Developmental regulation of cell-surface glycoproteins in clonal cultures of human skeletal muscle satellite cells

Pure populations of myogenic cells were obtained by cloning satellite cells from human skeletal muscle biopsies. Cell-surface glycoproteins at various stages of myogenesis were analysed by one- and two-dimensional gel electrophoresis. A total of 14 distinct proteins were detectable at the cell surface, on the basis of their susceptibility to desialation by exogenous neuraminidase or their iodination by exogenous lactoperoxidase. Reproducible changes in lectin binding or iodination of eight of these proteins occurred during myogenesis. Only two of the developmentally regulated proteins were components of the detergent-insoluble extracellular matrix fraction. Developmental regulation of these two proteins was unaffected by growth of cultures in 5-bromo-2'-deoxyuridine to inhibit myogenesis. In contrast, developmental regulation of the other cell-surface proteins was inhibited by growth in 5-bromo-2'-deoxyuridine, suggesting that changes in these proteins are tightly coupled to satellite cell differentiation. These studies represent the first systematic analysis of the surface proteins of pure, clonally derived, primary cultures of normal myogenic cells.     

cAMP analogues inhibit phosphatidylcholine biosynthesis in cultured rat hepatocytes

The effect of cAMP analogues on phosphatidylcholine formation via the CDP-choline pathway was investigated in cultured monolayers of rat hepatocytes. Treatment with chlorophenylthio-cAMP or the cAMP phosphodiesterase inhibitor, aminophylline, reduced the total uptake of [methyl-3H]choline by 32 and 26% (p less than 0.01), respectively. Chlorophenylthio-cAMP inhibited the incorporation of [methyl-3H]choline into phosphatidylcholine by 2.5-fold (p less than 0.001) and reduced the rate of phosphatidylcholine biosynthesis by approximately 40%. Aminophylline, 8-bromoadenosine 3':5'-monophosphate and N6,O2'-dibutyryladenosine 3':5'-monophosphate also inhibited [methyl-3H]choline incorporation into phosphatidylcholine. Although choline kinase and phosphocholinetransferase activities were stimulated by chlorophenylthio-cAMP treatment, CTP: phosphocholine cytidylyltransferase activity was reduced 46% (p less than 0.01). The results indicate that cytidylyltransferase may be phosphorylated and inhibited by cAMP-dependent protein kinases.     

Effects of human chorionic gonadotropin and N6,O2'-dibutyryladenosine 3' ,5'-monophosphate on phosphofructokinase activity in isolated rat testis.

Phosphofructokinase activity in rat testis is elevated by treatment in vitro with human chorionic godadotropin or N6,O2'-dibutyryladenosine 3' ,5'-monophosphate. Puromycin or actinomycin D suppresses the effect of the gonadotropin but does not affect the enzyme increase induced by the cyclic nucleotide. The possible cause for the divergent action of the two stimulatory agents are discussed.     

Effects of 5-bromo-2''-deoxyuridine on beating heart cell cultures from neonatal hamsters.

1. Primary heart cell cultures from neonatal hamsters yielded a heterogeneous cell population, containing muscle cells undergoing progressive differentiation, as well as non-muscle cells. 2. Addition of 5-bromo-2'-deoxyuridine, at an early stage, to such cultures enhanced the formation of beating sheets of differentiated muscle cells. Accumulation of myosin heavy chains and creatine kinase also occurred in the presence of the analogue. 3. To obtain these effects, the analogue had to be added during the initial rapid growth phase of the cells. Division of the treated cells then ceased when the cell numbers had approximately doubled. 4. Similar results were obtained with other inhibitors of DNA synthesis. Thus improved muscle cell cultures can be obtained by preventing non-muscle cells from overgrowing the cultures. 5. One effect caused only by 5-bromo-2'-deoxyuridine was a large increase in the Ca2+-stimulated ATPase (adenosine triphosphatase) activity which sedimented at low ionic strength. This increase was not due to a greater content of myofibrillar myosin, or to myosin isoenzyme changes, because purified myosin prepared from treated and untreated cultures did not exhibit the increased Ca2+-stimulated ATPase activity.     

Effect of cyclic nucleotides on the cyanide-insensitive respiration of polymorphonuclear leucocytes.

The effects of N6,O2'-dibutyryladenosine 3',5'-monophosphate (Bu2-cyclic AMP) and N2,O2'-dibutyrylguanosine 3',5'-monophosphate (Bu2-cyclic GMP) on the cyanide-insensitive respiration of guinea pig peritoneal exudate polymorphonuclear leucocytes were studied. Bu2-cyclic AMP inhibited the respiration induced both by phagocytosis of E. coli and by the interaction with trypsin-digested rat liver microsomes. The addition of theophylline gave rise to an inhibitory pattern similar to that with Bu2-cyclic AMP against both the respirations induced. On the other hand, Bu2-cyclic GMP did not affect the respiration induced by phagocytosis whereas it inhibited the respiration induced by the addition of myristic acid was inhibited by Bu2-cyclic AMP, which was similar to that with E. coli. The respiration induced by methylene blue was inhibited neither by Bu2-cyclic AMP nor by Bu2-cyclic GMP. These observations suggest that the cyanide-insensitive respiration of polymorphonuclear leucocytes may be classified into at least three types from the inhibitory pattern of cyclic AMP and cyclic GMP.     

Activity of protein kinase dependent on adenosine 3':5'-monophosphate and of its thermostable protein inhibitor in rat hepatoma (HTC) cells. Unlikely role in the permissive action of glucocorticoids.

Normal expression of a variety of hormonal effects which depend on cyclic AMP (adenosine 3':5'-monophosphate) requires the presence of glucocorticoids. Our hypothesis was that glucocorticoids control directly or indirectly the activity of cyclic-AMP-dependent protein kinase. This has been investigated in cultured hepatoma (HTC) cells in which N6,O2'-dibutyryladenosine 3':5'-monophosphate increases the activity of tyrosine transaminase only after glucocorticoid treatment. In these cells, we have determined the concentration and half-life of protein kinase, the sensitivity of this enzyme in vitro to cyclic AMP and to its thermostable protein inhibitor, the state of dissociation of protein kinase holoenzyme in vivo and its sensitivity, in the intact cell, to dibutyryladenosine 3':5'-monophosphate and to the inhibitor diamide, and we have also determined the concentration of endogenous thermostable protein inhibitor of protein kinase. None of these parameters were influenced by glucocorticoids under conditions where these hormones stimulate the activity of tyrosine transaminase and restore sensitivity to dibutyryladenosine 3':5'-monophosphate. It is concluded that the permissive action of glucocorticoids probably results from a control of cyclic-AMP-dependent processes exerted at a level beyond the protein kinase system.     

5-bromo-2''-deoxyuridine-stimulated calcium ion- or magnesium ion-dependent ecto-(adenosine triphosphatase) activity of cultured hamster cardiac cells.

1. Treatment of hamster heart cells in primary culture with 5-bromo-2'-deoxyuridine resulted in the greatly increased activity of a particulate Ca2+- or Mg2+-dependent ATPase (adenosine triphosphatase). 2. 5-Bromo-2'-deoxyuridine exerted these effects only when it was incorporated into cellular DNA, and then in a concentration-dependent manner. 3. Serially replated cells contained less of the activity (expressed as a function of total cell protein) than did the primary cultures, but the stimulation caused by 5-bromo-2'-deoxyuridine addition was much greater. 4. The affected enzyme was apparently localized in the plasma membrane of the cells with its active centre exposed to the outer environment [ecto-(ATPase) dependent on Ca2+ or Mg2+].5. The activity was unaffected by treatment with p-chloromercuriphenylsulphonate, ouabain andverapamil. 6. Ecto (5'-nucleotidase) activity was not increased by 5-bromo-2'-deoxyuridine treatment of cells, and ecto-(p-nitrophenyl phosphatase) activity was only slightly enhanced.     

Interaction of guanine-nucleotide-binding regulatory proteins with chemotactic peptide receptors in differentiated human leukemic HL-60 cells.

Human leukemic HL-60 cells were differentiated into neutrophil-like cells by treatment with dimethylsulfoxide (Me2SO) or N6,O2'-dibutyryladenosine 3',5'-phosphate (Bt2cAMP), and membrane fractions were prepared from the differentiated cells. Receptors for fMLF (fM,N-formylmethionine) and guanine-nucleotide-binding regulatory proteins (G proteins) serving as the substrate for pertussis toxin (islet-activating protein; IAP) were extracted from cell membranes then reconstituted into phospholipid vesicles. The binding of fMLF to the reconstituted vesicles (or the membranes) was determined with 10 nM [3H] fMLF. In both cases, high-affinity binding to vesicle preparations from the Me2SO- and Bt2cAMP-induced cells was abolished following treatment with IAP, suggesting that fMLF receptors were functionally coupled to IAP-sensitive G proteins in each of the two vesicle types. However, the high-affinity fMLF binding was much higher in vesicle preparations originating from Bt2cAMP-induced cells than in those from Me2SO-induced cells, although the amount of IAP-substrate G protein reconstituted into the each phospholipid vesicles preparation was not significantly different from the other. The G proteins of the two differentiated cells were both identified as inhibitory forms (Gi-2) based on their electrophoretic mobilities and immunoblot analyses. When purified Gi-2 from rat brain was reconstituted into the two IAP-treated vesicles, high-affinity fMLF binding was restored in a similar manner in both. IAP-substrate G proteins partially purified from the two differentiated HL-60 cells were also effective in restoring high-affinity fMLF binding to the IAP-treated vesicles. However, a significant difference was observed that the reconstituted binding was higher with the G-protein-rich fraction from Bt2cAMP-induced cells than with that from Me2SO-induced cells, with each of the two IAP-treated vesicle types. These results suggest that the different high-affinity binding of fMLF observed in the two differentiated HL-60 cells are due to a difference in the property of endogenous G proteins rather than fMLF receptors, though the two G proteins are indistinguishable from each other in terms of the subtype of G protein, Gi-2.     

Developmental regulation of neuraminidase-sensitive lectin-binding glycoproteins during myogenesis of rat L6 myoblasts.

Intact monolayers of L6 myoblasts were treated with neuraminidase, with the aim of selectively removing sialic acid residues of cell-surface glycoproteins. Neuraminidase treatment unmasked binding sites for Ricinus communis agglutinin I and peanut agglutinin, thus allowing the identification of the major binding proteins for these lectins. For Ricinus communis agglutinin I these neuraminidase-sensitive glycoproteins had apparent Mr values of 136000, 115000, 87000, 83000 and 49000. For peanut agglutinin the major neuraminidase-sensitive glycoproteins had apparent Mr values of 200000, 136000, 87000 and 83000. We found highly reproducible, developmentally regulated, changes in the lectin-binding capacity of certain of these glycoproteins as L6 myoblasts differentiated into myotubes. Coincident with myoblast fusion there was a co-ordinate decrease in Ricinus communis agglutinin I binding by glycoproteins of apparent Mr of 136000 and 49000. There was also a co-ordinate shift in mobility of the broad band of glycoprotein, centred at an apparent Mr of 115000 in myoblasts, to a new average apparent Mr of 107000 in mid-fusion cultures and myotube cultures. Peanut agglutinin binding by the major protein of apparent Mr 136000 also decreased at the mid-fusion stage of myogenesis, and was barely detectable in 7-day-old fused cultures. These developmentally regulated changes in neuraminidase-sensitive glycoproteins were all inhibited by growth of myoblasts in 6.4 microM-5-bromo-2'-deoxyuridine, indicating that they are associated with myoblast differentiation. In contrast, an increase in fibronectin was seen in mid-fusion cultures, which was not inhibited by growth of myoblasts in 5-bromo-2'-deoxyuridine. This initial increase in fibronectin is, therefore, unlikely to be directly related to myoblast fusion or differentiation.     

Induction and inhibition of Friend erythroleukemia cell differentiation by pyrimidine analogs: analysis of the requirement for intracellular accumulation and incorporation into DNA.

Alkyldeoxyuridines which differ from thymidine by a C5 substitution of straight or branched alkyl chains of two to six carbon atoms have been tested for their ability to be taken up, phosphorylated, and incorporated into DNA. Analysis of the uptake of 5-ethyl-2'-deoxyuridine and 5-propyl-2'-deoxyuridine (n-PrdU)--similar to both thymidine and 5-bromo-2'-deoxyuridine--indicates that transport is dependent upon a functional cellular thymidine kinase. All of the aforementioned pyrimidines with the exception of n-PrdU are phosphorylated to the triphosphate and incorporated into DNA. The homologs 5-iso-propyl-2'-deoxyuridine (iso-PrdU) and 5-hexyl-2'-deoxyuridine are neither transported into the cell, phosphorylated, nor incorporated into DNA. These analogs were tested (i) for their ability to induce in the absence of dimethyl sulfoxide and (ii) to determine whether they enhance or inhibit dimethyl sulfoxide-induced differentiation of Friend erythroleukemia cells. Inhibition of erythroid differentiation appears to require the incorporation of thymidine analogs into DNA, and thus only 5-ethyl-2'-deoxyuridine and 5-bromo-2'-deoxyuridine were effective in inhibiting dimethyl sulfoxide-induced differentiation. The observation that iso-PrdU, and to a lesser extent n-PrdU and 5-propyldeoxyuridine monophosphate, induce differentiation under conditions in which they are not detectable intracellularly is strong evidence that this class of inducer acts at the cell membrane.     

Protein phosphorylation of the smooth and rough endoplasmic reticulum in normal and neoplastic liver of the rat.

Smooth and rough endoplasmic reticulum from rat liver and hepatomas exhibited endogenous protein kinase activity independent of adenosine 3':5'-monophosphate. The phosphorylation of smooth membranes by this process was consistently higher than that of rough membranes. When histone was added along with the smooth endoplasmic reticulum, cyclic AMP stimulated protein phosphorylation. Analysis of membrane-phosphorylated proteins by gel electrophoresis showed 5 major phosphorylated bands with estimated molecular weights of 155 000, 62 000, 50 000, 46 000 and 43 000, whereas major bands having estimated molecular weights of 62 000, 50 000 and 43 000 were found in membranes of the smooth endoplasmic reticulum of the Morris hepatoma 5123 C. Since previous studies in this and other laboratories have demonstrated the similarity of the protein components of membranes of the endoplasmic reticulum of normal liver and hepatoma, our findings indicate an inability of the protein kinase of hepatoma intracellular membranes to phosphorylate protein species that are found in membranes of both liver and the neoplasm.     

Independent regulation of the nuclear and mitochondrial DNA synthesis induced by either Simian virus 40 or serum in mouse fibroblast 3T3 cells.

Resting cultures of 3T3 cells (an established line of mouse fibroblasts) were released from density inhibition by either infection with Simian virus 40 or addition of serum. The increased rate of thymidine incorporation into DNA, induced by these two agents, was measured in the presence and in the absence of three inhibitory conditions (cycloheximide or dibutyryladenosine 3':5'-monophosphate added to the medium, or lack of anchorage). The inhibition was found to be quite similar in cultures stimulated by virus or serum; under the same conditions, however, the incorporation into mitochondrial DNA was much less inhibited than that into nuclear DNA. The experiments also suggest that new protein synthesis may not be necessary, for either virus or serum, to start the inductive mechanism.     

Phospholipid metabolism and lysosomal enzyme secretion by leukocytes. Effects of dibutyryl cyclic adenosine 3':5'-monophosphate and ATP.

The effects of dibutyryl cyclic adenosine 3':5'-monophosphate and ATP on isotope incorporation into phospholipids and the release of beta-glucuronidase into the extracellular medium were studied in polymorphonuclear leukocytes from guinea pig peritoneal exudates. Exogenous dibutyryl cyclic adenosine 3':5'-monophosphate (0.1--1.0 mM) reduced beta-glucoronidase release induced by cytochalasin B in the absence of inert particles. It selectively inhibited 32Pi incorporation into phosphatidic acid and the phosphoinositides and the incorporation of myo-[2-3H]inositol into the phosphoinositides. Added ATP (0.1--1.0 MM), but not other nucleotides, was found to potentiate beta-glucuronidase release provoked by cytochasin B, but it impaired the labeling of the phosphoinositides by myo-[2-3H]inositol. The mechanism of the inhibition the isotope incarparation into these acidic phospholipids by the two mucleotides has not been defined. Dibutyryl cyclic adenosine 3':5'-monophosphate at 2--4 mM concentration was not found to appreciably alter the incorporation of [gamma-32P]ATP into phosphatidic acid, phosphatidylinositol, diphosphoinositide, and triphosphoinositide.     

Cytogenetic effect of the radiosensitizing action of 5-bromo-2'-deoxyuridine on eukaryotic cells at various periods of the DNA synthesis phase

The effect of 5-bromo-2'-deoxyuridine (BUDR) radiosensitizing action on meristematic cells of wheat seedlings and human peripheral blood lymphocytes synchronized with 5-fluoro-2'-deoxyuridine at the G1-S boundary was revealed. This effect was only found in the beginning of the S phase. We show that it was conditioned by incorporation of BUDR into special early replicating DNA sequences which are specific for chromosomal mutagenesis. Using the molecular biology methods non-random distribution of 3H-BUDR in the early replicating wheat DNA sequences with different degree of repeating was found.     

Dibutyryl cAMP-induced protein changes in differentiating mouse neuroblastoma cells.

Proteins from mouse neuroblastoma cells treated with dibutyryl adenosine-3',5'-monophosphate (B2cAMP) were analyzed by high resolution, two-dimensional gel electrophoresis. Quantitative changes in proteins and charge modifications of proteins apparently induced B2cAMP were detected by isoelectric focusing. Some proteins appeared to be modified and one protein was increased 7- to 8-fold in cells treated with B2cAMP. Since neuroblastoma cells differentiate when treated with B2cAMP, understanding the protein changes induced by B2cAMP may help to understand cellular differentiation in neural tissue.     

The incorporation of 5-iodo-5'-amino-2',5-dideoxyuridine and 5-iodo-2'-deoxyuridine into herpes simplex virus DNA. Relationship between antiviral activity and effects on DNA structure

Isopycnic centrifugation in CsCl gradients was used to quantify the incorporation of 5-iodo-5'-amino-2',5'-dideoxyuridine and 5-iodo-2'-deoxyuridine into herpes simplex virus type 1 DNA. A parallelism between the degree of incorporation into viral DNA and the inhibition of herpes simplex virus type I replication was found for both thymidine analogs. A concentration of 5-iodo-5'-amino-2',5'-dideoxyuridine approximately 100 times greater than 5-iodo-2'-deoxyuridine was required to achieve similar levels of antiviral activity. However, the inhibitory effects of these compounds are similar when compared with respect to the percent of substitution for thymidine in herpes simplex virus type I DNA. Damage to the viral DNA, as indicated by the presence of single or double-stranded breaks, was assessed by centrifugation in alkaline and neutral sucrose gradients. The incorporation of 5-iodo-5'-amino-2',5'-dideoxyuridine into herpes simplex virus type I DNA produced single and, to a lesser extent, double-stranded breaks in a dose-dependent manner. 5-Iodo-2'-deoxyuridine did not, however, induced DNA breakage. These data indicate that the additional presence of a phosphoramidate bond in the DNA produced the extensive damage detected under these conditions, but that such damage is not required for antiviral activity.     

Coordinate regulation of type IX and type II collagen synthesis during growth of chick chondrocytes in retinoic acid or 5-bromo-2'-deoxyuridine

Chondrocytes isolated from 15-day-old embryonic chick sterna were cultured as monolayers for 7 days in control medium or in medium supplemented with retinoic acid or 5-bromo-2'-deoxyuridine. Control cells exhibited characteristic polygonal morphology and maintained the synthesis of cartilage-specific collagens, i.e. type II, type IX, 1 alpha, 2 alpha, and 3 alpha chains, and 45 K (presumptive type X). Type IX was the second most prevalent collagen and represented 12-15% of the phenotype. When exposed to retinoic acid, chrondrocytes displayed a fibroblast-like morphology and decreased collagen synthesis by day 2. The synthesis of collagen types II and IX declined in parallel along with that of the other cartilage collagens and ceased by day 7. During the same period, the synthesis of collagen types I, III, and V and two unidentified collagen chains was initiated and stimulated. Similar changes in collagen expression were caused by 5-bromo-2'-deoxyuridine but were delayed, beginning after day 4. Type III collagen, however, was never detected in 5-bromo-2'-deoxyuridine or control cultures. Because two different agents and two rates of modulation produced parallel changes in the synthesis of collagen types II and IX, these collagens appear to be coordinately regulated.     

Apoptotic cell death, long-term persistence, and neuronal differentiation of aneuploid cells generated in the adult brain of teleost fish

Aneuploidy, caused by segregation defects during mitosis, has previously been identified in adult-born cells of mammals and teleosts. In the present study, we have examined the fate of these cells in the brain of the teleost fish Apteronotus leptorhynchus. By immunostaining against active caspase-3, we have shown that both cells with normal nuclear morphology and cells with mitotic segregation defects undergo apoptosis, but the relative number of apoptotic cells is higher among cells of the latter category. Long-term survival of cells with mitotic segregation defects could be demonstrated by incorporation of 5-bromo-2'-deoxyuridine into newly synthesized DNA during the S-phase of mitosis, and by employment of postadministration survival times of up to 860 days. Moreover, by combining 5-bromo-2'-deoxyuridine immunolabeling with immunostaining against the neuron-specific marker protein Hu, we have shown that among the long-term persistent cells with mitotic segregation defects a similar portion develops into neurons as does among the long-term persistent cells without such defects. It is possible that aneuploid cells play a role in the regulation of gene expression by somatic genomic alterations during postnatal development.     

Trypanosoma cruzi: adrenergic modulation of cyclic AMP role in proliferation and differentiation of amastigotes in vitro

Trypanosoma cruzi amastigotes, obtained from the supernatant of J774G-8 macrophage cultures infected with Y strain trypomastigotes, proliferated and differentiated into epimastigotes in Warren medium at 28-29 C. The basal level of adenosine 3':5'-monophosphate (cAMP) in recently harvested amastigotes was 0.12 pmole/10(7) cells, which could be increased in a dose-dependent manner to 0.62 pmole/10(7) cells with 1 mM of the adrenergic ligand isoproterenol plus 0.5 mM isobutyl methylxanthine. Isoproterenol inhibited [3H]thymidine incorporation into amastigote DNA, as well as the proliferation of amastigotes and newly transformed epimastigotes. Because dibutyryl cAMP had the same effect as isoproterenol on the cells, the experimental results suggest a role for cAMP, modulated by adrenergic ligands, in the control of proliferation and differentiation of amastigotes.