A20/TNFAIP3, a new estrogen-regulated gene that confers tamoxifen resistance in breast cancer cells.

The zinc-finger protein A20/TNFAIP3, an inhibitor of nuclear factor-kappaB (NF-kappaB) activation, has been shown to protect MCF-7 breast carcinoma cells from TNFalpha-induced apoptosis. As estrogen receptor (ER) status is an important parameter in the development and progression of breast cancer, we analysed the effect of 17beta-estradiol (E2) treatment on the expression of A20. We found that A20 is a new E2-regulated gene, whose expression correlates with ER expression in both cell lines and tumor samples. With the aim of investigating the impact of A20 expression on MCF-7 cells in response to ER ligands, we established stably transfected-MCF-7 cells overexpressing A20 (MCF-7-A20). These cells exhibited a phenotype of resistance to the 4-hydroxy-tamoxifen cytostatic and pro-apoptotic actions and of hyper-response to E2. Dysregulations in bax, bcl2, bak, phospho-bad, cyclin D1, cyclin E2, cyclin D2 and cyclin A2 proteins expression were shown to be related to the resistant phenotype developed by the MCF-7-A20 cells. Interestingly, we found that A20 was also overexpressed in MVLN and VP tamoxifen-resistant cell lines. Furthermore, high A20 expression levels were observed in more aggressive breast tumors (ER-negative, progesterone receptor-negative and high histological grade). These overall findings strongly suggest that A20 is a key protein involved in tamoxifen resistance, and thus represents both a new breast cancer marker and a promising target for developing new strategies to prevent the emergence of acquired mechanisms of drug resistance in breast cancer.

IL-6 regulates CD44 cell surface expression on human myeloma cells.

Multiple myeloma (MM) is a progressive B-lineage neoplasia characterized by the accumulation of slow proliferative malignant plasma cells in the bone marrow compartment where the microenvironment seems to be favorable for their growth and survival. Heparan sulfate proteoglycans such as syndecan-1 and CD44 are thought to play a central role in the survival signals provided by these bone marrow survival niches, which require complex interactions between myeloma cells, extracellular matrix, stromal cells and soluble factors. In this report, we demonstrate that interleukin-6 (IL-6), the main survival and growth factor for myeloma cells, strongly increases CD44 gene expression. In addition, we show that IL-6 modulates CD44 RNA alternative splicing and induces the overexpression of all CD44 variant exons. Finally, we show that IL-6-induced CD44 cell surface molecules have a functional polarized membrane distribution. As IL-6 secretion induced from bone marrow stromal cells by myeloma cells is partly mediated through direct cell-to-cell interaction involving CD44 adhesion molecules, our findings suggest that a CD44/IL-6 amplification loop plays a crucial role in myeloma cell survival.

Identifying intercellular signaling genes expressed in malignant plasma cells by using complementary DNA arrays.

In multiple myeloma (MM), the growth of primary plasma cells depends not only on interleukin-6 (IL-6), but also on additional unidentified signals delivered by the bone marrow environment. Using Atlas complementary DNA (cDNA) arrays comprising 268 genes coding for intercellular signaling molecules, this study identified genes that are overexpressed in myeloma cells compared to autologous B-lymphoblastoid cell lines. These genes encode the oncogenic Tyro3 tyrosine kinase receptor, the heparin-binding epidermal growth factor-like growth factor (HB-EGF) that is an epithelial autocrine tumor growth factor, the thrombin receptor (TR) that is linked to HB-EGF and syndecan-1 processing and to cell invasion, chemokine receptors CCR1 and CCR2, the Wnt pathway actor Frizzled-related protein (FRZB), and the Notch receptor ligand Jagged 2. These data, obtained with the Atlas cDNA array, were confirmed by reverse transcriptase-polymerase chain reaction or protein analysis or both. Furthermore, Tyro3, HB-EGF, TR, and FRZB gene expression was documented in purified primary malignant plasma cells from patients with plasma cell leukemia or MM. HB-EGF and FRZB were poorly expressed in purified polyclonal plasma cells. Finally, HB-EGF was proved to be an essential autocrine growth factor for the XG-1 myeloma cells. This study shows the potency and the biologic relevance of cDNA arrays used to analyze simultaneously a large panel of intercellular signaling genes and, by identifying several genes overexpressed in malignant plasma cells, opens new fields of investigation in MM biology. (Blood. 2001;98:771-780)

The human interferon- and estrogen-regulated ISG20/HEM45 gene product degrades single-stranded RNA and DNA in vitro.

The human ISG20/HEM45 gene was identified independently on the basis of its increased level of expression in response to either interferon or estrogen hormone. Notably, the encoded protein is homologous with members of the 3' to 5' exonuclease superfamily that includes RNases T and D, and the proofreading domain of Escherichia coli DNA polymerase I. We provide here direct biochemical evidence that Isg20 acts as a 3' to 5' exonuclease in vitro. This protein displays a pH optimum of approximately 7.0, prefers Mn2+ as a metal cofactor, and degrades RNA at a rate that is approximately 35-fold higher than its rate for single-stranded DNA. Along with RNase L, Isg20 is the second known RNase regulated by interferon. Previous data showed that Isg20 is located in promyelocytic leukemia (PML) nuclear bodies, known sites of hormone-dependent RNA polymerase II transcription and oncogenic DNA viral transcription and replication. The combined data suggest a potential role for Isg20 in degrading viral RNAs as part of the interferon-regulated antiviral response and/or cellular mRNAs as a regulatory component of interferon and estrogen signaling.

Hyaluronic acid induces survival and proliferation of human myeloma cells through an interleukin-6-mediated pathway involving the phosphorylation of retinoblastoma protein.

Originating from a post-switch memory B cell or plasma cell compartment in peripheral lymphoid tissues, malignant myeloma cells accumulate in the bone marrow of patients with multiple myeloma. In this favorable microenvironment their growth and survival are dependent upon both soluble factors and physical cell-to-cell and cell-to-extracellular matrix contacts. In this report we show that hyaluronan (HA), a major nonprotein glycosaminoglycan component of the extracellular matrix in mammalian bone marrow, is a survival and proliferation factor for human myeloma cells. The effect of HA is mainly mediated through a gp 80-interleukin 6 (IL-6) receptor pathway by a CD44-independent mechanism, suggesting that HA retains and concentrates IL-6 close to its site of secretion, thus favoring its autocrine activity. In addition, we show that HA-mediated survival and proliferation of myeloma cells is associated with a down-regulation in the expression of p27(kip1) cyclin-dependent kinase inhibitor and a hyperphosphorylation of the retinoblastoma protein (pRb). These data suggest that HA could be an important component in the myeloma cell physiopathology in vivo by potentiating autocrine and/or paracrine IL-6 activities.

The interferon-inducible Staf50 gene is downregulated during T cell costimulation by CD2 and CD28.

It is well established that interferons (IFN) exert potent regulatory effects on the immune system. We have recently isolated a new IFN-induced human cDNA coding for a member of the Ring finger B-box/B30.2 subfamily that localizes to the chromosome band 11p15. We have named it Staf50. We show in this report that Staf50 is expressed in resting T cells in the absence of exogenous IFN treatment and is strongly repressed during T cell activation by anti-CD28 and anti-CD2 monoclonal antibodies (mAb) at both messenger and protein levels. In addition, we show that several members of the Ring finger B-box/B30.2 subfamily, including the 52-kDa SSA/Ro autoantigen, localize to the same chromosome band, 11p15, and are upregulated by IFN. These data led us to define a family of IFN-induced genes clustered on chromosome 11p15 that may be involved in T cell regulatory processes.

A unique ISRE, in the TATA-less human Isg20 promoter, confers IRF-1-mediated responsiveness to both interferon type I and type II.

Interferons (IFNs) encode a family of secreted proteins involved in a number of regulatory functions such as control of cell proliferation, differentiation and regulation of the immune system. Their diverse biological actions are thought to be mediated by the products of specific but usually overlapping sets of cellular genes induced in the target cells. We have recently isolated a human cDNA encoding a new nuclear bodies-associated protein (PML-NBs), which we have termed Isg20. In this report, we describe the cloning and functional characterization of the Isg20 promoter region and the identification of sequence elements and trans-acting factors implicated in its regulation. In the absence of any recognizable TATA or CAAT elements, Isg20 promoter basal activity is dependent upon the positive transcription factors Sp-1 and USF-1. Interestingly, we demonstrate that a unique interferon stimulated response element (ISRE) mediates both IFN type I and type II Isg20 induction in the absence of functional gamma-activated sequence. These inductions are strictly dependent upon of the IFN regulatory factor 1 (IRF-1). In addition, we show that the ISRE is also implicated in the constitutive transcriptional activity of Isg20 gene.

Regulation of Bcl-2-family proteins in myeloma cells by three myeloma survival factors: interleukin-6, interferon-alpha and insulin-like growth factor 1.

As survival regulation is a key process in multiple myeloma biology, we have studied the Bcl-2 family proteins that can be regulated by three myeloma cell survival factors: interleukin-6 (IL-6), interferon-alpha (IFN-alpha) and insulin-like growth factor (IGF-1). Eleven myeloma cell lines, whose survival and proliferation are dependent on addition of IL-6, variably expressed 10 anti-apoptotic or pro-apoptotic proteins of the Bcl-2-family. When myeloma cells from four cell lines were IL-6 starved and activated with IL-6 or IFN-alpha, we observed that only Mcl-1 expression was up-regulated with myeloma cell survival induction. Nor was obvious regulation of these 10 pro-apoptotic or anti-apoptotic proteins found with IGF-1, another potent myeloma cell survival factor. Our results indicate that the myeloma cell survival activity of IL-6 linked to Bcl-xL regulation cannot be generalized and emphasize that Mcl-1 is the main target of IL-6 and IFN-alpha stimulation. However, other changes in the activity of the Bcl-2 protein family or other apoptosis regulators must be identified to elucidate the IGF-1 action mechanism. Cell Death and Differentiation (2000) 7, 1244 - 1252.

[Interferon signaling pathways]. / L'interféron: un mécanisme complexe de signalisation.

Interferons (IFNs) encode a large family of multifonctional secreted proteins that are involved in antiviral defense, the regulation of cell growth and modulation of the immune response. They are subdivided into two types that activate transduction pathways via different cell surface receptors. Binding of both IFN type I and II results in the differential activation of JAK (Janus kinases) that phosphorylate latent cytoplasmic transcription factors termed STATs (signal transducer and activator of transcription). Phosphorylated STATs translocate to the nucleus, bind specific DNA elements and direct transcription. Type I IFN induces the phosphorylation of STAT1 and STAT2 proteins by tyrosine phosphorylation involving the type I IFN receptor-associated tyrosine kinases TYK2 and JAK1. Following phosphorylation, STAT1 and STAT2 form the transcriptionally active IFN-stimulated gene factor 3 (ISGF3) by association with a protein of the IFN regulatory factor (IRF) family, p48. The specificity of the transcriptional activation by ISGF3 is mediated by specific elements termed IFN-stimulatory response element (ISRE) located in the promoter region of IFN-inducible genes. ISREs drive the expression of most IFN type I-regulated genes and a few IFN type II-regulated genes. Gene induction by type II IFN involves the phosphorylation of only STAT1 by JAK1 and Jak2 kinases. This phosphorylation generates a homodimer of STAT1 which is able to bind the IFNgamma-activated site (GAS) to activate transcription. This signaling is rapid and direct. Molecules involved in the IFN signaling pathways have been shown to be used by other polypeptide ligands in their own signal transduction pathways. Pathways other than JAK/STAT are also involved in IFN signaling, but their mechanisms are less clear. The best documented are the mitogen-activated protein kinase (MAPK) cascade, the components of the TCR (T cell receptor) signaling cascade and the Pi3 kinase pathway.

Molecular cloning of a new interferon-induced PML nuclear body-associated protein.

Transcriptional induction of genes is an essential part of the cellular response to interferons. We have established a cDNA library from human lymphoblastoid Daudi cells treated for 16 h with human alpha/beta-interferon (IFN) and made use of differential screening to search for as yet unidentified IFN-regulated genes. In the course of this study, we have isolated a human cDNA that codes for a 20-kDa protein sharing striking homology with the product of the Xenopus laevis XPMC2 gene. This new gene is induced by both type I and II IFNs in various cell lines and will be referred to as ISG20 for interferon-stimulated gene product of 20 kDa. Confocal immunofluorescence analysis of the subcellular localization of ISG20 protein reveals that it is closely associated with PML and SP100 gene products within the large nuclear matrix-associated multiprotein complexes termed the PML nuclear bodies.

Cloning and expression of an alternatively spliced mRNA encoding a soluble form of the human interleukin-6 signal transducer gp130.

The membrane-bound gp130 glycoprotein acts as an affinity converting and signal transducing receptor (R) for interleukin-6 and several other cytokines. In this work, we RT-PCR amplified gp130 cDNA using primers flanking the sequence encoding the transmembrane domain of gp130. We observed in blood mononuclear cells, in addition to the expected 333-bp length fragment, a second major band of 418 bp. Sequencing of the 418-bp fragment and its genomic counterpart showed a new 85-bp exon located in the sequence encoding the extracellular region of the gp130 protein. This exon is most likely due to alternative splicing and leads to a frame-shift resulting in a stop-codon 1 bp before the transmembrane coding region. Correspondingly, supernatants from chinese hamster ovary cells transfected with this cDNA contained 4-5 times more soluble (s) gp130 than supernatants from cells transfected with a cDNA encoding the membrane-bound gp130 protein. Both gp130 and alternatively spliced sgp130 were also transcribed by the myeloma cell lines XG-1, XG-2, XG-4, XG-4CNTF XG-6, XG-7, XG-9, XG-10, U266 and RPMI 8226. However, XG-4A cells derived from XG-4 cells, but growing independently of exogenous IL-6, did not transcribe sgp130 mRNA. A possible interference with intracrine stimulatory factors by alternatively spliced sgp130 needs to be further investigated.

Both human alpha/beta and gamma interferons upregulate the expression of CD48 cell surface molecules.

We have established a cDNA library from interferon (IFN)-treated human lymphoblastoid Daudi cells and made use of differential screening to search for yet unidentified IFN-regulated genes. In the course of these studies, we have isolated a human cDNA coding for the glycosyl-phosphatidylinositol-linked (GPI) membrane glycoprotein CD48 (TCT-1, Blast-1). Various studies demonstrated that the murine CD48 is the predominant counterreceptor for the mouse CD2 and is involved in the regulation of T cell activation. Since the murine CD48 is functionally homologous to the human CD2 ligand LFA-3 (CD48), the function of the human CD48 remains unknown. In this report, we show that both Hu-IFN-alpha/beta and Hu-IFN-gamma increase the level of CD48 mRNA and upregulate the expression of CD48 proteins at the surface of various cultured human cell lines. However, the IFN have no effect on the expression of LFA-3. In addition, we show that IFN increase CD48 expression on peripheral blood mononuclear CD3+, CD14+, and CD19+ subpopulations. These data suggest that in addition to modulation of the conventional MHC class I and class II-restricted interactions, the IFN might promote MHC-unrestricted interactions of target cells with the immune cells by inducing the expression of the cell surface CD48 molecule.

Molecular cloning of a new interferon-induced factor that represses human immunodeficiency virus type 1 long terminal repeat expression.

Transcriptional induction of genes is an essential part of the cellular response to interferons. To isolate yet unidentified IFN-regulated genes we have performed a differential screening on a cDNA library prepared from human lymphoblastoid Daudi cells treated for 16 h with human alpha/beta interferon (Hu-alpha/beta IFN). In the course of these studies we have isolated a human cDNA which codes for a protein sharing homology with the mouse Rpt-1 gene; it will be referred as Staf-50 for Stimulated Trans-Acting Factor of 50 kDa. Amino acid sequence analysis revealed that Staf-50 is a member of the Ring finger family and contains all the features of a transcriptional regulator able to initiate a second cascade of gene induction (secondary response). Staf-50 is induced by both type I and type II IFN in various cell lines and down-regulates the transcription directed by the long terminal repeat promoter region of human immunodeficiency virus type 1 in transfected cells. These data are consistent with a role of Staf-50 in the mechanism of transduction of the IFN antiviral action.

In vivo footprints between the murine c-myc P1 and P2 promoters.

Assuming that when transcription starts at the P2 promoter of the c-myc gene sites located immediately upstream from P2 are occupied whereas in the absence of initiation they are not, the polymerase chain reaction (PCR)-based method of Mueller & Wold [(1989). Science, 246, 780-786] was used to map in vivo footprints upstream from the P2 promoter in various mouse cell lines. In cultured Friend erythroleukemic cells induced to differentiate with dimethysulfoxide (DMSO), a clear protection corresponding to ME1a2 and E2F sites was observed, consistent with in vitro band-shift and footprint data. However, in cell lines in which the gene was either silent or truncated the footprints were no longer visible. Friend c-myc transcripts decreased to a barely detectable level after 3 h of DMSO treatment. Transcription, as measured by in vitro run-on, was turned off at the level of RNA polymerase elongation rather than initiation [Mechti N., Piechaczyk, M. Blanchard, J.-M., Marty, L., Bonnieu, A., Jeanteur, Ph. & Lebler, B. (1986). Nucleic Acids Res., 24, 9653-9666]. The state of occupancy of the sites did not vary from the first hours up to 9 days of DMSO treatment, suggesting that DNA occupancy per se cannot explain premature termination, which rather would involve a more complex phenomenon.

Sequence requirements for premature transcription arrest within the first intron of the mouse c-fos gene.

A strong block to the elongation of nascent RNA transcripts by RNA polymerase II occurs in the 5' part of the mammalian c-fos proto-oncogene. In addition to the control of initiation, this mechanism contributes to transcriptional regulation of the gene. In vitro transcription experiments using nuclear extracts and purified transcription templates allowed us to map a unique arrest site within the mouse first intron 385 nucleotides downstream from the promoter. This position is in keeping with that estimated from nuclear run-on assays performed with short DNA probes and thus suggests that it corresponds to the actual block in vivo. Moreover, we have shown that neither the c-fos promoter nor upstream sequences are absolute requirements for an efficient transcription arrest both in vivo and in vitro. Finally, we have characterized a 103-nucleotide-long intron 1 motif comprising the arrest site and sufficient for obtaining the block in a cell-free transcription assay.

Intracellular distribution of microinjected antisense oligonucleotides.

Antisense oligomers constitute an attractive class of specific tools for genetic analysis and for potential therapeutic applications. Targets with different cellular locations have been described, such as mRNA translation initiation sites, pre-mRNA splicing sites, or the genes themselves. However the mechanism(s) of action and the intracellular distribution of antisense oligomers remain poorly understood. Antisense oligomers conjugated with various fluorochromes or with BrdUrd were microinjected into the cytoplasm of somatic cells, and their cellular distribution was monitored by fluorescence microscopy in fixed and nonfixed cells. A fast translocation in the nuclei and a concentration on nuclear structures were observed whatever probe was used. Nuclear transport occurs by diffusion since it is not affected by depletion of the intracellular ATP pool, temperature, or excess unlabeled oligomer. Accumulation of the oligomers in the nuclei essentially takes place on a set of proteins preferentially extracted between 0.2 M and 0.4 M NaCl as revealed by crosslinking of photosensitive oligomers. The relationship between nuclear location of antisense oligomers and their mechanism of action remains to be ascertained and could be of major interest in the design of more efficient antisense molecules.