Adaptation of Saccharomyces cerevisiae cells to high ethanol concentration and changes in fatty acid composition of membrane and cell size

Background

Microorganisms can adapt to perturbations of the surrounding environment to grow. To analyze the adaptation process of the yeast Saccharomyces cerevisiae to a high ethanol concentration, repetitive cultivation was performed with a stepwise increase in the ethanol concentration in the culture medium.

Methodology/Principal Findings

First, a laboratory strain of S. cerevisiae was cultivated in medium containing a low ethanol concentration, followed by repetitive cultivations. Then, the strain repeatedly cultivated in the low ethanol concentration was transferred to medium containing a high ethanol concentration and cultivated repeatedly in the same high-ethanol-concentration medium. When subjected to a stepwise increase in ethanol concentration with the repetitive cultivations, the yeast cells adapted to the high ethanol concentration; the specific growth rate of the adapted yeast strain did not decrease during repetitive cultivation in the medium containing the same ethanol concentration, while that of the non-adapted strain decreased during repetitive cultivation. A comparison of the fatty acid composition of the cell membrane showed that the contents in oleic acid (C_18:1) in ethanol-adapted and non-adapted strains were similar, but the content of palmitic acid (C_16:0) in the ethanol-adapted strains was lower than that in the non-adapted strain in media containing ethanol. Moreover, microscopic observation showed that the mother cells of the adapted yeast were significantly larger than those of the non-adapted strain.

Conclusions

Our results suggest that activity of cell growth defined by specific growth rate of the yeast cells adapted to stepwise increase in ethanol concentration did not decrease during repetitive cultivation in high-ethanol-concentration medium. Moreover, fatty acid content of cell membrane and the size of ethanol-adapted yeast cells were changed during adaptation process. Those might be the typical phenotypes of yeast cells adapted to high ethanol concentration. In addition, the difference in sizes of the mother cell between the non-adapted and ethanol strains suggests that the cell size, cell cycle and adaptation to ethanol are thought to be closely correlated.     

Rat glioma cells (C6) cultured in serum-free defined medium

Rat glioma C₆ cells were adapted to and maintained in serum-free medium for 11 months. Morphological differentiation with extended dendrite processes was observed. This phenomenon is reversible if serum is re-supplemented and is protein or RNA synthesis dependent. The formed cytoplasmic processes rapidly retract when colchicine or vinblastine sulfate is added. db-cAMP is found able to stimulate the extension of cytoplasmic processes of cells cultured in medium containing serum, but no further stimulation was observed in cells adapted to serum-free medium. The serum-free adapted cells retain the ability to synthesize the acidic S-100 protein and the production rate is 25% higher than the cells cultured in serum-supplemented medium. The serum-free adapted cells have a longer population doubling time but the metaphase chromosomes show the same karyotype and modal number as that of C₆ cells continuously cultured in serum-supplemented medium.     

Differentiation markers of mouse C2C12 and rat L6 myogenic cell lines and the effect of the differentiation medium

Summary The differentiation grade of cells in culture is dependent on the composition of the culture medium. Two commonly used myogenic cell lines, mouse C₂C₁₂ and rat L₆, usually differentiate at a low concentration of horse serum. In this study we compared the effect of horse serum with a medium containing a low percentage of Ultroser G and rat brain extract. The maturation grade was evaluated on the basis of various biochemical, (immuno)histochemical and cell-physiological parameters. Substitution of horse serum by Ultroser G and rat brain extract during the differentiation phase resulted in a higher maturation grade of the myotubes of both cell lines, on the basis of creatine kinase activity and the diameter of the myotubes. In addition, the C₂C₁₂ myotubes display cross-striation, contain a higher percentage of creatine kinase muscle-specific isoenzyme MM, show a ninefold increase in acetylcholine receptor (AChR) clusters, form a continuous basement membrane, and have a lower resting cytosolic Ca²⁺ concentration. L₆ myotubes show a fivefold increase in AChR clusters and a twofold increase in the expression of the mRNA of the ɛ-subunit of AChR. C₂C₁₂ cells show spontaneous contraction and response of cytosolic Ca²⁺ to various stimulants in contrast to L₆ cells which do not. These studies established that the Ultroser G/brain extract medium leads to a higher differentiation grade of both cell lines, but parameters appropriate for use as differentiation markers appear to differ between both cell lines.     

Rat pituitary tumor cells in serum-free culture. I. Selection of thyroid hormone-responsive and autonomous cells

Summary The growth of GH₄C₁, GH₃, GH₁, and GH₃C₁₅ rat pituitary tumor cell lines was studied in a serum-free medium (designated TRM-1) formulated with 1∶1 (vol/vol) mixture of Ham's F12 nutrient mixture and Dulbecco's modified Eagle's medium (F12-DME) containing 15 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 50 μg/ml gentamicin supplemented with 10 μg/ml bovine insulin, 10 μg/ml human transferrin (Tf), 10 ng/ml selenous acid, 10 nM 3,5,3′-triiodothyronine (T₃), 50 μM ethanolamine (Etn), and 500 μg/ml bovine serum albumin. Of the lines evaluated, only the GH₁ failed to grow in TRM-1. Passage of the GH₄C₁ and GH₃ lines from serum-containing medium into TRM-1 caused an initial selection resulting in cells that grew progressively at higher rates and finally were maintained indefinitely in TRM-1. These populations showed a requirement for supraphysiologic concentrations of T₃ (1.0 to 10 nM). After adaptation of the GH₄C₁ line in TRM-1 for ≤20 generations, removal of components gave a less complex mixture containing 15 mM HEPES, 50 μ/ml gentamicin, 10 μg/ml Tf, 10 nM T₃, and 50 μM Etn (designated TRM-2) that supported serial passage of the cells. Under these conditions, thyroid hormone dependence was lost progressively. When T₃ was removed from TRM-2 adapted cells, a third population was selected that no longer required thyroid hormones and was only slightly stimulated by T₃. These studies demonstrated that the combination of serum-containing and serum-free conditions can be used to select pituitary cell populations that a) required both serum-factor(s) and T₃ for optimum growth, b) required supraphysiologic concentrations of T₃ without serum proteins other than Tf and albumin, and c) were completely autonomous in that they proliferated in medium supplemented only with Tf and nutrients without necessity of other serum factor(s) or T₃. This work was supported by grants CA-26617 and CA-38024 from the National Cancer Institute, Bethesda, MD, American Cancer Society grant BC-255, and grant 2225 from the Council for Tobacco Research, Inc., USA.     

Integration of cell line and process development to overcome the challenge of a difficult to express protein

This case study addresses the difficulty in achieving high level expression and production of a small, very positively charged recombinant protein. The novel challenges with this protein include the protein's adherence to the cell surface and its inhibitory effects on Chinese hamster ovary (CHO) cell growth. To overcome these challenges, we utilized a multi‐prong approach. We identified dextran sulfate as a way to simultaneously extract the protein from the cell surface and boost cellular productivity. In addition, host cells were adapted to grow in the presence of this protein to improve growth and production characteristics. To achieve an increase in productivity, new cell lines from three different CHO host lines were created and evaluated in parallel with new process development workflows. Instead of a traditional screen of only four to six cell lines in bioreactors, over 130 cell lines were screened by utilization of 15 mL automated bioreactors (AMBR) in an optimal production process specifically developed for this protein. Using the automation, far less manual intervention is required than in traditional bench‐top bioreactors, and much more control is achieved than typical plate or shake flask based screens. By utilizing an integrated cell line and process development incorporating medium optimized for this protein, we were able to increase titer more than 10‐fold while obtaining desirable product quality. Finally, Monte Carlo simulations were performed to predict the optimal number of cell lines to screen in future cell line development work with the goal of systematically increasing titer through enhanced cell line screening. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1201–1211, 2015     

Regulation of retinol-binding protein metabolism in cultured rat liver cell lines

Retinol-binding protein (RBP), the plasma transport protein for vitamin A, is synthesized and secreted by the liver. In vitamin A deficiency, RBP secretion is blocked, leading to low serum and high liver levels of RBP. Administration of retinol to the intact rat stimulates a rapid secretion of RBP from liver into serum. We explored the use of a liver cell culture system to study the regulation of the synthesis and secretion of RBP. We found two lines of differentiated rat hepatoma cells, MH₁C₁ and H₄ II EC₃ (H₄), that synthesized RBP during culture in vitro. The net synthesis of RBP was a function of the number of cells per dish and the duration of incubation. Both cell lines synthesized RBP when incubated in Neuman and Tytell's Serumless Medium (NTS medium), while the MH₁C₁ cells also synthesized RBP in Ham's F-12 medium with added serum. A relatively large proportion (14–56%) of the RBP was retained within the cells when they were incubated in the vitamin A-free NTS medium alone. Addition of serum to NTS medium stimulated the release of RBP from the cells into the medium and also increased the net synthesis of RBP. These effects were not due to the increased adhesion of the cells to the petri dish. Addition of retinol (at levels of 0.35 or 3.5 nmole/ml) to the NTS medium resulted in the stimulation of RBP secretion from the cells into the medium and an increase in the net synthesis of RBP. By contrast, retinol had no effect on either the net synthesis or the cell-to-medium distribution of rat serum albumin. The data from these cell lines in culture suggest that retinol has a specific regulatory effect on RBP metabolism. These cells thus resemble the normal rat liver cell in vivo in regard to the known regulation of RBP metabolism.     

Growth, metabolism and baculovirus production in suspension cultures of an Anticarsia gemmatalis cell line

The UFL-AG-286 cell line, established from embryonic tissue of the lepidopteran insect Anticarsia gemmatalis, has been identified as a good candidate to be used as a cellular substrate in the development of a process for in vitro production of the Anticarsia gemmatalis multicapsid nucleopolyhedrovirus, a baculovirus widely used as bioinsecticide. In order to characterize the technological properties of this cell line and evaluate its feasibility to use it for the large-scale production of Anticarsia gemmatalis multicapsid nucleopolyhedrovirus, UFL-AG-286 cells were adapted to grow as agitated suspension cultures in spinner-flasks. Batch suspension cultures of adapted cells in serum-supplemented TC-100 medium grew with a doubling time of about 29 h and reached a maximum cell density higher than 3.5 × 10⁶ viable cells ml⁻¹. At the end of the growth period glucose was completely depleted from the culture medium, but l-lactate was not produced. Amino acids, with the exception of glutamine, were only negligibly consumed or produced. In contrast to other insect cell lines, UFL-AG-286 cells appeared to be unable to synthesize alanine as a metabolic way to dispose the by-product ammonia. The synchronous infection of suspension cultures with Anticarsia gemmatalis multicapsid nucleopolyhedrovirus in the early to medium exponential growth phase yielded high amounts of both viral progenies per cell and reduced the specific demands of UFL-AG-286 cells for the main nutrients.     

Establishment of two chicken embryonic cell lines in a newly developed nutrient medium

Two cell lines, named KCEK and KCEL, were established from chicken embryonic kidney and lung. The basal culture medium was newly developed and the cell growth medium consisted of K1999 supplemented with 10% heat inactivated chicken serum. Both cells were well adapted to grow in vitro and more than 50 passages have been made so far. Once the cell lines were established the cells were easily adapted to grow in other growth media supplemented with fetal calf serum. Neither tumor formation in chicks nor P52 avian leucosis common antigen was detected in these cells. However, the oncogene analysis on these cells has not been performed yet. Both cells were permissive hosts for the Aujeszky's disease virus, Newcastle disease virus, and vesicular stomatitis virus.     

Selection of tomato tissue cultures able to grow on ribose as the sole carbon source

When tomato (Lycopersicon esculentum Mill.) callus or cell cultures were placed on media containing ribose as the sole carbon source, the tissues turned dark brown and ceased growth. However, after approximately 60 days bright green tissue able to grow on ribose emerged from 3 % of the brown necrotic callus tissue pieces plated. The selected tissue was highly organized, consisting of leafy primordia and associated meristematic tissues, sustained growth on ribose, and demonstrated the capacity to regenerate whole plants for at least 3 years. Cultures able to grow on ribose could not be selected from liquid suspension cultured tomato cells or from callus which had been mechanically macerated into cell aggregates containing less than approximately 100 cells. Plants regenerated from ribose adapted cultures were abnormal, having shortened internodes and thicker greener leaves. Regenerated plants were both male and female sterile.Abbreviations BAP N⁶-benzylaminopurine - CFM callusforming medium - IAA indole acetic acid - SDM shoot determination medium - RCM ribose containing medium     

Evidence for Inorganic Carbon Transport by Intact Chloroplasts of Chlamydomonas reinhardtii

Isolated intact chloroplasts from wall-less mutants of Chlamydomonas reinhardtii accumulate inorganic carbon (C_i) from the medium provided the cells had been adapted to low CO₂ photoautotrophic growth conditions. Chloroplasts from cultures grown on high (5%) CO₂ or photoheterotrophically with acetate did not accumulate inorganic carbon. Chloroplast C_i accumulation from low CO₂ grown cells was light dependent and was inhibited by uncouplers and inhibitors of electron transport. In a model for C_i accumulation by Chlamydomonas, it is proposed that CO₂ diffuses into the cell and C_i accumulation occurs in the chloroplast.     

Hormone-defined cell system for studying G-protein coupled receptor agonist-activated growth modulation: δ-Opioid and serotonin-5HT2C receptor activation show opposite mitogenic effects

G-protein-coupled receptor (GPCR) agonist-activated transformation of NIH/3T3 fibroblast cells has been documented by many workers. Our present interest is in the growth control exerted by these agonists. The mechanisms involved in GPCR agonist-activated growth regulation are not known and investigations using existing cell lines are complicated by the endogenous expression of numerous different GPCRs as well as by the fact that these cell lines are cultured in serum that contains naturally occurring agonists for these receptors. To study the agonist induced growth response of cells transfected with either δ-opioid or serotonin-5HT_2C neurotransmitter receptor genes, we have developed new clonal cell lines derived from NIH/3T3 mouse fibroblast cells. These new cell lines, designated with the suffix 3T3DA, can be cultured stably in serum-free, hormone-defined medium: insulin is the only exogenous growth factor added to the culture medium of proliferating 3T3DA cell lines, and their proliferation can be stopped and started by the respective removal or addition of insulin. Micromolar concentrations of agonists were used to activate the corresponding opioid and serotonin receptors over periods extending to 6 days. We observed distinct patterns of GPCR-specific, agonist-activated growth regulation in serum-free cultures, but not in serum-supplemented cultures. At concentrations > 10 μM, morphine inhibits growth of δ-opioid receptor-expressing cells by 40% with respect to normal 3T3DA cells. Opioid agonist induced inhibition of cyclic AMP (cAMP) production as well as growth down-regulation are pertussis toxin sensitive indicating that the exogenously expressed δ-opioid receptors demonstrate classical opioid receptor signaling. The presence of 1 μM serotonin stimulates growth of serotonin-5HT_2C receptor-expressing cells by approximately 100% with respect to normal 3T3DA cells. Neither the untreated nor the agonist-treated cells form colonies in soft agar, indicating that they retain anchorage-dependent growth control. These cell lines provide a simple system that could be used as a tool for probing the complex molecular mechanisms associated with GPCR agonist-activated growth control. J. Cell. Physiol. 171:61–74, 1997. © 1997 Wiley-Liss, Inc.     

Oxidation of Gaseous and Volatile Hydrocarbons by Selected Alkene-Utilizing Bacteria

Eleven strains of alkene-utilizing bacteria belonging to the genera Mycobacterium, Nocardia, and Xanthobacter were tested for their ability to grow with C₁ to C₆ alkanes, C₂ to C₆ alkenes, alkadienes, and monoterpenes furnished individually as sole sources of carbon and energy in a mineral salts medium. A limited number of alkenes and alkanes supported growth of the bacteria; some bacteria were unable to grow on any of the saturated hydrocarbons tested. Monoterpenes were frequently used as carbon and energy sources by alkene-utilizing bacteria belonging to the genera Mycobacterium and Nocardia. Washed cell suspensions of alkene-grown bacteria attacked the whole range of alkenes tested, whereas only three strains were able to oxidize alkanes as well. The alkenes tested were oxidized either to water and carbon dioxide or to epoxyalkanes. Few epoxides accumulated in stoichiometric amounts from the corresponding alkenes, because most epoxides formed were further converted to other compounds like alkanediols.     

The role of calmodulin in cell transformation

Two cell lines transformed with temperature sensitive retroviruses were examined for: their ability to grow in low Ca²⁺ medium, their calmodulin levels and changes in calmodulin acceptor proteins. Both cell lines grow in low Ca²⁺ medium at the permissive temperature 34°C while both lines did not replicate at the non-permissive temperature 39°C. The NRKLA23 cells have nearly twice as much calmodulin at the permissive temperature than they do at the non-permissive temperature while the 6M2 cells have an equal amount of calmodulin at both temperatures. Both cell lines exhibit changes in the calmodulin acceptor proteins going from the permissive to the non-permissive temperature. We suspect that the changes in the calmodulin acceptor proteins may be involved in the altered Ca²⁺-sensitivity of growth in the cells going from the permissive to non-permissive temperature.     

A serum-free,chemically-defined medium for function and growth of primary neonatal rat heart cell cultures

Summary A serum-free, chemically defined medium for supporting rhythmic contraction, maximum survival, and moderate growth of cardiac cells was achieved by using a combination of hormones and growth supplements in a mixture of equal volumes of Ham’s F12 and Dulbecco’s modified Eagle’s medium. The hormones and growth supplements included insulin, transferrin, selenium, fetuin, bovine serum albumin, hydrocortisone (HC),l-thyroxine (T₄), and epidermal growth factor (EGF). Cardiac cells were grown on fibronectin-precoated plates using the above serum-free medium. Cells grow in this medium exhibited a higher beating rate and were maintained for a longer time compared to those cells grown in serum. The effects of T₄, EGF, and HC on beating rate and survival time of both cultures of mixed cell population and enriched myoblast cell population were studied. In the enriched myoblast cell cultures grown in serum-supplemented medium, the beating rate ranged from 40 to 200 beats/min, and these cultures survived for 30 d. When these enriched cell cultures were grown in serum-free hormone-supplemented medium, the beating rate ranged from 190 to 240 beats/min, and these cultures survived for more than 90 d. These results show that some hormones affect growth, whereas others affect function.     

Thermotolerance and intracellular pH in two Chinese hamster cell lines adapted to growth at low pH

As an in vitro model for the low extracellular pH (pH_e) which has frequently been observed in tumors, cell lines have been grown in a low-pH medium in order to allow cell adaptation to that milieu. Two Chinese hamster cell lines [Chinese hamster ovary (CHO) and Chinese hamster ovarian carcinoma (OvCa)] were compared, both of which acquired thermotolerance during 42°C heating in pH_e = 7.3 buffer, but not in pH_e = 6.7 medium unless grown at that pH long enough to become adapted. CHO cells, even when acutely acidified, showed higher intracellular pH (pH_i) values in a suspension assay than OvCa cells, which confirmed the danger of comparing absolute values of pH_i between cell lines. Despite this fundamental difference, relative changes in pH_i were similar in that both lines showed a higher pH_i in adapted than in unadapted cells, over the range of pH_e values tested. The upregulation of pH_i was statistically significant, but the two lines differed in the time frame over which adaptation occurred. OvCa cells acquired an enhanced ability to develop tolerance to 42° heat at pH_e = 6.7 in 4 days, but the CHO cells acquired this ability more progressively, achieving a maximum ability at approximately 100 days. In contrast, both lines were able to upregulate their pH_i within 4 hours of being exposed to pH 6.7 medium. A further indication of different biochemical mechanisms at work was the opposite effects seen on pH_i in the two cell lines upon the removal of extracellular CO₂/HCO⁻₃. The differential between adapted and unadapted OvCa cells was enhanced by removal of bicarbonate, whereas CHO cells seemed less stable and the data with greater scatter failed to show any difference between adapted and unadapted cells. © 1996 Wiley-Liss, Inc.     

Improvement in the proliferative activity of human-human hybridomas at low cell density by transfection with bFGF gene

Highly purified recombiaant basic fibroblast growth factor (rbFGF) and acidic FGF (aFGF) stimulated the proliferation of human-human (h-h) hybridomas to the extent of over four-fold from a low cell density such as 1×10³ cells per ml in a serum-free medium in 24-well plates. The stimulatory effect of rbFGF was also observed in various lymphoid cell lines. Expecting that FGF could be an autocrine growth factor, we introduced bFGF gene into a h-h hybridoma using an expression plasmid induced by dexamethasone. The transformed cells thus obtained, HPO-75.11bbFGF-7, were able to grow well from a low inoculum density in a serum-free medium and antibody production was also increased when bFGF gene expression was induced. The transformed cells could grow at clonal density in a serum-free medium in 96-well plates, though the original cells could not. We also obtained a more practical transfectant, HPO-75.29-H74, using a high-shear stress adapted clone as the recipient and an expression plasmid having bFGF gene under the control of metallothioneine-I promoter. The HOP-75.29-H74 cells were capable of growing and producing human monoclonal antibody against hepatitis B virus surface antigen from an inoculum density of 1×10³ cells per ml in an agitation vessel without addition of an inducer.     

The culture of rat myeloma and rat hybridoma cells in a protein-free medium

Y0 is a rat x rat hybridoma cell line, which does not secrete immunoglobulin, produced using a fusion partner derived from the Y3 (Y3,Ag.1.2.3) rat myoloma cell line. Y0 and Y3 have both been widely used as fusion partners in the production of rat x rat hybridomas. Y0 has also been used in recombinant gene technology. Y0 cells grown in shake flask culture, using RPMI 1640 medium with 4mM l-glutamine and 5% foetal bovine serum, reached a maximal cell density of 1.5×10⁶ cells ml^–1 with 86% viability. Y0 cells which has been adapted to grow in ABC protein-free medium reached a maximal density, in shake flask culture, of 8.75×10⁵ cells ml^–1 with 79% viability. An improved protein-free medium, designated W38 medium, was developed. In shake flask culture, W38 medium supported Y0 cell growth to a density of 2.02×10⁶ cells ml^–1 with 96% viability. Two Y3 hybridomas, YID 13.9.4 cells and SAM 618 cells were adapted to growth in W38 medium. For both hybridomas, cell growth and product yield in shake flask culture using W38 medium was superior to that obtained with serum-containing RPMI 1640 medium.Abbreviations F12 Hams F12 medium - DMEM Dulbeccos medium - RPMI RPMI 1640 medium - FBS foetal bovine serum     

An increased requirement for methionine by transformed rat liver epithelial cells in vitro

The growth of two normal and four transformed rat liver epithelial cell lines in a methionine-containing medium and a methionine-deficient medium supplemented with homocysteine was examined. The growth rates of the normal cells on the homocysteine-supplemented medium were approximately one-half the growth rates shown by the same cells in the methionine-containing medium. In contrast, three of the four transformed cell lines studied showed virtually no growth on the homocysteine-supplemented medium, although they grew quite rapidly on the methionine-containing medium. The fourth, transformed by N-methyl-N-nitrosourea, was able to grow on the homocysteine-supplemented medium at about one-third the rate as on the methionine-containing medium. Thus, transformed rat liver epithelial cells resemble other malignant cells in their reduced capacity to grow on homocysteine in the absence of methionine.     

Two routes for synthesis of phosphoenolpyruvate from C4-dicarboxylic acids in Escherichia coli

Mutants of $\text{E. coli}$ defective in both phosphoenolpyruvate carboxykinase and phosphoenolpyruvate synthetase are unable to use C₄-dicarboxylic acids such as succinate and malate as carbon and energy sources for growth. Revertants that have restored function for either one of these enzymes can grow in a malate-mineral medium, but at a reduced rate compared with the growth of wild-type cells. $\text{E. coli}$ appears to use two pathways for synthesis of phosphoenolpyruvate from C₄-dicarboxylic acids. One of these involves decarboxylation of oxalacetate catalyzed by phosphoenolpyruvate carboxykinase. The second pathway makes use of the combined action of malic enzyme and phosphoenolpyruvate synthetase.     

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