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PubMed1977

Changes in ornithine decarboxylase activity and cyclic adenosine-3'-5'-monophosphate concentrations during the cell cycle of synchronized BHK cells.

BHK cells were synchronized by excess thymidine treatment, which resulted in approximately 90% synchrony. The activity of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, elevated in early S phase, decreased in G2 + M and G1 phase and then increased during late G1 approximately second round of early S phase. The concentration of cyclic adenosine-3'-5'-monophosphate (cAMP) gradually decreased during S approximately G2 + M phase and then increased during late G1 approximately second round of early S phase, preceding that of ODC activity. The data suggest that ODC activity might be regulated by cellular cAMP level.

Carboxy-Lyases↗
PubMed1979

Cell cycle regulation in mammalian cells: hormones and commitment to DNA synthesis.

The growth regulation of cultured mouse fibroblasts and functional adrenal cells was studied. Variants of mutants from these cell lines were obtained. The effects of classical hormones (insulin, hydrocortisone and adrenocorticotropin) and of growth factors (EGF and PF) were analysed. These hormones stimulate or inhibit the entry of cells into S phase. However G1 cells become irreversibly committed to DNA synthesis 5 hours before entering S phase.

Adrenal Glands↗
PubMed1978

Induction of ornithine decarboxylase activity in a temperature-sensitive cell cycle mutant of Chinese hamster cells.

We have investigated the induction of ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) activity in a temperature-sensitive cell cycle mutant of Chinese hamster fibroblasts. This activity is not induced at the nonpermissive temperature, although the synthesis of the majority of proteins is normal. From a combination of studies with inhibitors of mRNA synthesis and maturation (alpha-amanitin, and cordycepin) and of proteins synthesis (cycloheximide, diphtheria toxin, and emetine), we conclude that the temperature-sensitive block is at the level of translation of one or more specific mRNAs.

Carboxy-Lyases↗
PubMed1979

Cell-cycle inhibition by misonidazole of human cells cultivated in vitro under aerobic conditions.

By means of flow cytometric recording of DNA histograms and counting of cells in synchronized populations, we have found that misonidazole (MIS) in clinically relevant concentrations induces cell-kinetic changes in human cells (NHIK 3025) cultivated in vitro under aerobic conditions. The effect seems to be a general lengthening of the cell cycle, affecting all phases. However, induction of this effect is phase-dependent, since only cells exposed to MIS during mitosis and/or early G1 will suffer significant cell-cycle prolongation. In exponentially growing populations this effect of MIS leads to a transient increase in the fraction of G1 cells and a corresponding decrease in the fraction of S cells. The possible significance of this effect for the clinical use of MIS is discussed.

Aerobiosis↗
PubMed1978

[Automatic recognition of different phases of the cell cycle and automatic count of the cells by means of a computer coupled to a cytophotometer].

Existing relationships between cell cycle phases and chromatin scattering within the nucleus led us to state more precisely our first alogrithm for cell cycle phases recognition. These refundments give evidence for the accuracy and the improving performance of the densitometric and morphologic analysis system used in this study: the "Samba".

Animals↗
PubMed2026

PAFAH1B1 governs follicular development by modulating the protein complex of CCNE1-CDK2-CDK1 to induce cell cycle arrest.

BACKGROUND: Ovarian follicle development plays a crucial role in mammalian fertility, which is primarily regulated by granulosa cell (GC) proliferation and cell cycle. Cell cycle dysregulation collectively might drive follicular atresia through GC dysfunction. However, the underlying molecular mechanisms remain largely unexplored. METHODS: The scRNA-seq and integrative analysis revealed that PAFAH1B1 was involved in cell cycle. Functional assays, including overexpression/knockdown, flow cytometry, EdU, HE, and TUNEL, confirmed that PAFAH1B1 regulated cell cycle and follicular development in vitro and in vivo. CoIP showed that PAFAH1B1 bound CCNE1-CDK2-CDK1 to arrest G2/M phase. Chromatin accessibility and CRISPR/dCas9-TET1 demonstrated that DNA methylation modulated PAFAH1B1 transcription. RESULTS: A novel regulator of cell cycle, PAFAH1B1, was identified in Pig Genotype-Tissue Expression (PigGTEx). During GC proliferation, we found that PAFAH1B1 transcription was correlated with the distribution rate of G1 phase in GCs. PAFAH1B1 protein was confirmed to specifically bind to CCNE1-CDK2-CDK1 to arrest G2/M phase. Notably, PAFAH1B1 appeared to hinder the development of follicles. Furthermore, the demethylation significantly promoted the transcription activity and chromatin accessibility of CpG island (-7 bp to +170 bp) of PAFAH1B1. Taken together, PAFAH1B1 physically interacted with the CCNE1-CDK2-CDK1 complex to arrest G2/M phase and inhibit the GCs proliferation and follicular development. Additionally, demethylation of CpG island significantly promoted the transcription of PAFAH1B1. CONCLUSION: These findings not only advance understanding of cell proliferation and cycle regulation but also identify PAFAH1B1 as a candidate gene for further investigation in follicular development.

CCNE1-CDK2-CDK1 complex↗
PubMed1979

Reversible retention of synchronized HeLa cells in G2 of the mammalian cell cycle.

The rate of progression of cycling HeLa cells from G2 into mitosis is greatly reduced by the amino acid analogue, p-fluorophenylalanine, but a small percentage of cells continually escapes into mitosis. Most of a presynchronized population can be held for several h in G2, thereby facilitating analyses of cells at this late stage of the cell cycle. They are reversed from the block by the addition of phenylalanine and resume progression within about 1 h with predictable kinetics but without a significantly enhanced synchrony. The mechanism of action of the analogue is consistent with the hypothesis that synthesis of false proteins interferes with the correct assembly of division-relevant structures.

Cell Cycle↗
PubMed1976

Cell surface changes in HeLa cells as an indication of cell cycle events.

A HeLa cell line synchronized by double thymidine block and mitotic shake off was shown to have a characteristic surface morphology for each of the different cell cycle stages. Inhibitors of cell multiplication were used to arrest cells in specific cell cycle phases, and these cells had a surface morphology similar to that of synchronized cells in the same phase. The results indicated a close association between the cell surface topography and the cycles of DNA synthesis in the cell nucleus of this HeLa line.

Bleomycin↗
PubMed1977

Rapid cell cycle analysis by measurement of the radioactivity per cell in a narrow window in S phase (RCSi).

A new rapid method for the cell cycle analysis of asynchronously growing cells is presented. The new method is an alternative to the more time consuming and subjective fraction of labeled mitoses (FLM) method. Like the FLM method, all cells in the S phase of the cell cycle are marked by pulse labeling with a radioactive DNA precursor. The subsequent progress of the cohort of cells thus labeled is monitored through a narrow window in the cell cycle. The window is defined by a narrow range of DNA contents corresponding to cells in mid-S phase and is designated Si. The cellular DNA content is measured by flow cytometry and the cells in the window Si are selected by electronic cell sorting. The radioactivity per cell in Si (RCSi) is determined by liquid scintillation counting. The duration of S phase and of the total cycle and the dispersions therein are determined from the oscillation of the RCSi values with time. The complete cell cycle analysis can be accomplished in as little as 1 day following the collection of samples. Exponentially growing Chinese hamster ovary (CHO) cells were analyzed according to the RCSi method and the FLM method. It is demonstrated that the two techniques give essentially the same results.

Animals↗
PubMed1977

Commitment to deoxyribonulceic acid synthesis and the cell cycle in endotoxin-stimulated murine spleen cells.

Events associated with endotoxin-induced mitogenesis in murine spleen cells were investigated. Commitment to deoxyribonucleic acid (DNA) synthesis, the onset of DNA synthesis, and phases of cell cycle were timed. Increased levels of DNA synthesis in murine spleen cells stimulated with endotoxin were observed 12 to 16 h after the addition of the mitogen. The total cell cycle time of stimulated B-cells was 11 to 14 h. The S-phase was 8 h. The G2-phase was 1 h, and the combined M-plus G1-phase of cycling cells was 2 to 5 h. A 1- to 4-h exposure to lipopolysaccharide elicited a significant increase in DNA synthesis. Progressively longer exposures to lipopolysaccharide, up to 24 h, produced further increases in first-cycle DNA synthesis. Polymyxin B, when added with endotoxin to cultures from the outset, inhibited first-cycle DNA synthesis. However, if addition of the antibiotic was delayed, progressive increases in first-cycle dna synthesis were observed. These data indicate a heterogeneity among B-cells in their responsiveness to endotoxin.

Animals↗
PubMed1979

Cell-cycle distribution of urothelial tumour cells as measured by flow cytometry.

The fraction of cells in S + G2 + mitosis from 54 urothelial tumours was calculated by flow cytometry after acridine orange (AO) staining of cells obtained by bladder irrigation or biopsy. Fluorescence signals emitted by the AO-stained DNA and RNA of each cell were separated optically and measured for 5,000 cells per specimen. The patients were classified by the histology of their tumours and clinical data into 5 diagnostic categories: NED (no evidence of disease, but history of bladder tumour), 3; papilloma, 8; non-invasive papillary carcinoma, 8; carcinoma in situ, 17 and invasive carcinoma, 18. The fraction of cells with DNA values in S + G2 + M of the cell cycle varied between 7 and 57% of the total, with a wide range within each diagnostic category, but no statistically significant differences between the groups. The proportion of cells in S + G2 + M from an individual tumour was not correlated with histologic grade or clinical behaviour. The possibility that some tumour cells with DNA values above G1 level are quiescent cells arrested at S or G2 is discussed.

Cell Count↗
PubMed1978

Poly(A) polymerase activity during cell cycle and erythropoietic differentiation in erythroleukemic mouse spleen cells.

Poly(A) polymerase activity was studied in lysates of cultured murine erythroleukemic cells (Friend cells). Incorporation of ATP into acid-precipitable products is dependendent on the presence of Mn2+ or Mg2+ and of an RNA primer. The reaction is specific for ATP as the substrate (KM=290 290 micron, it is not inhibited by actinomycin D and only slightly interferred with by ethidium bromide. Cordycepin 5'-triphosphate and sodium pyrophosphate inhibit the enzyme activity. The chain length of the products of the reaction is dependent on the primer concentration and reaches up to 30 nucleotides. Poly(A) polymerase activity is low in resting (G1 phase) cells 75 nmol ATP incorporated/h per 10(6) cells) and increases to a level about twice as high in early S phase of the cell cycle. A possible model for regulation of enzyme activity is discussed. Polymerase activity in the early phase of erythropoietic differentiation of the cells induced by butyric acid does not show any difference in comparison to untreated controls. A decrease in enzyme activity to levels characteristic for cells in G1 phase accompanies shutdown of cell growth in the course of the ongoing differentiation. Analysis of the DNA content of the cells revealed that erythropoietic differentiation of Friend cells induced by butyric acid is characterized by arrest of the cells in G1 phase of the cell cycle. Poly(A) polymerase activity in erythroleukemic cells is thus controlled only by the phase of the cell cycle; it is not affected by changes in gene expression during erythroid differentiation.

Adenosine Triphosphate↗
PubMed1976

The significance of DNA damage in the cell cycle sensitivity of Chinese hamster ovary cells to bleomycin.

Chinese hamster ovary cells have been treated with bleomycin at various stages in the cell cycle. Mitotic cells, which exhibit least survival, show the greatest amount of DNA strand breakage and a marked inhibition of DNA replication in the subsequent S phase. Strand-rejoining experiments suggest that this is primarily due to differences in the amount of damage produced in the DNA but does not exclude the possibility that cells at the various cell stages also differ in their ability to repair this damage. DNA breakage also occurs in cells incubated in bleomycin at 4 degrees.

Bleomycin↗
PubMed1978

Regulatory substances produced by lymphocytes. VI. Cell cycle specificity of inhibitor of DNA synthesis action in L cells.

IDS inhibits DNA synthesis and mitosis of L cells only when present during the late G1 phase of the cell cycle, as shown with L cells synchronized by a variety of methods. This corresponds well with earlier findings that IDS inhibits DNA synthesis in mitogen-stimulated lymphocytes when present between 16 and 24 h after adding mitogen. In both cell types, the inhibition produced by IDS appears to be totally the result of elevation of cAMP level. Thus, inhibitors of cAMP phosphodiesterase work synergistically with IDS, and activators of cAMP phosphodiesterase overcome the inhibition by IDS. This paper shows that IDS raises cAMP levels in L cells only within a narrow interval of the cell cycle, around 6-8 h after mitosis. This cell cycle specificity, which may be related to appearance of receptors for IDS only at discrete times, may be important in limiting IDS action to suppression, as elevated cAMP levels have a variety of other effects during other phases of the cell cycle.

Animals↗
PubMed1978

Significance of the cell cycle in commitment of murine erythroleukemia cells to erythroid differentiation.

The relationship between differentiation of murine erythroleukemia cells (MEL) induced by DMSO and the cell division cycle has been analyzed. We demonstrate that incubation in the presence of DMSO increases the length of the G1 phase of the cell cycle. A method of synchronization of MEL cells by unit gravity sedimentation has been developed and characterized. Using this method, a series of synchronized cell populations covering the entire cell division cycle can be generated simultaneously. Cells synchronized by this technique were challenged with DMSO and analyzed for kinetics of commitment to the differentiation program. Our results indicate that populations of cells in G1 or G2 at the time of addition of inducer give rise to a greater proportion of committed cells than an unfractionated population, while cells in S phase result in a lower percentage of committed cells than the unfractionated population when cultured in DMSO.

Animals↗