Quantitation of cell kinetic responses using simultaneous flow cytometric measurements of DNA and nuclear protein

A rapid procedure was developed for the simultaneous flow cytometric analysis of nuclear protein using fluorescein isothiocyanate, and DNA using propidium iodide in isolated nuclei. The staining procedure did not involve centrifugation and was easily adapted to the staining of human peripheral blood lymphocytes stimulated with phytohemagglutinin, EL4 murine lymphoid tumor cells in suspension culture, and R3327-G rat prostatic adenocarcinoma solid tumor specimens. Histograms of unstimulated and PHA-stimulated HPBL perturbed by actinomycin D, hydroxyurea, 3H-TdR, colcemid, or hydroxyurea + colcemid showed that 1) resting, noncycling G1 (G1Q) cells are distinguished from late G1 (G1AB) cells, 2) early G2 (G2A) cells are distinguished from late G2 (G2B) cells, and 3) mitotic cells are distinguished from G2 cells. Treatment with hydroxyurea resulted in a build-up of cells having high nuclear protein content and 2C DNA content (G1AB), while incubation with 3H-TdR caused an increase in the number of cells with high nuclear protein content and 4C DNA content (G2B). Colcemid-blocked mitotic cells were identified as having low nuclear protein content (lower than G2A nuclei) and 4C DNA content. The nuclear DNA/protein histograms of untreated and colcemid-treated log-phase EL4 cells provided information concerning G1A, G1B, S, G2A, G2B, and M. The method was also used to quantitate the response of androgen-sensitive rat prostatic R3327-G tumors to androgen deprivation following castration. Sample preparation and staining for correlated nuclear DNA/protein measurements takes approximately the same amount of time as for single parameter nuclear DNA measurements.     

Human cytomegalovirus infection inhibits G1/S transition.

Cell cycle progression during cytomegalovirus infection was investigated by fluorescence-activated cell sorter (FACS) analysis of the DNA content in growth-arrested as well as serum-stimulated human fibroblasts. Virus-infected cells maintained in either low (0.2%) or high (10%) serum failed to progress into S phase and failed to divide. DNA content analysis in the presence of G1/S (hydroxyurea and mimosine) and G2/M (nocodazole and colcemid) inhibitors demonstrated that upon virus infection of quiescent (G0) cells, the cell cycle did not progress beyond the G1/S border even after serum stimulation. Proteins which normally indicate G1/S transition (proliferating cell nuclear antigen [PCNA]) or G2/M transition (cyclin B1) were elevated by virus infection. PCNA levels were induced in infected cells and exhibited a punctate pattern of nuclear staining instead of the diffuse pattern observed in mock-infected cells. Cyclin B1 was induced in infected cells which exhibited a G1/S DNA content by FACS analysis, suggesting that expression of this key cell cycle function was dramatically altered by viral functions. These data demonstrate that contrary to expectations, cytomegalovirus inhibits normal cell cycle progression. The host cell is blocked prior to S phase to provide a favorable environment for viral replication.     

Sequence and structural analysis of the rfb (O antigen) gene cluster from a group C1 Salmonella enterica strain.

The rfb (O antigen) gene cluster of a group C1 Salmonella enterica strain was sequenced; it comprised seven open reading frames which precisely replaced the 16 open reading frames of a group B strain. Two genes of the mannose biosynthetic pathway were present: rfbK (phosphomannomutase) had a G+C content of 0.61 and had only 40% identity to rfbK of group B but was very similar to cpsG of the capsular polysaccharide pathway with 96% identity, whereas rfbM [guanosine diphosphomannose (GDP-Man) pyrophosphorylase] had a G+C content of 0.39. Other genes had G+C contents ranging from 0.24 to 0.28. rfbM(C1) and rfbM(B) had 60% identity, which is much less than expected within a species, but nonetheless indicates a much more recent common ancestor than for rfbK. The other genes showed much lower or no similarity to rfb genes of other S. enterica strains. It appears that the gene cluster evolved outside of Salmonella in a species with low G+C content: the rfbM gene presumably derives from that period whereas the rfbK gene appears to have arisen after transfer of the cluster to S. enterica by duplication of the S. enterica cpsG gene, presumably replacing an rfbK gene of low G+C content.     

Differentiation of HL-60 myeloid leukaemia cells is associated with a transient block in the G2 phase of the cell cycle

The human promyelocytic leukaemia cell line HL-60 can be induced to differentiate towards mature granulocytes by treatment with dibutyryl cyclic adenosine-3',5'-monophosphate (dbcAMP). Differentiation begins within 16-24 h of treatment and is associated with a time- and dose-dependent accumulation of cells in the G0/G1 phase of the cell cycle with a concomitant decrease in the number of cells in the S and G2 + M phases. Using acridine orange staining, we found that the RNA content of the cells also decreased following differentiation. Stathmokinetic analysis of HL-60 cell populations following dbcAMP treatment showed no effect on the total number of cells in the G0/G1 or S phases, or the rate of progression of cells through these cell cycle compartments. In contrast, dbcAMP was found to induce a transient arrest of the cells in the G2 phase. We also found that differentiation induced by dbcAMP did not require progression of the cells through the cell cycle. Cells arrested in either G1/S by hydroxyurea or G2 + M by colcemid eventually expressed markers of mature granulocytes. These results demonstrate that dbcAMP modulates cell cycle progression. However, these cell cycle changes alone are insufficient to induce granulocytic differentiation of HL-60 cells.     

Dual effect of protein kinase C on the induction of DNA synthesis by colcemid in G0-arrested human diploid fibroblasts

G0-arrested human diploid fibroblasts, TIG-1, was stimulated to induce DNA synthesis by serum, epidermal growth factor (EGF), colchicine, colcemid, or 12-O-tetradecanoylphorbol-13-acetate (TPA). The induction of DNA synthesis was mediated by protein kinase C (PKC) when stimulated with TPA but not when stimulated with other agents. When TPA-stimulated cells were immediately treated with colcemid, induction of DNA synthesis was reduced. This reduction diminished when colcemid was added more than 6 h after TPA treatment. Conversely, when colcemid-stimulated cells were treated with TPA, induction of DNA synthesis was also reduced. This reduction was enhanced when the interval between the addition of two stimulants was extended. PKC-deprivation abolished both stimulatory and inhibitory effects of TPA on DNA synthesis. Staurosporine blocked an induction of DNA synthesis by TPA but appeared to be ineffective on the inhibitory action of TPA on DNA synthesis by colcemid. These results suggest that the inhibitory effect of TPA on the induction of DNA synthesis by colcemid is mediated by down regulation-sensitive and staurosporine-insensitive PKC.     

Incorporation of 35S-sulfate and 3H-glucosamine into heparan and chondroitin sulfates during the cell cycle of B16-F10 cells

Changes in glycosaminoglycan composition occurring during the cell cycle were determined in B16-F10 cells sorted flow cytometrically with respect to DNA content. Incorporation of 35S-sulfate into heparan sulfate and chondroitin sulfate of unsorted and G1,S, and G2 +M sorted cells was determined following chondroitinase ABC or nitrous acid treatment; the incorporation into surface material was measured as the difference between the radioactivity of control and trypsin-treated cells. Incorporation of 35S-sulfate and 3H-glucosamine into cetyl pyridinium chloride (CPC)-precipitable material was characterized before and after chondroitinase or nitrous acid treatment by Sephadex G50 chromatography. Long-term (48 h) and short-term (1 h) labeling studies demonstrate that (a) the amount of total cellular chondroitin sulfate is greater than that of heparan sulfate, with larger amounts of unsulfated heparan than chondroitin being present; (b) the rate of turnover of heparan sulfate is greater than that of chondroitin sulfate; (c) greatest short-term incorporation of 3H-glucosamine into CPC-precipitable material occurs during S phase; and (d) the rate of turnover of both heparan sulfate and chondroitin sulfate is decreased in S phase relative to G1 and G2 + M.     

A new method to discriminate G1, S, G2, M, and G1 postmitotic cells

A new flow cytometric method combining light scattering measurements, detection of bromodeoxyuridine (BrdU) incorporation via fluorescent antibody, and quantitation of cellular DNA content by propidium iodide (PI) allows identification of additional compartments in the cell cycle. Thus, while cell staining with BrdU-antibodies and PI reveals the G1, S, and G2 + M phases of the cell cycle, differences in light scattering allow separation of G2 phase cells from M phase cells and subdivision of G1 phase into two compartments, i.e., G1A representing postmitotic cells which mature to G1B cells ready to initiate DNA synthesis. The method involves fixation of cells in 70% ethanol, extraction of histones with HC1, and thermal denaturation of DNA. This treatment appears to enhance the differences in chromatin structure of cells in the various phases of the cell cycle to the extent that cells could be separated on the basis of the 90 degrees scatter. Mitotic cells show much lower scatter than G2 phase cells, and G1 postmitotic cells (G1A) show lower scatter than G1 cells about to enter the S phase (G1B). Light scattering is correlated with chromatin condensation, as judged by microscopic evaluation of cells sorted on the basis of light scatter. The method has the advantage over the parental BrdU/DNA bivariate analysis in allowing the G2 and M phases of the cell cycle to be separated and the G1 phase to be analyzed in more detail. The method may also allow separation of unlabeled S phase cells from mitotic cells and distinguish between labeled and unlabeled mitotic cells.     

c-Myc Overexpression Uncouples DNA Replication from Mitosis

c-myc has been shown to regulate G(1)/S transition, but a role for c-myc in other phases of the cell cycle has not been identified. Exposure of cells to colcemid activates the mitotic spindle checkpoint and arrests cells transiently in metaphase. After prolonged colcemid exposure, the cells withdraw from mitosis and enter a G(1)-like state. In contrast to cells in G(1), colcemid-arrested cells have decreased G(1) cyclin-dependent kinase activity and show hypophosphorylation of the retinoblastoma protein. We have found that overexpression of c-myc causes colcemid-treated human and rodent cells to become either apoptotic or polyploid by replicating DNA without chromosomal segregation. Although c-myc-induced polyploidy is not inhibited by wild-type p53 in immortalized murine fibroblasts, overexpression of c-myc in primary fibroblasts resulted in massive apoptosis of colcemid-treated cells. We surmise that additional genes are altered in immortalized cells to suppress the apoptotic pathway and allow c-myc-overexpressing cells to progress forward in the presence of colcemid. Our results also suggest that c-myc induces DNA rereplication in this G(1)-like state by activating CDK2 activity. These observations indicate that activation of c-myc may contribute to the genomic instability commonly found in human cancers.     

An investigation using inhibition of G2 repair of the molecular basis of lesions which result in chromosomal aberrations

Cultures of JU56 cells were irradiated with 2.5 Gy X-rays and 16 h later the cultures were exposed to a moderately inhibitory dose of 1-beta-D-arabinofuranosylcytosine (ara-C) or aphidicolin (APC) and to colcemid, for 2 h. The c-metaphases collected for examination had therefore been exposed to X-rays in G1 or early S, and to the repair inhibitors APC and ara-C during the latter half of G2. It was found that treatment of cells irradiated early in cell cycle, that is, in G1 and early S, with APC or ara-C in G2, (1) reduced the frequency of chromatid and chromosome exchanges below that of cells treated with X-rays alone, (2) produced no more chromatid breaks and gaps than were seen in unirradiated cells, (3) increased the number of chromosome fragments and gaps in a more than additive fashion, and (4) produced only an additive effect, by comparison with the effect of X-rays and drug given separately, on the total number of chromosomal aberrations.     

Regenerative proliferation of mouse epidermal cells following adhesive tape stripping. Micro-flow fluorometry of isolated epidermal basal cells combined with 3H-TdR incorporation and a stathmokinetic method (colcemid).

The proliferating cells of mouse epidermis (basal cells) can be separated from the non-proliferating cells (differentiating cells) Laerum, 1969) and brought into a monodisperse suspension. This makes it possible to determine the cell cycle distributions (e.g. the relative number of cells in the G1, S and (G1 + M) phases of the cell cycle) of the basal cell population by means of micro-flow fluorometry. To study the regenerative cell proliferation in epidermis in more detail, changes in cell cycle distributions were observed by means of micro-flow fluorometry during the first 48 hr following adhesive tape stripping. 3H-TdR uptake (LI and grain count distribution) and mitotic rate (colcemid method) were also observed. An initial accumulation of G2 cells was observed 2 hr after stripping, followed by a subsequent decrease to less than half the control level. This was followed by an increase of cells entering mitosis from an initial depression to a first peak between 5 and 9 hr which could be satisfactorily explained by the changes in the G2 pool. After an initial depression of the S phase parameters, three peaks with intervals of about 12 hr followed. The cells in these peaks could be followed as cohorts through the G2 phase and mitosis, indicating a partial synchrony of cell cycle passage, with a shortening of the mean generation time of basal cells from 83-3 hr to about 12 hr. The oscillations of the proportion of cells in G2 phase indicated a rapid passage through this cell cycle phase. The S phase duration was within the normal range but showed a moderate decrease and the G1 phase duration was decreased to a minimum. In rapidly proliferating epidermis there was a good correlation between change in the number of labelled cells and cells with S phase DNA content. This shows that micro-flow fluorometry is a rapid method for the study of cell kinetics in a perturbed cell system in vivo.     

Colcemid inhibits growth during early G1 in normal but not in tumorigenic lymphocytes

Mitogenically stimulated human and mouse lymphocytes enter the cell cycle (G0, G1A, G1B, S, G2+M) via a newly recognized subphase, G1'. This subphase precedes G1A and is distinct from G0. The G1' subphase is absent in immortalized and tumorigenic lymphoblastoid cell lines (LCLs) by cytofluorimetric criteria. Furthermore, colcemid inhibits transition through the G0/G1' as well as G2 phases in mitogen-stimulated lymphocytes and in LCLs. Tumorigenic LCLs are not sensitive to growth inhibition by colcemid during early G1. These observations suggest that a progressive series of changes have occurred during G0/G1' which lead to deregulation of growth control.     

Variation through the cell cycle in the dose-response of DNA neutral filter elution in X-irradiated synchronous CHO-cells

Dose-response curves for DNA neutral (pH 9.6) filter elution were obtained with synchronized CHO cells exposed to X-rays at various phases of the cell cycle. The dose response was similar in synchronized and plateau-phase G1 cells, as well as in cells that were arrested at the G1/S border using aphidicolin; it flattened as cells progressed into S phase and reached a minimum in the middle of this phase. An increase in DNA elution dose response, to values only slightly lower than those obtained with G1 cells, was observed as cells entered G2 phase. Significant alterations in the sedimentation properties of the DNA during S phase were also observed in Ehrlich ascites tumor cells using the neutral sucrose gradient centrifugation technique. A significant proportion of the DNA from S cells irradiated with 10 Gy sedimented at speeds (350S-700S) well above the maximum sedimentation speed expected for free sedimenting DNA molecules (Smax = 350S), indicating the formation of a DNA complex. DNA from G1, G1/S, or G2 + M cells sedimented as expected for free sedimenting molecules. These results indicate significant alterations in the physicochemical properties of the DNA--probably caused by DNA replication-associated alterations in DNA structure and chromatin conformation--as cells enter S phase, and are invoked to explain the observed variation in DNA elution dose response throughout the cycle. It is proposed that the formation of a complex DNA structure, resistant to the proteolytic enzymes and detergents used, affected the elution characteristics of the DNA and gave rise to the observed curvilinear DNA elution dose-response curves, as well as to the fluctuations in elution characteristics observed throughout the cell cycle.     

Nuclear non-histone protein content of G0/G1 cells as related to growth fraction in mouse mammary tumors.

In eight mouse mammary tumors with varying growth fractions DNA and non-histone nuclear protein (NHNP) were determined by absorption cytophotometry of Feulgen-Naphthol Yellow S stained, isolated cells. It was found that: 1. The mean NHNP content of cells with postmitotic DNA content (G0 + G1) increased with increasing growth fraction. 2. The mean NHNP content of S and G2 cells in the eight tumors did not vary significantly with growth fraction. 3. The frequency distributions of NHNP in G0/G1 cells were unimodal and right-skewed. The results are interpreted as follows: A) G0 cells differ from G1 cells by their lower content of NHNP. B). If it is assumed that the G0 and G1 compartments are arranged in series, the cells in the transition from G0 to late G1 may account for the unimodality and skewedness of the NHNP frequency distributions of postmitotic cells.     

S-phase-dependent cell cycle disturbances caused by Aleutian mink disease parvovirus.

We examined replication of the autonomous parvovirus Aleutian mink disease parvovirus (ADV) in relation to cell cycle progression of permissive Crandell feline kidney (CRFK) cells. Flow cytometric analysis showed that ADV caused a composite, binary pattern of cell cycle arrest. ADV-induced cell cycle arrest occurred exclusively in cells containing de novo-synthesized viral nonstructural (NS) proteins. Production of ADV NS proteins, indicative of ADV replication, was triggered during S-phase traverse. The NS+ cells that were generated during later parts of S phase did not undergo cytokinesis and formed a distinct population, termed population A. Formation of population A was not prevented by VM-26, indicating that these cells were arrested in late S or G2 phase. Cells in population A continued to support high-level ADV DNA replication and production of infectious virus after the normal S phase had ceased. A second, postmitotic, NS+ population (termed population B) arose in G0/G1, downstream of population A. Population B cells were unable to traverse S phase but did exhibit low-level DNA synthesis. Since the nature of this DNA synthesis was not examined, we cannot at present differentiate between G1 and early S arrest in population B. Cells that became NS+ during S phase entered population A, whereas population B cells apparently remained NS- during S phase and expressed high NS levels postmitosis in G0/G1. This suggested that population B resulted from leakage of cells with subthreshold levels of ADV products through the late S/G2 block and, consequently, that the binary pattern of ADV-induced cell cycle arrest may be governed merely by viral replication levels within a single S phase. Flow cytometric analysis of propidium iodide fluorescence and bromodeoxyuridine uptake showed that population A cells sustained significantly higher levels of DNA replication than population B cells during the ADV-induced cell cycle arrest. Therefore, the type of ADV-induced cell cycle arrest was not trivial and could have implications for subsequent viral replication in the target cell.     

Alterations of growth fraction and DNA content in K562 cells by differentiating agents

The growth fraction, estimated by the monoclonal antibody Ki-67 labeling, and DNA content, assessed by ethidium bromide staining, were determined simultaneously in K562 leukemic cells by flow cytometry. A multiparametric analysis enabled the fraction of the cell population with G1, S, and G2 + M contents in Ki-67-positive and Ki-67-negative cells to be evaluated. Butyric acid (BUT) was used as positive control. The fraction of Ki-positive cells decreased with the BUT concentration, while the proportion of cells with G1 DNA content increased only in the Ki-negative cells. Adriamycin, aclacinomycin A, and fagaronine induced differentiation, as assessed by benzidine staining and glycophorin A expression. These drugs decreased the fraction of Ki-positive cells by more than 50% for both anthracyclines and by 25% for fagaronine. Following treatment, Ki-negative cells displayed a G1, but also a G2 and a S DNA content in different proportions, indicating that induction of quiescent cells by differentiating agents is not a uniform process and is worthy of interest.     

Cell cycle phase, cellular Ca2+ and development in Dictyostelium discoideum

In Dictyostelium discoideum, the initial differentiation of cells is regulated by the phase of the cell cycle at starvation. Cells in S and early G2 (or with a low DNA content) have relatively high levels of cellular Ca2+ and display a prestalk tendency after starvation, whereas cells in mid to late G2 (or with a high DNA content) have relatively low levels of Ca2+ and display a prespore tendency. We found that there is a correlation between cytosolic Ca2+ and cell cycle phase, with high Ca2+ levels being restricted to cells in the S and early G2 phases. As expected on the basis of this correlation, cell cycle inhibitors influence the proportions of amoebae containing high or low Ca2+. However, it has been reported that in the rtoA mutant, which upon differentiation gives rise to many more stalk cells than spores (compared to the wild type), initial cell-type choice is independent of cell cycle phase at starvation. In contrast to the wild type, a disproportionately large fraction of rtoA amoebae fall into the high Ca2+ class, possibly due to an altered ability of this mutant to transport Ca2+.     

Dicentric chromosome frequency analysis using slit-scan flow cytometry

Slit-scan flow cytometry (SSFCM) was used to quantify the frequency of dicentric chromosomes in human lymphoblastoid cells following gamma irradiation. In this study, cultured human cells were irradiated with 0, 0.25, 0.5, 1.0, and 2.0 Gy of 0.66 MeV gamma-rays, cultured for an additional 11 h, and treated for 5 h with colcemid. Chromosomes were then isolated, stained with propidium iodide, and analyzed using SSFCM for total fluorescence and slit-scan profile. The frequency of chromosomes having DNA contents greater than once and less than twice the DNA content of the number 1 chromosome and producing trimodal profiles was determined at each dose. This frequency was used as an estimate of the relative dicentric chromosome frequency at that dose. The estimated dicentric chromosome frequency per cell, f(D), increased with dose, D, in a linear-quadratic manner according to the relation f(D) = 4.52 x 10(-5) + 5.72 x 10(-5) D + 1.19 x 10(-4) D2.     

Influence of cell cycle stage of the donor nucleus on development of nuclear transplant rabbit embryos.

We evaluated the influence of the stage of the cell cycle of the donor nucleus on development in vitro of nuclear transplant rabbit embryos. The developmental potential of nuclei in early, mid-, and late stages of the cell cycle was determined. Duration of the G1 phase in early embryos was determined, and a procedure for reversibly synchronizing donor embryos in the G1 phase was developed. In addition, the extent of development in vitro of nuclear transplant embryos with donor nuclei synchronized in the G1 phase was evaluated. Development to blastocysts was greatly affected by the stage of the cycle of the donor nucleus. Use of early-stage nuclei led to 59% nuclear transplant blastocysts, whereas 32% and 3% were obtained with mid- and late-stage nuclear donors, respectively (p less than 0.001). The short duration of the G1 phase in 16- and 32-cell-stage embryos (approximately 30 min) necessitated a procedure for synchronizing blastomeres in the G1 phase. This entailed, first, a 10-h incubation in 0.5 micrograms/ml colcemid to arrest embryos in metaphase. After release from colcemid, embryos were allowed to cleave in 0.1 microgram/ml of the DNA synthesis inhibitor, aphidicolin, and remained blocked at the G1/S transition. This treatment was reversible, as assessed by the resumption of DNA synthesis, cleavage rate, and development to blastocysts of treated embryos. The beneficial effect of using early-stage donor blastomeres was confirmed by the enhanced rate of development of manipulated embryos to blastocysts with donor nuclei in the G1 phase (71%), as opposed to the late S phase (15%, p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

用双参数流式细胞光度术(FCM)研究阿克拉霉素对L_(1210)白血病细胞周期的影响

本文用双参数FCM技术,对同一个细胞的DNA和RNA含量进行相关测量,比较了ACM B对小鼠L_(1210)白血病细胞周期和RNA含量的影响.结果发现在一次给药后8小时可导致早、中期S的积累,并抑制S期细胞的DNA合成;到24小时DNA合成恢复正常,并进入G_2期,但由于G_2期细胞进入M期受阻,造成G_2期细胞的积累,这时被阻断在G_2期的细胞RNA含量显著增加,形成正不平衡生长,而给药剂量较大的实验组(1/1.5LD_(50))S期细胞的RNA含量不随着DNA含量的增加而增加,形成负不平衡生长,ACM A和ACM B对体内Li_(210)细胞周期作用相同.     

The synergistic anti-proliferative effect of gamma-interferon and human lymphotoxin

Highly purified preparations of rHuIFN-gamma and HLT synergistically inhibit the in vitro proliferation of WI38VA13 cells, a SV40 virus-transformed derivative of the human cell line W138. The growth of nontumorigenic WI38 cells, established from normal diploid embryonic lung tissue, was not inhibited under identical incubation conditions; rMuIFN-gamma and HLT have the same in vitro effects on the murine melanoma B16. Thus, the combination of rMuIFN-gamma and HLT synergistically inhibits the growth of B16 cells, although incubation with either lymphokine alone has no significant anti-proliferative effect. Flow cytometric analysis of the cell cycle of asynchronous cells indicates that when B16 melanoma cells are treated with rMuIFN-gamma and HLT in combination, more cells accumulate in the G0/G1 phase than when the cells are treated with rMuIFN-gamma alone. It appears that populations of rapidly proliferating tumor cells, which normally contain a large percentage in the S+G2+M phases of the cell cycle, become arrested in the quiescent G0/G1 phase by the combination of IFN-gamma and HLT, resulting in the inhibition of cell growth.