Increased proline transport resulting from growth of normal and Kirsten sarcoma virus-transformed BALB 3T3 cells in the presence of N(6), O(2')-dibutyryl cyclic adenosine 3',5'-monophosphate.

Proline transport in Kirsten sarcoma virus-transformed BALB 3T3 (Ki-3T3) cells was increased approximately twofold by 0.5 mm dibutyryl cAMP (dbcAMP), and the increase was observed whether transport was assayed in the presence or absence of cycloheximide. Two days of exposure to the analog was required for maximum stimulation. Increased proline transport contributed almost entirely to the increased incorporation of [¹⁴C]proline into noncollagen protein but for only 13% of the increased incorporation into collagen of dbcAMP-treated Ki-3T3 cells. Proline transport was further characterized using an assay system containing 0.1 mm cycloheximide, which did not affect transport over a 30-min period. The K_m for proline was decreased from 6.5 to 3.4 mm by dbcAMP treatment of Ki-3T3. Proline transport in Ki-3T3 proceeds almost entirely via the A system, and the effect of dbcAMP appears to be on this system specifically since glycine and glutamine transport, which are heterogeneous, were not affected but transport of N-methylaminoisobutyrate, a specific A system substrate, was increased by dbcAMP treatment. Although 0.5 mm butyrate increased proline transport in Ki-3T3 cells to a similar degree as dbcAMP, the effect of the latter appeared related to its action as a cAMP analog since N⁶-monobutyryl cAMP, having a stable butyryl group, and 8-bromo-cAMP also increased proline transport while dbcGMP did not. The rate of proline transport in normal BALB 3T3 cells was only 30–40% lower than that of Ki-3T3 cells at various growth stages, and dbcAMP and 8-bromo-cAMP treatment also increased proline transport in the normal cells. The results of these studies suggest that dbcAMP and other cAMP analogs induce the synthesis of an altered component of the A system for amino acid transport and that the effect of these compounds is unrelated to the effect of transformation on proline transport.     

Retention of transformant specific type III collagen in dibutyryl cAMP treated Kirsten sarcoma virus transformed BALB 3T3 cells and in a flat revertant.

We previously reported that Kirsten sarcoma virus transformed BALB 3T3 (Ki-3T3) cell cultures contained mainly type I collagen and about 30% of another type designated by us as Y and which appears to be type III collagen, [α₁ (III)]₃. Clones of BALB 3T3 which exhibited contact-inhibition were found to contain mainly type I collagen [α₁(I)]₂α₂, and about 25% of another type (X) which was composed of three α₁ chains differing from those of type III (Hata, R. and B. Peterkofsky, 1977 Proc. Nat. Acad. Sci. (U.S.A.), 74: 2933—2937). Since dibutyryl 3′:5′ cyclic adenosine monophosphate (dbcAMP) increases collagen synthesis and alters other transformation specific properties of Ki-3T3 cells, we determined whether treatment of Ki-3T3 cells with this compound restored the normal collagen phenotype. We also analyzed the collagen of a revertant of Ki-3T3 which exhibits properties similar to those of the dbcAMP treated transformant. Procollagen labeled with radioactive proline was isolated from the medium or cells of cultures and was converted to collagen with pepsin; the collagen was analyzed by carboxymethyl cellulose (CMC) chromatography or gel electrophoresis under denaturing conditions. Ki-3T3 cells treated with 0.5 mM dbcAMP continued to accumulate type III collagen but there was an increase in the number of α₁ chains eluting from CMC columns in the same position as α₁ (I) suggesting increased accumulation of type X collagen. Although the revertant was similar to dbcAMP treated cells in that it exhibited a flattened morphology and a high relative rate of collagen synthesis, the collagen profile was similar to that of the transformant, consisting mainly of types I and III. These results indicate that accumulation of type III collagen is unaffected by dbcAMP but suggest that cAMP may be involved in the regulation of type X collagen. The failure of dbcAMP or reversion to affect the occurrence of type III collagen supports the mechanism of cell selection as a means of explaining the specific occurrence of type III collagen in sarcoma virus transformed 3T3 cells.     

Ascorbate-independent proline hydroxylation resulting from viral transformation of Balb 3T3 cells and unaffected by dibutyryl cAMP treatment.

Collagen synthesis, hydroxylation of proline in collagen, and collagen secretion were studied in the contact-inhibited mouse fibroblast line, Balb 3T3; the Kirsten virus transformed line, Ki-3T3; and dibutyryl cAMP (dbcAMP)-treated Ki-3T3 cells, during the various phases of the growth cycle. Transformed cells in both logarithmic and stationary phase produced lower levels of collagen than the parent line but 85-90% of the theoretically possible hydroxyproline residues of the collagen were formed even when ascorbic acid was not added to the culture medium. Moreover, the transformed cells showed only about a 20% increase of collagen secretion upon addition of ascorbate. This was in contrast to the ascorbate requirement for maximal proline hydroxylation and the 2-3 fold stimulation of collagen secretion by ascorbate in the parent Balb 3T3 cells. Although dbcAMP treatment caused Ki-3T3 cells to assume a more normal morphology and increased the relative rate of collagen synthesis to levels similar to that of 3T3, such treatment did not restore an ascorbate requirement for proline hydroxylation or collagen secretion. The specific activity of the enzyme prolyl hydroxylase also was not affected by dbcAMP treatment although collagen synthesis was increased by such treatment. In addition, it was found that ascorbic acid was not effective in activating prolyl hydroxylase derived from Ki-3T3 or dbcAMP-treated Ki-3T3 cell cultures either in logarithmic phase or stationary phase. Ki-3T3 cultures did not accumulate ascorbic acid in cells or medium nor was ascorbic acid synthesized from the precursor 14C-glucuronate in cell homogenates. The results suggest that virally transformed Balb 3T3 cells acquire the capacity to synthesize a reducing cofactor for prolyl hydroxylase and that this function may be related to the increased glycolytic metabolism of these cells since neither cellular metabolism nor ascrobate-independent hydroxylation was altered by treatment with dbcAMP.     

Dibutyryl cyclic AMP decreases glutamine synthetase in cultured 3T3-L1 adipocytes

Glutamine synthetase specific activity increases greater than 100-fold during the insulin-mediated differentiation of confluent 3T3-L1 cells into adipocytes. Incubation of the adipocytes for 22 h with 0.5 mM dibutyryl cyclic AMP plus 0.5 mM theophylline, 0.2 mM 8-bromo-cyclic AMP, 10 micro M epinephrine, or 1 microgram of alpha 1-24 adrenocorticotropic hormone/ml decreased glutamine synthetase by greater than 60%. During the same incubation period, there was no effect of these compounds on protein or on the specific activities of glucose-6-P dehydrogenase or hexokinase. In the presence of 0.5 mM theophylline, the dibutyryl cyclic AMP-mediated decrease in glutamine synthetase activity was half-maximal at 50 micro M dibutyryl cyclic AMP. Furthermore, between 10 micro M and 5 mM dibutyryl cyclic AMP, the dibutyryl cyclic AMP-mediated decrease in glutamine synthetase was similar in the absence or presence of 1 microgram of insulin/ml. Immunotitration of glutamine synthetase activity from 3T3 adipocytes indicates that the dibutyryl cyclic AMP-mediated decrease in the activity is due to a decrease in the cellular content of glutamine synthetase molecules. We studied the effects of dibutyryl cyclic AMP on the synthesis and degradation of glutamine synthetase. Synthesis rate was estimated from the incorporation of L-[35S]methionine into glutamine synthetase during a 60-min incubation period. Degradation rate was estimated from the first order disappearance of radioactivity from glutamine synthetase in 3T3 adipocytes previously incubated with L-[35S]methionine. Glutamine synthetase was isolated by immunoprecipitation followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Incubation of 3T3 adipocytes with dibutyrl cyclic AMP resulted in a rapid decline in the apparent synthesis rate of glutamine synthetase. In addition, dibutyryl cyclic AMP treatment increased the initial rate of glutamine synthetase degradation. The half-life of glutamine synthetase was 24.5 h in control cultures and 16 h in dibutyryl cyclic AMP-treated cultures. In contrast, dibutyryl cyclic AMP had little effect on the synthesis or degradation of soluble protein. Our data indicate that the dibutyryl cyclic AMP-mediated decrease in 3T3 adipocyte glutamine synthetase activity results from a decrease in the synthesis rate and an increase in the initial degradation rate of the enzyme.     

The effect of ascorbic acid on collagen polypeptide synthesis and proline hydroxylation during the growth of cultured fibroblasts

The rate of collagen synthesis relative to the rate of synthesis of noncollagen protein was determined in several lines of cultured fibroblasts using an assay which measures [¹⁴C]proline incorporation into the polypeptide chains of collagen. In this assay procedure, collagen is degraded by protease-free collagenase regardless of whether proline and lysine residues are hydroxylated, thus separating the process of polypeptide synthesis from hydroxylation. It was found that the relative rate of collagen synthesis in L-929 cells was approximately 0.8–1% at all stages of growth. There was no significant increase in the relative rate of collagen synthesis in stationary phase compared to log phase cells in the lines Balb 3T3, 3T6, 3T12, and Swiss mouse 3T6. In all cases, the absolute incorporation of [¹⁴C]proline into both collagen and noncollagen proteins expressed as radioactivity incorporated per milligram of cellular protein, was 2–10 times higher in log phase cells, depending on the line examined.     

Dibutyryl cyclic AMP decreases the rate of lipoprotein lipase synthesis in cultured adipocytes

The regulation of avian lipoprotein lipase by dibutyryl cyclic AMP in cultured adipocytes was studied with quantitative and specific methods for the measurements of enzyme catalytic activity, enzyme protein mass, and immunoadsorption of labeled enzyme. Incubation of adipocytes in 0.5 mM dibutyryl cyclic AMP plus 0.5 mM theophylline results in a time-dependent decrease in cell lipoprotein lipase catalytic activity. The activity is decreased by 70% in 4 h and over 90% by 12 h. The decrease in cellular catalytic activity is due to a decrease in both enzyme content and enzyme catalytic efficiency. 4 h after exposure of adipocytes to cAMP, enzyme protein was decreased from 3.58 +/- 0.5 to 1.92 +/- 0.1 ng/dish and specific activity from 15.1 +/- 2.1 to 8.4 +/- 1.1 nmol/ng. In the presence of 0.5 mM theophylline, the dibutyryl cyclic AMP-mediated decrease in lipoprotein lipase activity was half-maximal at less than 25 microM dibutyryl cyclic AMP. The rate of lipoprotein lipase synthesis was estimated by measuring the incorporation of L-[35S]methionine into enzyme protein during 30 min. A method for the quantitative immunoadsorption of lipoprotein lipase from cell lysates was developed. Utilizing this immunoadsorption technique, the rate of incorporation of L-[35S]methionine into lipoprotein lipase was 0.0026 +/- 0.002%, when expressed as a percentage of that incorporated into total trichloroacetic acid-precipitable counts. By 2 h after exposure of adipocytes to 0.5 mM dibutyryl cAMP, the relative synthesis rate had already decreased to 64 +/- 4% of the control rate. After 16 h the synthesis rate was 43.2 +/- 13.8% of the control rate. The observed decreased synthesis rate could account for most of the decreased cellular enzyme content and diminished enzyme secretion rate.     

Increased sensitivity of scleroderma fibroblasts in culture to stimulation of protein and collagen synthesis by serum

Serum effects on ¹⁴C-proline incorporation and ¹⁴C-hydroxyproline synthesis by normal and scleroderma fibroblasts in culture were studied. Serum resulted in 97% and 212% increases in ¹⁴C-proline incorporation in two lines of scleroderma fibroblasts while the increase in normal fibroblasts was only 53%. Effects on collagen synthesis were more pronounced. Addition of serum resulted in 124% and 445% increments in ¹⁴C-hydroxyproline synthesis in the scleroderma fibroblasts but only a 43% increment in the normal fibroblasts. The results indicate that cultured scleroderma fibroblasts have increased sensitivity to biosynthetic stimulation by serum and this mechanism may be of pathogenetic importance in the excessive collagen accumulation characteristic of the disease.     

IGF-I and MAP kinase involvement in the stimulatory effects of LNCaP prostate cancer cell conditioned media on cell proliferation and protein synthesis in MC3T3-E1 osteoblastic cells

Bone metastases from prostate cancer cause abnormal new bone formation, however, the factors involved and the pathways leading to the response are incompletely defined. We investigated the mechanisms of osteoblast stimulatory effects of LNCaP prostate carcinoma cell conditioned media (CM). MC3T3-E1 osteoblastic cells were cultured with CM from confluent LNCaP cells. LNCaP CM stimulated MAP kinase, cell proliferation (3H-thymidine incorporation), and protein synthesis (14C-proline incorporation) in the MC3T3-E1 cells. The increases in cell proliferation and protein synthesis were prevented by inhibition of the MAP kinase pathway. IGF-I mimicked the effects of the CM on the MC3T3-E1 cells and inhibition of IGF-I action decreased the LNCaP CM stimulation of 3H-thymidine and 14C-proline incorporation and MAP kinase activity. The findings indicate that IGF-I is an important factor for the stimulatory effects of LNCaP cell CM on cell proliferation and protein synthesis in osteoblastic cells, and that MAP kinase is a component of the signaling pathway for these effects.     

UDP-galactose inhibition of BALB/3T12-3 cell growth : Requirement for medium galactosyltransferase activity

Incubation of BALB/3T12-3 cells with uridine diphosphate galactose (UDP-gal) resulted in a concentration-dependent inhibition of cell growth when cells were cultured in calf serum-supplemented Dulbecco's modified Eagle medium (CS-DMEM). Cell growth was completely inhibited by 5 mM UDP-gal with an ID₅₀ of 0.75 mM. This inhibitory effect was reversible. Other nucleotide-sugars, as well as galactose, glucose, and galactose-1-phosphate had no effect on cell growth. UDP-gal had no effect on cell growth when cells were cultured in heat-inactivated calf serum containing DMEM (HICS-DMEM) suggesting that a serum enzyme activity was responsible for the inhibition observed in CS-DMEM. No significant difference could be detected by descending chromatography in the degradation of UDP-gal during 96 h of incubation in CS-DMEM and in HICS-DMEM. Furthermore, the potential breakdown products of UDP-gal had no effect on cell growth when added directly to 3T12 cultures. When cells were incubated with 5 mM UDP-gal+5 mM CDP-choline (a potent inhibitor of pyrophosphatase activity), complete inhibition of cell growth was still observed. However, if cells were incubated with 5 mM UDP-gal+UDP (which inhibited calf serum galactosyltransferase activity), no inhibition of cell growth was observed over that found for UDP alone, suggesting that galactosyltransferase and not pyrophosphatase activity mediated the effect of UDP-gal on cell growth. A direct effect of UDP-gal on cells was suggested by (a) normal growth of cells in UDP-gal-conditioned medium (preincubated with UDP-gal for 24 h followed by dialysis to remove UDP-gal); (b) 3-fold greater incorporation of [³H]galactose from UDP-[³H]gal into cells grown in CS-DMEM than in HICS-DMEM. These studies suggest that the inhibition of 3T12 cell growth by exogenous UDP-gal may be due to alteration of cell surface glycoconjugates by extracellular galactosyltransferase activity.     

Effect of dibutyryl cyclic adenosine monophosphate on granulosa cell proliferation, oocyte growth and meiotic maturation in isolated mouse primary ovarian follicles cultured in collagen gels.

Isolated primary follicles from 10-day-old mice were cultured in a collagen gel matrix for 6 days in Minimum Essential Medium + foetal calf serum, followed by culture in unsupplemented medium (control) or in medium containing hypoxanthine (2 mM) or dibutyryl cyclic adenosine monophosphate (dbcAMP, 0.25 mM) for a further 3 or 6 days. Less than 10% of oocytes resumed meiosis during the culture period in all groups. At recovery, the diameter of oocytes at the germinal vesicle stage was recorded and their ability to resume meiosis was determined. Hypoxanthine had little effect on oocyte growth and meiotic competence, but culture in dbcAMP resulted in oocytes that were larger (60.2 +/- 0.6 microns) than those of controls (55.8 +/- 0.5 microns) and more competent to resume meiosis than were controls (42.9% and 10.8%, respectively). The addition of dbcAMP to the culture medium induced a 4-5-fold increase in the number of granulosa cells oocyte compared with controls (3757 +/- 423 and 838 +/- 93, respectively). These results indicate that increased oocyte growth and meiotic competence is primarily mediated via dbcAMP effects on the granulosa cells.     

Effect of dibutyryl cyclic AMP on collagen synthesis in a clonal osteoblast-like cell line derived from newborn mouse calvaria

The effects of dibutyryl cyclic AMP (DBcAMP) and related compounds on collagen synthesis in a clonal osteoblast-like cell line, MC3T3-E1, were investigated. The addition of DBcAMP to cultures increased the hydroxyproline content of the cells. It also enhanced the incorporation of labeled proline into collagen and elevated the activity of prolyl hydroxylase, an enzyme involved in collagen synthesis. These effects were observed at concentrations of 0.1 to 2 mM DBcAMP. 8-Bromo cyclic AMP also increased the hydroxyproline content of the cells, while sodium butyrate and dibutyryl cyclic GMP had no such effect. These results suggest that the intracellular accumulation of cyclic AMP in osteoblasts leads to their active production of collagen, a major component of the organic matrix of bone.     

Changes in enzyme patterns produced by high potassium concentration and dibutyryl cyclic AMP in organ cultures of sympathetic ganglia

—Preliminary experiments had shown that acetylcholine, the putative mediator of trans-synaptic induction of tyrosine hydroxylase (TH) and dopamine β-hydroxylase (DBH) in vivo, did not lead to an increase in these enzyme activities in mouse superior cervical ganglia kept in organ culture. It was the aim of the present study to evaluate whether increases in tyrosine hydroxylase and dopamine β-hydroxylase evoked by other stimuli such as potassium or dibutyryl cyclic AMP in such an in vitro system are representative for in vivo trans-synaptic induction where changes in the levels of enzymes involved in norepinephrine synthesis or degradation are strictly confined to TH and DBH. In the presence of elevated concentrations of potassium or 5 mm dibutyryl cyclic AMP under organ culture conditions TH and DBH as well as DOPA decarboxylase and monoamine oxidase were significantly (P < 0.025) increased. The increase in total activities of TH and DBH were completely, those of DOPA decarboxylase and monoamine oxidase partially, inhibited by cycloheximide. In the presence of high concentrations of potassium, the total protein content of the ganglia was 28 per cent higher than in culture controls while dibutyryl cyclic AMP had no significant effect. Cycloheximide alone caused the protein content to fall to 70 per cent of that in control cultures. The loss of protein in the presence of cycloheximide was not accompanied by a simultaneous loss of TH, DOPA decarboxylase or monoamine oxidase, but DBH was decreased. Potassium was shown to increase the incorporation of [³H]leucine into TCA-insoluble protein during an early culture period but dibutyryl cyclic AMP showed no such effect. An increase in the rate of incorporation of [³H]leucine into protein was seen in both the control and elevated potassium cultures after 48 h. This increase did not occur in the presence of dbcAMP. The difference in enzyme patterns under conditions of elevated potassium and dibutyryl cyclic AMP and the fact that no changes in the levels of endogenous cyclic AMP were observed during exposure to 54 mm -potassium for a time period sufficient to initiate changes ultimately leading to elevated TH levels argues against the mediation of the potassium-induced enzyme increases by cAMP. Since changes in enzyme patterns caused by potassium and dbcAMP were not similar to patterns seen in vivo under conditions of trans-synaptic induction we conclude that use of this system as an in vitro model for in vivo trans-synaptic induction necessitates great caution.     

Effects of N6, 2′-O-dibutyryl cyclic adenosine monophosphate and the leukemia inhibitory factor on embryo development of the giant freshwater prawn,Macrobrachium rosenbergii

Summary

The effects of N⁶, 2′-O-dibutyryl cyclic adenosine monophosphate (dbcAMP) and the leukemia inhibitory factor (LIF) on embryo development of the giant freshwater prawn, Macrobrachium rosenbergii, were studied. Embryos 1.5 days old were cultured in vitro in 15% artificial seawater supplemented with 0.05 mM, 0.1 mM or 0.5 mM dbcAMP, or with 10 ng/ml, 20 ng/ml or 50 ng/ml LIF for 2 days. No differences in the morphology or growth of the embryos between the supplemented and the control groups were observed. The developmental rates as revealed by the eye formation rates were not altered with either supplement. The survival rates of the embryos declined during the course of development; and, again, the rates were not altered when supplemented with dbcAMP or LIF. Although there were no significant changes in the eye formation rate in embryos supplemented with 0.05 mM dbcAMP, an increase in the hatching rate was found. In contrast, supplementation with LIF did not improve the hatching rates. Germ cell development seemed to be most affected. The number of primordial germ cells (PGCs), the progenitors of gametes, was significantly increased with both dbcAMP and LIF supplementation. However, supplementation with 0.05 mM dbcAMP and 10 ng/ml LIF combined did not provide any additional effect on this feature.     

N6,O2′-dibutyryl adenosine 3′,5′-monophosphate-stimulated release of proteoglycans from cultured immature rabbit ear cartilage

The stimulatory effects of N⁶,O^2′-dibutyryl adenosine 3′,5′-monophosphate on proteoglycans released from immature rabbit ear cartilage were studied in vitro. Cartilage incubated in medium containing dibutyryl cyclic AMP resulted in a significant increase of proteoglycans released in concentrations above 0.5 mM. Theophylline (1 mM) which did not significantly stimulate proteoglycans released alone, was found to potentiate the action of this nucleotide. ATP, 5′-AMP and butyric acid in the presence of theophylline, did not stimulate proteoglycans released. The addition of protein or RNA synthesis inhibitors depressed proteoglycans released by dibutyryl cyclic AMP and theophylline.Gel chromatographic and chemical investigations of the proteoglycans released into the culture media in the presence of dibutyryl cyclic AMP indicated a reduction in the proportion of protein associated with these complexes. This result, together with enzyme inhibitor studies, leads us to speculate that the observed action of dibutyryl cyclic AMP on rabbit ear cartilages may be mediated by the neural proteases.     

Biochemical consequences of follicle-stimulating hormone binding to testicular macrophages in culture

The present studies demonstrate that testicular macrophages respond to follicle-stimulating hormone (FSH) by: 1) stimulating the rate of incorporation of amino acids into secreted proteins; 2) increasing the rate of incorporation of uridine into RNA; and 3) enhancing the accumulation of intracellular cyclic adenosine monophosphate (cAMP; which was potentiated by the addition of 1 mM 3-isobutyl-1-methylxanthine; MIX). In addition, dibutyryl cAMP (dbcAMP) enhanced the incorporation of amino acids into secreted proteins; however, this cAMP analog had no effect on the incorporation of uridine into RNA. Finally, it was demonstrated that testicular macrophages possess specific receptors with a high affinity for FSH.     

Curcumin treatment modulates collagen metabolism in isoprot3erenol induced myoxardial necrosis in rats

This study was carried out to evaluate whether curcumin, a potent antioxidant, had any specific role in the synthesis and degradation of collagen in rat heart with mocardial necrosis, induced by isoproterenol.HCI (ISO). Myocardial necrosis was induced by administration of ISO (30 mg/100 g body weight subcutaneously twice at an interval of 24 h) and studies on collagen metabolism were carried out with curcumin (200 mg/kg) pre-and co-treatment with ISO. The incorporation of ₁₄C-proline into collagen was studied as an index of collagen synthesis. The heart weight /body weight ratio,heart RNA/DNA ratio and protein were found to increase significantly in ISO administered animals. Curcumin pre- and co-treatment with ISO reversed these changes and attenuated the development of cardiac hypertrophy two weeks after the second dose of ISO. Increased fractional synthesis rate and enhanced degradation of newly synthesized collagen were observed in ISO administered animals. Curcumin pre- and co-treatment with ISO was noticed to decrease the degree of degradation of the existing collagen matrix and collagen synthesis, two weeks after the second dose of ISO. The observed effects could be due to free radical scavenging capacity and inhibition of lysosomal enzyme release by curcumin.     

Protein synthesis by astrocytes in primary cultures

Protein synthesis, measured as leucine incorporation into acid-precipitable proteins, was determined in astrocytes in primary cultures obtained from the cerebral hemispheres of newborn mice. As can be expected for eucaryotic, ribosomal protein synthesis, the incorporation was almost completely inhibited by cycloheximide (0.01 mM), but unaffected by chloramphenicol (0.03 mM). The rate of synthesis, measured during exposure to a high (0.8 mM) concentration of leucine was 5.4 nmol/hr/mg protein in mature (i.e., at least 4-week-old) cultures. This value is at least twice as high as the protein synthesis rates reported for the adult brain in vivo, suggesting that a very considerable part of the protein synthesis in the adult brain may take place in astrocytes. The molecular weight distribution of the synthesized proteins was determined by polyacrylamide gel electrophoresis, demonstrating synthesis of at least 50 different polypeptides, ranging in molecular weight between 190,000 and 27,000 daltons. The pattern of the synthesized proteins underwent considerable alteration with age in young cultures in which the total content of protein was still increasing, but it was remarkably stable after the age of two weeks. Exposure to dibutyryl cyclic AMP, which is known to alter morphology, content of glial fibrillary acidic protein (GFA), and activities of certain enzymes in the cultured astrocytes, caused marked alterations in the pattern of the synthesized proteins.     

Stimulation of growth of serum-deprived chick embryo fibroblasts by 3',5'-phosphodiesterase.

In chick embryo fibroblasts (CEF) deprived of serum, DNA synthesis is reduced to a basal level in about 12 h, cell division ceases after 24–36 h and their morphology changes to a rounded, less refringent form. During several days without serum the cAMP content of the cells showed a slow increase or a maintenance of the level found before serum was removed. When CEF deprived of serum for 24 h were treated with beef heart 3′,5′-phosphodiesterase (PHD) the cAMP level fell about 40% after 3 h, ³H-thymidine incorporation into DNA was strongly stimulated with a peak of incorporation at 12 h after the start of PHD treatment, cell morphology returned to that observed before serum deprivation, and at 24 h there was an evident growth in cell population, with a parallel increase in protein content. The growth stimulation by PHD is transitory: after cells had been deprived of serum for 4 days the PHD effect was no longer noticeable on the above parameters. Theophylline (1 mM and 4 mM) inhibited the PHD-mediated stimulation of ³H-TdR incorporation, this could well have been due to its general toxic effect on the cells (see Discussion).     

Stimulation of albumin synthesis in mouse hepatoma cells by Bt2cAMP: cell cycle specificity

Addition of 1 mm dibutyryl cyclic AMP (Bt₂cAMP) to cultures of mouse hepatoma cells, Hepa, specifically stimulates the synthesis of serum proteins including albumin. This stimulation is accompanied by an inhibition of cell proliferation. We have investigated these phenomena in synchronous cultures of Hepa. Proliferation of Hepa was arrested by isoleucine starvation. Synchronous growth was initiated by addition of complete growth medium or complete growth medium supplemented with 1 mm Bt₂cAMP. S phase and mitosis were estimated by determinations of [³H]thymidine incorporation and by cell numbers. The rate of albumin synthesis relative to total protein synthesis was measured by pulse labeling cultures for 30 min with [³H]leucine and comparing amounts of immunoprecipitable label with trichloroacetic acid-precipitable label. Treatment of synchronous cultures with Bt₂cAMP did not alter the duration of S phase or the onset of mitosis. The relative rate of albumin synthesis in Bt₂cAMP-treated culture began increasing after mitosis. The timing of the Bt₂cAMP stimulation of albumin synthesis was further investigated by adding Bt₂cAMP to cultures of Hepa at various times after the initiation of synchronous growth. The relative rate of albumin synthesis was then measured at a fixed postmitotic time. An increased relative rate of albumin synthesis was observed only in cultures exposed to Bt₂cAMP before or during S phase. Thus the postmitotic increase in the synthesis of albumin requires the presence of Bt₂cAMP during S phase.     

Optimal conditions for the study of growth control in BALB/c 3T3 fibroblasts

The effect of a Fibroblast Growth Factor (FGF) on the initiation of DNA synthesis in sparse populations of BALB/c 3T3 cells maintained quiescent in the presence of various serum concentrations has been investigated. The initiation of DNA synthesis, as measured by ³H-thymidine incorporation, is greatest in cultures maintained quiescent in the presence of 0.8% serum. Under these conditions, the cells are on the border between quiescence and growth. The minimal effective dose of FGF needed to increase DNA synthesis is 0.01 ng/ml and plateau values are obtained between 2.5 and 5 ng/ml. At plateau concentrations, FGF is 65% as effective as saturating concentrations of serum in the stimulation of DNA synthesis. When dexamethasone and insulin are present, FGF was 82% as effective. In contrast, cultures maintained in the presence of lower serum concentrations (0.2% and 0.4%) are much less responsive to the FGF. This can be attributed to the lack of supplemental factors which make the cells maximally responsive to growth stimulation and to degenerative changes that take place in the cells. Insulin and the glucocorticoid, dexamethasone, potentiated the response to FGF and delayed the degeneration of cells maintained in low serum.