inhibitors of DNA replication (thymidine, aphidicolin and hydroxyurea), at concentrations commonly used to synchronize cell populations, had histone H2AX phosphorylated on Ser-139. This modification of H2AX, a marker of DNA damage (induction of DNA double-strand breaks; DSBs), was most pronounced in S-phase cells, and led to their apoptosis. Thus, to a large extent, synchronization was caused by selective kill of DNA replicating cells through induction of replication stress. In fact, similar synchronization has been achieved by exposure of cells to the DNA topoisomerase I inhibitor camptothecin, a cytotoxic drug known to target S-phase cells. A large proportion of the surviving cells 'synchronized' by DNA replication inhibitors at the G1/S boundary had phosphorylated histone H2AX. Inhibitors of DNA replication, thus, not only selectively kill DNA replicating cells, induce growth imbalance and alter the machinery regulating progression through the cycle, but they also cause DNA damage involving formation of DSBs in the surviving ('synchronized') cells. The above effects should be taken into account when interpreting data obtained with the use of cells synchronized by inhibitors of DNA replication.

ATM activation and histone H2AX phosphorylation as indicators of DNA damage by DNA topoisomerase I inhibitor topotecan and during apoptosis.

Damage that engenders DNA double-strand breaks (DSBs) activates ataxia telangiectasia mutated (ATM) kinase through its auto- or trans-phosphorylation on Ser1981 and activated ATM is one of the mediators of histone H2AX phosphorylation on Ser139. The present study was designed to explore: (i) whether measurement of ATM activation combined with H2AX phosphorylation provides a more sensitive indicator of DSBs than each of these events alone, and (ii) to reveal possible involvement of ATM activation in H2AX phosphorylation during apoptosis. Activation of ATM and/or H2AX phosphorylation in HL-60 or Jurkat cells treated with topotecan (Tpt) was detected immunocytochemically in relation to cell cycle phase, by multiparameter cytometry. Exposure to Tpt led to concurrent phosphorylation of ATM and H2AX in S-phase cells, whereas G1 cells were unaffected. Immunofluorescence (IF) of the S-phase cells immunostained for ATM-S1981P and gammaH2AX combined was distinctly stronger compared to that of the cells stained for each of these proteins alone. However, because of the relatively high ATM-S1981P IF of G1 cells, the ratio of IF of S to G1 cells, that is, the factor that determines competence of the assay in distinction of cells with DSBs, was 2- to 3-fold lower for ATM-S1981P alone, or for ATM-S1981P and gammaH2AX IF combined, than for gammaH2AX alone. ATM activation concurrent with H2AX phosphorylation, likely triggered by induction of DSBs during DNA fragmentation, occurred during apoptosis. The data suggest that frequency of activated ATM and phosphorylated H2AX molecules, per apoptotic cell, is comparable.

Effects of retinoic acid versus dimethyl sulfoxide on Friend erythroleukemia cell growth. I. Cell proliferation, RNA content, and protein content.

The effect of all-trans-retinoic acid (RA), an oxidative product of vitamin A, on cell growth, cell cycle kinetics, RNA content, and protein content of exponentially growing Friend erythroleukemia (FL) cells was determined and compared with the results obtained with dimethyl sulfoxide [(DMSO) CAS: 67-68-5; methyl sulfoxide], an inducer of differentiation, and alpha-difluoromethylornithine (DFMO), a potent inhibitor of ornithine decarboxylase (EC 4.1.1.17) activity. Growth inhibition of FL cells was observed only during continuous treatment with RA. While RA did not prevent growth to a high cell density, if cultures were maintained in exponential growth, cell number was reduced by 37, 67.4, and 72.2% after 6-day exposure to 10(-7), 10(-6), and 10(-5) M RA, respectively. In comparison, 280 mM (2%) DMSO and 5 mM DFMO inhibited growth over the same time course by 92 and 97.3%, respectively. DMSO resulted in an early, transient (18-24 hr) accumulation of cells in G1 phase followed by a later (5 day), irreversible accumulation of G1 cells. RA required several cell generations (48-72 hr) before a dose-dependent G1 accumulation was observed. Two populations of RA-treated FL cells could be identified: one with an intermediate RNA content (T-cells) similar to near-plateau-phase control cultures and the other with low RNA content (Q-cells) similar to that observed for DMSO-differentiated (D) cells. The kinetics of the decrease in RNA content of Q-cells paralleled those of D-cells in DMSO-treated cultures; the proportion of Q- versus T-cells in RA-treated cultures was dependent on both concentration and length of exposure. DFMO treatment did not give rise to low-RNA-containing Q-cells. Protein content of RA-treated cells was also diminished and approached that observed for hemoglobin-containing D-cells. FL cells were recoverable from long-term (greater than 5 days) treatment with RA, though 2-3 days were required for reestablishment of exponentially growing cultures; apparently only moderate RNA-containing T-cells repopulated the culture. Neither RA nor DFMO treatment gave rise to benzedine-positive, hemoglobin-containing cells as compared to DMSO that induced differentiation in these cultures.

Effects of retinoic acid versus dimethyl sulfoxide on Friend erythroleukemia cell growth. II. Induction of quiescent, nonproliferating cells.

Treatment of Friend erythroleukemia (FL) cells in vitro with 10(-7) to 10(-5) all-trans-retinoic acid (RA) leads to a concentration-dependent accumulation of a subpopulation of quiescent cells. This subpopulation, termed "Q-cells," contained markedly reduced RNA and protein levels and had a cell cycle distribution with a predominance of cells in G1 phase, which was nearly identical to that found in fully differentiated dimethyl sulfoxide (CAS:67-68-5; methyl sulfoxide)-induced FL cultures. The G1 cells in this RA-induced subpopulation (G1Q cells), though viable, did not enter S-phase, whereas the small percentage of Q-cells with S and G2 DNA content progressed very slowly through the cycle. While the Q-cell population did not contain the differentiation-associated chromatin protein H1 degrees, the cells did manifest a more condensed nuclear chromatin, altered sensitivity to acid denaturation, and reduced accessibility of the DNA in chromatin to acridine orange. The extent of chromatin condensation and the number of free ribosomes versus polysomes in RA-treated FL cells were intermediate between those in untreated and fully differentiated cells, whereas viral budding and the number of nucleoli remained unchanged from those seen in the untreated cell state. The non-Q-cell population in RA-treated cultures, termed "T" (transitional) cells, had an intermediate RNA and protein content and a cell cycle distribution similar to those of control cultures nearing the plateau phase of growth. In the absence of any late markers of differentiation, the Q-cell population was tentatively identified as a unique, quiescent cell population not previously described in the FL cell system.