The drug resistance-related protein LRP is the human major vault protein.

Multidrug-resistant cancer cells frequently overexpress the 110-kD LRP protein (originally named Lung Resistance-related Protein). LRP overexpression has been found to predict a poor response to chemotherapy in acute myeloid leukaemia and ovarian carcinoma. We describe the cloning and chromosome localization of the gene coding for this novel protein. The deduced LRP amino acid sequence shows 87.7% identity with the 104-kD rat major vault protein. Vaults are multi-subunit structures that may be involved in nucleo-cytoplasmic transport. The LRP gene is located on chromosome 16, close to the genes coding for multidrug resistance-associated protein and protein kinase C-beta, and may mediate drug resistance, perhaps via a transport process.

Dysfunctional AMPK activity, signalling through mTOR and survival in response to energetic stress in LKB1-deficient lung cancer.

LKB1, mutated in Peutz-Jeghers and in sporadic lung tumours, phosphorylates a group of protein kinases named AMP-activated protein kinase (AMPK)-related kinases. Among them is included the AMPK, a sensor of cellular energy status. To investigate the relevance of LKB1 in lung carcinogenesis, we study several lung cancer cells with and without LKB1-inactivating mutations. We report that LKB1-mutant cells are deficient for AMPK activity and refractory to mTOR inhibition upon glucose depletion but not growth-factor deprivation. The requirement for wild-type LKB1 to properly activate AMPK is further demonstrated in genetically modified cancer cells. In addition, LKB1-deficient lung primary tumours had diminished AMPK activity, assessed by complete absence or low level of phosphorylation of its critical substrate, acetyl-CoA carboxylase. We also demonstrate that LKB1 wild-type cells are more resistant to cell death upon glucose withdrawal than their mutant counterparts. Finally, modulation of AMPK activity did not affect PI3K/AKT signalling, an advantage for the potential use of AMPK as a target for cancer therapy in LKB1 wild-type tumours. Thus, sustained abrogation of cell energetic checkpoint control, through alterations at key genes, appear to be an obligatory step in the development of some lung tumours.

The gene encoding the granulocyte colony-stimulating factor receptor is a target for deregulation in pre-B ALL by the t(1;19)-specific oncoprotein E2A-Pbx1.

Approximately 25-30% of childhood pre-B cell acute lymphoblastic leukemias (pre-B ALL) is characterized by the presence of a (1;19)(q23;p13.3) translocation. The presence of this translocation is generally accompanied by a poor prognosis. The chimeric gene resulting from this chromosomal rearrangement encodes a hybrid transcription factor, E2A-Pbx1. In an attempt to delineate the genetic cascade initiated by E2A-Pbx1, we sought to identify genes that are deregulated by this transcription factor in t(1;19) pre-B ALL. We show here that the gene encoding the granulocyte colony-stimulating factor receptor (G-CSFr) is specifically upregulated in pre-B cells expressing E2A-Pbx1. G-CSFr is also expressed in cell lines established from t(1;19) pre-B cell leukemia and on primary t(1;19) tumor cells, but not on control cells. These data indicate that G-CSFr gene is a target for deregulation by E2A-Pbx1.

Allogeneic bone marrow transplantation can restore CD4+ T-lymphocyte count and immune function in idiopathic CD4+ T-lymphocytopenia.

CD4+ T-lymphocytopenia in the absence of HIV infection is a heterogeneous disorder of unknown cause. Here we report a patient with idiopathic CD4+ T-lymphocytopenia, presenting with an opportunistic Rhodococcus equi infection. When aplastic anemia developed subsequently, allogeneic bone marrow transplantation was performed. Complete restoration of immune function was observed. We conclude that allogeneic bone marrow transplantation presents a potentially curative therapy for CD4+ T-lymphocytopenia.

Clustering of monoclonal antibodies recognizing different members of the WC1 gene family.

Mouse L cell lines expressing two different bovine WC1 glycoproteins were produced by transfection of the cells with the corresponding cDNAs. The cell lines were used to analyze the reactivities of 67 monoclonal antibodies (mAbs) which recognize bovine gamma/delta T cells. The results indicated that preliminary clustering of mAbs can be achieved based on their recognition of epitopes expressed on all gene products, or of epitopes encoded by individual members of the gene family. The studies also showed that at least three members of the WC1 gene family are expressed, although it is not yet known how many can be expressed by individual bovine gamma/delta T cells. Final clustering of the WC1 mAbs will not be possible until the exact number of expressed gene products is known, and the reactivities of the mAbs with these products have been analyzed.

Thoughts on the thymus and the T-lymphocyte repertoire. Relevance to the tolerance of the immune response.

The thymus is a central lymphoid organ, and has one important function in the immune system. It generates immunocompetent T (= thymus-dependent) cells. 'Immunocompetence' is the single most important characteristic acquired during the differentiation process of T-cells, from progenitor cell in the bone marrow to mature cell in peripheral lymphoid organs and blood. The basis of competence is recognition, mediated by the T-cell receptor on the cell surface. Using this receptor, T-cells recognize antigens only in the form of antigenic fragments complexed with molecules of the major histocompatibility complex. Within the thymus, progenitor cells rearrange gene segments encoding the various parts of the receptor molecule. After expression of all possible receptors (the potentially available repertoire) selection processes occur, yielding only those T-cells that have a receptor by which antigens can be recognized together with molecules of the major histocompatibility complex, and that do not recognize autoantigens (the actual repertoire). This process of selection is considered as a unique function of the thymus. In this review, first some histophysiological aspects of the thymus are described. Next, the events that occur during the sojourn of the lymphocytes in the thymus are presented. Finally, these data are considered with regard to thymic abnormalities. These include acute and age-associated involution, and the generation of autoimmune reactivity due to disturbed intrathymic selection.

Towards a molecular understanding of T-cell differentiation.

Lymphoid differentiation is one of the best studied examples of mammalian development. Here Hans Clevers and Michael Owen describe how the cloning of the genes that encode T-cell-specific membrane proteins allows the identification of transcription factors that control the expression of these T-cell genes. Such transcription factors play a key role in the development of the mature T-cell phenotype by functioning as 'master regulators of T-cell differentiation'.

Transcription factors in early T-cell development.

Characterization of the transcription control regions for most genes encoding the TCR-CD3 complex has been followed by identification of DNA-binding factors, some of which are restricted to lymphoid or T-lineage cells. The sequential expression of these factors is linked with the appearance of stage-specific T-cell markers, thus controlling the commitment of the elusive lymphoid progenitor cell and its progression along the T-lineage. Here, Hans Clevers, Mariëtte Oosterwegel and Katia Georgopoulos discuss recent progress made in the recognition of transcription factors that regulate these early events in T-cell ontogeny.

Distinct beta-catenins mediate adhesion and signalling functions in C. elegans.

In flies and vertebrates, Armadillo/beta-catenin forms a complex with Tcf/Lef-1 transcription factors, serving as an essential co-activator to mediate Wnt signalling. It also associates with cadherins to mediate adhesion. In Caenorhabditis elegans, three putative beta-catenin homologues have been identified: WRM-1, BAR-1 and HMP-2. WRM-1 and the Tcf homologue POP-1 mediate Wnt signalling by a mechanism that has challenged current views of the Wnt pathway. Here we show that BAR-1 is the only beta-catenin homologue that interacts directly with POP-1. BAR-1 mediates Wnt signalling by forming a BAR-1/POP-1 bipartite transcription factor that activates expression of Wnt target genes such as the Hox gene mab-5. HMP-2 is the only beta-catenin homologue that interacts with the single cadherin of C. elegans, HMR-1. We conclude that a canonical Wnt pathway exists in C. elegans. Furthermore, our analysis shows that the functions of C. elegans beta-catenins in adhesion and in signalling are performed by separate proteins.

Human CD3-epsilon gene contains three miniexons and is transcribed from a non-TATA promoter.

The antigen receptor of the T lymphocyte consists of two variable T-cell receptor chains (either TCR-alpha, TCR-beta or TCR-gamma, TCR-delta) noncovalently linked to four different invariant membrane proteins (CD3-gamma, CD3-delta, CD3-epsilon, and the CD3-zeta homodimer). The CD3 genes are expressed early in thymocyte development, preceding the rearrangement and expression of the T-cell receptor genes. Here we report the isolation and structural analysis of the human CD3-epsilon gene. The gene consisted of nine exons. Three exons, encoding the junction of leader peptide and mature protein, were extremely small (21, 15, and 18 base pairs, respectively). The murine gene contained only two such miniexons, the sequences of which were not homologous to those of the three human miniexons. But from comparisons of intron sequences the regions surrounding the human miniexons III and IV appeared to be closely related to those surrounding the murine miniexons III and IV. The most-3' miniexon in the human gene (IVa) had no murine counterpart and appeared not to duplicate any of the other miniexons. Sequence analysis of CD3-epsilon cDNA clones isolated from four independent libraries gave no evidence for alternative use of these miniexons. Like CD3-delta, the CD3-epsilon gene was transcribed from a weak, nontissue-specific, TATA-less promoter. Pulsed-field electrophoresis showed that the human CD3-epsilon gene was separated from the CD3-gamma, CD3-delta gene pair by at least 30 kilobases, but by no more than 300 kilobases.

Calcium ionophore A23187 induces interleukin 2 reactivity in human T cells.

In the present study the activation of purified human T lymphocytes by the calcium ionophore A23187 was analysed in the light of current concepts of receptor-linked inositol lipid metabolism. It was found that A23187 was only slightly mitogenic, with a narrow optimum at 400-500 nM. The proliferation could be blocked by anti-Tac ascites at 10(-3) dilution, suggesting an interleukin 2 (IL-2)-dependent pathway of activation. However, an unexpectedly large proportion of A23187-stimulated cells expressed the IL-2 receptor. Reculturing the cells with exogenous IL-2 after removal of A23187 resulted in strongly enhanced proliferation. Phorbol myristic acetate (PMA) at non-mitogenic concentrations exerted an extremely strong synergistic effect on A23187-induced cell proliferation, which was, again, mediated via an IL-2-dependent pathway. Supernatants of A23187-stimulated T cells did not contain detectable amounts of IL-2. Combination of PMA and A23187 resulted in considerable IL-2 production. It is concluded that A23187 induces the expression of IL-2 receptors without concurrent stimulation of IL-2 production, thus allowing only low levels of proliferation. Addition of exogenous IL-2 or of PMA restores the imbalance between the occurrence of IL-2 and its receptor and results in high rates of proliferation.

Ligands of surface Ig raise cytoplasmic free Ca++ in human B cells.

With the use of the fluorescent Ca++ indicator Quin-2, we have measured changes in intracellular calcium levels in human B cells in response to anti-Ig antibodies, to Staphylococcus aureus (Staph) or to protein A. Cells of an Epstein-Barr virus-transformed mu lambda-carrying B-cell line, AZU-1, increased free cytosolic calcium after addition of anti-mu or anti-lambda antibodies; F (ab')2 fragments with anti-mu specificity were equally effective. Fab fragments of sheep anti-Ig antibodies only induced a rise in calcium levels after addition of a second anti-sheep Ig antiserum. Cross-linking of non-Ig surface determinants did not influence calcium homeostasis. The calcium channel blockers verapamil (100 microM), nifedipine (20 microM), and LaCl3 (200 microM) inhibited the anti-mu-induced calcium influx. Peripheral blood B cells reacted in essentially the same way in response to anti-mu antibodies. The B cell mitogens protein A and Staph also induced a rise in intracellular calcium. These observations indicate that Ca++ may play a role as a messenger in the activation of human B cells via surface Ig.

A common ancestor of the mammalian transcription factors TCF-1 and TCF-1 alpha/LEF-1 expressed in chicken T cells.

Several mammalian T cell-specific transcription factors have been cloned recently. Two of these, TCF-1 and TCF-1 alpha/LEF-1, display a moderate level of overall sequence similarity, and contain virtually identical versions of a novel type of DNA-binding domain, the HMG box. To study evolutionary aspects of the TCF transcription factors in relation to lymphoid differentiation, we have isolated chicken TCF clones from a spleen cDNA library. Low-stringency screening with human probes as well as a polymerase chain reaction-aided strategy resulted in the cloning of a single chicken TCF (chTCF) gene. Sequence comparison revealed that chTCF contained a TCF-1 alpha-like N terminus, and a TCF-1-like C terminus. Furthermore, TCF-1 and TCF-1 alpha were more homologous to chTCF than to each other. We postulate that chTCF is the direct descendant of a single ancestral gene, which has been duplicated in mammals to yield TCF-1 and TCF-1 alpha.

The many faces of the tumor suppressor gene APC.

Inactivation of the tumor suppressor adenomatous polyposis coli (APC) protein is a critical early step in the development of familial and sporadic colon cancer. Close examination of the function of APC has shown that it is a multifunctional protein involved in a wide variety of processes, including regulation of cell proliferation, cell migration, cell adhesion, cytoskeletal reorganization, and chromosomal stability. Tantalizing clues to the different functions of APC have been provided by the identification of proteins interacting with several discrete motifs within APC. Each of these putative functions could link APC inactivation with tumorigenesis. Here, we will summarize recent findings regarding the diverse role of APC. We will emphasize the interaction of APC with different binding partners, the role of these complex interactions for normal functioning of the cell, and how disruption of these interactions may play a role in tumor development. The rapid progress made recently shows the many faces of APC, leading to a constant reappreciation of this multitasking tumor suppressor protein.

Transcriptional control of t lymphocyte differentiation.

Initiation of gene transcription by transcription factors (TFs) is an important regulatory step in many developmental processes. The differentiation of T cell progenitors in the thymus is tightly controlled by signaling molecules, ultimately activating nuclear TFs that regulate the expression of T lineage-specific genes. During the last 2 years, significant progress has been made in our understanding of the signaling routes and TFs operating during the earliest stages of thymic differentiation at the CD4(-)CD8(-) double negative stage. Here we will review the TF families that play an important role in differentiation of thymocytes, particularly focusing on recent new information with respect to the Tcf, bHLH, GATA, and CBF/HES TF families.

Sox-4 facilitates thymocyte differentiation.

The mouse Sry-like transcription factor Sox-4 is expressed in thymus, bone marrow, and gonads of adult mice. Sox-4-deficient mice die at embryonic day E14 due to cardiac malformation. In transfer experiments to irradiated recipients, B cell development was shown to be severely impaired in Sox-4-deficient progenitor cells. However, no drastic effects on T lymphocyte development were noted, despite the high level expression of the Sox-4 gene in the thymus of normal mice. Here, we report a detailed analysis of T cell development from Sox-4-deficient progenitors. Explanted fetal thymic organ cultures (FTOC) of Sox-4-deficient thymi yielded 10-50-fold fewer CD4 CD8 double-positive and single-positive cells than FTOC of littermates. This effect was T cell-autonomous, since similar observations were made when FTOC were performed by culturing of Sox-4-deficient progenitors in wild-type thymus lobes. When Sox-4-deficient fetal liver cells were injected together with normal cells intrathymically, they did not compete efficiently for reconstitution. It is concluded that Sox-4 facilitates thymocyte development.

Tcf-1-mediated transcription in T lymphocytes: differential role for glycogen synthase kinase-3 in fibroblasts and T cells.

Beta-catenin is the vertebrate homolog of the Drosophila segment polarity gene Armadillo and plays roles in both cell-cell adhesion and transduction of the Wnt signaling cascade. Recently, members of the Lef/Tcf transcription factor family have been identified as protein partners of beta-catenin, explaining how beta-catenin alters gene expression. Here we report that in T cells, Tcf-1 also becomes transcriptionally active through interaction with beta-catenin, suggesting that the Wnt signal transduction pathway is operational in T lymphocytes as well. However, although Wnt signals are known to inhibit the activity of the negative regulatory protein kinase glycogen synthase kinase-3beta (GSK-3beta), resulting in increased levels of beta-catenin, we find no evidence for involvement of GSK-3beta in Tcf-mediated transcription in T cells. That is, a dominant negative GSK-3beta does not specifically activate Tcf transcription and stimuli (lithium or phytohemagglutinin) that inhibit GSK-3beta activity also do not activate Tcf reporter genes. Thus, inhibition of GSK-3beta is insufficient to activate Tcf-dependent transcription in T lymphocytes. In contrast, in C57MG fibroblast cells, lithium inactivates GSK-3beta and induces Tcf-controlled transcription. This is the first demonstration that lithium can alter gene expression of Tcf-responsive genes, and points to a difference in regulation of Wnt signaling between fibroblasts and lymphocytes.