Events blocked in prereplicative phase in senescent human diploid cells, TIG-1, following serum stimulation

When senescent human diploid cells, TIG-1, were stimulated with serum at the end of their proliferative life span, such biochemical events as uptakes of 2-deoxyglucose and uridine, and expression of c-myc, were enhanced. However, RNA synthesis, polyamine accumulation, thymidine uptake and DNA synthesis were not enhanced at all. Protein synthesis increased only moderately as compared with that observed in younger cells. These results indicated that the events in prereplicative phase known to be independent on protein synthesis are induced in senescent cells after the stimulation with serum, whereas those required protein synthesis failed to increase to the same extent as seen in young cells.     

Down-regulation of mitochondrial cytochrome c oxidase in senescent porcine pulmonary artery endothelial cells

Cellular aging is associated with dysfunction of the mitochondrial respiration chain. Deficiency of mitochondrial cytochrome c oxidase (complex IV) plays a critical role in aging-induced mitochondrial dysfunction. We investigated whether in vitro cellular aging causes the downregulation of complex IV activity and gene expression using senescent (passage 45) and young (passage 3) pulmonary artery endothelial cells (PAEC). In senescent PAEC, the catalytic activity of complex IV decreased 84%, compared to that in young cells. Relative protein levels of complex IV subunits I and IV (complex IV S1 and S4) in senescent cells decreased 91%, compared to those in young cells. This suggests that lack of complex IV S1 and S4 in senescent cells may contribute to the deficiency of complex IV. Total steady state levels of mRNA for complex IV S1 and S4 in senescent cells were decreased to 20% and 18% of those in young cells. The relative rates of mRNA synthesis of complex IV S1 and S4 were decreased 46% and 37% in senescent cells, respectively, compared to young cells. The degradation of complex IV S1 and S4 was increased 76% and 64% in senescent cells, compared to young cells. These data indicate that mitochondrial DNA-encoded subunit I and nuclear DNA-encoded subunit IV of complex IV are downregulated through reduced synthesis and enhanced degradation of their mRNA, which may be responsible for the deficiency of complex IV in replicative senescent PAEC.     

Effects of contraction and insulin on protein synthesis, AMP-activated protein kinase and phosphorylation state of translation factors in rat skeletal muscle

In rat epitrochlearis skeletal muscle, contraction inhibited the basal and insulin-stimulated rates of protein synthesis by 75 and 70%, respectively, while increasing adenosine monophosphate-activated protein kinase (AMPK) activity. Insulin, on the other hand, stimulated protein synthesis (by 30%) and increased p70 ribosomal protein S6 kinase (p70S6K) Thr389, 40S ribosomal protein S6 (rpS6) Ser235/236, rpS6 Ser240/244 and eukaryotic initiation factor-4E-binding protein-1 (4E-BP1) Thr37/46 phosphorylation over basal values. Electrical stimulation had no effect on mammalian target of rapamycin complex 1 (mTORC1) signalling, as reflected by the lack of reduction in basal levels of p70S6K, rpS6 Ser235/236, rpS6 Ser240/244 and 4E-BP1 phosphorylation, but did antagonize mTORC1 signalling after stimulation of the pathway by insulin. Eukaryotic elongation factor-2 (eEF2) Thr56 phosphorylation increased rapidly on electrical stimulation reaching a maximum at 1 min, whereas AMPK Thr172 phosphorylation slowly increased to reach threefold after 30 min. Eukaryotic elongation factor-2 kinase (eEF2K) was not activated after 30 min of contraction when AMPK was activated. This could not be explained by the expression of a tissue-specific isoform of eEF2K in skeletal muscle lacking the Ser398 AMPK phosphorylation site. Therefore, in this skeletal muscle system, the contraction-induced inhibition of protein synthesis could not be attributed to a reduction in mTORC1 signalling but could be due to an increase in eEF2 phosphorylation independent of AMPK activation.     

IL2-dependent phosphorylation of 40S ribosomal protein S6 is controlled by PI-3K/mTOR signalling in CTLL2 cells

Growth factors and hormones activate global and selective protein translation by phosphorylation and therefore activation of p70 S6 kinase through a wortmannin-sensitive phosphoinositide-3 kinase (PI-3K) antiapoptotic pathway and a rapamycin-sensitive signalling pathway of mTOR. Here we demonstrate that the phosphorylation of 40S ribosomal protein S6, a physiological substrate p70 S6 kinase, was highly increased by growth-stimulation of the cytolytic T cells (CTLL2) with interleukin 2 (IL2), which was accompanied with the increased phosphorylation of p70 S6K. The activity of p70 S6K and phosphorylation of the S6 protein was completely blocked by rapamycin and significantly decreased upon treatment of the cells with wortmannin, indicating an involvement of the PI-3K pathway in concert with the signalling pathway of mTOR in IL2-dependent phos-phorylation of ribosomal protein S6. The phosphorylation and activity of PKB/Akt in IL2-stimulated CTLL2 cells were rapamycin-insensitive and reduced upon wortmannin treatment of the cells, confirming a requirement for PI-3K for Akt activity. The data support the hypothesis that Akt may act downstream to PI-3K and upstream to mTOR in an IL2-mediated signal transduction pathway that controls phosphorylation of the regulatory protein S6 in CTLL2 cells.     

Phosphorylation of ribosomal protein S6 and its regulation during differentiation of human leukemic cells.

We attempted to study the role of protein tyrosine kinase (PTK) and protein kinase C (PKC) in the cascade of phosphorylation of ribosomal protein S6 during differentiation of leukemic cells (HL-60, THP-1, and RWLeu-4). Neither activation nor inhibition of colony stimulating factor-1 (CSF-1) receptor''s PTK activity with CSF-1 or genistein respectively affected the phosphorylation of S6. However, vanadate which is a protein tyrosine phosphatase (PTP) inhibitor showed enhancement of S6 phosphorylation. Dimethylsulfoxide which does not affect either PTK or PKC demonstrated no change in S6 phosphorylation. PKC activation by acute 12-0-tetradecanoyl phorbol-13-acetate (TPA) treatment induced monocytic differentiation and S6 phosphorylation. Surprisingly, the more prominent phosphorylation of S6 protein was observed in PKC-depleted cells by prolonged TPA treatment. Our results suggest that PTK/PTP play a lesser role in S6 phosphorylation of HL-60 cells than PKC does. In addition, two different mechanisms seem to be involved in TPA-induced S6 phosphorylation during HL-60 differentiation: PKC activation by acute TPA treatment and PKC depletion which may lead to the synthesis of some endogenous protein responsible for the differentiation by chronic TPA treatment.     

Changes in protein synthesis and breakdown rates and responsiveness to growth factors with ageing in human lung fibroblasts

The effects of insulin, epidermal growth factor, insulin-like growth factor-2 and fetal bovine serum on protein synthesis and protein breakdown have been measured in mid-passage and senescent cultures of human diploid lung fibroblasts. Each of the individual growth factors was a potent inhibitor of protein breakdown with no difference in either the maximum response or sensitivity evident between senescent cells and mid-passage cultures. Binding of 125I-labelled epidermal growth factor per mg of cell protein similarly showed no difference between senescent and confluent mid-passage fibroblasts, although sparse mid-passage cells bound more of the ligand. These results indicate normal binding and normal responsiveness to growth factors in senescent cultures. However, rates of protein synthesis are higher in confluent mid-passage cells and especially so in sparse mid-passage cells than in senescent cells of intermediate density. Furthermore, senescent cells differ from either growth state of mid-passage cells because protein synthesis in aged cells is much less responsive to any of the growth factors or to fetal bovine serum. It is suggested from these results that the reduced ability of serum or growth factors to initiate DNA synthesis or growth in ageing cells may be a consequence of an unresponsive protein synthesis pathway rather than a generalised defect in growth factor action.     

Differential effects of resistance and endurance exercise in the fed state on signalling molecule phosphorylation and protein synthesis in human muscle

Resistance (RE) and endurance (EE) exercise stimulate mixed skeletal muscle protein synthesis. The phenotypes induced by RE (myofibrillar protein accretion) and EE (mitochondrial expansion) training must result from differential stimulation of myofibrillar and mitochondrial protein synthesis. We measured the synthetic rates of myofibrillar and mitochondrial proteins and the activation of signalling proteins (Akt–mTOR–p70S6K) at rest and after an acute bout of RE or EE in the untrained state and after 10 weeks of RE or EE training in young healthy men. While untrained, RE stimulated both myofibrillar and mitochondrial protein synthesis, 67% and 69% ( P < 0.02), respectively. After training, only myofibrillar protein synthesis increased with RE (36%, P = 0.05). EE stimulated mitochondrial protein synthesis in both the untrained, 154%, and trained, 105% (both P < 0.05), but not myofibrillar protein synthesis. Acute RE and EE increased the phosphorylation of proteins in the Akt–mTOR–p70S6K pathway with comparatively minor differences between two exercise stimuli. Phosphorylation of Akt–mTOR–p70S6K proteins was increased after 10 weeks of RE training but not by EE training. Chronic RE or EE training modifies the protein synthetic response of functional protein fractions, with a shift toward exercise phenotype-specific responses, without an obvious explanatory change in the phosphorylation of regulatory signalling pathway proteins.     

Receptor for epidermal growth factor retains normal structure and function in aging cells

Cellular responsiveness to epidermal growth factor (EGF) and the structure of the receptor for epidermal growth factor (EGF-R) were compared in young and senescent human fibroblast (HF) cells. Biosynthetic labeling of HF cells with [35S] methionine followed by immunoprecipitation with EGF-R antibody revealed the presence of Mr 170 000 EGF-R in cells from both stages. Autophosphorylation of EGF-R in response to EGF was identical in young and senescent cells. Phosphoamino acid analysis of the autophosphorylated EGF-R indicated that tyrosine residues were phosphorylated in each preparation. Two-dimensional peptide mapping of [125I]EGF-R from young and senescent cells showed essentially the same pattern, indicating that EGF-R does not apparently undergo detectable changes in senescent human fibroblasts. The responsiveness of aging HF cells to EGF for the induction of ornithine decarboxylase activity and for the production of secretory proteins was measured. Young and senescent HF cells showed about a three-fold induction of collagenase activity upon addition of EGF. Ornithine decarboxylase activity was also stimulated by EGF to a comparable level in young and senescent cells. Our results indicate that the responsiveness of HF cells to EGF for these two biochemical parameters does not decline with the loss of proliferative activity.     

Glutamate-dependent translational regulation in cultured Bergmann glia cells: involvement of p70S6K

Glutamate, the main excitatory amino acid transmitter in the vertebrate brain is involved in the dynamic changes in protein repertoire that underlie synaptic plasticity. Activity-dependent differential expression patterns occur not only in neurons but also in glial cells. In fact, a membrane to nuclei signaling has been described after ionotropic glutamate receptor stimulation in cultured chick cerebellar Bergmann glia cells. In order to characterize other levels of protein expression regulation, we explored the effect of glutamate treatment in [35S]-methionine incorporation into newly synthesized polypeptides. A time-dependent modification in protein synthesis was found. An important component of translational control is the ribosomal S6 protein kinase. Threonine phosphorylation renders the kinase active increasing translation initiation. Glutamate exposure results in ribosomal S6 protein kinase Thr389 phosphorylation in a dose and time-dependent manner that matches perfectly with the overall protein synthesis profile detected upon the excitatory amino acid. Pharmacological characterization of the receptors involved suggests the participation of both ionotropic as well as metabotropic glutamate receptors. The non-receptor tyrosine kinase Src, phosphatidylinositol 3-kinase, protein kinase B and the mammalian target of rapamycin are mediators of the glutamate effect. These results not only demonstrate that glutamate receptors activation is critically involved in translational control in glial cells adjacent to synaptic processes like cerebellar Bergmann glia cells, but also further strengthen the notion of an active participation of glial cells in synaptic transmission.     

The state of ribosomal protein phosphorylation during thioacetamide-induced liver injury

The development of thioacetamide (TAA)-induced liver injury is associated with a strong stimulation of phosphorylation of liver ribosomes. The phosphorylation of the 40 S ribosomal subunit was enhanced five- and ninefold 3 and 6 hr after the onset of injury, respectively. By two-dimensional gel electrophoresis and autoradiography it was shown that the increase was due to an eightfold-stimulated phosphorylation of protein S6 of the small ribosomal subunit. No significant change of the specific radioactivity of the 60 S subunit was detected. Two days after injection of TAA a tendency toward regeneration of normal (low phosphorylated) protein S6 was observed.     

Resistance exercise enhances mTOR and MAPK signalling in human muscle over that seen at rest after bolus protein ingestion

Aim: Feeding protein after resistance exercise enhances the magnitude and duration of myofibrillar protein synthesis (MPS) over that induced by feeding alone. We hypothesized that the underlying mechanism for this would be a greater and prolonged phosphorylation of signalling involved in protein translation. Methods: Seven healthy young males performed unilateral resistance exercise followed immediately by the ingestion of 25 g of whey protein to maximally stimulate MPS in a rested and exercised leg. Results: Phosphorylation of p70 ribosomal protein S6 kinase (p70S6K) was elevated (P < 0.05) above fasted at 1 h at rest whereas it was elevated at 1, 3 and 5 h after exercise with protein ingestion and displayed a similar post‐exercise time course to that shown by MPS. Extracellular regulated kinase1/2 (ERK1/2) and p90 ribosomal S6 kinase (p90RSK) phosphorylation were unaltered after protein ingestion at rest but were elevated (P < 0.05) above fasted early in recovery (1 h) and were greater for the exercised‐fed leg than feeding alone (main effect; P < 0.01). Eukaryotic elongation factor 2 (eEF2) phosphorylation was also less (main effect; P < 0.05) in the exercised‐fed leg than in the rested leg suggesting greater activity after exercise. Eukaryotic initiation 4E binding protein‐1 (4EBP‐1) phosphorylation was increased (P < 0.05) above fasted to the same extent in both conditions. Conclusion: Our data suggest that resistance exercise followed by protein feeding stimulates MPS over that induced by feeding alone in part by enhancing the phosphorylation of select proteins within the mammalian target of rapamycin (p70S6K, eEF2) and by activating proteins within the mitogen‐activated protein kinase (ERK1/2, p90RSK) signalling.     

Induction of host DNA synthesis in senescent human diploid fibroblasts by infection with human cytomegalovirus

A human diploid fibroblast strain, TIG -1, ceased to proliferate at about 60-62 population doubling level. The percentage of nuclei incorporating [3H]thymidine during 24-h culture in fresh medium containing 10% fetal bovine serum was less than 2% in the senescent cells used in this study. Infection of these cells with human cytomegalovirus (HCMV), strain AD-169, increased the percentage of [3H]thymidine-labeled cells by about ten-fold. Equilibrium density gradient centrifugation analysis of purified DNA from infected cells showed that cellular DNA synthesis was stimulated preceded by the viral DNA synthesis. Ultraviolet irradiation of HCMV reduced the ability to induce DNA synthesis. Equilibrium density gradient centrifugation analysis of DNA which was labeled with 5-bromodeoxyuridine indicated semiconservative replication rather than repair synthesis. These results suggested that in a considerable fraction of human senescent cells host DNA replication could be reinitiated by infection with HCMV, but not by the addition of fetal bovine serum.     

Phosphorylation of a ribosomal protein and of virus-specific proteins in cells infected with herpes simplex virus.

In cells infected with herpes simplex virus a protein associated with the small subunit of ribosomes became phosphorylated. It was not detectably labelled with 14C-amino acids added after infection and is therefore probably a cellular protein. The phosphorylated ribosomal proteins from HSV-1- and HSV-2-infected cells were indistinguishable electrophoretically and had an apparent mol. wt. of about 48 000. Phosphorylation of the 48K protein was detected 2 to 3 h after infection and reached a maximum rate at 4 to 5 h. It was prevented by adding cycloheximide at 2 h, or actinomycin at 1.5 h p.i., or azetidine at the beginning of infection. The phosphorylation did not occur on reversal of a cycloheximide block in the presence of actinomycin, confirming that it is not caused by a virus alpha-polypeptide. Virus that had been irradiated with u.v. light, although still able to suppress synthesis of cellular protein and DNA, did not induce phosphorylation of the 48K ribosomal protein. Therefore the phosphorylation is not responsible for the suppression of host synthesis. The alpha polypeptides ICP 4, 0, 22 and 27 are also phosphorylated but, in contrast to that of the ribosomal protein, their phosphorylation does not depend on the synthesis of beta and gamma polypeptides. It is probably mediated by a host enzyme.     

Loss of responsiveness in senescent human TIG-1 cells to the DNA synthesis-inducing effect of various growth factors

Responses of human diploid cells, TIG-1, were examined with respect to their ability to initiate DNA synthesis under the influence of various growth factors and their combinations. The following agents stimulated DNA synthesis in quiescent TIG-1 cells at 37-49 PDL (population doubling level) (66-79% of lifespan completed): fetal bovine serum; tumor-derived DNA synthesis factors such as those from rat rhodamine fibrosarcoma, human adenoma and from the conditioned medium of cultured human pituitary cells; human and mouse epidermal growth factors; tumor promotors such as 12-O-tetradecanoylphorbol 13-acetate and teleocidin; microtubule-disrupting agents as colchicine, vinblastine, podophyllotoxin and TN-16; melittin; and dexamethasone. Cells at 58-60 PDL (94-97% of lifespan completed) were stimulated to synthesize DNA by fetal bovine serum, tumor-derived DNA synthesis factors and epidermal growth factors, but not by other agents. Finally, in senescent cells at 62 PDL (100% of lifespan completed), any of these growth factors and of their combinations failed to induce DNA synthesis at all. These senescent cells, however, still retained the ability to initiate DNA synthesis following infection with SV40 as reported previously [Exp. Cell Res., 143 (1983) 343-349].     

Mechanism of interferon action. Kinetics of interferon action in mouse L929 cells: translation inhibition, protein phosphorylation, and messenger RNA methylation and degradation

The effect of the duration of interferon treatment of mouse L929 fibroblasts on the expression of ribosome-associated and cell-sap-localized translation inhibition, protein phosphorylation, and messenger RNA (mRNA) methylation and degradation was investigated. The following results were obtained: (1) Ribosomal salt-wash fractions prepared from L929 cells treated for 12 or 18 hr significantly inhibited the translation of methylated reovirus mRNA catalyzed by the cell-free system prepared from untreated ascites tumor cells. The translation of reovirus mRNA was slightly stimulated by the ribosomal salt-wash fractions prepared from untreated cells and cells treated for 2 hr and was slightly inhibited by the salt-wash fraction from cells treated for 6 hr. (2) Cell-sap fractions prepared from untreated cells and from cells treated for 2 or 6 hr did not appreciably affect the translation of reovirus mRNA in the absence of double-stranded RNA (dsRNA); cell-sap fractions prepared from cells treated for 12 or 18 hr were slightly inhibitory. (3) Neither reovirus genome dsRNA nor polyriboinosinic acid-polyribocytidylic acid inhibited the translation of reovirus mRNA by the untreated ascites system. The inhibitory activity of ribosomal salt-wash fractions from interferon-treated cells was dsRNA independent, whereas dsRNA enhanced the inhibitory activity of cell-sap fractions from interferon-treated cells. (4) Interferon treatment significantly enhanced a cell-sap-independent phosphorylation of at least three proteins present in the ribosomal salt-wash fractions. Phosphorylation of an approximately 50,000-dalton component was maximally enhanced after 12 hr of interferon treatment and was increased by dsRNA; phosphorylation of 30,000- and <20,000-dalton components was enhanced earlier and was dsRNA independent. (5) An increase in the nuclease-mediated degradation of [3H]uridine-labeled methylated reovirus mRNA catalyzed by ribosomal salt-wash fractions was detectable after 6 hr of interferon treatment and was maximally enhanced after 12 hr of interferon treatment to about threefold the level of the untreated ribosomal salt-wash fraction. The degradation was not enhanced by dsRNA. The levels of reovirus mRNA degradation catalyzed by cell-sap fractions from untreated cells and from cells treated with interferon for 2 to 18 hr were comparable. (6) No significant difference was observed in the ability of cell-sap fractions from untreated cells or from cells treated with interferon for 2 to 18 hr to catalyze the methylation of unmethylated reovirus mRNA. In addition, none of the ribosomal salt-wash fractions significantly inhibited reovirus mRNA methylation catalyzed by untreated ascites cell-free extracts. These results suggest that interferon treatment may mediate multiple biochemical changes in murine cells, including the specific phosphorylation of protein(s) associated with ribosomes that possess interferon-mediated inhibitor(s) of viral mRNA translation.     

Analysis of UV-induced damage and repair in young and senescent human dermal fibroblasts using the comet assay

A major cause of ageing is thought to be the accumulation of damage to macromolecules. Accumulation to DNA damage in cells therefore presupposes that aged cells are unable to repair this damage. We have used the in vitro model of cellular ageing to test the idea that senescent cells are deficient in some aspect of DNA repair. Using the alkaline single cell gel electrophoresis assay (comet assay), we have determined the responses of young and senescent human dermal fibroblasts to DNA damage caused by exposure to UVC light. At low doses of UVC, senescent cells generate smaller comets than young cells whilst at medium doses the situation is reversed. At high doses, young and senescent cells respond similarly to one another. Time course experiments revealing repair of DNA damage show that senescent cells generate larger comets than young cells at early stages of repair suggesting that either senescent cells bear more damage per genome than do young cells or that senescent cells are more efficient at excising bulky adducts from DNA. Cells maintained in low levels of serum irrespective of age are less able to repair DNA damage compared with cells maintained in high levels of serum, and furthermore young and senescent cells maintained in high levels of serum are equally able to repair DNA damage. Our data, therefore, reveal both age-dependent and age-independent responses to UV-induced DNA damage. Use of the comet assay highlights the heterogeneity of cellular responses to genotoxic stress.     

Endotoxicosis induced by Coxiella burnetii lipopolysaccharide stimulates a ribosomal protein S6 kinase: some properties of the partially purified enzyme.

Guinea pig endotoxicosis induced by lipopolysaccharide from Coxiella burnetii Nine Mile phase I stimulates phosphorylation of liver ribosomal protein S6, with a 50% increase at 12 h postinoculation. The responsible protein kinase (S6PK) has been partially purified from liver; its activity is independent of cyclic AMP and of Ca2+ plus phosphatidyl serine or diacylglycerol. The preparation has an apparent optimum concentration of 20 mM Mg2+, while Ca2+ and Mn2+ are each inhibitory at 2 mM. The apparent Km for ATP is 30 microM with intact ribosomes. Because of the central role of phosphorylation in metabolic regulation and a purported role of phosphorylated S6 in protein synthesis, the lipopolysaccharide-induced stimulation of S6PK suggests a significant regulatory role of such enzymes in the pathobiochemistry of Q fever infection and endotoxicosis.     

Immunochemical specificity of cytoplasmic inclusions induced by viruses in the potato Y group

Cultured tomato root tips were infected with tobacco mosaic virus (TMV). The synthesis of TMV-RNA and ribosomal RNA was measured in healthy and TMV-infected roots by ³²P labeling and polyacrylamide gel electrophoresis. The region of most active TMV-RNA synthesis was 20–60 mm behind the tip. TMV-RNA was just over 4% of the total nucleic acid present in the entire infected root culture. Incorporation of ³²P into ribosomal RNA was greatly stimulated by TMV infection. This is discussed in relation to the increased size of the ³²P pool in roots infected by TMV: it is concluded that TMV infection stimulates ribosomal RNA synthesis. The region of the infected root showing high stimulation of ribosomal RNA synthesis coincided with the area of high TMV-RNA synthesis.