Regulation of metallothionein synthesis in HeLa cells by heavy metals and glucocorticoids

Metallothioneins (MTs) are low molecular weight, cysteine-rich proteins that bind heavy metals. MT induction occurs in liver in response to either heavy metal (Zn++ or Cd++) administration or stress. The synthesis of MT can also be induced by either heavy metals or glucocorticoid hormones in HeLa cells cultured in serum-free medium. Induction of MT by zinc is subject to "desensitization." In contrast, dexamethasone (dex) induction results in a continued elevation in the rate of MT synthesis. The stability of MT is dependent on the availability of metal; consequently, MT induced by dex is degraded much more rapidly (half-life of 11 to 12 hours) than MT induced by elevated zinc levels (half-life of 36 to 38 hours). Removal of either inducer results in biphasic degradation curves, as apothionein and zinc come into balance. In contrast, deinduction kinetics for MT synthesis following removal of the two inducers (zinc and dex) are the same, with a half-life of two and one-half hours. Inhibition of RNA synthesis blocks deinduction after removal of inducer. Induction of MT occurs in a wide variety of species, from blue-green algae to man. This system should provide an excellent model for the comparative biochemistry of regulation of gene expression.     

Differential regulation of the synthesis of mannosylphosphoryl derivatives of dolichol and retinol in HeLa cells

We have shown earlier that in HeLa S3G cells, glucocorticoids stimulate the synthesis of dolichyl phosphorylmannose (Dol-P-Man) with a concomitant increase in the glycosylation of proteins (Ramachandran, C.K., Gray, S.L. and Melnykovych G. (1982) Biochem. J. 208, 47-52). Although controversial, there have been several lines of evidence suggesting that the synthesis of retinyl phosphorylmannose (Ret-P-Man) and Dol-P-Man may be carried out by the same enzyme. We examined this possibility and conclude that in HeLa S3G cells the syntheses of Dol-P-Man and Ret-P-Man are catalyzed by two different enzymes located in the same microenvironment. Our conclusion is based on the following observations: exogenously added dolichyl phosphate and retinyl phosphate did not compete with each other; when the cells were grown in the presence of 1 microM dexamethasone, the microsomal synthesis of Dol-P-Man was stimulated, without affecting the Ret-P-Man synthesis; Arrhenius plots on Ret-P-Man and Dol-P-Man synthesis showed breaks at 22 and 37.7 degrees C.     

Evidence for control of protein synthesis in HeLa cells via the elongation rate

The effects of fresh medium and serum on protein synthesis in suspension-cultured HeLa cells after growth to high cell density (>5 × 10⁵ cells/ml) were studied. Cells which were resuspended in fresh medium plus serum and grown for 24 hours (control) were compared with cells grown for 2 hours after resuspension (stimulated). The spectrum of proteins being synthesized by control and stimulated cells does not appear to be grossly different; that is, the weight and number average molecular weights of newly synthesized whole-cell protein are about the same in both cultures. Also, no significant differences were observed in the number of ribosomes per polysome or in the fraction of total ribosomes in polysomes. However, the transit times (combined elongation and termination times) were found to differ significantly; the average transit time for control cells was 2.24 minutes, while the average transit time for stimulated cells was 1.26 minutes. (An appendex evaluating the methodology involved in measuring the transit time is included.) In agreement with the difference in transit time, the absolute rate of protein synthesis in stimulated cells was approximately 1.8 times the rate measured in control cells. These data are taken as evidence that under certain conditions, the rate of elongtion and/or termination of polypeptide chains limits the overall rate of translation, and that cells can respond to growth conditions by changing the elongation and/or termination rate of protein synthesis.     

Studies on the stimulation of dolichol-mediated glycosylation by dexamethasone in HeLa cells.

The effect of the addition of 1 microM-dexamethasone on the temporal sequence of the glycosylation process has been studied in HeLa S3G cells. In the presence of delipidized serum, dexamethasone caused an increase in 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase and an accelerated synthesis of dolichols. These events were followed by an increase in the transfer of mannose from GDP-mannose to mannolipid. An increase in the rate of synthesis of lipid-linked oligosaccharides and a stimulation of glycosylation were observed in cells grown in the presence of delipidized serum in the culture medium. The data are consistent with the view that cellular syntheses of lipids and glycoproteins are co-ordinately controlled.     

Induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase in HeLa cells by glucocorticoids.

The activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase, EC 1.1.1.34) has been demonstrated both in homogenates and microsomes of the S3G strain of HeLa cells. It was increased 8- to 10-fold by the removal of serum from the growth medium. The presence of steroids, specifically of the glucocorticoid series, in the serum-less growth medium elicited an additional 100 to 345% increase over the serum-less control, whereas the addition of N6,O2'-dibutyryl adenosine 3':5'-monophosphate to the medium or dexamethasone to the assay mixture was without any stimulatory effect. Both inductions were blocked by cycloheximide and actinomycin D, suggesting a protein synthesis-dependent elevation of enzyme activity. Glucocorticoids were effective in the induction at concentrations ranging from 10(-6) to 10(-8) M and there was a demonstrated parallel between the magnitude of enzyme induction and glucocorticoid potency. The HMG-CoA reductase activities from steroid-induced and control cultures had identical assay characteristics (pH optima and apparent Km values for both NADPH and HMG-CoA). This induction of the rate-controlling enzyme of cholesterogenesis occurred despite the observation that glucocorticoids specifically depress the rate of acetate or water, but not mevalonate, incorporation into cholesterol.     

Uptake of dolichol by Vero cells.

Characterization and kinetics of dolichol uptake by a Vero cell line are reported. Vero cells incorporate dolichol in a time- and dose-dependent manner. Optimal uptake is found at 37 degrees C and at a pH of 7.4. In contrast to cholesterol, an inhibitory effect on the dolichol incorporation is found for farnesol, geraniol, and retinol. Long chain polyprenols were slightly stimulatory. The translocation seems not to be highly energy dependent. The lack of substantial inhibition by chloroquine does not plead for a receptor-mediated endocytosis. Incorporated dolichol was distributed over both membranes and supernatant fractions, paralleling the distribution of the lysosomal marker beta-N-acetylhexosaminidase. The incorporated dolichol is subject to a fast efflux process, which is potentiated by the presence of lipid acceptors in the extracellular medium.     

Evidence for poliovirus-induced cytoplasmic alkalinization in HeLa cells

During the early period after poliovirus infection of HeLa cells, cellular Na+/K+ ATPase activity is transiently activated. We investigated the possibility that Na+/K+ ATPase activation is a consequence of Na+/H+ antiporter activation. Increased uptake of the weak organic acid 5,5-dimethyloxazolidine-2,4-dione by infected cells around 2 h after infection suggested cytoplasmic alkalinization equivalent to pH 7.7 during the biosynthetic phase of viral replication. Consistent with the involvement of Na+/H+ antiporter activation in this phenomenon, it was found to be [Na+]-dependent and inhibited by 5-(N-ethyl-N-isopropyl)amiloride (EIPA). However, the pH increase was not associated with an increase in amiloride-sensitive Na+ uptake by infected cells predicted by this mechanism. By contrast, the alkalinization could be abolished with the anion-exchange inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), implicating an anion-exchange mechanism, such as Cl-/HCO3- exchange, in this process. In addition to abolishing virus-induced intracellular alkalinization, both EIPA and DIDS moderately inhibited viral replication. Manipulation of intracellular pH with nigericin in the incubation medium revealed that maximum viral replication required a pH of about 7.7 and that replication was significantly inhibited even at pH 7.3. Thus, the pH increase in infected cells appeared to be physiologically relevant. These findings represent the first demonstration of a biologically meaningful pH increase in cells infected with a lytic virus.     

beta-Adrenergic stimulation alters oligosaccharide pyrophosphoryl dolichol metabolism in rat parotid acinar cells.

beta-Adrenergic stimulation of rat parotid acinar cells markedly increases [3H]mannose incorporation into N-linked glycoproteins [Kousvelari, Grant, Banerjee, Newby & Baum (1984) Biochem. J. 222, 17-24]. More than 90% of this protein-bound [3H]mannose was preferentially incorporated into four secretory glycoproteins. The ratio of [3H]mannose/[14C]leucine present in these individual proteins was 1.7-4-fold greater with isoproterenol-treated cells than with untreated controls. In isoproterenol-stimulated cells, [3H]mannose incorporation into mannosylphosphoryl dolichol and oligosaccharide-PP-dolichol was increased 2-3-fold over that observed in unstimulated cells. Similarly, formation of mannosylated oligosaccharide-PP-dolichol was increased approx. 4-fold in microsomes prepared from isoproterenol-treated cells. Also, turnover of oligosaccharide-PP-dolichol was significantly increased (5-fold) by beta-adrenergic stimulation; the half-life for oligosaccharide-PP-dolichol decreased from 6 min in control cells to 1.2 min in isoproterenol-stimulated cells. By 15 min after isoproterenol addition to acinar cells, the specific radioactivity of parotid oligosaccharide moieties increased about 3-fold over the value observed in the absence of the agonist. Taken together, these results strongly suggest that elevation of N-linked protein glycosylation in rat parotid acinar cells after beta-adrenoreceptor stimulation resulted from significant enhancement in the synthesis of mannosylphosphoryl dolichol and oligosaccharide-PP-dolichol and the turnover of oligosaccharide-PP-dolichol.     

Regulation of protein synthesis in mitotic HeLa cells.

Mitotic HeLa cells (M cells) synthesize protein at about 25% of the rate of S phase cells. This decrease in protein synthesis is due to a reduction in the rate of initiation. However, extracts prepared from M cells are almost as active in protein synthesis as S cell extracts. Both cell extracts are quite active in in vitro initiation of protein synthesis. Moreover, two steps in initiation, binding of Met-tRNAf to 40S ribosomal subunits and binding of mRNA to ribosomes, show similar activity in both extracts. The difference in protein synthesizing activity observed in vivo is largely eliminated in the preparation of cell-free systems. The ribosomes of M cells contain small mol wt RNA, which inhibits protein synthesis in vitro. This RNA, which has possibly a nuclear origin, may be a cause of the reduction in the rate of protein synthesis in M cells.     

Evidence for multiple enzymes in the dolichol utilizing pathway of glycoprotein biosynthesis.

A comparison has been made of the enzymes catalyzing the transfer of mannose, glucose and N-acetylglucosamine from, respectively, GDPmannose, UDP-glucose and UDP-N-acetylglucosamine to endogenous dolichol phosphate (Dol-P) in liver Golgi membranes. Evidence is presented with suggests that all three reactions utilize the same pool of Dol-P. The transfer of mannose from GDP-Man to Dol-P is not inhibited by 0.1 mM UDP or UMP; 0.1 mM GDP did block the accumulation of mannose in Dol-P-Man. The net transfer of glucose and N-acetylglucosamine to Dol-P is prevented by 0.1 mM UDP but not 0.1 mM GDP. UDPglucose inhibits the reverse of the glucose transfer reaction but not the reverse of the N-acetylglucosamine or mannose trasfer reaction. On the basis of this, and other data, it is concluded that the three sugar transfer reactions utilize separate enzymes.     

Mutants in dolichol synthesis: conversion of polyprenol to dolichol appears to be a rate-limiting step in dolichol synthesis

Chinese hamster ovary (CHO) cells of the Lec9 recessive complementationgroup display a distinctive profile of resistance to a varietyof toxic lectins. In addition, they accumulate cis--unsaturatedpolyprenol and use mainly polyprenol rather than dolichol tosynthesize the glycosylated lipids used in asparagine-linkedglycosylation of proteins. The primary defect in these cellsis thought to result from a deficiency in polyprenol reductaseactivity. Three new mutants were isolated and determined tohave qualitatively, although not quantitatively, similar lectinresistance profiles to Lec9 cells. Two of these mutants (Abr^Rand Ric^R) also contained polyprenol rather than dolichol. Thelectin resistance profile of an independent mutant which accumulatespolyprenol, F2A8, was also found to be qualitatively similarto the Lec9 pattern. The relationship among these mutants wasanalysed in more detail by construction of cell—cell hybrids.Lectin resistance profiles of the hybrids demonstrated thatAbr^R, Ric^R and F2A8 fell into the Lec9 complementation group.Analysis of prenols in the hybrids also showed that F2A8 wasa member of the Lec9 group. Surprisingly, a significant fractionof the prenols found in Lec9 Parent hybrids was polyprenol(up to 30% of the neutral fraction), whereas the prenols foundin Parent Parent hybrids were nearly exclusively dolichol(97% of the neutral lipid fraction). Therefore, reduction ofpolyprenol to dolichol appears to be a rate-limiting step inthe synthesis of dolichol since hybrids with differing numbersof wild-type alleles can be biochemically distinguished. CHO cells dolichol lectins mutants polyprenol reductase     

Effect of dexamethasone on mannolipid synthesis by hepatocytes prepared from control and inflamed rats.

Hepatocytes were prepared from control and inflamed rats. Mannose incorporation into dolichol monophosphate mannose in homogenate and microsomal fraction of the hepatocytes was increased 2-fold over the controls 24 h after induction of inflammation by turpentine injection. Incubation of hepatocytes from both control and inflamed rats with 0.1-10 microM-dexamethasone produced a 1.5-fold increase of dolichol phosphate mannose formation, whereas, 100 microM-dexamethasone decreased its formation. The increase in the ratio of dolichol phosphate mannose formation in inflamed over controls was virtually eliminated when the cell homogenate assay mixtures included 30 nmol of exogenous dolichol phosphate. This supports the earlier suggestion that the increase in the enzyme activity in inflammation could be due to higher concentrations of endogenous dolichol phosphate [ Coolbear & Mookerjea (1981) J. Biol. Chem. 256, 4529-4535]. In contrast, the increase in the ratio of dolichol phosphate mannose formation between dexamethasone-treated and untreated hepatocytes remained unchanged when increasing concentrations of exogenous dolichol phosphate were added to the assays. This suggests that the increase in glycosylation of dolichol phosphate in dexamethasone-treated hepatocytes is probably due to the increased mannosyltransferase activity, rather than due to higher concentrations of endogenous dolichol phosphate in these cells.     

Evidence for long-range electrostatic repulsion between HeLa cells

Agglutination curves obtained on addition of low molecular weight poly-l-lysines (mol. wt 4 000–23 000) to HeLa cells, show a deviation from linearity at low polymer concentration. This probably indicates the existence of a ζ-potential which has to be lowered before agglutination can take place. Experiments with dilysine support the assumption that cell surface charge is lowered on addition of low concentrations of short chain poly-l-lysines.Long poly-l-lysine molecules (mol. wts 70 000; 100 000) yield linear agglutination curves already at the lowest polymer concentrations. This might indicate that these polymers are able to bridge the original repulsion gap between HeLa cells.After removal of peripheral sialic acid by neuraminidase, linear agglutination curves are obtained with all poly-l-lysines irrespective of their chain lengths. This is interpreted as evidence for involvement of sialic acid residues in the charge organization responsible for electrostatic repulsion.The magnitude of the presumed repulsion effect is shown to vary with the cell density at the time the HeLa cells were harvested from the culture. The largest repulsion effect is obtained with cells from density inhibited cultures which also have the lowest tendency for mutual adhesion. With fast growing cells from low density cultures linear agglutination curves are obtained with short chain poly-l-lysines. This is interpreted as evidence for a strong diminishment or absence of long-range electrostatic repulsion between such cells.     

RNA synthesis in permeable HeLa cells

The equilibria and kinetics are reported for the partial reactions of the catalytic cycle of the Ca²⁺ ionophore X537A in phospholipid vesicles. The analysis is based on the study of the behavior of the ionophore's intrinsic fluorescence in fluorescence lifetime, stopped-flow, temperature, and conventional steady-state fluorescence experiments. Binding to dimyristoyl phosphatidylcholine vesicles gives rise to an enhancement of the fluorescence. At the pH of study (7.4) this involves the singly negatively charged form (X^?). Complexation of the membrane-bound form (X_m^?) by monovalent (M⁺) or divalent (M²⁺) cations to give 1:1 (M-X)_m and (M-X)_m⁺ complexes, respectively, gives rise to a further fluorescence enhancement. No evidence could be found for stoichiometries other than 1:1 in the equilibrium experiments. The fluorescence of X537A in the presence of phosphatidic acid vesicles or phosphatidylcholine/ phosphatidylethanolamine or phosphatidylcholine/cholesterol mixtures is much smaller than for pure phosphatidylcholine. Fluorescence lifetime experiments show that this is due to a reduction in binding rather than a reduction of the quantum yield of the bound species. Fluorescence decay profiles from the above-mentioned membranes showed two exponential components indicating that there were two fluorescent species. The shorter-lived species had a lifetime of 3–5 ns and accounted for 80–90% of the membrane-bound ionophore. The longerlived species (9–13 ns) was estimated to account for the remaining 10–20%. This species enjoys a higher degree of hydrophobic shielding than the shorter-lived species. Possible interpretations in terms of the ionophore orientation in the membrane are discussed. Temperature-jump experiments show that the binding rate of the ionophore is fast. The binding and dissociation rate constants were ca. 2 × 10⁷m (PC)^?1 s^?1 and 2 × 10³ s^?1, respectively. Stopped-flow experiments gave evidence for a slower “insertion” process with a ca. 10-ms half-time. Analysis shows that this process is capable of transport of (K-X) across the membrane with a rate constant ≤ 69^?1. In the presence of divalent cations a slower process involving transport of M²⁺-ionophore complexes across the membrane can be observed. The dependence of the rate on the total ionophore concentration indicates that the transported species is a neutral (M-X₂) complex. The lower limit for the rate constant for transport of the (Ca-X₂) complex is 35 s^?1. The divalent cation specificity of the overall reaction was shown to be Mg²⁺ ? Ca² < Sr²⁺ < Ba²⁺. The rates of the overall transport at low ionophore concentration are limited by the equilibrium constant for formation of the (M-X₂)_m complex from the (M-X)_m⁺ complex.     

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.