Serious gastric perforation after second stereotactic body radiotherapy for peripheral lung cancer that recurred after initial stereotactic body radiotherapy: a case report

Background

In recent reports, re-irradiation with stereotactic body radiotherapy for lung tumors in patients previously treated with thoracic radiation therapy resulted in several serious toxicities. Serious non-lung toxicities were observed mostly in patients with central tumors, but we experienced a case of fatal gastric perforation after a second stereotactic body radiotherapy in a patient with a peripheral lung tumor.

Case presentation

An 83-year-old Asian man was diagnosed with T2N0M0 lung cancer in the form of squamous cell carcinoma in the lower lobe of his left lung. He was treated with stereotactic body radiotherapy of 40 Gy in 4 fractions and the tumor decreased in size in partial response. The local tumor recurred 8 months after the first stereotactic body radiotherapy, and he was re-irradiated with a second stereotactic body radiotherapy of 50 Gy in 4 fractions. A Sengstaken–Blakemore tube was inserted below his diaphragm by laparoscopic surgery before the second stereotactic body radiotherapy in order to reduce the stomach dose by keeping his stomach apart from the tumor. Two months after the second stereotactic body radiotherapy, he developed fatal gastric perforation and gastropleural fistula penetrating his diaphragm.

Conclusions

To the best of our knowledge, this is the first report about a gastric perforation after stereotactic body radiotherapy for lung tumors and it warns of serious complication of stereotactic body radiotherapy in not only centrally located but also peripherally located tumors like in this case.

     

Expression and distribution of Gpr119 in the pancreatic islets of mice and rats: predominant localization in pancreatic polypeptide-secreting PP-cells

The GPR119 was recently shown to be activated by oleoylethanolamide (OEA), a naturally occurring bioactive lipid with hypophagic and anti-obesity effects. In this study, we have cloned and characterized its murine counterpart, Gpr119. The full-length cDNA contained an open reading frame of 1008bp encoding a 335-amino acid protein. The genomic organization of Gpr119 was unique, having a 3'-untranslated second exon that was also involved in an alternative splicing event. Gene expression analyses confirmed its specific expressions in pancreatic islets and two endocrine cell-lines, MIN6 and alphaTC1. Immunohistochemistry and double-immunofluorescence studies using a specific antibody revealed the predominant Gpr119 localization in pancreatic polypeptide (PP)-cells of islets. No definitive evidence of Gpr119-immunoreactivity in adult beta- or alpha-cells was obtained. The Gpr119 mRNA levels were elevated in islets of obese hyperglycemic db/db mice as compared to control islets, suggesting a possible involvement of this receptor in the development of obesity and diabetes.     

Suppression of metastases of small cell lung cancer cells in mice by a peptidic CXCR4 inhibitor TF14016

CXCL12 is a chemokine essential for the organ-specific spread of a variety of cancers including small cell lung cancer (SCLC). Here, we examined the anti-metastatic efficacy of TF14016, a small peptidic inhibitor of CXCL12 receptor CXCR4, in SCLC. Treatment of mice with TF14016 significantly suppressed pulmonary metastases of CXCR4-expressing SCLC in size and number. Furthermore, histological examination revealed that the expression of vascular endothelial cell growth factor and the density of CD31-positive microvessels in metastatic foci were both significantly reduced in TF14016-treated mice. Collectively, CXCR4 could be an attractive target for anti-metastatic and anti-angiogenic therapy in SCLC.     

The specific expression of tenascin in the synovial membrane of the temporomandibular joint with internal derangement: An immunohistochemical study

 The expression of tenascin (TN) in the temporomandibular joint (TMJ) disc and synovial membrane was examined in 18 human TMJ samples from patients with internal derangement of the TMJ and ten control specimens by an immunohistological technique using paraffin-embedded tissue and specific anti-human TN monoclonal antibody (RCB-1). The expression of TN was observed in all 28 samples, but it was limited to the walls of blood vessels, the perineurium, and the surface of the TMJ disc. The expression of TN was diffuse in the stroma of mildly hypertrophic synovial membranes and focal in the surface of severely hypertrophic synovial membranes. The clinical symptoms of internal derangement of the TMJ are thought to be related to the degree of synovitis. The present study demonstrates that TN is expressed specifically in the portion of the TMJ synovial membrane affected with internal derangement. Accepted: 17 December 1996     

Characterization of PXK as a Protein Involved in Epidermal Growth Factor Receptor Trafficking

The phox homology (PX) domain is a phosphoinositide-binding module that typically binds phosphatidylinositol 3-phosphate. Out of 47 mammalian proteins containing PX domains, more than 30 are denoted sorting nexins and several of these have been implicated in internalization of cell surface proteins to the endosome, where phosphatidylinositol-3-phosphate is concentrated. Here we investigated a multimodular protein termed PXK, composed of a PX domain, a protein kinase-like domain, and a WASP homology 2 domain. We show that the PX domain of PXK localizes this protein to the endosomal membrane via binding to phosphatidylinositol 3-phosphate. PXK expression in COS7 cells accelerated the ligand-induced internalization and degradation of epidermal growth factor receptors by a mechanism requiring phosphatidylinositol 3-phosphate binding but not involving the WASP homology 2 domain. Conversely, depletion of PXK using RNA interference decreased the rate of epidermal growth factor receptor internalization and degradation. Ubiquitination of epidermal growth factor receptor by the ligand stimulation was enhanced in PXK-expressing cells. These results indicate that PXK plays a critical role in epidermal growth factor receptor trafficking through modulating ligand-induced ubiquitination of the receptor.Both constitutive endocytosis and activated endocytosis are highly regulated events by which cells take up nutrients and internalize receptors for recycling or degradation (47). Endocytosed molecules are delivered to early endosomes, where the components are sorted to the cell surface for recycling back to the plasma membrane, or to late endosomes to be degraded in lysosomes (17). The molecular mechanisms regulating these events are not fully understood.One of the major protein families involved in the trafficking of membrane compartments is sorting nexins (SNXs), which are characterized by the presence of phox homology (PX) domains (8, 65). The PX domain is a protein module which consists of approximately 130 amino acids with three β-strands followed by three α-helices forming a helical subdomain, and the general function of this module is to interact with the head groups of inositol phospholipids through which parental proteins are targeted to specific cellular compartments. Most of the SNXs examined to date specifically recognize phosphatidylinositol 3-phosphate [PtdIns(3)P], which is found predominately in early endosomes (11). The founding member of the SNX family, SNX1, was initially identified as an interaction partner of epidermal growth factor receptor (EGFR), and the expression of SNX1 enhanced lysosomal degradation of EGFR (38); therefore, SNXs are most likely to be involved in the trafficking of many different families of receptors which are recycled to the cell surface or sent to the lysosome for degradation (19). On the other hand, PX domain-containing proteins have also been reported to bind to phosphoinositides other than PtdIns(3)P and to have functions independent of receptor trafficking (54). For example, phospholipase D is a PX domain-containing protein that hydrolyzes phosphatidylcholine to produce a second-messenger molecule, phosphatidic acid. Interestingly, phospholipase D has been recently shown to accelerate EGFR endocytosis by activating dynamin GTPase through its PX domain but independently of lipase activity (39). Cytokine-independent survival kinase (CISK) is a PX domain-containing protein kinase that has also been shown to regulate sorting of a chemokine receptor CXCR4 through AIP4, the CXCR4 ubiquitin ligase (60). RGS-PX1, a GTPase-activating protein for Gαs of heterotrimeric GTP-binding proteins, and KIF16B, a PX domain-containing kinesin superfamily member, have been shown to regulate EGFR trafficking (27, 72) and are now grouped into the SNX family as SNX13 and SNX26, respectively.Another feature of the PX domain is a well-conserved polyproline sequence (PXXP) in the variable loop between α1 and α2 helices, which led to the original identification of the PX domain as a SH3 domain-binding partner (53). The physiological importance of both intermolecular and intramolecular interactions mediated by polyproline sequences has been shown in various molecules, including phospholipase D2 (33) and p47^phox (1). In mammals, there are currently 47 proteins harboring PX domains, and 30 proteins are termed SNXs (59). The functions of these proteins have just begun to be revealed.Actin cytoskeletal dynamics have been implicated not only in cell motility and cytokinesis but also in endocytic processes, although the necessity and role in endocytosis in higher eukaryotic cells remain ambiguous (12, 34, 35, 55). The WASP homology 2 (WH2) domain is known as an actin-binding motif found in regulators of the actin cytoskeleton, including Wiskott-Aldrich syndrome protein (WASP), Scar/WASP-family verprolin-homologous protein (WAVE), verprolin/WASP-interacting protein (WIP), missing in metastasis (MIM), and β-thymosins (52). Some proteins with WH2 domains, such as β-thymosin, prevent actin filament assembly by sequestering actin monomers, while others, such as N-WASP and the Drosophila protein Ciboulot participate in barbed-end actin assembly (52). Recently, the structural basis for these opposite functions of WH2 domains was demonstrated; the interaction of the C-terminal region of β-thymosin/WH2 domain with the pointed end of the actin monomer accounts for the switch in function from inhibition to promotion of actin assembly (26). WH2 domains exist in almost 20 proteins, whose functions remain to be clarified.In the present study, we isolated a new multimodular protein (termed PXK), conserved in multicellular organisms including humans through flies, which possesses a PX domain, a protein kinase-like domain, and a WH2 domain. We show that the PX and WH2 domains function as PtdIns(3)P and actin-binding domains, respectively. PXK expression in COS cells accelerated ligand-induced EGFR endocytosis and degradation that was dependent on a functional PX domain but independent of the WH2 domain. PXK also enhanced ubiquitination of EGFR induced by EGF stimulation in these cells. Based on these results, we propose that PXK is a functional sorting nexin that may play an additional role in cellular function via its interaction with the actin cytoskeleton.     

Effects of voltage-gated calcium channel subunit genes on calcium influx in cultured C. elegans mechanosensory neurons

Voltage-gated calcium channels (VGCCs) serve as a critical link between electrical signaling and diverse cellular processes in neurons. We have exploited recent advances in genetically encoded calcium sensors and in culture techniques to investigate how the VGCC alpha1 subunit EGL-19 and alpha2/delta subunit UNC-36 affect the functional properties of C. elegans mechanosensory neurons. Using the protein-based optical indicator cameleon, we recorded calcium transients from cultured mechanosensory neurons in response to transient depolarization. We observed that in these cultured cells, calcium transients induced by extracellular potassium were significantly reduced by a reduction-of-function mutation in egl-19 and significantly reduced by L-type calcium channel inhibitors; thus, a main source of touch neuron calcium transients appeared to be influx of extracellular calcium through L-type channels. Transients did not depend directly on intracellular calcium stores, although a store-independent 2-APB and gadolinium-sensitive calcium flux was detected. The transients were also significantly reduced by mutations in unc-36, which encodes the main neuronal alpha2/delta subunit in C. elegans. Interestingly, while egl-19 mutations resulted in similar reductions in calcium influx at all stimulus strengths, unc-36 mutations preferentially affected responses to smaller depolarizations. These experiments suggest a central role for EGL-19 and UNC-36 in excitability and functional activity of the mechanosensory neurons.     

Molecular pathogenesis of malignant melanoma: a different perspective from the studies of melanocytic nevus and acral melanoma

The Clark model for melanoma progression emphasizes a series of histopathological changes beginning from benign melanocytic nevus to melanoma via dysplastic nevus. Several models of the genetic basis of melanoma development and progression are based on this Clark’s multi-step model, and predict that the acquisition of a BRAF mutation can be a founder event in melanocytic neoplasia. However, our recent investigations have challenged this view, showing the polyclonality of BRAF mutations in melanocytic nevi. Furthermore, it is suggested that many melanomas, including acral and mucosal melanomas, arise de novo, not from melanocytic nevus. While mutations of the BRAF gene are frequent in melanomas on non-chronic sun damaged skin which are prevalent in Caucasians, acral and mucosal melanomas harbor mutations of the KIT gene as well as the amplifications of cyclin D1 or cyclin-dependent kinase 4 gene. Amplifications of the cyclin D1 gene are detected in normal-looking ‘field melanocytes’, which represent a latent progression phase of acral melanoma that precedes the stage of atypical melanocyte proliferation in the epidermis. Based on these observations, we propose an alternative genetic progression model for melanoma.     

A Decreased Level of Serum Soluble Klotho Is an Independent Biomarker Associated with Arterial Stiffness in Patients with Chronic Kidney Disease

Background

Klotho was originally identified in a mutant mouse strain unable to express the gene that consequently showed shortened life spans. In humans, low serum Klotho levels are related to the prevalence of cardiovascular diseases in community-dwelling adults. However, it is unclear whether the serum Klotho levels are associated with signs of vascular dysfunction such as arterial stiffness, a major determinant of prognosis, in human subjects with chronic kidney disease (CKD).

Methods

We determined the levels of serum soluble Klotho in 114 patients with CKD using ELISA and investigated the relationship between the level of Klotho and markers of CKD-mineral and bone disorder (CKD-MBD) and various types of vascular dysfunction, including flow-mediated dilatation, a marker of endothelial dysfunction, ankle-brachial pulse wave velocity (baPWV), a marker of arterial stiffness, intima-media thickness (IMT), a marker of atherosclerosis, and the aortic calcification index (ACI), a marker of vascular calcification.

Results

The serum Klotho level significantly correlated with the 1,25-dihydroxyvitamin D level and inversely correlated with the parathyroid hormone level and the fractional excretion of phosphate. There were significant decreases in serum Klotho in patients with arterial stiffness defined as baPWV≥1400 cm/sec, atherosclerosis defined as maximum IMT≥1.1 mm and vascular calcification scores of ACI>0%. The serum Klotho level was a significant determinant of arterial stiffness, but not endothelial dysfunction, atherosclerosis or vascular calcification, in the multivariate analysis in either metabolic model, the CKD model or the CKD-MBD model. The adjusted odds ratio of serum Klotho for the baPWV was 0.60 (p = 0.0075).

Conclusions

Decreases in the serum soluble Klotho levels are independently associated with signs of vascular dysfunction such as arterial stiffness in patients with CKD. Further research exploring whether therapeutic approaches to maintain or elevate the Klotho level could improve arterial stiffness in CKD patients is warranted.     

Skewed X-chromosome inactivation causes intra-familial phenotypic variation of an EBP mutation in a family with X-linked dominant chondrodysplasia punctata

X-linked dominant chondrodysplasia punctata (CDPX2) is a skeletal dysplasia characterized by stippled epiphyses, cataracts, alopecia and skin lesions, including ichthyosis. CDPX2 exhibits a number of perplexing clinical features, such as intra- and inter-familial variation, anticipation, incomplete penetrance and possible gonadal and somatic mosaicism. Recently, mutations in the gene encoding Delta8,Delta7 sterol isomerase/emopamil-binding protein (EBP) have been identified in CDPX2. To better understand the genetics of CDPX2, we examined the entire EBP gene by direct sequencing in four CDPX2 patients. We found EBP mutations in all four patients, including three novel mutations: IVS3+1G>A, Y165C and W82C. Surprisingly, a known mutation (R147H) was identified in a patient and her clinically unaffected mother. Expression analysis revealed the mutant allele was predominantly expressed in the patient, while both alleles were expressed in the mother. Methylation analysis revealed that the wild-type allele was predominantly inactivated in the patient, while the mutated allele was predominantly inactivated in her mother. Thus, differences in expression of the mutated allele caused by skewed X-chromosome inactivation produced the diverse phenotypes within the family. Our findings could explain some of the perplexing features of CDPX2. The possibility that an apparently normal parent is a carrier should be considered when examining seemingly sporadic cases and providing genetic counseling to CDPX2 families.     

Modulation of functional properties of KCNQ1 channel by association of KCNE1 and KCNE2

The KCNE proteins (KCNE1 through KCNE5) function as beta-subunits of several voltage-gated K(+) channels. Assembly of KCNQ1 K(+) channel alpha-subunits and KCNE1 underlies cardiac I(Ks), while KCNQ1 interacts with all other members of KCNE forming complexes with different properties. Here we investigated synergic actions of KCNE1 and KCNE2 on functional properties of KCNQ1 heterologously expressed in COS7 cells. Patch-clamp recordings from cells expressing KCNQ1 and KCNE1 exhibited the slowly activating current, while co-expression of KCNQ1 with KCNE2 produced a practically time-independent current. When KCNQ1 was co-expressed with both of KCNE1 and KCNE2, the membrane current exhibited a voltage- and time-dependent current whose characteristics differed substantially from those of the KCNQ1/KCNE1 current. The KCNQ1/KCNE1/KCNE2 current had a more depolarized activation voltage, a faster deactivation kinetics, and a less sensitivity to activation by mefenamic acid. These results suggest that KCNE2 can functionally couple to KCNQ1 even in the presence of KCNE1.