Cyclin B1 expression in response to abrogation of the radiation-induced G2/M block in HeLa cells

The G₂ block is a major response of cells to DNA damage and seem to be induced independently of p53 status. It is thought that the G₂ block has a protective function and allows cells to repair their DNA. The molecular events involved in the formation of the G₂ block therefore are of great interest. We have used pentoxifylline, a potent G₂ delay abrogator, to study the expression of an essential component of the mitosis promoting complex (MPF), cyclin B1. Cyclin B1/G₂ ratios are used to show that irradiation induces a decrease in cyclin B1 expression and that pentoxifylline restores cyclin B1 expression to control level. This confirms that suppression of cyclin B1 plays a role in the formation of the G₂ cell cycle delay, and that elevating cyclin B1 expression is part of the mechanism of action of pentoxifylline on G₂ blocked cells.     

Expression of G1 and G2 cyclins measured in individual cells by multiparameter flow cytometry: a new tool in the analysis of the cell cycle

Abstract. Multivariate analysis of the expression of cyclin proteins and DNA content has opened new possibilities for the study of the cell cycle. By virtue of their cell cycle phase specificity, the expression of cyclins may serve, in addition to DNA content, as another marker of a cell's position in the cycle, and provide information about the proliferative potential of cell populations. Several applications of the methodology based on bivariate analysis of DNA content v . expression of B, E and D type cyclins are reviewed: 1 expression of cyclins by individual cells during their progression through the cycle can be studied, using exponentially growing cells without the necessity of cell synchronization or other perturbations of the cycle; 2 cells having the same DNA content but residing in different phases of the cycle (e.g. G₂ diploid v. G₁ tetraploid) can be distinguished; 3 cell transition from G₀ to G₁ and progression through G₁ (e.g. mitogen stimulated lymphocytes) can be assayed; 4 the population of proliferating cells can be distinguished from noncycling cells based on dual cell labelling with a G₁ and G₂ cyclin antibody; 5 cyclin restriction points can serve as additional cell cycle landmarks to map the point of action of antitumour drugs; 6 unscheduled expression of cyclins (e.g. the presence of cyclin B1 during G₁ and S) can be detected in several tumour transformed cell lines, possibly indicating disregulation of the machmery of cell cycle progression. The last finding 6 is of special importance, because such disregulation may be of prognostic consequence in human tumours.     

Influence of the G2 cell cycle block abrogator pentoxifylline on the expression and subcellular location of cyclin B1 and p34cdc2 in HeLa cervical carcinoma cells

The progression of cells from G₂ into mitosis is mainly controlled by formation of the cyclin B1/p34^cdc2 complex. The behaviour of this complex in the irradiation-induced G₂ cell cycle delay is still unclear. A prior study demonstrated that the expression of the cyclin B1 protein is reduced by irradiation, and restored to control levels by the methylxanthine drug pentoxifylline, which is a potent G₂ block abrogator. The present study shows that irradiation, and 2 mM pentoxifylline affect the expression of the cyclin-dependent kinase p34^cdc2 in HeLa cells. Irradiation induces p34^cdc2 levels to increase and cyclin B1 levels to decrease. Addition of pentoxifylline at the G₂ maximum reverses these trends. This is also evident from the cyclin B1/p34^cdc2 ratios which decline after irradiation and are rapidly restored to control levels upon addition of pentoxifylline. It is concluded that cyclin B1 and p34^cdc2 protein expression are important events and act in concert to control the irradiation induced G₂ block. Analysis of cyclin B1 expression in whole cells and in isolated nuclei furthermore show that cyclin B1 is translocated from the nucleus into the cytoplasm when the G₂ block is abrogated by pentoxifylline.     

Unscheduled expression of cyclins D1 and D3 in human tumour cell lines

D-type cyclins are involved in regulation of cell traverse through G₁ primarily by activating the cyclin-dependent kinase 4 (CDK4) and targeting it to the retinoblastoma tumour suppressor protein. There is a vast body of evidence that defective expression of D-type cyclins is associated with tumour development and/or progression. Immunocytochemical detection of D cyclins combined with multiparameter flow cytometry makes it possible to measure the expression of these proteins in individual cells in relation to their cell cycle position without the need for cell synchronization. This approach was used in the present study to compare the cell cycle phase specific expression of cyclins D3 and D1 in human normal proliferating lymphocytes and fibroblasts, respectively, with nine tumour cell lines of different lineage. During exponential, unperturbed growth, expression of cyclin D1 in fibroblasts from donors of different age, or cyclin D3 in lymphocytes, was limited to mid-G₁ cells: Less than 7% of the cells entering S phase or progressing through S and G₂ were cyclin D positive. In contrast, expression of either cyclin D1 or cyclin D3 in tumour cell lines of different lineage was not limited to G₁ phase. Namely, over 80% of the cells in S and G₂+M were cyclin D positive in eight of the nine cell lines studied. The data indicate that while expression of cyclin D1 or D3 in normal cells is discontinuous, occurring transiently in G₁, these proteins are expressed in some tumour lines persistently throughout the cell cycle. This suggests that the partner kinase CDK4 is perpetually active throughout the cell cycle in these tumour lines.     

Monocytic differentiation of HL60 cells induced by potent analogs of vitamin D3 precedes the G1/G0 phase cell cycle block

Abstract. Differentiation of mammalian cells is accompanied by reduced rates of proliferation and an exit from the cell cycle. Human leukemic cells HL60 present a widely used model of neoplastic cell differentiation, and acquire the monocytic phenotype when exposed to analogs of vitamin D₃ (VD₃). The maturation process is accompanied by two blocks in the cell cycle: an arrest in the G₁/G₀ phase, and a recently described G₂+ M block. In this study we have analyzed the traverse of the cell cycle phases of the well-differentiating HL60-G cells exposed to one of ten analogs of VD₃, and compared the cell cycle effects of each compound with its potency as a differentiation-inducing agent. We found that in general there was a good correlation between the effects of these compounds on the cell cycle and on differentiation, but the best cell cycle predictor of differentiation potency was the extent of accumulation of the cells in the G₂ compartment. All analogs induced a marked decrease in the mitotic index, and polynucleation of HL60 cells was produced, especially by compounds which were effective as inducers of differentiation. Time course studies showed that induction of differentiation was accompanied by a transient increase of the proportion of cells in the G₂+ M compartment, but preceded the G₁ to S, and the G₂ compartment blocks. These studies indicate that complex changes in the cell cycle traverse accompany, but do not precede, the acquisition of the monocytic phenotype by HL60 cells.     

EFFECTS OF ACTINOMYCIN D AND PUROMYCIN ON THE CELL PROGRESS FROM M TO G1 AND S STAGES IN CULTURED MOUSE LEUKEMIA L5178Y CELLS

Actinomycin D (0.5 μg/ml) did not prevent M stage cells from entering G₁ stage, but blocked their progress from G₁ to S stage. The position of the block was approximately 1.4 hr before S stage or just after the beginning of G₁ stage. Actinomycin D in this concentration also significantly depressed uridine-³H uptake into G₁ stage cells, but did not suppress leucine-³H uptake by M and G₁ cells. This suggests that some proteins may be synthesized in M and G₁ stage cells by messenger RNA left over from the previous cell cycle. However, entry of G₁ cells into S stage would require synthesis of new messenger RNA near the beginning of G₁ stage. Puromycin (10 μg/ml) did not prevent M cells from entering G₁ stage, but blocked their progress from G₁ to S stage. The site of blockage was about 0.7 hr before S stage or in the first two-third of G₁ stage. This might be the site where the cells synthesize new G₁ proteins necessary for entry to S stage.
Comparison of sensitivities of G₁ and G₂ stages to the two antibiotics reveals that the puromycin sensitivity of G₁ cells was similar to that of G₂ cells, but the actinomycin D sensitivity of G₁ was greater than that of G₂ cells.     

Elucidation of the DNA Synthetic Cycle of Entamoeba Spp. Using Flow Cytometry and Mathematical Modeling

ABSTRACT. We developed a method to study the DNA synthetic cycles of Entamoeba histolytica and Entamoeba invadens by flow cytometry (FCM) based on a preparative procedure to reduce both high levels of natural fluorescence and non-specific adsorption of fluorochromes. We modeled G₁, S, and G₂ phases as a series of overlapping Gaussian curves. Both E. histolytica and E. invadens displayed G₁, S, and G₂ proportions that are consistent with eukaryotic cell populations in exponential or stationary growth phase. Exponential phase E. histolytica populations contained a hypodiploid subset with a mass of about 20% less than the diploid value which we estimate by FCM to be 24 × 10^-14 g DNA/cell. Exponential phase E. invadens populations contained a hypodiploid subset with a mass of about 6% less than the diploid value which we estimate by FCM to be 30 × 10^-14 g DNA/cell.     

Normal G1/S transition and prolonged S phase within one cell cycle after seeding cells in the presence of an ornithine decarboxylase inhibitor

Abstract. We have previously found that DNA replication was affected within one cell cycle after seeding Chinese hamster ovary (CHO) cells in the presence of the polyamine biosynthesis inhibitor 2-difluoromethylornithine (DFMO). We could, however, not rule out if this was due to an effect on the G₁/S transition and/or on DNA synthesis elongation. In the present paper, we use a bromodeoxyuridine-flow cytometric method to more specifically study the G₁/S transition, the S phase length, and the progression of cells from S phase through G₂+ M and into G₁, after seeding plateau phase CHO cells at low density in the absence or presence of 5 mM DFMO. We report here that DFMO-induced polyamine depletion increased the length of the S phase within one cell cycle after seeding of CHO cells in the presence of the inhibitor. No effect on the G₁/S transition was observed until 2 days after seeding, suggesting that a DFMO-induced lengthening of the G₁ phase occurred later than the effect on S phase progression. These results imply that the G₂+ M phase was not prolonged until 2 days after seeding CHO cells in the presence of DFMO.     

Method For Probing Cells In Radiation-Induced G2Arrest: Demonstration of Potentially Lethal Damage Repair

Abstract. Chinese hamster ovary cells were arrested in the G₂ phase of the cell cycle by X-irradiation. When subsequently treated with 5 mM caffeine the arrested population progressed into mitosis as a synchronous cohort where it was harvested by mitotic cell selection. This procedure provides a means to isolate cell populations treated in G₂, for the investigation of G₂ arrest. Comparisons were made of the number of cells retrieved from G₂ arrest with the number suffering arrest, as determined by flow cytometry and by matrix algebraic simulations of irradiated cell progression. the retrieved population was not significantly less than expected for doses up to 3.5 Gy, indicating that the retrieval process does not favour the isolation of any population subset below this dose. Cell populations retrieved from arrest at varying intervals (0-3 h) after irradiation (0-3.5 Gy) showed an increase in survival with increase in interval, consistent with repair of potentially lethal damage. the repair curves (surviving fraction us time) were each described by a single exponential. G₂ cells that were brought to mitosis without a period of arrest exhibited the same radiation response as cells irradiated in mitosis.     

Unbudded G2 as well as Gj arrest in the stationary phase of the basidiomycetous yeast Cryptococcus neoformans

Abstract Stationary-phase cells of Cryptococcus neoformans displayed two morphological characteristics: virtually all the cells were unbudded even in the early stationary phase and even when grown in rich media, and average cell size increased from that of exponential-phase cells. DNA contents for small and large stationary-phase cells were determined by quantitative fluorescence microscopy after DNA staining with propidium iodide or DAPI. Small cells contained G, DNA, whereas large unbudded cells had either a G₂ or G₁ DNA content, indicating that Cr. neoformans can enter into the stationary phase from either the G₁ or G₂ period.     

Are progenitor cells pre‐programmed for sequential cell cycles not requiring cyclins D and E and activation of Cdk2?

Abstract.   Objectives : Based on studies of unicellular organisms or cultured mammalian cells, the generally accepted model of cell-cycle regulation has been developed in which sequential (scheduled) expression of cyclins D, E, A and B and activation of Cdk2 and Cdk1 takes place. It is assumed that the same model is applicable both in vivo and in vitro. Materials and methods : In the present study, we compared proliferating marrow cells freshly isolated from healthy individuals with proliferating lymphocytes in cultures. Results : We demonstrate that during progression of freshly collected human bone marrow cells through G₁, S and G₂/M, only Cdk1 combined with cyclins A and B₁ was distinctly present and active, and its activity gradually increased. In contrast, in vitro growing mitogen-stimulated lymphocytes had perfectly scheduled sequential expression of all four cyclins and Cdk1 and Cdk2 activities. Conclusion : Our findings demonstrate that the pattern of cyclin expression and Cdk activity in bone marrow in vivo is distinctly different from the one observed for normal cells in vitro . Because proliferating bone marrow cells are predominantly expanding populations of committed progenitors, it is likely that during the expansion phase their cell-cycle progression is pre-programmed, being driven solely by Cdk1 combined either with cyclin A or with cyclin B₁. Expansion of progenitor cells thus may not require the early steps of cell-cycle regulation, associated with triggering progression by availability of growth factors and mitogens.     

Mice Deficient in Group IV Cytosolic Phospholipase A2 Are Resistant to MPTP Neurotoxicity

Abstract: Phospholipase A₂ (PLA₂) enzymes are critical regulators of prostaglandin and leukotriene synthesis, and they may also play an important role in the generation of intracellular free radicals. The group IV cytosolic form of phospholipase A₂ (cPLA₂) is regulated by changes in intracellular calcium concentration, and the enzyme preferentially acts to release arachidonic acid esterified at the sn -2 position of phospholipids. We examined the susceptibility of mice carrying a targeted mutation of the cPLA₂ gene to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity. Mutant mice have no functional cPLA₂ activity. Mice that were homozygous for the mutation (cPLA₂^−/−) were significantly resistant to MPTP-induced dopamine depletion as compared with littermate control (cPLA₂^+/+) and heterozygous mice (cPLA₂^+/−). These findings provide evidence that cPLA₂ plays a role in MPTP neurotoxicity and suggest that cPLA₂ may play a role in the development of Parkinson's disease in humans.     

Synthesis of protein and mRNA is necessary for transition of suspension-cultured Catharanthus roseus cells from the G1 to the S phase of the cell cycle

The effects of inhibition of the synthesis of protein, mRNA or rRNA on the progression of the cell cycle have been analyzed in cultures of Catharanthus roseus in which cells were induced to divide in synchrony by the double phosphate starvation method. The partial inhibition of protein synthesis at the G₁ phase by anisoniycio or cycloheximide caused the arrest of cells in the G₁ phase or delayed the entry of cells into the S phase. When protein synthesis was partially inhibited at the S phase, cell division occurred to about the same extent as in the control. When asynchronously dividing cells were treated with cycloheximide, cells accumulated in the G₁ phase, as shown by flow-cytometric analysis. The partial inhibition of mRNA synthesis by α-amanitin at the G₁ phase caused the arrest of cells in the G₁ phase, although partial inhibition of mRNA synthesis at the S phase had little effect on cell division. In the case of inhibition of synthesis of rRNA by actinomycin D at the G₁ phase, initiation of DNA synthesis was observed, but no subsequent DNA synthesis or the division of cells occurred. However, the addition of actinomycin D during the S phase had no effect on cell division. These results suggest that specific protein(s), required for the progression of the cell cycle, are synthesized in the G₁ phase, and that the mRNA(s) that encode these proteins are also synthesized at the G₁ phase.     

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.     

Heterogeneity in the mouse epidermal cell cycle analysed by computer simulations

Abstract. Different sets of cell kinetic data obtained over many years from hairless mouse epidermis have been simulated by a mathematical model including circadian variations. Simulating several independent sets of data with the same mathematical model strengthens the validity of the results obtained. The data simulated in this investigation were all obtained with the experimental system in a state of natural synchrony. The data include cell cycle phase distributions measured by DNA flow cytometry of isolated epidermal basal cells, fractions of tritiated thymidine ([³H]TdR) labelled cells within the cell cycle phases measured by cell sorting at intervals after [³H]TdR pulse labelling, bivariate bromodeoxyuridine (BrdUrd)/DNA data from epidermal basal cells isolated at intervals after pulse labelling with BrdUrd, mitotic rate and per cent labelled mitosis (PLM) data from histologic sections. The following main new findings were made from the simulations: the second PLM peak observed at about 35 h after pulse labelling is hardly influenced by circadian variations; the peak is mainly determined by persisting synchrony of a rapidly cycling population with a G₁-duration (T_G1) of 20 h to 30 h; and there is a highly significant population of slowly cycling G₁-cells (G_1σ). However, no significant circadian variations were found in the number of these cells.     

Influence of denervation on the regeneration of Pleurodele limbs

Abstract. A cytophotometric study of Feulgen-stained mesenchymal cell nuclei from regeneration blastemas of both innervated and denervated limbs over the 1st 7 days following the midbud stage showed a diminution of the percentage of cells in the S + G₂ phases and a corresponding augmentation of the percentage of cells in the G₀+ G₁ phases. This change, which was temporally correlated with the redifferentiation of the innervated blastemas, was greater in denervated blastemas, even though they do not redifferentiate. From these results, it is concluded that the denervation of midbud blastemas brings about either an extension of the G₁ phase or an exiting from the cell cycle to G₁ (G_0–1), or both phenomena.     

LOCATION OF THE G0-PHASE IN THE CELL CYCLE OF THE MOUSE HAEMOPOIETIC SPLEEN COLONY FORMING CELLS

Experiments in mice on the fraction of haemopoietic stem cells in S-phase after irradiation indicated that a large fraction of the cells resting in G₀ will enter S-phase after a very short interval of time.
After excluding alternative explanations it must be concluded that cells in G₀ have completed all preparations for going into S-phase or, in other words, that the localization of these G₀ cells in relation to other phases of the cell cycle must be between G₁ and S-phase.     

THE SUBUNIT STRUCTURE OF MAMMALIAN ACETYLCHOLINESTERASE: CATALYTIC SUBUNITS, DISSOCIATING EFFECT OF PROTEOLYSIS AND DISULPHIDE REDUCTION ON THE POLYMERIC FORMS

Abstract— An analysis of the [³H]DFP-labelled catalytic subunits of mammalian (bovine SCG) acetylcholinesterase (AChE, EC 3.1.1.7.) indicates a monomer molecular weight of 75,000. This is equivalent to the mass previously determined for the smallest active form and demonstrates that the globular, or G forms, are respectively monomeric (G₁ form, 4S), dimeric (G₂ form, 6.5S) and tetrameric (G₄ form, 10S). In the tetrameric G₄ form the catalytic chains are associated in dimers, by disulphide bonds.
The effect of reduction and proteolysis has shown that the dimeric form (G₂ form, 6.5S) is readily reduced into G₁, while the tetramer G₄ is very stable, being only dissociated by a combination of reduction and proteolysis by high concentration of trypsin. The asymmetric forms A₁₂ (16S), A₈ (13S) and A₄ (9S) are not sensitive to reduction, but are readily dissociated by low concentrations of trypsin, into each other, progressively liberating isolated tetramers. We obtained essentially identical results with AChE preparations from rat brain or superior cervical ganglion. These observations support a general model for the quaternary structure of acetylcholinesterase molecular forms.     

Manipulation of fatty acid amide hydrolase functional activity alters sensitivity and dependence to ethanol

The aim of this study was to examine the role of fatty acid amide hydrolase (FAAH) on ethanol sensitivity, preference, and dependence. The deletion of FAAH gene or the inhibition of FAAH by carbamoyl-biphenyl-3-yl-cyclohexylcarbamate (URB597) (0.1 mg/kg) markedly increased the preference for ethanol. The study further reveals that URB597 specifically acts through FAAH and that cannabinoid-1 (CB₁) receptor is critical for N -arachidonoyl ethanolamide (AEA) mediated ethanol-reinforced behavior as revealed by lack of URB597 effect in both FAAH and CB₁−/− mice compared with vehicle-treated −/− mice. The FAAH −/− mice displayed a lower sensitivity to hypothermic and sedative effects to acute ethanol challenge. The FAAH −/− mice also exhibited a reduction in the severity of handling-induced convulsions following withdrawal from chronic ethanol exposure. The CB₁ receptor and proenkephalin gene expressions, and CB₁ receptor and μ-opioid (MO) receptor-mediated G-protein activation were found to be significantly lower in the caudate-putamen, nucleus accumbens core and shell of FAAH −/− than +/+ mice. Interestingly, the MO receptor-stimulated G-protein signaling was greater in the striatum of FAAH −/− than +/+ mice following voluntary ethanol consumption. These findings suggest that an elevation in the AEA content and its action on the limbic CB₁ receptor and MO receptor might contribute to ethanol-reinforced behavior. Treatment with drugs that decrease AEA tone might prove useful in reducing excessive ethanol consumption.