Deficit in oxygen causes G2 budding and unbudded G2 arrest in Cryptococcus neoformans

Cryptococcus neoformans exhibited diphasic growth when grown under limited aeration. First, it grew exponentially, but at OD 1, the concentration of dissolved oxygen in culture decreased to 1 mg l(-1) and a second phase of slow growth was started. This phase was characterized by a shift of budding from S to G(2), a sharp decrease in budding index and a sharp increase in the proportion of unbudded G(2) cells to 80%. Thus, a deficit in oxygen was demonstrated to delay the timing of budding, prolong the G(2) phase and cause accumulation of cells after DNA synthesis, but before commitment to budding.     

Epidermis Extracts (Chalone) Inhibit Cell Flux At the G1-S, S-G2 and G2-M Transitions In Mouse Epidermis

Epidermal cell flux at the G₁-S, S-G₂ and G₂-M transition was examined during the first 4 hr after injection of epidermis extract. the flux parameters were estimated by a combination of several methods. the G₁-S and S-G₂ transit rates were calculated on the basis of a double labelling technique with [³H]TdR, the G₂-M flux by means of colcemid and the relative proportion of cells in the S or G₂ phase by means of flow cytometry. All experiments were performed both in early morning and late evening, corresponding to maximum and minimum rates of epidermal cell proliferation in the hairless mouse. the epidermis extract inhibited the S-G and G₂-M transit rates to the same degree, while the inhibition of cell flux at the G₁-S transit was consistently stronger. In general, the inhibition of cell flux at the different transitions was most pronounced when the rate of cell proliferation was low and vice versa.     

G2 Sub-Population In Mouse Liver Induced Into Mitosis By Lead Acetate

A single intracardiac dose of lead acetate (40 μ lead/g body weight) induced a 25-fold increase in mitosis of mouse hepatocytes 5 hr after injection, as determined by autoradiography. the prompt appearance of a mitotic wave and the relatively large number of mitoses suggest that the mitotic cells were derived from a hepatocyte sub-population arrested in the G₂ phase. the injection of lead also stimulated a small increase in labeled hepatocytes within 6 hr. Analysis of grain counts gave no evidence for unscheduled DNA synthesis. the incremental labeled cells may have originated from a small fraction of the G₁ population that was ready to enter the S phase without the usual pre-synthetic delay.     

Cultured Human Tumour Cells May Be Arrested In All Stages of the Cycle During Stationary Phase: Demonstration of Quiescent Cells In G1, S and G2 Phase

Six human colon carcinoma cell lines were induced to enter stationary phase of growth by nutrient deprivation and cell crowding. Growth kinetics parameters (cell number, flow cytometric analysis of DNA distribution, and labelling and mitotic indices) were measured sequentially for all lines during the various stages of in vitro growth. Our results demonstrated that a substantial fraction of cells (9–18%) were located in G₂, phase when they changed from an exponential to a stationary mode of growth. Moreover, a large number of cells in stationary phase of growth had an S-phase DNA content, as determined by flow cytometry, but failed to incorporate radioactive DNA precursors (up to 15-fold difference). to substantiate these findings. cells in stationary phase of growth were induced to enter exponential growth by re-seeding in fresh medium at a lower density. Subsequently observed changes in DNA-compartment distribution, and in labelling and mitotic indices were those expected from cells that had been arrested at different stages of the cycle during their previous stationary phase. Thus, the non-proliferating quiescent state (Q), traditionally located ‘somewhere’ in G₁, phase, appears to be composed also of cells that can be arrested at other stages of the cycle (Q_s, and Q_G). Although the proportion of such cells is rather small, their contribution to the growth kinetics behaviour of human in vivo tumours will become apparent following ‘recruiting’ or ‘synchronizing’ clinical manoeuvres and will prevent the formation of a clear-cut wave of synchronized cells.     

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 Hydroxyurea On the Mitotic Activity and the G2 Phase In Hairless Mouse Epidermis

Hairless mice were given 5 mg hydroxyurea (HU) intraperitoneally (i.p.) followed by 0.15 mg Colcemid® at various times after HU. the animals were killed at 2 and 4 hr after Colcemid, the epidermal mitotic counts in dorsal skin were determined and the mitotic rates calculated. These were compared with the normal mitotic rates, and the ratios between the results from HU-treated and -untreated animals were calculated. Hydroxyurea caused a considerable reduction in the mitotic rate with a trough at 6 hr, followed by a wave of increased mitotic rate with a peak at 14 hr, followed by a secondary drop at 20 hr, and then a return to normal. Another group of mice were given HU only, and the fraction of epidermal cells in G₂ was measured by flow cytometry. From these animals, without previous injection of Colcemid, we also determined the mitotic counts and calculated the mitotic durations. Cells piled up in G₂ for the first 6 hr after HU injection, then the G₂ compartment was emptied. the results are discussed in relation to previous results from this department showing the effect of the same dose of HU on DNA synthesis in the same mouse strain. It is concluded that HU not only blocks or retards DNA synthesis in epidermal cells, but also affects the movement of cells through G₂ and M. the cell kinetic effects of HU thus seem to be very complex.     

ON THE EXISTENCE OF 'ARRESTED G2 CELLS' IN MOUSE EPIDERMIS

It has been postulated that mouse epidermis contains two populations of resting cells, one of which is blocked at the G₁-S boundary and the other between G₂ and mitosis. the ‘arrested G₂ cells’ were estimated, by the labelled mitosis method, to comprise 510% of the epidermal population and presumed to function as a ‘reserve pool’ which could be activated by wounding. A comprehensive search has now been carried out for arrested G₂ cells in mouse epidermis using the direct methods of single cell and flow through cytophotometry. No evidence was obtained which supports the existence of such a cell compartment. Suitable control experiments were carried out to ensure that G₂ cells were not lost during the isolation of epidermal nuclei.     

Pertussis Toxin-Insensitive Signaling of the ORL1 Receptor: Coupling to Gz and G16 Proteins

Abstract: Nociceptin/OFQ is the endogenous ligand for the G protein-coupled opioid receptor-like (ORL₁) receptor. To elucidate the cellular functions of the ORL₁ receptor, we examined its ability to interact with G_z and G₁₆, two pertussis toxin (PTX)-insensitive G proteins that are known molecular partners for the opioid receptors. In HEK 293 cells transiently expressing the ORL₁ and dopamine D₁ receptors, nociceptin/OFQ dose-dependently inhibited dopamine-stimulated cyclic AMP (cAMP) accumulation in a PTX-sensitive manner. However, PTX failed to block the nociceptin/OFQ-induced inhibition of dopamine-stimulated cAMP accumulation in HEK 293 cells co-expressing the α-subunit of G_z. This result indicates functional interaction between the ORL₁ receptor and G_z. A similar result was obtained with retinoic acid-differentiated SH-SY5Y cells, which endogenously express both the ORL₁ receptor and G_z. When the ORL₁ receptor was transiently co-expressed in COS-7 cells with the α-subunit of G₁₆, nociceptin/OFQ dose-dependently stimulated the formation of inositol phosphates. Nociceptin-induced stimulation of phospholipase C was absolutely dependent on the co-expression of α₁₆ and exhibited the appropriate ligand selectivity. In terms of its ability to interact with PTX-insensitive G proteins, the ORL₁ receptor behaves very much like the opioid receptors.     

SEPARATION OF PROLIFERATING CFU FROM G2 CELLS OF MURINE BONE MARROW

The technique of velocity sedimentation was employed to fractionate mouse bone marrow CFU-s relative to two different phases of the cell cycle. CFU-s in DNA synthesis which are destroyed by hydroxyurea were separated from other smaller CFU-s which were shown to be insensitive to the drug and are presumably those CFU-s in the G₂ phase of cell cycle.     

Direct Measurement of the G1 Phase In Meristematic Cells At Two Different Temperatures

Abstract. The duration of the G₁ period in meristematic cells has been measured directly at two different temperatures by using a synchronous cell population 'labelled' as binucleate by treatment with caffeine. By studying the uptake of a radioactive DNA-precursor, it proved possible to determine the duration of the G₁ period in relation to the total interphase duration, at two temperatures tested, and to demonstrate that the relationship remained constant.     

HYDROXYUREA: USE IN DETERMINING THE DURATION OF G2 IN TETRAHYMENA

Observation of division of individual cells in microdrops, plus autoradiographic studies using tritiated thymidine and standard cell cycle analysis techniques, reveal that hydroxyurea (10 DIM) reversibly arrests the normal progression of exponentially growing Tetrahymena pyriformis through the initial 92 % of S-phase while not affecting cells in the terminal 8 % and in G₂ and division. Thus the fraction of the population of cells that is in G₂ can be approximately determined by the fraction of the population able to divide in the presence of hydroxyurea. This fraction can be related to the approximate duration of G₂ by calculations which compensate for the age gradient.     

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.     

Early and late G2 arrest of cells undergoing radiation-induced apoptosis or micronucleation

Conventional flow cytometric DNA measurements combined with the microscopic detection of cells in the late G₂ phase of the cell cycle (characterized by the occurrence of paired kinetochores) enabled us to differentiate and to quantify early and late G₂ cells 0–40 h after irradiation using a radioresistant (L929) and a radiosensitive (HL-60) cell line. This approach provided us with ( 1 ) a new kind of G₂ arrest characteristic revealing changes in the G₂ phase which can not be detected by flow cytometric DNA measurements, ( 2 ) cell line dependent differences in the radiation-induced transition through G₂, accompanied by the occurrence of micronucleation and apoptosis, and ( 3 ) the characterization of apoptotic cells occurring probably during early G₂ and bearing a rapidly reduced number of kinetochores in contrast to mitotic cells, suggesting processes different from those that operate in mitosis.     

Evidence for arrested G2 cell subpopulation in rat liver inducible to mitosis

Abstract The intraperitoneal administration of several substances (biliverdin, heat-killed bacteria and diatomaceous earth) to rats caused the prompt appearance of a mitotic wave in the liver. Autoradiographic analysis of livers of treated animals showed no evidence of [³H]-thymidine uptake by mitotic hepatocytes. In addition, livers from xenobiotic-treated rats showed a very low thymidine kinase activity, close to that found in normal livers. This excludes the possibility that non-cycling cells move to mitosis through the S phase. The results suggest that mitosis could be derived from a hepatocyte subpopulation arrested in the G₂ phase of the cell cycle, which is stimulated to divide by the xenobiotics.     

D2 Receptors May Modulate the Function of the Striatal Transporter for Dopamine: Kinetic Evidence from Studies In Vitro and In Vivo

Abstract: Recently it was hypothesized by others that the D₂dopamine receptor can regulate the uptake of dopamine. However, the evidence in support of this hypothesis, although compelling, was not based on observations related to direct measures of the kinetic activity of the transporter itself. Here kinetic evidence in support of this hypothesis is shown. The apparent time-resolved initial velocity of the transport of 1.0 μ M dopamine into striatal suspensions, measured using rotating disk electrode voltammetry, was found to increase in the presence of the D₂ receptor agonist, quinpirole, at 100 μ M . This effect was reversed by sulpiride. In separate studies it was shown that acute and chronic treatments with haloperidol at 0.5 mg/kg, i.p., reduced the reuptake transport of dopamine in vivo following intrastriatal stimulation of its release by K⁺. Thus, it appears that D₂ receptors may influence the functioning of the striatal transporter for dopamine. These results are consistent with a model in which presynaptically released dopamine may feed back onto the function of its transporter to increase the velocity of the clearance of synaptic dopamine following an action potential, suggesting the existence of a mechanism, in addition to release and synthesis modulation, for fine-tuning dopaminergic chemical signaling.     

Rat Serum Factors Inhibiting the G1-S Transition In Hepatocytes

An attempt was made to detect the serum factors inhibiting the G₁-S transition in synchronized, baby rat hepatocytes. In untreated adult rat serum, this inhibitory activity was always linked to high molecular weight (HMW) compounds. Incubation of serum with trypsin or chymotrypsin resulted in the formation of a low molecular weight (LMW) G₁-S inhibitory factor. the same result was obtained with fractions from adult rat liver but not with kidney or spleen fractions. Separation of the LMW factor by ultrafiltration increased its specific activity by about 10³. the active period in the cell cycle of both the LMW and HMW factors was the same: the late G₁ phase. However, the activity of the LMW factor was not blocked by the Kunitz factor. an enzymatic transformation of the HMW factor might be induced by liver cell membrane-bound proteases and constitute a mechanism regulating hepatocyte proliferation.     

Variation In G1 Transit Time Relative to the Cycloheximide and Actinomycin D Drug Restriction Points

The transit time distribution at various points in the cell cycle of synchronized Chinese hamster ovary cells was determined from the mitotic index, [³H]thymidine labeling index and increase in cell number monitored at regular intervals after mitotic selection. Variation in G₁ transit time compared with that for the total cell cycle indicates that variation in cell cycle transit time occurs mainly during G₁ phase. the cycloheximide (5.0 μg/ml) and actinomycin D (3.0 μg/ml) restriction points occur 0.2 and 1.7 hr prior to entry into S phase, respectively. the transit time distributions are further characterized by the moments of the distributions. the variance (2nd moment about the mean) of the transit time distribution at the actinomycin D restriction point is similar to the variance of the transit time distribution at the G₁/S border, thus variation in cell cycle transit time originates earlier than 1.7 hr prior to entry into S phase (i.e., the first 3/4 of G₁). If G₁ transit time variability and cell cycle control are related, then the results presented here indicate that the major regulatory events do not occur during late G₁ phase.     

In Vitro and In Vivo Irreversible Blockade of Cortical S2 Serotonin Receptors by N-Ethoxycarbonyl-2-Ethoxy-1,2-Dihydroquinoline: A Technique for Investigating S2 Serotonin Receptor Recovery

N-Ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) treatment, both in vitro and in vivo, results in an irreversible blockade of cortical S2 5-hydroxytryptamine (serotonin) receptors. Incubation of rat cortical homogenates with EEDQ in vitro results in a concentration-dependent (EC50 approximately 5 microM) and time-dependent decrease in the Bmax of [3H]ketanserin-labeled S2 serotonin receptors. Extensive washing of the homogenate following in vitro or in vivo EEDQ treatment does not result in an increase in the amount of [3H]ketanserin binding, indicating that EEDQ acts to modify irreversibly cortical S2 serotonin receptors. That the modification of S2 receptor binding by EEDQ occurs at the recognition site of the receptor is indicated by the finding that coincubation with the S2 receptor antagonist ketanserin, but not the D2 3,4-dihydroxyphenylethylamine (dopamine) receptor antagonist domperidone, selectively protects against the irreversible blockade of S2 serotonin receptors. Peripheral administration of EEDQ results in a dose-dependent reduction in cortical S2 serotonin receptors with maximal effects (approximately 90% reduction) observed following 10 mg/kg (i.p.). Seven days following peripheral administration of EEDQ there is a recovery of S2 serotonin receptors back to 74% of the original receptor population. These data demonstrate that EEDQ in vitro and in vivo acts as an irreversible antagonist of S2 serotonin receptors and that it can be used to investigate the recovery rate of these receptors.     

D2 Receptor Antagonists Do Not Modify Guanine Nucleotide-Sensitive Interactions Between Dopamine and D1 Dopamine Receptors Under In Vitro Conditions

Abstract: This study investigated possible D₁/D₂ interactions in rat and bovine striatal tissue by examining the effects of D₂ antagonists on the action of dopamine at D₁ dopamine receptors. In addition, the extent to which D₂ antagonists may induce an agonist low-affinity state of the D₁ receptor was evaluated in comparison with the effects of the guanine nucleotide analogue 5′-guanylylimidodiphosphate [Gpp(NH)p]. In saturation experiments dopamine caused a dose-dependent decrease in rat striatal and bovine caudate D₁ receptor density. This effect of dopamine, which has been shown to be sensitive to Gpp(NH)p, was not altered by pretreatment with either of the selective D₂ antagonists eticlopride (200 nM) or domperidone (200 nM). Results from displacement experiments show that the affinity of dopamine for D₁ receptors and the proportion of receptors in an agonist high-affinity state, are reduced by Gpp(NH)p (100 µM) but not by eticlopride. A molar excess of dopamine (100 µM) promotes the dissociation of (±)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepine-7-ol ([³H]SCH 23390) from rat striatal D₁ receptors at a rate that is significantly slower than when dissociation is initiated using 1 µM piflutixol. After pretreatment with Gpp(NH)p, [³H]SCH 23390 dissociation induced by dopamine occurred at an even slower rate. Pretreatment with eticlopride had no effect on the dopamine-induced rate of [³H]SCH 23390 dissociation. These results indicate that all experimental approaches detected dopamine effects at D₁ receptors that are Gpp(NH)p sensitive and D₂ antagonist insensitive and provide no evidence to support a D₁/D₂ link operating at the receptor level.