Co-active receptor tyrosine kinases mitigate the effect of FGFR inhibitors in FGFR1-amplified lung cancers with low FGFR1 protein expression.

Targeted therapies are effective in subsets of lung cancers with EGFR mutations and anaplastic lymphoma kinase (ALK) translocations. Large-scale genomics have recently expanded the lung cancer landscape with FGFR1 amplification found in 10-20% of squamous cell carcinomas (SCCs). However, the response rates have been low for biomarker-directed fibroblast growth factor receptor (FGFR) inhibitor therapy in SCC, which contrasts to the relatively high rates of response seen in EGFR mutant and ALK-translocated lung cancers treated with epidermal growth factor receptor (EGFR) inhibitors and ALK inhibitors, respectively. In order to better understand the low response rates of FGFR1-amplified lung cancers to FGFR inhibitors, relationships between gene copy number, mRNA expression and protein expression of FGFR1 were assessed in cell lines, tumor specimens and data from The Cancer Genome Atlas. The importance of these factors for the sensitivity to FGFR inhibitors was determined by analyzing drug screen data and conducting in vitro and in vivo experiments. We report that there was a discrepancy between FGFR1 amplification level and FGFR1 protein expression in a number of these cell lines, and the cancers with unexpectedly low FGFR1 expression were uniformly resistant to the different FGFR inhibitors. Further interrogation of the receptor tyrosine kinase activity in these discordant cell lines revealed co-activation of HER2 and platelet-derived growth factor receptor-α (PDGFRα) caused by gene amplification or ligand overexpression maintained phosphoinositide 3-kinase (PI3K) and MEK/ERK signaling even in the presence of FGFR inhibitor. Accordingly, co-inhibition of FGFR1 and HER2 or PDGFRα led to enhanced drug responses. In contrast, FGFR1-amplified high FGFR1 protein-expressing lung cancers are sensitive to FGFR inhibitor monotherapy by downregulating ERK signaling. Addition of a PI3K inhibitor to these high FGFR1 protein-expressing cancers further sensitized them to FGFR inhibitor. These data reveal that biomarker-directed trials for FGFR1-amplified SCC require assessment of FGFR1 protein expression and uncover novel therapeutic strategies for FGFR1-amplified SCC with low FGFR1 protein expression.

Increased plasma LIGHT levels in patients with atopic dermatitis.

LIGHT [the name of which is derived from 'homologous to lymphotoxins, exhibits inducible expression, competes with herpes simplex virus glycoprotein D for herpes simplex virus entry mediator (HVEM), and expressed by T lymphocytes'], is a member of the tumour necrosis factor superfamily that is involved in various inflammatory diseases. We aimed to estimate the relevance of plasma LIGHT levels as a biomarker for atopic dermatitis (AD). In order to understand the putative role of LIGHT in AD pathogenesis, we also investigate the effects of LIGHT on a monocytic cell line, human acute monocytic leukaemia cell line (THP-1). We examined plasma LIGHT levels, total serum IgE, serum value of CCL17 and peripheral blood eosinophil counts in patients with AD and healthy subjects. The effects of LIGHT on activation and apoptosis in THP-1 cells were also investigated. The plasma concentrations of LIGHT in AD patients were significantly higher than those in healthy individuals and the concentrations decreased as the symptoms were improved by treatment. The LIGHT plasma concentrations correlated with IgE levels and the Severity Scoring of AD (SCORAD) index. In addition, LIGHT stimulation increased expression of CD86 and induced production of interleukin-1ß in THP-1 cells. Apoptosis was inhibited, the Bcl-2 level increased and the caspase-3 level decreased in THP-1 cells stimulated with LIGHT, compared to unstimulated control cells. These results suggest that plasma LIGHT levels may be one of the promising biomarkers for AD.

Influence of leukotriene pathway polymorphisms on clinical responses to montelukast in Japanese patients with asthma.

WHAT IS KNOWN AND OBJECTIVE: Montelukast, a cysteinyl leukotriene receptor 1 antagonist, is safe and efficacious in patients with asthma. The mechanisms underlying the significant interpatient variability in response to montelukast are not clear but are believed to be, in part, because of genetic variability. METHODS: To examine the associations between polymorphisms in candidate genes in the leukotriene pathway and outcomes in patients with asthma on montelukast for 4-8 weeks, we evaluated the changes in peak expiratory flow (PEF), forced expiratory volume in 1 s (FEV(1·0) ) and patients' subjective symptom before and after montelukast treatment. DNA was collected from 252 Japanese participants. RESULTS AND DISCUSSION: Two single-nucleotide polymorphisms (SNPs) in the ALOX5 (rs2115819) and LTA4H (rs2660845) genes were successfully typed. There was no difference between members of the general population (n = 200) and patients (n = 52) in each genotype frequency. Significant associations were found between SNP genotypes in the LTA4H gene and changes in PEF and FEV(1·0) . The PEF and FEV(1·0) responses to montelukast in the A/A genotypes (n = 4) for the LTA4H SNP were significantly higher than those in the G allele carriers (A/G+G/G) (n = 17). WHAT IS NEW AND CONCLUSION: Despite the small sample size, our results suggest that genetic variation in leukotriene pathway candidate genes contributes to variability in clinical responses to montelukast in Japanese patients with asthma.

Gene expression-based pharmacodynamic biomarkers: the beginning of a new era in biomarker-driven anti-tumor drug development.

Pharmacodynamic (PD) biomarkers play a pivotal role in anti-tumor drug development as a biochemical measurement to estimate the level of drug interaction with the target, or to assess the downstream impact of its interaction with the target. Although immunohistochemistry (IHC)-based protein biomarkers have been conventionally used as PD biomarkers, gene expression-based PD biomarkers have recently emerged as quantitative biomarkers. This review introduces examples of gene expression-based mRNA biomarkers that have been validated in preclinical or clinical studies of several anti-tumor agents including HDAC, mTOR, and B-RAF inhibitors. The measurement of PD biomarker levels in tumors has proven to be ideal; however, in clinical studies, easily accessible surrogate tissues have been used for analysis. In the present review, we also discuss the advantages and disadvantages in using surrogate tissues, such as peripheral blood mononuclear cells (PBMCs), skin tissue, and circulating tumor cells, in the assessment of PD biomarkers. PD biomarkers are generally classified into two categories: 1) target engagement biomarkers and 2) early efficacy biomarkers. This classification depends on their respective distance from target intervention. The strategies used to identify and distinguish between these two types of PD biomarkers via expression profiling are also discussed. Finally, we propose two novel approaches for PD marker identification. One approach utilizes mRNA expression profiling of tumors prior to drug treatment rather than post-treatment samples. The second method involves the application of microRNA expression profiles to determine PD effects. In conclusion, the recent advances in mRNA and microRNA profiling and the identification of gene expression-based PD biomarkers may aid investigators to drive drug development through the establishment of quantitative PD effects.

Renal complications in two patients with dentatorubral-pallidoluysian atrophy.

Dentatorubral-pallidoluysian atrophy (DRPLA) is an autosomal dominant neurodegenerative disorder characterized by various combinations of myoclonus epilepsy, ataxia, choreoathetosis and dementia. No specific therapy has been established and renal complication is rare. We report two cases of DRPLA with renal complications. Hematuria and proteinuria had gradually progressed for 2 and 13 years in these patients. Renal biopsy findings revealed focal glomerulosclerosis in one case and end-stage kidney disease in the other case. Angiotensin-converting enzyme inhibitor and angiotensin receptor II antagonist were administered to both patients, resulting in improved proteinuria and preserved renal function in one patient, while renal function continued to deteriorate in the other patient. Although renal complication is rare in patients with DRPLA, the presence of renal disease has to be suspected in patients with persistent proteinuria.

RB silencing compromises the DNA damage-induced G2/M checkpoint and causes deregulated expression of the ECT2 oncogene.

As alterations in retinoblastoma (RB)/E2F pathway are commonly found in human cancers, the molecular mechanism underlying cell cycle deregulation caused by the mutations in the RB/E2F pathway needs to be investigated extensively. Compared with good understanding of RB/E2F functions in G1-S cell cycle progression, it is not fully understood how an abrogated RB pathway affects the G2-M phase of the cell cycle. Here, we report that disruption of RB accelerated G2-M progression in the presence of DNA damage by elevating the expression of a set of mitotic regulatory genes. We generated RB(+)- and (-)-matched cells using short hairpin RNA. In the RB(-) cells, the G2/M checkpoint mediated by a DNA-damaging agent was over-ridden. With microarray analysis, we found that the expression of key G2-M regulatory genes was upregulated in RB(-) cells. In particular, we demonstrated that the proto-oncogene ECT2 was directly regulated by E2Fs. Furthermore, suppression of ECT2 expression by small interfering RNA in RB(-) cells resulted in cytokinesis arrest, suggesting that RB(-) cells lack the regulation of E2F-mediated cytokinesis. These results indicate that aberrant ECT2 expression, observed in various human tumors, could be the direct result of RB/E2F pathway deficiency, thereby contributing to cell division in cancers.

Effect of serotonin on the differentiation of human monocytes into dendritic cells.

The local cytokine environment and presence of stimulatory signals determine whether monocytes acquire dendritic cell (DC) or macrophage characteristics and functions. Because enhanced platelet activation is reported in patients with many allergic disorders, such as atopic dermatitis, platelet-derived factors may influence monocytic differentiation into DC. In this study we examined the effect of serotonin, a prototypic mediator of allergic inflammation released mainly by activated platelets at the inflammatory site, on the granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4-driven differentiation of monocytes into monocyte-derived DC. Monocytes from healthy adult donors were cultured with GM-CSF and IL-4 in the presence or absence of serotonin, and the phenotypes and function of these cells were analysed. In the presence of serotonin, monocytes differentiated into DC with reduced expression of co-stimulatory molecules and CD1a, whereas expression of CD14 was increased. These serotonin-treated DC exhibited significantly reduced stimulatory activity toward allogeneic T cells. However, these cells showed enhanced cytokine-producing capacity, including IL-10 but not IL-12. There was no significant difference between both types of DC in phagocytic activity. Experiments using agonists and antagonists indicated that serotonin 5-hydroxytryptamine (5-HT) induced the alteration of their phenotype and reduction in antigen-presenting capacity were mediated via 5-HTR(1/7). It is therefore suggested that serotonin-driven DC may have a regulatory function in the inflammatory process.

Identification of breast cancer resistant protein/mitoxantrone resistance/placenta-specific, ATP-binding cassette transporter as a transporter of NB-506 and J-107088, topoisomerase I inhibitors with an indolocarbazole structure.

The antitumor drugs NB-506 and J-107088 are potent topoisomerase I inhibitors with an indolocarbazole structure. To clarify the factors involved in resistance to these drugs, we established two NB-506-resistant mouse fibroblast cell lines (LY/NR1 and LY/NR2), a human colon carcinoma cell line (HCT116/NR1), and a lung cancer cell line (PC13/NR1). These cell lines were highly resistant to NB-506 and J-107088, and LY/NR2 cells showed markedly reduced accumulation and strong efflux of NB-506, suggesting activation of a drug efflux pump in the resistant cells. To identify the molecules responsible for efflux of NB-506, we compared the gene expressions of the mouse resistant LY/NR1 cells, LY/NR2 cells, and their parental cells by oligonucleotide microarray. Of 34,020 genes analyzed, we found that an ATP-binding cassette transporter BCRP/MXR/ABCP (BCRP) gene showed the highest increase in the expression, 31-fold higher in the LY/NR2-resistant cells than in their parental cells. The selective overexpression of this gene was also detected in the two human resistant cell lines, suggesting the involvement of breast cancer resistant protein (BCRP) in the resistance and efflux of these drugs. Finally, a PC-13 cell line transfected with BCRP expression vector displayed 22- and 17-fold resistance to NB-506 and J-107088 and enhanced efflux activity of J-107088. However, the transfectants were not resistant to mitoxantrone or topotecan, the drugs previously thought to be the substrates of BCRP. Thus, our study presents a novel mechanism of drug resistance mediated by BCRP.

Recurrent dislocation of tibialis posterior tendon. A report of two cases.

We successfully treated two patients with recurrent dislocation of the tibialis posterior tendon by creating a bone block. Sudden resistive contraction of the tibialis posterior muscle is considered to be the mechanical cause of the initial traumatic injury, and a shallow tibialis posterior tendon sulcus may be the predisposing factor. Once the flexor retinaculum is torn during the initial trauma, recurrent dislocation is inevitable, and surgical treatment is mandatory. When treating patients with a complaint of long-standing pain around the medial malleolus, we must bear in mind the possible diagnosis of recurrent dislocation of the tibialis posterior tendon. If the patient can voluntarily dislocate the tendon by active plantar flexion and inversion of the ankle, the diagnosis is definitive.

The delta isoform of protein phosphatase type 1 is localized in nucleolus and dephosphorylates nucleolar phosphoproteins.

The immunolocalization and substrates of protein phosphatases present in nucleolus were investigated using Swiss 3T3 cells and Novikoff hepatoma ascites cells. The protein phosphatase activity was detected in the extract of the isolated nucleoli and its activity was inhibited by okadaic acid with IC50 value of 160 nM. Immunoblotting assay indicated that PP1c delta but not PP1c alpha, PP1c gamma 1, and PP2Ac was localized in the isolated nucleoli. Confocal microscopy showed that PP1c delta was localized in nucleoli, nuclei, and cytosol, though the intensity of fluorescence at the nucleoli was stronger than that of the cytosol or nuclei. PP1c delta was co-localized with the major nucleolar phosphoprotein B23 at nucleoli. The phosphatase was capable of dephosphorylating several proteins in the nucleolus, including B23. The Km of PP1 for the recombinant B23.1, phosphorylated by endogenous kinase(s), was 3.5 microM. These results indicate that PP1c delta is the major serine/threonine phosphatase present in nucleolus and it dephosphorylates nucleolar phosphoproteins, including B23.

Tomosyn: a syntaxin-1-binding protein that forms a novel complex in the neurotransmitter release process.

Syntaxin-1 is a component of the synaptic vesicle docking and/or membrane fusion soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) complex (7S and 20S complexes) in nerve terminals. Syntaxin-1 also forms a heterodimer with Munc18/n-Sec1/rbSec1 in a complex that is distinct from the 7S and 20S complexes. In this report, we identify a novel syntaxin-1-binding protein, tomosyn, that is capable of dissociating Munc18 from syntaxin-1 and forming a novel 10S complex with syntaxin-1, soluble N-etyhlmaleimide-sensitive factor attachment (SNAP) 25, and synaptotagmin. The 130 kDa isoform of tomosyn is specifically expressed in brain, where its distribution partly overlaps with that of syntaxin-1 in nerve terminals. High level expression of either syntaxin-1 or tomosyn results in a specific reduction in Ca2+-dependent exocytosis from PC12 cells. These results suggest that tomosyn is an important component in the neurotransmitter release process where it may stimulate SNARE complex formation.

Successful peripheral T-lymphocyte-directed gene transfer for a patient with severe combined immune deficiency caused by adenosine deaminase deficiency.

Ten patients with adenosine deaminase deficiency (ADA-) have been enrolled in gene therapy clinical trials since the first patient was treated in September 1990. We describe a Japanese ADA- severe combined immune deficiency (SCID) patient who has received periodic infusions of genetically modified autologous T lymphocytes transduced with the human ADA cDNA containing retroviral vector LASN. The percentage of peripheral blood lymphocytes carrying the transduced ADA gene has remained stable at 10% to 20% during the 12 months since the fourth infusion. ADA enzyme activity in the patient's circulating T cells, which was only marginally detected before gene transfer, increased to levels comparable to those of a heterozygous carrier individual and was associated with increased T-lymphocyte counts and improvement of the patient's immune function. The results obtained in this trial are in agreement with previously published observations and support the usefulness of T lymphocyte-directed gene transfer in the treatment of ADA-SCID.

Rho regulates association of both the ERM family and vinculin with the plasma membrane in MDCK cells.

Rho small G protein regulates various actin-dependent cell functions. As to the functioning sites of Rho, Rho regulates formation of stress fibers and focal adhesions in many types of cultured cells, whereas we have shown that the association sites of actin filaments with the plasma membrane controlled by the ERM (Ezrin, Radixin, Moesin) family are the functioning sites of Rho in MDCK cells stably expressing myc-RhoA. We have investigated here the effect of microinjection of Rho GDI, a negative regulator of Rho which inhibits activation of Rho, C3, an exoenzyme of Clostridium botulinum which ADP-ribosylates Rho and inhibits its functions, or guanosine 5'-(3-O-thio) triphosphate-bound active form of Rho on the intracellular localization of both the ERM family and vinculin, which is one of the structural proteins of focal adhesions, in wild type MDCK cells. The ERM family was preferentially localized at peripheral bundles of actin filaments which are localized at the outer edge of colonies of the cells, microvilli and low Ca2+-induced cortical bundles of actin filaments in wild type MDCK cells. Microinjection of Rho GDI or C3 inhibited the localization of the ERM family at both the peripheral bundles and the low Ca2+-induced cortical bundles. On the other hand, vinculin was localized at both focal adhesions and basal edges of the colonies of the cells, and microinjection of Rho GDI or C3 inhibited the localization of vinculin at both of these sites. These results indicate that activation of Rho is necessary for the association of both the ERM family and vinculin with the plasma membrane in wild type MDCK cells. Microinjection of the guanosine 5'-(3-O-thio) triphosphate-bound form of Rho induced an increase in the localization of vinculin at focal adhesions, but did not induce an increase in the localization of the ERM family at the plasma membrane, indicating that activation of Rho itself is sufficient only for the association of vinculin with the plasma membrane at focal adhesions.

Effect of low-power laser irradiation on impulse conduction in anesthetized rabbits.

Low-power laser analgesic effect was generally accepted in clinical cases, whereas there was no direct evidence to indicate that low-power laser irradiation suppressed an impulse conduction within a peripheral nerve. The effect of low-power laser irradiation on electrically evoked responses within the sural nerve was electrophysiologically analyzed in anesthetized rabbits. High threshold evoked responses (conduction velocity was about 11 m/sec, unmyelinated A delta), which were induced by an electrical stimulation to the peripheral stump of the nerve, were significantly suppressed (9 to 19% inhibition) during low-power laser irradiation, which applied to the exposed sural nerve between the stimulus site and the recording site. The suppressive effect was reversible and recovered to the control level after the irradiation. Experimental evidence indicated that low-power laser irradiation suppressed the impulse conduction of unmyelinated A delta afferents in peripheral sensory nerve, which caused a pain sensation. Our data suggest that low-power laser acts as a reversible direct suppressor of neuronal activity.

Effect of low-power laser irradiation on procollagen synthesis in human fibroblasts.

The conflicting views of the effect of low-power laser (LPL) irradiation on procollagen synthesis have existed at the present time, whereas many clinical studies have tested usefulness of LPL irradiation for the wound healing. To evaluate the effect of LPL irradiation on the procollagen synthesis of human fibroblasts in vitro, LPL irradiation on human fibroblast was carried out using two different culture medium, serum-starved medium and fetal calf serum (FCS)-contained medium. In addition, to investigate the mechanism of the LPL on the procollagen synthesis of human fibroblasts, dexamethasone and methylene blue contained medium were used for inhibition of procollagen product at the pretranslational level and cGMP-mediated processes, respectively. Enhanced effect of LPL was consistently observed in the serum-starved medium (50% increase by a 3 min irradiation), not in the FCS-contained medium. The LPL enhanced effect was not blocked by dexamethasone (3% inhibition) but methylene blue (40% inhibition). Our data suggest that some factors in FCS might interfere with the enhanced effect of LPL on procollagen synthesis and the LPL might act as a direct stimulator of the procollagen synthesis. It seems probable that the LPL enhanced effects might be occurred at the translational level or at the pretranslational level, which is not affected by dexamethasone and cGMP, might be involved in the LPL enhanced effect of the procollagen synthesis in fibroblast.

Retroviral transduction of CD34-enriched hematopoietic progenitor cells under serum-free conditions.

The use of defined or serum-free culture conditions during retroviral transduction of hematopoietic cells would be desirable for standardization and safety reasons, as well as potentially allowing greater expansion of progenitor cells. Retroviral vector supernatants were concentrated and purified via tangential flow filtration polyethylene glycol (PEG)-precipitation, and ultracentrifugation, allowing serum-free transductions at standard multiplicities of infection (moi). Protein content of transductions using these concentrated vectors was 5-6 logs lower than in standard transductions. Transduction efficiencies of these concentrated vector preparations added back to serum-free or serum-containing media were equivalent to standard retroviral supernatant transductions of CD34-enriched progenitors. Absolute progenitor (CFU-C) numbers at the end of transduction were higher in serum-free + concentrated virus transductions, as opposed to transductions in standard vector supernatants containing fetal calf serum.

Electron-microscopic and immunohistochemical studies of Langerhans cells and Thy-1-positive cells in mouse tongue epithelium subjected to local hyperthermia.

Local hyperthermia via skin has been used to treat cancer but may suppress local immune responses as a side-effect. To examine effects of heat on immunologically responsive cells in oral mucosa, mouse tongues were heated by an implant system at 43 degrees C for 20 min. The densities of Langerhans cells and Thy-1-positive cells rapidly increased within 3 h after the treatment, then returned to a normal level after 7 days. Electron microscopy confirmed that Langerhans cells in the tongue epithelium formed clusters with lymphocytic cells, suggesting an active response to the hyperthermia.

Posterior interosseous nerve paralysis with multiple constrictions.

We report four cases of posterior interosseous nerve paralysis with multiple constrictions. At surgery the constrictions were found between the arcade of Frohse and a point of bifurcation of the supinator motor branch. External neurolysis with epineurotomy using the microscope was performed in all cases, and full recovery was obtained.