Neurofibromatosis type 1 gene mutations in neuroblastoma.

The introduction of human chromosome 17 suppresses the tumourigenicity of a neuroblastoma cell line in the absence of any effects on in vitro growth and the neurofibromatosis type 1 (NF1) gene may be responsible. Here we report that 4 out of 10 human neuroblastoma lines express little or no neurofibromin and that two of these lines show evidence of NF1 mutations, providing further proof that NF1 mutations occur in tumours that are not commonly found in NF1 patients. We also show that NF1 deficient neuroblastomas show only moderately elevated ras-GTP levels, in contrast to NF1 tumour cells, indicating that neurofibromin contributes differently to the negative regulation of ras in different cell types.

Integrin cytoplasmic interactions and bidirectional transmembrane signalling.

Integrins are heterodimeric integral plasma membrane proteins containing extracellular, transmembrane, and cytoplasmic domains. These highly versatile receptors mediate not only cell adhesion and migration, but also the bidirectional transfer of information across the plasma membrane. The cytoplasmic domains of integrins are required for the transduction of this bidirectional information, and have recently been shown to participate in direct interactions with some novel cytoplasmic proteins, such as an ankyrin repeat containing serine/threonine protein kinase (integrin-linked kinase) and beta3 endonexin. New evidence also suggests that, via interactions with focal adhesion kinase, the integrin cytoplasmic domains can coordinate actin cytoskeletal organization and responses to growth factors. The elucidation of the signal transduction pathways activated by integrins is an intense area of investigation that has shown that integrins have some unique properties as signal transducing receptors.

Signal transduction by interferon-alpha through arachidonic acid metabolism.

Molecular mechanisms that mediate signal transduction by growth inhibitory cytokines are poorly understood. Type I (alpha and beta) interferons (IFNs) are potent growth inhibitory cytokines whose biological activities depend on induced changes in gene expression. IFN-alpha induced the transient activation of phospholipase A2 in 3T3 fibroblasts and rapid hydrolysis of [3H]arachidonic acid (AA) from prelabeled phospholipid pools. The phospholipase inhibitor, bromophenacyl bromide (BPB), specifically blocked IFN-induced binding of nuclear factors to a conserved, IFN-regulated enhancer element, the interferon-stimulated response element (ISRE). BPB also caused a dose-dependent inhibition of IFN-alpha-induced ISRE-dependent transcription in transient transfection assays. Specific inhibition of AA oxygenation by eicosatetraynoic acid prevented IFN-alpha induction of factor binding to the ISRE. Treatment of intact cells with inhibitors of fatty acid cyclooxygenase or lipoxygenase enzymes resulted in amplification of IFN-alpha-induced ISRE binding and gene expression. Thus, IFN-alpha receptor-coupled AA hydrolysis may function in activation of latent transcription factors by IFN-alpha and provides a system for studying the role of AA metabolism in transduction of growth inhibitory signals.

Rescue of a Drosophila NF1 mutant phenotype by protein kinase A.

The neurofibromatosis type 1 (NF1) tumor suppressor protein is thought to restrict cell proliferation by functioning as a Ras-specific guanosine triphosphatase-activating protein. However, Drosophila homozygous for null mutations of an NF1 homolog showed no obvious signs of perturbed Ras1-mediated signaling. Loss of NF1 resulted in a reduction in size of larvae, pupae, and adults. This size defect was not modified by manipulating Ras1 signaling but was restored by expression of activated adenosine 3', 5'-monophosphate-dependent protein kinase (PKA). Thus, NF1 and PKA appear to interact in a pathway that controls the overall growth of Drosophila.

Regulation of interferon receptor expression in human blood lymphocytes in vitro and during interferon therapy.

Interferons (IFN) elicit antiviral and antineoplastic activities by binding to specific receptors on the cell surface. The binding characteristics of IFN to human lymphocytes were studied using IFN alpha 2 labeled with 125I to high specific activity. The specific binding curves generated were analyzed by the LIGAND program of Munson and Rodbard to determine receptor numbers. The number of receptors in peripheral blood lymphocytes (PBL) and tonsillar B-lymphocytes (TBL) from normal individuals were 505 +/- 293 (n = 10) and 393 +/- 147 (n = 3) respectively. When these cells were preincubated in vitro with unlabeled IFN alpha 2, the receptor number decreased to 82 +/- 45 and 61 +/- 16 respectively. Receptor binding activities recovered gradually over a period of 72 h when the cells were incubated in IFN-free medium. This recovery of receptors could be blocked by the addition of actinomycin D to the incubation medium. A similar decrease in receptor expression was observed in vivo in PBL from patients being treated daily with 5 X 10(6) units/m2 per d of IFN alpha 2 by subcutaneous injection, for acute lymphoblastic leukemia or papilloma virus infections. Receptor numbers in PBL in vivo were further reduced concurrent with the progression of IFN therapy. Thus the reduction in IFN receptor expression observed in vitro can be demonstrated in vivo. These studies indicate that monitoring IFN receptor expression in vivo can provide information regarding the availability of IFN receptors at the cell surface for the mediation of IFN actions during the course of IFN therapy.

Enhancement by retinoic acid and dibutyryl cyclic adenosine 3':5'-monophosphate of the differentiation and gene expression of human neuroblastoma cells induced by interferon.

Human neuroblastoma cell lines are induced to differentiate and display neuronal phenotypes when treated with interferon (IFN)-alpha 2, retinoic acid (RA), or dibutyryl cyclic AMP (dbcAMP). We investigated the effects of combinations of these agents in induction-differentiation in the neuroblastoma cell line, NUB-6. The inductive effect of IFN-alpha 2 was markedly enhanced when used in combination with RA or dbcAMP. In parallel, RA or dbcAMP also enhanced the level of 2'-5'-oligoadenylate (2-5A) synthetase, and enzyme induced by IFNs and implicated in their biological action. The levels of another IFN-inducible enzyme, p68 kinase, were not enhanced by the combination treatments. The enhancement effects appeared to be exerted largely at the posttranscriptional level as both RA and dbcAMP stabilized IFN-induced 2-5A synthetase mRNA, resulting in increased enzyme activity. Thus, the 2-5A synthetase system is likely involved in mediating the IFN-alpha 2-induced differentiation of neuroblastoma cells and may also mediate the enhancement effects of RA and dbcAMP on IFN activity in these cells. These results also provide a rational basis for establishing a combination therapeutic approach for the treatment of neuroblastoma.

Protein kinase mediators of integrin signal transduction.

Protein kinases are important mediators of signal transduction initiated by soluble growth factors and cytokines. Cellular interactions with the extracellular matrix are mediated largely by members of the integrin class of cell adhesion molecules, which also subsume signal transduction functions required for cell growth, differentiation, and survival. Here we review the involvement of protein kinases in mediating integrin intracellular signal transduction and the possible role for these molecules in regulating integrin adhesion. Although in most cases mechanistic details are incomplete, the emerging theme of protein kinases mediating cross-talk between growth factor receptor and integrin signalling systems provides a timely backdrop against which to present new developments in this area. The contribution of the actin cytoskeleton to integrin signal transduction is discussed, with respect to the concept of 'solid-state' signalling providing a mechanism for imposing order on the protein-protein interactions which underlie signal discrimination. Moreover, we review evidence that dysregulated integrin signalling contributes to pathological processes including arthritis, thrombasthenia, leucocyte adhesion deficiencies, and tumour angiogenesis and invasion.

Complete human NF1 cDNA sequence: two alternatively spliced mRNAs and absence of expression in a neuroblastoma line.

Neurofibromatosis type 1 (NF1) is caused by mutations in a large gene on chromosome 17q11.2. Previously described partial cDNAs for this gene predicted a protein related to yeast IRA1/IRA2 and the mammalian RAS GTPase activator protein GAP. To initiate a detailed study of the role of this gene in NF1, we have characterized a set of overlapping cDNAs that represent its complete coding sequence. Our results show that two differentially expressed human NF1 mRNAs differ by a 63-bp insertion in the GAP-related domain. These mRNAs predict two 2,818- and 2,839-amino acid proteins with calculated molecular masses of approximately 317 and 319 kD. Extensive similarity to IRA proteins is evident in a 1,450-amino-acid central segment, roughly between amino acids 900 and 2,350. However, the remainder of the NF1 protein is not significantly similar to other proteins. Interestingly, the SK-N-SH human neuroblastoma line expresses no detectable NF1 mRNA, indicating that expression of NF1 is not essential for viability of this neural crest-derived tumor cell line.

Selective regulation of p38ß protein and signaling by integrin-linked kinase mediates bladder cancer cell migration.

Integrin-linked kinase (ILK) and p38(MAPK) are protein kinases that transduce extracellular signals regulating cell migration and actin cytoskeletal organization. ILK-dependent regulation of p38(MAPK) is critical for mammalian kidney development and in smooth muscle cell migration, however, specific p38 isoforms has not been previously examined in ILK-regulated responses. Signaling by ILK and p38(MAPK) is often dysregulated in bladder cancer, and here we report a strong positive correlation between protein levels of ILK and p38ß, which is the predominant isoform found in bladder cancer cells, as well as in patient-matched normal bladder and tumor samples. Knockdown by RNA interference of either p38ß or ILK disrupts serum-induced, Rac1-dependent migration and actin cytoskeletal organization in bladder cancer cells. Surprisingly, ILK knockdown causes the selective reduction in p38ß cellular protein level, without inhibiting p38ß messenger RNA (mRNA) expression. The loss of p38ß protein in ILK-depleted cells is partially rescued by the 26S proteasomal inhibitor MG132. Using co-precipitation and bimolecular fluorescent complementation assays, we find that ILK selectively forms cytoplasmic complexes with p38ß. In situ proximity ligation assays further demonstrate that serum-stimulated assembly of endogenous ILK-p38ß complexes is sensitive to QLT-0267, a small molecule ILK kinase inhibitor. Finally, inhibition of ILK reduces the amplitude and period of serum-induced activation of heat shock protein 27 (Hsp27), a target of p38ß implicated in actin cytoskeletal reorganization. Our work identifies Hsp27 as a novel target of ILK-p38ß signaling complexes, playing a key role in bladder cancer cell migration.

Serum integrin-linked kinase (sILK) concentration and survival in non-small cell lung cancer: a pilot study.

BACKGROUND: Integrin-linked kinase (ILK) is an intracellular signaling protein critically involved in cellular growth and motility. In non-small cell lung cancer (NSCLC), increased ILK expression has been associated with decreased recurrence-free and overall survival. Recently, ILK has also been detected in the serum of NSCLC patients. OBJECTIVE: To assess the prognostic impact of preoperative serum ILK (sILK) concentration on overall survival in surgically amenable NSCLC. PATIENTS AND METHODS: Preoperative sILK was quantified by ELISA in 50 newly diagnosed NSCLC patients. After surgery, patients were followed-up for a median interval of 2.5 years. RESULTS: Serum ILK concentrations ranged from 0 to 2.44 ng/ml. Mean sILK was around 2.3 times higher in the 16 patients who died as compared to the 34 patients who survived (1.04 vs. 0.45 ng/ml, p = 0.001). In univariate time-to-event analysis, increased sILK was associated with adverse survival [Hazard ratio (HR): 4.03, 95 % CI: 2.00-8.13, p < 0.001]. This association prevailed after multivariable adjustment for several clinical, demographic, and laboratory parameters (HR: 3.85, 95 % CI: 1.53-9.72, p = 0.004). CONCLUSIONS: Serum ILK shows potential as a novel strong and independent prognostic marker for postoperative survival in surgically amenable NSCLC.

Integrin-linked kinase: not so 'pseudo' after all.

Integrin-linked kinase (ILK) is a highly evolutionarily conserved intracellular protein that was originally identified as an integrin-interacting protein, and extensive genetic and biochemical studies have shown that ILK expression is vital during both embryonic development and tissue homeostasis. At the cellular and tissue levels, ILK regulates signaling pathways for cell adhesion-mediated cell survival (anoikis), apoptosis, proliferation and mitosis, migration, invasion, and vascularization and tumor angiogenesis. ILK also has central roles in cardiac and smooth-muscle contractility, and ILK dysregulation causes cardiomyopathies in humans. ILK protein levels are increased in several human cancers and often the expression level predicts poor patient outcome. Abundant evidence has accumulated suggesting that, of the diverse functions of ILK, some may require kinase activity whereas others depend on protein-protein interactions and are, therefore, independent of kinase activity. However, the past several years have seen an ongoing debate about whether ILK indeed functions as a protein serine/threonine kinase. This debate centers on the atypical protein kinase domain of ILK, which lacks some amino-acid residues thought to be essential for phosphotransferase activity. However, similar deficiencies are present in the catalytic domains of other kinases now known to possess protein kinase activity. Numerous studies have shown that ILK phosphorylates peptide substrates in vitro, corresponding to ILK-mediated phosphorylations in intact cells, and a recent report characterizing in vitro phosphotransferase activity of highly purified, full-length ILK, accompanied by detailed enzyme kinetic analyses, shows that, at least in vitro, ILK is a bona fide protein kinase. However, several genetic studies suggest that, not all biological functions of ILK require kinase activity, and that it can function as an adaptor/scaffold protein. Here, we review evidence for and against ILK being an active kinase, and provide a framework for strategies to further analyze the kinase and adaptor functions of ILK in different cellular contexts.

Integrin-linked kinase (ILK) is highly expressed in first trimester human chorionic villi and regulates migration of a human cytotrophoblast-derived cell line.

The placenta represents a critically important fetal-maternal interaction. Trophoblast migration and invasion into the uterine wall is a precisely controlled process and aberrations in these processes are implicated in diseases such as preeclampsia. Integrin-linked kinase (ILK) is a multifunctional, cytoplasmic, serine/threonine kinase that has been implicated in regulating processes such as cell proliferation, survival, migration, and invasion; yet the temporal and spatial pattern of expression of ILK in human chorionic villi and its role in early human placental development are completely unknown. We hypothesized that ILK would be expressed in trophoblast subtypes of human chorionic villi during early placental development and that it would regulate trophoblast migration. Immunoblot analysis revealed that ILK protein was highly detectable in placental tissue samples throughout gestation. In floating branches of chorionic villi, from 6 to 15 wk of gestation immunofluorescence analysis of ILK expression in placental tissue sections demonstrated that ILK was highly detectable in the cytoplasm and membranes of villous cytotrophoblast cells and in stromal mesenchyme, whereas it was barely detectable in the syncytiotrophoblast layer. In anchoring branches of villi, ILK was highly localized to plasma membranes of extravillous trophoblast cells. Transient expression of dominant negative E359K-ILK in the villous explant-derived trophoblast cell line HTR8-SVneo dramatically reduced migration into wounds compared to cells expressing wild-type ILK or empty vector. Therefore, our work has demonstrated that ILK is highly expressed in trophoblast subtypes of human chorionic villi during the first trimester of pregnancy and is a likely mediator of trophoblast migration during this period of development.

Induced factor binding to the interferon-stimulated response element. Interferon-alpha and platelet-derived growth factor utilize distinct signaling pathways.

Interferon-alpha (IFN alpha) and platelet-derived growth factor (PDGF) each rapidly stimulate binding of nuclear factors from Balb/c 3T3 fibroblasts, to a 29-base pair regulatory sequence derived from the 5' upstream region of the murine 2-5A synthetase gene. This regulatory sequence contains a functional interferon-stimulated response element (ISRE) and also functions as a PDGF-responsive sequence. We show that IFN alpha induces binding of a protein of molecular mass 65 kDa to the ISRE. Constitutively expressed ISRE-binding proteins of 98 and 150 kDa are also demonstrated. Binding of inducible factors to the ISRE increases significantly within 15 min of IFN alpha or PDGF treatment. PDGF-induced binding is not mediated by IFN beta. The protein kinase inhibitors, staurosporine and K252a, block PDGF-induced ISRE binding and 2-5A synthetase gene expression. IFN alpha-induced ISRE binding and gene activation are not blocked by these inhibitors. Treatment of cells with 12-O-tetradecanoyl-13-acetate or dibutyryl cyclic AMP does not activate ISRE binding factors or 2-5A synthetase gene expression. PDGF responsiveness of the ISRE in vivo is also sensitive to staurosporine, indicating that inhibition of a protein kinase activity blocks the PDGF-specific transcriptional signal. Our data indicate the signal transduction pathway for IFN alpha-induced, ISRE-dependent transcription is distinct from the PDGF-induced ISRE response and is likely independent of cyclic AMP-dependent protein kinase and protein kinase C activities.

Interferon-alpha activates binding of nuclear factors to a sequence element in the c-fos proto-oncogene 5'-flanking region.

Interferon-alpha (IFN-alpha) can regulate the expression of the c-fos proto-oncogene in different cell types. Here we show IFN-alpha-activated binding of murine and human fibroblast nuclear factors to a DNA sequence element located in the 5' upstream region (nucleotides -351/-337) of the c-fos gene. This element, like the conserved enhancer element, the IFN-stimulated response element (ISRE), that mediates transcriptional induction of IFN-alpha-inducible genes, also binds factors in response to platelet-derived growth factor (PDGF) and v-sis-conditioned medium (SCM). The IFN-inducible ISRE shares an 8-bp stretch of sequence homology with the IFN-responsive c-fos SCM element, and competes efficiently for binding of factors to the SCM. Protein-DNA cross-linking experiments with the SCM binding site identified an IFN-modulated nuclear protein of approximately 98 kD. This protein does not appear to be involved in transcription activation, since IFN-alpha failed to stimulate c-fos transcription in nuclear run-off assays, or the c-fos promoter in transient transcription assays of 3T3 fibroblasts. Our data nonetheless suggest the c-fos promoter may be an early target for signal transduction triggered by IFN alpha-receptor interaction.

Interferon and growth factor modulation of nuclear factors binding to 5' upstream elements of the 2-5A synthetase gene.

We assayed fragments of the 5' flanking sequence of the human 2-5A synthetase gene for their ability to respond to interferon-alpha (IFN) and platelet-derived growth factor (PDGF). Transient transfection assays identified a 40-base pair fragment, which, regardless of orientation, could confer IFN-inducibility on the thymidine kinase promoter. This same fragment was active in monkey and mouse cells and in the latter was responsive to PDGF. The effect of PDGF could be inhibited by anti-interferon antibodies. Gel retardation assays, using the 40-base pair probe, detected the presence of IFN-modulated DNA-binding factors in nuclear extracts from monkey cells. In mouse cells both IFN and PDGF induced the binding of nuclear factors to a synthetic 2-5A synthetase response sequence. Thus, both IFN and growth factors directly or indirectly modulate the binding of nuclear factors to the same region of the 2-5A synthetase gene.

Interferon-induced binding of nuclear factors to promoter elements of the 2-5A synthetase gene.

Fragments of the 5'-flanking sequence of a human 2-5A synthetase gene were assayed for their ability to respond to interferon-alpha (IFN). Transient transfection assays in monkey cells demonstrated that the 5' boundary of the sequence required for IFN-regulated transcription is, at most, 155 nucleotides upstream from the presumed translational initiation codon. The 3' boundary of this sequence lies within a region of multiple transcription start sites preceded by no obvious TATA box. Binding assays, using a 40-bp probe derived from this IFN-responsive sequence, demonstrated the presence of three IFN-modulated, DNA-factor band shifts using nuclear extracts prepared from human and monkey cells. The induction of these complexes in human cells by IFN occurs with kinetics which closely parallel those previously observed for the transcriptional activation of the 2-5A synthetase gene by IFN. In vivo competition assays showed that the same 40-bp region which bound IFN-modulated factors could decrease the IFN-induced activity of a co-transfected 2-5A synthetase promoter; this fragment, regardless of its orientation, could confer IFN-inducibility on a heterologous promoter.

Characterization and regulation of alpha-interferon receptor expression in interferon-sensitive and -resistant human lymphoblastoid cells.

Interferon-sensitive (IFN-S) and IFN-resistant (IFN-R) Daudi lymphoblastoid cells were studied for IFN-alpha receptor expression and regulation by steady state and kinetic procedures, utilizing a homogeneous 125I-IFN-alpha 2 probe. Heterogeneity in the binding of this probe to IFN-S cells was determined to result from negatively cooperative interactions between an initially homogeneous class of IFN receptor. No such heterogeneity was noted in the IFN-R cells, indicating an apparent difference in the interaction of IFN-alpha 2 with these cells. The apparent dissociation constants (Kd) for IFN-S cell receptors were calculated to be 1 X 10(-10) M and 1 X 10(-8) M, for the high and low affinity sites, respectively. The Kd for sites on the IFN-R cells was estimated to be 4 X 10(-9) M. IFN-R and IFN-S cells expressed 2.4 X 10(4) and 3.5 X 10(4) binding sites per cell, respectively, representing an increase of at least 6-fold over previous reports of IFN-S Daudi IFN receptor density. Both IFN-S and IFN-R cells were capable of down-regulating expression of the IFN-alpha receptor in response to low concentrations of IFN-alpha 2. Furthermore, both cell lines were shown to be capable of internalizing specifically bound 125I-IFN-alpha 2 to an equivalent degree. Accordingly, we propose that the relative insensitivity of the Daudi IFN-R phenotype involves the loss of a high affinity interaction between cellular receptors and IFN-alpha 2, in addition to the reduced level of expressed low affinity binding sites.

Modulation of human interferon-alpha receptor expression by human interferon-gamma.

The human neuroblastoma cell line T98G is sensitive to the antiproliferative activity of both alpha- and gamma-interferons (IFNs). Specific binding of a homogeneous 125I-IFN-alpha 2 probe to high-affinity membrane receptors (apparent Kd = 6.2 X 10(-10) M) can be decreased by pretreatment of these cells with human IFN-gamma (HuIFN-gamma, derived by recombinant DNA techniques). The regulation of HuIFN-alpha 2 binding by such pretreatment is reflected by subsequent inhibition of both the antiviral and antiproliferative activities of HuIFN-alpha 2 on T98G cells. Thus, binding of HuIFN-gamma to its distinct cell-surface receptor can modify expression of the separate IFN-alpha receptor, presumably through an indirect effect on the apparent Kd of the IFN-alpha 2 receptor-ligand interaction.

Differential human interferon alpha receptor expression on proliferating and non-proliferating cells.

The expression of interferon-alpha (IFN-alpha) receptors was studied on a variety of human cells, using monoiodinated IFN-alpha 2 probes. Steady-state binding at 4 degrees C revealed a single class of non-interacting IFN receptor on peripheral blood lymphocytes, and tonsillar B lymphocytes, which are both known to be G0/G1 resting cell populations. The binding affinity of this class of receptor was found to be on the order of 5 X 10(-10) M, expressed as an apparent dissociation constant (Kd). However, cells proliferating either in culture or in vivo were found to express a heterogeneity in IFN-alpha 2 binding. Such binding could be objectively resolved (by a version of the LIGAND program of P. Munson) into a two-site receptor model. Hill plots of binding to proliferating cells indicated a negative cooperativity in the interaction of IFN and receptor. The high-affinity component, expressed on proliferating cells, typically exhibits a Kd of (1-10) X 10(-11) M, while the lower-affinity component indicates a Kd of (1-10) X 10(-9) M. Furthermore, the low-affinity component is apparently expressed on the order of 10-200 times the copy number, per cell, of the high-affinity site. Affinity-labeling experiments revealed that, in addition to the 140-160-kDa IFN-binding complex reported by others, both the proliferating and non-proliferating cell populations possess a novel IFN-binding component of 60 kDa.

Roles of globotriosyl- and galabiosylceramide in verotoxin binding and high affinity interferon receptor.

The cellular specificity of the Escherichia coli-derived verotoxin is of particular interest because of its extreme toxicity and high selectivity toward certain primate cells. The human Burkitt lymphoma cell line (Daudi) is highly susceptible to the cytotoxicity of verotoxin and contains large amounts of the verotoxin-binding glycolipids on its surface. A mutant selected from Daudi cells for verotoxin resistance was found to be deficient in the verotoxin-binding glycolipids, globotriosylceramide and galabiosylceramide, and failed to bind verotoxin to its surface; interestingly, these mutant cells were found to be cross-resistant to inhibition of growth by alpha-interferon. Mutant cells also lack the high affinity component of alpha-interferon binding. These observations suggest that, in addition to providing the functional cell-surface receptor for verotoxin, these glycolipids may also play a role in the modulation of the affinity of alpha-interferon for its membrane protein receptors.