Lars Öhman

Molecular basis of agonism and antagonism in the oestrogen receptor.

Oestrogens are involved in the growth, development and homeostasis of a number of tissues. The physiological effects of these steroids are mediated by a ligand-inducible nuclear transcription factor, the oestrogen receptor (ER). Hormone binding to the ligand-binding domain (LBD) of the ER initiates a series of molecular events culminating in the activation or repression of target genes. Transcriptional regulation arises from the direct interaction of the ER with components of the cellular transcription machinery,. Here we report the crystal structures of the LBD of ER in complex with the endogenous oestrogen, 17β-oestradiol, and the selective antagonist raloxifene, at resolutions of 3.1 and 2.6 Å, respectively. The structures provide a molecular basis for the distinctive pharmacophore of the ER and its catholic binding properties. Agonist and antagonist bind at the same site within the core of the LBD but demonstrate different binding modes. In addition, each class of ligand induces a distinct conformation in the transactivation domain of the LBD, providing structural evidence of the mechanism of antagonism.

Physiology of cultured animal cells

The physiology of cultured animal cells, in particular hybridoma, myeloma and insect cells, with respect to growth and proliferation, amino acid metabolism, energy metabolism and cellular responses to environmental stress is discussed in this paper. The rate of proliferation of hybridoma cells in serum-containing media is limited by growth factors at a surprisingly early stage of growth. To maintain exponential growth in a batch culture, it is necessary to stimulate cell proliferation with repeated additions of serum or pure growth factor. It is further suggested that proliferation of Spodoptera frugiperda (Sf9 insect cells), a normal cell line able to grow in a serum-free medium without any added growth factors, is regulated by autocrine growth factors and possibly by other regulatory mechanisms, as Sf9 cells secrete a growth factor (IGF-I) and the medium still appears nutritionally sufficient at the time of cessation of growth. The uptake and metabolism of amino acids is one of the determinants of growth and production. Wasteful overproduction of amino acids in myeloma and hybridoma cells is a result of excess glutamine, and can be avoided by glutamine limitation. Synthesis of amino acids may be conditional, as in Sf9 cells which synthesise glutamine provided that ammonium is supplied to the medium; and cysteine (from methionine) provided that a sufficiently young inoculum is used. Uptake of amino acids in Sf9 cells appears regulated in relation to the proliferative status as there is a distinct cessation of uptake even before growth ceases. The energy metabolism in myeloma, hybridoma and insect cells is a typically substrate-concentration-dependent overflow metabolism. Substrate limitation (glucose and glutamine) decreases by-product formation and increases metabolic efficiency in all these cell lines. However, glutamine limitation, as used in fed-batch cultures (or chemostat cultures) provokes cell death (in parallel to growth) in hybridoma cells in the concentration range below 0.05 mM.

Autoantibodies against the tumour-associated antigen GA733-2 in patients with colorectal carcinoma

Abstract The tumour-associated antigen (TAA) GA733-2 is expressed as a non-secreted surface molecule on the majority of human colorectal carcinoma cells. The antigen has been used as a target for passive and active immunotherapy during the last decade. To determine the incidence of autoantibodies against this antigen, sera from 1068 patients with colorectal carcinoma were analysed for naturally occurring IgG antibodies against the baculovirus-produced GA733-2E protein. A total of 14.5% of the patients had IgG antibodies against the antigen. In 519 patients, sera were collected at the time of diagnosis and 15% of those patients had anti-GA733-2E IgG antibodies. There was a tendency to a higher frequency of patients with antibodies among those in the advanced Dukes stages: 11% in stage A and 32% in stage D respectively (P = 0.06). Antibodies could be detected for up to 10 years after the diagnosis. Patients with Crohns disease or colitis ulcerosa (n = 20) did not elicit anti-GA733-2E antibodies. No healthy control donor (n = 45) had detectable antibodies against the antigen. The specificity of GA733-2E-reactive serum IgG was indicated by significant inhibition of mAb17-1A (originally used to define GA733-2) binding to the GA733-2E antigen. Sera of positive patients bound to the GA733-2-expressing human colorectal carcinoma cell line, SW948. No significant correlation was found between the presence of antibodies and survival in the present patient population. However, the high incidence of autoantibodies against this tumour antigen in colorectal carcinoma patients confirms its antigenicity in humans and supports the use of the GA733-2 antigen as a target for immunotherapy.

Pathways of glutamine metabolism in Spodoptera frugiperda (Sf9) insect cells: evidence for the presence of the nitrogen assimilation system, and a metabolic switch by 1H/15N NMR☆

1H/15N and 13C NMR were used to investigate metabolism in Spodoptera frugiperda (Sf9) cells. Labelled substrates ([2-15N]glutamine, [5-15N]glutamine, [2-15N]glutamate, 15NH4Cl, [2-15N]alanine, and [1-13C]glucose) were added to batch cultures and the concentration of labelled excreted metabolites (alanine, NH4+, glutamine, glycerol, and lactate) were quantified. Cultures with excess glucose and glutamine produce alanine as the main metabolic by-product while no ammonium ions are released. 1H/15N NMR data showed that both the amide and amine-nitrogen of glutamine was incorporated into alanine in these cultures. The amide-nitrogen of glutamine was not transferred to the amine-position in glutamate (for further transamination to alanine) via free NH4+ but directly via an azaserine inhibitable amido-transfer reaction. In glutamine-free media 15NH4+ was consumed and incorporated into alanine. 15NH4+ was also incorporated into the amide-position of glutamine synthesised by the cells. These data suggest that the nitrogen assimilation system, glutamine synthetase/glutamate synthase (NADH-GOGAT), is active in glutamine-deprived cells. In cultures devoid of glucose, ammonium is the main metabolic by-product while no alanine is formed. The ammonium ions stem both from the amide and amine-nitrogen of glutamine, most likely via glutaminase and glutamate dehydrogenase. 13C NMR revealed that the [1-13C] label from glucose appeared in glycerol, alanine, lactate, and in extracellular glutamine. Labelling data also showed that intermediates of the tricarboxylic acid cycle were recycled to glycolysis and that carbon sources, other than glucose-derived acetylCoA, entered the cycle. Furthermore, Sf9 cell cultures excreted significant amounts glycerol (1.9-3.2 mM) and ethanol (6 mM), thus highlighting the importance of sinks for reducing equivalents in maintaining the cytosolic redox balance.

Glutamine is not an essential amino acid for Sf-9 insect cells

SummarySpodoptera frugiperda (Sf-9) insect cells are fully capable of growth and proliferation in a glutamine, glutamate and aspartate-free medium, provided that ammonium ions are supplied. S. frugiperda (Sf-21) and Mamestra brassicae cells (IZD-MB-0503) also grow in glutamine-free media but not Trichoplusia ni cells (BTI-TN 5B1-4). The yield of β-galactosidase in Sf-9 cells infected with a recombinant baculovirus under glutamine-free conditions was even higher than the yield obtained in glutamine containing cultures.

Cell Cycle Progression in Serum‐Free Cultures of Sf9 Insect Cells: Modulation by Conditioned Medium Factors and Implications for Proliferation and Productivity

Cell cycle progression was studied in serum‐free batch cultures of Spodoptera frugiperda (Sf9) insect cells, and the implications for proliferation and productivity were investigated. Cell cycle dynamics in KBM10 serum‐free medium was characterized by an accumulation of 50−70% of the cells in the G2/M phase of the cell cycle during the first 24 h after inoculation. Following the cell cycle arrest, the cell population was redistributed into G1 and in particular into the S phase. Maximum rate of proliferation (μN,max) was reached 24−48 h after the release from cell cycle arrest, coinciding with a minimum distribution of cells in the G2/M phase. The following declining μN could be explained by a slow increase in the G2/M cell population. However, at approximately 100 h, an abrupt increase in the amount of G2/M cells occurred. This switch occurred at about the same time point and cell density, irrespective of medium composition and maximum cell density. An octaploid population evolved from G2/M arrested cells, showing the occurrence of endoreplication in this cell line. In addition, conditioned medium factor(s) were found to increase μN,max, decrease the time to reach μN,max, and decrease the synchronization of cells in G2/M during the lag and growth phase. A conditioned medium factor appears to be a small peptide. On basis of these results we suggest that the observed cell cycle dynamics is the result of autoregulatory events occurring at key points during the course of a culture, and that entry into mitosis is the target for regulation. Infecting the Sf9 cells with recombinant baculovirus resulted in a linear increase in volumetric productivity of β‐galactosidase up to 68−75 h of culture. Beyond this point almost no product was formed. Medium renewal at the time of infection could only partly restore the lost hypertrophy and product yield of cultures infected after the transition point. The critical time of infection correlated to the time when the mean population cell volume had attained a minimum, and this occurred 24 h before the switch into the G2/M phase. We suggest that the cell density dependent decrease in productivity ultimately depends on the autoregulatory events leading to G2/M cell cycle arrest.

Metabolic Engineering of Animal Cellsa

Substrate-limited fed-batch cultures were used to study growth and overflow metabolism in hybridoma and insect cells. In hybridoma cells a glucose-limited fed-batch culture decreased lactate formation but increased glutamine consumption and ammonium formation. Glutamine limitation decreased ammonium and alanine formation but did not enhance glucose consumption. Instead lactate formation was reduced, indicating that glucose was used more efficiently. The formation of lactate, alanine, and ammonium was negligible in a dual substrate-limited fed-batch culture. The efficiency of the energy metabolism increased, as judged by the increase in the cellular yield coefficient for glucose of 100% and for glutamine of 150% and by the change in the metabolic ratios lac/glc, ala/gln, and NHx/gln, in the combined fed-batch culture. Insect cell metabolism was studied in Spodoptera frugiperda (Sf-9) cells. A stringent relation between glucose excess and alanine formation was found. In contrast, glucose limitation induced ammonium formation, while, at the same time, alanine formation was completely suppressed. Simultaneous glucose and glutamine limitation suppressed both alanine and ammonium formation. Alanine formation appears as wasteful as lactate formation because the growth rate of insect cells in substrate-limited cultures was the same as in batch cultures with substrate excess. In batch and fed-batch cultures of both cell lines, mu reaches it maximum early during growth and decreases thereafter so that no exponential growth occurs. The growth rate limiting factor for hybridoma cells was found to be a component of serum, because intermittent serum additions to batch cultures resulted in a high and constant growth rate. Insulin was identified as the main cause, inasmuch as intermittent insulin additions gave the same result as serum.