Detection of oestrogenic chemicals by assaying the expression level of oestrogen regulated genes.

The oestrogen receptor belongs to the superfamily of nuclear receptors. Classically, nuclear receptors are thought to reside either in the nucleus or in the cytoplasm where they interact with their ligand which induces a conformational change that exposes the DNA binding domain. This is followed by dimerisation and binding of their corresponding response elements. By interacting with the transcriptional apparatus they then either activate or repress the transcription of target genes. However, this is a highly simplified view, since the activated oestrogen receptor interacts with other signal transduction pathways and its intrinsic transcriptional activity is highly influenced by phosphorylation and by its interaction with other proteins. This is clearly observed when the oestrogenicity of antioestrogens is tested since some compounds activate the receptor in yeast, but not in mammalian cells. However, when specific kinases are activated antioestrogens can also function as oestrogens in mammalian cells. Moreover, components of the MAP kinase and perhaps the cAMP and other pathways are activated before the receptor even enters the nucleus. Thus, when analysing the effects of oestrogenic compounds, it is important to assay both their potency as activators of transcription as the effects caused by interactions with other signal transduction pathways. This may be possible by combining assay methods, such as direct in vitro measurement of interaction between a potential oestrogenic chemical and the receptor or the yeast E-screen, with methods that are based on mammalian cells or whole animals. An alternative is to assay gene expression directly by methods such as differential display, where the expression of both genes known to be regulated directly by the receptor and genes regulated by other pathways can be monitored. Thereby it may be possible to assign different responses to the activation of distinct pathways.

Down regulation of ribosomal protein mRNAs during neuronal differentiation of human NTERA2 cells.

We have analysed the expression of 32 ribosomal protein (RP) mRNAs during retinoic acid induced neuronal differentiation of human NTERA2 cells. Except for a new S27 variant (S27v), all were down regulated both in selectively replated differentiated neurons and the most differentiated continuous cultures, i.e., non-replated cultures. However, the expression profiles of the individual RP mRNAs were different, most (L3, L7, L8, L10, L13, L23a, L27a, L36a, L39, P0, S2, S3, S3a, S4X, S6, S9, S12, S13, S16, S19, S20, S23, and S27a) exhibited a constant down regulation, whereas a few were either initially constant (L11, L32, S8, and S11) or up regulated (L6, L15, L17, L31, and S27y) and then down regulated. The expression of S27v remained elevated in the most differentiated continuous cultures but was down regulated in replated differentiated neurons. The down regulation of RP mRNAs was variable: the expression levels in differentiated replated neurons were between 10% (S3) and 90% (S11) of the levels in undifferentiated cells. The ratio between rRNA and RP mRNA changed during the differentiation; in differentiated neurons there were, on average, about half the number of RP mRNAs per rRNA as compared to undifferentiated cells. The expression profiles of a few translation-related proteins were also determined. EF1alpha1, EF1beta1, and EF1delta were down regulated, whereas the expression of the neuron and muscle specific EF1alpha2 increased. The reduction in the expression of RP mRNAs was coordinated with a reduction in the expression level of the proliferation marker PCNA. The expression levels of most RP mRNAs were lower in purified differentiated post-mitotic neurons than in the most differentiated continuous cultures, despite similar levels of PCNA, suggesting that both the differentiation state and the proliferative status of the cells affect the expression of RP mRNAs.

Differential display competitive polymerase chain reaction: an optimal tool for assaying gene expression.

Gene discovery, i.e. detection of genes whose expression is affected in diseases or by different treatments of cells or animals, has become the focus of much genetic research. The technologies that are used to detect changes in expression level include polymerase chain reaction (PCR)-based subtraction methods, arrays of cDNA clones on chips or filters, serial analysis of gene expression, and differential display. In this paper we show that differential display can be used to investigate global gene expression in situations where a few genes change expression levels such as exposure of MCF7 cells to estradiol, and in more complex situations such as neuronal differentiation of human NTERA2 cells which affects a large number of genes. Furthermore, we show that differential display can replace Northern blotting and RNase protection as a tool to study the expression level of a specific gene in many samples. Results obtained by differential display can be stored in databases, where the identity of a band (gene or mRNA name) can be linked with information about the primer combination displaying the band and a gel image showing the band pattern, which is all the information that is needed to compare the expression level of this gene in other samples.

Human papillomavirus type 16 E7-regulated genes: regulation of S100P and ADP/ATP carrier protein genes identified by differential-display technology.

Human papillomavirus type 16 (HPV-16) is the dominant risk factor for the development of cervical cancer. The virus encodes three oncoproteins, of which the E7 oncoprotein is the major protein involved in cell immortalization and transformation. E7 is a multi-functional protein. It binds the retinoblastoma tumour-suppressor protein (pRb), which regulates progression through the G(1) restriction point in the cell cycle. The E7 protein interacts with transcription-regulatory proteins such as the TATA box-binding protein and with proteins of the AP1 transcription factor family. To identify additional proteins regulated by E7, differential-display PCR was used to identify differentially expressed mRNAs in cells containing an inducible E7 protein. It is reported that E7 expression leads to regulation of the genes encoding the calcium-binding protein S100P and the mitochondrial ADP/ATP carrier protein. These data identify new functions of the E7 protein and thus expand the number of routes by which HPV-16 influences cell growth control, although the function of S100P has still to be elucidated.

MAP-1, a novel proapoptotic protein containing a BH3-like motif that associates with Bax through its Bcl-2 homology domains.

A novel Bax-associating protein, named MAP-1 (Modulator of Apoptosis), has been identified in a yeast two-hybrid screen. MAP-1 contains a BH3-like (BH: Bcl-2 homology) motif and mediates caspase-dependent apoptosis in mammalian cells when overexpressed. MAP-1 homodimerizes and associates with the proapoptotic Bax and the prosurvival Bcl-2 and Bcl-X(L) of the Bcl-2 family in vitro and in vivo in mammalian cells. Mutagenesis analyses revealed that the BH3-like domain in MAP-1 is not required for its association with Bcl-X(L) but is required for association with Bax and for mediating apoptosis. Interestingly, in contrast to other Bax-associating proteins such as Bcl-X(L) and Bid, which require the BH3 and BH1 domains of Bax, respectively, for binding, the binding of MAP-1 to Bax appears to require all three BH domains (BH1, BH2, and BH3) of Bax, because point mutation of the critical amino acid in any one of these domains is sufficient to abolish its binding to MAP-1. These data suggest that MAP-1 mediates apoptosis through a mechanism that involves binding to Bax.