Protein degradation in 3T3 cells and tumorigenic transformed 3T3 cells

To study the relation of overall rates of protein degradation in the control of cell growth, we determined if transformation of fibroblasts to tumorigenicity affected their rates of degradation of short- and long-lived proteins. Rates of protein degradation were measured in nontumorigenic mouse Balb/c 3T3 fibroblasts, and in tumorigenic 3T3 cells transformed by different agents. Growing 3T3 cells, and cells transformed with Moloney sarcoma virus (MA-3T3) or Rous sarcoma virus (RS-3T3), degraded short- and long-lived proteins at similar rates. Simian virus 40 (SV-3T3)- and benzo(a)pyrene (BP-3T3)-transformed cells had slightly lower rates of degradation of both short- and long-lived proteins. Reducing the serum concentration in the culture medium from 10% to 0.5%, immediately caused about a twofold increase in the rate of degradation of long-lived proteins in 3T3 cells. Transformed lines increased their rates of degradation of long-lived proteins only by different amounts upon serum deprivation, but none of them to the same extent as did 3T3. Greater differences in the degradation rates of proteins were seen among the transformed cells than between 3T3 cells and some transformed cells. Thus, there was no consistent change in any rate of protein degradation in 3T3 cells due to transformation to tumorigenicity.     

Initial kinetics of Protein turnover in growing yeast

Proteins in yeast growing in a medium with glucose or ethanol as carbon source were pulse-labelled by a 20-min incubation with¹⁴C-leucine. The proteins in cells labelled and growing in a glucose medium were stable; when this population was transferred to the ethanol medium, the proteins were degraded at a rate of 1.1 %/h. The population labelled and growing in an ethanol medium displayed a fraction of short-lived proteins (about 4 %), decaying with a half-life of 0.5 h. The size of the short-lived protein fraction increased slightly after shifts to a glucose as well as to a starvation medium. The residual long-lived proteins underwent a turnover of 1.3 –1.4 %/h in the ethanol or the starvation medium and of 0.3 %/h in the glucose medium, respectively. Proteins labelled in the presence of canavanine or ethionine were degraded at only a slightly greater rate than the normal proteins. Participant of the UNESCO Postgraduate Course “On Modern Problems in Biology”.     

Polyadenylate-protein complexes in resting and growing 3T3 cells.

The properties of the ribonuclease resistant cytoplasmic ribonucleoprotein particles were studied in contact-inhibited and serum induced proliferating 3T3 cells. The RNP particles were fractionated by oligo (dT)-cellulose chromatography and banded in CsSO4 gradients. The main RNP fraction, eluted with 25% formamide, contained the major ribonuclease resistant RNA sequences in both resting and growing cells. The protein component of this fraction had a molecular weight of about 72,000 in contact-inhibited cells and 81,000 in serum induced cells.     

Protein translocation and turnover in eukaryotic cells

Although most eukaryotic proteins are synthesized in the cytoplasm from mRNAs originating in the nucleus, many function in specialized compartments and must be specifically translocated co- or post-translationally. A variety of signals contained within the amino acid sequence guide these processes as well as direct turnover by different proteolytic schemes. Several properties of the mature protein are determined as a result of translocation and can serve as predictors of cellular localization.     

S-Nitrosocysteine increases palmitate turnover on Ha-Ras in NIH 3T3 cells

Ha-Ras is modified by isoprenoid on Cys(186) and by reversibly attached palmitates at Cys(181) and Cys(184). Ha-Ras loses 90% of its transforming activity if Cys(181) and Cys(184) are changed to serines, implying that palmitates make important contributions to oncogenicity. However, study of dynamic acylation is hampered by an absence of methods for acutely manipulating Ha-Ras palmitoylation in living cells. S-nitrosocysteine (SNC) and, to a more modest extent, S-nitrosoglutathione were found to rapidly increase [(3)H]palmitate incorporation into cellular or oncogenic Ha-Ras in NIH 3T3 cells. In contrast, SNC decreased [(3)H]palmitate labeling of the transferrin receptor and caveolin. SNC accelerated loss of [(3)H]palmitate from Ha-Ras, implying that SNC stimulated deacylation and permitted subsequent reacylation of Ha-Ras. SNC also decreased Ha-Ras GTP binding and inhibited phosphorylation of the kinases ERK1 and ERK2 in NIH 3T3 cells. Thus, SNC altered two important properties of Ha-Ras activation state and lipidation. These results identify SNC as a new tool for manipulating palmitate turnover on Ha-Ras and for studying requirements of repalmitoylation and the relationship between palmitate cycling, membrane localization, and signaling by Ha-Ras.     

Prostaglandin F2 alpha stimulates phosphatidylinositol turnover and increases the cellular content of 1,2-diacylglycerol in confluent resting Swiss 3T3 cells

Prostaglandin F2 alpha (PGF2 alpha); which stimulates DNA synthesis in resting 3T3 cells, also stimulates the incorporation of [32P]PO4 into phosphatidylinositol. The effect is selective for PGF2 alpha when compared with PGE1, PGE2, and PGF2 beta. Epidermal growth factor (EGF) also stimulates DNA synthesis but does not affect phosphatidylinositol turnover. PGE1, which acts synergistically with PGF2 alpha to enhance DNA synthesis, does not affect the ability of PGF2 alpha, to enhance the incorporation of [32P]PO4 into phosphatidylinositol. PGF2 alpha, also causes a small increase in the cellular content of 1,2-diacylglycerol. This effect is not shared by EGF or PGE1. Stimulation of phosphatidylinositol metabolism resulting in an increase in the cellular content of 1,2-diacylglycerol may thus constitute an event in the pathway leading to the initiation of DNA synthesis in which PGF2 alpha differs in its action from EGF.     

Metabolic turnover of proliferation-related nuclear proteins in serum-stimulated Swiss mouse 3T3 cells

Stimulation of quiescent Swiss mouse 3T3 cells either by serum or by pure growth factors induces DNA synthesis after a lag period of about 15 h. Following restimulation by serum or by growth factors there is an overall increase of 2-4-fold in the rate of biosynthesis of nuclear proteins. Two nuclear polypeptides show specific temporal correlations with the transition from quiescence to proliferation. The synthesis of p30 (30 kDa, pI 5.2) is at a maximum within 5 h of restimulation, while the synthesis of p36 (36 kDa, pI 4.25) is first seen at 10-12 h after restimulation. The synthesis of p36 correlates well with the initiation of DNA biosynthesis. The metabolic turnover of both of these proteins has been estimated by pulse-chase and by cycloheximide inhibition experiments. They both have a half-life of 10-15 h and appear to be cell-cycle related.     

Protein turnover in exponential and stationary phase Vibrio cells

Vibrio strain 14 supports phage alpha 3a growth in standing stationary phase cells but not in shaking (aerated) stationary phase cells. In exponential cells, protein was turned over at 1.8% h-1, and the rate was increased by starvation or inhibition of protein synthesis. In shaking stationary phase cells the rate of protein turnover was low (1.0% h-1) for proteins synthesised during growth but high (20% h-1) for recently synthesised proteins. In contrast recently synthesised proteins in standing stationary phase cells were stable over 60 min and proteins synthesised during growth were turned over at 2.9% h-1. ppGpp and pppGpp were detected in exponential cells, but were not detected in stationary phase cells.     

Lysosomal turnover, but not a cellular level, of endogenous LC3 is a marker for autophagy

During starvation-induced autophagy in mammals, autophagosomes form and fuse with lysosomes, leading to the degradation of the intra-autophagosomal contents by lysosomal proteases. During the formation of autophagosomes, LC3 is lipidated, and this LC3-phospholipid conjugate (LC3-II) is localized on autophagosomes and autolysosomes. While intra-autophagosomal LC3-II may be degraded by lysosomal hydrolases, recent studies have regarded LC3-II accumulation as marker of autophagy. The effect of lysosomal turnover of endogenous LC3-II in this process, however, has not been considered. We therefore investigated the lysosomal turnover of endogenous LC3-II during starvation-induced autophagy using E64d and pepstatin A, which inhibit lysosomal proteases, including cathepsins B, D and L. We found that endogenous LC3-II significantly accumulated in the presence of E64d and pepstatin A under starvation conditions, increasing about 3.5 fold in HEK293 cells and about 6.7 fold in HeLa cells compared with that in their absence, whereas the amount of LC3-II in their absence is cell-line dependent. Morphological analyses indicated that endogenous LC3-positive puncta and autolysosomes increased in HeLa cells under starvation conditions in the presence of these inhibitors. These results indicate that endogenous LC3-II is considerably degraded by lysosomal hydrolases after formation of autolysosomes, and suggest that lysosomal turnover, not a transient amount, of this protein reflects starvation-induced autophagic activity.     

Binding of isolated 3T3 surface membranes to growing 3T3 cells and their effect on cell growth

We have quantitated by autoradiography the binding of [¹²⁵I]labeled 3T3 plasma membrane fragments to 3T3 cells growing on the surface of plastic dishes; ie, the same conditions in which these membranes specifically arrest the growth of 3T3 cells early in the G₁ phase of the cell cycle. We have been able to demonstrate that binding of membranes to cells is coincidental with the expression of the growth inhibitory activity of protein(s) present in the membrane fragments. Treatments that reduce binding (heat denaturation of the membranes or culture in the presence of high scrum) also reduce growth inhibitory activity. [¹²⁵I]labeled membranes bound to cells are located primarily on the cell surface (as determined by electron microscope autoradiography) and are exchangeable with unlabeled membranes. We conclude that binding of membranes to cells is necessary but may not be sufficient for the expression of the growth inhibitory activity of these membranes. This approach provides information not only on the average level of binding of membranes to cells, but also provides a quantitative assessment of the variation of the level of membrane to cell binding between different cells in the population.     

Surface exposure of glycosaminoglycans in resting, growing and virus transformed 3T3 cells.

Glycosaminoglycans (GAG's) were released by trypsin from the surface of cultured mouse cells (3T3) in two different growing states: during log-growth phase and during resting due to serum starvation. Doubly labelled molecules from resting cells were compared with those from growing as well as from trnsformed cells. Reproducible differences in the elution pattern during ion exchange chromatography and in susceptibility to specific hydrolytic enzymes have been demonstrated: the GAGs pattern of growing normal cells is similar to the pattern of the cells transformed by either Polyoma or SV-40 viruses and very different from the pattern of resting cells. Growing and transformed 3T3 show a relatively low amount of trypsin removable heparan sulphate (HS) and a relatively high amount of hyaluronic acid (HA) while resting cells exhibit an opposite ratio between the two GAG'S. The lowering of HS and the increase of HA in the cell coat is therefore suspected to be more dependent upon growth than upon transformation.     

Protein synthesis in transformed 3T3 cells permeabilized by exogenous ATP

Exogenous ATP has been shown to cause a rapid and reversible increase in permeability in transformed 3T3 cells (3T6 and SV3T3) but not in untransformed 3T3 cells. The cells remain viable, but lose intracellular acid-soluble pools. Treatment of transformed cells with ATP greatly reduces incorporation of ¹⁴C-leucine into protein, which is restored by the incubation of the cells with Dulbecco's modified Eagle's medium or by the external additions of certain ions and energy sources. tRNA is not required for the restoration of protein synthesis. In the permeabilized cells the energy for protein synthesis can be provided by glycolysis, oxidative phosphorylation, or direct addition of ATP. These studies demonstrate the usefulness of this method for studying the control of metabolism and macromolecular synthesis in monolayer cultures of transformed mammalian cells.     

Protein turnover and proliferation. Failure of SV-3T3 cells to increase lysosomal proteinases, increase protein degradation and cease net protein accumulation

The contrasting control of lysosomal proteinases, protein turnover and proliferation was studied in 3T3 and SV-3T3 (SV-40-virus-transformed 3T3) cells. 1. In 3T3 cells, net protein accumulation proceeded from 5%/h (doubling time, T(d)=14h) in growing cells to 0%/h as cells became quiescent. SV-3T3 cells never ceased to gain protein, but rather decreased their protein accumulation rate from 6-7%/h (T(d)=10-12h) to 2%/h (T(d)=35-40h) just before culture death in unchanged medium. 2. In both cell types the rates of protein synthesis per unit of protein (a) were proportional to the initial serum concentration from 0 to 6%, and (b) declined under progressive depletion of undefined serum growth factors. In depleted growth medium, leucine incorporation per unit of protein in 3T3 and SV-3T3 cells declined to almost equal synthetic rates while the 3T3 cell existed in a steady state of zero net gain, and the SV-3T3 cell continued to gain protein at a rate of 2%/h. 3. Whereas a large fraction of the control of 3T3-cell net protein accumulation can be accounted for by an increase in degradation from 1%/h to 3%/h, the SV-3T3 cell did not exhibit a growth-related increase in degradation appreciably above 1%/h. 4. Thus, by using first-order kinetics, the continued net protein accumulation of the transformed cell can be accounted for by a failure to increase protein degradation, whereas fractional synthesis can be made to decline to a rate similar to that in the quiescent non-transformed cell. 5. Upon acute serum deprivation, both cell types similarly exhibited small rapid increases in proteolysis independent of cell growth state or lysosomal enzyme status. 6. The 3T3 cell increased its lysosomal proteinase activity in conjunction with increase in the growth-state-dependent proteolytic mechanism; however, the SV-3T3 cell failed to increase lysosomal proteinases or the growth-state-dependent proteolytic mechanism.     

Anisomycin and cycloheximide, like growth factors, stimulate rapidly ATP turnover in 3T3 cells

Addition of EGF and insulin to quiescent cultures of 3T3 cells induce a rapid stimulation of ATP turnover. We show that, like growth factors, anisomycin and cycloheximide, two inhibitors of protein synthesis, induce in 3T3 cells, a rapid increase in ATP turnover. However, this effect was not the result of the inhibition of protein synthesis since puromycin, in the same experimental conditions, did not stimulate ATP turnover.