Flow cytometric analysis of the recruitment of G0 cells in human epidermis in vivo following tape stripping

Abstract. Tape stripping of human skin elicits a proliferative response of a synchronously-dividing group of cells. The progress of this cohort of cells has been monitored using two windows in the cell cycle, one located in mid-S phase and the other centred around G₂+ M. The cellular DNA is measured with flow cytometry, the windows are defined by two ranges in the DNA histogram.
The cohort can be described as the recruitment of cells from a pre-existing G₀ compartment which consists of 76% of all proliferative cells. The duration of the S phase is calculated to be 10.2 hr and G₂+ M phase 5.1 hr. The cell cycle time of 39 hr for normal human keratinocytes derived from these figures is in line with recent values obtained by different techniques.     

The differentiation of T-lymphocytes. III. The behaviour of subpopulations of mouse thymus cells in short-term cell culture.

Seven human cultured lymphoblastoid cell lines (CLL) were divided into two major groups based on studies of their cell cycle characteristics and surface Ig. CLL I (lines CL, MW, HH and TM) had generation times ranging from 25–40 hr, S phase times of 10–12 hr, G₂ + M times of 6–8 hr, and demonstrated sharp differences between the percentage of SIg(+) cells in different phases of the cell cycle. Line TM was particularly discordant with the highest percentage of SIg(+) cells in G₂ + M. CLL II (lines PS, JR and HT) demonstrated generation times ranging from 18–21 hr, S phase times of 7–10 hr and G₂ + M phase times of 2 hr. In this second group, two of the three CLLs had no differences between cells taken from different points of the cell cycle. DNA synthesis and cell density could not be correlated with either of the above major parameters, i.e. cell cycle times or SIg expression. The results suggest that human CLLs fall into subgroups in which specific patterns of cellular and immune functions may predominate.     

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.     

Non-circadian periodicity in the duration of the cell cycle and the G1 phase in the hindlimb epidermis of the anuran tadpole, Rana pipiens

Using the percentage labeled mitoses method, seven cell cycle determinations were initiated at 6-hr intervals over a 36-hr span in order to see if the cell cycle in the tadpole hindlimb epidermis varied with time or showed rhythmicity. There was a pattern of two long cell cycles followed by a shorter one. Total cell cycle length (Tc) and the length of the G1 phase plus one-half of the mitotic time (TG1 + 1/2M) fluctuated the most, although only TG1 + 1/2M varied significantly with the Chi-square test. The proportion of TC spent in each phase was also calculated. Only TG1 + 1/2M/TC had statistically significant fluctuations with time. Rhythmicity was analyzed by a computer program using the method of least squares for cosine curve fitting. Statistically significant ultradian rhythms of 18.4 hr in TC, 18.5 hr in TG1 + 1/2M and 18.6 hr in TG1 + 1/2M/TC and the length of the DNA synthetic phase/total cell cycle length (TS/TC) were found. Circadian rhythmicity was not observed. The acrophases of the ultradian rhythms of TC and TG1 + 1/2M coincided, suggesting that the rhythm of TC was due mainly to variation in TG1 + 1/2M. In the absence of significant variation in TS, the longest phase of the cell cycle, whenever G1 + 1/2M was short, TS/TC increased, so that the 18.6 hr rhythm in TS/TC was also a result of the periodicity in TG1 + 1/2M.     

Ploidy of endothelium in high-grade astrocytomas

To determine the ploidy and proliferative activity of the endothelium in high-grade astrocytomas, the nuclear DNA content of 11 high-grade astrocytomas, including two gliosarcomas, was measured by cytophotometry. This technique allowed comparison of the endothelial population with the astrocytic population. In all cases, the endothelium was diploid, with an average of 24.6% of cells in the S + G2M phases of the cell cycle. In contrast, the astrocytic population displayed marked DNA abnormalities. The two gliosarcomas had a marked difference in proliferative activity of the endothelium with 4% and 40% of cells, respectively, in the S + G2M phases. These data indicate that the vast majority of endothelial cells compromising the vascular hypercellularity observed in high-grade astrocytomas are in the normal cell cycle whereas in many cases the malignant astrocytes are not. The nuclear DNA content of gliosarcomas appears to be similar to other high-grade astrocytomas.     

Cell kinetics in mouse epidermis studied by bivariate DNA/bromodeoxyuridine and DNA/keratin flow cytometry.

Hairless mice were injected intraperitoneally with bromodeoxyuridine (Brd-Urd). Basal cells were isolated from epidermis, fixed in 70% ethanol, and prepared for bivariate BrdUrd/DNA flow cytometric (FCM) analysis. Optimum detection of incorporated BrdUrd in DNA was obtained by combining pepsin digestion and acid denaturation. The cell loss was reduced to a minimum by using phosphate-buffered saline containing Ca2+ and Mg2+ to neutralize the acid. The percentage of cells in S phase and the average uptake of BrdUrd per labelled cell in eight consecutive windows throughout the S phase were measured after pulse labelling at intervals during a 24 h period. Furthermore, the cell cycle progression of a pulse-labelled cohort of cells was followed up to 96 h after BrdUrd injection. In general the results from both experiments were in good agreement with previous data from 3H-thymidine labelling studies. The percentage of cells in S phase was highest at night and lowest in the afternoon, whereas the average uptake of BrdUrd per labelled cell showed only minor circadian variations. There were no indications that BrdUrd significantly perturbed normal epidermal growth kinetics. A cell cycle time of about 36 h was observed for the labelled cohort. Indications of heterogeneity in traverse through G1 phase were found, and the existence of slowly cycling or temporarily resting cells in G2 phase was confirmed. There was, however, no evidence of a significant population of temporarily resting cells in the S phase. Bivariate DNA/keratin FCM analysis revealed a high purity of basal cells in the suspensions and indicated that the synthesis of the differentiation-keratin K10 was turned on only in G1 phase and after the last division.     

Cell cycle perturbation of cultured C6 glioma cells following short-term contact with a low dose of ACNU

The purpose of this study was to investigate the cell cycle perturbation of cultured C6 rat glioma cells induced by 1-(4-amino-2-methyl-5-pyrimidyl)methyl-3-(2-chloroethyl)3-nitrosourea hydrochloride (ACNU) using simultaneous flow cytometric measurements of DNA and bromodeoxyuridine (BrdU) content. A new graphic computer program permitted the quantification of cell density in hexagonal subareas and allowed the fraction of BrdU-labeled cells with mid-S phase DNA content (FLS) to be defined in a narrow window. The cell kinetic parameters such as cell cycle time (Tc) and S phase time (Ts) were estimated from a manually plotted FLS curve at 18 and 6 hr, respectively. The major effect of ACNU on the cell cycle was an accumulation of the cells in the G2M phase 12 to 24 hr posttreatment when compared to G2M traverse of untreated cells. For the two-dimensional analysis, cells were labeled with BrdU and then treated with ACNU, or treated with ACNU and then labeled with BrdU. It was concluded that the cells in the S and G2M phases at the time of ACNU administration progressed to mitosis but that the G1 phase cells accumulated in the subsequent G2M phase. Two-dimensional FCM analysis using BrdU provided a useful tool in studying cell cycle perturbation.     

Flow cytometric analysis of the cell cycle: Mathematical modeling and biological interpretation

Estimation of the repartition of asynchronous cells in the cell cycle can be explained by two hypotheses: the cells are supposed to be distributed into three groups: cells with a 2c DNA content (G0/1 phase), cells with a 4c DNA content (G2 + M phase) and cells with a DNA content ranging from 2c to 4c (S phase); there is a linear relationship between the amount of fluorescence emitted by the fluorescent probe which reveals the DNA and the DNA content. According to these hypotheses, the cell cycle can be represented by the following equation: [formula: see text] All the solutions for this equation are approximations. Non parametric methods (or graphical methods: rectangle, peak reflect) only use one or two phase(s) of the cell cycle, the remaining phase(s) being estimated by exclusion. In parametric methods (Dean & Jett, Baisch II, Fried), the DNAT(x) distribution is supposed to be known and is composed of two gaussians (representative of G0/1 and G2 + M) and a P(x,y) function representative of S phase. Despite the generality, these models are not applicable to all sample types, particularly heterogeneous cell populations with various DNA content. In addition, the cell cycle is dependent on several regulation points (transition from quiescence to proliferation, DNA synthesis initiation, mitosis induction) and biological perturbations can also lead to cytokinesis perturbations. Before the emergence of flow cytometry, the current view of cell cycle resided in the assessment of cell proliferation (increase in cell number) or the kinetic of molecules incorporation (DNA precursors).(ABSTRACT TRUNCATED AT 250 WORDS)     

Macrophage-mediated cytostatic activity blocks lymphoblast cell cycle progression independently in both G1 phase and S phase

Recent work has shown that macrophage-mediated cytostatic activity inhibits cell cycle traverse in G1 and/or S phase of the cell cycle without affecting late S, G2, or M phases. The present report is directed at distinguishing between such cytostatic effects on G1 phase or S phase using the accumulation of DNA polymerase alpha as a marker of G1 to S phase transition. Quiescent lymphocytes stimulated with concanavalin A undergo a semisynchronous progression from G0 to G1 to S phase with a dramatic increase in DNA polymerase alpha activity between 20 and 30 hr after stimulation. This increase in enzyme activity was inhibited, as was the accumulation of DNA, when such cells were cocultured with activated murine peritoneal macrophages during this time interval. However, if mitogen-stimulated lymphocytes were enriched for S-phase cells by centrifugal elutriation and cocultured with activated macrophages for 4-6 hr, DNA synthesis was inhibited but the already elevated DNA-polymerase activity was unaffected. Similar results were obtained when a virally transformed lymphoma cell line was substituted as the target cell in this assay. These results show that both G1 and S phase of the cycle are inhibited and suggest that inhibition of progression through the different phases may be accomplished by at least two distinct mechanisms.     

Mode of estrogen action on cell proliferation in CAMA-1 cells: II. Sensitivity of G1 phase population

The mammary cancer cell line CAMA-1 synchronized at the G1/S boundary by thymidine block or at the G1/M boundary by nocodazole was used to evaluate 1) the sensitivity of a specific cell cycle phase or phases to 17 beta-estradiol (E2), 2) the effect of E2 on cell cycle kinetics, and 3) the resultant E2 effect on cell proliferation. In synchronized G1/S cells, E2-induced 3H-thymidine uptake, which indicated a newly formed S population, was observed only when E2 was added during, but not after, thymidine synchronization. Synchronized G2/M cells, enriched by Percoll gradient centrifugation to approximately 90% mitotic cells, responded to E2 added immediately following selection; the total E2-treated population traversed the cycle faster and reached S phase approximately 4 hr earlier than cells not exposed to E2. When E2 was added during the last hour of synchronization (ie, at late G2 or G2/M), or for 1 hr during mitotic cell enrichment, a mixed response occurred: a small portion had an accelerated G1 exit, while the majority of cells behaved the same as controls not incubated with E2. When E2 addition was delayed until 2 hr, 7 hr, or 12 hr following cell selection, to allow many early G1 phase cells to miss E2 exposure, the response to E2 was again mixed. When E2 was added during the 16 hr of nocodazole synchronization, when cells were largely at S or possibly at early G2, it inhibited entry into S phase. The E2-induced increase or decrease of S phase cells in the nocodazole experiments also showed corresponding changes in mitotic index and cell number. These results showed that the early G1 phase and possibly the G2/M phase are sensitive to E2 stimulation, late G1, G1/S, or G2 are refractory; the E2 stimualtion of cell proliferation is due primarily to an increased proportion of G1 cells that traverse the cell cycle and a shortened G1 period, E2 does not facilitate faster cell division; and estrogen-induced cell proliferation or G1/S transition occurs only when very early G1 phase cells are exposed to estrogen. These results are consistent with the constant transition probability hypothesis, that is, E2 alters the probability of cells entering into DNA synthesis without significantly affecting the duration of other cell cycle phases. Results from this study provide new information for further studies aimed at elucidating E2-modulated G1 events related to tumor growth.     

Circadian rhythms in mouse epidermal basal cell proliferation. Variations in compartment size, flux and phase duration.

Several kinetic parameters of basal cell proliferation in hairless mouse epidermis were studied, and all parameters clearly showed circadian fluctuations during two successive 24 hr periods. Mitotic indices and the mitotic rate were studied in histological sections; the proportions of cells with S and G2 phase DNA content were measured by flow cytometry of isolated basal cells, and the [3H]TdR labelling indices and grain densities were determined by autoradiography in smears from basal cell suspensions. The influx and efflux of cells from each cell cycle phase were calculated from sinusoidal curves adapted to the cell kinetic findings and the phase durations were determined. A peak of cells in S phase was observed around midnight, and a cohort of partially synchronized cells passed from the S phase to the G2 phase and traversed the G2 phase and mitosis in the early morning. The fluctuations in the influx of cells into the S phase were small compared with the variations in efflux from the S phase and the flux through the subsequent cell cycle phases. The resulting delay in cell cycle traverse through S phase before midnight could well account for the accumulation of cells in S phase and, therefore, also the subsequent partial synchrony of cell cycle traverse through the G2 phase and mitosis. Circadian variations in the duration of the S phase, the G2 phase and mitosis were clearly demonstrated.     

The early (18-hr) human mixed lymphocyte reaction: identification and isolation of activated T-cell clones

This study provides direct evidence that de novo expression of the activation antigens B1 49.9 (49.9) (interleukin-2 receptor) and 4F2 enables identification of alloactivated cells within 18 hr of initiation of human mixed lymphocyte reactions (MLR). Using a dual-parameter flow cytometer (simultaneous assessment of immunofluorescence and DNA content on the same cell), it was demonstrated that these activation antigens emerge before activated cells enter into S/G2/M phase of the cell cycle. Family studies illustrate that early activation antigen appearance occurs in response to a mismatch at chromosome 6, and invariably heralds the proliferative outcome at 6 days of MLR. In order to directly study the small alloactivated T-cell population, 49.9-positive cells were isolated using a cell sorter after 18 hr of MLR and cloned by limiting dilution using purified recombinant interleukin-2 (rIL-2). Antigen-specific T4-positive T-cell clones were isolated. Analysis of these clones demonstrates that antigen-specific reactivity is acquired within 18 hr in the MLR. These methods should permit a dissection of the early events of alloactivation.     

Endothelial cell subpopulations in vitro: cell volume, cell cycle, and radiosensitivity

Vascular endothelial cells (EC) are important clinical targets of radiation and other forms of free radical/oxidant stresses. In this study, we found that the extent of endothelial damage may be determined by the different cytotoxic responses of EC subpopulations. The following characteristics of EC subpopulations were examined: 1) cell volume; 2) cell cycle position; and 3) cytotoxic indexes for both acute cell survival and proliferative capacity after irradiation (137Cs, gamma, 0-10 Gy). EC cultured from bovine aortas were separated by centrifugal elutriation into subpopulations of different cell volumes. Through flow cytometry, we found that cell volume was related to the cell cycle phase distribution. The smallest EC were distributed in G1 phase and the larger cells were distributed in either early S, middle S, or late S + G2M phases. Cell cycle phase at the time of irradiation was not associated with acute cell loss. However, distribution in the cell cycle did relate to cell survival based on proliferative capacity (P less than 0.01). The order of increasing radioresistance was cells in G1 (D0 = 110 cGy), early S (135 cGy), middle S (145 cGy), and late S + G2M phases (180 cGy). These findings 1) suggest an age-related response to radiation in a nonmalignant differentiated cell type and 2) demonstrate EC subpopulations in culture.     

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.     

Cell-mediated inhibition of proliferation and activation of alloreactive cytotoxic lymphocytes: maintenance of response potential of precursors and dissociation between proliferation and effector function of activated cytotoxic lymphocytes

Adherent layers of macrophages (M phi-c) generated in vitro from splenic precursors inhibit lymphoproliferative responses to mitogen and to alloantigen without inhibiting the production of interleukin-2 (IL-2). Analysis of spleen cells stimulated for 48 hr in the presence of M phi-c indicated that both blastogenesis (increased cell mass) and expression of IL-2 receptors (7D4 determinants) were reduced. Analysis of BrdU incorporation (frequency of S-phase cells) and total cellular DNA revealed that the M phi-c inhibited the progression from G1 to S phase of cell cycle. The M phi-c not only inhibited the proliferative response to alloantigen but also prevented the generation of alloreactive cytotoxic T cells. The M phi-c were shown not to inhibit CTL responses by eliminating the stimulators or by inactivating precursors or inducing suppressors. The M phi-c were affecting the induction of CTL activity since the M phi-c did not affect the expression of cytolytic activity by activated CTL. The M phi-c did inhibit the proliferation of the activated CTL, suggesting that although cytolytic activity can be expressed in G1 phase of cell cycle, the activation of cytolytic activity in CTL-P may require a G1 to S phase transition. The cells recovered from 5-day MLC incubated in the presence of M phi-c were fully capable of generating a subsequent CTL response. This is in contrast to cells recovered from unstimulated cultures (no M phi-c) which have lost the ability to generate CTL responses. The M phi-c therefore prevent the generation of CTL responses in a totally reversible fashion, so as to allow activation and proliferation of CTL-P which have been removed from the influence of the M phi-c. These observations are discussed in the context of the currently hypothesized role of tissue macrophages in microenvironmental regulation.     

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.     

Cell cycle dependence of Hoechst 33342 dye cytotoxicity on sorted living cells

The effect of staining cellular DNA with the bisbenzimidazole dye Hoechst 33342 on the colony forming efficiency of Chinese Hamster Ovary Cells in different cell cycle phases has been studied. Exposures of 90 and 120 min to 5 microM Hoechst 33342 provided a considerable loss of clonogenicity depending on the cycle phase at staining procedure. The G2+M cells reveal to be the most sensitive fraction followed by the G1 cells. The highest resistance was found on S-phase cells with a colony forming efficiency exceeding that of the G2+M fraction by a factor of two.     

Cell kinetics of hypoxic cells in a murine tumour in vivo: flow cytometric determination of the radiation-induced blockage of cell cycle progression

Cells from the small cell population of viable cells in the large necrotic centre of murine M8013 tumours were investigated with respect to their cell kinetics. Flow cytometry (FCM) of this part of subcutaneously transplanted tumours revealed the presence of tumour cells with G1, S and G2 + M phase DNA-contents. These severely hypoxic cells could have stopped cell cycle progression due to the nutritional deprivation, irrespective of their position within the cell cycle. Labelling methods, used to disclose the cell kinetics of this cell population, are hampered by the absence of a transport system in these large necrotic areas. Therefore, FCM was used to monitor radiation-induced changes in the cell cycle distribution. From this investigation it was concluded that hypoxic cells in the necrotic centre of the M8013 tumour progress through the cell cycle. As well as a cell population with a cell cycle time (Tc) of approximately 84 hr, a subpopulation with a Tc of approximately 21 hr occurred.     

Cell cycle-dependent expression of cyclooxygenase-2 in human fibroblasts.

The purpose of this investigation is to determine whether the levels of cyclooxygenase-2 (COX-2) expression are cell cycle dependent. We used a serum-starved human foreskin fibroblast model to determine changes in COX-2 mRNA, protein, and promoter activity in response to stimulation with interleukin-1b (IL-1b) and phorbol 12-myristate 13-acetate (PMA) at G0, G1, S and G2/M phases of the cell cycle. IL-1b (1 ng/ml) and PMA (100 nM) induced robust COX-2 expression in the G0 cells, and the level of COX-2 expression declined progressively after the cells had entered the cell cycle. The COX-2 mRNA level at G1, S and G2/M phases of the cell cycle was 76%, 46%, and 30% of that at G0, respectively. A 5-flanking promoter fragment of COX-2 constructed into a luciferase expression vector was transfected into cells. The promoter activity in response to PMA stimulation was significantly higher in G0 than in S phase cells. These results imply that G0 cells are the key players in inflammation and other COX-2-dependent pathophysiological processes. When the cells are in the proliferative phase, COX-2 inducibility becomes restrained probably by an endogenous control mechanism to avoid COX-2 mediated oxidative DNA damage.