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.     

Differentiation-arrested rat fetal lung in primary monolayer cell culture

Summary Differentiation-arrested lung cell cultures were developed from fetal rats of various gestational ages. In contrast to previously published observations with cultures in a pO₂ of ∼142 mm Hg, cultures developed in a pO₂ of ∼30 mm Hg, close to the normal fetal arterial pO₂, have improved plating efficiency and a slightly increased growth rate. They did not, however, show gestation-dependent increases of choline incorporation into phospholipids, nor did immature lung cell cultures respond to dexamethasone or triiodothyronine, singly or in combination, by increased choline incorporation into saturated lecithin. The incorporation of choline and glycerol into lipids suggested a mature rate of lipid synthesis by immature cultures at a pO₂ ∼30 mm Hg, despite preservation of an immature morphology. Electron microscope observations revealed no gross differences between immature cultures developed at either pO₂. The cellular mechanisms underlying these differences are unclear but suggest that oxygen tension may significantly influence results obtained with in vitro studies of lipid synthesis by immature lung. This work was supported by grants from the Medical Research Council of Canada, the Ontario Thoracic Society, and the Physicians' Services Incorporated Foundation.     

MITOSIS AND THE PROCESSES OF DIFFERENTIATION OF MYOGENIC CELLS IN VITRO

The relation between the mitotic cycle and myoblast fusion has been studied in chick skeletal muscle in vitro. The duration of the cell cycle phases was the same in both early and late cultures. By tracing a cohort of pulse-labeled cells, it was found that myoblast fusion does not occur in S, G₂, or M. Cell surface alterations required for fusion are dependent upon the position of the cell in the division cycle. In early cultures, fusion takes place only after a minimum delay of 5 hr from the time the cell has entered G₁. The mitosis preceding fusion may condition the cell for the abrupt shift in synthetic activity that occurs in the subsequent G₁. In older cultures fusion of labeled cells is diminished. Two factors account for the cessation of fusion in older cultures. First, the number of myogenic stem cells declines, but these cells do not disappear as the cultures mature. Their persistence was demonstrated by labeling dividing mononucleated cells in older cultures and challenging them with nascent myotubes. Some of these labeled cells were incorporated into the forming myotubes. Second, a block to fusion develops during myotube maturation. Well developed myotubes challenged with labeled competent myogenic cells failed to incorporate the labeled nuclei.     

Lack of a correlation between polyamine synthesis and DNA synthesis by cultured rat liver cells and fibroblasts

Growth stimulation of either fetal rat liver cells or rat embryo fibroblasts in culture results in considerable increases in intracellular polyamine levels as cells proceed through the cell cycle. Treatment of such cell cultures with appropriate levels of two inhibitors of polyamine synthesis, namely α-hydrazino ornithine and methylglyoxal bis(guanylhydrazone), can essentially completely block these increases in cellular polyamine content. Under such conditions, where the elevation in intracellular polyamine content is prevented, cell cultures are nevertheless able to initiate DNA synthesis and subsequently synthesize DNA at rates comparable to untreated control cultures that have been growth-stimulated. These two cell types therefore contain sufficient polyamines when in a resting state (G₁) to enable them to enter from G₁ into S phase and traverse S phase at normal rates in the absence of further polyamine synthesis. The recruitment of cells into the first cell cycle, through serum stimulation of growth, therefore appears not to be mediated or regulated by the increases in intracellular levels of polyamines that occurs under these conditions. Conversely, the arrest of growth of these cell types resulting from serum deprivation is not mediated by a limitation of intracellular polyamine content.     

Limitation of macrophage production in long-term marrow cultures containing prostaglandin E

Addition of prostaglandin E₂ (PGE₂) significantly altered the cellular composition of murine long-term bone marrow cultures. After 4–5 weeks of culture, increased cellularity in the suspension phase was observed in all cultures containing prostaglandin. These suspension cells contained markedly higher proportions of differentiated neutrophils than did cells cultured in the absence of PGE₂. Granulocyte-macrophage progenitor cell levels in the suspension layer were increased 3–20 fold after five weeks in prostaglandin-containing cultures compared with control cultures. Fewer cells comprised the adherent layer in cultures containing prostaglandin. The number of macrophages in this layer was reduced 3–8 fold in these cultures compared with control cultures, while the number of granulocytes was increased 2–3 fold. The progenitor cells biased toward macrophage development were selectively inhibited in the cultures with PGE₂. There was no significant effect of PGE₂ on pluripotent stem cell levels or on the longevity of the cultures. It is concluded that excessive monopoiesis in bone marrow may be limited by PGE₂ without influencing either stem cell maintenance or the development of other marrow-derived cell types.     

Antiproliferative Effect of the Tripeptide PyroGlu-Phe-GlyNH2on Murine Melanocytes: Transitory Delay of Cell Growthin Vitroand the Cell Cycle Specificity

Cell growth and differentiation in melanocyte cell populations are regulated by a wide range of bioactive substances. Recently, the tripeptide pyroGlu-Phe-GlyNH₂which inhibits melanocyte growthin vitrowas identified in both murine nontransformed melanocytes and malignant melanoma cells. The present study was undertaken to investigate the cell cycle specificity as well as the growth inhibitory profile of the tripeptide after a single or repeated administration to melanocyte cultures. Dose-related effects of the peptide were studied using three different bioassay systems: estimation of cell number, DNA synthesis, and cell flux into mitosis. Growth of melanocyte cultures as well as melanocyte mitotic activity were found to be reduced significantly by the tripeptide at two separate dose levels (10⁻¹¹and 10⁻¹⁴–10⁻¹⁵M). Growth inhibition of melanocyte population did not last long: less than 36 h after the first and less than 24 h after the second peptide addition to the cultures. The level of DNA synthesis in melanocytes remained unchanged after a single peptide administration. The findings indicate that the tripeptide pyroGlu-Phe-GlyNH₂causes transitory delay of cell growth in cultured melanocyte population resulting from a reversible inhibition of melanocyte transition from the G₂-phase of the cell cycle into mitosis.     

Kinetic studies of the localization of aminopeptidase N in monolayer and in follicle-associated cultures of porcine thyroid cells

Summary The fate of aminopeptidase N (AP_N) was investigated by means of immunofluorescence in porcine thyroid-cell cultures. At day 1, control cells cultured without TSH formed flat, cylinder-like aggregates in which AP_N was mainly located on the lateral surface. At day 2, the fluorescence completely disappeared. At days 3 and 4, AP_N appeared in the apical pole of monolayer cells directed toward the medium and in intracellular organelles. The cilium in each cell was intensely labelled and located in the center of the apical pole. In thyrotropin-stimulated cell cultures, the fluorescence at days 1 and 2 was located over the entire plasma membrane. At days 3 and 4, morphological polarization of cells, reorganized into follicles, occurs, AP_N being located either in the apical or basolateral regions and in intracellular vesicles. Between day 4 and 6, segregation of the enzyme to the apical membrane occurred. Between day 6 and 14, fluorescence was generally retained in the apical region and in an intracellular pool. In growing cell cultures, after 2 to 5 subcultures, the cells exposed AP_N in a pattern similar to primary monolayers. After conversion of monolayer cells into follicles, the antigen was recovered in the apical pole of cells facing the follicular lumen.     

Diethylene glycol disulfide from castor bean cell suspension cultures

Diethylene glycol disulfide was isolated from castor bean cell suspension cultures. Incubation of suspension cultures with Na₂³⁵SO₄ resulted in the incorporation of radioactivity into the isolated diethylene glycol disulfide. Diethylene glycol disulfide was detected in cells (430 nmol/g cells) and in cell-free growth medium (41.5 nmol/ml).     

Temperature response of the cell cycle of Haplopappus gracilis in suspension culture and its significance to the G1 transition probability model

The effects of temperature on the cell cycle of Haplopappus gracilis suspension cultures were analysed by the fraction of labelled mitoses method. Sphase in these cultures shows a different temperature optimum as compared to optima derived for G₂ and mitosis. G₁ phase has a much lower Q₁₀ than the other cell cycle phases and shows no temperature optimum between 22 and 34° C. These results are discussed in relation to a transition probability model of the cell cycle proposed by Smith and Martin (Proc. Natl. Acad. Sci. USA 70, 1263–1267, 1973), in which each cell has a time independent probability of initiating the transition into another round of DNA replication and division. The implications of such a model for cell cycle analysis are discussed and a tentative model for a probabilistic transition trigger is advanced.Abbreviations FLM Fraction of labelled mitoses - T_B Total B-phase     

Density dependent regulation of growth in suspension cultures of L-929 cells

Suspension cultures of L-929 fibroblasts grown to densities of 6 to 10 × 10⁶ cells/ml through daily centrifugation and resuspension in fresh media, have been maintained for periods up to five months without change in viability or cell size. DNA synthesis and mitosis in these cultures is limited to 5% of the cells per day, a fraction very nearly equal to the fraction of cells rendered nonviable, most likely during the manipulations associated with medium renewal. The kinetics of the flow of cells into the S and M periods following (a) renewal of the medium and (b) dilution of the high density cultures, suggest that the large majority of the cells are in a G₀ or early G₁ phase, resuming growth readily in response to decreased cell density. This is further indicated by the sequence of the marked shifts occurring in the cell volume distribution spectrum of the high density cultures after dilution. Long term, steady state regulation of growth with retention of intact viability was thus demonstrated in the case of a long established aneuploid cell line. The fact that this occurs in suspension but not in attached cultures, supports the concept that impairment of growth control in such cells affects predominantly regulatory mechanisms located at the cell surface rather than those concerned with intracellular synthesis and metabolism.     

OBSERVATIONS ON CELL DEVELOPMENT IN CHLAMYDOMONAS SEGNIS (CHLOROPHYCEAE) AT LOW AND HIGH CARBON DIOXIDE TENSION1

Some cell cycle events were compared in Chlamydomonas segnis Ettl during its development in synchronous cultures (12:12 LD) supplied with air and air enriched with 5% CO₂. In cultures bubbled with air, growth resulted in production of 2 relatively small zoospores. In cultures provided with 5% CO₂, 4 large zoospores were formed but not released in darkness unless the cultures were bubbled with CO₂-free air and/or exposed to light. Respiration in zoospores was inhibited by high CO₂ tension. In cultures maintained under continuous illumination for one cell cycle, provision of 5% CO₂ led to enhanced growth, a relatively long S-phase and a 4 h delay of the second cell division. In such cultures, the DNA content of parental cells (12 h L) was insufficient to support two cell divisions. The RNA/DNA ratio of the resulting zoospores was 10 compared to 4 in air cultures. These results provided evidence that the delay of the second cell division was a consequence of the delay in DNA production.     

INFLUENCE OF PHOSPHORUS LIMITATION ON TOXICITY AND PHOTOSYNTHESIS OF ALEXANDRIUM MINUTUM (DINOPHYCEAE) MONITORED BY IN‐LINE DETECTION OF VARIABLE CHLOROPHYLL FLUORESCENCE1

The effects of phosphorus (P) limitation on growth, toxicity, and variable chl fluorescence of Alexandrium minutum were examined in batch culture experiments. Cell division was greatly impaired in P‐limited cultures, but P spiking of these cultures after 9 days stimulated high levels of cell division equivalent to P‐replete cultures. The cellular concentration of paralytic shellfish toxins was consistent over the growth cycle of control cultures from lag phase into logarithmic growth phase, with toxins repeatedly lost to daughter cells during division. The low level of cell division in P‐limited cultures resulted in a 10‐fold increase of cellular toxin compared with controls, but this dropped upon P spiking due to increased rates of cell division. The history of phosphorus supply had an important effect on toxin concentration, with the P‐limited and the P‐spiked cultures showing values 2‐fold higher than the P‐replete cultures. Toxin profiles of the A. minutum strain used in these experiments were dominated by the N₁‐hydroxy toxins, gonyautoxins (GTX) GTX1 and GTX4, which were approximately 40 times more abundant than their analogues, GTX2 and GTX3, in P‐limited cultures. The dominance of the N₁‐hydroxy toxins increased significantly in control cultures as they advanced through logarithmic growth. In‐line measurements of the variable chl fluorescence of light‐adapted cells indicated consistent photochemical efficiency under P‐replete conditions. P limitation induced a drop in fluorescence‐based photochemical efficiency that was reversible by P spiking. There was an inverse linear relationship between in‐line fluorescence and cell toxin quota (r = ?0.88). Monitoring fluorescence in‐line may be valuable in managing efficient biotechnological production of toxins.     

P2X5 and P2X7 receptors in human warts and CIN-612 organotypic raft cultures of human papillomavirus infected keratinocytes

Purinergic receptors, which bind adenosine 5′-triphosphate (ATP), are expressed on human cutaneous keratinocytes and in squamous cell carcinomas. Studies on normal human epidermis and primary keratinocyte cultures have suggested that P2X₅ receptors are likely to be involved in keratinocyte differentiation and P2X₇ receptors are likely to be part of the machinery of end stage terminal differentiation/apoptosis of keratinocytes. P2X₇ receptor agonists can significantly reduce primary keratinocyte cell numbers in culture. Human papillomaviruses are increasingly recognised as important human carcinogens in the development of non-melanoma skin cancers. In our study, immunohistochemical analysis for P2X₅ and P2X₇ receptors was performed on paraffin sections of normal human skin, warts, raft cultures of normal human keratinocytes and raft cultures of CIN 612 cells, a model of keratinocytes infected with human papillomavirus type 31. In warts there was up-regulation of the expression of P2X₅ receptors. A similar pattern was seen in the CIN 612 raft cultures. Both P2X₅ and P2X₇ receptors were found in the nuclei of koilocytes, abnormal keratinocytes characteristic of human papillomavirus infection. P2X₅ and P2X₇ receptors may provide a new focus for therapeutic research into treatments for warts because these receptors can induce cell differentiation and cell death.An erratum to this article can be found at     

Fatty acid composition of lipids from differentiated tissues and cell cultures of Euonymus europaeus

The fatty acid patterns of Euonymus europaeus callus cultures and cell suspension cultures were analysed at the beginning of stationary growth phase and compared with those from the respective differentiated tissues. The lipid and fatty acid patterns in cell cultures differed remarkably from those in the tissues of the mother plant. No glycerol triacetate was detected in the callus cultures derived from differentiated tissues whereas in seeds this lipid compound amounts to 29%. In addition to fatty acids normally occurring in differentiated tissues, lipids in cultured cells also contained short-chain (C₁₂–C₁₄) as well as very long-chain fatty acids (C₂₀–C₂₄). In tissue culture cells the major fatty acids were found to be saturated, whereas in the mother cells unsaturated fatty acids were predominant. Palmitic acid is the most abundant fatty acid in most of the cultures. Lauric, myristic and palmitic acid amount to 50% in lipids of cell suspension cultures.     

Strontium ranelate increases the formation of bone-like mineralized nodules in osteoblast cell cultures and leads to Sr incorporation into the intact nodules

We describe effects of strontium ranelate treatment on intact mineralized nodules produced in osteoblast cell cultures. We analyzed the matrix directly at the cell culture surfaces following treatment with 0.05 and 0.5 mM Sr²⁺. This method allowed for data to be obtained from intact nodules, rather than from extracted samples. The bone-like nature of the matrix was evaluated by using attenuated total reflection Fourier transform infrared spectroscopy and the incorporation of Sr into the nodules was investigated by using both energy dispersive X-ray spectroscopy and synchrotron radiation micro X-ray fluorescence. We observed typical mineralized nodules in all of the cell cultures. However, the formation of these nodules was markedly increased in cultures treated with 0.5 mM Sr²⁺. In all of the cultures, the nature of the intact matrix was similar to that described in native bone tissue, being comprised of a poorly crystalline CO₃ ^2?-containing apatite and a collagenous matrix. This indicated that treatment had no deleterious effects on the matrix. Moreover, the nodules presented Ca and P as the main chemical components, confirming their bone-like mineralized nature. The incorporation of Sr into the nodules was clearly observed in the treated cultures, with their relative Sr content [Sr/(Ca+Sr) ratio] being markedly increased in a dose-dependent manner. Thus, strontium ranelate promoted an increase in the formation of mineralized nodules in osteoblast cell cultures while preserving the bone-like nature of the matrix at the tissue level. We further demonstrated that Sr was incorporated into the intact nodules formed during treatment.     

A diploid rat liver cell culture

Summary Chronic exposure of a cloned rat hepatocyte culture (RL-PR-C) to a subtoxic, sublethal dose of aflatoxin B₁ resulted in malignant transformation. Continuous exposure to aflatoxin B₁ caused increasing tumorigenic potential as tested by back injection into isogenic animals. Control cultures exhibited spontaneous transformation, although approximately 20 passages beyond the chemically induced event. Neither aflatoxin-treated nor control cultures exhibited cytopathological morphology, formation of cell foci, growth in soft agar, or irregular fibroblast-like growth patterns that could be specifically related to the onset of tumorigenic potential. In general, those parameters commonly used to monitor fibroblast cultures for transformation in vitro were not applicable for assessing the tumorigenic potential of these epithelial cells. Karyotypic analyses revealed no specific chromosomal aberrations associated with aflatoxin treatment; however, chromosomal instability was a property of the tumorigenic cell populations. Injection of both aflatoxin-treated and control cultures at passage 56 resulted in tumors indicative of both carcinoma and sarcoma indicating to us the multipotency of these epithelial cells transformed in vitro.     

A NanoDrop-based method for rapid determination of viability decline in suspension cultures of animal cells

We describe a rapid method for monitoring the cell growth and decline phases in suspension cultures of animal cells. During the cell growth phase, ultraviolet (UV)-absorbing components in the medium are consumed, but at later times as cells begin to die, UV-absorbing molecules such as proteins are released into the medium. Measuring the absorbance at 280 nm (A₂₈₀) with a NanoDrop spectrophotometer, an inverse correlation between the onset of the cell decline phase and A₂₈₀ was observed. This simple method can be applied to quickly determine the beginning of the decline phase of cultures of mammalian and insect cells in suspension.     

TES and HEPES buffers in mammalian cell cultures and viral studies: Problem of carbon dioxide requirement

In order to evaluate TES and HEPES as a buffer system for cell culture, the proliferative capacities of cells of several mammalian cell lines in the medium buffered with either of these compounds were examined in cultures in stoppered and open flasks at high and low cell densities. When cultivated in stoppered flasks, cells grew equally well or even better in TES- and HEPES-buffered medium than in NaHCO₃-buffered medium irrespective of cell culture density. In open flasks or Petri dishes in TES- or HEPES-buffered medium, however, the proliferative capacity of cells in low density cultures was limited. The inhibition of cell growth in the latter condition was restored (1) as the cell density of the cultures increased; (2) by feeding continuously the cultures with the gas produced by high density cultures; (3) by introducing a small amount of CO₂ to the environment.These and other evidences presented suggest that, in agreement with the prevailing notions, CO₂ is required by cells as an essential nutrient for growth, and that the desired level of CO₂ in culture can be maintained efficiently by its production by even a small number of cells in culture as long as the culture flasks are stoppered. If flasks are not stoppered, however, the level of CO₂ tension is determined by an equilibrium between the rate of its production by the cells and that of escape from culture to air, resulting in the observed failure in growth of cells in TES- and HEPES-buffered medium at low cell densities unless cultures were further supplemented with added CO₂.     

Characterization of the cell cycle of cultured human diploid cells: Effects of aging and hydrocortisone

Age-related changes in the cytokinetics of human diploid cells in vitro have been compared in normal cultures and in cultures in which lifespan has been prolonged by the addition of hydrocortisone to the medium. For both cultures, with advancing age the fraction of cells in the actively proliferating pool decreased and the intercellular variation in cell cycle times increased. The average cell cycle time was prolonged during aging due almost entirely to changes in the duration of G₁. The duration of S remained constant, while a small delay in G₂ was observed in late passage cells near the end of their lifespan. Although the same pattern of change in proliferative parameters occurred in both control and hydrocortisone-treated cultures, the changes were somewhat delayed in the presence of the steroid. The results are interpreted in terms of several cell cycle models and suggest that the events controlling cell proliferation are sensitive to hydrocortisone modulation during the G₁ and possibly the G₂ periods.     

Gametic differentiation in Chlamydomonas reinhardi: cell cycle dependency and rates in attainment of mating competency

Withdrawal of a utilizable nitrogen source during mid G₁ of the cell cycle induces gametic differentiation in synchronously grown vegetative cultures of Chlamydomonas reinhardi. Cell division accompanies gametic differentiation in such cultures, and the ability of mid G₁ vegetative cells to form gametes is matched by their ability to undergo a round of cell division after nitrogen withdrawal. Synchronously grown cultures require up to 19 hr in nitrogen-free medium to complete a round of division and to form mating-competent cells. Asynchronously grown liquid cultures require less time after nitrogen withdrawal (generally 5–8 hr) to achieve mating competency. In these cultures cell division did not necessarily accompany gametic differentiation since gametic differentiation took place in induced cultures at high cell concentrations which prevented cell division. Maximum mating competency was achieved in less than 2 hr after induction of vegetative cells grown on agar plates. Little cell division was observed during that short induction interval. The relationship between the attainment of mating competency (gametogenesis) and other physiological events resulting from nitrogen withdrawal is discussed.