Changes in subcellular localisation of MI-ER1 alpha, a novel oestrogen receptor-alpha interacting protein, is associated with breast cancer progression.

The oestrogen receptor-alpha (ER alpha) plays a key role in breast development and tumorigenesis and inhibiting its activity remains a prime strategy in the treatment of ER alpha-positive breast cancers. Thus, elucidation of the molecular mechanisms responsible for regulating ER alpha activity may facilitate the design of new, more effective breast cancer therapies. The MI-ER1 alpha is a novel transcriptional repressor that contains an LXXLL motif for interaction with nuclear hormone receptors. We investigated the ability of MI-ER1 alpha to bind to ER alpha in HEK293 and MCF-7 breast carcinoma cells, using co-immunoprecipitation assays. In both cell lines, MI-ER1 alpha interacted with ER alpha in the presence and absence of oestrogen, but the interaction was stronger in the absence of ligand. Functional analysis revealed that overexpression of MI-ER1 alpha in T47D breast carcinoma cells results in inhibition of oestrogen-stimulated anchorage-independent growth, suggesting that MI-ER1 alpha may play a role in regulating breast carcinoma cell proliferation in vivo. To explore this further, we performed an immunohistochemical analysis of normal breast tissue and breast carcinoma; a total of 110 cases were examined in whole tissue sections and 771 cases were analysed in tissue microarrays. No consistent difference in the MI-ER1 alpha expression level between normal breast tissue and breast carcinoma was discernible. However, there was a dramatic shift in the subcellular localisation: nuclear MI-ER1 alpha was detectable in 75% of normal breast samples and in 77% of hyperplasia, but in breast carcinoma, only 51% of DCIS, 25% of ILC and 4% of IDC contained nuclear staining. This shift from nuclear to cytoplasmic localisation of MI-ER1 alpha during breast cancer progression suggests that loss of nuclear MI-ER1 alpha might contribute to the development of invasive breast carcinoma.

X chromosome reactivation in mouse embryonal carcinoma cells.

The embryonal carcinoma cell line, C86S1, carries two X chromosomes, one of which replicates late during S phase of the cell cycle and appears to be genetically inactive. C86S1A1 is a mutant which lacks activity of the X-encoded enzyme, hypoxanthine phosphoribosyltransferase (HPRT). Treatment of C86S1A1 cells with DNA-demethylating agents, such as 5-azacytidine (5AC), resulted in (i) the transient expression in almost all cells of elevated levels of HPRT and three other enzymes encoded by X-linked genes and (ii) the stable expression of HPRT in up to 5 to 20% of surviving cells. Most cells which stably expressed HPRT had two X chromosomes which replicated in early S phase. C86S1A1 cells which had lost the inactive X chromosome did not respond to 5AC. These results suggest that DNA demethylation results in the reactivation of genes on the inactive X chromosome and perhaps in the reactivation of the entire X chromosome. No such reactivation occurred in C86S1A1 cells when the cells were differentiated before exposure to 5AC. Thus, the process of X chromosome inactivation may be a sequential one involving, as a first step, methylation of certain DNA sequences and, as a second step, some other mechanism(s) of transcriptional repression.

Intracellular signalling pathways involved in mesoderm induction by FGF.

We have examined the possible role of two signal transducing mechanisms, tyrosine phosphorylation and activation of protein kinase C (PKC), during fibroblast growth factor (FGF)-induced mesoderm induction in Xenopus. Tyrosine phosphorylation was examined through the use of a monoclonal anti-phosphotyrosine antibody. This antibody was shown to recognize the FGF receptor crosslinked to radioiodinated FGF. We also studied the response of Xenopus ectodermal explants to sodium orthovanadate, a compound that has been shown to elevate intracellular phosphotyrosine levels. Thirty percent of explants cultured in 100 microM vanadate were induced. In addition, vanadate synergized with FGF to give inductions that were more dorsal in nature than either vanadate or FGF alone. The role of PKC was evaluated by measuring PKC activity during mesoderm induction by FGF and by examining the effect of the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) on explants. TPA did not induce mesoderm, however, activation of PKC was detected in FGF-treated explants. Therefore, activation of the PKC pathway alone is not sufficient for mesoderm induction. Simultaneous treatment with TPA and FGF resulted in a significant inhibition of mesoderm induction by FGF, suggesting that activation of PKC could be part of a negative feedback mechanism. In contrast, TPA had no effect on induction by activin A.

Differential nuclear localization of ER1 protein during embryonic development in Xenopus laevis.

The er1 gene is a novel fibroblast growth factor (FGF)-regulated immediate-early gene, first isolated from Xenopus blastulae, that encodes a nuclear protein with potent transcription transactivational activity (Paterno et al., 1997). We report here the expression pattern of the ER1 protein during Xenopus embryonic development. ER1 protein is present in the early embryo but does not begin to appear in the nucleus until the mid-blastula stage. The first cells to show nuclear localization of ER1 are the presumptive mesodermal cells of the stage 8 blastula. ER1 gradually becomes localized to the nucleus of the remaining cells, first in the presumptive ectoderm and finally, in the presumptive endoderm such that by late blastula, all nuclei in the animal hemisphere are stained. By early gastrula, nuclear staining is ubiquitous. During subsequent development, ER1 protein gradually disappears from the nuclei of various tissues. In tailbud stages, ER1 begins to disappear from the nucleus of ectodermally-derived tissues, such as epidermis and brain, while remaining localized in the nucleus of endodermal cells and of mesodermal tissues, such as somites and notochord. In tadpoles, ER1 is no longer detectable in the nucleus of any cells, except for a few endodermal cells. Cytoplasmic staining, on the other hand, is observed in some mesodermal tissues, including somites and muscle cells. Neural tissue is largely unstained except for weak cytoplasmic staining in the eye.

Sequence of a cDNA encoding a mouse cyclin B protein.

We report here the nucleotide sequence of a cDNA encoding a mouse (Mus musculus) cyclin B protein. The deduced protein shows 84%, 66% and 49% similarity with human cyclin, Xenopus cyclin B1 and B2, respectively.

Molecular cloning of human er1 cDNA and its differential expression in breast tumours and tumour-derived cell lines.

We recently cloned and characterized a novel immediate-early gene, called er1, from Xenopus embryos whose expression levels were increased during mesoderm induction by fibroblast growth factor (FGF). We describe here the isolation and expression pattern of the human er1 sequence. Human ER1 and Xenopus ER1 proteins display 91% similarity; the amino acid sequence motifs, including the putative DNA-binding SANT domain, the predicted nuclear localization signals (NLS) and the putative SH3 binding domain share 100% identity. er1 mRNA expression was negligible in all 50 normal human tissues analyzed. Examination of nine breast carcinoma-derived cell lines and eight breast tumour tissue samples by reverse transcription-polymerase chain reaction (RT-PCR) revealed that human er1 was consistently expressed in all tumour cell lines and tumour tissue while remaining undetectable in normal breast cell lines and breast tissue. These data suggest that er1 expression is associated with the neoplastic state in human breast carcinoma.

Nuclear localization signals in the Xenopus FGF embryonic early response 1 protein.

Xenopus early response 1 (XER1) is a fibroblast growth factor-inducible transcription factor whose developmentally regulated nuclear localization is thought to be important in the control of cell differentation during embryonic development [Luchman et al., Mech. Dev. 80 (1999) 111-114]. Analysis of the XER1 amino acid sequence revealed four regions which contain potential nuclear localization sequences (NLSs). Using mutant XER1 proteins and portions of XER1 fused to green fluorescent protein (GFP) transfected into NIH 3T3 cells, we have determined that only one of these, NLS4, located near the carboxy-terminus of XER1, is necessary and sufficient for targeting exclusively to the nucleus. Of the other three predicted NLS sequences, only NLS1, consisting of the sequence (138)RPRRCK(143) was shown to function as a cryptic, weak NLS. NLS4 contains a core region consisting of the sequence (463)RPIKRQRMD(471) which is similar to the core NLS directing the human c-MYC protein to the nucleus. The core sequence is flanked by a predicted cdc2/protein kinase A phosphorylation motif, however mutation of the serine(472) to alanine or aspartic acid had no detectable effect on accumulation of GFP-XER1 fusion proteins in the nucleus, demonstrating that this putative phosphorylation site plays no role in regulating nuclear transport.

Localization of 5-HT2A receptor mRNA by in situ hybridization in the olfactory bulb of the postnatal rat.

The olfactory bulb receives a dense serotonergic input and appears to require serotonergic input in early olfactory associational learning. However, it is not known which cell types receive the serotonergic input or whether the cells express markers for the input throughout life. These issues need resolution in order for the mechanisms of serotonergic interactions to be better understood. The mRNA for the 5-HT2A receptor was localized in the olfactory bulb of postnatal day 1, 2, 14 and 9-month-old Sprague-Dawley rats as well as in the bulb of adult (6 months) and aged (22-30 month) Fisher 344 rats by in situ hybridization using an 35S-labelled 5-HT2A- specific oligonucleotide probe mixture. In all animals, hybridization was observed in mitral cells which are the major output cells of the main olfactory bulb. Tufted cells, located in the external plexiform layer and juxtaglomerular region, were readily observed in adult and aged animals and were also observed, albeit not as readily, in neonate pups. Quantitative analysis of the silver grain density over cells confirmed qualitative observations and showed that mitral and tufted cells were labelled in the neonate as well as in adult and aged animals. Labelled cells were also numerous in the external division of the anterior olfactory nucleus in all animals. 5-HT2A receptor mRNA could not be detected either qualitatively or quantitatively by in situ hybridization in the accessory olfactory bulb, nor could it be observed in the olfactory epithelium.(ABSTRACT TRUNCATED AT 250 WORDS)

Developmental expression of functional GABAA receptors containing the gamma 2 subunit in neurons derived from embryonal carcinoma (P19) cells.

The expression of the gamma 2 subunit into functional GABAA receptors has been examined in the embryonal carcinoma (EC) cell line P19, a pluripotent cell line which differentiates into a neuronal phenotype after exposure to retinoic acid. Whole-cell voltage-clamp recordings were used to examine the characteristics of the GABA receptors expressed in P19 cells at different times after exposure to retinoic acid. Messenger RNA for both the gamma 2L and gamma 2S splice variants of the GABAA receptor increased dramatically following differentiation of P19 EC cells with retinoic acid. By 12 days after retinoic acid treatment, while both mRNAs were present, there was an approximately 10-fold greater abundance of gamma 2S mRNA compared to gamma 2L. However, at this same time point neurons derived from P19 cells stained intensely with a polyclonal antibody raised against a peptide fragment specific for the gamma 2L subunit. A significant increase in both the affinity for GABA and the maximum current amplitude elicited by GABA occurred between 7 and 12 days after retinoic acid treatment. In contrast, the ability of the benzodiazepine agonist flurazepam to potentiate GABA-induced membrane current was the same at 7 and 12 days after retinoic acid treatment. These data suggest that the gamma 2 subunit of the GABAA receptor is expressed early following differentation of P19 cells into a neuronal phenotype, and that this subunit is incorporated into functional GABAA receptors. Moreover, the gamma 2S and gamma 2L splice variants of this subunit may be co-expressed in neurons derived from P19 cells. The observed affinity change for GABA may reflect a time-dependent change in the expression of alpha and/or beta subunits of the GABAA receptor, as occurs in developing neuronal tissue both in vitro and in vivo.

Regulation of neurofilament L, M and H gene expression during retinoic acid-induced neural differentiation of P19 embryonal carcinoma cells.

We have investigated the regulation of neurofilament gene expression during retinoic acid (RA)-induced neural differentiation of P19 embryonal carcinoma (EC) cells. Western blot analysis demonstrated that P19 EC cells contain significant levels of NF-L protein in the insoluble fraction but undetectable levels of NF-M and NF-H protein in either the insoluble or total cell fractions. However, immunocytochemical detection of NF-L protein in P19 EC cells showed diffuse staining within the majority of cells, rather than association with intermediate filament-like structures or staining within a subpopulation of differentiated neurons. Detectable levels of both NF-L and NF-M mRNA were present in P19 EC cells whereas NF-H mRNA remained below levels of detection, even by RT-PCR analysis. When RA-treated aggregates of P19 cells were cultured under conditions permissive for neurite outgrowth, we observed a significant increase in the amount of detectable NF-L protein localized within morphologically distinct neurons. Differentiation was also accompanied by the appearance of both the NF-M and NF-H subunits. Northern analysis revealed that this differentiation was accompanied by coincident increase in the steady-state levels of the mRNA for all three subunits and that the temporal pattern of increase was similar to what has been observed in the fetal and neonatal brain. The increase in NF-L and NF-M mRNA levels were accompanied by a concomitant increase in the rate of transcription, however, our results suggest that additional post-transcriptional mechanisms may be involved in regulating NF gene expression during the differentiation of pluripotent P19 cells.

Neurons derived from embryonal carcinoma (P19) cells express multiple GABAA receptor subunits and fully functional GABAA receptors.

The embryonal carcinoma (EC) cell line P19 can be induced to differentiate into neurons and glia by treatment with retinoic acid. Neuronal cells derived from P19 stem cells were found to express messenger RNAs for alpha, beta, and gamma 2 subunits of the GABAA receptor-chloride channel complex. Whole-cell voltage-clamp recording in differentiated P19 cells revealed that these cells possess GABA receptor-activated chloride currents which are blocked by bicuculline and potentiated by flurazepam. P19 EC cells thus represent a stable neuronal cell line which expresses functional GABAA receptors with all of the characteristics of native GABAA receptors.

Fibroblast growth factor and transforming growth factor beta in early embryonic development.

Growth factors are known to have pleiotropic effects on many cell types ranging from the control of cell proliferation to inducing cell differentiation. FGF and TGF beta are members of two growth factor families which are thought to be involved in embryogenesis of the frog, Xenopus laevis. These two growth factors are equivalent to the embryonic "morphogen(s)" which induce one of the first differentiation events during embryogenesis, the formation of the mesoderm. Embryonic induction events are crucial for the development of most organisms and, therefore, these growth factors may be involved in induction events during mammalian embryogenesis. Thus, the structure and function of TGF beta and FGF molecules appear to be conserved throughout vertebrate evolution and during ontogeny, growth factors and their signalling pathways may be used for different functions depending upon the nature of the target cell.

X chromosome inactivation during induced differentiation of a female mouse embryonal carcinoma cell line.

P10 is a line of embryonal carcinoma cells with a euploid female karyotype. By making use of variant alleles of X-linked genes and of cytogenetic procedures, we have previously shown that the P10 cells have two genetically active X chromosomes. We show here that P10 cells rapidly differentiate into a cell type resembling extraembryonic endoderm when cultured in the presence of retinoic acid. This differentiation is accompanied by X chromosome inactivation as determined by the appearance of a late-replicating X chromosome. Analysis of the X-linked variant alleles indicated that the P10-derived endoderm did not preferentially inactivate paternally derived X chromosomes. This result is in contrast to the situation in normal extraembryonic endoderm, and suggests that the X inactivation process in differentiating P10 cultures resembles that which occurs in normal embryonic rather than extraembryonic tissues.

Cloning of a fibroblast growth factor receptor 1 splice variant from Xenopus embryos that lacks a protein kinase C site important for the regulation of receptor activity.

A cDNA clone, predicted to encode a variant form of the type 1 fibroblast growth factor receptor (FGFR1) containing a dipeptide Val-Thr (VT) deletion at amino acid positions 423 and 424 located within the juxtamembrane region, was isolated from a Xenopus embryo (stage 8 blastula) library. Sequence analysis of genomic DNA encoding a portion of the FGFR1 juxtamembrane region demonstrated that this variant form arises from use of an alternative 5' splice donor site. RNase protection analysis revealed that both VT- and VT+ forms of the FGFR1 were expressed throughout embryonic development, the VT+ being the major form. Amino acid position 424 is located within a consensus sequence for phosphorylation by a number of Ser/Thr kinases. We demonstrate that a VT+ peptide was specifically phosphorylated by protein kinase C (PKC) in vitro, but not by protein kinase A (PKA). A VT- peptide, on the other hand, was not a substrate for either enzyme. Phosphorylation levels of in vitro synthesized FGFR-VT+ protein by PKC were twice that of FGFR-VT- protein. In a functional assay, Xenopus oocytes expressing FGFR-VT- or FGFR-VT+ protein were equally able to mobilize intracellular Ca2+ in response to basic fibroblast growth factor (bFGF). However, pretreatment with phorbol 12-myristate 13-acetate significantly reduced this mobilization in oocytes expressing FGFR-VT+ while having little effect on oocytes expressing FGFR-VT-. These findings demonstrate that alternative splicing of Val423-Thr424 generates isoforms which differ in their ability to be regulated by phosphorylation and thus represents an important mechanism for regulating FGFR activity.

Analysis of competence: receptors for fibroblast growth factor in early Xenopus embryos.

Xenopus ectodermal cells have previously been shown to respond to acidic and basic FGF by differentiating into mesodermal tissue. In the present study, ectodermal explants from Xenopus blastulae were shown to have high affinity binding sites for 125I-aFGF (Kd = 1.4 X 10(-10) M). The total number of sites, determined by Scatchard analysis, was 3 X 10(8) per explant (surface area of approximately 1 mm2). Two putative receptors of relative molecular mass 130,000 and 140,000 were identified by chemical crosslinking to 125I-aFGF. Both acidic and basic FGF, but not TGF beta 2, could compete for affinity labelling of these bands. The receptor density at the cell surface parallels the developmental competence of Xenopus animal pole cells to respond to FGF. Receptors are present at highest density in the marginal zone but are not restricted to cells in this region.

Cellular differentiation and pattern formation in the absence of morphogenesis in the cellular slime mould Polysphondylium pallidum: evidence for a biochemical tip (organizer) in submerged aggregates.

When amoebae of Polysphondylium pallidum WS320 are placed in nonnutrient buffer in roller tube culture they form spherical or ellipsoidal aggregates. At first the aggregates demonstrate a "loose" morphology but by 12 h, with the formation a a cellulose-containing, peripheral sheath, they become "tight" aggregates. At this time stalk differentiation begins. Using various methods for the resolution of prespore (ultrastructure, spore antigen immunofluorescence, periodic acid - Schiff staining) and prestalk (ultrastructure, alkaline phosphatase histochemistry, neutral red staining, Calcofluor fluorescence) cell localization, the pattern of cell differentiation in submerged aggregates was shown to be essentially identical to that of normal pseudoplasmodia. Furthermore, using a cAMP bioassay it was revealed that the submerged aggregates, while devoid of a morphological tip, do possess a biochemical tip which is correlated with sites of neutral red staining and stalk cell differentiation. As a result of these studies, an earlier argument that the tip of the pseudoplasmodium is not essential for the establishment of pattern or in the "organization" of cellular differentiation during slime mould development is contradicted.

Identification of phosphorylated proteins associated with the fibroblast growth factor receptor type I during early Xenopus development.

Signaling through the FGF receptor (FGFR) is required for mesoderm induction in Xenopus. Some of the downstream signaling molecules implicated in this developmental process include Ras, Raf and MAP kinase. In a previous report, we demonstrated that PLC gamma 1, Grb-2, SOS and Nck were associated with activated FGFR1s in a signaling complex in Xenopus blastulae. In addition, several unidentified phosphotyrosylproteins were present in the FGFR1 complex. Here we identify three of these proteins as Ras-GAP, the p85 of P13'K and SHP2, while demonstrating that c-Src and She were not associated with the FGFR1. Furthermore, we show that three additional phosphotyrosylproteins from the FGFR1 complex specifically bound to the adaptor molecule Nck.

An embryonic DNA-binding protein specific for a region of the human IFN beta 1 promoter.

Embryonal carcinoma (EC) cells are unable to make interferon in response to inducing agents. This block disappears after differentiation. We have found that nuclear extracts from undifferentiated P19 EC cells contain a DNA-binding activity which specifically recognizes a region within the human interferon-beta 1 promoter. This activity is absent from differentiated cell types, both of EC and non-EC origin. The binding of the factor in undifferentiated EC cells leads to dramatic changes in the overall protein binding pattern of the interferon promoter as compared with differentiated cells, and may be responsible for repression of the endogenous interferon-beta gene prior to differentiation.

Isolation and characterisation of a murine cDNA clone highly homologous to the yeast L29 ribosomal protein gene.

We report here the isolation of a murine cDNA clone (cCL3) which is homologous to the mRNA of the yeast ribosomal protein L29. Comparison of the deduced amino-acid composition of cCL3 to those known for rat ribosomal proteins indicates that this cDNA codes for mammalian ribosomal protein L27'. The gene corresponding to the cDNA is present at approximately 15 copies per genome, some of these probably representing processed pseudogenes. The cDNA hybridizes to an mRNA of 600 nucleotides from various mammals at high stringency, and to an avian transcript of the same size at low stringency. It has been suggested that L29 is involved in peptidyl transferase activity. The strong homology of mammalian L27' to yeast L29 suggests a function which has been conserved throughout evolution, and thus L27' may also be involved in peptidyl transferase activity.

Expression of multiple heparin-binding growth factor species by murine embryonal carcinoma and embryonic stem cells.

Culture medium conditioned by P19 embryonal carcinoma (EC) cells contains potent mitogenic activity which is markedly potentiated when the medium is conditioned in the presence of heparin. Fractionation of P19 medium conditioned in the presence of heparin reveals the existence of two biochemically distinct growth factor species both of which exhibit high affinity for immobilised heparin and significant activity as amphibian mesoderm-inducing agents. One of the species is recovered as a single polypeptide of apparent Mr = 15,000. This molecule is immunologically related to the protein product of the human K-FGF proto-oncogene. Transcripts derived from the murine K-FGF gene are also expressed by both differentiated and undifferentiated EC cells and embryonic stem cells. The second heparin-binding growth factor is recovered as a complex of four polypeptides, the largest of which has an apparent Mr = 17,000. This agent is immunologically and biochemically distinct from both acidic and basic fibroblast growth factor as well as K-FGF, and represents the predominant mitogenic activity in EC-cell-conditioned medium.