Glutamine and the regulation of DNA replication and cell multiplication in fibroblasts

Several studies indicate that glutamine is a critical requirement for cell growth in vitro. Growing and quiescent (serum-starved) 3T3-fibroblasts were exposed to media (Dulbecco's modified Eagle's minimal essential medium) in which the concentration of the 13 essential amino acids had been lowered to 1/100 or 1/1,000 of that in DMEM - either all together or one by one. The effects on DNA synthesis were measured by autoradiographic determinations of the percentage of labeled cells after 24 hours exposure to ³H-thymidine. A reduction of all 13 essential amino acids to 1/100 or 1/1,000 of the normal concentration in the medium resulted only in a minor growth inhibitory effect during the first cell cycle. A similar growth inhibitory effect was caused by the depletion of one of the 13 essential amino acids (except glutamine) from the medium. However, a depletion of glutamine from the medium resulted in a marked inhibition of growth. Conversely, a relative excess of glutamine, when the other 12 amino acids were lowered to 1/1,000 of the normal concentration, counteracted the growth inhibitory effect of serum starvation. It was even possible to stimulate quiescent cells to undergo DNA synthesis by exposing them to a serum-depleted (0.5% serum) medium with a relative excess of glutamine.     

Control of cell multiplication in vitro. IV. Isolation and characteristics of C3H10T1/2-line cells with decreased dependence for multiplication on blood serum

Mouse embryo fibroblasts of C3H10T1/2 cell line were selected in the medium with a low serum content. The frequency of clone occurrence was about 1.10(-5) (for 0.5% serum). The treatment of cells by N-methyl-N'-nitrosoguanidine significantly increased the frequency of occurrence of these clones. The obtained spontaneous variants were assayed for stability of growth characteristics. The phenotypic analysis of clones with low serum growth dependence discovered at least two cell types differing in some morphological and growth characteristics. The former did not differ from the parental line C3H10T1/2 in any phenotype characteristics but their serum growth dependence; whereas, the latter were poorly spread over the substrate and showed an enhanced saturation density and a decreased population doubling time. These cells also differed in their growth dependence on the homologous conditioned medium.     

H1N1流感病毒在微载体培养MDCK细胞上增殖的研究

目的探索MDCK细胞在微载体上的培养条件,并研究H1N1型流感病毒在MDCK细胞上的增殖条件。方法在微载体上培养好MDCK细胞上用H1N1型流感病毒在不同的病毒感染复数(MOI)、胰酶浓度两个关键的病毒增殖条件进行流感病毒在细胞上的增殖研究。结果微载体质量浓度为6 g/L时,MDCK细胞培养密度可以达到4.5×106cells/mL。在MOI为0.05接种流感病毒,胰酶质量浓度4μg/mL,流感病毒在MDCK细胞上可获得较高的滴度。结论 MDCK细胞用微载体培养可以达到较高的细胞密度,可以作为规模化生产新型流感病毒疫苗的主要细胞基质进行进一步的研究。     

Effect of prostaglandins on cell function and multiplication of parainfluenza 3 viruses in WISH cells.

Cytotoxic effect of prostaglandins E2 and F2alpha on cells grown in vitro and the influence of these compounds on multiplication of myxovirus parainfluenza 3 were investigated. The prostaglandins were added to culture medium (0-01-10 mug/ml) 24 hr before virus infection, or for 2 and 48 hr after inoculation with viruses. WISH cells and monkey kidney cell cultures were used. No direct cytotoxic effect of prostaglandins at concentrations 0-01-1 mug/ml was found (viability, supravital staining, phase-contrast system, Nitro-BT reduction and succinic dehydrogenase tests), whereas the concentration of 10 mug/ml within 48 hr led to reduction and succinic dehydrogenase tests), whereas the concentration of 10 mug/ml within 48 hr led to partial injury of the cell population with symptoms of damage to mitochondria. Prostaglandins E2 and F2alpha inhibited multiplication of parainfluenza 3 virus at concentrations 0-1-10 mug/ml. The inhibitory effect was most pronounced if prostaglandins were added to medium for the whole period of virus multiplication i.e. for 48 hr but little or no effect was found if they were added prior to inoculation or for 2 hr after it. Inhibitory effect of prostaglandins on replication phase of viruses is suggested.     

Dissimilar peptide growth factors can induce normal human mesothelial cell multiplication

Summary Quiescent normal human mesothelial (NHM) cells will undergo one round of DNA synthesis when they are incubated in a defined medium consisting of LHC basal medium supplemented with hydrocortisone, insulin, transferrin, and one of the following peptide mitogens: epidermal growth factor; transforming growth factor beta (1 or 2); platelet derived growth factor (a,b heterodimer or b,b homodimer); fibroblast growth factor (acid or basic forms); interleukin 1 (alpha or beta forms); interleukin 2; interferon gamma; interferon beta; or cholera toxin. However, sustained cell multiplication does not occur unless the medium contains hydrocortisone, insulin, transferrin, any one of the above-listed peptide growth factors and high density lipoproteins. Growth can be increased twofold if the medium contains certain combinations of these mitogens and high density lipoproteins. The finding that NHM cells can respond to a broad spectrum of growth factors supports the possibility that an autocrine mechanism may be part of the mechanism that leads to transformation of these cells by asbestos.     

Co-stimulation of T cell proliferation by transforming growth factor-beta 1.

Transforming growth factor-beta (TGF-beta) exhibits diverse regulatory roles in the immune system and has been described as a potent inhibitor of lymphocyte and hemopoietic progenitor cell growth. The present studies investigated the effects of TGF-beta 1 on murine T cell proliferation triggered through the T cell receptor/CD3 complex. In contrast to previously reported T cell growth inhibition, TGF-beta 1 costimulated splenic T cell proliferation in the presence of immobilized anti-CD3 antibody 2C11, with maximal effect at anti-CD3 concentration of 50 micrograms/ml. Although TGF-beta 1 induced a modest increase in IL-2R display, TGF-beta 1 co-stimulated proliferation was largely independent of IL-2 and/or IL-4. Anti-IL-2 and/or anti-IL-4 antibody did not significantly block the TGF-beta 1 co-stimulated T cell growth, and no IL-2 or IL-4 bioactivity was detected in TGF-beta 1 co-stimulated cultures. TGF-beta 1 did not enhance IL-2 mRNA expression beyond control levels. However, TGF-beta 1 co-stimulation caused an accelerated evolution of a memory or mature T cell population phenotype. Both CD4+ and CD8+ T cell subsets were significantly enriched for cells of the mature CD45RBloPgp-1hi phenotype, in comparison with T cells stimulated by anti-CD3 alone or with PMA, and CD8+ T cells predominated. These results thus provide initial evidence that TGF-beta 1 is capable of bifunctional T cell growth regulation, which occurs largely via an IL-2- and IL-4-independent pathway.     

Synergistic actions of cytokines and growth factors in enhancing porcine granulosa cell growth.

In our studies of the growth-promoting effect of a cytokine, interleukin-1 (IL-1), on cultured porcine granulosa cells, we found that the potency of IL-1 action correlated with the serum concentration in the culture medium and that IL-1 acted synergistically with insulin to increase the number of cells in the presence of low serum concentrations (0.1-1%). With granulosa cells maintained in a quiescent state under serum-free conditions, we therefore examined the effects of combined treatment with IL-1 and peptide growth factors, including insulin, on [3H]thymidine incorporation by these cells. IL-1 by itself enhanced [3H]thymidine incorporation in a concentration-dependent manner. Moreover, IL-1 acted synergistically with insulin, epidermal growth factor (EGF), or fibroblast growth factor (FGF) to enhance [3H]thymidine incorporation. Combinations of maximally effective concentrations of insulin (1 micrograms/ml), EGF (1 ng/ml), or FGF (50 ng/ml) with the maximally effective concentration of IL-1 (10 ng/ml) increased the levels of [3H]thymidine incorporation to 10-, 22-, and 20-fold, respectively, over the control values. Whereas IL-2 (0.1-100 ng/ml) did not affect [3H]thymidine incorporation, tumor necrosis factor alpha (TNF alpha) stimulated [3H]thymidine incorporation by itself and reproduced the actions of IL-1 to act synergistically with insulin, EGF, or FGF. When IL-1 and TNF alpha were added together in relatively low concentrations (1 ng/ml each), the combination had synergistic effects in enhancing [3H]thymidine incorporation. The present study demonstrates that cytokines and peptide growth factors act synergistically to markedly enhance porcine granulosa cell growth in vitro.     

Role of the cell surface in the stimulation of cell multiplication

Fertilization of eggs and effects of many growth factors on the membrane receptors of the cultured somatic cells induce similar changes in the plasma membrane transport properties, which determine changes in Ca2+ and H+ concentrations in the cytoplasm. The data are discussed which favour the concept that Ca2+ and H+ are secondary messengers of growth-stimulating influences and control many intracellular processes related to cell multiplication.     

Coordinate control of Balb/c3T3 cell survival and multiplication by serum or calcium pyrophosphate complexes.

Balb/c3T3 cells in crowded cultures detach from the dish when deprived of serum, and the survivors incorporate 3H-thymidine at a reduced rate. The detachment becomes pronounced two hours after removal of serum, and reaches its maximum rate between two and four hours. Cells in sparse culture are not detached by serum removal, and their rate of 3H-thymidine incorporation is only slightly reduced. As the sparse cultures grow into more crowded cultures, and the serum is depleted, increasing numbers detach. The detached cells are incapable of reattaching when placed in a new dish with ample fresh serum. The cells are leaky to cellular constituents and appear to be dead. Detachment is a consequence rather than the cause of cell death, and can be produced by agents which inhibit cellular energy metabolism. The cells on the dish which survive serum deprivation are fully viable and grow rapidly when serum is added. When they become crowded they are as sensitive to serum deprivation as was the original population. They are therefore not selected for a low serum requirement but apparently survive because they spread into the space vacated by the detaching cells and then behave as sparse cultures in response to serum variations. Insoluble complexes of Ca2+ and pyrophosphate (Ca2+-PPi) show the same concentration dependence in promoting cell survival as in stimulating 3H-thymidine incorporation, showing that a single substance can be responsible for both activities. It is concluded that survival and growth are part of the coordinate response of 3T3 cells to single external effectors. The results are discussed in terms of a simple model in which the coordinate response is regulated by the availability of Mg2+ for transphosphorylation reactions within the cell, and the availability depends on the binding affinity of cellular membranes for Mg2+. The difference between survival and multiplication is postulated to be in the intensity and duration rather than the kind of stimulus.     

Comprehension of attachment and multiplication properties by observing individual cell behaviors in anchorage-dependent culture

The behavioral properties of cell attachment and division were characterized by direct observation of individual cells in the culture of murine fibroblasts. At the cell attachment stage in the culture for early 10 h, the extent of cell spreading, which was defined as a ratio of the projected area of each cell against its saturated value, had a relatively broad distribution at 0.25 h, and it shifted to a higher level with elapsed time up to 10 h with narrowing in the distribution. The critical value of the extent of cell spreading was determined to be 0.54 as a threshold at which a cell is assumed to complete its adhesion to culture surface. The ratio of the number of cells with the extent of cell spreading over 0.54 against the total number of examined cells fairly followed the profile of cell adhesion which was obtained by measuring the number of adherent cells on culture surface.

At the cell growth stage in the culture for 20–64 h, doubling time of cell population increased gradually as the culture progressed toward confluence. Generation times (or cell-dividing spans) of individual cells, however, did not show a discriminating dependency on cell concentration and culture time. To clarify the influence of local congestion on the cell division, the generation time was formulated as a function of the number of contact cells around each target cell. Applying the cell placement growth model to estimating the extent of contact inhibition, the reciprocal value of doubling time could be correlated with the average of reciprocal generation times, implying that the doubling time on a cell-population basis is explained by considering the variation in dividing spans of individual cells affected by local contact environment.     

Characterization of the effects of butyric acid on cell proliferation, cell cycle distribution and apoptosis

We examined concentration-dependent changes in cell cycle distribution and cell cycle-related proteins induced by butyric acid. Butyric acid enhanced or suppressed the proliferation of Jurkat human T lymphocytes depending on concentration. A low concentration of butyric acid induced a massive increase in the number of cells in S and G2/M phases, whereas a high concentration significantly increased the accumulation of cells in G2/M phase, suppressed the accumulation of cells in G0/G1 and S phases, and induced apoptosis that cell cycle-related protein expression in Jurkat cells treated with high levels of butyric acid caused a marked decrease in cyclin A, cyclin E, cyclin-dependent kinase 2 (CDK2), CDK4 and CDK6 protein levels in G0/G1 and S phases, with apoptosis induction, and a decrease in cyclin B, Cdc25c and p27KIP1 protein levels, as well as an increase in p21CIP1/WAF1 protein level, in the G2/M phase. Taken together, our results indicate that butyric acid has bimodal effects on cell proliferation and survival. The inhibition of cell growth followed by the increase in apoptosis induced by high levels of butyric acid were related to an increase in cell death in G0/G1 and S phases, as well as G2/M arrest of cells. Finally, these results were further substantiated by the expression profile of butyric acid-treated Jurkat cells obtained by means of cDNA array.     

Extracellular regulation of fibroblast multiplication. Quantitative differences in nutrient and serum factor requirements for multiplication of normal and SV40 virus-transformed human lung cells

The principles of Henri-Michaelis-Menten kinetic analysis were applied to directly relate the concentration of serum growth factors and individual nutrients in the culture medium to the multiplication rate of a population of normal (N-HLF) and SV40 virus-transformed (SV-HLF) human lung fibroblasts. When all nutrient concentrations were optimal and in steady state, the concentration of serum factors that was required to support a half-maximal rate of proliferation of both N-HLF and SV-HLF was similar. When the serum factor concentration was optimal and constant, SV-HLF cells exhibited a reduced requirement (p less than 0.001) for 12 of 27 individual nutrients that were examined. Serum factors control the cellular requirement for Ca2+, K+, Mg2+, phosphate ions, and 2-oxocarboxylic acids for multiplication of N-HLF (McKeehan, W. L., and McKeehan, K. A. (1980) Proc. Natl, Acad, Sci, U. S. A. 77, 3417-3421). SV-HLF exhibited a constitutively reduced requirement for Ca2+, K+, and Mg2+ which partially removed the requirement for the 3 ions for multiplication of SV-HLF from the control of serum factors. The results suggest that SV40 virus transformation confers a growth advantage on human lung fibroblasts by alteration of their quantitative requirements for specific nutrients.     

Dual effects of estradiol on normal and tumor pituitary cell multiplication

We have compared the effects of estradiol on the [3H]thymidine (TdR) incorporation into the DNA of 2 rat tissues whose growth is controlled by estradiol in vivo in 2 opposite directions: the normal anterior pituitary and the MtF4 pituitary tumor transplanted under the kidney capsule. Small pieces of pituitary or tumor from Fischer rats, treated or not by estradiol in silastic tubing, were incubated in vitro with [3H]TdR. The [3H]TdR incorporated per microgram DNA was decreased in tumor after 2 to 8 day-estradiol treatment while simultaneously, in the same rats, it was increased in the pituitary. In addition, we studied the effect of estradiol in vitro on the F4C1 cell line obtained from the MtF4 tumor. A dose-dependent decrease of both the [3H]TdR incorporated into DNA and the DNA amount was observed between 10(-6) and 10(-5) M estradiol. These results suggest that the control of the pituitary or MtF4 tumor growth by estradiol in vivo is in part due to an inhibition of cell multiplication. Although estradiol inhibits the growth of a clone of MtF4 tumor cells in vitro we cannot decide whether or not the in vivo effect of estradiol is direct.     

Semliki Forest virus multiplication in clones of Aedes albopictus cells.

A total of 115 clones of Aedes albopictus cells were examined for their response to infection with Semliki Forest virus. Virus yield and cytopathology showed a bimodal distribution. More than 68% of the clones gave low yields of virus (between 8 x 10(6) and 2 x 10(8) PFU/ml) with no discernable cytopathology, and 30% gave high yields of virus (between 1 x 10(9) and 8 x 10(9) PFU/ml) and showed moderate to severe cytopathology. To determine the level at which restriction in virus growth occurs in the low-virus-producing clones, we compared the nature and extent of several virus-directed events in selected low-virus-producing clones with the same events in high-virus-producing clones. Specifically, we compared virus-specified polypeptide synthesis, positive- and negative-strand RNA synthesis, adsorption, uncoating, and transfection with virion 42S RNA. These studies showed that whereas events before negative-strand RNA synthesis and all subsequent virus-specified events were markedly reduced in the low-virus-producing lines, compared with the high-virus-producing lines. Thus, the restriction in virus growth in the low-virus-producing lines occurs at the level of synthesis of negative-strand RNA. The consequence of this restriction in an early step in the virus multiplication cycle is discussed in terms of the survival of invertebrate cells after alphavirus infection.     

Variations in tumor cell growth rates and metabolism with oxygen concentration, glucose concentration, and extracellular pH.

Tumors and multicellular tumor spheroids can develop gradients in oxygen concentration, glucose concentration, and extracellular pH as they grow. In order to calculate these gradients and assess their impact on tumor growth, it is necessary to quantify the effect of these variables on tumor cell metabolism and growth. In this work, the oxygen consumption rates, glucose consumption rates, and growth rates of EMT6/Ro mouse mammary tumor cells were measured at a variety of oxygen concentrations, glucose concentrations, and extracellular pH levels. At an extracellular pH of 7.25, the oxygen consumption rate of EMT6/Ro cells increased by nearly a factor of 2 as the glucose concentration was decreased from 5.5 mM to 0.4 mM. This effect of glucose concentration on oxygen consumption rate, however, was slight at an extracellular pH of 6.95 and disappeared completely at an extracellular pH of 6.60. The glucose consumption rate of EMT6/Ro cells increased by roughly 40% when the oxygen concentration was reduced from 0.21 mM to 0.023 mM and decreased by roughly 60% when the extracellular pH was decreased from 7.25 to 6.95. The growth rate of EMT6/Ro cells decreased with decreasing oxygen concentration and extracellular pH; however, severe conditions were required to stop cell growth (0.0082 mM oxygen and an extracellular pH of 6.60). Empirical correlations were developed from these data to express EMT6/Ro cell growth rates, oxygen consumption rates, and glucose consumption rates, as functions of oxygen concentration, glucose concentration, and extracellular pH. These empirical correlations make it possible to mathematically model the gradients in oxygen concentration, glucose concentration, and extracellular pH in EMT6/Ro multicellular spheroids by solution of the diffusion/reaction equations. Computations such as these, along with oxygen and pH microelectrode measurements in EMT6/Ro multicellular spheroids, indicated that nutrient concentration and pH levels in the inner regions of spheroids were low enough to cause significant changes in nutrient consumption rates and cell growth rates. However, pH and oxygen concentrations measured or calculated in EMT6/Ro spheroids where quiescent cells have been observed were not low enough to cause the cessation of cell growth, indicating that the observed quiescence must have been due to factors other than acidic pH, oxygen depletion, or glucose depletion.     

In vitro effect of insulin and insulin-like growth factor-I on cell multiplication and adrenocorticotropin responsiveness of fetal adrenal cells

The present study examined the effects of both insulin and insulin-like growth factor-I (IGF-I) on cell division and specific functions of cultured adrenocortical cells from 100- to 122-day-old ovine fetuses. When culture was performed in a serum-free medium containing transferrin and ascorbic acid, the number of cells increased only slightly (1.2-fold) over a 4-day period. Addition of insulin or IGF-I in the culture medium enhanced the number of cells counted on Day 5. The effect of both peptides was dose-dependent, but 10 ng/ml IGF-I was as potent as 10 micrograms/ml insulin. The acute cyclic adenosine 3',5'-monophosphate (cAMP) and steroidogenic responses to adrenocorticotropin (ACTH1-24) decreased in fetal cells cultured in the absence of insulin or ACTH. Insulin at micromolar concentrations not only prevented this decrease but enhanced the acute ACTH1-24-induced cAMP output on Day 5 over that observed on Day 2. Treatment of fetal cells for 4 days with increasing concentrations of insulin or IGF-I enhanced the acute cAMP and steroidogenic responses (3- to 4-fold) to ACTH1-24 over that of control cells. The ED50 of IGF-I was about 3 ng/ml (congruent to 0.4 nM) whereas that of insulin was about 10 ng/ml (1.7 nM). However, a second plateau was apparent at concentrations of insulin above 1 microgram/ml. The acute cholera toxin stimulation of cAMP production of cells cultured in the absence of insulin or ACTH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Long-term multiplication of the Chinese hamster ovary (CHO) cell line in a serum-free medium

Summary A new synthetic medium (referred to as GC₃) that supports the growth of the Chinese hamster ovary cell line has been developed. It is composed of a 1∶1 mixture of Ham's F12 and modified Eagle's minimum essential (MEM.S) mediums supplemented with transferrin (10 μg/ml), insulin (80 mU/ml), and selenium (1×10⁻⁷ M). Other more simple supplementations of our basal medium MEM.S/F12 (transferrin+insulin, transferrin+selenium, ferrous iron+selenium) also give good cell growth responses. Fibronectin or serum pretreatment is not needed for cellular attachment and spreading. Our culture system is characterized by a continuous serum-free cultivation (more than 200 doublings), a clonal growth, a high density proliferation, and a rapid growth rate near that of cells in serum-supplemented medium.     

Culture of the astaxanthin-producing green algaHaematococcus pluvialis 1. Effects of nutrients on growth and cell type

The freshwater green algaHaematococcus pluvialis (Strain Vischer 1923/2) grows best at high nitrate concentrations (about 0.5 to 1.0 g 1^–1 KNO₃), intermediate phosphate concentration (about 0.1 g 1^–1 K₂HPO₄) and over a wide range of Fe concentrations. Low nitrate or high phosphate induce the formation of reddish palmella cells and aplanospores. Mixotrophic growth with acetate improves growth rate and final cell yield, and also stimulates the formation of the astaxanthin-containing palmella cells and aplanospores.H. pluvialis cannot grow above about 28 °C, or above a salinity of approximately 1% w/v NaCl. An increase in temperature or the addition of NaCl also stimulates the formation of palmella cells and aplanospores.     

Block to multiplication of adenovirus serotype 2 in monkey cells.

The block to adenovirus 2 (Ad2) multiplication in monkey cells can be overcome by coinfection with simian virus 40 (SV40). To identify this block we have compared the synthesis of Ad2 proteins in monkey cells infected with Ad2 alone (unenhanced) or with Ad2 plus SV40 (enhanced). Synthesis of viral proteins in enhanced cells was virtually identical to that found for permissive infection of human cells by Ad2 alone. In contrast, the unenhanced cells were strikingly deficient in the production of the IV (fiber) and 11.5K proteins whereas the synthesis of 100K and IVa2 was normal. Synthesis of a number of other proteins such as II, V, and P-VII was partially reduced. A similar specific reduction in synthesis of these proteins was found when their messages were assayed by cell-free translation. This result suggests that the block to Ad2 protein synthesis is at the RNA level rather than with the translational machinery of monkey cells. Analysis of the complexity and the concentration of Ak2-specific RNAs, using hybridization of restriction endonuclease fragments of the Ad2 genome to increasing concentrations of RNA, shows that although all species of late Ad2 mRNA are present, the concentration of several species is reduced sevenfold or more in unenhanced monkey cells as compared with enhanced cells. These species come from regions of the genome known to encode the deficient proteins. A model for the failure of adenovirus to multiply in monkey cells, based on abnormal processing of specific adenovirus messages, is presented.     

Effect of liver cell coat acid mucopolysaccharide on the appearance of density-dependent inhibition in hepatoma cell growth.

Yoshida ascites hepatoma 66 (AH 66) cells grown in monolayer cultures show a lack of density-dependent inhibition of growth. When acid mucopolysaccharide (AMPS) isolated from rat liver cell coats is added to growing cultures at concentrations of 50–100 μg/ml, AH 66 cell cultures became markedly inhibited and exhibited density-dependent inhibition of growth at a cell density of 19 × 10⁴ cells/cm². Inhibition reached 84% below control levels. Inhibition is a density-related phenomenon since cells at densities below 19×10⁴ cells/cm² do not exhibit inhibition of growth. AH 66 cells inhibited in the plateau state are capable of resuming growth when AMPS is removed from the cultures. When AMPS is added at a concentration of 0.5 μg/ml, growth of tumor cells is promoted. Promotion reaches 78% above control levels. It is suggested that AMPS may play an important part in the regulation of cellular proliferation.