Modulation of Adenylylcyclase by Protein Kinase C in Human Neurotumor SK-N-MC Cells: Evidence that the α Isozyme Mediates Both Potentiation and Desensitization

Abstract: Exposure of human SK-N-MC neurotumor cells to 4β-phorbol 12-myristate 13-acetate (PMA) increased isoproterenol stimulation of cyclic AMP levels by severalfold. This potentiation was blocked by inhibitors of protein kinase C (PKC) and did not occur in cells in which PKC had been down-regulated. PMA treatment also enhanced the stimulation by dopamine, cholera toxin, and forskolin. Thus, the effect of PMA on the adenylylcyclase system was postreceptor and involved either the guanine nucleotide binding regulatory (G) proteins or the cyclase itself. As PMA treatment did not impair the inhibition of isoproterenol stimulation by neuropeptide Y, an involvement of the inhibitory G protein G_i was unlikely. Cholate extracts of membranes from control and PMA-treated cells were equally effective in the reconstitution of adenylylcyclase activity in S49 cyc^? membranes, which lack the stimulatory G protein subunit G_sα; thus, G_s did not appear to be the target of PMA action. Membranes from PMA-treated cells exhibited increased adenylylcyclase activity to all stimulators including Mn²⁺ and Mn²⁺ plus forskolin. In addition, activity was increased when control membranes were incubated with ATP and purified PKC from rat brain. This is consistent with a direct effect of PKC on the adenylylcyclase catalyst in SK-N-MC cells. PMA treatment also resulted in a shift to less sensitivity in the K_act for isoproterenol but not for dopamine or CGP-12177 (a β₃-adrenergic agonist) stimulation. Thus, the β₁ but not the D₁ or β₃ receptors were being desensitized by PKC activation. Analysis of SK-N-MC cells by western blotting with antibodies against different PKC isozymes revealed that both the α and ζ isozymes were present in these cells. Whereas PKC-α was activated and translocated from cytosol to membrane by phorbol esters, the ζ isozyme was not. Thus, PKC-α, which has been implicated in desensitization in other cell lines, also appears to potentiate adenylylcyclase activity.     

cAMP and PMA enhance the effects of IGF-I in the proliferation of endometrial adenocarcinoma cell line HEC-1-A by acting at the G1 phase of the cell cycle

The present study was undertaken to determine whether endometrial cancer cell line HEC-1-A differ from nontransformed cells, in that the cAMP and protein kinase C pathways may enhance IGF-I effects in mitogenesis by acting at the G₁ phase of the cell cycle instead of G₀. Immunofluorescence staining of HEC-1-A cells using the proliferating cell nuclear antigen (PCNA) monoclonal antibody and flow cytometric analysis determined that HEC-1-A cells do not enter the G₀ phase of the cell cycle when incubated in a serum-free medium. Approximately 51% of the cells were in G₁, 12% were in S and 37% in G₂ phase of the cell cycle prior to treatment. Forskolin and phorbol-12-myristate 13-acetate (PMA) were used to stimulate cAMP production and protein kinase C activity, respectively. IGF-I, forskolin and PMA each increased (P <0.01) [³H]-thymidine incorporation in a dose and time dependent manner. The interaction of forskolin and PMA with IGF-I was then determined. Cells preincubated with forskolin or PMA followed by incubation with IFG-I incorporated significantly more (P <0.01) [³H]-thymidine into DNA than controls or any treatment alone. It is concluded that forskolin and, to a lesser extent, PMA exert their effect at the G₁ phase of the cycle to enhance IGF-I effects in cell proliferation.     

Effects of p21Cip1/Waf1 at Both the G1/S and the G2/M Cell Cycle Transitions: pRb Is a Critical Determinant in Blocking DNA Replication and in Preventing Endoreduplication

It has been proposed that the functions of the cyclin-dependent kinase inhibitors p21^Cip1/Waf1 and p27^Kip1 are limited to cell cycle control at the G₁/S-phase transition and in the maintenance of cellular quiescence. To test the validity of this hypothesis, p21 was expressed in a diverse panel of cell lines, thus isolating the effects of p21 activity from the pleiotropic effects of upstream signaling pathways that normally induce p21 expression. The data show that at physiological levels of accumulation, p21, in addition to its role in negatively regulating the G₁/S transition, contributes to regulation of the G₂/M transition. Both G₁- and G₂-arrested cells were observed in all cell types, with different preponderances. Preponderant G₁ arrest in response to p21 expression correlated with the presence of functional pRb. G₂ arrest was more prominent in pRb-negative cells. The arrest distribution did not correlate with the p53 status, and proliferating-cell nuclear antigen (PCNA) binding activity of p21 did not appear to be involved, since p27, which lacks a PCNA binding domain, produced similar arrest Bs. In addition, DNA endoreduplication occurred in pRb-negative but not in pRb-positive cells, suggesting that functional pRb is necessary to prevent DNA replication in p21 G₂-arrested cells. These results suggest that the primary target of the Cip/Kip family of inhibitors leading to efficient G₁ arrest as well as to blockade of DNA replication from either G₁ or G₂ phase is the pRb regulatory system. Finally, the tendency of Rb-negative cells to undergo endoreduplication cycles when p21 is expressed may have negative implications in the therapy of Rb-negative cancers with genotoxic agents that activate the p53/p21 pathway.     

REGULATION OF INITIATION OF DNA SYNTHESIS IN CHINESE HAMSTER CELLS : II. Induction of DNA Synthesis and Cell Division by Isoleucine and Glutamine in G1-Arrested Cells in Suspension Culture

Suspension cultures of Chinese hamster cells (line CHO), which had stopped dividing and were arrested in G₁ following growth to high cell concentrations in F-10 medium, could be induced to reinitiate DNA synthesis and to divide in synchrony upon addition of the appropriate amounts of isoleucine and glutamine. Both amino acids were required to initiate resumption of cell-cycle traverse. Deficiencies in other amino acids contained in F-10 medium did not result in accumulation of cells in G₁, indicating a specific action produced by limiting quantities of isoleucine and glutamine. In the presence of sufficient glutamine, approximately 2 x 10^-6 M isoleucine was required for all cells to initiate DNA synthesis in a population initially containing 1.5 x 10⁵ cells/ml. Under similar conditions, about 4 x 10^-6 M isoleucine was required for all G₁-arrested cells to progress through cell division. In contrast, 1 x 10^-4 M glutamine was necessary for maximum initiation of DNA synthesis in G₁ cells, along with sufficient isoleucine. A technique for rapid production of G₁-arrested cells is described in which cells from an exponentially growing population placed in F-10 medium deficient in both isoleucine and glutamine or isoleucine alone accumulated in G₁ after 30 hr.     

Induction of cell division in BALB/c-3T3 cells by phorbol myristate acetate or bovine serum: Effects of inhibitors of cyclic AMP phosphodiesterase and Na+−K+-ATPase

Summary Cell division is induced in stationary cultures of BALB/c-3T3 mouse embryo cells without renewal of medium by addition of the tumor promoter, phorbol myristate acetate (PMA), or bovine serum. The addition of dbcAMP (10⁻³ m) or other inhibitors of cAMP phosphodiesterase, papaverine (6.7×10⁻⁶ m), Persantin (5×10⁻⁵ m) or RO-20-1724 (10⁻⁴ m), prevents cell replication induced by PMA or serum. In contrast, ouabain (10⁻⁴ m) and N,N′-dicyclohexylcarbodiimide (10⁻⁵ m), inhibitors of Na⁺−K⁺-ATPase activity, block the PMA-stimulated effect but do not inhibit serum-stimulated cell division. Several stages in the cell cycle are sensitive to dbcAMP addition. One is early in the G₁ phase at the time of reinitiation of the cell cycle from a stationary (G₀) phase, a second is associated with the G₁-S transition, and a third with passage of cells from a post-S phase to mitosis. Based on observations of early morphological changes, responses of plasma membrane ezymes and effects of enzyme inhibitors, the stimulation of cell division in BALB/c-3T3 cells by PMA or serum appears to involve several membrane functions which may act in a cooperative manner. This work was supported by a USPHS Research Grant CA12503, and a Center Grant ES-00260 awarded to the Institute of Environmental Medicine. Mrs. Susan Kulina provided the consistent and excellent technical aid necessary to perform this work. Note added in proof: During the preparation and review of this paper, Boynton reported that PMA appears to sensitize BALB/c-3T3 cells to calcium ion which may play a critical role in the regulation of the DNA synthesis (36).     

Changes in the phosphorylation of non-histone chromatin proteins during the cell cycle of HeLa S 3 cells

The phosphorylation of non-histone chromatin proteins in synchronized HeLa S₃ cells was studied in 5 phases of the cell cycle: mitosis, G₁, early and late S, and G₂. The rate of non-histone chromatin protein phosphorylation was found to be maximal during G₁ and G₂, somewhat decreased during S phase, and almost 90% depressed during mitosis. Analysis of the phosphorylated non-histone chromatin proteins by SDS-acrylamide gel electrophoresis showed a heterogeneous pattern of phosphorylation as measured by labeling with ³²P. Significant variations in the labeling pattern were seen during different stages of the cell cycle, and particular unique species appeared to be phosphorylated selectively during certain stages of the cycle.     

Histones from exponential and stationary L-cells. Evidence for metabolic heterogeneity of histone fractions retained after isolation of nuclei

Histones of exponential and G₁-arrested mouse L-cells were double-labeled with either [³H]lysine and [³²P]phosphate or with [¹⁴C]arginine and [³H]acetate in order to investigate cell cycle-dependent changes in rates of synthesis and metabolic modifications as well as the effect of the method of nuclear isolation on retention of the labeled fractions. Results indicate that newly synthesized lysine-rich histones incorporate ³²P at the highest rate. However, phosphorylation can also be detected in G₁-arrested cells and in the case of F_2b, F_1⁰, and F_1¹, with no detectable new synthesis.On the other hand, acetylation proceeded at similar rates in both exponential and stationary cells with the exception of F_{2a 2} and F₃ which showed higher acetylation levels in the latter.In relation to the method used for nuclear isolation, we observed that, even in cases where no difference in the relative amount of a given histone could be detected, the species recovered were not identical. We conclude that none of the methods commonly employed retains all of the histones. In general, the acetylated species appear to be best preserved at a higher divalent cation concentration while the newly synthesized ones are better conserved at lower ionic strengths.     

Frequency of N-benzyladenine incorporation into major RNA fractions of tobacco cell suspensions grown at stimulatory or cytostatic cytokinin concentration

The frequency of incorporation of the cytokinin N⁶-[p-³H]benzyladenine into major RNA species of tobacco (Nicotiana tabacum cv W 38) cells steadily increased as a function of its concentration in the culture medium, up to a 10 micromolar cytostatic overdose. During a 55-hour incubation of cells with 0.4 micromolar benzyladenine (BA), which is the optimal concentration for cell division, the incorporation frequency increased to one BA per 1.5 to 2.0 × 10⁴ conventional bases in total RNA. Frequencies of BA incorporation into 18S and 25S rRNA and into RNA precursors were very similar, 2- to 3-fold higher than the frequency of BA incorporation into the 4S + 5S RNA fraction. In cells incubated with 10 micromolar BA, the rate of RNA synthesis between 24 and 55 hours was lower than at optimal growth conditions; 18S and 25S rRNA synthesis was depressed more than the synthesis of 4S + 5S RNA. At 55 hours, BA was incorporated into total RNA at the steady state frequency of one per 1,300 conventional bases. All major RNA species were BA-labeled to approximately the same level, except that the labeling of the RNA precursors was 2-fold higher than the labeling of mature RNA species. These results may reflect an alteration in the processing of the RNA precursors at supra-optimal cytokinin concentration.     

Cell cycle parameters of 3T3 cells cultured as aggregates

Summary BALB/c 3T3 cells cultured as aggregates were examined by two independent techniques to determine whether or not cells accumulated at a specific point in the cell cycle, and if so to determine the point at which they accumulate. Replating cells onto dishes followed by pulse labeling with [³H]thymidine and autoradiography indicated that aggregate-cultured cells were in the same phase of the cell cycle as cells cultured as confluent monolayers. Flow microfluorometry confirmed that 75% of the aggregate-maintained cells were arrested in G₀ or G₁, with 25% distributed throughout the rest of the cell cycle. Labeling and mitotic indices of cells in aggregates were also consistent with about 20 to 25% of the cells being in S+G₂=M phases of the cell cycle at any time. This work was supported by PHS Grant CA20323 and NSF Grant PCM 74-15092 to H. G., who is also a Harry H. Pinney Cancer Scholar.     

Effect of transient overexpression of Gqα on soluble and polymerized tubulin pools in GH3 and AtT-20 cells

In order to study G_q-tubulin interaction in the cytosol, GH₃ and AtT-20 cells (stably expressing TRH receptor) were transiently transfected with G_qα cDNA. Forty-eight hours after transfection, thyrotropin-releasing hormone (TRH)-stimulated prolactin (PRL) secretion by G_qα-transfected GH₃ cells increased by 90% compared to mock-transfected cells. In addition, using immunocytochemistry it was observed that G_qα-specific staining was much more prominent in G_qα-transfected GH₃ and AtT-20 cells (also transfected with G_qα) compared to mock-transfected cells. Thus, transfection resulted in successful overexpression of functional G_qα. Forty-eight hours after transfection, cells were processed to obtain soluble and polymerized tubulin fractions. Tubulin levels were determined in these fractions by immunoblotting using polyclonal anti-tubulin antibodies. Compared to mock-transfected cells soluble tubulin levels decreased in G_qα-transfected GH₁ and AtT-20 cells, by 33 and 52%, respectively. Moreover, compared to mock-transfected cells a 50% reduction in the ratio (an index of the flux between tubulin pools) of soluble and polymerized tubulin levels was observed in G_qα-transfected GH₃ and AtT-20 cells. To determine whether these effects on tubulin were mediated by G_q directly, we examined the influence of purified G_q on tubulin polymerization. G_q (0.5 μM) inhibited polymerization of crude tubulin (present in GH₃ cell cytosol) by 53%. In contrast to its effects on GH₃ cell cytosol tubulin, G_q stimulated purified tubulin polymerization by 160%. These results suggest that G_q modulates the polymerization and depolymerization cycles of tubulin and that this modulation is in turn influenced by other unknown cellular components. © 1996 Wiley-Liss, Inc.     

Role of Vitamin D3 Receptor in the Synergistic Differentiation of WEHI-3B Leukemia Cells by Vitamin D3 and Retinoic Acid

WEHI-3B D⁻ cells differentiate in response to 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) but not to all-trans-retinoic acid (RA) or other inducing agents. Combinations of RA with 1,25-(OH)₂D₃ interact to produce synergistic differentiation of WEHI-3B D⁻ cells. To determine factors involved in the synergistic interaction, expression of the 1,25-(OH)₂D₃ receptor (VDR) and retinoid receptors, RARα and RXRα, was measured. No VDR was detected in untreated WEHI-3B D⁻ cells; however, RA and 1,25-(OH)₂D₃ when used as single agents caused a slight induction of the VDR and in combination produced a marked increase in the VDR. In contrast, no changes in RARα and RXRα were initiated by these compounds. An RAR-selective agonist combined with 1,25-(OH)₂D₃ produced synergistic differentiation of WEHI-3B D⁻ cells, whereas an RXR-selective agonist did not. To gain information on the role of the VDR in the synergistic interaction, the VDR gene was transferred into WEHI-3B D⁺ cells, in which no VDR was detected and no synergism was produced. Expression of the VDR conferred differentiation responsiveness to 1,25-(OH)₂D₃ in WEHI-3B D⁺ cells. These findings suggest that (a) induction of VDR expression is a key component in the synergistic differentiation induced by 1,25-(OH)₂D₃ and RA and (b) RAR and not RXR must be activated for enhanced induction of the VDR and for the synergistic differentiation produced by RA and 1,25-(OH)₂D₃.     

Uptake of inorganic carbon by isolated chloroplasts from air-adapted dunaliella

Neither Dunaliella cells grown with 5% CO₂ nor their isolated chloroplasts had a CO₂ concentrating mechanism. These cells primarily utilized CO₂ from the medium because the K_(0.5) (HCO₃⁻) increase from 57 micromolar at pH 7.0 to 1489 micromolar at pH 8.5, where as the K_(0.5) CO₂ was about 12 micromolar over the pH range. After air adaptation for 24 hours in light, a CO₂ concentrating mechanism was present that decreased the K_0.5 (CO₂) to about 0.5 micromolar and K_0.5 (HCO₃⁻) to 11 micromolar at pH 8. These K_0.5 values suggest that air-adapted cells preferentially concentrated CO₂ but could also use HCO₃⁻ from the medium. Chloroplasts isolated from air-adapted cells had a K_(0.5) for total inorganic carbon of less than 10 micromolar compared to 130 micromolar for chloroplasts from cells grown on high CO₂. Chloroplasts from air-adapted cells, but not CO₂-grown cells, concentrate inorganic carbon internally to 1 millimolar in 60 seconds from 240 micromolar in the medium. Maximum uptake rates occurred after preillumination of 45 seconds to 3 minutes. The CO₂ concentrating mechanism by chloroplasts from air-adapted cells was light dependent and inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) or flurocarbonyl-cyamidephenylhydrazone (FCCP). Phenazine-methosulfate at 10 micromolar to provide cyclic phosphorylation partially reversed the inhibition by DCMU but not by FCCP. One to 0.1 millimolar vanadate, an inhibitor of plasma membrane ATPase, inhibited inorganic carbon accumulation by isolated chloroplasts. Vanadate had no effect on CO₂ concentration by whole cells, as it did not readily cross the cell plasmalemma. Addition of external ATP to the isolated chloroplast only slightly stimulated inorganic carbon uptake and did not reverse vanadate inhibition by more than 25%. These results are consistent with a CO₂ concentrating mechanism in Dunaliella cells which consists in part of an inorganic carbon transporter at the chloroplast envelope that is energized by ATP from photosynthetic electron transport.     

The metabolism of histone fractions. VI. Differences in the phosphorylation of histone fractions during the cell cycle

Phosphorylation of histone fractions in the presence and absence of DNA synthesis was measured using the new “isoleucine-limiting” method for synchronizing Chinese hamster cells in early G₁-phase. Using preparative electrophoresis, histone f1 phosphorylation was found to be dependent upon cell-cycle position, being absent in G₁-arrested and G₁-traversing cells and active in the S-phase. The absence of f1 phosphorylation in G₁-arrested cells, which are known to exhibit f1 turnover, indicates that f1 phosphorylation is not an obligatory part of the f1 turnover process. In contrast to histone f1, it was found that histone f2a2 phosphorylation is independent of cell-cycle position, occurring with equal magnitude in the G₁-traversing state when DNA synthesis is essentially absent and in the S-phase when DNA synthesis is active. When cells were arrested in the G₁-state by isoleucine deprivation, f2a2 phosphorylation continued to be active, occurring at 56% of the rate observed in the G₁-traversing state. These results indicate that phosphorylation of histone f2a2 is independent of f2a2 synthesis, independent of DNA synthesis, and independent of histone f1 phosphorylation. Because f2a2 is actively phosphorylated in G₁-arrested cells known to be active in the synthesis of various types of RNA (including messenger) as well as in G₁-traversing and S-phase cells, we feel that phosphorylation of histone f2a2 should continue to be considered in models concerning activation of DNA template activity.     

The synthesis of acidic chromosomal proteins during the cell cycle of HeLa S-3 cells. II. The kinetics of residual protein synthesis and transport

The kinetics of acidic residual chromosomal protein synthesis and transport were studied throughout the cell cycle in HeLa S-3 cells synchronized by 2 mM thymidine block and selective detachment of mitotic cells. Pulse labeling the cells with leucine-³H for 2 min and then "chasing" the radioactive proteins for up to 3 hr showed that the amount of protein synthesized, transported, and retained in the acidic residual chromosomal protein fraction is greater immediately after mitosis and later in G₁ than in the S or G₂ phases of the cell cycle. During S, only 20–25% of the proteins synthesized and transported to the acidic residual chromosomal protein fraction are chased during the first 2 hr after pulse labeling, whereas up to 40% of the material entering the residual nuclear fraction in mitosis, G₁, and G₂ leaves during a 2 hr chase. Polyacrylamide gel electrophoretic profiles of these proteins, at various times after pulse labeling, reveal that the turnover of individual polypeptides within this fraction has kinetics of synthesis and turnover which are markedly different from one another and undergo stage-specific changes.     

Mechanism of C4 photosynthesis in Chloris gayana: pool sizes and kinetics of 14CO2 incorporation into 4-carbon and 3-carbon intermediates.

In one group of C₄ species, including Chloris gayana, C₄ acids are decarboxylated via phosphoenolpyruvate carboxykinase to give phosphoenolpyruvate as the initial C₃ product. This paper presents an analysis of the kinetics of labeling of various photosynthetic intermediates in Chloris gayana leaves exposed to ¹⁴CO₂, and the pool sizes of these intermediates, primarily to provide information about the subsequent metabolism of phosphoenolpyruvate. Saturation labeling of the C-4 of aspartate and malate, and the C-1 of 3-phosphoglycerate, indicated photosynthetically active pools of 0.45, 0.22, and 0.95 μol/mg chlorophyll, respectively. For aspartate and 3-phosphoglycerate, the total leaf pools and the photosynthetic pools were of similar size, but the total pool of malate was about 100 times larger than the photosynthetically active pool. From the relative rates of labeling of phosphoenolpyruvate, pyruvate, alanine, and C-1, C-2 plus C-3 of aspartate, during steady-state ¹⁴CO₂ assimilation, relative pool sizes were calculated to be about 10:11:78:100, respectively. Pulse/chase labeling of leaves provided estimates of relative photosynthetic pool sizes in the ratio of about 6:15:90:100, respectively, where aspartate is arbitrarily assigned a value of 100 in both cases. Notably, labeling of alanine was consistent with its derivation from the C-1, C-2 plus C-3 carbons of aspartate, and the alanine pool was at least eight times larger than the phosphoenolpyruvate pool that showed similar labeling kinetics. Results were consistent with the view that at least most of the phosphoenolpyruvate produced by C₄ acid decarboxylation is metabolized via alanine.     

STUDIES ON THE DIVISION CYCLE OF MAMMALIAN CELLS : V. Modifications of Time Parameters by Different Steady-State Culture Conditions

In cultures of murine neoplastic mast cells, the duration of different phases of the division cycle (G₁, S, G₂, and mitosis [M]) was determined under optimal and several well-defined suboptimal growth conditions. Two methods of evaluation were applied to the same culture system: first, the relative number of G₁, S, G₂, and M cells was determined by pulse labeling of samples with thymidine-³H and subsequent radioautography in conjunction with a microfluorometric technique permitting rapid measurements of cellular DNA content; second, after pulse labeling with thymidine-³H, the variations with time of the mitotic labeling index were analyzed. Suboptimal culture conditions were obtained by reducing the concentration of single essential medium components (leucine, glucose, or serum) or by the addition of specific metabolic inhibitors (actinomycin D, amethopterin). Growth-limiting culture conditions resulted in increased generation times. Even under control conditions, the cell number doubling time exceeded the generation time, and this difference was more pronounced in suboptimal media. Under most of the suboptimal conditions tested, the increase in generation time was attributable primarily to an extended duration of the G₁ phase. Under certain growth-limiting conditions, however, other phases were also prolonged. In addition, the variabilities of the generation time and of certain cell cycle phases were increased under suboptimal culture conditions. Results obtained by the two methods of evaluation were, in general, in good agreement with each other. Some differences were, however, observed and interpreted in terms of cell death and/or asymmetric frequency distributions of cell cycle parameters.     

CELL CYCLE-SPECIFIC CHANGES IN HISTONE PHOSPHORYLATION ASSOCIATED WITH CELL PROLIFERATION AND CHROMOSOME CONDENSATION

Preparative polyacrylamide gel electrophoresis was used to examine histone phosphorylation in synchronized Chinese hamster cells (line CHO). Results showed that histone f1 phosphorylation, absent in G₁-arrested and early G₁-traversing cells, commences 2 h before entry of traversing cells into the S phase. It is concluded that f1 phosphorylation is one of the earliest biochemical events associated with conversion of nonproliferating cells to proliferating cells occurring on old f1 before synthesis of new f1 during the S phase. Results also showed that f3 and a subfraction of f1 were rapidly phosphorylated only during the time when cells were crossing the G₂/M boundary and traversing prophase. Since these phosphorylation events do not occur in G₁, S, or G₂ and are reduced greatly in metaphase, it is concluded that these two specific phosphorylation events are involved with condensation of interphase chromatin into mitotic chromosomes. This conclusion is supported by loss of prelabeled ³²PO₄ from those specific histone fractions during transition of metaphase cells into interphase G₁ cells. A model of the relationship of histone phosphorylation to the cell cycle is presented which suggests involvement of f1 phosphorylation in chromatin structural changes associated with a continuous interphase "chromosome cycle" which culminates at mitosis with an f3 and f1 phosphorylation-mediated chromosome condensation.     

Stimulation of heparan sulfate proteoglycan synthesis and secretion during G1 phase induced by growth factors and PMA

Fetal calf serum (FCS) and PMA (phorbol 12-myristate-13-acetate) specifically stimulate the synthesis of heparan sulfate proteoglycan in endothelial cells. Staurosporine and n-butanol, kinase inhibitors, abolish the PMA effect. Forskolin and 8-bromo adenosine 3′:5′-cyclic monophosphate, activators of, respectively, adenylate cyclase and protein kinase A cannot reproduce the PMA effect. The kinetics of cell entry into S phase of the endothelial cells was determined by DNA synthesis ([³H]-thymidine and Br-dU incorporation), and flow cytometry. The mitogenic effect of fetal calf serum is abolished by PMA. Also, PMA pre-treatment inhibits the enhanced synthesis of heparan sulfate proteoglycan after a second PMA exposure. Remarkably, the stimulation of heparan sulfate proteoglycan synthesis by fetal calf serum and PMA seems to be mainly restricted to G₁ phase. Therefore fetal calf serum and PMA cause an enhanced synthesis of heparan sulfate proteoglycan, and PMA causes a cell cycle block at G₁ phase. J. Cell. Biochem. 70:563–572, 1998. © 1998 Wiley-Liss, Inc.     

Expression of the μ-Opioid Receptor in CHO Cells: Ability of μ-Opioid Ligands to Promote α-Azidoanilido[32P]GTP Labeling of Multiple G Protein α Subunits

Abstract: The identities of heterotrimeric G proteins that can interact with the μ-opioid receptor were investigated by α-azidoanilido[³²P]GTP labeling of α subunits in the presence of opioid agonists in Chinese hamster ovary (CHO)-MORIVA3 cells, a CHO clone that stably expressed μ-opioid receptor cDNA (MOR-1). This clone expressed 1.01 × 10⁶μ-opioid receptors per cell and had higher binding affinity and potency to inhibit adenylyl cyclase for the μ-opioid-selective ligands [d -Ala²,N-MePhe⁴,Gly-ol]-enkephalin and [N-MePhe³,d -Pro⁴]-morphiceptin, relative to the δ-selective opioid agonist [d -Pen²,d -Pen⁵]-enkephalin or the κ-selective opioid agonist U-50,488H. μ-Opioid ligands induced an increase in α-azidoanilido[³²P]GTP photoaffinity labeling of four Gα subunits in this clone, three of which were identified as G_i3α, G_i2α, and G_o2α. The same pattern of simultaneous interaction of the μ-opioid receptor with multiple Gα subunits was also observed in two other clones, one expressing about three times more and the other 10-fold fewer receptors as those expressed in CHO-MORIVA3 cells. The opioid-induced increase of labeling of these G proteins was agonist specific, concentration dependent, and blocked by naloxone and by pretreatment of these cells with pertussis toxin. A greater agonist-induced increase of α-azidoanilido[³²P]GTP incorporation into G_i2α (160–280%) and G_o2α (110–220%) than for an unknown Gα (G_?α) (60%) or G_i3α (40%) was produced by three different μ-opioid ligands tested. In addition, slight differences were also found between the ability of various μ-opioid agonists to produce half-maximal labeling (ED₅₀) of any given Gα subunit, with a rank order of G_i3α > G_o2α > G_i2α = G_?α. In any case, these results suggest that the activated μ-opioid receptor couples to four distinct G protein α subunits simultaneously.