Surface ATPase activity at cell-cell contacts in hepatic parenchymal cells and in cAMP-treated hepatoma cells in monolayer culture

Summary Histochemical investigation shows that ATPase activity is located intensively on the surface of cell contacts in hepatoma cells grown in confluent monolayer culture. Dibutyryl cyclic AMP and theophylline-treated hepatoma cells which exhibit contact-inhibited growth show the absence of localization of intense ATPase activity at cell-cell contacts. However, after removal of these additives the activity fully recovers to the intense level of control cells, when the release of cells from contact inhibition of growth occurs. Cultured hepatic parenchymal cells in monolayer have little or no ATPase activity at their surface immediately after contacts are established, and again after growth to a confluent state. In a different type of hepatoma cell which is less malignant and forms a small tissue mass or island, cell surface ATPase activity at cell-cell contacts in an island is very weak or scarcely detected even when cells are not treated with dibutyryl cyclic AMP and theophylline.     

Effects of db-cAMP and theophylline on cell surface adenosine triphosphatase activity in cultured hepatoma cells

The surface ATPase activity in monolayer of the Yoshida ascites hepatoma 66(AH66) cells was compared with the activity in dibutyryl cyclic AMP(db-cAMP) and theophylline-treated monolayer. Control, sparse cells had a very small K_m for ATP and a small V_max. When these cells were grown to a dense state or treated with db-cAMP and theophylline, the cells behaved in an identical manner for K_m and V_max at the time the surface ATPase activity increased. Electron microscope cytochemistry showed that strong ATPase activity was associated at the region of contact between two control cells. In contrast, the lower activity was distributed universally both at the upper surface and at the intercellular space in the treated cells.     

The influence of culture conditions on the induction of tyrosine aminotransferase by cyclic nucleotides in rat hepatoma cells.

The increase in tyrosine aminotransferase activity which occurs in rat hepatoma tissue culture (HTC) cells in response to cyclic AMP analogs has been shown to be an enzyme induction, similar to the larger response observed in certain other hepatoma cells and in liver. A specific antibody to tyrosine aminotransferase has been used to show that the number of enzyme molecules and the rate of enzyme synthesis are increased by N6,O2'-dibutyryl cyclic AMP in HTC cells. The effect on tyrosine aminotransferase is also produced by various 8-substituted derivatives of cyclic AMP and occurs whether or not the enzyme has been preinduced with a glucocorticoid. The response of the enzyme is greater when HTC cells are maintained in monolayer than in suspension cultures. Neither cell growth nor serum is required for the response.     

Dibutyryl cyclic AMP modulation of gap junctions in SW-13 human adrenal cortical tumor cells

Cultured human adrenal cortical adenocarcinoma cells (SW-13) form a confluent monolayer of epithelial-like cells when seeded into culture flasks. Following a 24-48 hr non-mitotic period, cells begin to divide and become confluent within a week after seeding at 5 X 10(4) cells/cm2. The SW-13 cells were exposed to dibutyryl cyclic AMP (DbcAMP), cyclic AMP (cAMP), sodium butyrate, and adrenocorticotropin (ACTH). The rate of SW-13 cell proliferation was measured with a DNA microfluorometric assay, as well as by procedures measuring the incorporation of 3H-thymidine. In addition, following administration of ACTH and DbcAMP, the fractional area of membrane covered by gap junctions was quantitated with freeze-fracture electron microscopic techniques. Dibutyryl cyclic AMP at a concentration of 1 X 10(-3) M decreased the growth rate of the cell population. There was a corresponding increase in the fractional area of gap junctions found on the cell membrane in 96-hr DbcAMP-treated cultures. ACTH (40 mU/ml) exposure failed to produce an increase in the fractional area of gap junctions or to alter the rate of cell proliferation. From these data it can be suggested that elevations in cAMP levels within the cell can be related to both the proliferation of gap junctions and the decrease in cell proliferation in the SW-13 tumor cell.     

A further study on the regulation of cyclic nucleotide phosphodiesterase activity in neuroblastoma cells: effect of growth.

Adenosine 3',5'-cyclic monophosphate (cyclic AMP) phsophodiesterase activity in mouse neuroblastoma cells in culture markedly increased during exponential growth and reached a maximal level at confluency; whereas guanosine 3'5'-cyclic monophosphate (cyclic GMP) phosphodiesterase activity only slightly but significantly increased under a similar experimental condition. The increase in cyclic AMP phosphodiesterase activity was blocked by both cycloheximide and dactinomycin, whereas the increase in cyclic GMP phosphodiesterase was blocked by only cycloheximide. When the confluent cells were replated at low density, the cyclic nucleotide phosphodiesterase activity decreased; however, when they were plated at high cell density which equaled confluency, the enzyme activity did not decrease. Unlike cyclic AMP phosphodiesterase activity, cyclic GMP phosphodiesterase activity did not change significantly in prostaglandin E1-treated cells, but decreased in cells treated with the inhibitor of phosphodiesterase. Like cyclic AMP phosphodiesterase activity, cyclic GMP phosphodiesterase activity also did not change in cells treated with serum-free medium, X-irradiation, sodium butyrate and 6-thioguanine.     

Induction of alkaline phosphatase in cultured human fibroblasts. Comparison of normal cells and those from patients with cystic fibrosis.

The membrane glycoprotein enzyme, alkaline phosphatase was induced in cultured human fibroblasts by dibutyryl cyclic AMP, sodium butyrate, the serum glycoprotein fetuin, the Tamm-Horsfall urinary glycoprotein, and by a number of inhibitors of DNA synthesis. The uninduced basal enzyme activity increased at later stages of growth when the cells became confluent. Induction by dibutyryl cyclic AMP or fetuin was most effective when the agents were added after the cells had reached stationary phase and was maximal after at least two days of exposure. The levels of induction resulting from the addition of pairs of the agents, dibutyryl cyclic AMP, n-butyrate and fetuin were additive indicating that these have different modes of action. The inhibitors of DNA synthesis, cytosine arabinoside, hydroxyurea, and methothrexate were less effective inducers. Bromodeoxyuridine which also has non-DNA mediated effects induced to the same extent as dibutyryl cyclic AMP. Similar experiments with sex- and age-matched cell strains derived from patients with cystic fibrosis failed to detect differences in the levels of induction from those observed in normal cells. In addition, the combined inductive effects of Tamm-Horsfall glycoprotein, isoproterenol and theophylline, were similar with normal and cystic fibrosis cells.     

Induction of alkaline phosphatase in cultured human fibroblasts: Comparison of normal cells and those from patients with cystic fibrosis

The membrane glycoprotein enzyme, alkaline phosphatase was induced in cultured human fibroblasts by dibutyryl cyclic AMP, sodium butyrate, the serum glycoprotein fetuin, the Tamm-Horsfall urinary glycoprotein, and by a number of inhibitors of DNA synthesis. The uninduced basal enzyme activity increased at later stages of growth when the cells became confluent. Induction by dibutyryl cyclic AMP or fetuin was most effective when the agents were added after the cells had reached stationary phase and was maximal after at least two days of exposure. The levels of induction resulting from the addition of pairs of the agents, dibutyryl cyclic AMP, n-butyrate and fetuin were additive indicating that these have different modes of action. The inhibitors of DNA synthesis, cytosine arabinoside, hydroxyurea, and methothrexate were less effective inducers. Bromodeoxyuridine which also has non-DNA mediated effects induced to the same extent as dibutyryl cyclic AMP.Similar experiments with sex- and age-matched cell strains derived from patients with cystic fibrosis failed to detect differences in the levels of induction from those observed in normal cells. In addition, the combined inductive effects of Tamm-Horsfall glycoprotein, isoproterenol and theophylline, were similar with normal and cystic fibrosis cells.     

A further study on the regulation of cyclic nucleotide phosphodiesterase activity in neuroblastoma cells: Effect of growth

Summary Adenosine 3′,5′-cyclic monophosphate (cyclic AMP) phosphodiesterase activity in mouse neuroblastoma cells in culture markedly increased during exponential growth and reached a maximal level at confluency; whereas guanosine 3′, 5′-cyclic monophosphate (cyclic GMP) phosphodiesterase activity only slightly but significantly increased under a similar experimental condition. The increase in cyclic AMP phosphodiesterase activity was blocked by both cycloheximide and dactinomycin, whereas the increase in cyclic GMP phosphodiesterase was blocked by only cycloheximide. When the confluent cells were replated at low density, the cyclic nucleotide phosphodiesterase activity decreased; however, when they were plated at high cell density which equaled confluency, the enzyme activity did not decrease. Unlike cyclic AMP phosphodiesterase activity, cyclic GMP phosphodiesterase activity did not change significantly in prostaglandin E₁-treated cells, but decreased in cells treated with the inhibitor of phosphodiesterase. Like cyclic AMP phosphodiesterase activity, cyclic GMP phosphodiesterase activity also did not change in cells treated with serum-free medium, X-irradiation, sodium butyrate and 6-thioguanine. This work was supported by USPHS NS-09230, and DRG-1273 from Damon Runyon-Walter Winchell Cancer Fund.     

Cellular interactions and tubulin detyrosination in fibroblastic and epithelial cells

In mammalian cells most microtubules are enriched in tyrosinated alpha-tubulin (tyr-tubulin). Other subclasses of microtubules are present in variable amounts and some are enriched in detyrosinated alpha-tubulin (glu-tubulin). We examined the effect of cell-cell interactions on the level of glu-tubulin in microtubules. This was studied by quantitative immunofluorescence using antibodies against tyr- and glu-tubulin. We found that in cells which have established cell-cell contacts, the ratio of glu-/tyr-tubulin is higher than in isolated cells. We also examined the effect of cell-cell interactions on the glu-/tyr-tubulin ratio by using the antibody blocking method of Schulze and Kirschner [42]. Microtubules containing mainly tyr-tubulin had been blocked first by a polyclonal antibody against tyr-tubulin and several layers of secondary antibodies. The unblocked microtubules were then labeled by a monoclonal antibody against alpha-tubulin. Since the coating efficiency of microtubules by the anti-tyr tubulin depends on the amount of tyr-tubulin in each microtubule, this procedure allows the visualization of microtubules enriched or depleted in tyr-tubulin in specific domains of each cell. Microtubules were more extensively blocked in subconfluent than in confluent cells and preferentially at the periphery of the cytoplasm. In cells present at the margin of an artificial wound produced in a confluent monolayer, the amount of blocked microtubules increased slowly with time (between 2 and 4 h). These results are consistent with the hypothesis that cell-cell contacts lead to increased tubulin dytyrosination both in fibroblastic and epithelial cells.     

Dibutyryl cyclic AMP resistant MDCK cells in serum free medium have reduced cyclic AMP dependent protein kinase activity and a diminished effect of PGE1 on differentiated function

Prostaglandin E1 (PGE1) has a stimulatory effect both on the growth and the expression of differentiated function of Madin Darby Canine Kidney (MDCK) cells in a hormonally defined medium (Medium K-1). While the stimulatory effect of PGE1 on MDCK cell growth is observed in subconfluent cultures, the effect of PGE1 on differentiated function (i.e., dome formation) is observed at confluency. PGE1 may possibly affect growth and such differentiated functions by separate mechanisms. In order to examine this possibility, dibutyryl cyclic AMP resistant variants of MDCK were selected. All of the variants were partially resistant to the growth inhibitory effects of dibutyryl cyclic AMP and theophylline. The cyclic AMP dependent protein kinase activity of four of the five variant clones studied was significantly reduced as compared with normal MDCK cells. The dependence of the kinase activity of several of the dibutyryl cyclic AMP resistant variants (DBr2 and DBr3) on the cyclic AMP concentration in the reaction mixture was compared with that of normal MDCK cells. At all of the cyclic AMP concentrations tested DBr2 and DBr3 cells had reduced protein kinase activity as compared with normal MDCK cells. This reduced activity could be attributed to a decrease in the Vmax for kinase in the two variants, rather than to a change in the Km of kinase for cyclic AMP. The cyclic AMP phosphodiesterase activity of dibutyryl cyclic AMP resistant variants was also studied. Unlike PGE1 independent clone 1, DBr2 and DBr3 cells did not differ significantly from normal MDCK cells with regard to their ability to degrade cyclic AMP. The growth and functional responsiveness of DBr2 and DBr3 cells to PGE1 was also examined. DBr2 and DBr3 cells were shown to retain a normal growth response to PGE1. However the capacity of DBr2 and DBr3 cells to form domes in response to PGE1 was dramatically reduced as compared with normal MDCK cells. Nevertheless DBr3 cells were shown to still retain the capacity to form domes in response to other inducers. The effect of PGE1 on one of the functional parameters involved in dome formation (the activity of the Na+/K+ATPase) was examined. The rate of ouabain-sensitive Rb+ uptake was observed to be elevated in confluent monolayers of normal MDCK cells maintained in Medium K-1, as compared with monolayers maintained in Medium K-1 minus PGE1.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cell-cell contacts mediated by E-cadherin (uvomorulin) restrict invasive behavior of L-cells

L-cells were cotransfected with plasmids coding for mouse E-cadherin (uvomorulin) and the neophosphotransferase gene, and stable transfectants expressing E-cadherin at the cell surface were selected and cloned. Control transfection was done with the neophosphotransferase gene alone. The invasive migration of transfected and untransfected L-cells into three-dimensional collagen gels was then analyzed. L-cells not expressing E-cadherin migrated efficiently into the gels, whereas invasion of the E-cadherin-expressing L-cells was restricted in a cell density dependent manner. At sparse density, when the cells exhibited little cell-cell contacts, no difference was observed between the level of invasion of the cadherin-expressing cells and the control cells. However, with increasing cell density, decreasing amounts of the cadherin-expressing cells but increasing amounts of the control cells migrated into the gels. At confluent density hardly any cadherin-expressing cells were able to migrate into the gels. The inhibition of the invasion of the cadherin-expressing cells could be reverted if confluent cells were cultured in the presence of monoclonal antibodies against E-cadherin. Since the expression of E-cadherin did not influence the invasive mobility of single cells, these results indicate that E-cadherin-mediated cell-cell contacts inhibited invasive cellular migration. Time-lapse videoscopy and studies of cell migration from a monolayer into a cell-free area demonstrated that the restricted invasion could be explained by contact inhibition of cell movement of the cadherin-expressing cells.     

Levels of cyclic AMP in murine L5178Y lymphoblasts grown in different concentrations of serum

Murine L5178Y lymphoblasts were grown in different concentrations of horse and fetal calf serum. Intracellular levels of cyclic AMP, determined throughout the growth period of these cells, did not vary significantly. Furthermore, logarithmically growing cells that became stationary did not show the pronounced increase in levels of cyclic AMP that is characteristic of other cell types when they become confluent or quiescent. These results demonstrate that the unrestricted growth of lymphoblasts is independent of the serum concentration used and may be due to an aberration in the cyclic AMP system.     

Overexpression of vinculin suppresses cell motility in BALB/c 3T3 cells.

The content of vinculin, a cytoplasmic protein found in focal contacts and cell-cell junctions, was increased in BALB/c 3T3 cells by gene transfection. The vinculin expressed from the full length chicken cDNA, incorporated into focal contacts and its pattern was identical to that of the endogenous protein. Cells stably expressing vinculin by 20% over the endogenous level had altered locomotory properties. In these cells, the ability to migrate into a wound formed in a confluent monolayer and the locomotion of individual cells were drastically reduced. The results provide direct evidence that cell locomotion can be regulated by modulating vinculin expression.     

Hormonal regulation of the differentiation of rat adipocyte precursor cells in primary culture

A reproducible cell culture system is described that allows the study of adipose conversion in fibroblast-like cells isolated by collagenase digestion of epididymal and perirenal adipose tissue from male rats weighing 70-200 g. Adipose conversion as measured by lipid accumulation and increase in glycerophosphate dehydrogenase (GPDH) activity during differentiation strongly depends on the density at which cells are inoculated and starts only when cells are confluent and when physiological amounts of corticosterone and insulin are added. beta-Estradiol, testosterone, thyroxine, triiodothyronine, and growth hormone do not affect the differentiation process. Methylisobutylxanthine added during the first 2 days after confluence, added with insulin and corticosterone, potentiates the effect of insulin on GPDH activity and accelerates triglyceride accumulation. The effect of methyl-isobutylxanthine seems to be mediated by increased cyclic AMP concentrations, inasmuch as it may be replaced by forskolin.     

Inhibition of hepatoma cell growth by analogs of adenosine and cyclic AMP and the influence of enzymes in mammalian sera

The following evidence suggests that inhibition of hepatoma cell (HTC) growth by cyclic nucleotides is an adenosine-like effect that is greatly modified by the type and treatment of serum used in the culture medium and is probably not mediated by cyclic AMP-dependent protein kinase: 1) Heating serum reduces its phosphodiesterase content, thereby slowing metabolism of cyclic AMP and reducing the inhibition of HTC cell growth by cyclic AMP; 2) Using medium that contains phosphodiesterase but lacks adenosine deaminase causes adenosine to accumulate from cyclic AMP and increases the toxicity of cyclic AMP; 3) Uridine or cytidine reverses the growth inhibition caused by adenosine, 5'-AMP or cyclic AMP; 4) adenosine, 5'-AMP and N6-(delta 2-isopentenyl) adenosine are more toxic for HTC cells than is cyclic AMP, and N6,O2-dibutyryl cyclic AMP is not toxic; and 5) N6,O2'-dibutyryl cyclic AMP inhibits growth of Reuber H35 cells, but uridine prevents this inhibition of growth. We conclude that most, if not all, of the inhibitory effects of cyclic AMP and N6,O2'-dibutyryl cyclic AMP on HTc and Reuber H35 hepatoma cell growth are due to the generation of toxic metabolites.     

Effects of growth conditions on cyclic nucleotide phosphodiesterases of cultured fibroblasts.

Cyclic nucleotide phosphodiesterase activities of baby hamster kidney cells (BHK) grown in surface cultures were altered by modifying growth conditions. The untransformed BHK cells grown in medium containing 10% fetal calf serum showed non-linear LineweaverBurk plots for cyclic AMP phosphodiesterase activity with apparent Michaelis constants for cyclic AMP of approximately 5 and 30 muM. When these cells were placed in medium containing 1% fetal calf serum, linear kinetic plots for cyclic AMP phosphodiesterase with an apparent Km for cyclic AMP of approximately 20 muM were obtained. Modification of the apparent Km of BHK cell phosphodiesterase was detectable within 20 minutes after dillution of cells grown in 10% serum into fresh medium containing 1% serum. With the BHK cell line transformed with Rous sarcoma virus, differences in enzyme kinetics were not seen when these cells were diluted in 1% or 10% serum. In addition to the serum induced differences in the apparent Km of cyclic AMP phosphodiesterases of BHK cells, total cyclic AMP and cyclic GMP phosphodiesterase activities were also modified by growth conditions. BHK cells grown to high cell densities had three to five-fold higher total cyclic AMP activity than did the cells in less dense cultures. When the dense cell cultures were diluted into fresh medium containing 10% serum, total enzyme activities fell to levels comparable to those found in the rapidly growing cells at low cell densities. The reduction in total enzyme activity after dilution of BHK cells occurred rapidly and was influenced by cell density. A similar reduction of total enzyme activity was also seen in diluted RSV cells; however, the time course of the response differed from that seen in the untransformed cells.     

Effect of cyclic AMP on chromatin-bound protein kinases in WI-38 fibroblasts stimulated to proliferate.

When resting confluent monolayers of WI-38 fibroblasts are stimulated to proliferate by serum, DNA synthesis begins to increase between 15-18 h after stimulation. Chromatin-bound protein kinase activity increases in stimulated cells within 1 h after the nutritional change, concomitant with an increase in the template activity of nuclear chromatin. Addition of dibutyryl 3' : 5'-cyclic adenosine monophosphate (dibutyryl cyclic) AMP to the stimulating medium inhibits the entrance of cells into S phase, but only if dibutyryl cyclic AMP (5-10(-4) M) is added before the onset of DNA synthesis. The increases in chromatin template activity and in the chromatin-bound kinase activity are not inhibited by dibutyryl cyclic AMP in the early hours after stimulation, but are completely inhibited after the 5th hour from the nutritional change. This seems to indicate that in stimulated WI-38 cells, dibutyryl cyclic AMP exerts its inhibitory action somewhere between 5 and 12 h after stimulation. A number of protein kinase activities were extracted from chromatin with 0.3 M NaCl and partially resolved on a phosphocellulose column. Two distinct peaks of protein kinase activity appeared to be markedly increased in WI-38 cells 6 h after serum stimulation. Both peaks of increased activity were inhibited by dibutyryl cyclic AMP in vivo. Adenosine, sodium butyrate and adenosine 5'-monophosphate (AMP) do not inhibit the increase in DNA synthesis nor the increase in protein kinase activity. The results suggest that stimulation of cell proliferation in confluent monolayers of WI-38 cells causes an increase (or the new appearance) of certain chromatin-bound protein kinases, and that this increase is inhibited by cyclic AMP in vivo.     

Growth dependence of phosphoglyceric acid dehydrogenase activity in cultured rat liver cells.

Rat liver epithelial cells in culture (WIRL-3C) have the enzymes that synthesize serine from 3-phophoglyceric acid. Both phosphoglyceric acid dehydrogenase (PGAD) and serine-phosphate (serine-P) forming activities fluctuate with time after subculture and are higher in growing than confluent cells. This activity pattern was not common for other dehydrogenases in WIRL-3C cells, nor was it common for PGAD activity in other cultured cells. At time of subculture, cells are removed from spent medium, treated with trypsin, and fed fresh medium. None of these parameters causes the rise in activity; in contrast, reduction in cell density and the accompanying stimulation of growth do. PGAD activity decreases when growth is slowed either as the cells progress to the end of the culture cycle, when cells are treated with dexamethasone-phosphate (Dx-P) or dibutyryl cyclic AMP(cAMP) and theophylline or when the serum concentration of the medium is reduced to 0.2%. Under these conditions, decreased PGAD activity is paralleled by a decline in growth and DNA accumulation. PGAD activity in WIRL-3C cells is regulated in a manner closely resembling what has been observed previously in rat liver from the whole animal. The possible use of this system in studying regulation of gene expression in mammalian cells is discussed.     

Cyclic AMP and growth of Ehrlich ascites tumor cells. Lack of cyclic AMP elevation in nutritionally deprived cells and mechanism of retardation of growth by dibutryl cyclic AMP.

Cyclic AMP levels in Ehrlich ascites tumor cells changed little after deprivation of cells of essential nutrients, serum, glucose and amino acids, deprival of each of which leads to marked inhibition of growth and protein synthesis. Cyclic AMP levels also changed little after the addition of these nutrients to deprived cells. Thus cyclic AMP is not likely to be the intracellular mediator for growth regulation by these three nutrients. Elevation of cyclic AMP levels for short periods by exposure of cells to choleratoxin or theophylline produced only slight changes in parameters of protein synthesis (polyribosome pattern and rate of [3H]leucine incorporation). An exposure for 1 day to dibutyryl cyclic AMP did not inhibit cell growth. However, prolonged exposure to dibutyryl cyclic AMP inhibited the multiplication of Ehrlich ascites cells both in suspension and in stationary cultures. No morphological effects were evident in the former; in the latter, cells attached firmly to the substratum and formed elongated cytoplasmic processes. Inhibition of cell multiplication by dibutyryl cyclic AMP was related to cell density and to serum concentration. Cells in dibutyryl cyclic AMP-containing media plated at low cell densities multiplied as rapidly as control cells. The final densities cells reached were determined by the serum concentration; in dibutyryl cyclic AMP-containing media these densities were about one-half those of respective control cells. Limitation of cell multiplication by dibutyryl cyclic AMP was reversed by the addition of serum, by resuspending cells at lower densities, or by resuspending cells in media without dibutyryl cyclic AMP. These findings suggested that dibutyryl cyclic AMP may affect the utilization of serum factors by cells. Dibutyryl cyclic AMP did not inactivate serum factors and did not change the rate at which cells depleted the growth medium of serum factors. Dibutyryl cyclic AMP may limit cell multiplication by increasing the cellular requirement for serum factors.