The antagonistic effect of dexamethasone and insulin on alpha-fetoprotein secretion by cultured H4-II-E-C3 cells derived from the Reuber H-35 hepatoma.

Non-confluent monolayers of H4-II-E-C3 cells were maintained in serum-free media. Dexamethasone alone (5 × 10^?7M) stimulated α-fetoprotein secretion 2- to 4-fold while insulin alone (8.7 × 10^?8M) inhibited α-fetoprotein secretion by 20%. When dexamethasone (5 × 10^?7 to 5 × 10^?9M) and insulin (8.7 × 10^?8 to 8.7 × 10^?11M) were added simultaneously, insulin diminished the stimulatory effect of dexamethasone. When α-fetoprotein secretion was elevated by dexamethasone and the medium was replaced by media containing either insulin or no hormones, the rate of α-fetoprotein secretion diminished more rapidly with the insulin-supplemented medium. Alone or in combination, insulin and dexamethasone had little effect on albumin secretion.     

Effect of tunicamycin and monensin on secretion of thyroxine-binding globulin by cultured human hepatoma (Hep G2) cells

We have reported in the preceding paper that human hepatoma (Hep G2) cells synthesize thyroxine-binding globulin (TBG). In this paper, we evaluated the kinetics of secretion of the protein and the effects produced by the ionophore monensin and the glycosylation inhibitor tunicamycin. Cells were pulse labeled with [35S]methionine and then chased after addition of excess unlabeled methionine. TBG appeared in the medium after 10 min, and 50% of the protein was secreted after 45 min. After 2 h, more than 85% of TBG had been released. The rate of secretion of TBG was much slower than that of albumin, 50% of which was secreted after 20 min. Monensin, 1 microM, caused a marked delay in TBG secretion, with 50% released after 80 min. After 2 h, less than 60% had been released and a plateau was approached. Endoglycosidase H (endo H) treatment of intracellular and secreted TBG showed no alteration in the rate of conversion of TBG oligosaccharide units from high-mannose type (endo H-sensitive) to complex type (endo H-resistant), thus suggesting that monensin impeded the exit of TBG from the Golgi apparatus without affecting the terminal glycosylation of the protein. Tunicamycin, 5 micrograms/ml, completely blocked glycosylation and markedly affected TBG secretion, almost doubling the time required for the secretion of 50% of the protein. The effect was specific for TBG, since it was not observed in the case of albumin. After 2 h, only 56% of the protein had been released. Analysis of intracellular and extracellular immunoprecipitated products revealed the presence of aggregates (Mr greater than 100,000). The lack of carbohydrates, although not preventing TBG secretion, had marked quantitative effects, and increased the susceptibility to aggregation.     

Insulin modulation of hepatic synthesis and secretion of apolipoprotein B by rat hepatocytes

Insulin inhibition of apolipoprotein B (apoB) secretion by primary cultures of rat hepatocytes was investigated in pulse-chase experiments using [35S]methionine as label. Radioactivity incorporation into apoBH and apoBL, the higher and lower molecular weight forms, was assessed after immunoprecipitation of detergent-solubilized cells and media and separation of the apoB forms using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Hepatocyte monolayers were incubated for 12-14 h in medium with and without an inhibitory concentration of insulin. Cells were then incubated for 10 min with label, and, after differing periods of chase with unlabeled methionine, cellular medium and media labeled apoB were analyzed; greater than 90% of labeled apoB was present in cells at 10 and 20 min after pulse, and labeled apoB did not appear in the medium until 40 min of chase. Insulin treatment inhibited the incorporation of label into total apoB by 48%, into apoBH by 62%, and into apoBL by 40% relative to other cellular proteins. Insulin treatment favored the more rapid disappearance of labeled cellular apoBH with an intra-cellular retention half-time of 50 min (initial half-life of decay, t1/2 = 25 min) compared with 85 min in control (t1/2 = 60 min). Intracellular retention half-times of labeled apoBL were similar in control and insulin-treated hepatocytes and ranged from 80 to 100 min. After 180 min of chase, 44% of labeled apoBL in control and 32% in insulin-treated hepatocytes remained cell associated. Recovery studies indicated that insulin stimulated the degradation of 45 and 27% of newly synthesized apoBH and apoBL, respectively. When hepatocyte monolayers were continuously labeled with [35S]methionine and then incubated in chase medium with and without insulin, labeled apoBH was secreted rapidly, reaching a plateau by 1 h of chase, whereas labeled apoBL was secreted linearly over 3-5 h of chase. Insulin inhibited the secretion of immunoassayable apoB but not labeled apoB. Results demonstrate that 1) insulin inhibits synthesis of apoB from [35S]methionine, 2) insulin stimulates degradation of freshly translated apoB favoring apoBH over apoBL, and 3) an intracellular pool of apoB, primarily apoBL, exists that is largely unaffected by insulin. Overall, insulin action in primary hepatocyte cultures reduces the secretion of freshly synthesized apoB and favors secretion of preformed apoB enriched in apoBL.     

Inhibition of synthesis of alpha-fetoprotein by glucocorticoids in cultured hepatoma cells

alpha-Fetoprotein (AFP) synthesis was studied in the presence and absence of glucocorticoids in rat hepatoma Mc-A-RH-7777 cells. Radioimmunoassay of media from cell cultures grown in the presence of glucocorticoid (dexamethasone or cortisol) showed a reduction in AFP, an increase in albumin, and no significant change in transferrin accumulation, as compared to controls. Labeling experiments with L-[35S]methionine indicated that in both cells and media of dexamethasone-treated cultures there was a 50--80% reduction in polypeptide precipitated by anti-AFP serum, as compared with controls; no change was seen in polypeptide precipitated by anti-transferrin serum. Pulse and pulse-chase experiments demonstrated that dexamethasone inhibited the synthesis of AFP but not its secretion. The half-time for secretion of AFP in the presence and absence of dexamethasone was 43 min.     

Synthesis and secretion of albumin by a synchronized rat hepatoma cell line.

The rat hepatoma cell H4-12 which synthesizes and secretes albumin was synchronized by growth in isoleucine-deficient medium followed by a second block with excess thymidine. Albumin synthesis and secretion was measured in the synchronized cells at different time intervals representative of early S, late S, G2, mitosis, early G1 and late G1 phases of the cell cycle. Maximal albumin synthesis occurred during G1 although significant synthesis also occurred during the other cell cyle phases. Most (75--80%) of the radioactive albumin produced during a 15 min pulse incubation with L-[4,5-3H] leucine was found in the microsomal cell fraction and this nascent albumin was secreted into the incubation medium during a 160 min chase period. Fifty percent of the nascent albumin was secreted by 50--55 min and this pattern of secretion did not change during the cell cycle. These data indicate that albumin synthesis occurs throughout the cell cycle but that it is preferred during G1. The rate of intracellular transport and secretion of albumin does not vary during the different phase of the cell cycle.     

Growth and hepatospecific gene expression of human hepatoma cells in a defined medium

Summary The production of albumin, α-fetoprotein (AFP), and α-1 antitrypsin has been compared among human hepatoma cells cultured in medium containing serum, medium containing hormones and growth factors, and a basal medium containing selenium as the only supplement. Growth is sustained in all three media, and the expression of all three proteins was maintained for over 4 mo. in the various media. However, the quantitative production of albumin and AFP were dramatically different in the three media. Two hormones, insulin and triiodothyronine, influenced the level of secreted proteins. Triiodothyronine increases the amount of secreted albumin whereas insulin at 10 μg/ml reduced the level of total secreted protein.     

Effects of hexamethylene bisacetamide onα-fetoprotein,albumin, and transferrin production by two rat hepatoma cell lines

Summary α-Fetoprotein (AFP), albumin, and transferrin production by two rat hepatoma cell lines, McA-RH 7777 (7777) and McA-RH 8994 (8994), was determined after treatment with hexamethylene bisacetamide (HMBA, 2 to 6 mM). Radioimmunoassays were used to determine the levels of both secreted and intracellular AFP, albumin, and transferrin. Line 7777 normally produces large quantities of AFP and small quantities of albumin, thus resembling the less differentiated fetal liver with respect to the synthesis of these two proteins. Line 8994 normally produces small quantities of AFP and relatively larger amounts of albumin, thus resembling hepatic functions characteristic of a more differentiated state. After treatment with HMBA for a period of 28 to 96 h a threefold increase in AFP secretion by 7777 and a dose related increase in AFP, albumin, and transferrin secretion by 8994 were observed. In contrast, the secretion of albumin and transferrin in 7777 was inhibited by 60 and 40%, respectively, following treatment with HMBA. The intracellular concentrations of AFP in 7777 and AFP, albumin, and transferrin in 8994 were increased by treatment with HMBA indicating that HMBA is able to stimulate the synthesis of these proteins. The intracellular concentration of AFP, albumin, and transferrin in 7777, when expressed as a percentage of the extracellular concentration of these proteins, did not change significantly during HMBA treatment, indicating that the observed decrease in secreted albumin and transferrin by 7777 is due to decreased synthesis. Similarly, in Line 8994, when the intracellular concentration of the three proteins was expressed as percentage of the extracellular concentration, the only significant change observed was an increase in AFP after 72 h of HMBA (5 mM) treatment. The observed changes in the synthesis of AFP, albumin, and transferrin in both 7777 and 8994 after HMBA treatment were reversible, as judged by the return to control values upon removal of HMBA from the culture medium. Thus, HMBA stimulates synthesis of the oncofetal protein AFP, a result that appears to be independent of the stage of differentiation of the cell. However, its effect on the synthesis of albumin and transferrin are opposite in the two cell lines, suggesting that the regulation of the synthesis of these two proteins is controlled by factors or conditions that are dependent upon the stage of differentiation of the hepatoma cell lines.     

A study on the intracellular transport of prothrombin, albumin and transferrin in rat

The intracellular transport of prothrombin in rat has been studied and compared with the transport of albumin and transferrin. The proteins were immunoisolated from plasma samples after pulse labelling with [3H]leucine and the secretion kinetics were determined. The half-times for secretion (t1/2) were approx. 30, 53 and 75 min for albumin, prothrombin and transferrin, respectively, whereas the minimal transit time for prothrombin was approx. 30 min, and those for albumin and transferrin 15-20 min. After injection of vitamin K-1 into warfarin-treated rats, the accumulated prothrombin precursor was gamma-carboxylated and secreted with a t1/2 of 37 min. This indicates that the gamma-carboxylation of prothrombin in rough endoplasmic reticulum cannot account for the delay in the transport of prothrombin as compared to albumin. Comparison of the incorporation of [3H]leucine and [3H]glucosamine into plasma prothrombin and transferrin suggested that transferrin is secreted randomly from an intracellular pool, whereas prothrombin is transported in a more orderly sequence. Moreover, treatment of rough microsomes with 0.05% sodium deoxycholate indicated that prothrombin is more tightly associated with the membranes of rough endoplasmic reticulum than albumin and transferrin.     

Stimulation of apolipoprotein secretion in very-low-density and high-density lipoproteins from cultured rat hepatocytes by dexamethasone.

The effects of dexamethasone (a synthetic glucocorticoid) and insulin on the secretion of very-low-density lipoprotein (VLDL) and high-density lipoprotein (HDL) were investigated. Rat hepatocytes in monolayer culture were preincubated for 15 h in the presence or absence of combinations of 100 nM-dexamethasone and 2 nM-, 10 nM- or 50 nM-insulin. Dexamethasone increased [3H]oleate incorporation into secreted triacylglycerol by 2.7-fold and the mass of triacylglycerol secreted by 1.5-fold. Insulin alone decreased these parameters and antagonized the effect of dexamethasone. Dexamethasone increased the secretion of [3H]leucine in apolipoprotein (apo) E, and in the large (BH) and small (BI) forms of apo B in VLDL by about 7.1-, 3.6- and 4.0-fold respectively. Insulin alone decreased the secretion of these 3H-labelled apolipoproteins in VLDL. However, 2 nM-insulin with dexamethasone increased the secretion of 3H-labelled apo BH and apo BL by a further 0.8- and 3.2-fold respectively; 50 nM-insulin decreased the secretions of apo E, apo BH and apo BL in VLDL. Similar effects for dexamethasone or insulin alone were also obtained for the masses of apo E and apo BL + H secreted in VLDL. Albumin secretion was not significantly altered by either dexamethasone or insulin alone, but in combination they stimulated by 2.1-2.6-fold. Insulin or dexamethasone alone had little effect on the secretion of apolipoproteins in the HDL fraction. However, dexamethasone plus 2 nM-insulin increased the incorporation of [3H]leucine into apo AI, apo AH plus apo C, apo AIV and apo E of HDL by about 1.8-, 1.6-, 1.7- and 2.0-fold respectively. The apo E in the bottom fraction represented about 69% of the total 3H-labelled apo E secreted. The responses in the total secretion of apo E from the hepatocytes resembled those seen in HDL. The interactions of insulin and dexamethasone are discussed in relation to the general regulation of lipoprotein metabolism, the development of hyperlipidaemias and the predisposition to premature atherosclerosis.     

Processing and secretion of insulin-related peptides in an insulinoma cell line

Certain classes of prohormones and other neuroendocrine or endocrine-derived secretory proteins are post-translationally modified in the secretory storage granules. If such molecules were to be biosynthesized to acceptable quantity and yield using endocrine-derived cell lines, it would be important to understand the relationship between the secretory dynamics and the conversion and release of the immature and mature forms of the molecule. We studied aspects of such a relationship using the endocrine-derived cell line betaTC-3, which synthesizes murine proinsulin, sequesters it into secretory granules, and converts it into mature insulin. In T-flask experiments with confluent cultures of betaTC-3 cells, intracellular and secreted (pro)insulin was sampled before and after episodes of stimulated exocytosis and recharging and quantified by radioimmunoassay and reversed-phase high-performance liquid chromatography (HPLC). Under conditions of steady-state secretion in glucose-rich growth medium the cells turned over their (pro)insulin inventory (90 +/- 5% mature insulin) at 2-3% per hour through secretion of (pro)insulin which was less than 70% mature. During an episode of hyperstimulated exocytosis induced by the combined secretagogues carbachol (1 muM) and isobutylmethylxanthine (1 mM), approximately 80% of the intracellular (pro)insulin stores were depleted within 2 h and 84 +/- 4% of the secreted (pro)insulin was in the mature form. Following the discharging episode, exocytosis was suppressed to 10% of its steady-state rate with a treatment which attenuated calcium influx (20 muM verapamil with reduced levels of calcium in the medium). Under this condition the secreted protein was only approximately 50% converted to mature insulin, but 85 +/- 10% of the net (pro)insulin accumulating within the intracellular stores was converted to the mature form. The inverse relationship between rate of secretion and degree of conversion of secreted (pro)insulin is consistent with a previously observed phenomenon of preferential basal secretion from immature secretory granules. This tends to enrich the secreted peptides in immature forms relative to the total intracellular pool. Preferential early secretion can best be overcome by rapid discharging of the long-term and predominantly mature stores. Thus, a cyclic controlled secretion process wherein product is collected during intermittent discharging episodes would provide a better yield of mature product than would steady-state secretion. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 283-289, 1997.     

Primary cultures of rat pancreatic acinar cells in serum-free medium

Summary Rat pancreatic acinar cells were isolated and cultured in Ham's F12 medium with 15% bovine calf serum. Caerulein, insulin, somatostatin, and dexamethasone (DEX) had no effect on intracellular or secreted amylase in these cultured cells. A serum-free medium, using Waymouth's MB 752/1 supplemented with albumin, epidermal growth factor (EGF), DEX, and HEPES, was then developed to avoid serum factors that might mask hormonal effects. In this SF medium, pancreatic acinar, cells maintained the morphological and ultrastructural characteristics of freshly isolated cells and secreted amylase in response to the secretagogue, carbamyl choline. Insulin, at a concentration of 1 μg/ml, significantly increased intracellular and secreted amylase activity after 3 d. This model cell system can be used to study the regulation of the synthesis of amylase and other pancreatic enzymes in vitro.     

Differential arrest of secretory protein transport in cultured rat hepatocytes by azide treatment

The effect of reduced cellular ATP content on intracellular transport of two secretory proteins, albumin and haptoglobin, in isolated rat hepatocytes was studied. The cells were labeled with [35S]methionine and the cellular ATP content was then rapidly reduced to different stable levels by incubation with azide at different concentrations (2.0- 10 mM). The amount of the radioactively labeled secretory proteins in the cells and in the medium after 150 min of incubation was determined by immunoprecipitation followed by gel electrophoresis, fluorography, and densitometry. At progressively lower ATP levels, down to 50% of normal, the protein secretion was unaffected, whereas at even lower levels an increasing portion of the proteins remained in the cells; at 30 and 10% of normal ATP level, 25 and 75% of albumin, respectively, was arrested intracellularly. Analysis of the carbohydrate structure of intracellularly arrested haptoglobin showed that in cells with an ATP level of approximately 30% of normal, the majority of haptoglobin molecules (55%) were fully or partially resistant to endoglycosidase H. This result indicates that exit from the medial and/or the trans part of the Golgi complex (GC) was inhibited under these conditions. It also shows that the protein had accumulated in the GC, since under normal conditions the fraction of the intracellular haptoglobin that is endoglycosidase H resistant is approximately 10%. By similar criteria it was found that at ATP levels below 10% of normal transport of haptoglobin from the endoplasmic reticulum to the medial GC (and possibly also to the cis GC) as well as from the trans GC to the medium were blocked.     

Production of albumin and α-fetoprotein in primary culture of fetal human liver cells on collagenous substrata in the presence of hydrocortisone

Summary When primary cultures of fetal human liver cells established on type I collagen gels were compared to sister cultures developed on tissue culture plastic, the cells in contact with type I collagen secreted albumin at a higher rate than those without contact. The albumin secretion was dependent on the presence of hydrocortisone (HC) in the medium. Also, α-fetoprotein (AFP), of which the level decreased gradually and became undetectable after 6 d regardless of the presence or absence of HC in the cells cultured on plastic, was maintained for longer periods of time by plating the cells on type I collagen gels in the presence of HC. Different secretion rates of albumin and AFP were observed after Day 13 and Day 16, respectively, between cells maintained on type I collagen gels and those on film plastic. The cells secreted larger amounts of both albumin and AFP in plates coated with type IV or I collagens than with fibronectin after Day 10. The cells cultured on type I collagen gels were cuboidal in shape, whereas those on plastic were flattened in cultures with HC. These data indicate that the secretion of human albumin and AFP is facilitated by synergies between HC and collagenous substrata.     

Secretion of proalbumin by canavanine-treated Hep-G2 cells

The two processing sites in the conversion of preproalbumin to albumin are marked by arginine residues. Therefore, to study the mechanisms of albumin processing and secretion, the arginine residues of nascent albumin were replaced with canavanine by the incubation of Hep-G2 cells with this arginine analog. During a 4-h interval, canavanine inhibited (67%) the secretion of nascent albumin and increased the intracellular transit time of albumin secretion from 24 to 39 min. At 1 h, canavanine inhibited total protein synthesis by 19% and albumin synthesis by about 40%. Both the intracellular and secreted albumin produced by canavanine-treated cells were analyzed by DEAE-cellulose chromatography and were found to be more acidic than normal proalbumin and albumin. Further analysis on sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that the albumin produced and secreted by canavanine-treated cells appeared to have a larger molecular weight (by 4000) than serum albumin. The canavanine-treated cells were incubated with L-[3H]leucine and L-[3H]phenylalanine and the location of radioactive L-leucine and L-phenylalanine in the 30 NH2-terminal amino acid residues of secreted albumin was determined. The results indicated that canavanine-treated cells secreted proalbumin (79%) and also some fully processed albumin (21%). Preproalbumin was not secreted. Untreated Hep-G2 cells mostly secreted fully processed serum albumin (93%) with only traces of proalbumin (7%).     

Secretion of chondroitin SO4 by monolayer cultures of chick embryo chondrocytes

Monolayer cultures of chick embryo tibial chondrocytes incorporate 35SO42- into chondroitin SO4 which is rapidly secreted from the cells into two extracellular pools. Part of the extracellular chondroitin SO4 is recovered in a soluble form in the culture medium, and the remainder is associated with the cell matrix from which it is released by isotonic trypsinization. At 38 degrees C labeled chondroitin SO4 appears in the cell matrix fraction within 5 min after addition of 35SO42- and in the culture medium fraction 15 min after 35SO42- is added. The intracellular pool of labeled chondroitin SO4 reaches a steady state level of 150 to 200 pmol of bound SO4 per 10(6) cells in 60 min, while the cell matrix and medium fractions increase at rates of 3 and 1 nmol of bound SO4 per h per 10(6) cells, respectively. After 4 h of labeling, less than 20% of the newly synthesized cell-associated chondroitin SO4 is in the intracellular fraction. By labeling cells for 15 min at 25 degrees C 80% of the cell-associated chondroitin 35SO4 is obtained in the intracellular fraction. This material is chased without lag into both the cell matrix fraction and the medium fraction. A mixture of NaF and NaCN, both at 30 mM, lowers the cellular ATP level to 15% of normal and blocks secretion of the intracellular chondroitin SO4 into both extracellular fractions. Colchicine at 10(-6) M gives a partial inhibition of both synthesis and secretion of chondroitinSO4. Sucrose density gradient sedimentation analysis of the intracellular chondroitin SO4 and the two extracellular fractions shows that all three fractions contain both a heavy and light proteoglycan fraction. The intracellular light proteoglycan fraction is secreted preferentially into the culture medium where it represents 30% of the total culture medium pool. The ratio of 6-sulfated GalNAc to 4-sulfated GalNAc in the heavy proteochondroitin SO4 fraction is approximately twice that found for the light fraction.     

Origin of corticosteroid-binding globulin in fetal rat. Comparative dynamics of corticosteroid-binding globulin, alpha-fetoprotein, and albumin secretion in primary cultures of fetal rat hepatocytes

The origin of corticosteroid-binding globulin (CBG) and its evolution in comparison with alpha-fetoprotein (AFP) and albumin synthesis, during early development of rat liver (days 13 and 15 of fetal life), have been investigated using cultured fetal hepatocytes. Synthesis and secretion of CBG, AFP, and albumin is evidence by cycloheximide-sensitive [14C]leucine incorporation into immunoprecipitable polypeptides secreted by cultured hepatocytes into the medium, two-dimensional immunoelectrophoretic and autoradiographic identification of newly synthesized labeled proteins, corticosterone and estradiol-17 beta binding to CBG and AFP, respectively, and indirect immunofluorescence localization of AFP, albumin, and CBG in cultured fetal hepatocytes. CBG, albumin, and AFP accounted for 6, 11, and 25% (in 13-day-old rat fetuses) and 5, 15, and 28% (15-day-old rat fetuses), respectively, of the total secreted proteins in the culture medium. The rates of CBG, AFP, and albumin (counts/minute of secretion [14C]leucine incorporated per milligram of cell protein/hour of culture) in the hepatocytes of 15-day-old rat fetuses were 1.48-, 2.1-, and 2.57-fold higher, respectively, than in the 13-day-old rat fetuses. These results indicate that fetal liver is also active in CBG synthesis, along with AFP and albumin, as early as day 13 of fetal life and that the synthetic rates of these secretory proteins depend upon the developmental stage of the fetal liver. This developmental related change in the rate of synthesis of CBG by the fetal hepatocytes may regulate the level of free (active) glucocorticoid in the fetal circulation and thereby the initiation and regulation of glucocorticoid-dependent processes during the crucial stages of the differentiation of fetal liver and other developing tissues.     

Dexamethasone and retinyl acetate similarly inhibit and stimulate EGF- or insulin-induced proliferation of prostatic epithelium

Prostatic epithelium proliferates in a defined medium consisting of basal medium RPMI1640 containing transferring (1 microgram/ml), EGF (10 ng/ml), and insulin (3.7 micrograms/ml or 0.1 IU/ml). Although neither dexamethasone nor retinyl acetate affected the proliferation of prostatic epithelium in RPMI1640 containing transferrin alone, they modify the mitogenic effect of EGF and insulin. Dexamethasone at 10(-10) M or retinyl acetate at about 3 X 10(-9) M inhibits proliferation stimulated by EGF. Higher concentrations of dexamethasone (10(-8) - 10(-6) M) or retinyl acetate (3 X 10(-8) - 10(-7) M) enhance the mitogenic activity of EGF. Dexamethasone had a similar effect in the presence of insulin. However, retinyl acetate stimulated, but did not significantly inhibit, proliferation in the presence of insulin. These results suggest that both dexamethasone and retinyl acetate, and possibly other glucocorticoids and retinoids, may regulate the proliferation of prostate epithelium by a dose-dependent modification of the activity of insulin and EGF.     

The effects of cycloheximide on the biosynthesis and secretion of proteoglycans by chondrocytes in culture.

Proteoglycans synthesized by rat chondrosarcoma cells in culture are secreted into the culture medium through a pericellular matrix. The appearance of [35S]sulphate in secreted proteoglycan after a 5 min pulse was rapid (half-time, t 1/2 less than 10 min), but that of [3H]serine into proteoglycan measured after a 15 min pulse was much slower (t 1/2 120 min). The incorporation of [3H]serine into secreted protein was immediately inhibited by 1 mM-cycloheximide, but the incorporation of [35S]sulphate into proteoglycans was only inhibited gradually(t 1/2 79 min), suggesting the presence of a large intracellular pool of proteoglycan that did not carry sulphated glycosaminoglycans. Cultures were pulsed with [3H]serine and [35S]sulphate and chased for up to 6 h in the presence of 1 mM-cycloheximide. Analysis showed that cycloheximide-chased cells secreted less than 50% of the [3H]serine in proteoglycan of control cultures and the rate of incorporation into secreted proteoglycan was decreased (from t 1/2 120 min to t 1/2 80 min). Under these conditions cycloheximide interfered with the flow of proteoglycan protein core along the route of intracellular synthesis leading to secretion, as well as inhibiting further protein core synthesis. The results suggested that the newly synthesized protein core of proteoglycan passes through an intracellular pool for about 70-90 min before the chondroitin sulphate chains are synthesized on it, and it is then rapidly secreted from the cell. Proteoglycan produced by cultures incubated in the presence of cycloheximide and labelled with [35S]sulphate showed an increase with time of both the average proteoglycan size and the length of the constituent chondroitin sulphate chain. However, the proportion of synthesized proteoglycans able to form stable aggregates did not alter.     

Multihormonal induction of alpha 2u-globulin in an established rat hepatoma cell line

A subclone of the FU5-5 rat hepatoma cell line has been isolated which is inducible more than several hundred fold for the 20,000 dalton form of the major rat urinary protein alpha 2u-globulin. The basal relative synthetic rate (RSR) in growth medium containing 10% fetal calf serum was less than 2 X 10(-6) of total protein synthesis. Both dexamethasone and insulin were necessary for induction, and yielded a maximum induced RSR of 4-8 X 10(-3). Triiodothyronine (T3), dihydrotestosterone (DHT), rat growth hormone (GH), and estrogen, all of which have been shown to influence the induction of alpha 2u-globulin in the intact rat, were without effect on the cell line. A factor present in fetal calf serum was also necessary for maximum induction, since dexamethasone plus insulin in serum-free medium raised the RSR to only 3 X 10(-5); exogenous T3, GH, and DHT could not substitute for this serum factor. The kinetics of induction by dexamethasone were slow, with a lag of approximately 48 hr followed by a period of increasing RSR for 6-20 days. Removal of dexamethasone from induced cells led to an exponential decline in the RSR (t 1/2 15 hr). The concentrations of dexamethasone and insulin that could yield half maximum induction were 5 X 10(-8)M and 3 X 10(-11)M, respectively. Higher concentrations of insulin, although still in physiological range (10(-9)M), inhibited induction. At yet higher insulin levels, beyond the physiological range, alpha 2u-globulin synthesis returned to maximum values. The lack of DHT, T3, and GH requirement for alpha 2u-globulin induction in this cell line may mean that a regulatory aberrancy has occurred in this transformed cell line, or, alternatively, that these hormones act indirectly in the intact animal. This cell line should prove useful for the study of the molecular events associated with alpha 2u-globulin induction and for genetic approaches to the problem of multihormonal regulation of gene expression.