Melanoma cells exhibit strong intracellular TASK-3-specific immunopositivity in both tissue sections and cell culture.

Amplification of the kcnk9 gene and overexpression of the encoded channel protein (TASK-3) seems to be involved in carcinogenesis. In the present work, TASK-3 expression of melanoma cells has been studied. For the investigation of TASK-3-specific immunolabelling, a monoclonal antibody has been developed and applied along with two, commercially available polyclonal antibodies targeting different epitopes of the channel protein. Both primary and metastatic melanoma cells proved to be TASK-3 positive, showing prominent intracellular TASK-3-specific labelling; mostly concentrating around or in the proximity of the nuclei. The immunoreaction was associated with the nuclear envelope, and with the processes of the cells and it was also present in the cell surface membrane. Specificity of the immunolabelling was confirmed by Western blot and transfection experiments. As TASK-3 immunopositivity of benign melanocytes could also be demonstrated, the presence or absence of TASK-3 channels cannot differentiate between malignant and non-malignant melanocytic tumours.

A RUNX/AML-binding motif residing in a novel 13-bp DNA palindrome may determine the expression of the proximal promoter of the human uPA gene.

Urokinase-type plasminogen activator (uPA) is a multifunctional extracellular serine protease implicated in different events including fibrinolysis, tissue remodeling, and hematopoiesis. The human uPA gene contains a major promoter region at around 2000 bp upstream from the transcription start site (+1), and a second regulatory region spanning nucleotides -90/+32 within the proximal promoter. Here, an inspection of this region revealed a novel 13-bp palindrome residing at position +8/+20. Interestingly, the palindrome contains the DNA consensus-binding hexamer for the RUNX/AML family of transcription factors that play a role in hematopoiesis, leukemia, and several developmental processes. Measuring the expression for promoter-reporter constructs after transfection revealed that deletion of the palindrome abrogated most of the proximal promoter activity in 293A cell. Additionally, electrophoretic mobility shift assays have shown that the palindrome could bind the RUNX1 component in nuclear extracts of myeloid cell lines exclusively through its RUNX motif. The palindrome was found in five additional human genes, two of which (MYH11 and MLLT1) have been linked to chromosomal rearrangements leading to leukemia. The data presented here have implicated, for the first time, RUNX/AML in the regulation of the uPA gene. The significance of the novel palindrome regarding gene regulation through the RUNX motif deserves further investigation.

Coordinate regulation of RARgamma2, TBP, and TAFII135 by targeted proteolysis during retinoic acid-induced differentiation of F9 embryonal carcinoma cells.

BACKGROUND: Treatment of mouse F9 embryonal carcinoma cells with all-trans retinoic acid (T-RA) induces differentiation into primitive endodermal type cells. Differentiation requires the action of the receptors for all trans, and 9cis-retinoic acid (RAR and RXR, respectively) and is accompanied by growth inhibition, changes in cell morphology, increased apoptosis, proteolytic degradation of the RARgamma2 receptor, and induction of target genes. RESULTS: We show that the RNA polymerase II transcription factor TFIID subunits TBP and TAFII135 are selectively depleted in extracts from differentiated F9 cells. In contrast, TBP and TAFII135 are readily detected in extracts from differentiated F9 cells treated with proteasome inhibitors showing that their disappearance is due to targeted proteolysis. This regulatory pathway is not limited to F9 cells as it is also seen when C2C12 myoblasts differentiate into myotubes. Targeting of TBP and TAFII135 for proteolysis in F9 cells takes place coordinately with that previously reported for the RARgamma2 receptor and is delayed or does not take place in RAR mutant F9 cells where differentiation is known to be impaired or abolished. Moreover, ectopic expression of TAFII135 delays proteolysis of the RARgamma2 receptor and impairs primitive endoderm differentiation at an early stage as evidenced by cell morphology, induction of marker genes and apoptotic response. In addition, enhanced TAFII135 expression induces a novel differentiation pathway characterised by the appearance of cells with an atypical elongated morphology which are cAMP resistant. CONCLUSIONS: These observations indicate that appropriately timed proteolysis of TBP and TAFII135 is required for normal F9 cell differentiation. Hence, in addition to transactivators, targeted proteolysis of basal transcription factors also plays an important role in gene regulation in response to physiological stimuli.

Dimerization with retinoid X receptors and phosphorylation modulate the retinoic acid-induced degradation of retinoic acid receptors alpha and gamma through the ubiquitin-proteasome pathway.

In eukaryotic cells, the ubiquitin-proteasome pathway is the major mechanism for targeted degradation of proteins. We show that, in F9 cells and in transfected COS-1 cells, the nuclear retinoid receptors, retinoic acid receptor gamma2 (RARgamma2), RARalpha1, and retinoid X receptor alpha1 (RXRalpha1) are degraded in a retinoic acid-dependent manner through the ubiquitin-proteasome pathway. The degradation of RARgamma2 is entirely dependent on its phosphorylation and on its heterodimerization with liganded RXRalpha1. In contrast, RARalpha1 degradation can occur in the absence of heterodimerization, whereas it is inhibited by phosphorylation, and heterodimerization reverses that inhibition. RXRalpha1 degradation is also modulated by heterodimerization. Thus, each partner of RARgamma/RXRalpha and RARalpha/RXRalpha heterodimers modulates the degradation of the other. We conclude that the ligand-dependent degradation of RARs and RXRs by the ubiquitin-proteasome pathway, which is regulated by heterodimerization and by phosphorylation, could be important for the regulation of the magnitude and duration of the effects of retinoid signals.

Retinoic acid induces proteasome-dependent degradation of retinoic acid receptor alpha (RARalpha) and oncogenic RARalpha fusion proteins.

Analyzing the pathways by which retinoic acid (RA) induces promyelocytic leukemia/retinoic acid receptor alpha (PML/RARalpha) catabolism in acute promyelocytic leukemia (APL), we found that, in addition to caspase-mediated PML/RARalpha cleavage, RA triggers degradation of both PML/RARalpha and RARalpha. Similarly, in non-APL cells, RA directly targeted RARalpha and RARalpha fusions to the proteasome degradation pathway. Activation of either RARalpha or RXRalpha by specific agonists induced degradation of both proteins. Conversely, a mutation in RARalpha that abolishes heterodimer formation and DNA binding, blocked both RARalpha and RXRalpha degradation. Mutations in the RARalpha DNA-binding domain or AF-2 transcriptional activation region also impaired RARalpha catabolism. Hence, our results link transcriptional activation to receptor catabolism and suggest that transcriptional up-regulation of nuclear receptors by their ligands may be a feedback mechanism allowing sustained target-gene activation.

The expression of the H19 gene and its function in human bladder carcinoma cell lines.

The human H19 gene is a paternally imprinted oncofetal gene, highly expressed in several fetal tissues, down-regulated in nearly all adult tissues but re-expressed in carcinomas of tissues which express the gene in fetal life. It has no known protein product and till today, no function could be designated to H19 RNA. Cells derived from bladder carcinomas and hepatocellular carcinomas were transfected with plasmids carrying a luciferase reporter gene under the control of a 800 nucleotides long promoter region of the H19 gene either alone or together with different parts of a 5 kb downstream region, previously shown to possess enhancer activity. Our results provide evidence that three regions of the 3' downstream sequence can independently stimulate the H19 promoter activity in a tissue and cell specific manner. The growth rate of two cell populations, both derived from the same bladder carcinoma cell line and which differ in their H19 RNA content, were compared. The cells with a high H19 RNA level stopped their proliferation after 48 h when cultivated in a low serum containing media while the cells lacking H19 RNA continued their proliferation for at least an additional 48 h period.

The effect of retinoic acid on the activation of the human H19 promoter by a 3' downstream region.

The human H19 is paternally imprinted (maternally expressed). It is transcribed by RNA pol II, but has no protein product. Its function is unknown. We showed that the transcription of the human H19 gene is under the simultaneous control of both a 5' upstream (promoter) region and a 3' downstream region in cell lines derived from human choriocarcinomas. Moreover, the activation of the H19 promoter by retinoic acid in cells derived from human testicular germ cell tumors is dependent upon the 3' downstream region. The possibility that the action of retinoic acid on the H19 promoter is an indirect one and involves a member of the AP2 transcription factor family is discussed.

The 3'-untranslated region of the urokinase gene enhances the expression of chimeric genes in cultured cells and correlates with specific brain expression in transgenic mice.

Transgenic mice were previously described, carrying the cDNA of the human or murine protease urokinase-type plasminogen activator (uPA) while linked to the cell-specific promoter of the alphaA-crystallin gene. Surprisingly, these mice produced transgenic uPA in an ectopic manner specifically in the brain. Here we tested the possibility that this ectopic expression could have been contributed primarily by the uPA transgenic moiety. Several experimental approaches have been used. (a) Constructs consisting of uPA cDNA linked to the cell-specific promoters of the alphaA-crystallin or insulin genes yielded active uPA after transfection into cells where these promoters are thought to be inactive. (b) When reporter genes were inserted into these constructs between the promoter and the cDNA, the cDNA enhanced the chimeric reporter expression 5-50-fold. This effect was obtained upon stable or transient construct transfection into four different cell types. (c) Reporter enhancement also took place in the presence of the homologous uPA gene promoter. (d) Mapping of the cDNA through deletion-substitution analysis has detected fragments mediating positive or negative effects on reporter expression, all fragments residing in the 3'-untranslated region (3'UTR) of the uPA gene that was included in the cDNA. Some fragments exhibited cell-specific effects. One fragment (2002/2187) behaved like a classical transcriptional enhancer, enhancing reporter expression from different positions and orientations. (e) Transgenic mice have now been generated that carry a transgene consisting of the alphaA-crystallin promoter, the luciferase reporter gene and mouse uPA cDNA. Among four transgenic lines producing luciferase activity in the eye lens, three lines exhibited ectopic luciferase activity exclusively in the brain, where luciferase mRNA was localized through in situ hybridization. From these results we conclude that the 3'UTR of the uPA gene contains sequences capable of exerting variable effects on gene expression, including transcriptional enhancement. In addition, uPA cDNA correlates with transgenic brain expression. Therefore, we suggest that the 3'UTR of the uPA gene is involved in brain expression of the transgenes containing uPA cDNA as well as of the normal uPA gene.

The expression of the imprinted H19 and IGF-2 genes in human bladder carcinoma.

The imprinted H19 gene is highly expressed in human embryos, fetal tissues and is nearly completely shut off in adults. However, it is reexpressed in a number of tumors including bladder carcinoma, demonstrating that H19 RNA is an oncofetal RNA. Tumors induced by injection of bladder carcinoma cell lines express H19 in contrast to the cells before injection. These observations support the notion of a positive correlation between H19 expression and bladder carcinoma. Loss of imprinting of H19 and IGF-2 was observed in samples of human bladder carcinoma.

Ipsilateral cortical connections from the second and fourth somatic sensory areas in the cat.

The ipsilateral corticocortical connections of the second and fourth somatic sensory areas (SII and SIV) were traced with the aid of anterograde or retrograde axonal transport techniques involving horseradish peroxidase conjugated to wheat germ agglutinin (HRP-WGA) or tritiated amino acids. The injections were placed into physiological defined components of the body representation in SII or SIV. The results from cases with localized injections into SII showed precise topographically organized, reciprocal connections with SI and motor cortex area 4. The distribution of connections in SI included areas 3a, 3b, and 1-2. A uniform pattern of cell and fiber labeling was seen across area 3b and 1 within the zones that were homotypical to the injection site in SII as though only a single representation of the cutaneous surface of the body existed in SI. Intrinsic connections within SII were also topographically arranged. Additional areas found to be interconnected with SII included, in decreasing order of density: area 5, insula, perirhinal cortex (area 36), and ventrolateral orbital cortex. SII connections with area 6 were seen only in the region of the lateral bank of the presylvian sulcus. There may be interconnections between SII and SIV but these were from possible local intrinsic connections in the AEG. The results from injections involving SIV showed reciprocal connections with area 5, the suprasylvian fringe, insula, dorsolateral orbital area, and area 6. The densest connections for SIV were with area 5. No topography was noted in the connections for SIV.

Connections between the thalamus and the somatosensory areas of the anterior ectosylvian gyrus in the cat.

The thalamocortical and corticothalamic connections of the second somatic sensory area (SII) and adjacent cortical areas in the cat were studied with anterograde and retrograde tracers. Injections consisted of horseradish peroxidase conjugated to wheat germ agglutinin (HRP-WGA) or a mixture of equal parts of tritiated leucine and proline. The cortical regions to be injected were electrophysiologically studied with microelectrodes to determine the localization of the selected components of the body representation in SII. The distribution of recording points was correlated in each case with the extent of the injection mass in the cortex. Distributions of retrograde and anterograde labeling in the thalamus were reconstructed from serial coronal sections. The results from cases with injections of tracers exclusively confined to separate parts of the body map in SII indicated a fairly precise topographical organization of projections from the ventrobasal complex (VB) to SII. The labeled cells and fibers were located within a series of lamella-like rods that curved throughout the dorsoventral and rostrocaudal axis of VB. The position and extent of these lamellae shifted from medial and ventral, in the medial subdivision of ventral posterior lateral nucleus (VPLm) for radial forelimb digit zones of SII, to dorsal, posterior, and lateral, in the lateral subdivision of ventral posterior lateral nucleus (VPL1) for proximal leg and trunk regions in SII. For every injected area in SII the densest clustering of labeled cells and fibers was usually more posteriorly represented in VB. The distribution in these dense zones of labeling often extended through the central core of VB. SII projecting neurons were also consistently noted in the extreme rostral portion of the medial subdivision of the posterior nuclei (Pom) that lies dorsal to VB. Corticothalamic and thalamocortical connections for SII were entirely reciprocal. Injections of tracers into cortical areas surrounding SII labeled other parts of the posterior complex but failed to label any part of VB except when the injection mass also diffused into SII. Injections into the somatic sensory cortex located lateral to SII, within the lips and depth of the upper bank of the anterior ectosylvian sulcus (AES), heavily labeled the central and posterior portions of Pom. Substantial labeling was noted in the lateral (Pol) and intermediate (Poi) divisions of Po only when the injections involved some part of the auditory areas located immediately posterior to SII. This region included the anterior auditory area that occupies the most posterior part of the AEG and both banks of the immediately adjoining AES. The magnocellular nucleus of the medial geniculate (MGmc) was labeled only when some part of the auditory cortex was injected. The suprageniculate nucleus (SG) was labeled from the insula and lower bank of the AES. These results indicated that the medial (rostral and caudal Pom) and lateral components (Poi, Pol, MGmc) of the posterior complex have separate cortical projection zones to somatic sensory and auditory cortical regions, respectively. SIV and the lateral extent of area 5a located in the medial bank of the anterior suprasylvian sulcus sent projections to the deep layers of the superior colliculus and the ventrolateral periaqueductal gray. No cortico-tectal projections were seen from SII.

Glomus tumor treated by prostaglandin inhibition. Report of a case.

Glomus tumor is a very painful pericytal lesion of the arteriovenous anastomotic complex that controls circulation in a limb. Glomus tumor usually involves a digit. Prostaglandin inhibition may control the glomus tumor pain, but surgical removal is the cure. When the condition is discovered early, or when there is no gross evidence of tumor, thermography and localized anesthetic blocks are invaluable in arriving at the proper diagnosis.