Hdm2- and proteasome-dependent turnover limits p21 accumulation during S phase

Double-strand DNA breaks detected in different phases of the cell cycle induce molecularly distinct checkpoints downstream of the ATM kinase. p53 is known to induce arrest of cells in G₁ and occasionally G₂ phase but not S phase following ionizing radiation, a time at which the MRN complex and cdc25-dependent mechanisms induce arrest. Our understanding of how cell cycle phase modulates pathway choice and the reasons certain pathways might be favored at different times is limited. In this report, we examined how cell cycle phase affects the activation of the p53 checkpoint and its ability to induce accumulation of the cdk2 inhibitor p21. Using flow cytometric tools and centrifugal elutriation, we found that the p53 response to ionizing radiation is largely intact in all phases of the cell cycle; however, the accumulation of p21 protein is limited to the G₁ and G₂ phase of the cell cycle because of the activity of a proteasome-dependent p21 turnover pathway in S-phase cells. We found that the turnover of p21 was independent of the SCF^skp2 E3 ligase but could be inhibited, at least in part, by reducing hdm2, although this depended on the cell type studied. Our results suggest that there are several redundant pathways active in S-phase cells that can prevent the accumulation of p21.     

Phase-Specific Polypeptides and Poly(A) RNAs during the Cell Cycle in Synchronous Cultures of Catharanthus roseus Cells

This study shows an overall analysis of gene expression during the cell cycle in synchronous suspension cultures of Catharanthus roseus cells. First, the cellular cytoplasmic proteins were fractionated by two-dimensional gel electrophoresis and visualized by staining with silver. Seventeen polypeptides showed qualitative or quantitative changes during the cell cycle. Second, the rates of synthesis of cytoplasmic proteins were also investigated by autoradiography by labeling cells with [³⁵S]methionine at each phase of the cell cycle. The rates of synthesis of 13 polypeptides were found to vary during the cell cycle. The silverstained electrophoretic pattern of proteins in the G₂ phase in particular showed characteristic changes in levels of polypeptides, while the rates of synthesis of polypeptides synthesized during the G₂ phase did not show such phase-specific changes. This result suggests that posttranslational processing of polypeptides occurs during or prior to the G₂ phase. In the G₁ and S phases and during cytokinesis, several other polypeptides were specifically synthesized. Finally, the variation of mRNAs was analyzed from the autoradiograms of in vitro translation products of poly(A)⁺ RNA isolated at each phase. Three poly(A)⁺ RNAs increased in amount from the G₁ to the S phase and one poly (A)⁺ RNA increased preferentially from the G₂ phase to cytokinesis.     

The synthesis of acidic chromosomal proteins during the cell cycle of HeLa S-3 cells. II. The kinetics of residual protein synthesis and transport

The kinetics of acidic residual chromosomal protein synthesis and transport were studied throughout the cell cycle in HeLa S-3 cells synchronized by 2 mM thymidine block and selective detachment of mitotic cells. Pulse labeling the cells with leucine-³H for 2 min and then "chasing" the radioactive proteins for up to 3 hr showed that the amount of protein synthesized, transported, and retained in the acidic residual chromosomal protein fraction is greater immediately after mitosis and later in G₁ than in the S or G₂ phases of the cell cycle. During S, only 20–25% of the proteins synthesized and transported to the acidic residual chromosomal protein fraction are chased during the first 2 hr after pulse labeling, whereas up to 40% of the material entering the residual nuclear fraction in mitosis, G₁, and G₂ leaves during a 2 hr chase. Polyacrylamide gel electrophoretic profiles of these proteins, at various times after pulse labeling, reveal that the turnover of individual polypeptides within this fraction has kinetics of synthesis and turnover which are markedly different from one another and undergo stage-specific changes.     

Inference of cell cycle-dependent proteolysis by laser scanning cytometry

Mechanisms that couple protein turnover to cell cycle progression are critical for coordinating the events of cell duplication and division. Despite the importance of cell cycle-regulated proteolysis, however, technologies to measure this phenomenon are limited, and typically involve monitoring cells that are released back into the cell cycle after synchronization. We describe here the use of laser scanning cytometry (LSC), a technical merger between fluorescence microscopy and flow cytometry, to determine cell cycle-dependent changes in protein stability in unperturbed, asynchronous, cultures of mammalian cells. In this method, the ability of the LSC to accurately measure whole cell fluorescence is employed, together with RNA fluorescence in situ hybridization and immunofluorescence, to relate abundance of a particular RNA and protein in a cell to its point at the cell cycle. Parallel monitoring of RNA and protein levels is used, together with protein synthesis inhibitors, to reveal cell cycle-specific changes in protein turnover. We demonstrate the viability of this method by analyzing the proteolysis of two prominent human oncoproteins, Myc and Cyclin E, and argue that this LSC-based approach offers several practical advantages over traditional cell synchronization methods.     

Mitochondrial protein synthesis in chinese hamster cells (line CHO) synchronized by isoleucine deprivation

Mitochondrial protein synthesis was measured in line CHO cells after phases of the cell cycle were synchronized by isoleucine deprivation or mitotic selection. Maximum incorporation of [³H] leucine into mitochondrial polypeptides occurred within 2 hours after isoleucine was added to initiate G₁ traverse. In cells synchronized in G₁ by mitotic selection, the rate of mitochondrial protein synthesis was fairly constant throughout the cell cycle. SDS-polyacrylamide gel electrophoretic profiles of labeled mitochondrial polypeptides were similar in cells synchronized by either isoleucine deprivation or mitotic selection. Obvious changes in the distribution of polypeptides were not detected during various phases of the cell cycle. The increased rate of incorporation of [³H] leucine into mitochondrial polypeptides after reversal of G₁-arrest may indicate that mitochondrial protein synthesis and possibly mitochondrial biogenesis are synchronized in CHO cells deprived of isoleucine.     

High-resolution two-dimensional electrophoretic analysis of acidic,basic and surface proteins of mouse neutrophils

The proteins from murine neutrophils have been examined using isoelectric focusing and non-equilibrium pH gradient electrophoresis in the first dimension and sodium dodecyl sulfate-polyacrylamide electrophoresis as a second dimension. The major protein, actin, dominates the protein profiles and it appears to be one of the few proteins being synthesised rapidly. In the presence of protease inhibitors, neutrophil (a homogeneous, non-dividing cell population) lysates gave extremely reproducible two-dimensional electrophoretic patterns both with Coomassie blue staining (approx. 200 proteins detected) and with fluorography or autoradiography after [³⁵S]methionine biosynthetic labelling (approx. 450 proteins detected between pH 4 and 7). Biosynthetic labelling was more sensitive than protein staining for some components, although the mature neutrophils did not synthesis certain cellular proteins (e.g., granule proteins such as lactoferrin). Surface labelling of neutrophils (as indicated by the absence of ¹²⁵I associated with actin) yielded more than 20 major ¹²⁵I-labelled proteins on high-resolution electrophoretic maps. The major ¹²⁵I-labelled protein (M_r ≈ 90 kdalton) focused at the acidic end of the gels near pH 4.1. This protein could also be detected after [³⁵S]methionine biosynthetic labelling. All of the high molecular weight components focused over a broad pH range (0.2 pH units). At lease one of the surface components appeared to consist of several discrete charge entities.     

Studies on the turnover of plasma membranes in cultured mammalian cells.: II. Demonstration of heterogeneous rates of turnover for plasma membrane proteins and glycoproteins

The relative rate of turnover of individual membrane proteins and glycoproteins in exponentially growing and contact-inhibited MK₂ cells was investigated. Plasma membranes were isolated from cells that had been sequentially labelled with ¹⁴C and ³H isotopes of leucine and glucosamine. The membranes were then solubilized in sodium dodecylsulfate and their polypeptides separated by acrylamide gel electrophoresis. The ³H/¹⁴C ratios of the individual polypeptides reflected their relative rates of turnover. The proteins and glycoproteins of the exponentially growing cells exhibited markedly heterogeneous rates of turnover. In contrast, polypeptides in membranes of contact-inhibited cells exhibited a lesser degree of heterogeneity of turnover. In both exponential and contacted cell membranes a glycoprotein with a high apparent molecular weight exhibited the fastest rate of turnover.     

Modulation of synthesis of specific proteins in endothelial cells by copper,cadmium, and disulfiram: An early response to an angiogenic inducer of cell migration

Copper, cadmium, and disulfiram (an ionophore for copper) modulate the synthesis of several polypeptides in two clonal lines of bovine aortal endothelial cells. After treatment of type 1 endothelial cells with 10^?3 M CuSO₄ or 10^?5 M CdCl₂ four cell-associated polypeptides (M_r = 28,000, 32,000, 73,000, and 83,000 daltons) were induced. In contrast, in Type 2 endothelial cells, which have cultural characteristics distinct from Type 1, only one new cell-associated protein (M_r = 32,000 and 40,000 daltons) was induced. Other differences are revealed by analyses of proteins secreted into the growth medium. In particular low levels of only CuSO₄ (10^?6 M) enhanced the synthesis in Type 2 cells of a protein (M_r = 220,000 daltons) identified as fibronectin. Since only copper ions induced fibronectin, we propose that the mechanism of induction of fibronectin synthesis, in contrast to the induction of cell?associated polypeptides, does not involve a sulphydryl?containing receptor molecule. It is suggested that the specific enhancement of fibronectin synthesis by copper ions may be a controlling event in the stimulation by copper ions of endothelial cell migration and angiogenesis.     

Envelope synthesis during the cell cycle in Escherichia coli B/r

The rate of synthesis of envelope proteins and phospholipids during the cell cycle of Escherichia coli B/r has been studied using both synchronous cultures and random cultures, first labelled and then subsequently fractionated on an age basis by the membrane elution technique. The rate of total protein synthesis and of phospholipid synthesis, measured by incorporation of [2-³H]glycerol into whole cells, was found to increase exponentially throughout the cell cycle. Total envelope protein was also synthesized continuously throughout the cycle, but the rate of synthesis showed a stepwise pattern with a discrete doubling in rate in the first half of the cycle. Analysis of the pattern of synthesis of about 29 individual envelope polypeptides by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and autoradiography revealed that the great majority followed the pattern of the bulk measurements, with a discrete increase in rate of synthesis early in the cycle. One envelope polypeptide, molecular weight 76,000, was, however, only synthesized during a brief period, near the time of division of the bacteria. Pulse-chase studies of envelope polypeptide synthesis in synchronous cultures demonstrated that (1) synthesis and insertion of polypeptide into the envelope was always completed within the pulse period; (2) no post-synthetic modification of polypeptides was detected; (3) one group of polypeptides, including a major outer membrane protein, maintained a stable association with the envelope, whilst a second group displayed considerable “turnover”; (4) about 70% of newly synthesized 76,000 molecular weight protein was lost from the envelope during the succeeding generation.     

The program of protein synthesis during heterocyst differentiation in nitrogen-fixing blue-green algae

The program of protein synthesis that accompanies cellular differentiation following transfer of the blue-green alga Nostoc muscorum from nitrogen-containing to nitrogen-free medium has been determined by polyacrylamide gel electrophoresis of whole cell proteins labeled with ³⁵SO₄⁼ during successive intervals of the differentiation. Differentiating cells (proheterocysts, which become heterocysts) are distinguished from vegetative cells on the basis of the latter's susceptibility to lysis with lysozyme.At least ten sets of proteins can be distinguished on the basis of the time at which they are synthesized or the type of cell in which they are located. Regulation of most of these sets can be accounted for by classical induction or repression involving NH₄⁺ or a simple derivative of NH₄⁺. An additional mechanism is required to explain how the synthesis of several sets of proteins is initiated in all cells following transfer to nitrogen-free medium, but is permitted to continue only in developing proheterocysts. The structural polypeptides of the nitrogenase enzyme complex are members of the latter set.In differentiated filaments, very few proteins are synthesized in both vegetative cells and heterocysts. The qualitatively different pattern of protein synthesis is established very early, within the first 9 hr after transfer. Moreover, the proteins present in proheterocysts at that time are already qualitatively different from those of vegetative cells. Rapid turnover of vegetative cell proteins appears to be a characteristic of the early development of proheterocysts.     

Mathematical model analysis of mouse epidermal cell kinetics measured by bivariate DNA/anti-bromodeoxyuridine flow cytometry and continuous [3H]-thymidine labelling

Abstract. In a previous study the epidermal cell kinetics of hairless mice were investigated with bivariate DNA/anti-bromodeoxyuridine (BrdU) flow cytometry of isolated basal cells after BrdU pulse labelling. The results confirmed our previous observations of two kinetically distinct sub-populations in the G₂ phase. However, the results also showed that almost all BrdU-positive cells had left S phase 6–12 h after pulse labelling, contradicting our previous assumption of a distinct, slowly cycling, major sub-population in S phase. The latter study was based on an experiment combining continuous tritiated thymidine ([³H]TdR) labelling and cell sorting. The purpose of the present study was to use a mathematical model to analyse epidermal cell kinetics by simulating bivariate DNA/BrdU data in order to get more details about the kinetic organization and cell cycle parameter values. We also wanted to re-evaluate our assumption of slowly cycling cells in S phase. The mathematical model shows a good fit to the experimental BrdU data initiated either at 08.00 hours or 20.00 hours. Simultaneously, it was also possible to obtain a good fit to our previous continuous labelling data without including a sub-population of slowly cycling cells in S phase. This was achieved by improving the way in which the continuous [³H]TdR labelling was simulated. The presence of two distinct sub-populations in G₂ phase was confirmed and a similar kinetic organization with rapidly and slowly cycling cells in G₁ phase is suggested. The sizes of the slowly cycling fractions in G₁ and G₂ showed the same distinct circadian dependency. The model analysis indicates that a small fraction of BrdU labelled cells (3–5%) was arrested in G₂ phase due to BrdU toxicity. This is insignificant compared with the total number of labelled cells and has a negligible effect on the average cell cycle data. However, it comprises 1/3 to 1/2 of the BrdU positive G₂ cells after the pulse labelled cells have been distributed among the cell cycle compartments.     

The role of p38 kinase in the activation of the premature senescence program in transformed mouse fibroblasts

The role of p38α stress-kinase in the regulation of the premature senescence program induced by the histone deacetylase inhibitor sodium butyrate (NaB) was studied in rodent transformed cell lines. The study was carried out on E1A+cHa-ras transformants obtained from mouse embryonic fibroblasts null for the Mapk14 gene encoding p38α stress-kinase (the mERasp38^?/? cell line), or for the PPM1D gene encoding the Wip1 phosphatase (the mERas Wip1^?/? cell line), whose absence led to constitutive activation of p38α kinase. It was found that after NaB treatment both cell lines completely stopped proliferation due to irreversible G₁/S cell cycle arrest. In both lines a marker of senescence appeared—the activity of β-galactosidase (SA-β-Gal). As well, treatment of the cells with NaB for several days led to morphological cell changes, such as partial readjustment of the actin cytoskeleton, spreading on the substrate, and heterochromatin focus formation (SAHF) in the senescent cell nuclei. These data allow us to suggest that, in the absence of functionally active p38α kinase, the NaB-induced irreversible process of cellular senescence may occur via alternative pathways for downregulation of the cell cycle.     

Cell kinetic studies on the JB-1 ascites tumour of the mouse

Abstract. The FLM method, modified by double labelling with [³H]- and [¹⁴C]-thymidine, has been applied to the 4-day old JB-1 ascites tumour of the mouse. It results in well separated waves of purely [³H]- and purely [¹⁴C]-labelled mitoses, which show a remarkable asymmetry with long tails to the right. The following values for the mean transit times of the cells have been derived from this FLM curve, for a tumour age of 4–6 days: T_C= 32.5 hr, T_S= 16.7 hr, T_G1= 3.7 hr, T_G1= 11.0 hr and T_M= 1.1 hr. A further evaluation of the FLM curve, however, is difficult, due to the non-stationary growth of the tumour. A number of other experimental findings (growth curve, decrease of the labelling and mitotic index with increasing tumour age, two single-labelled FLM curves starting 4 and 6 days after tumour inoculation) indicate that the cell cycle time increases during the experimental period of the double-labelled FLM curve (about 2 days). A lengthening of the cycle time should result in an increasing enlargement of the areas under the waves of the modified FLM curve. However, such an increase in area has not been found; the areas are constant. All the results of the present cell kinetic studies would be consistent if it were postulated that the cell cycle time lengthens with increasing tumour age up to about 4 days after inoculation, then remains relatively constant at between 4 and 6 days and thereafter increases again. Short-term double labelling experiments suggest that this is actually the case. Under the assumption of nearly constant phase durations during the 5th and 6th day of tumour growth further conclusions can be drawn from the modified FLM curve. In particular, it follows that the transit times of the cells through successive cycle phases are uncorrelated and the variances of the transit times through a cycle phase are proportional to the duration of this phase.     

Turnover of plasma membrane polypeptides in nonproliferating cultures of Chinese hamster ovary cells and human skin fibroblasts.

When cultures of Chinese hamster ovary cells were maintained in stationary phase on medium deficient in l-isoleucine (A) or low in serum (B), active protein turnover occurs. These cells can be acetylated with trace levels of radioactive acetic anhydride in order to incorporate label into all of the major species of polypeptides of the plasma membrane. Four days following acetylation with [³H]acetic anhydride and removal from medium A containing l-[¹⁴C]leucine, the specific ³H and ¹⁴C radioactivities of the plasma membrane proteins had fallen 15- and 7-fold respectively. The lower value obtained with the radioactive leucine is probably due to reutilization of this amino acid. The ³H and ¹⁴C radioactivity profiles for the polypeptides separated by discontinuous gel electrophoresis, however, showed little qualitative change over the course of the experiment, suggesting that differential rates of protein turnover were not occurring. These results were confirmed in experiments with cells using both the above culture conditions in which two acetylations were carried out, one with ³H at time zero and the other with contrasting ³C label up to 96 h later. Two methods for plasma membrane isolation and a number of electrophoretic conditions were employed. Again, however, the radioactivity profiles along the gels coincided almost exactly, even though the ³H specific radioactivity had fallen several fold. Similar results have been obtained with confluent human skin fibroblasts. We suggest that the major proteins in the plasma membranes of cultured mammalian cells do not show markedly heterogeneous rates of turnover. In particular, larger species of polypeptides do not appear to have shorter half-lives than smaller ones.     

Analyzing folding and degradation of metabolically labelled polypeptides by conventional and diagonal sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Efficient protein folding and quality control are essential for unperturbed cell viability. Defects in these processes may lead to production of aberrant polypeptides that are either degraded leading to “loss-of-function” phenotypes, or deposited in or outside cells leading to “gain-of-toxic-function” phenotypes. Elucidation of molecular mechanisms regulating folding and quality control of newly synthesized polypeptides is therefore of greatest interest. Here we describe protocols for metabolic labelling of transfected/infected mammalian cells with [³⁵S]-methionine and [³⁵S]-cysteine, for immunoisolation from detergent extracts of the selected model proteins and for the investigation of the model polypeptide’s intracellular fate in response to chaperone-deletions or to cell exposure to folding or degradation inhibitors.     

Patterns of post-infectional protein synthesis in barley carrying different genes for resistance to the powdery mildew fungus

Summary Pairs of susceptible and resistant, near-isogenic cultivars ofHordeum vulgare which differ for the Mla, Mlk and Mlp genes for resistance toErysiphe graminis f. sp.hordei were inoculated with race 3 of this pathogen and patterns of protein synthesis associated with primary infection mapped using pulse-labelling with L-[³⁵S]methionine and 2-dimensional electrophoresis. Extraction of proteins with buffer containing detergent revealed the enhanced synthesis of 5 and 8 polypeptides at 25 and 30 h respectively after inoculation of barley carrying the Mla gene (cvMla). The enhanced synthesis of these same polypeptides together with 11 additional polypeptides was observed at 48 h and 72 h after inoculation of barley carrying either the Mlp (cvMlp) or Mlk (cvMlk) genes. The labelling of several major constitutive polypeptides was suppressed in cvMla at 24 h after inoculation; the labelling of six of these polypeptides was also suppressed in both cvMlp and cvMlk but not until 48 and 72 h after inoculation. These results indicate that changes occur in the synthesis of some common polypeptides following infection of cultivars carrying different resistance genes but the timing and extent of these changes varies with the resistance gene in the host.     

Impact of oxidative stress on the function,abundance, and turnover of the Arabidopsis 80S cytosolic ribosome

Abiotic stress in plants causes accumulation of reactive oxygen species (ROS) leading to the need for new protein synthesis to defend against ROS and to replace existing proteins that are damaged by oxidation. Functional plant ribosomes are critical for these activities, however we know little about the impact of oxidative stress on plant ribosome abundance, turnover, and function. Using Arabidopsis cell culture as a model system, we induced oxidative stress using 1 µm of H₂O₂ or 5 µm menadione to more than halve cell growth rate and limit total protein content. We show that ribosome content on a total cell protein basis decreased in oxidatively stressed cells. However, overall protein synthesis rates on a ribosome abundance basis showed the resident ribosomes retained their function in oxidatively stressed cells. ¹⁵N progressive labelling was used to calculate the rate of ribosome synthesis and degradation to track the fate of 62 r‐proteins. The degradation rates and the synthesis rates of most r‐proteins slowed following oxidative stress leading to an ageing population of ribosomes in stressed cells. However, there were exceptions to this trend; r‐protein RPS14C doubled its degradation rate in both oxidative treatments. Overall, we show that ribosome abundance decreases and their age increases with oxidative stress in line with loss of cell growth rate and total cellular protein amount, but ribosome function of the ageing ribosomes appeared to be maintained concomittently with differences in the turnover rate and abundance of specific ribosomal proteins. Data are available via ProteomeXchange with identifier PXD012840.     

Thylakoid membrane protein phosphorylation in correlation with photosynthetic membrane activation

The phosphorylation of five $\text{E.}\text{gracilis}$ thylakoid membrane polypeptides was studied, in isolated chloroplasts. Using [³²P] labelling, in the light, we found that phosphorylation was inhibited by ethanol and DCMU. Inhibition curves were characteristic of photosynthetic inhibition. [γ-³²P] ATP labelling was used to distinguish between two groups of phosphoproteins: the first one, includes protein I, II, V which require only ATP for phosphorylation while the second one includes protein III and IV whose phosphorylation is light-requiring. Phosphorylation of protein III and IV was inhibited by CCCP, NH₄Cl and DCMU, and was reversible in the dark.