Interplay of transcription factors STAT3, STAT1 and AP-1 mediates activity of the matrix metallo-proteinase-1 promoter in colorectal carcinoma cells.

Signal Transducers (STATs) 1 and 3 and Activator Protein 1 (AP-1) are transcription factors involved in the development of malignancy in colorectal carcinoma (CRC). Matrix Metalloproteinase 1 (MMP-1) is a protease frequently dysregulated in de-differentiated and invasive cancer cells. Its expression is influenced by STAT and AP-1 transcription factors. We studied their contributions to transcriptional regulation of MMP-1 in colorectal carcinoma (CRC) cells. Both STAT3 and AP-1 contribute individual expression-inducing and additive effects and interact with the MMP-1 promoter. DNA binding of AP-1 protein c-Jun is stimulation-independent but modulated by STAT3 and a STAT recognition DNA element. Activated STAT3 showed a suppressive effect on AP-1-mediated MMP-1 mRNA upregulation as shown by STAT3 knockdown. Surprisingly, activated STAT1 overcame STAT3-dependent repression of AP-1-driven MMP-1 expression. Moreover, combined STAT3, STAT1 and AP-1 activities evoked maximal MMP-1 mRNA levels in a synergistic manner. Our results suggest a dominant role of AP-1 in transcriptional upregulation of MMP-1 in CRC cells which is modulated by joint functions of STAT3 and STAT1. The individual and combinatorial activity of these factors is of diagnostic and prognostic interest.

Hedgehog/GLI and PI3K signaling in the initiation and maintenance of chronic lymphocytic leukemia.

The initiation and maintenance of a malignant phenotype requires complex and synergistic interactions of multiple oncogenic signals. The Hedgehog (HH)/GLI pathway has been implicated in a variety of cancer entities and targeted pathway inhibition is of therapeutic relevance. Signal cross-talk with other cancer pathways including PI3K/AKT modulates HH/GLI signal strength and its oncogenicity. In this study, we addressed the role of HH/GLI and its putative interaction with the PI3K/AKT cascade in the initiation and maintenance of chronic lymphocytic leukemia (CLL). Using transgenic mouse models, we show that B-cell-specific constitutive activation of HH/GLI signaling either at the level of the HH effector and drug target Smoothened or at the level of the GLI transcription factors does not suffice to initiate a CLL-like phenotype characterized by the accumulation of CD5(+) B cells in the lymphatic system and peripheral blood. Furthermore, Hh/Gli activation in Pten-deficient B cells with activated Pi3K/Akt signaling failed to enhance the expansion of leukemic CD5(+) B cells, suggesting that genetic or epigenetic alterations leading to aberrant HH/GLI signaling in B cells do not suffice to elicit a CLL-like phenotype in mice. By contrast, we identify a critical role of GLI and PI3K signaling for the survival of human primary CLL cells. We show that combined targeting of GLI and PI3K/AKT/mTOR signaling can have a synergistic therapeutic effect in cells from a subgroup of CLL patients, thereby providing a basis for the evaluation of future combination therapies targeting HH/GLI and PI3K signaling in this common hematopoietic malignancy.

Imiquimod directly inhibits Hedgehog signalling by stimulating adenosine receptor/protein kinase A-mediated GLI phosphorylation.

Imiquimod (IMQ), a nucleoside analogue of the imidazoquinoline family, is used in the topical treatment of basal cell carcinoma (BCC) and other skin diseases. It is reported to be a TLR7 and TLR8 agonist and, as such, initiates a Th1 immune response by activating sentinel cells in the vicinity of the tumour. BCC is a hedgehog (HH)-driven malignancy with oncogenic glioma-associated oncogene (GLI) signalling activated in a ligand-independent manner. Here we show that IMQ can also directly repress HH signalling by negatively modulating GLI activity in BCC and medulloblastoma cells. Further, we provide evidence that the repressive effect of IMQ on HH signalling is not dependent on TLR/MYD88 signalling. Our results suggest a mechanism for IMQ engaging adenosine receptors (ADORAs) to control GLI signalling. Pharmacological activation of ADORA with either an ADORA agonist or IMQ resulted in a protein kinase A (PKA)-mediated GLI phosphorylation and reduction in GLI activator levels. The activation of PKA and HH pathway target gene downregulation in response to IMQ were abrogated by ADORA inhibition. Furthermore, activated Smoothened signalling, which positively signals to GLI transcription factors, could be effectively counteracted by IMQ. These results reveal a previously unknown mode of action of IMQ in the treatment of BCC and also suggest a role for ADORAs in the regulation of oncogenic HH signalling.

Interaction between the TP63 and SHH pathways is an important determinant of epidermal homeostasis.

Deregulation of the hedgehog (HH) pathway results in overexpression of the GLI target BCL2 and is an initiating event in specific tumor types including basal cell carcinoma of the skin. Regulation of the HH pathway during keratinocyte differentiation is not well understood. We measured HH pathway activity in response to differentiation stimuli in keratinocytes. An upregulation of suppressor of fused (SUFU), a negative regulator of the HH pathway, lowered HH pathway activity and was accompanied by loss of BCL2 expression associated with keratinocyte differentiation. We used in vitro and in vivo models to demonstrate that ΔNp63α, a crucial regulator of epidermal development, activates SUFU transcription in keratinocytes. Increasing SUFU protein levels inhibited GLI-mediated gene activation in suprabasal keratinocytes and promoted differentiation. Loss of SUFU expression caused deregulation of keratinocyte differentiation and BCL2 overexpression. Using in vivo murine models, we also provide evidence of GLI-mediated regulation of the TP63 pathway. p63 expression appears essential to establish an optimally functioning HH pathway. These observations present a regulatory mechanism by which SUFU acts as an interacting node between the HH and TP63 pathways to mediate differentiation and maintain epidermal homeostasis. Disruption of this regulatory node can be an important contributor to multistep carcinogenesis.

GLI1-dependent transcriptional repression of CYLD in basal cell carcinoma.

CYLD is a deubiquitination enzyme that regulates different cellular processes, such as cell proliferation and cell survival. Mutation and loss of heterozygosity of the CYLD gene causes development of cylindromatosis, a benign tumour originating from the skin. Our study shows that CYLD expression is dramatically downregulated in basal cell carcinoma (BCC), the most common cancer in humans. Reduced CYLD expression in basal cell carcinoma was mediated by GLI1-dependent activation of the transcriptional repressor Snail. Inhibition of GLI1 restored the CYLD expression-mediated Snail signaling pathway, and caused a significant delay in the G1 to S phase transition, as well as proliferation. Our data suggest that GLI1-mediated suppression of CYLD has a significant role in basal cell carcinoma progression.

Inhibition of GLI, but not Smoothened, induces apoptosis in chronic lymphocytic leukemia cells.

The Hedgehog (Hh) pathway regulates cell proliferation and survival and contributes to tumorigenesis. We investigated the expression and function of this pathway in B-cell chronic lymphocytic leukemia (CLL) cells and in healthy B lymphocytes. Profiling of cognate Hh pathway members revealed reduced expression of two key Hh signaling effectors, Smoothened (SMOH) and GLI, in CLL cells, whereas transcription levels of other investigated members resembled normal B-lymphocyte levels. Examining the functional role of SMOH and GLI in cell survival, we found that CLL cells were hardly sensitive toward specific SMOH inhibition, but showed an unspecific decline in cell viability in response to high concentrations of the SMOH antagonist cyclopamine. In contrast, treatment with the novel GLI antagonist GANT61 reduced expression of the target gene Patched and preferentially decreased the viability of malignant cells. Specific RNA interference knockdown experiments in a CLL-derived cell line confirmed the autonomous role of GLI in malignant cell survival. GANT61-induced apoptosis in primary leukemic cells was partly attenuated by protective stromal cells, but not soluble sonic hedgehog ligand. In summary, our data show a downregulation of the classical Hh pathway in CLL and suggest an intrinsic SMOH-independent role of GLI in the ex vivo survival of CLL cells.

Array-based profiling of ragweed and mugwort pollen allergens.

BACKGROUND: Ragweed (Ambrosia artemisiifolia) and mugwort (Artemisia vulgaris) pollen is the main cause of allergic reactions in late summer and autumn. The differential diagnosis between ragweed and mugwort pollen allergy is a frequent problem encountered by allergologists in areas where both plants are present due to shared antigenic structures and overlapping flowering seasons. OBJECTIVE: To evaluate the sensitization pattern of weed allergic patients towards a large panel of purified allergens in the microarray format and by enzyme-linked immunosorbent assay (ELISA). METHODS: Eight ragweed and six mugwort pollen allergens were purified from natural source or expressed as recombinant proteins in Escherichia coli. Allergens were spotted on protein microarray slides or coated onto ELISA plates. Sera from 19 ragweed and/or mugwort allergic individuals were used to determine the reactivity towards single molecules in both assays. RESULTS: All ragweed allergic individuals were sensitized to Amb a 1, among them 30% were monosensitized to the major ragweed allergen. Art v 1 and Art v 3 were recognized by 89% of mugwort pollen-allergic patients. Extensive cross-reactivity was observed for both patient groups mainly involving the pan-allergens profilin and nonspecific lipid transfer proteins. Comparable IgE profiles were obtained with both allergen microarray and ELISA methods. CONCLUSIONS: Molecule-based diagnosis provides essential information for the differential diagnosis between ragweed and mugwort pollen allergy and for the selection of the appropriate allergen source for specific immunotherapy.

Analysis of gene expression using high-density and IFN-gamma-specific low-density cDNA arrays.

The combination of high and low density cDNA filter array technology potentially permits both the identification of subsets of induced genes and convenient and rapid multisample expression profiling of such subsets under a variety of conditions. The JAK/STAT1 pathway for IFN-gamma signaling in human cells has been well characterized, but the extent and importance of additional pathways remain to be established. Here, using high-density filter arrays of the RZPD UniGene set, we identified 18 novel IFN-gamma-inducible genes. Expression profiling was carried out using low-density arrays representing both novel and known IFN-gamma-inducible genes. Initial experiments failed to detect evidence for any novel non-JAK-dependent pathways in cells expressing a kinase-dead JAK2. The data, however, validated the potential of the combined methods in establishing rapid and convenient expression profiling of several hundred genes in response to any ligand of choice.

Dopamine transporter expression distinguishes dopaminergic neurons from other catecholaminergic neurons in the developing zebrafish embryo.

To characterize the formation of the dopaminergic system in the developing zebrafish CNS, we cloned cDNAs encoding tyrosine hydroxylase (th), an enzyme in dopamine synthesis, and the dopamine transporter (dat), a membrane transport protein which terminates dopamine action by re-uptake. Dopaminergic neurons are first detected between 18 and 19 h post-fertilization in a cluster of cells in the ventral diencephalon. Subsequently, th and dat detection identifies dopaminergic neurons in the olfactory bulb, the pretectum, the retina and the locus coeruleus. Neurons expressing th but not dat are adrenergic or noradrenergic, and are found in the locus coeruleus, the medulla, the likely analog of the carotid body, and precursors of the enteric and sympathetic nervous system.

Clinical application of a new disposable lithotripter: a prospective multicenter study.

BACKGROUND: Mechanical lithotripsy has become a well-accepted method of bile duct stone fragmentation and removal. The Olympus lithotripter (Olympus American, Melville, NY) is the standard reusable lithotripter at the institutions that participated in this study. A disposable device with a preassembled pistol grip may perform equally well and facilitate operation. METHODS: Twenty patients with bile duct stones were evaluated as part of a multicenter prospective study. Data were obtained regarding stone size and number, bile duct diameter, and configuration, ease of cannulation, basket function, stone capture and crushing success, and complications. RESULTS: The maximum stone size averaged 16.5 +/- 1.2 mm (range 10 to 30 mm). Sixteen patients had multiple stones (median 5, range 2 to 12). The mean bile duct diameter was 20.5 +/- 1.5 mm (range 12 to 38 mm). Cannulation was successful in all within 5 attempts. Basket deployment failed in 1 patient because of stone size and the basket was misshapen in 14. Bile duct clearance was complete in 16 subjects (80%), incomplete in 2 patients, and failed in 2 patients. Abnormal duct configuration (sigmoid, stricture) was noted in 2 of 4 patients with failed capture and 7 of 16 patients with successful clearance. No statistically significant difference was observed between the bile duct diameter, maximum stone size, number of stones, and successful clearance. CONCLUSION: The disposable lithotripter is easy to use and, compared with the published results for the reusable lithotripter, performs almost as well.

Anterior specification of embryonic ectoderm: the role of the Xenopus cement gland-specific gene XAG-2.

In a search for novel developmental genes expressed in a spatially restricted pattern in dorsal ectoderm of Xenopus we have identified XAG-2, a cement gland-specific gene with a putative role in ectodermal patterning. XAG-2 encodes a secreted protein, which is expressed in the anterior region of dorsal ectoderm from late gastrula stages onwards. Activation of XAG-2 transcription is observed in response to organizer-secreted molecules including the noggin, chordin, follistatin and cerberus gene products. Overexpression of XAG-2 but not of the related cement gland marker XAG-1 induces both cement gland differentiation and expression of anterior neural marker genes in the absence of mesoderm formation. Further, we show that XAG-2 signaling depends on an intact fibroblast growth factor (FGF) signal transduction pathway and that XAG-2-induced anterior neural fate of ectodermal cells can be transformed to a more posterior character by retinoic acid. Based on these findings we propose a role for XAG-2 in the specification of dorsoanterior ectodermal fate, i.e. in the formation of cement gland and induction of forebrain fate of Xenopus.

Granulomatous appendicitis: Crohn's disease, atypical Crohn's or not Crohn's at all?

BACKGROUND: Crohn's disease isolated to the appendix has primarily been documented in case reports. We contribute a series with longterm followup and a literature review. STUDY DESIGN: A retrospective review of 1,133 consecutive appendectomy specimens over the 6-year period ending in 1994 identified seven patients with isolated granulomatous appendicitis. Two patients presented before the review period. These nine patients are reviewed and 156 patients identified in the world literature. RESULTS: Granulomatous appendicitis usually presents as an indolent course of appendicitis. No patient developed enterocutaneous fistula after appendectomy in our series. A mean followup of 7.3 years in our patients revealed no evidence of Crohn's disease. CONCLUSIONS: Granulomatous inflammatory disease isolated to the appendix differs from typical Crohn's disease with a decreased occurrence of enterocutaneous fistulas and rare recurrence. Consequently, isolated granulomatous appendicitis without small bowel or cecal involvement may not represent true Crohn's disease. Patients can be treated with minimal morbidity by appendectomy alone. If isolated granulomatous appendicitis does represent Crohn's disease, its longterm course in the majority of patients is extremely benign.

A member of the Met/HGF-receptor family is expressed in a BMP-4-like pattern in the ectoderm of Xenopus gastrulae.

The importance and involvement of growth factors and their corresponding receptors in embryonic induction has been more and more recognized during the past decade, in particular by loss-of-function experiments using dominant negative receptors. Here, we report the isolation of XHR, a Xenopus receptor-type tyrosine kinase, with homology to members of the Met/hepatocyte growth factor (HGF)-receptor family. Sequence comparison of XHR with other members of the Met/HGF-receptor family as well as in situ expression analyses suggest that XHR represents a novel member of this family of receptor-type tyrosine kinases. As could be shown by whole-mount in situ analysis, XHR transcripts are first expressed in the entire ectoderm at the onset of gastrulation. As gastrulation proceeds, XHR-transcription is turned off in cells induced by dorsal mesoderm to form neural tissue and thus, becomes predominantly confined to prospective epidermis. The strikingly similar expression patterns of XHR and Bone Morphogenetic Protein-4 (BMP-4), an inducer of epidermis and inhibitor of neural development, suggest an involvement of XHR signalling in the early cell-fate decision of ectodermal cells to form either neural derivatives or epidermis.

The Xenopus homologue of hepatocyte growth factor-like protein is specifically expressed in the presumptive neural plate during gastrulation.

Using a RT-PCR approach, we were able to isolate a cDNA encoding the Xenopus homologue of hepatocyte growth factor-like protein, which we have termed accordingly Xhl. The deduced Xhl protein consists of 717 amino acids, contains four putative kringle domains and a serine protease-like domain characteristic for mammalian HGF and HGF-like protein. The mRNA of Xhl is exclusively expressed in the midline of the prospective neural plate during the period of neural induction, only. Ectopic expression of Xhl causes a 'spina bifida'-like phenotype with enlargement of neural tissue. Activation of Xhl mRNA transcription can be induced by delayed reaggregation of animal caps and appears to require vertical rather than planar signals from the organizer. These data suggest that Xhl is involved in the formation of the embryonic nervous system of Xenopus.

Protein synthesis in murine organs during postimplantation development detected by two-dimensional gel electrophoresis.

Mouse embryos were isolated from the uterus on days 10 to 11 of gestation and incubated in Dulbecco's modified Eagle's medium (DMEM) with [35S]methionine for 4 h. Subsequently, their hearts and the brains were dissected. The brain was divided into three parts, containing the telencephalon, mesencephalon, and myelencephalon. These tissues were then processed for two-dimensional (2-D) gel electrophoresis. Protein synthesis of the isolated tissues was analyzed for organ-and cell lineage-specific patterns. We studied proteins with isoelectric points (pI) ranging from 4 to 10 and relative molecular weights (M(r)) varying from 10000 to 200000 and found several significant quantitative and qualitative differences between the tissues and the developmental stages analyzed. In particular, we were able to distinguish between protein spots that we now attribute putatively to the corresponding embryonic organs. These differences may reflect some of the organ- and cell lineage-specific changes in protein synthesis and gene expression during early mammalian differentiation.