Changes in eIF-4D hypusine modification or abundance are not correlated with translational repression in HeLa cells

Initiation factor eIF-4D is represented by about 11 X 10(6) molecules/HeLa cell (0.45% of the cytoplasmic protein molecules). The fraction of eIF-4D that contains the post-translational modification of lysine converted to hypusine is not regulated with respect to translation rate in HeLa cells. It is proportional to the rate of eIF-4D synthesis in exponentially growing cells (maximal protein synthesis rates) as well as in serum-depleted cells (protein synthesis rates depressed about 6-8-fold). In cells in which protein synthesis is arrested by cycloheximide, no hypusine addition or exchange is detected. During rapid repressions of protein synthesis due to either heat shock or hypertonic shock there is no change in the extent of eIF-4D containing hypusine. These results are most consistent with an eIF-4D biogenesis in which all molecules are modified to contain hypusine during or shortly after the translation process itself, and the modification state is not regulated thereafter.     

Posttranslational formation of hypusine in a single major protein occurs generally in growing cells and is associated with activation of lymphocyte growth

Growing lymphocytes perform a novel chemical modification of a single protein (Hy+: approximately 18 kd, pI approximately 5.1), resulting in the formation of the unusual amino acid, hypusine (N epsilon-[4-amino-2-hydroxybutyl]lysine). This posttranslational event occurs only following activation of lymphocyte growth. Hypusine formation increases at a rate parallel to protein synthesis during the first 24 hr of growth stimulation, beginning before 6 hr of growth. At all times, hypusine is restricted primarily to the single protein, Hy+. In resting cells, the unmodified substrate protein, Hy0, is continuously synthesized and maintained in a steady-state pool of significant size. In several other cell lines, hypusine formation was also observed in a single protein of approximately 18 kd, pI approximately 5.1, indistinguishable electrophoretically from the lymphocyte protein. Thus Hy+ is a ubiquitous protein showing significant conservation among divergent species. Maintenance by resting lymphocytes of a pool of unmodified protein and early activation during growth of the hypusine-forming enzyme system suggest that this posttranslational modification may be of importance to lymphocyte activation.     

Functional significance of eIF5A and its hypusine modification in eukaryotes

The unusual basic amino acid, hypusine [N^ε-(4-amino-2-hydroxybutyl)-lysine], is a modified lysine with the addition of the 4-aminobutyl moiety from the polyamine spermidine. This naturally occurring amino acid is a product of a unique posttranslational modification that occurs in only one cellular protein, eukaryotic translation initiation factor 5A (eIF5A, eIF-5A). Hypusine is synthesized exclusively in this protein by two sequential enzymatic steps involving deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH). The deoxyhypusine/hypusine synthetic pathway has evolved in archaea and eukaryotes, and eIF5A, DHS and DOHH are highly conserved suggesting a vital cellular function of eIF5A. Gene disruption and mutation studies in yeast and higher eukaryotes have provided valuable information on the essential nature of eIF5A and the deoxyhypusine/hypusine modification in cell growth and in protein synthesis. In view of the extraordinary specificity and functional significance of hypusine-containing eIF5A in mammalian cell proliferation, eIF5A and the hypusine biosynthetic enzymes are novel potential targets for intervention in aberrant cell proliferation.     

The identification of an eukaryotic initiation factor 4D precursor in spermidine-depleted Chinese hamster ovary cells

When Chinese hamster ovary cells are incubated with [terminal methylenes-3H]spermidine, radioactivity is incorporated into a single cellular protein, eukaryotic initiation factor 4D (eIF-4D), through posttranslational synthesis of the amino acid hypusine (N epsilon-(4-amino-2-hydroxybuyly)lysine). The effect of spermidine depletion on this protein modification reaction was studied by high resolution two-dimensional gel electrophoresis. Factor eIF-4D containing both [3H]lysine and [3H]hypusine was detected as one of the major labeled cellular proteins on the fluorographic map of the proteins from Chinese hamster ovary cells that had been incubated with [3H]lysine. When these cells were depleted of spermidine by the use of DL-alpha-difluoromethylornithine before addition of [3H]lysine, no radiolabeling of this mature eIF-4D (hypusine form, Mr approximately 18,000; pI approximately 5.3) occurred. Instead, a new radiolabeled protein (Mr 18,000; pI 5.1) that contained [3H]lysine but no [3H]hypusine or [3H]deoxyhypusine was seen. This protein was identified as an eIF-4D precursor by comparison of the two-dimensional map of its tryptic peptides with that of the tryptic peptides from [3H]lysine-labeled eIF-4D. Further comparisons also suggest that additional post-translational modification processes are involved in the biogenesis of eIF-4D.     

Inactivation of eukaryotic initiation factor 5A (eIF5A) by specific acetylation of its hypusine residue by spermidine/spermine acetyltransferase 1 (SSAT1)

eIF5A (eukaryotic translation initiation factor 5A) is the only cellular protein containing hypusine [N?-(4-amino-2-hydroxybutyl)lysine]. eIF5A is activated by the post-translational synthesis of hypusine and the hypusine modification is essential for cell proliferation. In the present study, we report selective acetylation of the hypusine and/or deoxyhypusine residue of eIF5A by a key polyamine catabolic enzyme SSAT1 (spermidine/spermine-N1-acetyltransferase 1). This enzyme normally catalyses the N1-acetylation of spermine and spermidine to form acetyl-derivatives, which in turn are degraded to lower polyamines. Although SSAT1 has been reported to exert other effects in cells by its interaction with other cellular proteins, eIF5A is the first target protein specifically acetylated by SSAT1. Hypusine or deoxyhypusine, as the free amino acid, does not act as a substrate for SSAT1, suggesting a macromolecular interaction between eIF5A and SSAT1. Indeed, the binding of eIF5A and SSAT1 was confirmed by pull-down assays. The effect of the acetylation of hypusine on eIF5A activity was assessed by comparison of acetylated with non-acetylated bovine testis eIF5A in the methionyl-puromycin synthesis assay. The loss of eIF5A activity by this SSAT1-mediated acetylation confirms the strict structural requirement for the hypusine side chain and suggests a possible regulation of eIF5A by hypusine acetylation/deacetylation.     

The post-translational synthesis of a polyamine-derived amino acid, hypusine, in the eukaryotic translation initiation factor 5A (eIF5A)

The eukaryotic translation initiation factor 5A (eIF5A) is the only cellular protein that contains the unique polyamine-derived amino acid, hypusine [Nepsilon-(4-amino-2-hydroxybutyl)lysine]. Hypusine is formed in eIF5A by a novel post-translational modification reaction that involves two enzymatic steps. In the first step, deoxyhypusine synthase catalyzes the cleavage of the polyamine spermidine and transfer of its 4-aminobutyl moiety to the epsilon-amino group of one specific lysine residue of the eIF5A precursor to form a deoxyhypusine intermediate. In the second step, deoxyhypusine hydroxylase converts the deoxyhypusine-containing intermediate to the hypusine-containing mature eIF5A. The structure and mechanism of deoxyhypusine synthase have been extensively characterized. Deoxyhypusine hydroxylase is a HEAT-repeat protein with a symmetrical superhelical structure consisting of 8 helical hairpins (HEAT motifs). It is a novel metalloenzyme containing tightly bound iron at the active sites. Four strictly conserved His-Glu pairs were identified as iron coordination sites. The structural fold of deoxyhypusine hydroxylase is entirely different from those of the other known protein hydroxylases such as prolyl 4-hydroxylase and lysyl hydroxylases. The eIF5A protein and deoxyhypusine/hypusine modification are essential for eukaryotic cell proliferation. Thus, hypusine synthesis represents the most specific protein modification known to date, and presents a novel target for intervention in mammalian cell proliferation.     

Growth inhibition and cytotoxicity of tumor necrosis factor in L929 cells is enhanced by high cell density and inhibition of mRNA synthesis

L929 cells were growth-inhibited after 1 to 2 days of treatment with human recombinant tumor necrosis factor (rTNF). This effect of rTNF was largely reversible, and L929 cells resumed normal growth when rTNF was removed. The rTNF showed growth inhibitory and cytotoxic activity when L929 cells approached a high cell density and grew slowly. This was shown in experiments in which L929 cells approached confluency at different times after being seeded at increasing initial densities. The rTNF had little effect on the growth of cells seeded at the lowest density tested. L929 cells cultured to high density synthesized RNA at a reduced rate. This suggested that a reduced rate of RNA synthesis may be at least in part responsible for the growth inhibitory and cytotoxic activities of rTNF on cells grown to high density. Treatment with inhibitors of RNA synthesis potentiated the cytotoxic activity of rTNF. Inhibition of mRNA synthesis was apparently responsible for the enhanced sensitivity to rTNF, as shown by experiments with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, an inhibitor of the synthesis of poly(A)-containing RNA.     

A novel mouse model for inhibition of DOHH-mediated hypusine modification reveals a crucial function in embryonic development,proliferation and oncogenic transformation

The central importance of translational control by post-translational modification has spurred major interest in regulatory pathways that control translation. One such pathway uniquely adds hypusine to eukaryotic initiation factor 5A (eIF5A), and thereby affects protein synthesis and, subsequently, cellular proliferation through an unknown mechanism. Using a novel conditional knockout mouse model and a Caenorhabditis elegans knockout model, we found an evolutionarily conserved role for the DOHH-mediated second step of hypusine synthesis in early embryonic development. At the cellular level, we observed reduced proliferation and induction of senescence in 3T3 Dohh^−/− cells as well as reduced capability for malignant transformation. Furthermore, mass spectrometry showed that deletion of DOHH results in an unexpected complete loss of hypusine modification. Our results provide new biological insight into the physiological roles of the second step of the hypusination of eIF5A. Moreover, the conditional mouse model presented here provides a powerful tool for manipulating hypusine modification in a temporal and spatial manner, to analyse both how this unique modification normally functions in vivo as well as how it contributes to different pathological conditions.KEY WORDS: Hypusine modification, Translational control, Cancer, Mouse models     

The effect of hypusine modification on the intracellular localization of eIF5A

Eukaryotic translation initiation factor 5A (eIF5A) is a highly conserved protein essential for eukaryotic cell proliferation and is the only protein containing hypusine, [N^ε-(4-amino-2-hydroxybutyl)lysine]. eIF5A is activated by the post-translational synthesis of hypusine. eIF5A also undergoes an acetylation at specific Lys residue(s). In this study, we have investigated the effect of hypusine modification and acetylation on the subcellular localization of eIF5A. Immunocytochemical analyses showed differences in the distribution of non-hypusinated eIF5A precursor and the hypusine-containing mature eIF5A. While the precursor is found in both cytoplasm and nucleus, the hypusinated eIF5A is primarily localized in cytoplasm. eIF5A mutant proteins, defective in hypusine modification (K50A, K50R) were localized in a similar manner to the eIF5A precursor, whereas hypusine-modified mutant proteins (K47A, K47R, K68A) were localized mainly in the cytoplasm. These findings provide strong evidence that the hypusine modification of eIF5A dictates its localization in the cytoplasmic compartment where it is required for protein synthesis.     

Inflating bacterial cells by increased protein synthesis

Understanding how the homeostasis of cellular size and composition is accomplished by different organisms is an outstanding challenge in biology. For exponentially growing Escherichia coli cells, it is long known that the size of cells exhibits a strong positive relation with their growth rates in different nutrient conditions. Here, we characterized cell sizes in a set of orthogonal growth limitations. We report that cell size and mass exhibit positive or negative dependences with growth rate depending on the growth limitation applied. In particular, synthesizing large amounts of “useless” proteins led to an inversion of the canonical, positive relation, with slow growing cells enlarged 7‐ to 8‐fold compared to cells growing at similar rates under nutrient limitation. Strikingly, this increase in cell size was accompanied by a 3‐ to 4‐fold increase in cellular DNA content at slow growth, reaching up to an amount equivalent to ~8 chromosomes per cell. Despite drastic changes in cell mass and macromolecular composition, cellular dry mass density remained constant. Our findings reveal an important role of protein synthesis in cell division control.     

Posttranslational synthesis of hypusine: evolutionary progression and specificity of the hypusine modification

Summary. A naturally occurring unusual amino acid, hypusine [N ^ɛ-(4-amino-2-hydroxybutyl)-lysine] is a component of a single cellular protein, eukaryotic translation initiation factor 5A (eIF5A). It is a modified lysine with structural contribution from the polyamine spermidine. Hypusine is formed in a novel posttranslational modification that involves two enzymes, deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH). eIF5A and deoxyhypusine/hypusine modification are essential for growth of eukaryotic cells. The hypusine synthetic pathway has evolved in eukaryotes and eIF5A, DHS and DOHH are highly conserved, suggesting maintenance of a fundamental cellular function of eIF5A through evolution. The unique feature of the hypusine modification is the strict specificity of the enzymes toward its substrate protein, eIF5A. Moreover, DHS exhibits a narrow specificity toward spermidine. In view of the extraordinary specificity and the requirement for hypusine-containing eIF5A for mammalian cell proliferation, eIF5A and the hypusine biosynthetic enzymes present new potential targets for intervention in aberrant cell proliferation.     

Replacement perfusion of cultured eucaryotic cells: A method for the accurate measurement of the rates of growth,protein synthesis,and protein turnover

The fractional rates of protein synthesis (k_s) and degradation (k_p) were studied in the myeloma cell line SP2/0-AG14 grown at different rates (k_g). Cells in spinner flask suspension cultures were maintained at constant cellular density for prolonged periods by replacement perfusion of labeling medium at a rate equivalent to the rate of growth. Total protein synthesis was calculated from the specific-radioactivity of labeled L-leucine in the precursor (medium) and cellular protein. Fractional synthesis rates determined by approach to equilibrium labeling were the same as those determined by equilibrium-pulse labeling kinetics and pulse-chase kinetics. The rate of protein degradation was determined from the established relationship k_g = k_s – k_p. Protein synthesis rates remained constant over a threefold range in the rate of cell growth. At relatively slow growth rates (k_g = 0.017/hr) turnover represented a major fraction of total synthesis (k_p = 0.032/hr = 0.65ks). At rapid growth rates (k_g = 0.058/hr) the value of k_p was less than 0.005/hr. No major difference was observed between the k_s determined for individual cellular proteins (separated by SDS-polyacrylamide (7.5%) gel electro-phoresis) from rapid- and slow-growing cultures. Thus, with an invariable k_s, any change in growth rate is due to an inverse change in the rate of turnover. Since turnover is the balance between synthesis and degradation and since synthesis is unchanging then changes in the growth rate of SP2/0-AG14 should be due to changes in the rate of protein degradation. Experiments were therefore performed to determine the origin of the degradative machinery, ie, cytosolic or lysosomal; autolysis of prelabeled cellular protein (in vitro) was observed only at acidic pH (4.2) and WUS totally inhibited by addition of lcupcptin (10 μM) and pepstatin (2 μM), the specific inhibitors of lysosomal cathepsins B (L) and D, respectively. Since growth rate appears to be regulated by the alterations in the rate of protein degradation and degradation (in vitro) in SP2/0-AG14 appearsto be lysosomal, then one should be able to alter the rate of cellular growth by interfering with rate of lysosomal proteolysis. Indeed, when the lysosomotropic amine NH ₄Cl (10 mM) is added to cells growing with a k_g of 0.018/hr ± 0.001 (k_s = 0.050/hr ± 0.002) the growth rate increased to 0.051/hr ± 0.002 without change in the rate of protein synthesis (k_s = 0.049/hr ± 0.003). It is suggested from our data that the cellular growth rate of SP2/0-AG14 is regulated by the lysosomal apparatus; whether this regulation is itself regulated by either a specific compartmentalization of the lysosomal proteinases and/or their substrates or by endogenous protease inhibitors, should prove to be an exciting area for future investigation.     

Protein synthesis initiation factor eIF-4D. Functional comparison of native and unhypusinated forms of the protein

Protein synthesis initiation factor eIF-4D is a relatively abundant protein in mammalian cells and possesses a unique amino acid residue, hypusine. The role of the hypusine modification in eIF-4D function was addressed by studying the function of eIF-4D variants lacking hypusine. The cloned human cDNA encoding eIF-4D was overexpressed in Escherichia coli and a precursor form lacking hypusine was purified. This protein fails to stimulate methionyl-puromycin synthesis in vitro, nor does it significantly inhibit the action of native eIF-4D. Mammalian expression vectors were constructed with the wild-type cDNA and a mutant form in which the codon for lysine-50 (the residue hypusinated) was altered by site-directed mutagenesis to that for arginine. Transient co-transfection of COS-1 cells with the eIF-4D vector and a vector expressing dihydrofolate reductase led to strong synthesis of both eIF-4D and dihydrofolate reductase. This indicates that normal cellular levels of eIF-4D are saturating in these cells and that excess levels of eIF-4D are not detrimental. Cotransfection with the eIF-4D arginine variant caused no effect on dihydrofolate reductase synthesis, in agreement with the in vitro experiments. The inability of the unhypusinated eIF-4D variants to stimulate methionyl-puromycin synthesis in vitro and to affect protein synthesis in vivo strongly suggests that the hypusine modification is required for eIF-4D activity and for its interaction with the 80 S initiation complex in protein synthesis.     

真核翻译起始因子5A在蛋白质合成中的功能及机制

在蛋白质合成过程中,除核糖体、氨酰 tRNA和mRNA外,还有多种翻译因子参与其中。真核翻译起始因子5A（eukaryotic translation initiation factor 5A, eIF5A）是维持细胞活性必不可少的翻译因子,在进化上高度保守。eIF5A是真核细胞中唯一含有羟腐胺赖氨酸（hypusine）的蛋白质,该翻译后修饰对eIF5A的活性至关重要。1978年,人们首次鉴定出eIF5A,认为它在翻译起始阶段促进第1个肽键的形成。直到2013年才证实它主要在翻译延伸阶段调控含多聚脯氨酸基序蛋白质的翻译。在经过四十多年研究后,人们对eIF5A的功能有了新的认识。近期基于核糖体图谱数据的分析表明,eIF5A能够缓解翻译延伸过程中核糖体在多种基序处的停滞,并不局限于多聚脯氨酸基序,并且它还能够通过促进肽链的释放增强翻译终止。此外,eIF5A还可以通过调控某些蛋白质的翻译,间接影响细胞内的各种生命活动。本文综述了eIF5A的多种翻译后修饰、在蛋白质合成和细胞自噬过程中的调控作用以及与人类疾病的关系,并与细菌及古细菌中的同源蛋白质进行了比较,探讨了该因子在进化中的保守性,以期为相关领域的研究提供一定的理论基础。     

Microbial Phospholipid Synthesis as a Marker for Microbial Protein Synthesis in the Rumen

Phosphate uptake into intracellular inorganic phosphorus and cellular phospholipids and the relationship between cell growth and phospholipid synthesis were studied with suspensions of washed ruminal bacteria in vitro with (33)P-phosphorus. It was shown that ruminal bacteria accumulated inorganic phosphate at a low rate when incubated without substrate. Upon the addition of substrate, the rate of inorganic phosphorus uptake into the cells increased markedly, and phospholipid synthesis and cell growth commenced. There was a highly significant relationship (r = 0.98; P < 0.01) between phospholipid synthesis and cell growth. The specific activity of the intracellular inorganic phosphorus did not equilibrate with phosphorus medium. When ruminal contents from sheep fed a high or low protein diet were incubated in vitro, the rate of (33)P incorporation into microbial phospholipids was higher for the high protein diet. Since there was a high relationship between phospholipid synthesis and growth, rumen contents were collected before and various times after feeding and incubated with (33)P-phosphorus in vitro. The short-term, zero time approach was used to measure the rate of microbial phospholipid synthesis in whole rumen contents. In these studies the average specific activity of the intracellular inorganic phosphorus was used to represent the precursor pool specific activity. Microbial phospholipid synthesis was then related to protein (N x 6.25) synthesis with appropriate nitrogen-to-phospholipid phosphorus ratios. Daily true protein synthesis in a 4-liter rumen was 185 g. This represents a rate of 22 g of protein synthesized per 100 g of organic matter digested. These data were also corrected for ruminal turnover. On this basis the rate of true protein synthesis in a 4-liter rumen was 16.1 g of protein per 100 g of organic matter digested. This value represents a 30-g digestible protein-to-Mcal digestible energy ratio which is adequate for growing calves and lambs.     

Synthesis of cytoskeletal and contractile proteins by cultured IMR-90 fibroblasts

Models of the assembly of cytoskeletal and contractile proteins of eukaryotic cells require quantitative information about the rates of synthesis of individual component proteins. We applied the dual isotope technique of Clark and Zak (1981, J. Biol. Chem., 256:4863-4870) to measure the synthesis rates of cytoskeletal and contractile proteins in stationary and growing cultures of IMR-90 fibroblasts. Fibroblast proteins were labeled to equilibrium with [14C]leucine over several days, at the end of which there was a 4-h pulse with [3H]leucine. Fractional synthesis rates (percent per hour) were calculated from the 3H/14C ratio of cell protein extracts or protein purified by one- or two-dimensional polyacrylamide gel electrophoresis and the 3H/14C ratio of medium-free leucine. The average fractional synthesis rate for total, SDS- or urea-soluble; Triton-soluble; and cytoskeletal protein extracts in stationary cells each was approximately 4.0%/h. The range of values for the synthesis of individual proteins from total cell extracts or cytoskeletal extracts sliced from one-dimensional gels was similar, though this range was greater than that for major proteins of Triton-soluble protein extracts. Three specific cytoskeletal proteins--actin, vimentin, and tubulin--were synthesized at similar rates that were significantly slower than the average fractional synthesis rate for total protein. Myosin, on the other hand, was synthesized faster than average. Synthesis rates were the same for beta-and gamma-actin and polymerized (cytoskeletal extract) vs. Triton-soluble actin. The same was true for alpha- and beta-tubulin and two different forms of vimentin. Synthesis rates were uniformly higher in growing cells, though the same pattern of differential rates was observed as for stationary cells. Synthesis rates in growing cells were higher than the rate necessary to maintain the growth rate, even for those cytoskeletal proteins being synthesized slowly. Therefore, there appears to be some turnover of these cytoskeletal elements even during growth. We conclude that proteins in cytoskeletal extracts may have nonuniform rates of synthesis, but at least one important subclass of cytoskeletal proteins that comprise filament subunits have the same synthesis rates.     

Protein turnover and cellular autophagy in growing and growth-inhibited 3T3 cells

The relationship between growth, protein degradation, and cellular autophagy was tested in growing and in growth-inhibited 3T3 cell monolayers. For the biochemical evaluation of DNA and protein metabolism, growth-inhibited 3T3 cell monolayers with high cell density and growing 3T3 cell monolayers with low cell density were labeled simultaneously with [14C]thymidine and [3H]leucine. The evaluation of the DNA turnover and additional [3H]thymidine autoradiography showed that 24 to 5% of 3T3 cells continue to replicate even in the growth-inhibited state, where no accumulation of protein and DNA can be observed. Cell loss, therefore, has to be assumed to compensate for the ongoing cell proliferation. When the data of protein turnover were corrected for cell loss, it was found that the rate constant of protein synthesis in nongrowing monolayers was reduced to half the value found in growing monolayers. Simultaneously, the rate constant of protein degradation in nongrowing monolayers was increased to about 1.5-fold the value of growing monolayers. In parallel to the increased rate constant of protein degradation, the cytoplasmic volume fraction of early autophagic vacuoles (AVs) as determined by electron microscopic morphometry was found to be increased twofold in nongrowing 3T3 cell monolayers when compared with the volume fraction of early AVs in growing 3T3 cell monolayers. These data are in agreement with the assumption that cellular autophagy represents a major pathway of regulating protein degradation in 3T3 cells and that the regulation of autophagic protein degradation is of relevance for the transition from a growing to a nongrowing state.     

Relationship of transformation, cell density, and growth control to the cellular distribution of newly synthesized glycosaminoglycan

Mouse 3T3 cells and their Simian Virus 40-transformed derivatives (3T3SV) were used to assess the relationship of transfromation, cell density, and growth control to the cellular distribution of newly synthesized glycosaminoglycan (GAG). Glucosamine- and galactosamine- containing GAG were labeled equivalently by [3H=A1-glucose regardless of culture type, allowing incorporation into the various GAG to be compared under all conditions studied. Three components of each culture type were examined: the cells, which contain the bulk of newly synthesized GAG and are enriched in chondroitin sulfate and heparan sulfate; cell surface materials released by trypsin, which contain predominantly hyaluronic acid; and the media , which contain predominantly hyaluronic acid and undersulfated chondroitin sulfate. Increased cell density and viral transformation reduce incorporation into GAG relative to the incorporation into other polysaccharides. Transformation, however, does not substantially alter the type or distribution of newly synthesized GAG; the relative amounts and cellular distributions were very similar in 3T3 and 3T3SV cultures growing at similar rates at low densities. On the other hand, increased cell density as well as density-dependent growth inhibition modified the type and distribution of newly synthesized GAG. At high cell densities both cell types showed reduced incorporation into hyaluronate and an increase in cellular GAG due to enhanced labeling of chondroitin sulfate and heparan sulfate. These changes were more marked in confluent 3T3 cultures which also differed in showing substantially more GAG label in the medium and in chondroitin-6-sulfate and heparan sulfate at the cell surface. Since cell density and possibly density- dependent inhibition of growth but not viral transformation are major factors controlling the cellular distribution and type of newly synthesized GAG, differences due to GAG's in the culture behavior of normal and transformed cells may occur only at high cell density. The density-induced GAG alterations most likely involved are increased condroitin-6-sulfate and heparan sulfate and decreased hyaluronic acid at the cell surface.     

Induction of an oligodendroglial enzyme in C-6 glioma cells maintained at high density or in serum-free medium.

The relationship between cell density and the activity of 2':3'-cyclic nucleotide 3'-phosphohydrolase (CNP), an enzyme believed to be specific to oligodendroglial cells and myelin in the brain, has been studied in cultured C-6 glioma cells. Over a 12-day period, the specific activity of CNP underwent a 4-fold increase in conjunction with an increase in the cell density (total protein/flask) and a decline in the growth rate of the cultures. In contrast, the specific activity of Na+,K+-ATPase was not influenced by cell density. Experiments with cultures seeded at different initial densities indicated that the increase in CNP activity coincided with the attainment of a specific cell density rather than with the length of time that the cells were maintained in culture. Arrest of cell proliferation in non-confluent C-6 cells by means of thymidine blockade was not sufficient to cause an increase in the activity of CNP; however, removal of serum from the culture medium resulted in a 3-fold induction of the enzyme in the absence of a high degree of cell contact. The induction of CNP in cells maintained in serum-free medium paralleled the development of a series of distinct morphological changes reminiscent of glial differentiation, which occurred within 48 hours after removal of the serum. Inhibition of protein synthesis by cycloheximide prevented the induction of CNP in serum-free cultures. The demonstration that an enhancement of an oligodendroglial characteristic in C-6 glioma cells can be obtained by growing the cells to high density or by removing serum from the medium, provides further support for the suggestion that these cells may be analogous to the glial stem cells present in the developing brain.