Effects of serum and conditioned medium on protein degradation, migration of nonhistone proteins to the nucleus, and DNA synthesis in transformed cells

Stimulation of resting transformed cells (Chang liver cells), prelabeled with [3H] leucine, with fetal calf serum, caused increased nuclear translocation of [3H] nonhistone proteins ([3H] NHP) and DNA synthesis and a parallel inhibition of proteolysis of cellular proteins. [3H] NHP migration was independent of protein synthesis. Fractionation of the nuclear proteins in a pH gradient of 2.5-6.5, showed that [3H] NHP fractions with high degradation rates in resting cells corresponded to the [3H] NHP fractions with high migration rates in stimulated cells, suggesting that degradation and migration of [3H] NHP are linked. Conditioned medium (COM) produced by Chang cells had similar effects as serum, suggesting that factors produced by these transformed cells, control cell growth by a mechanism that is similar to serum. The lysosomotropic amine eserine had similar effects as serum and COM. Based on the similarity of the effects, it would appear that serum and COM inhibit lysosomal proteolysis. It is proposed that serum and COM induce NHP migration to the nucleus by inhibiting lysosomal degradation of these proteins. Serum and COM caused also migration of [3H] histones to the nucleus, however the mechanism is not clear.     

Effects of platelet-derived growth factor on degradation, nuclear translocation of nonhistone proteins, and DNA synthesis.

Stimulation of resting normal rat kidney fibroblasts, prelabeled with [3H]leucine, by platelet-derived growth factor (PDGF) caused inhibition of cellular protein degradation and a parallel increased nuclear translocation of 3H-labeled nonhistone proteins (3H-NHP) and DNA synthesis. Nuclear translocation of these proteins was independent of protein synthesis. Fractionation of the nuclear 3H-NHP in a pH gradient of 2.5-6.5 showed that the protein fractions with a high degree of proteolysis in resting cells corresponded to the protein fractions with a high extent of translocation in stimulated cells, suggesting that degradation and translocation of these proteins may be related. PDGF inhibited cellular uptake of [3H]chloroquine, suggesting that PDGF inhibits NHP degradation via the lysosomal pathway. These observations support the hypothesis that PDGF induces NHP translocation to the nucleus by inhibiting lysosomal degradation of these proteins.     

The effects of lysosomotropic amines on protein degradation, migration of nonhistone proteins to the nucleus, and cathepsin D in lymphocytes

Various lysosomotropic amines have two parallel effects in human lymphocytes: they inhibit the degradation of cellular proteins and increase the migration of nonhistone proteins (NHP) from the cytoplasm to the nucleus. The increased nuclear level of NHP is associated with increased cellular binding of [3H] actinomycin D, indicating an altered structure of chromatin. The agents inhibit the degradation of short- and long-lived proteins equally. Fractionation of the [3H] NHP of the nucleus according to pH 2.5-6.5 shows that [3H] NHP with a high rate of degradation in untreated cells correspond to [3H] NHP with a high rate of migration in cells treated with the agents. Eserine, amantadine, nicotine, atropine, benzylamine, and propranolol inhibit cathepsin D in concentrations causing proteolytic inhibition in cell cultures or in concentrations believed to be attained in lysosomes. The agents strongly inhibit the cellular accumulation of [3H] chloroquine. The data support the proposal that the migration of NHP from the cytoplasm to the nucleus is the direct consequence of inhibited degradation of these proteins in lysosomes by the amines.     

The effects of concanavalin A and other agents on protein degradation and migration of non-histone proteins (NHP) to the nucleus in lymphocytes

Concanavalin A (conA) inhibits the degradation of [3H]leucine-labeled cellular proteins of human lymphocytes. The lectin also stimulates the migration of non-histone proteins (NHP) from the cytoplasm to the nucleus. The increased nuclear level of NHP is associated with increased cellular binding of [3H]actinomycin D [(3H]AD). Decreased protein breakdown and increased migration of NHP are parallel events, i.e. both changes occur as a function of the lectin concentration and display a similar time course, suggesting that these events could be related. Similar effects are observed with fluoride, chloroquine and iodoacetate: these agents simultaneously decrease proteolysis and increase the nuclear level of NHP, associated with increased cellular [3H]AD binding. Fractionation of the acidic NHP according to pH 2.5-6.5 shows that proteins with a high degree of degradation in unstimulated cells correspond to proteins with a high degree of migration in conA-stimulated cells. A similar correlation was observed in fluoride-treated lymphocytes. conA, fluoride and iodoacetate decrease cellular [3H]chloroquine [(3H]CQ) accumulation, indicating a lysosomotropic effect. These and previously reported data suggest, but do not prove that conA inhibits degradation of cellular proteins via the lysosomal pathway. Ammonium chloride, methylamine and sodium azide also inhibit proteolysis and increase cellular [3H]AD binding; however, their effects are weak. On the basis of these observations it appears that lysosomal degradation and migration of NHP to the nucleus are linked; however, the mechanism of the linkage is unknown.     

Demonstration that some of the nonhistone proteins, inducible to translocate into the nucleus, are glycosylated

Con A, NaF, and eserine (lysosomotropic agents) induced marked translocation of acidic [3H] nonhistone proteins (NHP) from the cytoplasm to the nucleus in lymphocytes prelabeled with [3H]-2-mannose. The nuclear [3H] NHP contents were 38-120% higher in cells treated with these agents than in control cells. Tunicamycin, a strong inhibitor of N-glycosylation via the dolichol pathway, caused a concentration-dependent inhibition of [3H]-2-mannose incorporation into the nuclear [3H] NHP. Considerable amounts of nuclear [3H] NHP from lymphocytes labeled with either [3H]-2-mannose or [3H] leucine, bound specifically to Con A-Sepharose and could be eluted by alpha-methyl mannoside. Con A and NaF caused also nuclear translocation of acidic [3H] NHP in cells labeled with [3H] glucosamine, [3H] galactose, or [3H] fucose. Fractionation of the nuclear proteins by isoelectric focusing in a pH gradient of 2.5-6.5 showed that multiple species of acidic NHP were labeled with each of the four 3H-sugars. These results indicate that a fraction of the acidic nuclear NHP are N-glycosylated proteins and that gene activation and mitogenesis are associated with the translocation of these glycoproteins to the nucleus. Considering the known intracellular traffic of nascent glycoproteins our results suggest that at least some of the acidic NHP are synthesized and glycosylated in the endoplasmic reticulum and the Golgi (secretory pathway). It is likely that these proteins, after completion of synthesis and glycosylation, emerge from the trans-stack of the Golgi packaged in vesicles and accumulate in the cytoplasm. Induction of nuclear translocation of such NHP by various agents may be mediated by a vesicular transport mechanism.     

Endocytosed non-histone proteins translocate in part to the nucleus in lymphocytes

[3H]Non-histone proteins ([3H]NHP), dissolved in the culture medium, are endocytosed by lymphocytes and equilibrate rapidly between the cytoplasm and the nucleus. During incubation, the proteins are gradually degraded in the lysosomes. The lysosomotropic agents conA, NaF, eserine and atropine have two parallel effects on resting lymphocytes, after they have endocytosed [3H]NHP: inhibition of degradation and increased translocation of [3H]NHP from the cytoplasm to the nucleus. This indicates that lysosomal degradation and translocation of [3H]NHP to the nucleus are linked and suggests that this translocation may be the result of inhibited lysosomal degradation of the [3H]NHP. The behaviour of endocytosed [3H]NHP appears similar to that of endogenous [3H]NHP in cells prelabeled with [3H]leucine, when subjected to the same lysosomotropic agents, reported previously (Polet, H, Exp cell res 148 (1983) 345). This observation may provide a model to study the mechanism(s) controlling nucleo-cytoplasmic traffic of NHP.     

Cytochalasin D inhibits nuclear translocation of nonhistone proteins induced by lectin and other agents in lymphocytes.

Cytochalasin D (CD), known to interfere with microfilament activity, inhibits RNA and protein synthesis and the cellular uptake of [3H]actinomycin D in concanavalin A-stimulated lymphocytes. It also inhibits the nuclear translocation of nonhistone proteins (NHP) induced by the lectin. Since NHP mediate RNA and protein synthesis and [3H]actinomycin D binding, inhibited nuclear translocation of NHP can explain the inhibition of the former events. CD also inhibits nuclear translocation of NHP caused by NaF or chloroquine. The mechanism of how CD inhibits nuclear translocation of the NHP is obscure. Since NaF and chloroquine enter cells by diffusion, it would appear that CD acts on an intracellular target, rather than abrogating a cell surface-mediated signal generated by binding of the lectin to a cell surface receptor.     

Role of nuclear proteins on [H]actinomycin D binding during lymphocyte mitogenesis

The uptake of [³H]actinomycin D ([³H]AD) by ConA-stimulated lymphocytes was followed during 96 h of incubation and correlated with the level of nuclear proteins in the nucleus, DNA synthesis and the degree of AD-induced inhibition of RNA and DNA synthesis. During the first 48 h there is a parallel increase of drug binding to cells and a rising level of non-histone proteins (NHP) in the nucleus. During the next 48 h, DNA synthesis occurs, drug uptake decreases and the nuclear level of NHP continues to rise. The level of histones remains constant during 96 h. The variations in cellular [³H]AD uptake during 96 h are not due to changes in cell membrane permeability, since similar variations in drug binding are observed in isolated cell nuclei. NHP, obtained as 0.25 M NaCl extracts of cell nuclei, increase binding of [³H]AD to nuclei isolated from non-stimulated lymphocytes, while histones have no such effect. NHP extracted with phenol, after washing the nuclei with salt and acid solutions, or extracted with 0.25 M NaCl from non-stimulated and stimulated lymphocytes and Chang liver cells are equally active to bind [³H]AD to nuclei of non-stimulated lymphocytes. NHP from Chang cells, purified by DNA-cellulose chromatography using calf thymus DNA, stimulated [³H]AD binding to lymphocyte nuclei, indicating that the drug-binding activity is due to proteins binding to DNA. NHP increase binding of [³H]AD to pure DNA in the absence of histones. The degree of [³H]AD binding to ConA-stimulated lymphocytes during 96 h correlated with the degree of inhibition of RNA and DNA synthesis by AD.     

Role of nuclear proteins on [3H]actinomycin D binding during lymphocyte mitogenesis

The uptake of [³H]actinomycin D ([³H]AD) by ConA-stimulated lymphocytes was followed during 96 h of incubation and correlated with the level of nuclear proteins in the nucleus, DNA synthesis and the degree of AD-induced inhibition of RNA and DNA synthesis. During the first 48 h there is a parallel increase of drug binding to cells and a rising level of non-histone proteins (NHP) in the nucleus. During the next 48 h, DNA synthesis occurs, drug uptake decreases and the nuclear level of NHP continues to rise. The level of histones remains constant during 96 h. The variations in cellular [³H]AD uptake during 96 h are not due to changes in cell membrane permeability, since similar variations in drug binding are observed in isolated cell nuclei. NHP, obtained as 0.25 M NaCl extracts of cell nuclei, increase binding of [³H]AD to nuclei isolated from non-stimulated lymphocytes, while histones have no such effect. NHP extracted with phenol, after washing the nuclei with salt and acid solutions, or extracted with 0.25 M NaCl from non-stimulated and stimulated lymphocytes and Chang liver cells are equally active to bind [³H]AD to nuclei of non-stimulated lymphocytes. NHP from Chang cells, purified by DNA-cellulose chromatography using calf thymus DNA, stimulated [³H]AD binding to lymphocyte nuclei, indicating that the drug-binding activity is due to proteins binding to DNA. NHP increase binding of [³H]AD to pure DNA in the absence of histones. The degree of [³H]AD binding to ConA-stimulated lymphocytes during 96 h correlated with the degree of inhibition of RNA and DNA synthesis by AD.     

Post-translational changes of chromosomal proteins in rat cerebellum during postnatal development

Acetylation, phosphorylation and methylation of nuclear proteins in rat cerebellum at 10 and 30 days of age were investigated in vitro. Isolated nuclei were incubated in the presence of [1-14C]acetyl CoA, S-adenosyl [methyl-3H]methionine and [gamma-32P]ATP and then separated into histones and non histone proteins (NHP), which were further fractionated by polyacrylamide gel electrophoresis. The results obtained indicate that acetylation, phosphorylation and methylation of both basic and acidic proteins decrease from 10 to 30 days of age. Electrophoretic analysis of histones shows that the decrease mainly concerns H1, H3, and H2b fractions. The H3 fraction is always more labeled than the other fractions and shows the major changes during postnatal development. Phosphorylation of H2a and H4 fractions increases from 10 to 30 days of age, whereas acetylation and methylation of these fractions do not show significant changes from 10 to 30 days. The densitometric and radioactive patterns of NHP show considerable changes between 10 and 30 days, especially in the high molecular weight region. The incorporation of 14C-acetyl and 3H-methyl groups and of 32P phosphate appears to be generalized throughout the molecular weight range and decreases from 10 to 30 days of age. The methylation of an as yet unidentified protein with a molecular weight of approximately 110,000 daltons occurred at both ages.     

Exogenous nucleosides stimulate RNA synthesis in resting cells of a culture of scarlet rose

RNA synthesis in response to exogenous nucleoside precursors was studied in a suspension culture of rose cells. Exponentially growing and resting cells were prelabeled with [3H] uridine, an excess of unlabeled uridine added, and subsequent isotopic incorporation into nuclear and ribosomal fractions measured. The data were compared to control values in cells continuously labeled in the absence of unlabeled uridine. Addition of uridine to the growing culture reduced the further uptake, and incorporation of [3H] uridine into RNA. In contrast, in resting cells, the addition of uridine (or, purine nucleosides) enhanced the apparent utilization of [3H] uridine in RNA synthesis by 2- to 4-fold.     

The effects of amino acids on protein degradation and translocation of non-histone proteins to the nucleus in lymphocytes

Tryptophan, phenylalanine and leucine have two parallel effects in cultured lymphocytes, they inhibit cellular proteolysis and increase the translocation of non-histone proteins to the nucleus. The latter is associated with an increased cellular binding of [3H]actinomycin D, indicating an altered structure of chromatin. The amino acids also inhibit the cellular uptake of [3H]chloroquine, suggesting that inhibited protein degradation is lysosomal. Several amine catabolites of tryptophan and phenylalanine, some of which are known to play a role as biogenic amines, have similar actions, and can explain, at least in part, the effects of their parent amino acids. Fractionation of the nuclear 3H-labeled non-histone proteins according to pH 2.5-6.5 shows that such proteins with a high rate of degradation in untreated cells correspond to the 3H-labeled non-histone proteins with a high rate of translocation in tryptophan treated cells. These data suggest that the degradation and the translocation of the non-histone proteins are linked and that the increased translocation of the non-histone proteins to the nucleus may be the consequence of inhibited lysosomal degradation of these proteins by the amino acids.     

Chromatin proteins as a possible target for antitumour agents: alterations of chromatin proteins in dibromodulcitol treated Yoshida tumors

The effect of dibromodulcitol (DBD) on Yoshida sarcoma chromatin components has been investigated. Measurements on the radioactivity of nuclear components after in vivo treatment with [3H]DBD for 1 h indicated preferential drug binding to the high molecular weight component of the nuclear residual acidic protein (non-histones) and also to Histone 1 (H1) (very lysine rich, F1). Two-hour DBD treatment resulted in partial degradation and reduced [3H]leucine incorporation into the same fractions of chromatin. However, 6 h after DBD treatment, the synthesis of the degraded chromatin proteins began and by 24 h was completed. During the same treatment period the composition of chromatin showed a remarkable alteration; 2 h after DBD treatment the amount of the nuclear residual acidic proteins relative to DNA decreased by approx. 50%, but returned to control value 24 h after drug treatment. This in conjunction with the data on [3H]leucine incorporation suggests that certain chromatin proteins are degraded and subsequently newly synthesised after DND treatment resulting in an exchange of chromatin components. The formation of a nucleohistone complex between H1 and DNA was inhibited by pretreatment of H1 and DBD, dianhydrodulcitol (DAD) and bischloroethylnitrosourea (BCNU).     

Cell cycle-related transfer of proteins between the nucleus and cytoplasm of Physarum polycephalum

The proteins of wild-type and polyploid plasmodia of P. polycephalum were prelabelled with [³H]leucine and [¹⁴C]leucine. The two types of plasmodia were then fused for 2 h. Following fusion the nuclei were isolated and the smaller wild-type cell nuclei separated from the larger polyploid cell nuclei. The proteins were isolated from the recipient cell nuclei and the recipient nuclear proteins extracted. Ratios of ³H/¹⁴C in the various nuclear protein fractions show that during fusion differential transfer of labelled preformed proteins from the donor cell into the recipient cell nucleus occurs. The quantity of proteins transferred varies among the different fractions and with the phase of the cell cycle. Isotopic dilution experiments indicate that these differences in protein transfer are, in part, due to a high rate of synthesis and turnover of the nuclear proteins.     

Early DNA synthesis during the germination of wheat embryos

Both [³H]thymidine and [³H]deoxyadenosine were found to be incorporated into the nuclear DNA of wheat embryos immediately after dry embryos were allowed to imbibe aqueous solutions of the radioactive precursors. The early labelled DNA sedimented in a manner suggesting that replicative intermediates were already formed within the first 90 min of germination. However, aphidicolin remained without any effect on this early DNA synthesis. Likewise, a cell-free system derived from early embryos incorporated [³H]dCTP into DNA independently of the presence of aphidicolin. On the contrary, dideoxyTTP inhibited the DNA synthesis considerably. It is concluded that a proportion of the resting wheat embryo cells is able to initiate a replicative DNA synthesis immediately upon imbibition. The synthesis seems, however, to proceed with the participation of a γ-like, rather than an α-like, DNA polymerase.     

Nuclear Nonhistone Proteins in Murine Melanoma Cells: 1. Quantitative Isolation and Fractionation

Nuclear nonhistone proteins (NHP's) have been implicated as regulatory agents involved in controlling genetic expression. Utilizing murine melanoma cells, we describe a method for isolating and fractionating NHP's which greatly increases the yield of these proteins as well as the level of resolution required for detecting small differences in particular NHP's. Mouse melanoma cells were grown in medium labeled with [³H]leucine. Following 48 hr of incubation, the cells were harvested and nuclei isolated. The NHP's were extracted from the nuclei in a series of steps which yielded four major fractions: NHP₁, NHP₂, NHP₃, NHP₄. This method solubilized 80-90% of the protein from the nuclear homogenate. The NHP fractions were then separated on DEAE-cellulose columns in a series of salt steps increasing in concentration from 0.05 to 0.50 M NaCl, followed by steps of 2 M NaCl and 4 and 7 M guanidine-hydrochloride. The 40 NHP fractions eluted from these columns were further separated on polyacrylamide-SDS gels and ranged in molecular weight from 9000 to 110,000 daltons. Differences were observed in the electrophoretic pattern of each of these 40 fractions. The high resolution of these fractionation procedures greatly enhances the possibility of observing small changes in proteins which may play a role in gene regulation.     

Metabolic activities of histones in rat liver and spleen.

Synthesis and turnover of histone I and II in normal rat liver and spleen were studied by Amberlite CG 50 column chromatography. Histone I was separated into three or four subfractions, each of which showed a different rate of incorporation of [3H]lysine. This was verified by a more shallow gradient chromatography developed by Kinkade and Cole [3] for very lysine-rich histone (F1), which showed tissue specific differences between liver and spleen in both the elution pattern and synthetic rates. These subfractions were distinguished from each other by dodecylsulphate electrophoresis. The turnover, or disassociation of histone I and II in chromatin was measured by double-labelling of normal rat liver with [3H] and [14C]lysine. A good correspondence was found between the synthesis and turnover patterns of individual histone I fractions, while the histone II synthesized was conserved for over a month. From consideration of the turnover in relation to the cell population of normal liver tissue, which consists of a very small fraction of growing cells and a very large fraction of resting ones, it was concluded that turnover of histone I must occur even in resting cells. When DNA synthesis in the spleen was completely inhibited by hydroxyurea, the synthesis of histone II was inhibited but that of histone I was only partially inhibited. The remaining synthesis seemed to occur in cells in the resting state. It was concluded tentatively, the continuous replacement of very lysine-rich histones of chromatin must occur even in resting cells in which DNA synthesis has ceased. The biological significance of disassociation of histones from chromatin was discussed.     

Localization of the estrogen receptor in uterine cells by affinity labeling with [3H]tamoxifen aziridine

The possibility that estrogen receptors may exist in uterine plasma membranes was investigated by covalent labeling of estrogen receptors in mouse uterine cells with [3H]tamoxifen aziridine (TA). Isolated epithelial and stromal cells of immature mice were incubated with [3H]TA in the presence or absence of unlabeled tamoxifen, homogenized and separated into nuclear, cytosolic and microsomal fractions by differential centrifugation. These fractions were subjected to SDS-polyacrylamide gel electrophoresis and the proteins labeled covalently with TA were visualized by autoradiography. Proteins labeled specifically with [3H]TA were observed almost exclusively in the nuclear fraction of both epithelial and stromal cells. In contrast, very little labeled protein was detected in the cytosolic or microsomal fraction. Although these data do not preclude the possibility that estrogen binding sites are present in plasma membranes of uterine cells, this cellular fraction is definitely not labeled to a significant extent by [3H]TA. Thus, if membrane estrogen binding sites exist, their structural conformations may be different from that of nuclear estrogen receptors.     

Depression of cytochrome P-450 and alterations of protein metabolism in mice treated with the interferon inducer polyriboinosinic acid X polyribocytidylic acid

Treatment of mice with the interferon inducer polyriboinosinic acid X polyribocytidylic acid [poly(IC)] results in the depression of several hepatic proteins. In this study we examined synthesis and degradation of the proteins of liver cell organelles in mice treated with poly(IC). Effects on synthesis were determined by using [14C]- and L-[3H]leucine incorporation into control and poly(IC)-treated mice, respectively. At selected times after poly(IC) treatment the 3H/14C ratio was established for preparations of nuclei, mitochondria, lysosomes, smooth endoplasmic reticulum, rough endoplasmic reticulum, and 105,000g supernatant (cytosol). Time-dependent alterations in de novo protein synthesis were greatest in lysosomal and rough endoplasmic reticular fractions; both were depressed 9 h after treatment. The effects of poly(IC) on protein degradation were determined with [14C]bicarbonate. Poly(IC) treatment decreased the time required for disappearance of 50% of 14C-labeled protein (t1/2) of smooth and rough endoplasmic reticula. Examination of endoplasmic reticulum marker enzymes showed depression of cytochromes P-450 and b5 from 9 h onward after poly(IC) administration. Tyrosine aminotransferase activity was elevated 6 h after treatment with poly(IC), and then depressed after 9 h. The other organelle marker enzymes were not affected significantly. We conclude that poly(IC) decreases the content of proteins of the hepatic endoplasmic reticulum, including certain cytochrome P-450 isozymes, by decreasing rates of protein synthesis and increasing rates of protein degradation.     

Degradation of proteins in steady-state cultures of Escherichia coli.

The degradation of proteins in Escherichia coli was investigated in cells grown under steady-state conditions in a glucose-limited chemostat. During the first 24 h, approximately 25% of pulse-labeled proteins were degraded and after 72 h up to 58% of the proteins were broken down. To examine the stability of subcellular components steady-state cultures were labeled with an initial pulse of [14C]leucine, 24 h were allowed for turnover of these proteins, and the cells were then labeled with a short pulse of [3H]leucine. By this double-label protocol, the labile proteins were preferentially labeled with [H]leucine and had high 3H/14C ratios, while the more stable proteins had lower 3//14C ratios. The 3/-labeled proteins were degraded approximately five times as rapidly as the 14C-labeled proteins in exponentially growing cells. The relative stability of subcellular fractions was determined by comparing their 3H/14C ratios to the ratio of the cells at harvest. The soluble fraction contained the most labile proteins, while the ribosomal and membrane fractions were at least as stable as the average cell protein.