Use of global gene expression patterns in mechanistic studies of oestrogen action in MCF7 human breast cancer cells.

Over the years, the MCF7 human breast cancer cell line has provided a model system for the study of cellular and molecular mechanisms in oestrogen regulation of cell proliferation and in progression to oestrogen and antioestrogen independent growth. Global gene expression profiling has shown that oestrogen action in MCF7 cells involves the coordinated regulation of hundreds of genes across a wide range of functional groupings and that more genes are downregulated than upregulated. Adaptation to long-term oestrogen deprivation, which results in loss of oestrogen-responsive growth, involves alterations to gene patterns not only at early time points (0-4 weeks) but continuing through to later times (20-55 weeks), and even involves alterations to patterns of oestrogen-regulated gene expression. Only 48% of the genes which were regulated > or =2-fold by oestradiol in oestrogen-responsive cells retained this responsiveness after long-term oestrogen deprivation but other genes developed de novo oestrogen regulation. Long-term exposure to fulvestrant, which resulted in loss of growth inhibition by the antioestrogen, resulted in some very large fold changes in gene expression up to 10,000-fold. Comparison of gene profiles produced by environmental chemicals with oestrogenic properties showed that each ligand gave its own unique expression profile which suggests that environmental oestrogens entering the human breast may give rise to a more complex web of interference in cell function than simply mimicking oestrogen action at inappropriate times.

Structure and function of the protein kinase R.

The protein kinase R (PKR) is an intracellular sensor of stress, exemplified by viral infection. Double-stranded (ds) RNA produced during viral replication activates PKR, which in turn arrests protein synthesis by phosphorylating the alpha subunit of the translation initiation factor eIF2. As well as dsRNA, two additional ligands, PACT and heparin, directly activate the kinase. These mediate the response of PKR to additional indirect stimuli, including bacterial lipopolysaccharides, ceramide and polyanionic molecules. This responsiveness to multiple stimuli advocates a broader role for PKR as a signalling molecule for diverse physiological stresses. Appropriately, a number of other protein substrates have been reported for PKR. These substrates support additional roles for PKR in the regulation of transcription and signal transduction in infected cells, as well as uninfected but diseased tissues, such as in tumorigenesis and neurodegenerative diseases. Finally, PKR plays a role in normal cell differentiation in platelet-derived growth factor signalling and in osteoblast-mediated calcification.

Comparison of the global gene expression profiles produced by methylparaben, n-butylparaben and 17beta-oestradiol in MCF7 human breast cancer cells.

Since the alkyl esters of p-hydroxybenzoic acid (parabens) can be measured intact in the human breast and possess oestrogenic properties, it has been suggested that they could contribute to an aberrant burden of oestrogen signalling in the human breast and so play a role in the rising incidence of breast cancer. However, although parabens have been shown to regulate a few single genes (reporter genes, pS2, progesterone receptor) in a manner similar to that of 17beta-oestradiol, the question remains as to the full extent of the similarity in the overall gene profile induced in response to parabens compared with 17beta-oestradiol. The GE-Amersham CodeLink 20 K human expression microarray system was used to profile the expression of 19881 genes in MCF7 human breast cancer cells following a 7-day exposure to 5 x 10(-4) M methylparaben, 10(-5) M n-butylparaben and 10(-8) M 17beta-oestradiol. At these concentrations, the parabens gave growth responses in MCF7 cells of similar magnitude to 17beta-oestradiol. The study identified genes which are upregulated or downregulated to a similar extent by methylparaben, n-butylparaben and 17beta-oestradiol. However, the majority of genes were not regulated in the same way by all three treatments. Some genes responded differently to parabens from 17beta-oestradiol, and furthermore, differences in expression of some genes could be detected even between the two individual parabens. Therefore, although parabens possess oestrogenic properties, their mimicry in terms of global gene expression patterns is not perfect and differences in gene expression profiles could result in consequences to the cells that are not identical to those following exposure to 17beta-oestradiol.

Changes in oestrogen receptor-alpha and -beta during progression to acquired resistance to tamoxifen and fulvestrant (Faslodex, ICI 182,780) in MCF7 human breast cancer cells.

Cell culture models of antioestrogen resistance often involve applying selective pressures of oestrogen deprivation simultaneously with addition of tamoxifen or fulvestrant (Faslodex, ICI 182,780) which makes it difficult to distinguish events in development of antioestrogen resistance from those in loss of response to oestrogen or other components. We describe here time courses of loss of antioestrogen response using either oestrogen-maintained or oestrogen-deprived MCF7 cells in which the only alteration to the culture medium was addition of 10(-6) M tamoxifen or 10(-7) M fulvestrant. In both oestrogen-maintained and oestrogen-deprived models, loss of growth response to tamoxifen was not associated with loss of response to fulvestrant. However, loss of growth response to fulvestrant was associated in both models with concomitant loss of growth response to tamoxifen. Measurement of oestrogen receptor alpha (ERalpha) and oestrogen receptor beta (ERbeta) mRNA by real-time RT-PCR together with ERalpha and ERbeta protein by Western immunoblotting revealed substantial changes to ERalpha levels but very little alteration to ERbeta levels following development of antioestrogen resistance. In oestrogen-maintained cells, tamoxifen resistance was associated with raised levels of ERalpha mRNA/protein. However by contrast, in oestrogen-deprived MCF7 cells, where oestrogen deprivation alone had already resulted in increased levels of ERalpha mRNA/protein, long-term tamoxifen exposure now reduced ERalpha levels. Whilst long-term exposure to fulvestrant reduced ERalpha mRNA/protein levels in the oestrogen-maintained cells to a level barely detectable by Western immunoblotting and non-functional in inducing gene expression (ERE-LUC reporter or pS2), in oestrogen-deprived cells the reduction was much less substantial and these cells retained an oestrogen-induction of both the ERE-LUC reporter gene and the endogenous pS2 gene which could still be inhibited by antioestrogen. This demonstrates that whilst ERalpha can be abrogated by fulvestrant and increased by tamoxifen in some circumstances, this does not always hold true and mechanisms other than alteration to ER must be involved in the development of antioestrogen resistant growth.

Cerebral asymmetries in inspection time?

Recent studies have suggested that there are low-level processing asymmetries across the cerebral hemispheres, with a right visual field-left hemisphere advantage in tasks involving temporal resolution. In the present report, one such task, inspection time, was measured separately for each cerebral hemisphere in 10 right-handed male subjects over 5 days. A number of methodological improvements were made on previous studies in which a general right visual field-left hemisphere advantage had been found relative to the left visual field-right hemisphere in inspection time performance. The present results suggest that there is no general left hemisphere advantage in inspection time, although there might be asymmetries in practice effects across the hemispheres. The findings also suggest the existence of individual differences in the extent and direction of hemispheric specialisation for this task (ranging from left hemisphere dominance to marked right hemisphere dominance for some subjects) even in right-handed subjects.

Decrease in the protein kinase C-catalyzed phosphorylation of an endogenous lung protein (Mr 36,000) following treatment of mice with the tumor-modulatory agent butylated hydroxytoluene.

Butylated hydroxytoluene (BHT) causes transient lung damage in mice, and it can either inhibit or enhance carcinogen induction of tumors in internal organs, such as urethan-induced lung adenomas. Since protein kinase C (Pk-C) may mediate the action of one class of tumor-modulatory agents, the phorbol esters which promote skin tumorigenesis, we are examining the hypothesis that Pk-C is involved in the modulatory effects of BHT on internal organs. Endogenous phosphorylation of a Mr 36,000 cytosolic protein (p36) with a pI of 5.7 was demonstrable in extracts from lung and spleen but not from brain or heart. Phosphorylation required the presence of both Ca2+ and phosphatidylserine, and phosphate was incorporated into seryl and threonyl residues but not into tyrosyl residues. This reaction thus has the characteristics of Pk-C-dependent catalysis. A single i.p. injection of BHT (400 mg/kg body weight) decreased p36 phosphorylation severalfold in both BALB/cByJ and A/J mice. This decrease correlated with the extent of BHT-induced lung damage with regard to both the time course following BHT administration and the dose dependence of BHT. All of the pulmonary effects of BHT are abolished if the mice are pretreated with cedrene, an inducer of drug-detoxifying enzymes. Such treatment with cedrene prevented any BHT-induced decrease in p36 phosphorylation. A decrease in Pk-C specific activity, as measured using histone as an exogenous substrate, which resulted upon BHT treatment may provide a mechanism for decreased p36 phosphorylation. The specificity of this toxicity-related effect of BHT is emphasized by the fact that urethan injection did not detectably affect the phosphorylation of any lung proteins. Both p36 phosphorylation and Pk-C specific activity increased as a function of postnatal age. Thus the extent of p36 phosphorylation was inversely related to the extent of lung cell proliferation in two different physiological states, postnatal growth and regenerative repair following BHT-induced toxic injury. A single BHT injection is sufficient to cause lung toxicity, tumor prophylaxis, or cocarcinogenesis, while tumor promotion requires chronic treatment. P36 phosphorylation also decreased when mice were given multiple BHT injections over a period of 5 weeks. These results are consistent with a hypothesis that decreased Pk-C-dependent phosphorylation of p36 is involved in lung tumor modulation by BHT.

Protein conformation and reversed-phase high-performance liquid chromatography.

The structure of a series of proteins has been investigated by circular dichroism, fluorescence and visible spectroscopy as well as by differential scanning calorimetry under reversed-phase high-performance liquid chromatography elution conditions. These studies show that 1-propanol, a typical eluent, induces a reversible conformational change in proteins to an apparently ordered, helical form. This structural transition occurs in the range of propanol concentrations that produces elution of a particular protein. The possible relationship between this conformational change and protein elution is considered.

Near-infrared photoacoustic spectroscopy of proteins.

A major problem encountered with the use of electronic spectroscopy in the analysis of biological materials in the ultraviolet, visible, and infrared region involves the limited range of the physical state of samples that can be examined. In an attempt to expand this range, photoacoustic spectra of both solid- and solution-state proteins have been obtained in the near-infrared region. Solid proteins generate detailed spectra in the region 1.0-2.6 micron, resulting primarily from hydrogenic overtone and combinational modes. Harmonics and combinations of amide group frequencies which display significant spectral complexity are observed between 1.4 and 1.7 micron, although they appear to manifest only limited conformational sensitivity. Solution spectra in D2O are of much lower resolution. Assignments of peaks for both solution- and solid-state proteins are presented and the advantages and disadvantages of the use of near-infrared photoacoustic spectroscopy with proteins are discussed.

A simple experimental model for hydrophobic interactions in proteins.

The compound N-cyclohexyl-2-pyrrolidone contains a substantial apolar region as well as a peptide bond-like moiety. This solvent, therefore, provides a useful model for protein interiors. Under certain conditions of temperature and salt concentration, cyclohexylpyrrolidone forms a two-phase system with water. This permits partition coefficients and subsequent free energies of transfer of amino acid side chains from cyclohexylpyrrolidone to water to be simply determined. Free energies of transfer measured in this manner for 21 amino acids are found to be substantially less than those obtained from the commonly used ethanol/water solubility model. This suggests less of a contribution of hydrophobic interactions to the stabilization of protein structure than is conventionally assumed.