The platelet-derived growth factor

Formation of MacroH2A-Containing Senescence-Associated Heterochromatin Foci and Senescence Driven by ASF1a and HIRA

1
Protein degradation in isolated rat hepatocytes was measured as the release of [14C]valine from pre-labelled protein. To reduce background radioactivity, the intracellular [14C]valine pool was depleted by serial extraction at 37 °C, effecting equilibration between the intracellular pool and the valine-free extracellular medium. After extraction, a small, non-equilibrating intracellular [14C]valine pool remained; this pool could only be labelled in the presence of ongoing protein synthesis, and might represent valine and valine-containing oligopeptides derived from protein degradation.

2
The [14C]valine released from degraded protein was not significantly re-utilized for protein synthesis intracellularly (no effect of cycloheximide or high concentrations of unlabelled valine), reflecting the low rate of protein synthesis and the rapid transport of valine into the extracellular medium, both characteristic of isolated hepatocytes.

3
From cells pre-labelled for 24 h in vivo, [14C]valine was released linearly at a rate of 5%/h, probably representing the true over-all protein degradation rate. The lysosomotropic inhibitor ammonia (10 mM NH4Cl) inhibited 70% of the degradation, presumably the contribution by the lysosomal pathway.

From 1-h pre-labelled cells, [14C]valine was released at a declining rate, and ammonia inhibited degradation only by 45%, consistent with the view that the majority of short-lived proteins are degraded by the non-lysosomal pathway(s).

4
Chloroquine and methylamine, which accumulate in lysosomes by virtue of their weak base properties, inhibited hepatocytic protein degradation to the same extent as ammonia, with no additivity. These compounds therefore seem to block the lysosomal pathway of protein degradation selectively and completely. Leupeptin, which binds to and inhibits the activity of certain lysosomal proteases, also inhibited protein degradation almost to the same extent as ammonia, but with a small part of the effect (< 20%) being additive to the NH3 effect and thus probably reflecting a slight inhibition of non-lysosomal protein degradation as well.

5
Of the four inhibitors tested, only the effect of ammonia was rapidly reversible within the experimental period (2 h). Leupeptin, on the other hand, was the only degradation inhibitor which did not also affect protein synthesis. Chloroquine caused significant cell death at concentrations above 0.2 mmol/l in this protein-free medium, i.e. in the concentration range needed for maximal inhibition of protein degradation.

6
Incubation of hepatocytes under anoxic conditions resulted in an inhibition of protein degradation which was greater than, and partially additive to, the effect of ammonia, i.e. most of the degradation by the lysosomal pathway and more than one-half of the degradation by the non-lysosomal pathways appears to be energy-dependent.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1979.tb12956.x

Inhibition of the Lysosomal Pathway of Protein Degradation in Isolated Rat Hepatocytes by Ammonia, Methylamine, Chloroquine and Leupeptin

Kinetic analysis of cellular response to serum deprivation or inhibition of protein synthesis was performed on Swiss 3T3 cells Time-lapse cinematographic analysis of individual cells transiently exposed to serum-free medium (with or without the addition of purified growth factors) or cycloheximide enabled a detailed mapping of the magnitude and variability of cellular response in different parts of the cell cycle In all cells, in all stages of the cell cycle, serum deprivation resulted in inhibition of protein synthesis, but only in postmitotic cells in the first 3-4 hr of G1 (here denoted the G1pm phase) did it produce cell-cycle arrest During G1pm, the cells are highly dependent on the continuous presence of serum growth factors and a high level of protein synthesis in order to progress toward mitosis A 1-hr exposure to serum-free medium or to cycloheximide was sufficient to force most G1pm cells into a state of quiescence (G0), from which the cells required 8 hr to return to G1pm During G1pm the cells complete the growth factor-dependent processes leading to commitment for proliferation Thereafter they enter the growth factor-independent pre-DNA-synthetic part of G1 (here denoted G1ps) The commitment process in G1pm could be successfully completed in the presence of platelet-derived growth factor as the only supplied growth factor Epidermal growth factor and insulin were insufficient for the completion of the commitment processes in G1pm, although they were able to temporarily prevent the G1pm cells from entering G0 during serum starvation Under conditions optimal for proliferation, the cells complete the commitment processes in G1pm within a remarkably constant time period Almost all cells in the population left G1pm and entered G1ps between the third and fourth hour after mitosis The duration of G1ps, on the other hand, showed a large intercellular variability consistent with a transition-probability event In fact, G1ps accounts for most of the variability in G1 and cell cycle time

https://www.pnas.org/content/pnas/82/16/5365.full.pdf

Kinetic analysis of regulatory events in G1 leading to proliferation or quiescence of Swiss 3T3 cells

Simultaneous measurements of cellular DNA and RNA as well as estimates of the sensitivity of DNA in situ to denaturation by acid (which correlates with the degree of chromatin condensation) and cell ability to incorporate 5-bromodeoxyuridine (BUdR), performed on over 40 different cell systems enabled us to subclassify cells into 12 functionally distinct cell cycle compartments. Quiescent cells had low RNA values, DNA very sensitive to denaturation, and they did not incorporate BUdR. Although in most cell systems they had 2C DNA content (G1Q), quiescent cells with higher DNA values (SQ and G2Q) were also seen. The G1 phase of exponentially growing cells had two distinct compartments, A and B. G1 cells entered S phase only from the B compartment. An increase in RNA and a decrease in the sensitivity of DNA to acid denaturation beyond a specific level characterized the transition of cells from the A to B compartments. Since the G1A to G1B transition was not linear but exponential, it may be assumed that a non-deterministic event triggering cell progression into the cycle resides within G1A. Under adverse growth conditions the probability of a G1A to G1B transition decreased. Cells In G2 had DNA more sensitive to acid denaturation than S or G1 phase cells. Mitotic cells had the most condensed chromatin and their RNA content was twice that of G1A cells. Differentiated cells were characterized by 2C DNA (G1D) but, depending on the cell type, had varying RNA content and different degrees of chromatin condensation. A hitherto undescribed category of cells undergoing transition from quiescence to the cycle (or vice versa) was distinguished based on their intermediate values of RNA, sensitivity of DNA to acid denaturation and inability to incorporate BUdR at the rate characteristic for S cells. Depending on their DNA content (C) at the time of transition these cells could be classified as G1T (2C), ST (4>C>2) or G2T (4C).

/pdf/new-cell-cycle-compartments-identified-by-multiparameter-59wtcs6k1y.pdf

New cell cycle compartments identified by multiparameter flow cytometry

The effect of serum on the growth properties of non-transformed Balb 3T3 A31 and SV40-transformed Balb 3T3 A31 was studied. The concentration of serum in the growth medium of non-transformed cells had little effect on the initial population doubling time, but did regulate the cell density at which the population became quiescent in G/sub 1/. The doubling time of transformed cells, however, was increased significantly as the concentration of serum was decreased below 4 percent. This effect on the growth of transformed cells was seen at serum concentrations so low that non-transformed cells did not complete one population doubling. Flow microfluorometric analysis of these populations indicated that the primary effect of different serum concentrations on the non-transformed cells was to modulate the average residence time in G/sub 1/, whereas, all the cell cycle phases of the transformed cells were affected by serum. At saturation densities, the non-transformed cells became quiescent in G/sub 1/, but the transformed cells still traversed the cell cycle and their saturation density appeared to be a balance between cell production and cell death occurring primarily in the G/sub 1/ phase of the cell cycle.

/pdf/effect-of-serum-on-the-growth-of-balb-3t3-a31-mouse-54eci16bcu.pdf

Effect of serum on the growth of balb 3t3 a31 mouse fibroblasts and an sv40-transformed derivative.

https://escholarship.org/content/qt4hq131x9/qt4hq131x9.pdf?t=p2ase5

Leucine in cultured cells: Its metabolism and use as a marker for protein turnover

The metabolism of leucine by normal and virally transformed mouse fibroblasts in culture has been investigated. Cultures pulsed with (U--/sup 1/C)leucine were studied for the distribution of radioactivity in the free amino acid pool, cellular protein, lipid and CO/sub 2/. Less than 1% of the label taken up was found in lipid and no radioactivity was recovered as CO/sub 2/ when cultures were pulsed in growth medium. That these cells retain the capacity to oxidize leucine was shown by experiments in which cultures pulsed in a balanced salt solution (Krebs--Ringer bicarbonate) showed linear evolution of CO/sub 2/. Leucine proved to be a desirable marker for protein turnover studies due to its minimal metabolism when labeling was performed in growth medium. The appearance of radioactive leucine in the medium of cells previously labeled with (/sup 14/C)leucine was shown to be directly correlated with loss of radioactivity in cellular protein. A class of rapidly degrading proteins and a second group of proteins with a slower rate of turnover could be distinguished when cells were labeled with (/sup 14/C) and (/sup 3/H)leucine for varying lengths of time. Density inhibited cultures degraded both these classes of proteins about twice as rapidly as did growingmore » cells.« less

Leucine in cultured cells. [/sup 14/C tracer technique]

Flow microfluorometry has been used to characterize the effects of serum concentration and cell density on the initiation of cell cycle transit of stationary phase (G0) human diploid fibroblasts (strain WI-38). The concentration of serum used to stimulate these cultures had no effect on the time cells began appearing in S (the DNA synthetic period), nor on the synchrony with which they moved around the cell cycle. However, as the serum concentration increased, the fraction of the stationary phase population released from G0 increased. Cell density modulated the ability of serum to stimulate cell cycle traverse. For example, at a cell density of 1.81 X 10(4) cells/cm2, 78% of the population was sensitive to serum stimulation; whereas, when the density was increased to 7.25 X 10(4) cells/cm2, only 27% of the population could be stimulated. This effect of cell density on the serum response is not simply the result of changing the ratio of serum concentration to cell density, but appears to reflect a true modulation of the population's sensitivity to serum stimulation. These results are consistent with the interpretation that the primary action of serum is to determine the transition of cells from a non-cycling G0 state to a cycling state and that cell density determines the proportion of the population capable of undergoing this transition.

/pdf/stimulation-of-wi-38-cell-cycle-transit-effect-of-serum-284i1nj7gq.pdf

P.A. Ross

Papers

Effect of serum on the growth of balb 3t3 a31 mouse fibroblasts and an sv40-transformed derivative.

Leucine in cultured cells: Its metabolism and use as a marker for protein turnover

Leucine in cultured cells. [/sup 14/C tracer technique]

Stimulation of WI‐38 cell cycle transit: Effect of serum concentration and cell density