New analysis of the temperature-dependent threshold density for electron self-trapping in gaseous helium.

We present the results of a new analysis of the literature data on electron mobility µ in dense helium gas aimed at determining the existence of a threshold density for electron self-trapping in gaseous helium as a function of temperature. We have investigated the density dependence of µ and, when available, its dependence on the electric field. The experimental data are favorably rationalized by minimizing the excess free energy of the self-localized states within the optimum fluctuation model. It is shown that the formation of electron bubbles via the self-trapping phenomenon is determined by the delicate balance between the electron thermal energy, the density dependence of the electron energy at the bottom of the conduction band in the gas, and the work necessary to expand the bubble. We show that the self-trapping phenomenon is not limited to low temperatures but occurs at any temperatures for large enough densities.

Stem cell-like transcriptional reprogramming mediates metastatic resistance to mTOR inhibition.

Inhibitors of the mechanistic target of rapamycin (mTOR) are currently used to treat advanced metastatic breast cancer. However, whether an aggressive phenotype is sustained through adaptation or resistance to mTOR inhibition remains unknown. Here, complementary studies in human tumors, cancer models and cell lines reveal transcriptional reprogramming that supports metastasis in response to mTOR inhibition. This cancer feature is driven by EVI1 and SOX9. EVI1 functionally cooperates with and positively regulates SOX9, and promotes the transcriptional upregulation of key mTOR pathway components (REHB and RAPTOR) and of lung metastasis mediators (FSCN1 and SPARC). The expression of EVI1 and SOX9 is associated with stem cell-like and metastasis signatures, and their depletion impairs the metastatic potential of breast cancer cells. These results establish the mechanistic link between resistance to mTOR inhibition and cancer metastatic potential, thus enhancing our understanding of mTOR targeting failure.

Electron mobility in liquid and supercritical helium measured using corona discharges: a new semi-empirical model for cavity formation.

Electron mobilities in supercritical and liquid helium were investigated as a function of the density. The mobilities were derived from I(V) curves measured in a high-pressure cryogenic cell using a corona discharge in point-plane electrode geometry for charge generation. The presented data spans a wide pressure and temperature range due to the versatility of our experimental set-up. Where data from previous investigations is available for comparison, very good agreement is found. We present a semi-empirical model to calculate electron mobilities both in the liquid and supercritical phase. This model requires the electron-helium scattering length and thermodynamic state equations as the only input and circumvents any need to consider surface tension. Our semi-empirical model reproduces experimental data very well, in particular towards lower densities where transitions from localised to delocalised electron states were observed.

Biological reprogramming in acquired resistance to endocrine therapy of breast cancer.

Endocrine therapies targeting the proliferative effect of 17ß-estradiol through estrogen receptor α (ERα) are the most effective systemic treatment of ERα-positive breast cancer. However, most breast tumors initially responsive to these therapies develop resistance through molecular mechanisms that are not yet fully understood. The long-term estrogen-deprived (LTED) MCF7 cell model has been proposed to recapitulate acquired resistance to aromatase inhibitors in postmenopausal women. To elucidate this resistance, genomic, transcriptomic and molecular data were integrated into the time course of MCF7-LTED adaptation. Dynamic and widespread genomic changes were observed, including amplification of the ESR1 locus consequently linked to an increase in ERα. Dynamic transcriptomic profiles were also observed that correlated significantly with genomic changes and were predicted to be influenced by transcription factors known to be involved in acquired resistance or cell proliferation (for example, interferon regulatory transcription factor 1 and E2F1, respectively) but, notably, not by canonical ERα transcriptional function. Consistently, at the molecular level, activation of growth factor signaling pathways by EGFR/ERBB/AKT and a switch from phospho-Ser118 (pS118)- to pS167-ERα were observed during MCF7-LTED adaptation. Evaluation of relevant clinical settings identified significant associations between MCF7-LTED and breast tumor transcriptome profiles that characterize ERα-negative status, early response to letrozole and tamoxifen, and recurrence after tamoxifen treatment. In accordance with these profiles, MCF7-LTED cells showed increased sensitivity to inhibition of FGFR-mediated signaling with PD173074. This study provides mechanistic insight into acquired resistance to endocrine therapies of breast cancer and highlights a potential therapeutic strategy.

Gene expression profiling integrated into network modelling reveals heterogeneity in the mechanisms of BRCA1 tumorigenesis.

BACKGROUND: Gene expression profiling has distinguished sporadic breast tumour classes with genetic and clinical differences. Less is known about the molecular classification of familial breast tumours, which are generally considered to be less heterogeneous. Here, we describe molecular signatures that define BRCA1 subclasses depending on the expression of the gene encoding for oestrogen receptor, ESR1. METHODS: For this purpose, we have used the Oncochip v2, a cancer-related cDNA microarray to analyze 14 BRCA1-associated breast tumours. RESULTS: Signatures were found to be molecularly associated with different biological processes and transcriptional regulatory programs. The signature of ESR1-positive tumours was mainly linked to cell proliferation and regulated by ER, whereas the signature of ESR1-negative tumours was mainly linked to the immune response and possibly regulated by transcription factors of the REL/NFkappaB family. These signatures were then verified in an independent series of familial and sporadic breast tumours, which revealed a possible prognostic value for each subclass. Over-expression of immune response genes seems to be a common feature of ER-negative sporadic and familial breast cancer and may be associated with good prognosis. Interestingly, the ESR1-negative tumours were substratified into two groups presenting slight differences in the magnitude of the expression of immune response transcripts and REL/NFkappaB transcription factors, which could be dependent on the type of BRCA1 germline mutation. CONCLUSION: This study reveals the molecular complexity of BRCA1 breast tumours, which are found to display similarities to sporadic tumours, and suggests possible prognostic implications.

Two mutations affecting conserved residues in the Met receptor operate via different mechanisms.

We have investigated the mechanism by which two oncogenic mutations (M1268T and D1246H/N; Amino-acids are numbered according to Schmidt et al., 1999) affecting conserved residues in the catalytic domain of the Met receptor, activate its transforming potential. Both mutations were previously found in tumorigenic forms of the Ret and Kit receptors, respectively. The mutated residues are located either in the P+1 loop (M) or within the activation loop (A-loop) (D), which in a number of receptor tyrosine kinases harbors a pair of tandem tyrosines (Y1252-1253 in Met). Ligand-induced dimerization promotes their phosphorylation, and locks the A-loop into an open conformation. When unphosphorylated, the tandem tyrosines inhibit enzymatic activity by blocking the active site. Upon Y-->F mutation of Y1252-1253, neither ligand binding nor Tpr-mediated dimerization can release this block. Here we show that the M1268T mutation partially rescues the kinase activity (and the transforming ability) of the Y1252-1253F Tpr-Met mutant, but is completely dependent on dimerization for its effect. In contrast, the two D1246H/N mutants strictly depend on Y1252-1253 for activity. Surprisingly, however, they constitutively activate the isolated cytoplasmic TK domain of Met (Cyto-Met). These data indicate that the two mutations operate via distinct mechanisms.

Nuclear import and the evolution of a multifunctional RNA-binding protein.

La (SS-B) is a highly expressed protein that is able to bind 3'-oligouridylate and other common RNA sequence/structural motifs. By virtue of these interactions, La is present in a myriad of nuclear and cytoplasmic ribonucleoprotein complexes in vivo where it may function as an RNA-folding protein or RNA chaperone. We have recently characterized the nuclear import pathway of the S. cerevisiae La, Lhp1p. The soluble transport factor, or karyopherin, that mediates the import of Lhp1p is Kap108p/Sxm1p. We have now determined a 113-amino acid domain of Lhp1p that is brought to the nucleus by Kap108p. Unexpectedly, this domain does not coincide with the previously identified nuclear localization signal of human La. Furthermore, when expressed in Saccharomyces cerevisiae, the nuclear localization of Schizosaccharomyces pombe, Drosophila, and human La proteins are independent of Kap108p. We have been able to reconstitute the nuclear import of human La into permeabilized HeLa cells using the recombinant human factors karyopherin alpha2, karyopherin beta1, Ran, and p10. As such, the yeast and human La proteins are imported using different sequence motifs and dissimilar karyopherins. Our results are consistent with an intermingling of the nuclear import and evolution of La.

Karyopherin beta2 mediates nuclear import of a mRNA binding protein.

We have cloned and sequenced cDNA for human karyopherin beta2, also known as transportin. In a solution binding assay, recombinant beta2 bound directly to recombinant nuclear mRNA-binding protein A1. Binding was inhibited by a peptide representing A1's previously characterized M9 nuclear localization sequence (NLS), but not by a peptide representing a classical NLS. As previously shown for karyopherin beta1, karyopherin beta2 bound to several nucleoporins containing characteristic peptide repeat motifs. In a solution binding assay, both beta1 and beta2 competed with each other for binding to immobilized repeat nucleoporin Nup98. In digitonin-permeabilized cells, beta2 was able to dock A1 at the nuclear rim and to import it into the nucleoplasm. At low concentrations of beta2, there was no stimulation of import by the exogenous addition of the GTPase Ran. However, at higher concentrations of beta2 there was marked stimulation of import by Ran. Import was inhibited by the nonhydrolyzable GTP analog guanylyl imidodiphosphate by a Ran mutant that is unable to hydrolyze GTP and also by wheat germ agglutinin. Consistent with the solution binding results, karyopherin beta2 inhibited karyopherin alpha/beta1-mediated import of a classical NLS containing substrate and, vice versa, beta1 inhibited beta2-mediated import of A1 substrate, suggesting that the two import pathways merge at the level of docking of beta1 and beta2 to repeat nucleoporins.

The association of HIV-1 Tat with nuclei is regulated by Ca2+ ions and cytosolic factors.

Human immunodeficiency virus-1 (HIV-1) Tat, a nuclear transcription factor, has been shown to function extracellularly, implying that some Tat molecules escape nuclear import and are secreted. This raises the question of what regulates, in HIV-1-infected cells, the nuclear targeting of the polypeptide. Here we show that cytosolic components activated by Ca2+ ions are required to reveal the karyophilic properties of Tat: in vitro translated Tat molecules do not associate with isolated nuclei unless preincubated with Ca2+. Moreover, Ca2+ ions induce karyophilicity of chemically synthesized Tat molecules only upon addition of cytosolic extracts. The Ca2+-induced karyophilicity is prevented by inhibitors of either tyrosine kinases (herbimycin A and genistein) or tyrosine phosphatases (vanadate), suggesting the involvement of Ca2+-dependent phosphorylation/dephosphorylation events. In line with these observations, the transcriptional activity of Tat is inhibited by treatment with either vanadate or genistein. The same occurs with Tat mutants lacking either one or both the two tyrosine residues (positions 26 and 47). Hence, Ca2+-dependent tyrosine kinase(s) and phosphatase(s) act on accessory cellular protein(s), which in turn are responsible of Tat karyophilicity.

Nuclear translocation of an exogenous fusion protein containing HIV Tat requires unfolding.

OBJECTIVE: To characterize the transcellular transport of HIV-1 Tat. HIV-1 Tat contains a putative localization signal and no leader peptide; however, it can be released from virus-infected cells and taken up by uninfected cells. DESIGN AND METHODS: We constructed a chimeric protein between Tat and dihydrofolate reductase (DHFR), a cytosolic enzyme that binds tightly to the folate analogue methotrexate (MTX). As confirmed by protease sensitivity assays, binding to MTX results in stabilization of the three-dimensional structure of the DHFR domain. The nuclear translocation of recombinant proteins was monitored by both functional [transcellular transactivation of a long terminal repeat-chloramphenicol acetyl transferase (LTR-CAT) reporter gene] and biochemical (subcellular localization in HeLa cells of exogenous radiolabelled proteins) assays and the effects of MTX-induced stabilization were evaluated. RESULTS: When in vitro translated proteins are added to HeLa cells in culture, both wild-type Tat and the chimeric protein Tat-DHFR are taken up by target cells and accumulate in the nucleus, unlike wild-type DHFR. Cells transfected with Tat-DHFR, when co-cultured with cells harbouring a LTR-CAT gene, induce transactivation of the reporter gene to the same extent as cells expressing wild-type Tat. These findings indicate that Tat can mediate the internalization of unrelated polypeptides. Pre-treatment of Tat-DHFR with MTX blocks the nuclear translocation of the chimeric protein. MTX has no effect on wild-type Tat. CONCLUSION: HIV-1 Tat can act as a vector to drive polypeptides into the nucleoplasm of living cells. The inhibitor effects of MTX on the nuclear localization of Tat-DHFR suggest that an unfolding step is required for the internalization of exogenous Tat.

High rates of thioredoxin secretion correlate with growth arrest in hepatoma cells.

Thioredoxin (TRX), a disulfide-reducing intracellular dithiol enzyme, is synthesized by both normal liver cells and the hepatocarcinoma cell line HepG2. Only the former, however, secrete abundant TRX extracellularly. When cultured in mild reducing conditions, HepG2 cells but not normal hepatocytes increase the rate of TRX secretion and undergo growth inhibition accompanied by morphological changes. Also, recombinant TRX inhibits proliferation of HepG2 cells. In contrast, exogenous thiols and TRX stimulate proliferation of a B-cell lymphoma line, indicating that different cell types respond differently to variations in the extracellular redox potential.