Identification of albumin-synthesizing polysomes from mouse liver and a mouse hepatoma cell line.

Albumin-synthesizing polysomes from mouse liver and mouse hepatoma cells in in tissue culture have been localized on sucrose gradients with ¹²⁵I-labeled antimouse serum albumin used as a marker. Competition studies show that the ¹²⁵I-labeled antibody binds specifically to albumin-synthesizing polysomes from both tissues. The ¹²⁵I-labeled polysomes from liver and hepatoma cells have identical sedimentation properties on sucrose gradients, which indicates that the polysomes range in size from 9–14 ribosomes. This is comparable in size to polysomes from rat liver and Morris hepatoma. One significant difference between these albumin-synthesizing polysomes is that those extracted from hepatoma cells bind 70% less antibody than equivalent amounts of polysomes from liver cells. Since the level of albumin synthesis in the hepatoma cells is comparable to the level of albumin synthesis $\text{in vivo}$, this difference in antibody-binding capacity is not likely to be due to differences in polysomal content, but appears to be a characteristic difference between hepatoma and normal mouse liver cells.     

Stimulative effect of serum on the growth of HeLa cells suppressed in a K+-deficient chemically defined medium

Growth of HeLa cells cultured with a chemically defined medium was slightly stimulated in the presence of 5% dialyzed calf serum. The growth-promoting action of serum was more conspicuous when cell growth was suppressed in the same medium, in which K⁺ was replaced by Rb⁺ to various ratios. The growth-promoting factors(s) of serum was heat-labile. Upon addition of dialyzed serum, passive K⁺ or Rb⁺ influx was increased, whereas the active cation uptake was unaffected and cell K⁺ was rather decreased. The serum did not alter uptake of [³H] amino acids. Also, protein synthesis inhibited in the Rb⁺-substituted medium was not stimulated significantly, except that observed only when the external K⁺/Rb⁺ ratio was $\text{1}\text{5}$. From the distinct effects of serum on cell growth and protein synthesis, we conclude that (i) the serum-induced stimulation of cell growth, which is suppressed in the Rb⁺-substituted medium, is not a result of the direct effect of serum on synthesis of bulk protein, but a reflection of the effect on another mechanism(s) required for cell growth; and that (ii) this action is basically identical with the growth-promoting action on cells cultured in the normal medium.     

Altered growth factor requirements and cell cycle control in rat hepatoma cells versus adult rat hepatocytes in culture

Adult rat hepatocytes multiply in primary cultures when incubated in arginine-free MX-83 medium supplemented with dialyzed fetal calf serum, insulin, glucagon, hydrocortisone, epidermal growth factor, and transferrin. In the absence of mitogens, the fraction of the cells engaged in DNA synthesis dropped sharply. However, cells initiated DNA synthesis in response to the mitogenic mixture indicating that hepatocyte proliferation is controlled by G1----S transition rates. In contrast, rat hepatoma line DTH-3, derived from Morris 7777 "minimal deviation" hepatoma, required only insulin for proliferation in chemically defined MX-83 medium. The lengths of their cell cycle phases varied with the growth rate. The phases of the growth cycle were proportionately shortened (expanded) when the growth rate was increased (decreased). It is concluded that DTH-3 hepatoma cells, which display a decreased growth factor requirement as compared with adult rat hepatocytes differ from normal hepatocytes by fundamental alterations in the mechanisms controlling the progression of the cell cycle.     

Chymotrypsin stimulated development of delayed implantation mouse blastocysts

Chymotrypsin-like enzyme activity increases transiently in the uterine lumen of ovariectomized mice upon administration of progesterone and estrogen (1). This is one of the few known macromolecular changes associated with conditions which result in activation of delayed implantation blastocysts $\text{in}$$\text{utero}$. $\text{In}$$\text{vitro}$, α-chymotrypsin (100 μg/ml) was found to shorten the time required for these embryos to attach to the glass culture dish and then form outgrowths in fetal calf serum-supplemented medium. Higher concentrations of the enzyme (250 μg/ml) prevented embryo attachment probably by digesting the fetuin present in fetal calf serum. Nevertheless, 250 μg/ml α-chymotrypsin could apparently replace fetal calf serum as a stimulator of development during the first 24 hours of culture. In contrast, bovine serum albumin (3.0 mg/ml) seemed to slow development of blastocysts $\text{in}$$\text{vitro}$. It is suggested that chymotrypsin-like enzyme activity may stimulate development of delayed implantation blastocysts $\text{in}$$\text{utero}$ (a) indirectly by removing inhibitory proteins such as albumin and (b) by directly affecting these embryos in a manner yet to be determined.     

Depression of amino acid transport in cultured rat hepatocytes by purified enterotoxin from Clostridiumperfringens

Rat liver parenchymal cells (hepatocytes) were isolated by a collagenase perfusion technique and maintained as monolayers in serum-free medium in collagen-coated culture dishes. Glucagon, in combination with dexamethasone, induced α-aminoisobutyric acid transport in these cells. Addition of purified $\text{Clostridium}\text{perfringens}$ enterotoxin to hepatocytes preinduced by glucagon and dexamethasone rapidly depressed (but did not abolish) α-aminoisobutyric acid transport. The toxin effect was dose dependent: 1000 or 300 ng/ml produced maximal depression whereas 100 or 40 ng/ml were without effect in 120 minutes. The effect was eliminated by pretreating the toxin with heat or specific antisera. The effect of enterotoxin on α-aminoisobutyric acid transport in two cultured rat hepatoma cell lines (H4-II-E-C3 and McA-RH 7777) was also investigated. Only the McA-RH 7777 cells were sensitive to the toxin suggesting that the enterotoxin may interact with specific membrane components of normal rat liver cells which are also present on some (but not all) cancerous rat liver cells.     

Metabolism of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine by cell cultures

The metabolism of 1-alkyl-2-acetyl-$\text{sn}$-glycero-3-phosphocholine (platelet-activating factor) was studied using various cultured cell lines. All incubations were done in the presence of bovine serum albumin and serum-free media, since albumin eliminates the adsorption of 1-alkyl-2-acetyl-$\text{sn}$-glycero-3-phosphocholine to cultureware and serum enzymes interfere. Human leukemia (HL-60) cells, MDCK canine kidney cells, and transformed and nontransformed clones of mouse C3H1OT1/2 cells display varying rates of uptake, degradation, and capacities for reacylation of 1-alkyl-2-acetyl-$\text{sn}$-glycero-3-phosphocholine. HL-60 cells displayed the highest uptake rate (24.6 pmol/mg cell protein/15 min). Whereas C3H10T1/2 cells in culture showed uptake rates comparable to other cells tested, they displayed a relative metabolic inertness towards 1-alkyl-2-acetyl-$\text{sn}$-glycero-3-phosphocholine.     

Nucleolar localization of protein B23 (375.1) by immunocytochemical techniques

Highly acidic phosphoprotein B23 ($\text{37}\text{5.1}\text{;}\text{M.W. x}\text{10}{}^3\text{/}\text{pI}$) which is in preribosomal RNP particles in nucleoli of Novikoff hepatoma cells (1) was found to be one of the two major silver staining nucleolar proteins (2). An improved isolation method was developed for protein B23 which included 4 M urea/3 M LiCl extraction of nucleoli, dialysis of the extract against 4 M urea/20 mM Tris-malate/pH 5.5 and DEAE-cellulose chromatography. For studies on cellular localization of this protein, highly purified protein B23 was used to produce anti- B23 antibodies in rabbits. The specificity of the anti- B23 antibodies was demonstrated by formation of immunoprecipitin bands with the purified antigen and crude nucleolar extracts from Novikoff hepatoma cells. With the indirect peroxidase immunostaining method, a specific localization of protein B23 was demonstrated in the nucleoli of normal rat liver, thioacetamide-treated rat liver and Novikoff hepatoma cells.     

Reduction of lactic acid secreted from SV3T3 cells by a serum factor and biotin and its relationship to cell growth

Supplementation of media containing a low concentration (0.15–0.30% $\text{v}\text{>v}$) of calf serum with biotin or a low molecular weight serum growth factor (Peak III) reduces the amount of lactic acid secreted by simian virus 40-transformed 3T3 cells. While biotin and Peak III (which has been tentatively identified as biotin) can stimulate “stationary phase” cells to resume viable cell division, this growth promotion is not due to an alleviation of lactic acid toxicity per se. This conclusion is based on the finding that, although higher concentrations of lactic acid are cytotoxic, lactic acid added at concentrations found during “stationary phase” to cells plated in fresh medium is not growth inhibitory. These results suggest, instead, a possible major role for biotin and Peak III in energy production.     

Effect of amino acid and serum deprivation on the regulation of RNA synthesis in cultured Chinese hamster ovary cells

In a proline-requiring Chinese hamster ovary cell line, if both proline and serum are removed from the culture medium, net RNA synthesis is reduced to about 12 % of the unstarved control. This reduction in RNA synthesis is comparable to the stringent regulation of RNA in bacteria. A beta-globulin carbohydrate containing (3.5 % $\text{w}\text{w}$) protein factor was isolated and partially purified from fetal calf serum. The isolated serum factor is able to replace whole serum in stimulating cellular RNA synthesis and has an RNAase inhibitory effect in vitro. The effect of proline starvation and serum factor deprivation on RNA synthesis are independent and additive; each regulates about half of the total RNA synthesized. The regulation appears to affect the synthesis of all species of cytoplasmic RNA.     

Steroid induction of nerve growth factor synthesis in cell culture

Nerve growth factor (NGF) is a peptide hormone which is necessary for the development of sympathetic neurons. Exposing a rat central nervous system glioma cell line (C-6) to the steroid hormone 17β-estradiol increases the amount of NGF secreted by these cells into the surrounding medium. This induction is highly specific to 17β-estradiol in that similar steroids do not increase NGF levels. Both NGF activity and protein levels increase upon estradiol stimulation and there is a parallel increase in NGF $\text{de}$$\text{novo}$ synthesis. The estradiol effect can be blocked with actinomycin D but not with puromycin or cycloheximide. This is the first report demonstrating regulation of NGF synthesis by a steroid hormone in a clonal cell line of glial origin. We propose this system as a model system for the study of the regulation of NGF synthesis and the isolation and analysis of putative precursors to the NGF molecule.     

Suppression of rat mammary tumor cell growth in vitro by glucocorticoids requires serum proteins. Characterization of wild type and glucocorticoid-resistant epithelial tumor cells

CON8 is a single-cell derived subclone of the 13762NF transplantable, hormone-responsive rat mammary tumor that proliferates rapidly in serum-free medium. Addition of either glucocorticoids or calf serum alone caused a slight stimulation of CON8 proliferation. However, glucocorticoids required the presence of specific serum proteins to strongly suppress CON8 cell growth. Furthermore, the anchorage-independent growth of CON8 cells was significantly reduced in the presence of glucocorticoids and serum. We have designated this serum activity GMGSF, for glucocorticoid modulating growth suppression factor. Inhibition of cell growth was limited to steroids with strong glucocorticoid biological activity, while exposure to the glucocorticoid antagonist RU38486 prevented this response. Half-maximal growth inhibition and half-maximal expression of a glucocorticoid-inducible gene product (2 nM) occurred slightly below the half-maximal receptor binding of [3H]dexamethasone (10nM). We have also selected a variant mammary epithelial tumor cell line, derived from CON8, denoted 8RUV7, whose proliferation and soft agar colony formation failed to be suppressed by glucocorticoids in the presence of serum. These glucocorticoid-resistant variant cells possess functional glucocorticoid receptors, competently produce the glucocorticoid-responsive gene product plasminogen activator inhibitor, and along with CON8 cells express milk fat globule protein antigens on their cell surface, indicative of their mammary epithelial cell character. We are using this variant line to genetically dissect the molecular mechanism of the glucocorticoid/GMGSF growth suppression pathway in mammary epithelial tumor cells.     

An 18 000-dalton protein metabolically labeled by polyamines in various mammalian cell lines

The possible role of polyamines in the covalent modification of cellular protein(s) was investigated by studying the metabolic labeling of NB-15 mouse neuroblastoma cells by [¹⁴C]putrescine in fresh Dulbecco's medium followed by separation of cellular proteins through sodium dodecyl sulfate-polyacrylamide gel electrophoreses. Under such incubation conditions, a single protein band with an apparent molecular weight of 18 000 was radioactively labeled. [¹⁴C]Spermidine also specifically labeled this protein. The majority of the radioactivity covalently linked to the 18-kDa protein was recovered as hypusine. The radioactive labeling of this protein was stimulated 1.3-fold by 1 mM dibutyryl cAMP and 2.8-fold by 4% fetal calf serum. Fetal calf serum also stimulated the labeling of many other cellular proteins. This may be due to the conversion of putrescine to amino acids via the formation of γ-aminobutyric acid. Aminoguanidine, a potent inhibitor of diamine oxidase, completely inhibited the fetal calf serum-stimulated labeling of these cellular proteins but had no effect on the labeling of the 18-kDa protein. The specific labeling of the 18-kDa protein by [¹⁴C]putrescine occurred in various mammalian cells examined including the N-18 mouse neuroblastoma cells, 3T3-L1 murine preadipocytes, and H-35 rat hepatoma cells. The specificity of labeling of the apparently ubiquitous 18-kDa protein and the stimulation of this labeling by fetal calf serum suggest that this protein may be important in mediating some of the actions of polyamines in cell growth regulation.     

Differences in chromatin proteins of growing and non-growing tissues.

The non-histone chromatin proteins of growing and non-growing tissues were compared by two-dimensional polyacrylamide gel electrophoresis. The tissues studied were normal rat liver, regenerating rat liver, thioacetamide-treated rat liver, normal rat kidney, Novikoff hepatoma and Walker 256 carcinosarcoma. Although most of the protein components were common to all of the tissues studied, the densities and sizes of spots C18, CP, C21, C25, CQ, CR, CS and CT were greater in the growing tissues than in the non-growing tissues, including the thioacetamide-treated liver. In the latter, the increased densities and sizes of spots C18, C21 and CQ are presumably related to the markedly increased nucleolar size rather than to cell division. Accordingly the increases in sizes and densities of spots C25, CP, CR, CS and CT are apparently of importance to the growth processes of normal and tumor tissues. The number of tissue specific proteins was small compared with the number of proteins in this fraction and includes BP and CBL for normal liver, BJ′ for kidney and CG′, CH′ and CP$\text{\$}\text{?}$for the tumors.     

EGFR expression and EGF stimulation of proliferation in human liver carcinoma cells

Epidermal growth factor receptor (EGFR) gene expression and growth stimulation of EGF on human hepatoma cells of cell lines BEL-7404 and SMMC-7721 were studied. 125I-EGF binding assay was used to measure the binding characteristics and the amounts of EGFR on these cells. The binding time course and the binding competition assay showed that the binding of 125I-EGF to 7404 cells was saturable and specific. Scatchard analysis of EGF binding curve indicated that 7404 and 7721 cells expressed approximately 1.1 x 10(5) and 0.7 x 10(5) EGFRs per cell with binding affinity (Kd) 2.1 nM and 1.8 nM respectively. Northern hybridization and immunoblotting analysis showed the EGFR gene expression products in 7404 and 7721 cells were 5.6 Kb mRNA and 170 Kilo-dalton glycoprotein. Anchorage-dependent growth of 7404 and 7721 cells was stimulated in the presence of nanogram quantities of EGF in medium containing 10% calf serum or 0.5% calf serum. The factors in serum appeared to act synergitically in stimulating of cell proliferation. EGF also stimulated the anchorage-independent growth of 7404 and 7721 cells in soft agar. The results suggest that EGFR is actively expressed in human hepatoma 7404 and 7721 cells and EGF may be one of the mitogens needed for the growth of hepatoma cells.     

Cell growth factor activity: New quantitative method in cell culture assay

This quantitative method for the assay of growth factor activity in vitro was developed empirically. Based on the empirical equations, it seems logical to express the cell growth rate by cell duplication frequency, f, and the growth factor activity by the normalized cell growth rate: $\text{1}\text{(T-T}\text{min}\text{)}$. The latter is recommended as the basis of defining an arbitrary unit of growth factor activity. For the in vitro growth factor activity assay, the quantitative method based on the empirical equations has two important features: (1) it appears to express linearly the growth factor concentration in assay, and (2) it is not affected by the initial cell counts and the length of cultivation.The view that these two features overcome the apparent defects associated with the expression systems that use the cell count ratio and net cell count is discussed. The quantitative method based on the empirical equations has been verified with experiments and compared with expression systems using the cell count ratio and net cell count. The experiments, which verified the quantitative method based on the empirical equations, were carried out with four selected cell lines: aortic endothelial cells, mouse 3T3 fibroblasts, Walker 256 carcinoma, and Chang's liver cells, using serum as the source of growth factor(s) for all cell lines. Vitreous, retinal extract, and Walker 256 carcinoma extract were also used for aortic endothelial cells.     

Cell surface antigens on erythroid cells: A comparison of normal and anemic (WW) mice

Cell surface antigens of normal and anemic ($\text{W}\text{W}$) mouse erythroid cells have been examined in cytotoxicity assays with two rat antisera. When tested on fetal liver cells, a rat anti-erythroblast serum recognized antigen(s) present on erythroid cells early in development, while rat anti-adult red blood cell serum recognized antigen(s) present on mature erythroid cells. Each of these sera had different activity on normal (+/+ or $\text{W}\text{+}$) as compared to anemic ($\text{W}\text{W}$) erythroid cells.     

In vitro synthesis of nerve growth factor related glioma C6 cell polypeptides.

Rat glioma C₆ cell polyribosomal preparations were tested in a heterologous $\text{in vitro}$ system for their ability to direct the synthesis of nerve growth factor related polypeptides. Two major polypeptides of MW ～ 21,000 and ～ 43,000 respectively were found, both of which were immunoprecipitable with specific anti-mouse 2.5S nerve growth factor serum. After incubation of $\text{in vitro}$ synthesized proteins with submaxillary gland extract the bulk of these protein species was converted into immunoprecipitable material of MW ～ 13,000, which comigrated in sodium dodecyl sulfate/polyacrylamide gel electrophoresis with mouse 2.5S nerve growth factor.     

Rat alpha-fetoprotein: in vitro production of four molecular variants by clonal cell lines.

Mass and six clonal cultures derived from Morris hepatoma 7777 by standard tissue culture techniques synthesize and secrete alpha-fetoprotein $\text{in vitro}$. During serial passage, the alpha-fetoprotein which accumulates in the media of these cultures contains two concanavalin A-affinity molecular variants. Each of the concanavalin A-affinity molecular variants shows two electrophoretic variants. Mass and clonal cell populations of hepatoma cells continue to secrete $\text{in vitro}$four molecular variants of rat alpha-fetoprotein known to occur $\text{in vivo}$. These results demonstrate for the first time that individual hepatoma cells have the potential to synthesize four molecular variants of alpha-fetoprotein and that this phenotypic property is maintained during serial subculture $\text{in vitro}$.     

Effect of iron on the relative abundance of two large polypeptides of the Escherichiacoli outer membrane

The relative abundance of two polypeptides of the $\text{Escherichia}\text{coli}$ outer membrane is affected by the growth medium. The polypeptides have molecular weights of 85,000 and 95,000 and, in cells grown in medium containing low concentrations of iron, are dominant outer membrane proteins.