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PubMed2005

Metalloproteinase inhibitors for the disintegrin-like metalloproteinases ADAM10 and ADAM17 that differentially block constitutive and phorbol ester-inducible shedding of cell surface molecules.

The transmembrane metzinkin-proteases of the ADAM (a disintegrin and a metalloproteinase)-family ADAM10 and ADAM 17 are both implicated in the ectodomain shedding of various cell surface molecules including the IL6-receptor and the transmembrane chemokines CX3CL1 and CXCL16. These molecules are constitutively released from cultured cells, a process that can be rapidly enhanced by cell stimulation with phorbol esters such as PMA. Recent research supports the view that the constitutive cleavage predominantly involves ADAM10 while the inducible one is mediated to a large extent by ADAM17. We here describe the discovery of hydroxamate compounds with different potency against ADAM10 and ADAM17 and different ability to block constitutive and inducible cleavage of IL6R, CX3CL1 and CXCL16 by the two proteases. By screening a number of hydroxamate inhibitors for the inhibition of recombinant metalloproteinases, a compound was found inhibiting ADAM10 with more than 100-fold higher potency than ADAM17, which may be explained by an improved fit of the compound to the S1' specificity pocket of ADAM10 as compared to that of ADAM17. In cell-based cleavage experiments this compound (GI254023X) potently blocked the constitutive release of IL6R, CX3CL1 and CXCL16, which was in line with the reported involvement of ADAM10 but not ADAM17 in this process. By contrast, the compound did not affect the PMA-induced shedding, which was only blocked by GW280264X, a potent inhibitor of ADAM17. As expected, GI254023X did not further decrease the residual release of CX3CL1 and CXCL16 in ADAM10-deficient cells verifying that the compound's effect on the constitutive shedding of these molecules was exclusively due to the inhibition of ADAM10. Thus, GI254023X may by of use as a preferential inhibitor of constitutive shedding events without effecting the inducible shedding in response to agonists acting similar to PMA.

ADAM Proteins↗

PubMed1999

[A study on the role of the transmembrane V to VI regions of the human luteinizing hormone receptor in its constitutive activation].

Gonadotropin (luteinizing hormone: LH, and follicle stimulating hormone: FSH) receptors are members of the seven-transmembrane (TM) receptor family. Recently, point mutations have been identified in the LH receptor gene, leading to constitutive activation (ligand-independent activation) of the receptor in patients with familial male-limited precocious puberty. Most of such point mutations were found in the coding region consisting of TM V, VI and the intracellular loop 3 (i3). Because the primary structures of gonadotropin receptors are highly conserved, in vitro mutagenesis was performed to introduce the amino acid substitution in the i3 domain (Asp-->Gly) of FSH receptor, corresponding to the point mutation leading to constitutive activation in LH receptor. However, the FSH receptor mutant showed almost similar basal cAMP production to wild type counterpart. For investigation of this difference in constitutive activation between two receptors, the author designed chimeric LH/FSH receptors with or without an amino acid substitution in the i3 (Asp-->Gly) to identify the region in the LH receptor which is important for its constitutive activation. Introduction of the amino acid substitution into a chimeric receptor containing only TM V to VI from the LH receptor led to major increases in ligand-independent cAMP production. Furthermore, the chimeric receptor with only TM V and VI derived from the LH receptor was constitutively activated by the amino acid substitution in the i3 from the FSH receptor. These results that interactions between TM V and VI of the FSH receptor are essential for maintaining the receptor conformation constrained, whereas interactions between these domains of the LH receptor are required for constitutive activation by the amino acid substitution in the i3.

Animals↗

PubMed2001

Constitutive activity of the human beta(1)-adrenergic receptor in beta(1)-receptor transgenic mice.

We tested the hypothesis that the human beta(1)-adrenergic receptor displays constitutive activity and that beta-adrenergic antagonists differ in their ability to modulate this constitutive activity. Transfection of the cDNAs of the human beta(1)- and beta(2)-adrenergic receptors into COS-7 cells caused increases in basal cAMP that were proportional to the receptor levels, thus demonstrating constitutive activity for both subtypes. At comparable receptor levels, the increase in basal cAMP was about 5-fold higher for the beta(2)- than for the beta(1)-subtype. As a model for enhanced beta-adrenergic signaling at the whole-organ level, we used transgenic mice with heart-specific overexpression of the human beta(1)-adrenergic receptor. In this model, the beta(1)-adrenergic receptor displayed constitutive activity as evidenced by a higher spontaneous beating rate of isolated right atria from beta(1)-transgenic versus wild-type mice. This difference was abolished by the addition of CGP20712A, demonstrating inverse agonist properties of this compound. We then tested whether various beta-adrenergic antagonists currently in clinical use for the treatment of heart failure differ in their ability to modulate constitutive activity of the cardiac beta(1)-adrenergic receptor. The beta(1)-selective antagonists metoprolol and bisoprolol showed significant inverse agonist activity at the beta(1)-adrenergic receptor. Carvedilol behaved as a neutral antagonist and xamoterol displayed marked partial agonist activity. We conclude that the human beta(1)-adrenergic receptor displays constitutive activity that is considerably lower than that of the beta(2)-subtype. beta-Adrenergic antagonists currently in clinical use differ in their ability to exert inverse agonist activity at the human beta(1)-adrenergic receptor, which may contribute to their therapeutic effects.

Adrenergic Antagonists↗

PubMed1992

Structural constraints within a trimeric transcriptional regulatory region. Constitutive and interferon-gamma-inducible expression of the HLA-DRA gene.

Constitutive and inducible transcription of the major histocompatibility class II HLA-DRA gene involves the upstream S element and the conserved X and Y elements. In this report we have addressed the roles of spatial constraints and stereospecific alignment between the upstream S and X elements, and the X and Y elements, in both constitutive and interferon-gamma (gamma-IFN)-induced expression. Analysis of the constitutive expression in B cell lines (B-LCL) has previously shown that the X and Y elements must be stereoaligned. Further study reveals that any spacing changes between S and X, regardless of the helical alignment of these two elements, is not tolerated. These same restraints are involved in an inducible system, because the response to gamma-IFN treatment requires both stereo alignment between the X and Y elements and precise spacing between the S and X elements. Neither constitutive nor inducible expression can be restored by correcting the distance and spacing between only the S and Y elements with misalignment of X. These results reveal a common pathway for constitutive and inducible expression that may require either direct or indirect protein complex formation among proteins bound to three highly conserved regulatory elements. We have also evaluated the role of the A/T-rich sequence located immediately 5' of the Y element and show that it exerts little effect on constitutive and gamma-IFN induced DRA expression.

Base Sequence↗

PubMed1989

Comparative mapping of the constitutional and tumor-associated 11;22 translocations.

The reciprocal t(11;22)(q23;q11) is the most common non-Robertsonian constitutional translocation in humans. The tumor-associated 11;22 rearrangement of Ewing sarcoma (ES) and peripheral neuroepithelioma (NE) is cytologically very similar to the 11;22 constitutional rearrangement. Using immunoglobulin light-chain constant region, ETS1 probes, and the technique of in situ hybridization, we previously were able to show that the constitutional and ES/NE breakpoints are different. As a first step toward isolating these translocation junctions and to further distinguish between them, we have made somatic cell hybrids. Cells from a constitutional 46,XX,inv(9),t(11;22) carrier and from an ES cell line with a t(11;22) were separately fused to a hypoxanthine-guanine phosphoribosyltransferase-deficient Chinese hamster cell line (RJK88). Resulting clones were screened with G-banding and Southern hybridization. Hybrid clones derived from the constitutional t(11;22) were established which contained the der(22) and both the der(22) and the der(11). Hybrid clones derived from the ES cell line containing the der(11) were isolated. Using the technique of Southern hybridization we have sublocalized the loci; ApoA1/C3, CD3D, ETS1, PBGD, THY1, D11S29, D11S34, and D11S147 to the region between the two breakpoints on chromosome 11 and V lambda I, V lambda VI, V lambda VII, and D22S10 to the region between the breakpoints on chromosome 22. Using anonymous DNA probes, we found that D22S9 and D22S24 map proximal to the constitutional breakpoint and that D22S15 and D22S32 map distal to the ES breakpoint on chromosome 22.

Animals↗

PubMed1994

Three distinct regions within the constitutive activation domain of cAMP regulatory element-binding protein (CREB) are required for transcription activation.

The transcription factor cAMP regulatory element-binding protein (CREB) mediates both constitutive and cAMP-induced gene expression through distinct, independently acting domains. The constitutive activation domain (CAD) (amino acids (aa) 165-252) encompasses and overlaps exon 9 of the CREB gene (E9, aa 180-243). In the present study, deletion of either the CAD or exon 9 from CREB-GAL4 (CRG) reduced constitutive activity to less than 2-fold, without affecting kinase inducible activity. However, fusion of the CAD to the GAL4 DNA binding domain (CAD-G4) stimulated transcription, whereas fusion of exon 9 sequences did not. Deletion of the amino-terminal flanking region of exon 9 (aa 165-180), but not COOH-terminal flanking sequences (aa 243-252), decreased constitutive activation in either the CAD-G4 or CRG background. Deletion of the previously characterized glutamine-rich region (Q3, aa 218-252) or of a region containing a hydrophobic cluster of amino acids (HC, aa 180-218) also reduced constitutive activation by either CAD-G4 or CRG. No single mutation of hydrophobic residues within HC impaired activity of the CAD, but double and triple mutations did, suggesting that multiple weak interactions are involved in function of the HC region. Thus, exon 9 of the CREB gene is necessary but not sufficient for constitutive activation. The CAD requires three distinct regions for function, suggesting that CREB may interact with multiple targets in the RNA polymerase II complex.

Amino Acid Sequence↗

PubMed1993

Identification and use of constitutive proteins for the normalization of high resolution electrophoretograms.

Quantitative inter-gel comparisons of proteins separated by high resolution two-dimensional protein electrophoresis present a number of problems. These problems may arise from: variations in pipetting and other mechanical manipulations of samples, protein loss during transfer from the first to the second gel dimension, variations in staining, and/or variations in film development during autoradiography, in the case of radioactively labeled proteins. This study presents a discussion of these issues and a normalization algorithm to deal with variations, which relies on a class of proteins present in most biological samples which by their nature may be considered internal standards. This class consists of proteins which are controlled by constitutive genes. Constitutive genes are genes that are expressed constantly. We have developed an algorithm which is currently available as a subroutine, 'FINDCONS', in the computerized densitometry and protein comparison analysis program, developed by Olson & Miller (1988). This algorithm identifies potentially 'constitutive' proteins. A normalization method employing these potentially 'constitutive' proteins was compared to several others by examining 2D-electrophoretograms of proteins from developing gypsy moths (Lymantria dispar L.) insect tissue. Following normalization, inter-gel comparisons of spots, which were 'identified' as 'constitutive', were observed to vary less in density than when no normalization method was used, or when normalization based on total integrated spot density was used. In addition to its use as a normalization tool, this algorithm and the subroutine FINDCONS may be useful as an aid in biological studies to identify 'constitutive' proteins.

Algorithms↗

PubMed1996

Constitutive activation of STAT proteins in primary lymphoid and myeloid leukemia cells and in Epstein-Barr virus (EBV)-related lymphoma cell lines.

Although various molecular mechanisms of STAT protein (signal transducers and activators of transcription) activation have been identified, little is known about the functional role of STAT-dependent transcriptional activation. Herein we report the constitutive nuclear localization, phosphorylation, and DNA-binding activity of STAT proteins in leukemia cells and lymphoma cell lines. With the use of oligonucleotide probes derived from the Fc gamma RI promoter, the beta-casein promoter and a STAT-binding element in the promoter of the Bci-2 gene constitutive activation of STAT proteins was detected in untreated acute T- and C/B-leukemia cells (3 of 5 and 12 of 19 patients, respectively). Supershift analyses using Stats 1-6 specific antisera showed the constitutive DNA binding activity of Stat5 in these cells. Confocal microscopy revealed the nuclear localization of Stat5 and Western blot analyses showed tyrosine phosphorylation of Stat5 in nuclear extracts of acute leukemia cells. In contrast, peripheral blood mononuclear cells did not display constitutive STAT-DNA interaction. Further studies were performed on freshly isolated acute myeloid leukemia cells as well as on cell line derived K562, lymphoblastoid cells (LCL), and Burkitt's lymphoma cells (BL). Fluorescence microscopy, gelshift, and supershift experiments showed the nuclear localization and constitutive DNA-binding activity of Stat5 in K562 cells. Stat1 and Stat3 were constitutively activated in freshly isolated AML cells (10 of 14 patients) and in Epstein Barr virus-positive or interleukin-10 expressing permanent LCL and BL cells. Thus, these data indicate a differential pattern of STAT protein activation in lymphoid or myeloid leukemia and in lymphoma cells.

Acute Disease↗

PubMed1976

Correlation coefficients for electrocardiographic and constitutional variables.

The influence of noncardiac factors such as constitutional variables, sex, and age upon the orthogonal electrocardiogram (ECG) and vectorcardiogram was investigated in 300 normal Japanese subjects. Constitutional variables included body height (HT), body weight (WT), deviation from ideal body weight (DIW), chest transverse diameter (TD), chest sagittal diameter (SD), SD/TD ratio, and chest circumference (CC). Among these constitutional variables, HT was found to be the most important determinant of ECG, followed by WT, TD, CC, SD, SD/TD ratio, and DIW, in that order. An increase in HT resulted in the augmentation of magnitudes of a large number of the QRS and ST-T parameters. WT showed almost the same trends as HT. Of the four measurements of the chest configuration under study, TD proved to be the most powerful determinant. An increase in TD led to a significant increase in QRS duration and to a superior and anterior shift of the maximal T vector. The remaining constitutional variables revealed far less significant correlation with ECG measurements than did HT and WT. Besides the constitutional variables, sex and age were also proved to be important ECG determinants. Advancing age had the effect of reducing the magnitude of many of the QRS and ST-T measurements. It is also of interest that a majority of the QRS and ST-T parameters were larger in male than in female patients. It should be emphasized from the results of this study that normal ECG criteria must be established for each ECG determinant.

Adult↗

PubMed1998

Personal constitution and health status among Chinese elderly in Taipei and Los Angeles.

This paper reports ethnographic and statistical data on the indigenous Chinese concept of constitution (ti-zhi) and its association with health status. The data were obtained through in-depth interviews with 203 Chinese elderly residing in Taipei and Los Angeles. The informants used several dimensions to characterize their constitutions. They also described how their constitutions originated and were expressed over the lifecourse. The association between these characterizations of constitution and reported number of symptoms/diseases, self-rated health status and satisfaction with health were examined statistically. These analyses indicate that characterizations of constitution as strong/weak or as hot/cold, have significant but moderate associations with the indicators of health status.

Aged↗

PubMed2004

Agonist-dependent and agonist-independent transactivations of the human constitutive androstane receptor are modulated by specific amino acid pairs.

The constitutive androstane receptor (CAR) is an interesting member of the nuclear receptor superfamily because of its exceptionally high constitutive activity due to ligand-independent interaction of the ligand-binding domain with co-activator proteins. This study compares the agonist-dependent and agonist-independent activities of human CAR with those of mouse CAR and the vitamin D receptor and demonstrates that the constitutive activity of CAR is mediated by at least three contacts between the amino acids of helix 12, partner amino acids in helices 4 and 11, and a charge clamp between helices 12 and 3. The stabilization of helix 12 by a contact between its C terminus and the lysine of helix 4 has the same impact in human and mouse CARs. In addition, the charge clamp between the glutamate in helix 12 and the lysine in helix 3 is also important for the constitutive activity of both receptor orthologs but less critical for the agonist-dependent stabilization of their respective helices 12. Interestingly, Cys-357 in mouse CAR has significantly more impact on the stabilization of helix 12 than does the orthologous position Cys-347 in human CAR. This deficit appears to be compensated by a more dominant role of Ile-330 in human CAR over Leu-340 in mouse CAR because it is more efficient than Cys-347 in controlling the flexibility of helix 12 in the presence of an agonist. The constitutive activity of other members of the nuclear receptor superfamily could be explained by a homologous hydrophobic interaction between large, non-polar amino acids of helices 11 and 12.

Amino Acid Sequence↗

PubMed2004

Protein kinase C regulates alpha(2A/D)-adrenoceptor constitutive activity.

We investigated a possible role for protein kinases in the constitutive activity of alpha(2A/D) adrenoceptors in membranes from transfected PC12 cells, using a [35S]GTPgammaS binding assay. After treatment of intact cells with various protein kinase inhibitors, constitutive activity was assessed by the reduction in basal GTP binding caused by the inverse agonist rauwolscine (RAU). Inhibitors of protein kinase C (PKC) caused the loss of RAU-sensitive GTP binding, while inhibitors of other protein kinases were ineffective. Anti-G(alpha) antibody treatments showed that constitutive alpha(2A/D)-receptor activity is directed toward different G proteins than agonist-stimulated activity. T373A mutant receptors exhibited increased constitutive activity, including a component that was insensitive to PKC inhibition. Since T373 is located within a putative G(i/o) activator sequence, these results suggest that PKC-dependent phosphorylation of T373 increases alpha(2A/D)-adrenergic receptor constitutive activity and causes a switch in G protein preference.

Adrenergic alpha-Antagonists↗

PubMed2001

Constitutive activation of the Wnt signaling pathway by CTNNB1 (beta-catenin) mutations in a subset of human lung adenocarcinoma.

Constitutive activation of the Wnt signaling pathway as a result of genetic alterations of APC, AXIN1, and CTNNB1 has been found in various human cancers, including those of the colon, liver, endometrium, ovary, prostate, and stomach. To investigate the pathogenetic significance of constitutive activation of the Wnt signaling pathway in human lung carcinogenesis, CTNNB1 alterations in exon 3, a region known to represent a mutation hot spot, were screened in 46 lung cancer cell lines and 47 primary lung cancers. Missense mutations causing substitutions of Ser/Thr residues critical for regulation by GSK-3beta were detected in one (2%) of the cell lines, A427, and two (4%) of the surgical specimens. The three lung cancers with CTNNB1 mutations were adenocarcinomas. To explore the prevalence of constitutive activation of the Wnt signaling pathway in human lung cancer, we assessed 15 lung cancer cell lines representing major histological subtypes of lung cancers for constitutive Tcf transcriptional activity (CTTA). CTTA was observed only in the A427 adenocarcinoma cell line, but not in the remaining 14 cell lines. The data indicate that constitutive activation of the Wnt signaling pathway caused by CTNNB1 mutation is involved in the development and/or progression of a subset of lung carcinoma, preferentially in adenocarcinoma.

Adenocarcinoma↗

PubMedDate not supplied

Constitutive fragile sites in fra(X) individuals.

Recently, it was proposed that the constitutive fragile site at 3p14 be used as an "internal control" to indicate the effectiveness of the FUdR fragile site induction system. We have tested this hypothesis by determining the frequency of constitutive fragile sites at 1p31, 3p14, and 16q23 in cultures from 42 known fra(X) individuals. At least 50 cells were analyzed from each case. Seventy-four percent (31/42), 95% (40/42) and 90% (38/42) of the fra(X) individuals exhibited frequencies of less than 4% at constitutive fragile sites 3p14, 1p31 and 16q23, respectively. Of the 42 individuals tested, 12 or 28.6% showed no fragility at any of the 3 sites studied. On the other hand, at least one constitutive fragile site was observed in 50 cells studied from over 70% of the 42 people studied. It is suggested that "positive controls" continue to be used, while at the same time recording all fragile sites to identify a combination of constitutive fragile sites that may serve as an internal control indicator, and that DNA marker studies be used to complement cytogenetic testing.

Chromosome Fragile Sites↗

PubMed2002

Cartilage-specific constitutive expression of TSG-6 protein (product of tumor necrosis factor alpha-stimulated gene 6) provides a chondroprotective, but not antiinflammatory, effect in antigen-induced arthritis.

OBJECTIVE: To study the chondroprotective effect of constitutively expressed TSG-6 protein (tumor necrosis factor alpha-induced protein 6; Tnfip6) in cartilage, using antigen-induced arthritis (AIA) in mice. METHODS: Transgenic mice constitutively expressing TSG-6 protein in cartilage were generated. Cartilage-specific constitutive expression of TSG-6 protein was confirmed by in situ hybridization, Western blot analysis, and immunohistochemistry. Control and transgenic mice were immunized with methylated bovine serum albumin (mBSA), and arthritis was induced by the intraarticular injection of mBSA. Mice were monitored up to day 35 after the challenge, and knee joint sections were examined for loss of cartilage proteoglycan (aggrecan) using Safranin O staining and antibodies to neoepitopes generated by various metalloproteinases (MPs). The loss of aggrecan in Safranin O-stained sections was quantified by morphometric methods. RESULTS: Tsg6/tnfip6 transgenic mice constitutively expressed tsg6/tnfip6 messenger RNA and corresponding TSG-6 protein in cartilage from embryonic life through adulthood, without any phenotypic abnormalities. These mice were used for AIA studies. Intraarticular injection of mBSA uniformly induced severe inflammation both in control (wild-type and an irrelevant transgenic line) mice and in tsg6/tnfip6 transgenic mice. In contrast to the mBSA-injected knee joints of control animals that were heavily damaged from day 5, the cartilage of transgenic mice that constitutively expressed TSG-6 protein remained intact for at least 1 week, and this was followed by a relatively reduced loss of aggrecan. Concomitant with the loss of aggrecan, MP-generated neoepitopes accumulated in unprotected joints. By day 35, the proteoglycan content returned to nearly normal levels in tsg6/tnfip6 transgenic mice, whereas it remained low in MP-damaged knee cartilage of control mice. CONCLUSION: TSG-6 protein is known to form a complex with inter-alpha-inhibitor (IalphaI), a potent serine protease inhibitor, which may be immobilized via the hyaluronan (HA)-binding domain of TSG-6 protein in the HA-rich extracellular matrix of cartilage. Thus, the local accumulation of TSG-6 protein and TSG-6 protein-bound IalphaI in tsg6/tnfip6 transgenic mice may inhibit serine proteases and subsequent activation of MPs. It is suggested that this mechanism might protect cartilage from extensive degradation even in the presence of acute inflammation.

Animals↗

PubMed1988

Establishment of mouse cell lines which constitutively secrete large quantities of interleukin 2, 3, 4 or 5, using modified cDNA expression vectors.

Mouse cell lines of different lineages have been established which constitutively secrete large quantities of recombinant mouse interleukins (mIL2, mIL3, mIL4 or mIL5). An existing bovine papilloma virus-based expression vector, pBV-1MTHA, was modified to allow transformed X63Ag8-653 myeloma cells, NIH 3T3 fibroblasts and C127 mammary tumor cells to stably carry multiple copies of the vector, to express the inserted cDNA encoding a single interleukin constitutively, and to secrete the interleukin in high quantities. Cell lines transformed with mIL2 cDNA stably carried 30-100 copies of the plasmid per cell and constitutively secreted biologically active mIL2 in quantities similar to those produced by murine EL4 thymoma cells or rat spleen cells stimulated with mitogens. Deletion of the 3' untranslated region containing AT-rich sequences from the mIL2 cDNA resulted in a 100-fold increase in the constitutive production and secretion of mIL2 by the transformants. Addition of a heavy metal further increased the production 2 to 6-fold. Cells transformed with 3'-deleted mIL3 cDNA constitutively secreted 300-1000 times higher activities of mIL3 than the myelomonocytic leukemia line WEHI3. mIL4 produced by the similar transformants induced [3H]thymidine uptake of a T cell line, a mast cell line and B leukemia cells, and enhanced the production of IgG1 by B cells. IL4 titers were 150 times higher than those produced by the concanavalin A-stimulated T cell line 2.19. mIL5 was secreted by similar transformants at 10-fold higher titers than those produced by concanavalin A-stimulated 2.19 T cells, as judged by the proliferation and maturation of B cell leukemia BCL1. The expression vectors should be useful in establishing eukaryotic cell lines producing proteins from full length cDNA clones at higher rates. The established cell lines secreting IL2, 3, 4 or 5 at high rate should be useful sources for these interleukins in the investigation of their function in the immune system.

Animals↗

PubMed2003

Mitogenic and antiapoptotic role of constitutive NF-kappaB/Rel activity in pancreatic cancer.

The transcription factor NF-kappaB/Rel was found to be constitutively activated in human pancreatic cancer. RelA is present in the nucleus in primary human pancreatic cancer samples as well as in pancreatic cancer cell lines. NF-kappaB/Rel-binding activity consists of NF-kappaB1(p50) and RelA(p65). Constitutive NF-kappaB/Rel activity correlates with IkappaB kinase (IKK) activity and can be blocked by dominant negative mutants of IKKbeta and to a lesser extent by IKKalpha. Constitutive NF-kappaB/Rel activity and the transactivation potential of RelA(p65) can be inhibited by dominant negative mutant Ras, the PI3 kinase inhibitor LY294002, or dominant negative mutant Akt kinase. Transfection of a dominant negative mutant epidermal growth factor receptor (EGF-R), EGF-R kinase inhibitor Tyrphostin and LY 294002 blocked IKK activity and NF-kappaB-dependent transcription. Inhibition of constitutive IKK or NF-kappaB/Rel activity increased the number of apoptotic cells. Stably expressing a nondegradable form of IkappaBalpha inhibited anchorage-dependent and -independent proliferation in MiaPaCa2 and Panc1 cells. Our data demonstrate that an EGF-R/Ras/PI3 kinase/Akt/IKK-dependent pathway contributes to constitutive NF-kappaB/Rel activity in pancreatic cancer. Inhibition of NF-kappaB/Rel activity reveals a mitogenic and antiapoptotic role for NF-kappaB/Rel in pancreatic cancer.

Adenocarcinoma↗

PubMed1995

Constitutional and somatic mutations in the WT1 gene in Wilms' tumor patients.

Wilms' tumor development, like most tumors, involves multiple genetic alterations affecting diverse genes. Only one of these has thus far been identified, the Wilms' tumor 1 (WT1) gene on 11p13, which functions as a tumor suppressor gene. We assessed the involvement of the WT1 gene constitutionally and somatically in 26 Wilms' tumor patients. Of these, the clinical data suggest a constitutional pre-disposition in 12 bilateral cases and 7 cases with early onset. We employed Southern blot analysis and PCR-based markers for analyses of somatic allelic losses in chromosome bands 11p13, 11p15 and 16q and screened for point mutations in exons 2-10 of the WT1 gene with denaturing gradient gel electrophoresis (DGGE). Of the 12 cases with multiple tumors, 1 exhibited a constitutional 11p13 deletion and a somatic stop mutation in exon 4 of the WT1 gene and 2 harbored constitutional mutations in the WT1 gene: a pre-mature stop codon in exon 6 in a boy with bilateral cryptorchidism and bilateral Wilms' tumors and an intragenic deletion in a girl with bilateral WT. Three additional bilateral tumors displayed WT1 rearrangements or allelic losses with 11p13 markers. Four of 7 patients with an early onset of unilateral tumor had losses of 11p13, though no WT1 mutations were detected. Two of the remaining cases that did not show any somatic or constitutional 11p13 alterations had Beckwith-Wiedemann syndrome, known to involve the 11p15 region.

Alleles↗