ANTAGONISM BY DIBUTYRYL ADENOSINE CYCLIC 3',5'-MONOPHOSPHATE AND TESTOSTERONE OF CELL ROUNDING REACTIONS

In an attempt to understand further the mechanism of the morphological and functional "reverse transformation" of CHO-K1 cells induced by dibutyryl adenosine cyclic 3',5'-monophosphate (cAMP) and testosterone, the kinetics of variation in the susceptibility of cells to rounding after the addition or deletion of dibutyryl cAMP and testosterone have been investigated. Changes in susceptibility to cell rounding upon removal of divalent cations or pulse exposure to concanavalin A were complete within 0.5–1 h after addition or deletion of drug. In comparison, the gross conversion of CHO-K1 cells from epithelial- to fibroblast-like morphology after drug treatment or the converse change after drug removal required 8 or 4 h, respectively. The effects on cell rounding are not caused by an effect of dibutyryl cAMP upon cell growth rate. Inhibitor experiments indicate that the changes investigated do not require continued RNA or protein synthesis and are not prevented by agents which depolymerize microtubules.     

Actin paracrystal induction by forskolin and by db-cAMP in CHO cells

Forskolin, a hypotensive diterpine, is assumed to be a potent activator of adenylate cyclase leading to increased levels of cAMP. When this drug is used at 10(-5) M on CHO-C14 cells in culture, it induces within 15 min actin paracrystals in all cells. At this time the paracrystals are mostly situated close to the cell periphery. Electron microscopy (EM) shows structures typical of actin paracrystals. Scanning electron microscopy (SEM) reveals a reduction in surface microvilli and blebs. Identical results can be obtained by adding 1 mM db-cAMP to the culture medium directly. The paracrystals are observed within 15 min and thus represent one of the earliest ultrastructural changes so far described for reverse transformation of CHO cells by db-cAMP. The microtubular and vimentin profiles appear unchanged by forskolin treatment of CHO-K1 cells. Out of currently unknown reasons forskolin does not induce the actin transformation in several other commonly used cell lines.     

Cyclic AMP-protein-DNA complex formation in mammalian cell-free systems.

A cytoplasmic protein fraction from KB and Chinese hamster ovary cells (CHO-Kl) was shown to bind in vitro to cAMP and subsequently to DNA-cellulose. This protein complex was not found in DE-52 purified CHO-K1 cAMP-dependent protein kinases. The complex appeared to exist as a small fraction of the total cAMP binding proteins, preferred native to denatured DNA and exhibited multiple sedimentation coefficients in glycerol gradients. This complex, after elution from the DNA cellulose column, was shown to have bound specifically to [3H]-cAMP which could be displaced by non-radioactive cAMP in competitive binding assays.     

Carbachol-induced reverse transformation of Chinese hamster ovary cells transfected with and expressing the m5 muscarinic acetylcholine receptor

Reverse transformation was induced in Chinese hamster ovary (CHO) cells transfected with and stably expressing the m5 subtype of the muscarinic acetylcholine receptor when stimulated with the muscarinic agonist, carbachol. Atropine, a muscarinic antagonist, blocked the carbachol-stimulated reverse transformation. CHO cells not transfected with the muscarinic receptor did not change with added carbachol. PMA induced reverse transformation without increasing cAMP accumulation in CHO cells. Carbachol, prostaglandin E2, and cholecystokinin increased cAMP accumulation but only carbachol caused reverse transformation. Carbachol-stimulated cAMP accumulation occurred at a higher concentration (EC50 10 microM) than did carbachol-stimulated reverse transformation (EC50 63 nM). Muscarinic m5 acetylcholine receptor transfected into CHO cells can induce reverse transformation which may be independent of cAMP.     

Recovery of hormonal regulation in protein kinase defective adrenal cells through DNA-mediated gene transfer

A cAMP-resistant mutant (Kin-8) isolated from Y1 mouse adrenocortical tumor cells harbors a specific lesion in the regulatory subunit of the type 1 cAMP-dependent protein kinase. This mutant also is resistant to the effects of corticotropin and cAMP on steroidogenesis, growth and morphology, suggesting an obligatory role for the protein kinase in regulation of adrenocortical functions. In this study, the cAMP-resistant phenotype of the Kin-8 mutant was reverted by transformation with DNA from cAMP-responsive Y1 cells, and the biochemical basis of the transformation was explored. Initially, Y1 mouse adrenocortical tumor cells were evaluated for their competence as recipients in DNA-mediated transformation experiments, by measuring their ability to incorporate and express a bacterial gene (neo) encoding resistance to neomycin. Y1 cells were transfected with the plasmid pSV2-neo (an SV40-neo hybrid vector designed for expression in animal cells) and screened for resistance to the neomycin analog, G418. Neomycin-resistant transformants were recovered from Y1 cells at a frequency of approximately one per 10(3) cells per 10 micrograms of DNA, and had specific neo sequences integrated into their high molecular weight (mw) DNA. The Y1 mutant, Kin-8, then was transformed with pSV2-neo DNA plus high mw DNA prepared from cAMP-responsive Y1 cells. Cells competent for transformation were recovered by selective growth in the neomycin analog G418, and these transformants were screened for recovery of morphological responses to cAMP. Several colonies capable of rounding up in the presence of cAMP were recovered after transformation with DNA from Y1 cells. These transformants also recovered the ability to round up in the presence of corticotropin, and were able to respond to both corticotropin and cAMP with increased steroidogenesis. Transformants generated from either Y1 or Kin-8 cells were unstable. Y1 cells lost resistance to neomycin when grown in the absence of G418 at a frequency of 4% per generation. Similarly, Kin-8 transformants lost their sensitivity to cAMP in subsequent culture passages. In some of the cAMP-responsive transformants, cAMP-dependent protein kinase activity was recovered and approached the activity seen in cAMP-responsive Y1 cells. The recovery of a normal protein kinase by transformation appeared to have been sufficient to reverse the cAMP-resistant phenotype of Kin-8 cells. In other cAMP-responsive transformants, protein kinase activity was not appreciably affected by cAMP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Site-selective cAMP analogs induce nuclear translocation of the RII cAMP receptor protein in Ha-MuSV-transformed NIH/3T3 cells

Site-selective cAMP analogs, depending on the position of their substituents on the adenine ring, selectively bind to either site 1 or site 2 of the known cAMP binding sites of protein kinase. Treatment of Harvey murine sarcoma virus-transformed NIH/3T3 cells with such site-selective analogs results in growth inhibition and phenotypic reversion, and the combination of a C-8 thio or halogen analog (site 1 selective) with an N6 analog (site 2 selective) produces a synergistic effect. We report here that the growth inhibitory effect of the analogs correlates with the nuclear translocation of the RII cAMP receptor protein, the regulatory subunit of protein kinase type II. The transformed NIH/3T3 cells contained no detectable level of RII in the nucleus, whereas nontransformed NIH/3T3 cells exhibited a high level of nuclear RII. Within 30 min after treatment of the transformed cells with the site-selective analogs, immunofluorescence against the RII protein markedly increased in the cell nucleus. The nuclear translocation of the RII cAMP receptor protein is an early event in the reverse transformation of the fibroblasts treated with site-selective cAMP analogs.     

Role of the microfibrillar system in knob action of transformed cells

Transformed cells often display knobs (or blebs) distributed over their surface throughout most of interphase. Scanning electron microscopy (SEM) and time-lapse cinematography on CHO-K1 cells reveal roughly spherical knobs of 0.5–4 μm in diameter distributed densely around the cell periphery but sparsely over the central, nuclear hillock and oscillating in and out of the membrane with a period of 15–60 sec. Cyclic AMP derivatives cause the phenomenon of reverse transformation, in which the cell is converted to a fibroblastic morphology with disappearance of the knobs. A model was proposed attributing knob formation to the disorganization of the jointly operating microtubular and microfilamentous structure of the normal fibroblast. Evidence for this model includes the following: (1) Either colcemid or cytochalasin B (CB) prevents the knob disappearance normally produced by cAMP, and can elicit similar knobs from smooth-surfaced cells; (2) knob removal by cAMP is specific, with little effect on microvilli and lamellipodia; (3) immunofluorescence with antiactin sera reveals condensed, amorphous masses directly beneath the membrane of CB-treated cells instead of smooth, parallel fibrous patterns of reversetransformed cells or normal fibroblasts; (4) transmission electron microscopy (TEM) of sections show dense, elongated microfilament bundles and microtubules parallel to the long axis of the reverse-transformed CHO cell, but sparse, random microtubules throughout the transformed cell and an apparent disordered network of 6-nm microfilaments beneath the knobs; (5) cell membranes at the end of telophase, when the spindle disappears and cleavage is complete, display typical knob activity as expected by this picture.     

Cell type-specific regulation of B-Raf kinase by cAMP and 14-3-3 proteins

Cyclic AMP can either activate or inhibit the mitogen-activated protein kinase (MAPK) pathway in different cell types; MAPK activation has been observed in B-Raf-expressing cells and has been attributed to Rap1 activation with subsequent B-Raf activation, whereas MAPK inhibition has been observed in cells lacking B-Raf and has been attributed to cAMP-dependent protein kinase (protein kinase A)-mediated phosphorylation and inhibition of Raf-1 kinase. We found that cAMP stimulated MAPK activity in CHO-K1 and PC12 cells but inhibited MAPK activity in C6 and NB2A cells. In all four cell types, cAMP activated Rap1, and the 95- and 68-kDa isoforms of B-Raf were expressed. cAMP activation or inhibition of MAPK correlated with activation or inhibition of endogenous and transfected B-Raf kinase. Although all cell types expressed similar amounts of 14-3-3 proteins, approximately 5-fold less 14-3-3 was associated with B-Raf in cells in which cAMP was inhibitory than in cells in which cAMP was stimulatory. We found that the cell type-specific inhibition of B-Raf could be completely prevented by overexpression of 14-3-3 isoforms, whereas expression of a dominant negative 14-3-3 mutant resulted in partial loss of B-Raf activity. Our data suggest that 14-3-3 bound to B-Raf protects the enzyme from protein kinase A-mediated inhibition; the amount of 14-3-3 associated with B-Raf may explain the tissue-specific effects of cAMP on B-Raf kinase activity.     

Activation of MAP kinase by MC4-R through PI3 kinase

The melanocortin 4 receptor (MC4-R) is a G_s-coupled receptor known to increase cAMP production following agonist stimulation. We demonstrate that the mitogen-activated protein kinases p42 (ERK2) and p44 (ERK1) are also activated by MC4-R following treatment with the MC4-R agonist NDP--MSH in stably transfected CHO-K1 cells. This time- and dose-dependent response is abolished by the MC4-R antagonist SHU-9119. p42/p44 MAPK activation is blocked by the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002 but not by the protein kinase A (PKA) inhibitor Rp-cAMPS, indicating that that signal activating the p42/p44 MAPK pathway is conveyed through inositol triphosphate.     

Glucose-dependent insulinotropic polypeptide activates the Raf-Mek1/2-ERK1/2 module via a cyclic AMP/cAMP-dependent protein kinase/Rap1-mediated pathway

The gastrointestinal hormone, glucose-dependent insulinotropic polypeptide (GIP), is one of the most important regulators of insulin secretion following ingestion of a meal. GIP stimulates insulin secretion from the pancreatic beta-cell via its G protein-coupled receptor activation of adenylyl cyclase and other signal transduction pathways, but there is little known regarding subsequent protein kinase pathways that are activated. A screening technique was used to determine the relative abundance of 75 protein kinases in CHO-K1 cells expressing the GIP receptor and in two pancreatic beta-cell lines (betaTC-3 and INS-1 (832/13) cells). This information was used to identify kinases that are potentially regulated following GIP stimulation, with a focus on GIP regulation of the ERK1/2 MAPK pathway. In CHO-K1 cells, GIP induced phosphorylation of Raf-1 (Ser-259), Mek1/2 (Ser-217/Ser-221), ERK1/2 (Thr-202 and Tyr-204), and p90 RSK (Ser-380) in a concentration-dependent manner. Activation of ERK1/2 was maximal at 4 min and was cAMP-dependent protein kinase-dependent and protein kinase C-independent. Studies using a beta-cell line (INS-1 clone 832/13) corroborated these findings, and it was also demonstrated that the ERK1/2 module could be activated by GIP in the absence of glucose. Finally, we have shown that GIP regulation of the ERK1/2 module is via Rap1 but does not involve Gbetagamma subunits nor Src tyrosine kinase, and we propose that cAMP-based regulation occurs via B-Raf in both CHO-K1 and beta-cells. These results establish the importance of GIP in the cellular regulation of the ERK1/2 module and identify a role for cAMP in coupling its G protein-coupled receptors to ERK1/2 activity in pancreatic beta-cells.     

Use of biocarrier beads and flow cytometry for single-cell studies of fibronectin gene regulation in dibutyrl cyclic AMP reverse transformed CHO-K1 cells

The protease sensitivity of a number of cell surface or cytoskeletal components and the relationship of these to the substratum in attached cells has prevented the quantitative measurement of their expression by flow cytometry. Using traditional cell sorting techniques, cells must be treated with a protease to detach them from a substrate in order to produce a single-cell suspension. Unfortunately, proteolytic treatment alters or destroys a number of cellular proteins. Fibronectin either on the cell surface or as part of the substratum laid down by the cell is particularly sensitive to proteases, preventing its quantitative study by flow cytometry. To circumvent these problems and produce a single cell suspension necessary for flow cytometric analysis, CHO-K1, a Chinese hamster ovary cell line, were grown in suspension on specially-treated 25 μm biocarrier beads. The CHO-K1 cell line is composed of transformed epithelial-like cells that have lost the fibronectin deposit around their cell membranes. To restore the typical fibroblastic deposit of fibronectin, the cells attached to beads were induced by dibutyrl cAMP to undergo a reverse transformation reaction to restore fibroblastic morphology and the typical fibroblastic deposite of fibronectin on the cell surface and substratum. The cells attached to beads were then immunofluorescently labeled for the protease-sensitive, extracellular matrix component, fibronectin, and examined on a flow cytometer. Cell surface fibronectin heterogeneity was then examined on a cell-by-cell basis as a function of cell cycle using Hoechst 33342 dye that binds to AT base pairs of cellular DNA. Dual laser measurement and multiparameter list mode data analysis were used to study the relationship between cell surface fibronectin of biocarrier bead attached cells and cell cycle.     

Modulation of Intracellular Cyclic AMP Levels by Different Human Dopamine D4 Receptor Variants

Abstract: To investigate whether polymorphic forms of the human dopamine D4 receptor have different functional characteristics, we have stably expressed cDNAs of the D4.2, D4.4, and D4.7 isoforms in several cell lines. Chinese hamster ovary CHO-K1 cell lines expressing D4 receptor variants displayed pharmacological profiles that were in close agreement with previous data from transiently expressed D4 receptors in COS-7 cells. Dopamine stimulation of the D4 receptors resulted in a concentration-dependent inhibition of the forskolin-stimulated cyclic AMP (cAMP) levels. The potency of dopamine to inhibit cAMP formation was about twofold reduced for D4.7 (EC₅₀ of ∼37 n M ) compared with the D4.2 and D4.4 variants (EC₅₀ of ∼16 n M ). Antagonists block the dopamine-mediated inhibition of cAMP formation with a rank order of potency of emonapride > haloperidol = clozapine ≫ raclopride. There was no obvious correlation between the efficacy of inhibition of forskolin-stimulated cAMP levels and the D4 subtypes. Dopamine could completely reverse prostaglandin E₂-stimulated cAMP levels for all three D4 receptor variants. Deletion of the repeat sequence does not affect functional activity of the receptor. The data presented indicate that the polymorphic repeat sequence causes only small changes in the ability of the D4 receptor to block cAMP production in CHO cells.     

Use of biocarrier beads and flow cytometry for single-cell studies of fibronectin gene regulation in dibutyryl cyclic AMP reverse transformed CHO-K1 cells

The protease sensitivity of a number of cell surface or cytoskeletal components and the relationship of these to the substratum in attached cells has prevented the quantitative measurement of their expression by flow cytometry. Using traditional cell sorting techniques, cells must be treated with a protease to detach them from a substrate in order to produce a single-cell suspension. Unfortunately, proteolytic treatment alters or destroys a number of cellular proteins. Fibronectin either on the cell surface or as part of the substratum laid down by the cell is particularly sensitive to proteases, preventing its quantitative study by flow cytometry. To circumvent these problems and produce a single cell suspension necessary for flow cytometric analysis, CHO-K1, a Chinese hamster ovary cell line, were grown in suspension on specially-treated 25 microns biocarrier beads. The CHO-K1 cell line is composed of transformed epithelial-like cells that have lost the fibronectin deposit around their cell membranes. To restore the typical fibroblastic deposit of fibronectin, the cells attached to beads were induced by dibutyryl cAMP to undergo a reverse transformation reaction to restore fibroblastic morphology and the typical fibroblastic deposite of fibronectin on the cell surface and substratum. The cells attached to beads were then immunofluorescently labeled for the protease-sensitive, extracellular matrix component, fibronectin, and examined on a flow cytometer. Cell surface fibronectin heterogeneity was then examined on a cell-by-cell basis as a function of cell cycle using Hoechst 33342 dye that binds to AT base pairs of cellular DNA. Dual laser measurement and multiparameter list mode data analysis were used to study the relationship between cell surface fibronectin of biocarrier bead attached cells and cell cycle.     

A role for cAMP in angiotensin II mediated inhibition of cell growth in AT1A receptor-transfected CHO-K1 cells

G-protein coupled Angiotensin II receptors (AT_1A), mediate cellular responses through multiple signal transduction pathways. In AT_1A receptor-transfected CHO-K1 cells (T3CHO/AT_1A), angiotensin II (AII) stimulated a dose-dependent (EC₅₀=3.3 nM) increase in cAMP accumulation, which was inhibited by the selective AT₁, nonpeptide receptor antagonist EXP3174. Activation of protein kinase C, or increasing intracellular Ca²⁺ with ATP, the calcium ionophore A23187 or ionomycin failed to stimulate cAMP accumulation. Thus, AII-induced cAMP accumulation was not secondary to activation of a protein kinase C- or Ca²⁺/calmodulin-dependent pathway. Since cAMP has an established role in cellular growth responses, we investigated the effect of the AII-mediated increase in cAMP on cell number and [³H]thymidine incorporation in T3CHOA/AT_1A cells. AII (1 M) significantly inhibited cell number (51% at 96 h) and [³H]thymidine incorporation (68% at 24 h) compared to vehicle controls. These effects were blocked by EXP3174, confirming that these responses were mediated through the AT₁ receptor. Forskolin (10 M) and the cAMP analog dibutyryl-cAMP (1 mM) also inhibited [³H]thymidine incorporation by 55 and 25% respectively. We extended our investigation on the effect of AII-stimulated increases in cAMP, to determine the role for established growth related signaling events, i.e., mitogen-activated protein kinase activity and tyrosine phosphorylation of cellular proteins. AII-stimulated mitogen-activated protein kinase activity and phosphorylation of the 42 and 44 kD forms. These events were unaffected by forskolin stimulated increases in cAMP, thus the AII-stimulated mitogen-activated protein kinase activity was independent of cAMP in these cells. AII also stimulated tyrosine phosphorylation of a number of cellular proteins in T3CHO/AT_1A cells, in particular a 127 kD protein. The phosphorylation of the 127 kD protein was transient, reaching a maximum at 1 min, and returning to basal levels within 10 min. The dephosphorylation of this protein was blocked by a selective inhibitor of cAMP dependent protein kinase A, H89-dihydrochloride and preexposure to forskolin prevented the AII-induced transient tyrosine phosphorylation of the 127 kD protein. These data suggest that cAMP, and therefore protein kinase A can contribute to AII-mediated growth inhibition by stimulating the dephosphorylation of substrates that are tyrosine phosphorylated in response to AII.     

The role of butyrate in the reverse transformation reaction in mammalian cells.

The reverse transformation reaction of Chinese hamster ovary cells from compact, epithelial-like, randomly growing, heavily knobbed, lectin reactive cells into stretched, tighly adherent, smooth-surfaced, lectin resistant, fibroblast-like cells normally elicited by dibutyryl cAMP can be produced to its complete extent by N6-monobutyryl cAMP or 8-bromo-cAMP, O2'-monobutyryl cAMP is ineffective as is cAMP itself in the absence of an inhibitor of phosphodiesterase activity. In the presence of a phosphodiesterase inhibitor, cAMP is fully effective. These results indicate that the role of the butyryl groups of dibutyryl cAMP and, especially, the N6-butyryl, in the reverse transformation reaction is protection of the cAMP analogue from degradation. Butyrate at concentrations of about 1 mM does produce a response which to some extent mimics that of cAMP analogues. The cells, however, fail to assume a fibroblastic-like shape, but rather become flattened. The butyrate effect is much slower and less readily reversible than that evoked by cAMP analogues. Butyrate produces an approximately 2-fold increase in intracellular cAMP levels. These results are consistent with the hypothesis that butyrate effects, in part, are mediated by AMP.     

Deorphanization of Dresden G Protein-Coupled Receptor for an Odorant Receptor

Dresden G protein-coupled receptor (D-GPCR) is one of orphan G protein-coupled receptors (GPCR). Here we report the identification of the ligands and the characterization of D-GPCR. We investigated over 5000 compounds to evoke the response mediated by D-GPCR and identified 3-methyl-valeric acid and 4-methyl-valeric acid as agonists using a cAMP assay. It is of interest that they dramatically enhanced the intracellular cAMP accumulation and the CRE-luciferase activity in CHO-K1 cells and HEK293 cells expressing the chimeric protein of D-GPCR with a rhodopsin-tag at its N-terminus. Our results established new characteristics of D-GPCR as an olfactory receptor. First, agonists of D-GPCR belong to odorants. Second, D-GPCR mRNA is expressed in the olfactory bulb. In addition, D-GPCR was reported to have similar sequences and its genome locus nearby other olfactory receptors. These results suggest D-GPCR is an olfactory receptor.     

Isoproterenol stimulation of the repair of single-stranded DNA breaks in Chinese hamster cells induced by gamma irradiation

The action of the increased intracellular content of adenosine monophosphate (cAMP) in CHO-K1 cells (clones 773 and ADr112eb), treated with isoproterenol, on gamma-induced DNA single-strand breaks repair has been investigated. The hormonal treatment stimulates gamma-induced (180 Gr) DNA single-strand repair during the post-irradiation incubation (45 min) by 75 +/- 16%. The results show the involvement of the cAMP system in radiosensitivity of cultured mammalian cells and in regulation of cellular mechanisms of DNA repair.