The inhibition of mitosis in tissue culture cells by blue light

Blue light (wavelength 350-480 nm) irradiation of the early mitotic (prophase and prometaphase) tissue culture cells at the dose of 50-3000 J/cm2 delay mitosis or completely block it at the metaphase. Cell sensitivity to the near UV light (wavelength 360 nm) was few times more as compared with the sensitivity to the visible light (wavelength 400-480 nm). Mitotic cells irradiated with the green light (wavelength more than 500 nm; dose up to 7500 J/cm2) completed division normally. The effect of the blue light did not depend on the presence of phenol red in tissue culture medium. Rhodamin 123 staining did not show any changes in the mitochondrial system in the irradiated mitotic cells. Blue light irradiation with the dose enough for the induction of mitotic delay appears to be insufficient to affect the proliferation of interphase cells.     

Methemoglobin is a supplement for in vitro culture of human nasopharyngeal epithelial cells transformed by human Papillomavirus type 16 DNA

NPC-N cells were normal human nasopharvngeal epithelial cells transformed by transfection with human papillomavirus type 16 deoxyribonucleic acid. Bovine pituitary extract (BPE) was one of the indispensable ingredients for in vitro culture of NPC-N cells in a serum-free medium. Chromatographic fractionation of BPE and subsequent immunoblotting analyses identified the hemoglobin growth-stimulating factor. Methemoglobin (metHb) was then synthesized, and also found to be growth stimulating. The growth-stimulating effect of metHb was abolished when NPC-N cells were cultured in a medium that also contained haptoglobin, a molecule that binds to hemoglobin. A defined medium consisting of insulin and metHb was then developed for optimal growth of NPC-N cells. MetHb kept under the conditions identical to those of cell culture released hemin which also enhanced the cell growth. Though all the degradation products of hemin are currently known to be physiologically significant. only ferric iron derived from metHb or hemin could stimulate the growth of NPC-N cells. Abnormal vasculature showing leaky walls and hemorrhage is a common feature of malignant tumors. Hemoglobin originating from extravasated red blood cells and subsequently oxidized to metHb because of the presence of activated inflammatory cells might contribute to the increased proliferation of cancerous cells.     

Aluminum ions stimulate the oxidizability of low density lipoprotein by Fe2+: implication in hemodialysis mediated atherogenic LDL modification

Objective: Al³⁺ stimulates Fe²⁺ induced lipid oxidation in liposomal and cellular systems. Low-density lipoprotein (LDL) oxidation may render the particle atherogenic. As elevated levels of Al³⁺ and increased lipid oxidation of LDL are found in sera of hemodialysis patients, we investigated the influence of Al³⁺ on LDL oxidation.

Materials and methods: Using different LDL modifying systems (Fe²⁺, Cu²⁺, free radical generating compounds, human endothelial cells, hemin/H₂O₂ and HOCl), the influence of Al³⁺ on LDL lipid and apoprotein alteration was investigated by altered electrophoretic mobility, lipid hydroperoxide-, conjugated diene- and TBARS formation.

Results: Al³⁺ could stimulate the oxidizability of LDL by Fe²⁺, but not in the other systems tested. Al³⁺ and Fe²⁺ were found to bind to LDL and Al³⁺could compete with Fe²⁺ binding to the lipoprotein. Fluorescence polarization data indicated that Al³⁺ does not affect the phospholipid compartment of LDL.

Conclusions:The results indicate that increased LDL oxidation by Fe²⁺ in presence of Al³⁺ might be due to blockage of Fe²⁺ binding sites on LDL making more free Fe²⁺ available for lipid oxidation.     

Effect of insulin on a serum-free hybridoma culture

Insulin is often included in serum-free media for animal cell cultivation. However, the necessity of insulin for a specific cell line is rather uncertain. In this article we report the effects of insulin on the cultivation of a hybridoma cell line in a serum-free medium. It was found that insulin affected neither the cell growth nor the antibody production. The specific growth rate and specific antibody production rate were very similar in the cultures with or without insulin. However, the presence of insulin affected the nutrient consumption rate and cell metabolism. Including insulin in the medium resulted in a higher specific glucose consumption rate, a shorter exponential growth stage, and a lower final antibody concentration. The elimination of insulin from the medium allowed antibody to accumulate to a concentration substantially higher than that in the insulin-containing cultuvre. (c) 1995 John Wiley & Sons, Inc.     

Effect of TSH in human thyroid cells: evidence for both mitogenic and antimitogenic effects.

The well-known mitogenic effects of TSH observed in vivo on the thyroid are not always reproducible of human thyroid cells in vitro where conflicting results have been obtained. In order to clarify this issue, we have used primary cultures of human thyroid cells obtained from normal tissue and maintained in serum-free medium for several days. In this in vitro model we have studied the effect of TSH on growth by measuring three different parameters: [3H]-thymidine incorporation, cell counts, and DNA measurement. Monolayer cultures were plated at both low and high cell density (2 x 10(4) and 8 x 10(4) cells/25 mm well, respectively). Although at either cell density cultures were equally able to functionally respond to TSH in terms of cAMP accumulation a significant growth response to TSH was observed only in low density cultures. In high density cultures TSH had an antimitogenic effect. Moreover, TSH potentiated the mitogenic effect of insulin only in low density cultures. In contrast to TSH, FCS induced a similar proliferative response at both high and low cell density. Following TSH stimulation, cAMP content was always increased, paralleling the effect of growth in low density but not in high density cultures. The cAMP analogues dibutyryl-cAMP and 8-bromo-cAMP, as well as cholera toxin and forskolin, did not mimic the mitogenic effect of TSH but had an antiproliferative effect. In addition, these agents blunted the proliferative effect of insulin. These data suggest that in thyroid cells TSH is able to elicit both a mitogenic and an antimitogenic effect depending on the environmental conditions such as cell density.(ABSTRACT TRUNCATED AT 250 WORDS)     

A new serum-free method of measuring growth factor activities for human breast cancer cells in culture

Summary Growth of the MCF-7, T47D, and ZR-75-1 human breast cancer cells was established in a serum-free defined medium (MOM-1) composed of a 1∶1 (vol/vol) mixture of Ham's F12 medium and Dulbecco's modified Eagle's medium containing 15 mM HEPES (pH 7.2), 2 mM 1-glutamine, 20 μg/ml glutathione, 10 μg/ml insulin, 10 μg/ml transferrin (Tf), 10 ng/ml selenous acid, 0.3 nM triiodothyronine, 50 μg/ml ethanolamine, 20 ng/ml epidermal, growth factor, 2.0 nM 17β-estradiol, and 1.0 mg/ml bovine serum albumin (BSA). Proliferation in MOM-1 was 50 to 70% of the serum stimulated rate. Deletion of components from MOM-1 gave a medium (Tf-BSA) containing only HEPES, 10 μg/ml Tf, and 200 μg/ml BSA, which sustained MCF-7 and T47D cells in a slowly dividing and mitogen responsive state; ZR-75-1 cells required Tf plus 1.0 mg/ml BSA. In Tf-BSA, insulin and insulin-like growth factor I(IGF-I) were mitogenic with ED₅₀ values of 2 to 3 ng/ml and 30 to 150 pg/ml, respectively, with MCF-7 cells. The T47D cells were responsive to these factors in Tf-BSA but required 10-fold higher concentrations for ED₅₀. At saturating concentrations, insulin and IGF-I promoted 1.5 to 3.5 cell population doublings over controls in 8 d. At≤ng/ml concentrations, epidermal growth factor, insulin-like growth factor II, and basic fibroblast growth factor were mitogenic for human breast cancer cells in Tf-BSA. Mitogen activities in uterus and pituitary extracts were assayed readily in Tf-BSA. This new method offers a convenient means of comparing the potencies of growth-promoting factors on human breast cancer cells without interfering activities known to be present in serum. This work was supported by grants CA-38024 and CA-26617, from the National Cancer Institute, Bethesda, MD, and by American Cancer Society grant BC-255 and grant 2225 from the Council for Tobacco Research, USA, Inc.     

Prolongation of murine hybridoma cell survival in stationary batch culture by Bcl-xL expression

While the ectopic expression of the anti-apoptoticprotein Bcl-2 has been shown to significantly increaseboth cell viability and antibody production in batchculture, some cell lines are refractory to thesemanipulations. For example, the NS/O and theP3x63Ag8.653 murine myelomas, which express highendogenous levels of the Bcl-2 homologue Bcl-xL, areboth resistant to the anti-apoptotic effect of Bcl-2.This indicates that, in these cells, Bcl-2 and Bcl-xLmay be functionally redundant. In order to define therole which Bcl-xL plays in hybridoma cultures, we usedthe Sp2/0-Ag14 cell line. This murine hybridomaexpresses low levels of Bcl-xL and is highly sensitiveto apoptosis induction by cycloheximide (CHX) and byamino acid depletion. Bcl-xL-transfected Sp2/0-Ag14cells were more resistant than the wild type and theplasmid-containing cells to apoptosis induced by CHXand by glutamine depletion. Moreover, when compared tothe vector-transfected control, Bcl-xL-Sp2/0 cellsexhibited a substantial increase in viability instationary batch culture. Interestingly, Sp2/0-Ag14cells overexpressing Bcl-xL showed a growth behaviourthat was similar to the parent myeloma cell lineP3x63Ag8.653. Our results suggest that Bcl-xLexpression levels are sufficient to account for therelative robustness of some hybridoma cell lines instationary batch cultures.     

Aluminum ions stimulate DNA synthesis in quiescent cultures of Swiss 3T3 and 3T6 cells

Micromolar concentrations of AI3+ are shown to be strongly mitogenic for quiescent cultures of Swiss 3T3 and 3T6 cells. AI3+ caused a striking shift in the dose-response curve for the effect of fetal bovine serum on 3H-thymidine incorporation. In the absence of serum the mitogenic effect of aluminum was greatly potentiated by insulin or cholera toxin, but not epidermal growth factor or 12-0-tetradecanoyl-phorbol-13-acetate. The stimulation of DNA synthesis was maximal by 15-20 microM AI3+ X AI3+ at 100 microM had no inhibitory effect on DNA synthesis. AI3+ had no significant effect on cellular cyclic adenosine monophosphate in the presence or absence of insulin or an inhibitor of cyclic nucleotide phosphodiesterases.     

Control of DNA synthesis in tissue culture cells

Summary Eukaryotic DNA is functionally divided into thousands of replicons, each of which may be duplicated at a characteristic time within the DNA synthetic (S) period. Our approach toward an understanding of the molecular mechanisms which control orderly eukaryotic DNA synthesis has been: (a) to devise a method of cell synchrony in a suitable tissue culture system wherein all cells in the population enter and traverse the S period with a high degree of synchrony; (b) to determine, utilizing this system, precisely when during the S period critical events and macromolecular syntheses occur; and (c) to examine, by polyacrylamide-gel electrophoresis, the spectrum of proteins which become associated with chromatin during the S period in such a way as to suggest their involvement with DNA synthesis. Possible mechanisms for control are discussed based on the results presented here. Presented in the formal symposium on Mechanisms of Cellular Control at the 28th Annual Meeting of the Tissue Culture Association, New Orleans, Louisiana, June 6–9, 1977. The work reported in this communication was supported by NCI Grant CA 18612 to A.B.P.     

Hormonal growth control of cells in culture

Summary Serum is the last undefined component in cell culture media. Our results indicate that the primary role of serum is to provide hormones and that serum can be replaced by a group of hormones. A rat pituitary cell line, GH₃, can grow in serum-free medium if the medium is supplemented with 3,3′,5-triiodothyronine, TSH-releasing hormone, transferrin, parathyroid hormone, insulin and three isoelectric focusing fractions of blood meal. The blood-meal components can be replaced by fibroblast growth factor and somatomedin C. The growth rate of GH₃ cells in hormone-supplemented serum-free medium is equal to that in serum-supplemented medium, and subculture in such medium is also possible. These results indicate that the replacement of the serum component is complete in the GH₃ system. The hormonal requirements of GH₃ cells and those of HeLa and mouse melanoma, M2R, were compared. Two generalizations could be made: (a) All three cell lines require insulin and transferrin. (b) There is a requirement for a hormone which localizes in the nucleus for each cell line. These generalizations seem to hold true for most of the other cell lines for which the hormonal requirements have been partially worked out. Since insulin is one of the universally required hormones, its effects on GH₃, HeLa and M2R were compared. Insulin stimulates glycogen synthesis in all three cell lines and facilitates fatty-acid synthesis in GH₃ and M2R. However, there is a difference in the effect of insulin on growth among the three cell lines. Insulin is an absolute requirement for GH₃ cells without which the cells cannot survive, whereas this is not the case for HeLa and M2R. The most stringent requirement for HeLa cells is for hydrocortisone, and for M2R, it is for transferrin. These results indicate that even though the necessity for some hormones is common, the degree of requirement may vary from one cell line to another. Whether this difference reflects the difference in the primary mode of action of the hormone on each cell type needs further investigation. Presented in the Opening Symposium on Nutritional Factors and Differentiation at the 28th Annual Meeting of the Tissue Culture Association, New Orleans, Louisiana, June 6–9, 1977. This work was supported by NIH Grant GM 17019. J. Larner was supported by Josiah Macy Foundation     

Cerebral microvessels and derived cells in tissue culture

Summary An endothelial cell line has been established from a primary culture of cerebral microvessels isolated from Swiss-Webster mice. The microvessels were isolated by a mechanical dispersion and filtration technique. The cells that emerged from these microvessels, maintained in organoid cultures, proliferated and formed plaques of a single or mixed cell type. The endothelial cell line, designated ME-2, was isolated from one such morphologically homogeneous cell plaque, using both cloning ring techniques and C₆ glioma-conditioned medium. An endothelial specific antiserum was made in rabbits and was used immunocytochemically to confirm the cell type of origin of the ME-2 cell line. Not only did the cell type specific antiserum react exclusively with endothelial cells in vivo, but in the brain the antiserum localized preferentially to the luminal membrane of the endothelium. The ME-2 endothelial cells have retained several of their unique properties such as cytomorphology, growth characteristics, and cell type specific surface antigens throughout the life of the line (in one case 40 passages before senescence). This work was supported in part by an Arteriosclerosis Specialized Center of Research grant from the National Heart, Lung and Blood Institute, National Institutes of Health, Grant HL-14230, and Grant 584-127703 from the Veterans Adminsitration. This paper is dedicated to the memory of Steve Frommes, Electron Microscopist and Photographer.     

Cartilage tissue differentiation from mesenchymal cells derived from mature muscle in tissue culture

Summary Under the influence of biochemical components of bone matrix gelatin (BMG), cartilage differentiates in tissue culture from the connective tissue cell outgrowths of mature muscle. Proliferation and differentiation begin within 24 hr with synthesis of hyaluronate, continue with high levels of synthesis of DNA and hyaluronidase, and culminate in production of large quantities of chondroitin sulfate. The addition of hyaluronic acid to the culture medium during the first 48 hr of culture depresses, whereas chondroitin sulfate enhances, subsequent production of cartilage. These observations on the cell biosynthetic products prior to the appearance of mature cartilage suggest that the BMG-modified connective tissue outgrowths of mature muscle exhibit the developmental potential of embryonic axial mesenchyme. Whether muscle harbors embryonic cells in a programmed but not yet activated readiness (protodifferentiated state) to differentiate into cartilage, or simply contributes a population of temporarily dedifferentiated fibroblasts, is not known, but in any event, BMG switches the pathway of further development from fibrous connective tissue to cartilage. These investigations were supported by grants-in-aid from the USPHS, National Institute of Dental Research (DE-2103-01). Drs. Terashima and Nakagawa received a research fellowship from the Solo Cup Corporation. Charles Stamos was a Eugene and Marion Bailey Summer Student Research Fellow.     

Normal murine melanocytes in culture

Summary A major obstacle to applying the techniques of molecular biology to the genetics and cell biology of pigmentation has been our inability to grow normal murine melanocytes in culture. We report here the establishment and characterization of continuously proliferating cultures of cutaneous pigment cells from seven strains of mice. Melanocytes were grown from the dermis of newborn mice in medium containing 12-0-tetradecanoyl-13-phorbol-acetate; a substance, such as melanotropin, that raises intracellular levels of cyclic AMP; and an extract made from human placenta. This work was supported by Grant R01 CA04679 from the U.S. National Institutes of Health and a fellowship to Dr. A. Tamura from Mr. and Mrs. Allen Locklin. The chromosome studies were carried out in the laboratory of Dr. Uta Francke, Department of Human Genetics, Yale University. JCM was supported by NIH contract number N01-CP-21037.     

Cadmium produces a delayed mitogenic response and modulates the EGF response in quiescent NRK cells

Recent studies have shown that cadmium, at subtoxic levels, may induce a response characteristic of that elicited by a type of growth factor that supports the anchorage independent growth of cells that are not fully transformed. That is, Cd⁺⁺ was found to replace transforming growth factor beta in supporting soft agar growth of NRK-49F cells. To tes the extent to which Cd⁺⁺ further mimics transforming growth factor beta in its effects and to establish response patterns that suggest possible molecular mechnisms of action, we have determined the effects of Cd⁺⁺ and/or epidermal growth factor (EGF) on DNA synthesis in quiescent NRK-49F cells. We found that subtoxic doses of Cd⁺⁺ modulate EGF-induced DNA synthesis in a dose-dependent fashion. Although Cd⁺⁺ effects on early (16–24 hr) EGF-induced DNA synthesis are primarily inhibitory, later effects involve stimulation as well. Subtoxic doses of Cd⁺⁺ did not stimulate DNA synthesis in quiescent cells within 24 hr of addition. At later times (40 or 64 hr), however, an increase in DNA synthesis of up to threefold was induced by 0.25 M Cd⁺⁺. This pattern of mitogenic response, involving inhibition of early growth-factor induced DNA synthesis and stimulation of late DNA synthesis, is consistent with that reported to be effected in some instances by transforming growth factor beta. Because a defined pattern of gene expression also is associated with the mitogenic responses to transforming growth factor beta, future studies at the molecular level can definitively test the degree to which Cd⁺⁺ and transforming growth factor beta effects are common.Abbreviations CFE colony forming efficiency - EGF epidermal growth factor - MT metallothionein - PGDF paltelet derived growth factor - TGF transforming growth factor     

New approaches to insect tissue culture

Conclusion Current methods of insect cell culture have produced a limited variety of cell types in an ever expanding list of insect cell lines. In developing midgut epithelial cell lines, we found that traditional methods in insect cell culture failed to provide healthy cells from mature tissues. Examination of mammalian cell culture literature for this particular cell type provided the insight required to successfully develop a cell-specific line (Baines et al., 1994). The potential applications for cell-specific lines from insects are numerous. This paper is a compilation of ideas that will hopefully enable other researchers to develop additional cell-specific lines.     

Primary culture of normal rat adrenocortical cells

Summary Rat adrenocortical cells retiained their differentiated characteristics over 2 wk in culture without a specific requirement for additives other than inorganic salts, amino acids, vitamins, and fetal bovine serum. The cells were maintained free from fibroblast overgrowth by substitution ofd-valine in place ofl-valine in the medium. Corticotropin (ACTH) inhibited the growth of adrenocortical cells in this medium and the effect was reversible. The adrenocortical cells had a limited capacity for growth as reflected by total cell counts and [³H]thymidine uptake with cells from young animals demonstrated a greater potential for DNA synthesis than cells obtained from mature animals. A very sensitive assay for ACTH using a small number of cells in primary culture also is described. This work was supported by Grant CA-16417 from the National Cancer Institute.