Sclerosing pseudovascular rhabdomyosarcoma-immunohistochemical, ultrastructural, and genetic findings indicating a distinct subtype of rhabdomyosarcoma.

Sclerosing (pseudovascular) rhabdomyosarcoma in adults has been described as a rare variant of rhabdomyosarcoma characterized by extensive hyaline fibrosis and pseudovascular growth patterns. We describe another case of this rhabdomyosarcoma subtype including ultrastructural and genetic findings-the lesion presented in a 62-year-old male patient in the left lower leg. The tumor was located within the deep soft tissue with maximum diameter of 11.8 cm and skin ulceration. Ultrastructural analysis revealed irregularly distributed disorganized filaments without clear evidence of Z-bands and a richly collagenized matrix. Using comparative genomic hybridization, a sharply delineated loss of chromosomal region 10q22, loss of chromosome Y, and a gain of chromosome 18 (trisomy) were detected. Reciprocal translocations t(1;13) and t(2;13)(q35;q14) which are characteristic of alveolar rhabdomyosarcoma could be excluded. These findings, while showing a relation to other rhabdomyosarcoma subtypes, represent a relatively circumscribed genetic defect pattern in sclerosing (pseudovascular) rhabdomyosarcoma that is somewhat different from patterns described in most other rhabdomyosarcoma subtypes. Six months after tumor resection, the patient presented with metastatic disease. Further studies should concentrate on the identification of genes especially on chromosomal region 10q22 to elucidate more aspects in the pathogenesis of this rhabdomyosarcoma subtype.

Nucleotide binding and filament assembly of recombinant yeast septin complexes.

Septins are filament-forming GTPases involved in cytokinesis and cortical organization. In the yeast Saccharomyces cerevisiae, the septins encoded by CDC3, CDC10, CDC11, and CDC12 form a high-molecular-weight complex, localized at the cytoplasmic face of the plasma membrane in the mother-bud neck. While septin function at the cellular level is fairly well understood, progress on structure-function analysis of these proteins has been slow and limited by the lack of large amounts of pure complex. While monomeric septins form apparently non-native aggregates, stable recombinant complexes of two, three, or four yeast septins can be produced by co-expression from bi-cistronic vectors in E. coli. The septin polypeptides show various degrees of saturation with guanine nucleotides in different complexes. The binary core Cdc3p-Cdc12p complex contains no bound nucleotide. While ternary complexes are partially saturated and can bind extraneously added nucleotide with micromolar affinity, only the complete four-component septin complex is fully coordinated with tightly bound GDP/GTP after chromatographic purification. We show here that the nucleotide-binding sites of the septins show drastic changes on formation of higher oligomers. Although the binary core Cdc3p-Cdc12p complex does not form filaments, the ternary and quaternary complexes form bundles of paired filaments. In the case of ternary complexes, filament formation is stimulated by guanine nucleotide, but is not dependent on the presence or absence of the gamma-phosphate.

Different modes of sodium-D-glucose cotransporter-mediated D-glucose uptake regulation in Caco-2 cells.

We recently reported that a considerable amount of the sodium-d-glucose cotransporter SGLT1 present in Caco-2 cells, a model for human enterocytes, is located in intracellular compartments attached to microtubules. A similar distribution pattern was also observed in enterocytes in thin sections from human jejunum, highlighting the validity of the Caco-2 cell model. Fluorescent surface labeling of live Caco-2 cells revealed that the intracellular compartments containing SGLT1 were accessible by endocytosis. To elucidate the role of endosomal SGLT1 in the regulation of sodium-dependent d-glucose uptake into enterocytes, we compared SGLT1-mediated D-glucose uptake into Caco-2 cells with the subcellular distribution of SGLT1 after challenging the cells with different stimuli. Incubation (90 min) of Caco-2 cells with mastoparan (50 microM), a drug that enhances apical endocytosis, shifted a large amount of SGLT1 from the apical membrane to intracellular sites and significantly reduced sodium-dependent alpha-[(14)C]methyl-D-glucose uptake (-60%). We also investigated the effect of altered extracellular D-glucose levels. Cells preincubated (1 h) with d-glucose-free medium exhibited significantly higher sodium-dependent alpha-[(14)C]methyl-D-glucose uptake (+45%) than did cells preincubated with high d-glucose medium (100 mM, 1 h). Interestingly, regulation of SGLT1-mediated d-glucose uptake into Caco-2 cells by extracellular D-glucose levels occurred without redistribution of cellular SGLT1. These data suggest that, pharmacologically, d-glucose uptake can be regulated by a shift of SGLT1 between the plasma membrane and the endosomal pool; however, regulation by the physiological substrate d-glucose can be explained only by an alternative mechanism.

Murine Nr4a1 and Herpud1 are up-regulated by Wnt-1, but the homologous human genes are independent from beta-catenin activation.

The Wnt signal transduction pathway regulates morphogenesis and mitogenesis of cells in multicellular organisms. A major downstream consequence of Wnt-1 signalling is the activation of beta-catenin/T-cell factor (TCF)-mediated transcription. We compared Wnt-1-transformed murine mammary epithelial cells with control cells by subtractive hybridization. We found the two genes Nr4a1 and Herpud1 to be overexpressed in Wnt-1-transformed cells. Remarkably, the transcription levels of the two homologous human genes NR4A1 and HERPUD1 are neither activated in cells with activated beta-catenin/TCF-mediated transcription nor can be induced by beta-catenin transfection. These results indicate different regulation mechanisms of the two genes in murine and human cells.

Inhibition of Na(+)-dependent transporters in cystine-loaded human renal cells: electrophysiological studies on the Fanconi syndrome of cystinosis.

Cystinosis is the most common cause of the renal Fanconi syndrome in children, leading to severe electrolyte disturbances and growth failure. A defective lysosomal transporter, cystinosin, results in intralysosomal accumulation of cystine. Loading cells with cystine dimethyl ester (CDME) is the only available model for this disease. This model was used to present electrophysiologic studies on immortalized human kidney epithelial (IHKE-1) cells that had been derived from the proximal tubule with the slow whole-cell patch clamp technique. Basal membrane voltages (V(m)) of IHKE-1 cells were -30.7 +/- 0.4 mV (n = 151). CDME concentration-dependently altered V(m) with an initial depolarization (2.7 +/- 0.2 mV;n = 76; 1 mM CDME) followed by a more pronounced hyperpolarization (-9.9 +/- 1.0 mV;n = 49). Three Na(+)-dependent transporters were examined. Alanine (1 mM) depolarized IHKE-1 cells by 17.6 +/- 0.7 mV (n = 59), and phosphate (1.8 mM) depolarized by 9.7 +/- 1.1 mV (n = 18). Acidification of IHKE-1 cells with propionate (20 mM) resulted in a depolarization of V(m) by 7.1 +/- 0.3 mV (n = 21) followed by a repolarization by 2.9 +/- 0.3 mV/min (n = 17), reflecting Na(+)/H(+)-exchanger activity. Acute addition of 1 mM CDME did not alter the alanine- and propionate-induced changes in V(m), but it reduced the phosphate-induced depolarization by 37 +/- 9% (n = 10). Incubation with 1 mM CDME reduced the activity of all three transporters. Depolarizations by alanine and phosphate and the repolarization after propionate were inhibited by 57 +/- 4% (n =30), 45 +/- 9% (n = 9), and 78 +/- 15% (n = 8), respectively. In conclusion, this study demonstrates that CDME acutely alters V(m) of IHKE-1 cells and that at least three Na(+)-dependent transporters are inhibited, the Na(+)-phosphate cotransporter most sensitively. This might suggest that phosphate depletion and dissipation of the Na(+)-gradient are involved in the development of the Fanconi syndrome of cystinosis.

Pigmented (melanotic) neurofibroma. Report of an unusual case with immunohistochemical, ultrastructural and cytogenetic analyses.

In the spectrum of neurofibromas, pigmented tumors are rare variants usually showing only faint, macroscopically obvious pigmentation. We report a case of a huge pigmented neurofibroma with extended, macroscopically striking pigmentation in a patient with stigmata of neurofibromatosis. The immunohistochemical and ultrastructural findings support a melanotic line of differentiation besides schwann cell differentiation and indicate a phenotypic neoplastic spectrum between tumorous schwann cells and melanocytes. Using comparative genomic hybridization, striking chromosomal aberrations were not detected. High level amplifications of the known chromosomal regions, including genes of major enzymes responsible for melanin synthesis, appear to be unlikely. However, smaller chromosomal defects might have been overlooked by the limited resolution of this screening method. Therefore, other mechanisms up-regulating melanogenesis, such as mutations in regulatory genes, have to be considered.

The new sulindac derivative IND 12 reverses Ras-induced cell transformation.

The nonsteroidal anti-inflammatory drug Sulindac has chemopreventive and antitumorigenic properties. Its metabolites induce apoptosis and inhibit signaling pathways critical for malignant transformation, including the Ras pathway. Here we show that the new Sulindac derivative IND 12 reverses the phenotype of Ras-transformed MDCK-f3 cells and restores an untransformed epithelioid morphology characterized by growth in monolayers with regular cell-cell adhesions. Moreover, IND 12 treatment induces the expression at membranes of the cell adhesion protein E-cadherin and increases the level of the E-cadherin-bound beta-catenin. As a consequence, IND 12-treated MDCK-f3 cells lose their invasion capacity and regain the ability to aggregate. In the presence of IND 12, MDCK-f3 cells show regenerated expression and activity ratios of the small GTPases Rac and Rho normally found in untransformed MDCK cells. Strikingly, IND 12 treatment decreases the levels of phosphorylated mitogen-activated protein kinases, which are downstream substrates of the Ras-regulated Raf/mitogen-activated protein kinase pathway, and the level of Ras-induced activation of gene expression. Our findings identify a novel drug with high potential in cancer therapy by targeting Ras-induced cell transformation.

A non-radioactive protein truncation test for the sensitive detection of all stop and frameshift mutations.

A new method for mutation detection is described, which is a technical advancement of the protein truncation test. The new technique is non-radioactive and highly sensitive for detection of virtually all sequence mutations, which lead to a stop signal or to the shift of the translation frame. The method includes four steps: 1) capture of the interesting sequence copies out of the sample by binding to an immobilized complementary sequence, 2) PCR amplification of the gene fragment to be analyzed with primers coding both for amino- and carboxy-terminal tags, 3) in vitro transcription and translation, and 4) analysis of the translation products by Western blot. As an evaluation of the new method, we detected mutated gene copies at a dilution of 1 to 40 compared to the non-mutated gene. Using the method, we were able to detect a mutation in the adenomatous polyposis coli tumor suppressor gene (APC) in a stool sample of a colorectal cancer patient. This mutation could not be detected by direct sequencing of the amplified APC gene fragment.

The activation of RalGDS can be achieved independently of its Ras binding domain. Implications of an activation mechanism in Ras effector specificity and signal distribution.

Small GTPases of the Ras family are major players of signal transduction in eukaryotic cells. They receive signals from a number of receptors and transmit them to a variety of effectors. The distribution of signals to different effector molecules allows for the generation of opposing effects like proliferation and differentiation. To understand the specificity of Ras signaling, we investigated the activation of RalGDS, one of the Ras effector proteins with guanine-nucleotide exchange factor activity for Ral. We determined the GTP level on RalA and showed that the highly conserved Ras binding domain (RBD) of RalGDS, which mediates association with Ras, is important but not sufficient to explain the stimulation of the exchange factor. Although a point mutation in the RBD of RalGDS, which abrogates binding to Ras, renders RalGDS independent to activated Ras, an artificially membrane-targeted version of RalGDS lacking its RBD could still be activated by Ras. The switch II region of Ras is involved in the activation, because the mutant Y64W in this region is impaired in the RalGDS activation. Furthermore, it is shown that Rap1, which was originally identified as a Ras antagonist, can block Ras-mediated RalGDS signaling only when RalGDS contains an intact RBD. In addition, kinetic studies of the complex formation between RalGDS-RBD and Ras suggest that the fast association between RalGDS and Ras, which is analogous to the Ras/Raf case, achieves signaling specificity. Conversely, the Ras x RalGDS complex has a short lifetime of 0.1 s and Rap1 forms a long-lived complex with RalGDS, possibly explaining its antagonistic effect on Ras.