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R M Niles

Publications and source records attributed to R M Niles.

At least 55 records · Page 3Linked to original sources
PubMed1986

The effect of retinoic acid on protein phosphorylation in mouse melanoma cells.

Vitamin A inhibits growth and increases the activity of cAMP-dependent protein kinase in B16 mouse melanoma cells. In this report we show that retinoic acid (RA) treatment of intact cells alters their subsequent in vitro protein phosphorylation, but we could not demonstrate any changes in in vivo protein phosphorylation. A 48-h treatment with RA results in a concentration-dependent decrease of protein phosphorylation of a 95K molecular weight (MW) protein in both supernatant and particulate fractions. The phosphorylation of this protein does not appear to be regulated by cAMP. Proteins at 92K and 82K MW in the supernatant fraction are increased in phosphorylation. The former (but not the latter) is regulated by cAMP. In the particulate fraction a variety of proteins 12K-68K MW are increased in phosphorylation, as the cells are treated with increasing amounts of RA. The phosphorylation of most of these proteins is regulated by cAMP. Another inhibitor of B16 cell growth, melanocyte-stimulating hormone (MSH) also alters protein phosphorylation. At short incubation periods (1 h), this hormone stimulates phosphorylation of a number of proteins (17-40K MW), while in longer incubation periods (48 h) phosphorylation is inhibited. All of these phosphorylations appear to be regulated by cAMP. We attempted to repeat these observations using intact-cell phosphorylation with 32PO4. In two experiments we saw small changes in the phosphorylation of proteins. In most experiments, however, we could find no change in the phosphoproteins. Further experiments have led us to question the in vivo phosphorylation, since treatment of the cells with MSH, cholera toxin, or db-cAMP also did not affect intact-cell protein phosphorylation. We have previously documented that under these latter conditions cAMP levels are greatly elevated and cAMP-dependent protein kinase is activated. The in vitro phosphorylation results suggests that in RA-treated cells, kinase activities and/or protein substrate levels are changing. However, the physiological significance of the particular MW phosphoproteins changes we have described must await resolution of the in vivo phosphorylation data.

Animals↗
PubMed1986

Serine proteases stimulate mucous glycoprotein release from hamster tracheal ring organ culture.

The effects of several neurohumoral agents and serine proteases on glycoconjugate release from hamster tracheal organ cultures were assessed. The beta-adrenergic agonist isoproterenol inhibited glycoconjugate release, and its effect was abolished by the specific beta-blocking agent propranolol. A cholinergic agonist, pilocarpine, marginally increased glycoconjugate release, and its effect was abolished by the antagonist atropine. Human neutrophil elastase and porcine pancreatic trypsin consistently increased glycoconjugate release by 1.8 to 2.8-fold. When the proteases were inactivated, they were no longer effective in stimulating glycoconjugate release. Histologic and electron microscopic analysis of the protease-treated organ cultures revealed no discernible toxic reaction. In addition, organ cultures prelabeled with chromium 51 did not release an increased amount of radioactivity when treated with the proteases. Biochemical analysis of the glycoconjugates released into the culture medium showed them to be of high molecular weight (90% eluted in the void volume of a Sepharose 6B column) and to be resistant to digestion with hyaluronidase and heparinase, properties consistent with mucous glycoproteins. The mechanism of protease-induced glycoconjugate release is unknown. We speculate that stimulation of airway secretory cells by serine proteases of neutrophilic or other inflammatory cell origin may play a role in the increased airway secretion that is characteristic of acute tracheobronchitis.

Animals↗
PubMed1984

The effect of retinoic acid on cyclic-AMP-binding proteins in mouse melanoma cells.

We have previously reported [(1980) J. Biol. Chem. 255, 5999-6002] that retinoic acid inhibited growth and increased cyclic-AMP-dependent protein kinase activity in mouse melanoma cells. A variant melanoma line having depressed levels of cyclic-AMP-dependent protein kinase was not growth-inhibited by retinoic acid. In this report we describe the effect of retinoic acid on cyclic AMP binding proteins in B16 mouse melanoma cells. Using the technique of photoaffinity labeling, we found three major proteins of Mr 49 000, 52 000, and 55 000 which were specifically labeled with 8-N3-[32P]AMP in both control and treated cells. Based upon their molecular weight, relative affinity for 8-N3-[32P]AMP and comigration with standards, we have designated the 49 000-Mr and 55 000-Mr species as RI and RII respectively. The position of the intermediate band (Mr 52 000) was not affected by pre-incubation with ATP or alkaline phosphatase, and two-dimensional gel analysis indicated that it had the same pI as RI. Retinoic acid increased the 8-N3-[32P]AMP labeling of RI within 24 h, reaching a maximal six fold increase by 48 h. These increases were limited to the 40 000 X g supernatant fraction and occurred prior to any growth inhibition. By using increasing concentrations of 8-N3-cAMP we were able to construct a saturation curve for RI binding. Calculation of apparent Kd values from these curves showed nearly identical affinities for RI binding of 8-N3-cAMP from control and retinoic-acid-treated cells. Therefore we conclude that retinoic acid is increasing the amount of RI rather than altering its properties. Corroboration of these results was obtained by DEAE-cellulose chromatography. Peak I (corresponding to type I protein kinase) from retinoid-treated cells was increased about six fold in binding activity.

Affinity Labels↗
PubMed1984

In vitro characteristics of the lipid-filled interstitial cell associated with postnatal lung growth: evidence for fibroblast heterogeneity.

This study explores the in vitro modulation of the lipid-filled phenotype of the lipid interstitial cell (LIC) isolated from the developing rat lung. Isolated LIC lose their cytoplasmic lipid droplets when cultured in fetal bovine serum (FBS) but retain their potential for lipid storage, since they rapidly reaccumulate lipid when subcultured in neonatal rat serum (NRS) and to a lesser extent in adult rat serum (ARS). The return of LIC to a lipid-filled state may not represent cell differentiation, since it occurs in the presence of bromodeoxyuridine. NRS contains twice the free fatty acids (FFA) of FBS and ARS, and doubling the FFA concentration of FBS and ARS increases LIC storage lipids. Serum triglyceride (TG) is 10 times higher in ARS and 23 times higher in NRS than in FBS. Since LIC lipoprotein lipase (LPL) activity is in the range of 3T3-L1 adipocytes (0.56 vs. 1.72 units/mg DNA), the LIC has the potential of incorporating serum lipoprotein-triglyceride. The LPL activity of LIC is 9-12 times that of fetal and adult rat lung fibroblasts and 50 times that of human lung, trachea, or skin fibroblasts; LIC are probably a source of endothelial LPL in the developing lung. The response of LIC and ARLF cyclic-AMP to hormones known to influence lipid synthesis or degradation showed that: only LIC responded to glucagon; prostaglandin E1 was a more potent stimulus to LIC; isoproterenol was a more potent stimulus to ARLF; and neither cell responded to ACTH. The unique nature of LIC tends to support further the concept of fibroblast heterogeneity within tissues.

Animals↗
PubMed1981

Differential sensitivity of normal and chemically transformed epithelial cells to cholera toxin.

We have been studying the regulation of growth by cyclic adenosine 3':5'-monophosphate (cyclic AMP) and other factors in untransformed (K16) and chemically transformed (W8) rat liver epithelial cells. Initially, we found that 8-bromocyclic adenosine 3':5'-monophosphate was a more potent inhibitor of cell replication in K16 than in W8 cells. In addition, the phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine (MIX) caused marked growth inhibition in K16 but not in W8 cells. Through the use of cholera toxin (CT) with or without MIX, we elevated intracellular cyclic AMP levels in a quantifiable fashion. With CT alone or combined with MIX, we observed a dose-dependent morphological change in W8 cells, which consisted of extensive "process" formation. K16 morphology was not altered at any concentration of CT +/- MIX tested. K16 cell growth was only marginally inhibited by CT alone, but markedly inhibited by CT plus MIX. W8 cell growth was moderately inhibited by CT alone or combined with MIX. Analysis of cyclic AMP levels revealed that, at all concentrations of CT +/- MIX and at all time periods tested, W8 cells produced significantly more cyclic AMP than K16 cells. It appears that morphological changes and growth inhibition are not necessarily linked and that MIX may inhibit K16 cell replication by means other than its ability to increase intracellular cyclic AMP levels.

Animals↗
PubMed1980

Retinoic acid increases cyclic AMP-dependent protein kinase activity in murine melanoma cells.

The influence of all trans-retinoic acid on cyclic AMP metabolism was examined in B16-F1 mouse melanoma cells. Treatment of these cells with retinoic acid resulted in a dose-dependent inhibition of cell growth which was accompanied by a concentration-dependent increase in both basal and cyclic AMP-stimulated protein kinase activity, Intracellular levels of cyclic AMP, however, were not altered by retinoid treatment. A protein kinase-deficient variant of B16-F1 (MR-4) did not exhibit decreased growth or increased protein kinase activity in response to retinoic acid treatment. At least 24 h of incubation was required before increased protein kinase activity could be detected in treated B16-F1 cells. Retinoic acid treatment increased the Vmax of protein kinase, but the Ka for cyclic AMP activation was not altered. These findings suggest that in B16 mouse melanoma cells, cyclic AMP-dependent protein kinase may be a target for the growth inhibitory effects of the retinoid.

Animals↗
PubMed1979

Regulation of phosphatidylcholine metabolism by cyclic AMP in a model alveolar type 2 cell line.

The influence of cyclic AMP on the metabolism of phosphatidylcholine, the major component of pulmonary surfactant was examined in a cell line (A549) with type 2 pneumonocyte characteristics. It was found that cyclic AMP increased both the total amount of phosphatidylcholine and disaturated phosphatidylcholine as well as the incorporation of [3H]choline into these fractions. The effect was specific for cyclic AMP since 5'-AMP, adenosine, and cyclic GMP did not alter phosphatidylcholine or disaturated phosphatidylcholine levels. Cyclic AMP had no effect on phosphatidylcholine and disaturated phosphatidylcholine metabolism in another non-type 2 human epithelial cell line (MA-160). Since the ability of various cyclic AMP analogs to increase phosphatidylcholine and disaturated phosphatidylcholine levels was correlated with their ability to activate protein kinase, it seems likely that a protein phosphorylation mechanism is involved in controlling phosphatidylcholine metabolism.

Animals↗
PubMed1979

Isolation and characterization of a variant of B16-mouse melanoma resistant to MSH growth inhibition.

A variant of B-16 F1 mouse melanoma was selected for its ability to survive and replicate in the presence of melanocyte-stimulating hormone (MSH). Although the variant (MR-4) was completely resistant to growth inhibition of MSH, cyclic AMP was still able to block cell replication. Tyrosinase activity in MR-4 cells was considerably lower than in B-16 F1 cells. MSH induced a two fold to three-fold increase in tyrosinase activity in both cell types, but the absolute activity in MR-4 remained significantly less than in the parental cells. MR-4 cells were also found to have a markedly depressed cyclic AMP-dependent protein kinase activity relative to B-16 F1 cells. The protein kinase from both cell types was stimulated by cyclic AMP, but the level of MR-4 kinase activity at maximal cyclic AMP concentrations remained considerably lower than B-16 F1 kinase activity under the same conditions. In both cell types adenylate cyclase activity was markedly stimulated by MSH. When equal numbers of viable F1 and MR-4 cells were injected subcutaneously into C57/B1 mice, the MR-4 cells formed tumors earlier and killed the host sooner than the parental F1 cells. We conclude that the biochemical alteration which allows MR-4 cells to replicate in the presence of MSH is a low level of tyrosinase activity, which in turn may be the result of low cyclic AMP-dependent protein kinase activity.

Animals↗
PubMed1979

Differential growth inhibition in two human carcinoma cell lines by cyclic adenosine 5'-monophosphate analogs.

The inhibition of cell replication in two human carcinoma cell lines by various cyclic AMP analogs was explored. In all instances, the addition of the cyclic nucleotide phosphodiesterase inhibitor 1-methyl-3-isoburylxanthine resulted in synergistic growth inhibition by the analogs. A correlation was found between an analog's ability to inhibit growth and its ability to activate protein kinase. A differential effect of the breakdown product 8-bromo-AMP (8-BrAMP) on cell replication in the two cell lines was observed; i.e., one cell type was extremely sensitive to inhibition by 8-BrAMP and the growth inhibition could not be reversed by uridine, whereas the other cell type was less sensitive to 8-BrAMP and the growth inhibition was significantly reversed by uridine.

1-Methyl-3-isobutylxanthine↗
PubMed1978

Hormonal activation of adenylate cyclase in mouse melanoma metastatic variants.

The ability of melanocyte stimulating hormone (MSH), adrenocorticotropic hormone (ACTH), and prostaglandin E1 (PGE1) to stimulate the accumulation of cyclic AMP was examined in intact mouse melanoma cells of varying metastatic potential. F1 cells (low metastatic potential) had significantly greater cyclic AMP levels in response to all three hormones than F5 (intermediate metastatic potential) and F10 (high metastatic potential) cells. The ranking of the response was as follows: MSH, F1 greater than F5 greater than F10, ACTH, F1 greater than F5 greater F10, PGE, F1 greater than F10 greater F5. In contrast to the above, the degree of hormonal stimulation of adenylate cyclase in broken cell preparations was virtually identical in all three melanoma cell lines. Control enzyme activity was depressed in both F5 and F10 relative to F1. The conflicting results between studies of intact vs. broken cell preparations could not be explained by increased cyclic AMP phosphodiesterase activity in F5 and F10 cells. We conclude that as the melanoma cells increase in metastatic potential, there is a significant loss in the ability of their cyclic AMP system to respond appropriately to hormonal stimuli.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1978

Control of melanogenesis in mouse melanoma cells of varying metastatic potential.

The control of melanin production, tyrosinase activity, and cell replication by melanocyte-stimulating hormone (MSH) and cyclic AMP (cAMP) was examined in differentially metastasizing B16 mouse melanoma variants. In B16-F1 cells (low metastatic potential), MSH or cAMP greatly elevated tyrosinase activity and melanin content while inhibiting cell replication. The same parameters in B16-F5 cells (intermediate metastatic potential) were altered to a much lesser degree, whereas B16-F10 cells (high metastatic potential) were not significantly affected by MSH or cAMP. Therefore, a correlation exists between loss of hormonal regulation and increased metastatic potential.

Animals↗
PubMed1977

Adenylate cyclase activity in cultured epithelial cells.

The cyclic AMP metabolism of cultured epithelial cells was investigated. Epinephrine or 1-methyl,3-isobutylxanthine (MIX) alone had no effect on cyclic AMP levels in intact cells, whereas the combination of the two agents yielded a 6- to 10-fold increase in cyclic AMP levels. Both basal and stimulated cyclic AMP levels decreased with increasing cell density. Cell-free adenylate cyclase preparations were stimulated markedly by epinephrine or isoproterenol in the absence of MIX. Since the epithelial cells were found to have a relatively small amount of cyclic nucleotide phosphodiesterase (PDE) activity, the requirement for MIX to visualize intact cell responsiveness to epinephrine could be explained only partially by its PDE inhibitory properties.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗