Proportion Of Cells In G1 Research Articles

Growth inhibitory effects of 5-aza-2′-deoxycytidine in HL-60 promyelocytic leukemia cells resistant to differentiation induction

In the original HL-60 cells (HL-60-S) and an HL-60 subline (HL-60-R) respectively susceptible and resistant to induction of differentiation by retinoic acid or dimethyl sulfoxide, 5-aza-2'-deoxycytidine inhibited growth equally but induced differentiation to a greater extent in HL-60-S. Flow cytometry showed that 5-aza-2'-deoxycytidine produced in both HL-60 lines an increased proportion of cells in G2+M rather than G0/G1 as with retinoic acid. 5-aza-2'-deoxycytidine may have a differentiation-inducing effect in HL-60 provided cells have the competence to differentiate, indicating the importance of an alternate mechanism of action.

Methylmercury effects on cell cycle kinetics.

Methylmercury (MeHg) effects on cell cycle kinetics were investigated to help identify its mechanisms of action. Flow cytometric analysis of normal human fibroblasts grown in vitro in the presence of BrdU allowed quantitation of the proportion of cells in G1, S, G2 and the next G1 phase. This technique provides a rapid and easily performed method of characterizing phase lengths and transition rates for the complete cell cycle. After first exposure to MeHg the cell cycle time was lengthened due to a prolonged G1. At 3 microM MeHg the G1 phase length was 25% longer than the control. The G1/S transition rate was also decreased in a dose-related manner. Confluent cells exposed to MeHg and replated with MeHg respond in the same way as cells which have not been exposed to MeHg before replating. Cells exposed for long times to MeHg lost a detectable G1 effect, and instead showed an increase in the G2 percentage, which was directly related to MeHg concentration and length of exposure. After 8 days at 5 microM MeHg, 45% of the population was in G2. The G2 accumulation was reversible up to 3 days, but at 6 days the cells remained in G2 when the MeHg was removed. Cell counts and viability indicated that there was not a selective loss of cells from the MeHg. MeHg has multiple effects on the cell cycle which include a lengthened G1 and decreased transition probability after short term exposure of cycling cells, and a G2 accumulation after a longer term exposure. There were no detectable S phase effects. It appears that mitosis (the G2 accumulation) and probably synthesis of some macromolecules in G1 (the lengthened G1 and lowered transition probability) are particularly susceptible to MeHg.

Early effects of preoperative irradiation upon the cell cycle composition in rectal adenocarcinomas. A flow-cytometric DNA investigation.

The DNA patterns were studied by means of flow cytometric analysis in 43 rectal adenocarcinomas. Ploidy level and cell cycle distribution were related to clinical stage and histopathology. The frequency of grossly aneuploid tumours and tumours with multiple aneuploid cell populations increased with more advanced clinical stages and with the degree of dedifferentiation. In 15 cases the DNA pattern was studied before and after preoperative irradiation. The ploidy level was not affected by irradiation. A pronounced increase in the proportion of G2 cells was found after irradiation. This G2 blockage was proportional to the amount of S-phase cells before irradiation. Since following irradiation the proportion of S-phase cells was low and the proportion of G1 cells unchanged, the existence of a high fraction of resting G1 tumour cells can be assumed.

Three years of experience with the Gn-RH analogue Buserelin in 100 patients with advanced prostate cancer

The floral organs of Tradescantia clone 4430 were used to investigate, in terms of cell cycle parameters, cellular behaviour during the maturation of a terminally differentiating system. Petals were sampled at different stages of development for (a) cell number; (b) nuclear DNA content by cytophotometry; (c) [3H]thymidine incorporation into nuclei by autoradiography; and (d) pigment production by spectrophotometry. DNA synthesis was confirmed by measurement of [3H]thymidine incorporation into TCA-insoluble material and changes in DNA content by colorimetric estimation of DNA extracts by diphenylamine. The development of the petal involved four sequential steps. First, there was an increase in cell number, an event characterized by mitoses, DNA synthesis, a few cells in G2 and a predominance in G1. Second, there was a cessation of cell division and DNA synthesis when all the cells accumulated in G1. Third, there was a shift of a large proportion of the total cell population from G1 to the G2 stage of the cell cycle and finally, there was pigment production. In addition, cytophotometric analysis of individual tissues in the mature petal revealed tissue specific differences in the proportion of cells in G2.

Constancy of cell buoyant density for cultured murine cells.

The relationship between cell cycle and cell density was determined for three different lines of mouse cells by equilibrium centrifugation of suspension cultures. The mean cell densities of the three lines differed significantly, with values of 1.0622, 1.0678, 1.0540 gm/ml for 70Z/3, S 107, and ABE 8, respectively. However, the density distributions within each of the three lines were indistinguishable, with an average coefficient of variation about 5% of the mean reduced density (i.e., density minus one). Quantitative DNA analysis of the cells separated by density showed that the proportion of cells in G1, S, and G2 + M phase of the cell cycle changed very little or not at all with cell density. In addition, cells separated by size (and therefore by phase of the cell cycle) using velocity sedimentation had the same means and distributions of densities. These results indicate that there is little or no change in cell density as the cells traverse the life cycle and that buoyant density appears to be a constant property of a cell type.

The effects of ionizing radiation on cell cycle progression in ataxia telangiectasia

Although ataxia telangiectasia (AT) cells are more sensitive than normal cells to killing by ionizing radiation, their DNA synthesis is more resistant to inhibition by radiation. It was thought that this anomaly in DNA synthesis was likely to perturb cell cycle progression. Flow cytometry and the fraction of labelled mitoses (FLM) were used to investigate effects of irradiation in normal and AT cell lines. The FLM indicated that radiation apparently induced a longer G2 delay in normal cells than in AT cells. However, flow cytometry showed that radiation induced much larger and more prolonged increases in the proportion of G2 cells in AT than in normals. AT populations also showed much larger postirradiation decreases in viable cell numbers. These data suggest that a large proportion of the radiosensitive AT cells are not reversibly blocked in G2 but die there, and never proceed through mitosis. The less radiosensitive normal cells are delayed in G2 and then proceed through mitosis. We suggest that the apparently shorter radiation-induced mitotic delay seen in AT cells by FLM is not real but is an artifact arising from perturbation of steady state conditions by selective elimination of a particular cohort of AT cells. Accumulation of AT cells in G2 is compatible with radiosensitivity of these cells and may arise from a defect in DNA repair or an anomaly in DNA replication.

Several cytokinetic methods for showing circadian variation in normal murine tissue and in a tumor

Circadian rhythmicity in cell proliferation was studied by several cytokinetic techniques in 8-week-old CD2F1 and Swiss male mice. In separate experiments on four different dates, subgroups of six or seven mice (fed ad libitum and standardized to 8 hours light alternating with 16 hours of darkness) were killed at 3-hour intervals over a period of 24-48 hours. Cosinor analyses were used to determine the parameters of circadian periodicity in the data. Flow cytometric (FCM) analyses of DNA synthesis in tongue and stomach were compared with estimates of tritiated thymidine (3HTdR) incorporation into the DNA in these tissues. Circadian variation in tongue epithelium was reproducible in phasing and in range-of-change (100-140%) in seven 24-hour studies. The proportion of cells in G1 and G2 in tongue epithelium also demonstrated a circadian periodicity. When DNA synthesis in stomach was analyzed by the two methods, the results were entirely different and thus not comparable. The circadian rhythms in 3HTdR uptake agreed with those reported previously, but results of analyses by FCM were less certain, since epithelium of this organ could not be analyzed separately. Circadian rhythms were detected by FCM in G2, S, and G2M in Ehrlich ascites carcinoma (EAC). The mitotic index of this tumor varied with a circadian periodicity similar to that of the FCM-derived rhythm in G2M. This study validates the reliability of older techniques, such as 3HTdR uptake and mitotic indices, and suggests that circadian rhythms can be important in studies of cellular properties using FCM.

Variation in cell surface morphology of two 90Sr-induced osteosarcomas serially transplanted in CBA mice.

The induction of skeletal tumours, which can be classified as osteosarcomas of many different types, is considered to be the primary carcinogenic effect of radiostrontium. In the present report the cell surface morphology in vivo of 90Sr-induced osteosarcoma cells was investigated, since a variety of tumour cells--and especially those investigated in vitro--have been shown to possess morphologic changes compared with their normal counterparts. Using scanning electron microscopy, variations in cell surface morphology were observed in 2 tumour series, which were serially transplanted in mice for 45 and 60 transfer generations, respectively. The slow-growing osteosarcoma cells of the early transfer generations, of osteoblastic as well as fibroblastic type, seemed to have more cytopodia than the fast-growing osteosarcoma cells from later generations. This may be due to the fact that slowly growing cell populations have a large proportion of cells in G1, in which stage there is a higher frequency of cellular cytopodia.

Growth Kinetics of Bp8 Mouse Ascites Sarcoma after Single Doses of Whole Body Irradiation: II. Analysis of the Progression of Cells Through the Cell Cycle

Bp8 ascites sarcoma cells growing in vivo were whole body irradiated with doses of 1.75 to 8 Gy. The inflow and outflow rates of cells in the various compartments of the cell cycle were estimated on the basis of sequential analysis of the total number of cells and from the proportion of cells in G1, S-phase, G2 and M. The flow through the compartments was calculated from these data and from the sizes of the cell pools. The durations of the mitotic delay, the G1-depletion and the early G2 blockage were linearly related to the dose. After release of the mitotic division delay the accumulation rate of cells in M and G1 decreased linearly with the dose; for G2, plateau values for the accumulation rate were found after 2.5 Gy. The outflow rates from G2 and M after release of the G2 blockage showed a shoulder type of dose-response with a D0 of 2.5 Gy and a n value of 1.5. In addition to an increase in the duration of G2, generally responsible for the increase in the total cell cycle time at about 24 hours after irradiation and an increase in the duration of the S-phase up to 12 hours after irradiation, an early increase in the duration of G1 and M was observed.

Behaviour of the Bp8 mouse ascites sarcoma after partial body irradiation.

Mice bearing the Bp8 mouse ascites sarcoma were 4 days after inoculation irradiated with 2.5, 5 and 8 Gy. By shielding the head and the upper part of the thorax the mice survival, compared with whole body irradiation, was markedly prolonged and the behaviour of the ascites tumour could be evaluated up to 30 days after inoculation. While about 10 days after irradiation the total number of tumour cells started to decrease and amounted to only 4 per cent 20 days later, the ascites volume increased up to 30 ml. Using flow-cytometric cellular DNA analysis, the proportions of cells in the various parts of the cell cycle were determined. After an initial G2 blockage, a partial blockage with an increase proportion of G2 cells was found up to 20 days after inoculation. While the total number of G1, S-phase and mitotic cells started to decrease on day 10, the total number of G2 cells was independent of the dose and was constant up to day 18. This behaviour may indicate specific regulating processes linked to G2 cells.

G2 arrest and differentiation in the petal of Tradescantia clone 4430

The floral organs of Tradescantia clone 4430 were used to investigate, in terms of cell cycle parameters, cellular behaviour during the maturation of a terminally differentiating system. Petals were sampled at different stages of development for ( a) cell number; ( b) nuclear DNA content by cytophotometry; ( c) [ 3H]thymidine incorporation into nuclei by autoradiography; and ( d) pigment production by spectrophotometry. DNA synthesis was confirmed by measurement of [ 3H]thymidine incorporation into TCA-insoluble material and changes in DNA content by colorimetric estimation of DNA extracts by diphenylamine. The development of the petal involved four sequential steps. First, there was an increase in cell number, an event characterized by mitoses, DNA synthesis, a few cells in G2 and a predominance in G1. Second, there was a cessation of cell division and DNA synthesis when all the cells accumulated in G1. Third, there was a shift of a large proportion of the total cell population from G1 to the G2 stage of the cell cycle and finally, there was pigment production. In addition, cytophotometric analysis of individual tissues in the mature petal revealed tissue specific differences in the proportion of cells in G2.

Cell volume decrease during Friend leukemia cell differentiation.

Friend leukemia cells (GM86, clone 745) were induced to differentiate with dimethyl sulfoxide or butyric acid. The kinetics of induction were measured by cell growth, cell volume distributions, and [3H]thymidine incorporation. From the volume distributions, it was found that the rate of induction was both agent sensitive and concentration dependent. The changes in volume distributions occurred approximately 4 hr earlier with dimethyl sulfoxide induction relative to butyric acid induction. However, the changes with the butyric acid induction were more dependent on concentration. A decrease in labeling indices during the 12- to 20-hr time period was correlated to a decrease in mean cell volume and an increase in the proportion of G1 cells. After the 20-hr time period of induction, an increase in labeling indices and in the percentage of large cells was observed. The data suggest that a transient block of cells in G1 occurred between 12 to 20 hr, and that the early differentiation involved a volume decrease which was related to a redistribution of cell cycle stages. The study was also shown that the changes of cell volume are a rapid monitor to determine the early events of the differentiation process.

G2+M arrest of cultured mammalian cells after incorporation of tritium-labeled nucleosides.

Novikoff rat hepatoma cells were propagated in suspension cultures containing 0.5 to 10 muC of 3H-methyl-thymidine, 3H-5-uridine, 3H-G-adenosine or 3H-8-adenine. The presence of the 3H-labeled precursors caused an inhibition of cell replication which was due to a delay or arrest of the cells in G2 and M. The degree of inhibition was proportional to the amount of radioactivity incorporated into nucleic acids. Almost immediate and complete inhibition resulted from incubation with 10 muC 3H-thymidine/ml. The presence of 0.5 muC 3H-thmidine/ml caused a significant increase in the relative proportion of cells in G2+M, even though the population doubling time of the culture appeared to be unaltered.

Effect of Azathioprine on the Cell Cycle of Haemopoietic Cells

The megaloblastic erythropoiesis encountered in 3 azathioprine-treated patients with renal transplants was found to be associated with a marked increase in the proportion of early polychromatic erythroblasts in the G2 phase of the cell cycle. By contrast, there was only a slight increase in the proportion of G2 cells in the neutrophil promyelocyte-myelocyte pool of these patients.

Effect of cell population density on G2 arrest in Tetrahymena.

The ciliated protozoan, Tetrahymena pyriformis strain GL-C, has been used to study the effect of cell population density during starvation on the synchrony obtained after refeeding and on the number of cells arrested in G2 phase of the cell cycle. At high cell densities two peaks of division indices were observed after refeeding while only one was observed at low cell densities. Cell division began earlier in cultures starved at high cell densities. Most importantly, the proportion of cells in G2 was considerably higher in populations starved at high cell densities. When tritiated thymidine was present during the refeeding period, radioautographs of cell samples at different times showed that the first cells to exhibit division furrows contained unlabeled nuclei. The first peak in the division index after refeeding was observed only at higher cell densities and is attributed to the cells arrested in G2. These results suggest that Tetrahymena is an excellent organism to study the concept of resting stages in the cell cycle and their control.

A Study of Erythropoiesis by Combined Morphologic, Quantitative Cytochemical and Autoradiographic Methods: Normal Human Bone Marrow, Vitamin BI2 Deficiency and Iron Deficiency Anemia

Abstract The relationship between the morphology of human erythropoietic cells and their position in interphase has been studied. In normal bone marrow, pro-normoblasts, basophilic normoblasts and early polychromatic normoblasts were present in all stages of interphase. It has been shown that a significant increase in nuclear size occurs as a cell moves through its cell cycle, in both normal and megaloblastic erythropoiesis. The relative distribution of the basophilic erythropoietic cells and the dividing polychromatic cells, in the various stages of interphase, has been determined in normal bone marrow, vitamin B12 deficiency and iron deficiency anemia. In vitamin B12 deficiency, associated with moderate or severe anemia, there was an increased proportion of cells in G2, and there were several cells with DNA contents that were between the 2c and 4c values, which were not in DNA synthesis as judged by 3H-TdR labeling. These abnormalities were most pronounced in the dividing polychromatic cell group. Similar abnormalities were not present in iron deficiency anemia, indicating that these disturbances were not produced by the presence of anemia per se. The possible relationship between these changes in the cell cycle and the ineffective erythropoiesis seen in anemia due to vitamin B12 deficiency has been discussed.