Density dependent proliferation of human glia cells stimulated by epidermal growth factor.

Epidermal growth factor (EGF) isolated from mouse salivary glands, enhanced the multiplication and [³H]TdR incorporation of human normal glia cells in serum-free medium supplemented with human serum albumin. Optimal dose was 2 ng/ml for both dense and sparse cultures but dense cultures were stimulated by EGF to a much less extent than sparse cultures. Data are presented that make the possibility unlikely that the density dependent inhibition of the EGF response is due to depletion of EGF in the medium or a local, juxtacellular starvation for the factor.     

The importance of cell contact for the proliferation of neuroblasts in culture and its stimulation by meningeal extract

The influence of cell density and cell contacts on the proliferation of neuroblasts in culture and its stimulation by meningeal extract were investigated. Dissociated brain cells from 6-day-old chick embryos were cultured under 3 different culture conditions to obtain dense or sparse brain cell cultures, as well as cultures of isolated neuronal cells. The proliferation of neuroblasts, shown by morphological observations, cell counts, determinations of DNA content and measurements of [³H]thymidine incorporation, was found to be the highest in cultures where cell density and cellular contacts were greatest. The addition of meningeal extract stimulated the multiplication of neuroblasts only in cultures where the cells were in closer contact with each other. The results suggested, therefore, that cell density and cell-cell interactions are of importance and favored neuroblast proliferation.     

PANETH AND GOBLET CELL RENEWAL IN MOUSE DUODENAL CRYPTS

Proliferation of Paneth and goblet cells of mouse duodenal crypts was studied by high resolution light microscope radioautography. In one group of mice, blood levels of thymidine-³H were sustained for up to 12 hr by repeated injections of isotope to facilitate identification of proliferating cells. In these animals, many goblet cell nuclei incorporated thymidine-³H whereas only 1 of 6261 tabulated Paneth cells was labeled. Cells intermediate in structure between undifferentiated and goblet cells and between undifferentiated and Paneth cells were identified and their light and electron microscopic features are described. A significant number of these "intermediate" cells incorporated thymidine-³H into their nuclei. Another group of mice received a single injection of thymidine-³H. These animals were killed 4 hr to 29 days after isotope administration. Goblet cells and intermediate cells with labeled nuclei were identified 4 hr after thymidine-³H but could not be seen after 15 days. In contrast, Paneth cells with labeled nuclei were not observed until 24 hr after thymidine-³H but were still present at 29 days, long after labeled undifferentiated, goblet, and intermediate cells had disappeared. We conclude that differentiated Paneth cells in mouse duodenum do not normally proliferate, but, instead, arise by differentiation from undifferentiated crypt cells or from intermediate cells. Moreover, once formed, Paneth cells persist in crypts for a prolonged period. In contrast, intermediate cells and crypt goblet cells proliferate actively and are less stable cell populations than differentiated Paneth cells. The precise function of the intermediate cells is not known, but they may represent transition forms between undifferentiated cells and the more matrure secretory cells. Damage of crypt epithelial cells, thought to be due to radiation effects, was evident in both groups of mice.     

Author index

Binding of biologically active [¹²⁵I]thrombin to several normal and transformed human and chicken cell lines was found to depend on cell density; more [¹²⁵I]thrombin per cell was bound to sparse than to dense cultures. When normal and transformed cells were compared at equal densities the previously reported difference in [¹²⁵I]thrombin binding was not evident.     

Binding of thrombin to normal and transformed fibroblasts depends on cell density

Binding of biologically active [¹²⁵I]thrombin to several normal and transformed human and chicken cell lines was found to depend on cell density; more [¹²⁵I]thrombin per cell was bound to sparse than to dense cultures. When normal and transformed cells were compared at equal densities the previously reported difference in [¹²⁵I]thrombin binding was not evident.     

Capacity for tumor cell implantation as a function of in vitro cell density

Implantation properties of two melanoma cell lines, line 26 (low rate of implantation) and line 37 (high rate of implantation) were studied as a function of the cell density of the cells grown in monolayer in vitro. Sparse cultures (collected at a density of 0.8 × 10³ cells cm^?2) of line 37 produced 7.7 times as many lung tumor foci as those of line 26. Confluent cultures (collected at a density of 40 × 10³ cells cm^?2) resulted in greater numbers of tumor foci for both cell lines, but line 37 produced only 3.1 times as many tumor foci as did line 26 cells. Thus the high implantation line (37) has a much greater ability to implant when grown in the sparse state and injected than the low implantation line (26), but both lines have high implantation rates when injected as confluent cells.     

Changes in RNA-, DNA- and proteinsynthetic activity during the formation of analfin processes in ethisterone-treated females of Oryzias latipes

The incorporations of uridine-³H, thymidine-³H, and leucine-³H were studied in the process-forming regions of the anal-fin rays of the ethisterone-treated females of Oryzias latipes. The activity of alkaline phosphatase was also studied. The increased incorporation of uridine-³H was detected between 12 and 24 hours of ethisterone treatment, attaining the maximum at 24 hours. The percentage of thymidine-³H labeled nuclei increased rapidly between 48 and 84 hours. The incorporation of leucine-³H was found to increase during the first 12 hours, attaining a constant level at 24 hours. An additional increase in incorporation of leucine-³H took place at 60 hours, the incorporation coming up to the maximum at 72 hours. In the horny substance secreted by the scleroblast mass, grains in the autoradiograph were detected at and after 72 hours. Alkaline phosphatase activity was manifested between 48 and 72 hours. These results seem to correspond to the histological changes, such as the appearance of the precursor cells of scleroblasts at 48 hours, the formation of scleroblast mass during the next 24 hours, and the initiation of horny substance secretion at 72 hours.     

Density dependent inhibition of cell growth in cultures of primary and established lines of cells

The effect of cell crowding on DNA synthesis (incorporation of ³HTdR and ³²PO₄) was studied by an improved method in monolayers of secondary cells and established cell lines, either normal or transformed by viruses or carcinogens. The method was based mainly on pulse labeling of cultures of cells a few hours after their seeding in equal numbers onto areas of different size in identical dishes, a condition which ensured equal physiological conditions and different degrees of crowding of cells. DNA synthesis was hardly inhibited in crowded monolayers of secondary chick, mouse and hamster embryo cells. The incorporation of radioactive thymidine and phosphate into DNA of cell lines such as BHK 21, 3T3/SV40 and L929 was strongly inhibited. An SV40-transformed line of hamster kidney cells (HKT7) synthetized DNA equally well in sparse as in crowded monolayers. In lines of human amnion (FL) and BHK 21 cells which were more extensively studied the degree of inhibition of DNA synthesis was inversely proportional to their density. Autoradiography after ³HTdR pulse-labeling indicated that the same proportion of cell nuclei were labeled in sparse and in crowded cultures. The extent of labeling (number of grains per nucleus) was lower in crowded cultures of those cells that also showed inhibition of incorporation of this label as measured by scintillation. The inhibition is thus expressed in retardation of DNA synthesis in cells in S phase rather than arresting it in a larger percentage of cells.     

Replication of nucleolus-associated DNA during "G2 phase" in Physarum polycephalum

In the myxomycete, Physarum polycephalum, the bulk of nuclear DNA replication occurs during a period of a few hours immediately following upon mitosis. During the remainder of the intermitotic period, incorporation of thymidine-³H continues at a low rate in the region of the nucleolus (radioautographs). A few nuclei incorporated thymidine-³H into the extranucleolar chromatin at a high rate at all times of the intermitotic period. These nuclei were exceptionally large and they frequently contained several small nucleoli of different sizes rather than the one, central nucleolus which is characteristic of a normal interphase nucleus.     

The pattern of density dependent growth inhibition in murine fibroblasts

Observations on the pattern of nuclear incorporation of ³H-TdR in long term (8-day) and short term (3-day) 3T3 cultures with local cell densities between 0.2 × 10⁴ and 6.2 × 10⁴ cells/cm² are reported. Contrary to a number of previous studies our observations indicate that density dependent inhibition is exhibited in relatively sparse cultures, commencing at 0.5 × 10⁴ cells/cm². Various possible mechanisms which could have caused the observed pattern of density-dependent regression in labelling index are discussed.     

Density-dependent inhibitory effect of transforming growth factor-β on human fibroblasts involves the down-regulation of platelet-derived growth factor α-receptors

We have previously found that transforming growth factor-β1 (TGF-β1) inhibits the mitogenic activity of platelet-derived growth factor (PDGF) in cultures of human neonatal fibroblasts in a density-dependent fashion. In the present investigation we determined the effect of TGF-β1 on the PDGF α-receptor, which binds all PDGF isoforms, as well as on the β-receptor, which binds only PDGF-BB with high affinity. We found that the inhibitory effect of TGF-β1 on PDGF-AA-induced mitogenesis was density-dependent; when dense cell cultures were preincubated with TGF-β1, there was an complete inhibition of ³H-thymidine incorporation, whereas the effect was less in sparse cultures. A similar density-dependent effect of TGF-β1 was seen in PDGF-BB treated cells, although less pronounced. The binding of ¹²⁵I-labeled PDGF-AA and PDGF-BB to the α-receptor was significantly reduced after treatment with TGF-β1 in dense cultures, whereas the sparse cultures were less affected. A decrease of α-receptor mRNA was also seen. The levels of β-receptor protein and mRNA were unaffected. We conclude that the growth inhibitory effect of TGF-β1 is cell density-dependent and involves down-regulation of PDGF α-receptors. © 1993 Wiley-Liss, Inc.     

Alterations in the characteristics of 2-deoxy D-glucose transport into cultured human glioma cells during cell growth.

The uptake of 2-deoxy-d-glucose (2-DG) into human glioma cells (138 MG) was related to cell growth. The uptake of 2-DG was high at confluency but low in both rapidly growing sparse cultures and in growth-inhibited dense cultures. Lineweaver-Burke plots of uptake at different cell densities showed changes in V_max; K_m, however, remained constant. Dibutyryl cyclic-AMP (db-cAMP) doubled the uptake of 2-DG into rapidly growing sparse cultures but lacked effect at higher cell density. Independent of their density, cells treated with db-cAMP attained the characteristic morphology of differentiated glial cells.     

Cultivation of avian malaria parasites in mammalian liver cells

The exoerythrocytic stages of Plasmodium lophurae and P. fallax were grown in cell cultures derived from embryonic mouse livers. Liver cell monolayers were maintained in continuous culture with frequent subculturing for extended periods of time. Morphologically, the main cell type was a large, flat cell which closely resembled in shape the mouse parenchymal cell, although small colonies of spindle-shaped cells could also be found. Mammalian cells were labelled with thymidine-³H prior to infection with avian merozoites so they could be positively distinguished from any avian cells which might be present in the merozoite inoculum as contaminants. Merozoites were observed inside the labelled mammalian cells within three hours and large forms were seen at 48 hr. The mammalian cells supported parasite growth comparable to that observed in avian cell cultures.     

NuSerum,a synthetic serum replacement,supports chondrogenesis of embryonic chick limb bud mesenchymal cells in micromass culture

Summary In an effort to establish a more chemically defined culture system to study the regulation of chondrogenic differentiation in vitro, two commercially available serum replacements, NuSerum and NuSerum IV, were tested on embryonic limb mesenchyme. Limb bud (LB) mesenchymal cells were isolated from Hamilton-Hamburger stage 23–24 chick embryos and plated at various densities (1, 5, 10, or 20 × 10⁶ cells/ml) in micromass culture for 4 days in media supplemented with 10% fetal bovine serum (FBS), NuSerum or NuSerum IV. Cell growth was assessed by the incorporation of [³H]leucine and [³H]thymidine. Chondrogenesis was determined by the incorporation of [³⁵S]sulfate and by the number of Alcian blue-staining cartilage nodules. In high density (20 × 10⁶ cells/ml) cultures, which favored chondrogenic differentiation, both serum replacements supported protein synthesis and chondrogenesis equally well as FBS. In cultures plated at 5 × 10⁶ cells/ml, a cell density in which was chondrogenesis-limiting, both NuSerum and NuSerum IV significantly enhanced incorporation of [³⁵S]sulfate (2.6-fold), [³H]leucine (1.4-fold), and [³H]thymidine (1.9-fold), compared to FBS. Enhancement of chondrogenesis was also apparent by the increases in the number of Alcian blue-staining cartilage nodules and the ratio of sulfate: leucine incorporation in cultures plated at 5 × 10⁶ cells/ml. Interestingly, the localization of cartilage nodules was extended out to the periphery of micromass cultures fed with NuSerum or NuSerum IV. The observed effects of NuSerum and NuSerum IV may be attributed to a combination of factors, including lower concentrations of serum and its associated proteins, as well as supplemented growth factors and hormones known to promote cell proliferation and differentiation. Therefore, NuSerum and NuSerum IV are excellent, low-cost replacements for FBS in maintaining cellular growth and promoting chondrogenesis in LB mesenchymal cell cultures in vitro.     

Incorporation of thymidine-3H into the activated sea urchin egg with special reference to subthreshold stimulation with an activating reagent

It was ascertained that thymidine-³H added exogenously is incorporated periodically into the nucleus of the fertilized and the artificially activated eggs of Pseudocentrotus, Temnopleurus and Anthocidaris. Eggs stimulated insufficiently with butyric acid did not show any enhancement of incorporation of thymidine-³H. However, repetition of an insufficient stimulation induced incorporation of thymidine to some degree, although no visible cortical changes occurred.     

Myelin Gene Expression in Immortalized Schwann Cells: Relationship to Cell Density and Proliferation

Abstract: Myelin gene expression was investigated in the immortalized S16 Schwann cell line grown in the presence and absence of serum and at different densities. Protein expression was monitored by western blotting, and message levels were determined by RNase protection assays. To study cell proliferation rates at different cell densities and serum conditions. [³H]thymidine uptake assays and cell counts were performed. Although serum deprivation decreased cell proliferation as expected, the proliferation of S16 cells was unchanged or slightly increased at high density under the conditions of our experiments in either serum-containing or serum-free medium. This increased cell division at high density appeared to be due to greater release of an autocrine growth factor to the medium by dense cell populations. For both sparse and dense cells, substantially more P₀ glycoprotein (P₀) and myelin-associated glycoprotein (MAG) per milligram of total cellular protein were expressed when the cells were proliferating slowly in defined medium in comparison with more rapidly proliferating cells in serum-containing medium. Furthermore, in both serum-containing and defined media, dense cell populations expressed more MAG and P₀ than sparse ones. P₀ mRNA and MAG mRNA levels generally paralleled protein levels. The level of mRNA for peripheral myelin protein-22 (PMP-22) was also increased at high cell density but did not change much when proliferation was decreased by serum deprivation. PMP-22 protein was not detected under any of the growth conditions. The changes in expression of these genes with growth conditions may be specific for myelin proteins, because the expression of a nonmyelin glycoprotein, L1, remained constant. The level of cyclic AMP in the cells did not change with the different growth conditions tested. The results indicate that the S16 Schwann cell line mimics primary or secondary Schwann cells by down-regulating myelin gene expression when it proliferates more rapidly in the presence of serum. Furthermore, in both the presence and absence of serum, there was greater expression of myelin genes at high cell density that was not associated with a decreased proliferative rate. Because evidence for a role of secretory factors in affecting myelin gene expression was not obtained by treating sparse S16 cells with medium conditioned by dense S16 cells, the results suggest that the higher expression of myelin genes at high density may be mediated by cell-to-cell contact.     

Role of β-GP-derived pi in mineralization via ecto-alkaline phosphatase in cultured fetal calvaria cells

The permissive effect of β-GP on mineralization in cultured rat fetal calvaria cells was investigated in relationship with phosphohydrolase activity of ecto-ALP at physiological pH range. β-GP present in the culture medium for 8 days exerted a stimulatory effect on ⁴⁵Ca incorporation into matrix cell layers while the ecto-ALP activity level measured on intact cells with a saturating concentration of p cells grown either in the presence or absence of β-GP. In both types of cultures, β-GP addition inhibited pNPP hydrolysis in a competitive and reversible manner and increased Pi concentration in the medium. The dose dependency of the effect of β-GP on ⁴⁵Ca incorporation and generation of Pi was similar (kϕ = 3 mM). Levamisole, but not dexamisole, inhibited both pNPP and β-GP hydrolyses, which were likely catalyzed by the same ecto-enzyme. The rate of ⁴⁵Ca incorporation into matrix cell layers, which was high (0.90 μmol/4h/mg cell protein) in cells grown in the absence of β-GP, was inhibited by 50% by levamisole. In cells grown in the absence of β-GP, the ⁴⁵Ca incorporation rate increased progressively after β-GP addition, reaching after 12 h the value of cultures grown in the presence of β-GP, the increase being totally inhibited by levamisole. In both types of cells, addition of exogenous Pi at concentrations corresponding to medium levels of β-GP-derived Pi rapidly led to high ⁴⁵Ca incorporation rate which was unaffected by levamisole. β-GP removal from cultures grown in its presence reduced by 50% the ⁴⁵Ca incorporation rate which recovered the initial value after exogenous Pi addition independently of levamisole presence. Thus, mineral deposition did not affect the level and catalytic efficiency of ecto-ALP to hydrolyze β-GP in cultured fetal calvaria cells, yet it influenced the β-GP-stimulatory effect on mineralization so as to render this process not sensitive to high medium Pi levels. © 1996 Wiley-Liss, Inc.     

Effect of insulin at dilution endpoint concentrations on macromolecular synthesis in normal mouse mammary and human breast cancer epithelial cells

Employing defined media conditions, the insulin sensitivities of mouse mammary gland epithelial cells in primary culture and MCF-7 human mammary epithelial cells were determined. Insulin stimulated the rates of [³H]uridine incorporation into RNA and [³H]leucine incorporation into protein in both primary mouse mammary gland epithelial cell cultures and MCF-7 cell cultures at concentrations approximating the dilution endpoint of the hormone (10⁻²¹ M). Insulin stimulated the rate of [³H]thymidine incorporation into DNA in primary mouse mammary gland epithelial cells at the dilution endpoint concentrations. However, MCF-7 cells required insulin concentrations 100–1000-times that necessary in mouse mammary epithelial cultures to elicit an increased rate of [³H]thymidine incorporation into DNA. Evidence is presented which suggests that the increased rates of uptake of [³H]uridine, [³H]thymidine and [³H]leucine into their respective precursor pools is not responsible for the apparent stimulatation of RNA, DNA and protein synthesis.     

Alterations of the incorporation of [3H]thymidine in cells in culture regulated by nucleoplasmic extract

Porcine skin nucleoplasmic extract (PSNE) was shown to alter the incorporation of [³H]thymidine into DNA of selected porcine, bovine, and human cell populations in culture. PSNE stimulated incorporation of [³H]thymidine into DNA of porcine and bovine dermal cells an average of 300 and 200% of control value, respectively. When porcine and bovine epidermal cells were exposed to PSNE the treatment inhibited [³H]thymidine incorporation into DNA by an average of 48 and 45%, respectively. Similar inhibitions were observed for porcine and bovine kidney, porcine lung, and human KB cells. Thus, the effect of PSNE on the incorporation of [³H]thymidine into DNA of various cultured cells was either stimulatory to dermal cells or inhibitory to a variety of other cell types, including skin epidermal cells. The stimulatory and inhibitory effects of PSNE were abolished by heating PSNE for 5 min in boiling water before its addition to cell cultures. This suggests that macromolecular structure is important in the action of PSNE. This project was supported by a grant from the Research Advisory Board, University of Nevada, Reno, NV.     

Cyclic AMP and growth of Ehrlich ascites tumor cells. Lack of cyclic AMP elevation in nutritionally deprived cells and mechanism of retardation of growth by dibutryl cyclic AMP.

Cyclic AMP levels in Ehrlich ascites tumor cells changed little after deprivation of cells of essential nutrients, serum, glucose and amino acids, deprival of each of which leads to marked inhibition of growth and protein synthesis. Cyclic AMP levels also changed little after the addition of these nutrients to deprived cells. Thus cyclic AMP is not likely to be the intracellular mediator for growth regulation by these three nutrients. Elevation of cyclic AMP levels for short periods by exposure of cells to choleratoxin or theophylline produced only slight changes in parameters of protein synthesis (polyribosome pattern and rate of [3H]leucine incorporation). An exposure for 1 day to dibutyryl cyclic AMP did not inhibit cell growth. However, prolonged exposure to dibutyryl cyclic AMP inhibited the multiplication of Ehrlich ascites cells both in suspension and in stationary cultures. No morphological effects were evident in the former; in the latter, cells attached firmly to the substratum and formed elongated cytoplasmic processes. Inhibition of cell multiplication by dibutyryl cyclic AMP was related to cell density and to serum concentration. Cells in dibutyryl cyclic AMP-containing media plated at low cell densities multiplied as rapidly as control cells. The final densities cells reached were determined by the serum concentration; in dibutyryl cyclic AMP-containing media these densities were about one-half those of respective control cells. Limitation of cell multiplication by dibutyryl cyclic AMP was reversed by the addition of serum, by resuspending cells at lower densities, or by resuspending cells in media without dibutyryl cyclic AMP. These findings suggested that dibutyryl cyclic AMP may affect the utilization of serum factors by cells. Dibutyryl cyclic AMP did not inactivate serum factors and did not change the rate at which cells depleted the growth medium of serum factors. Dibutyryl cyclic AMP may limit cell multiplication by increasing the cellular requirement for serum factors.