Nobutake Akiyama

Identification of a Series of Transforming Growth Factor β-Responsive Genes by Retrovirus-Mediated Gene Trap Screening

ABSTRACT Transforming growth factor β (TGF-β) plays important roles in the regulation of proliferation, differentiation, apoptosis, and carcinogenesis. To identify genes responsible for maintaining the phenotype induced by TGF-β, we performed a retrovirus-mediated gene trap screening designed to isolate TGF-β-responsive genes in human lung carcinoma cell line A549. After screening 249 trap lines, 21 were found to express the reporter β-galactosidase gene in a TGF-β-responsive manner. Interestingly, in large proportions of these trap lines, the reporter gene was responsive also to phorbol ester and was suppressed by gamma interferon. Fragments of all these trapped genes were recovered by 5′- and 3′-rapid amplification of cDNA ends (RACE), and in 15 out of 21 cases (71%), the TGF-β responsiveness of the endogenous genes was confirmed by RNA blot hybridization. In at least five cases, the TGF-β-induced upregulation was found to be cycloheximide resistant, suggesting the roles of the genes in the TGF-β-induced primary responses. Sequence analyses revealed that 43% (9 of 21) of the trapped genes were novel and that the remainder included genes previously reported to be upregulated by TGF-β, such as epidermal growth factor receptor and β1 integrin, documenting the validity of this approach. Other known genes include the ones encoding the proteins associated with cell proliferation (ribosomal proteins S15a, hNRP/NAP-1, and lipocortin II), focal adhesions (paxillin), and transcriptional regulation (thyroid hormone receptor activator molecule 1 [TRAM-1]).

A single dose of interleukin-31 (IL-31) causes continuous itch-associated scratching behaviour in mice

We investigated the effects of a single dose of mouse interleukin‐31 (IL‐31) on scratching behaviour in comparison with spontaneous skin‐lesion‐ or serotonin (5‐HT)‐ induced scratching behaviour in NC/Nga and BALB/c mice. Intradermal (i.d.) injection of IL‐31 caused a gradual increase in long‐lasting scratching (LLS, over 1.5 s) about 3 h after administration followed by a gradual decrease for over 24 h after administration. I.d. injection of IL‐31 significantly increased the total LLS counts/24 h but not short‐lasting scratching (SLS, 0.3–1.5 s). In skin‐lesioned NC/Nga mice, the LLS but not SLS counts were significantly higher than those in non‐skin‐lesioned NC/Nga mice. We also investigated 5‐HT‐induced scratching in BALB/c mice, SLS but not LLS increased immediately after the injection and then decreased to baseline after at 20 min. These results suggest that IL‐31 may participate in the sensation of itching and promote scratching behaviour in skin‐lesioned NC/Nga mice, an animal model of atopic dermatitis (AD).

Expression of hydroxyindole-O-methyltransferase enzyme in the human central nervous system and in pineal parenchymal cell tumors.

Pineal parenchymal tumor (PPT) cells usually show immunoreactivity for synaptophysin, neuron-specific enolase, neurofilament protein, class III &bgr;-tubulin, tau protein, PGP9.5, chromogranin, serotonin, retinal S-antigen, and rhodopsin, but these markers are not specific for PPTs. Melatonin is produced and secreted mainly bypineal parenchymal cells; hydroxyindole-O-methyltransferase (HIOMT) catalyzes the final reaction in melatonin biosynthesis. We hypothesized that HIOMT could serve as a tumor marker of PPTs, and we investigated HIOMT localization and HIOMT expression in samples of normal human tissue and in PPTs, primitive neuroectodermal tumors, and medulloblastomas. In normal tissue, HIOMT was expressed in retinal cells, pineal parenchymal cells, neurons of the Edinger-Westphal nucleus, microglia, macrophages, thyroid follicular epithelium, principal and oxyphil cells of parathyroid gland, adrenal cortical cells, hepatic parenchymal cells, renal tubule epithelium, and enteroendocrine cells of stomach and duodenum. The HIOMT was also expressed in all 46 PPTs studied. The proportions of HIOMT-immunoreactive cells successively decreased in the following tumors: pineocytoma, pineal parenchymal tumor of intermediate differentiation, and pineoblastoma. A few HIOMT-immunoreactive cells were observed in one of 6 primitive neuroectodermal tumors and 23 of 42 medulloblastomas. These results indicate that HIOMT immunohistochemistry may be useful for the diagnosis of PPTs and be a prognostic factor in PPTs.

Human Shugoshin Mediates Kinetochore-Driven Formation of Kinetochore Microtubules

The conserved protein Shugoshin (Sgo) plays a role in the maintenance of centromeric cohesion in mitosis and meiosis. Human Shugoshin (hSgo) was first identified as an overexpressed protein in breast cancers. Here we demonstrate that hSgo mediates kinetochore-driven formation of kinetochore microtubules (MTs) during bipolar spindle assembly. The regulated overexpression of full-length hSgo, or of truncated proteins containing both the conserved N-terminal coiled-coil domain and C-terminal basic domain, resulted in hSgo localization at centromere at early mitosis and was associated with aberrant nucleation and formation of bundles of kinetochore MTs. The mid-portion of hSgo, between the N- and C-terminal domains, includes both a functional domain for centromeric cohesion and a regulatory domain for spindle assembly. The cells overexpressing natural alternative splicing isoforms, which are almost completely defective for the mid-portion of the hSgo protein, showed premature centromere separation (PCS) and aberrant MT connections. These isoforms are mildly overexpressed in HEK293 cells. On the other hand, cells expressing a truncated protein, defective in the lysine-rich region of the mid-portion, arrested at mitosis due to persistent abnormal MT connections and not because of PCS. Aberrant MT connections were predominantly observed in spindle regions where chromosomes were clustered. Interestingly, we also found that hSgo is rapidly exchanged at kinetochores at early mitosis. Based on these results, we conclude that hSgo may be diffusible and have a role in proper kinetochores-MTs attachment.

Both NUP98/TOP1 and TOP1/NUP98 transcripts are detected in a de novo AML with t(11;20)(p15;q11).

The NUP98 gene is involved in several chromosomal abnormalities associated with acute leukemia. The recurrent t(11;20)(p15;q11) chromosomal translocation results in generation of the NUP98/TOP1 chimeric gene. This abnormality has been observed primarily in therapy‐related leukemias, and TOP1/NUP98 transcripts have not been demonstrated. We describe a case of de novo acute myeloid leukemia with t(11;20)(p15;q11), with no known history of exposure to chemicals. The translocation occurred in intron 13 of NUP98 and intron 7 of TOP1, as in the three previously reported cases. The breakpoint in NUP98 was exactly the same as that found in a previously reported case. In addition, a reciprocal TOP1/NUP98 transcript was detected for the first time in our patient.

Repeated administration of IL-31 upregulates IL-31 receptor A (IL-31RA) in dorsal root ganglia and causes severe itch-associated scratching behaviour in mice

We investigated the effects of repeated administration of interleukin‐31 (IL‐31) on itch‐associated scratching counts (long‐lasting scratching, LLS) and IL‐31‐related receptor mRNA expression in mice. Intra‐dermal (i.d.) injection of IL‐31 (100 and 300 ng/site) every 12 h for 3 days significantly increased LLS. Repeated administration of IL‐31 also increased the expression of IL‐31 receptor A (IL‐31RA) and oncostatin M receptor beta (OSMRβ) in dorsal root ganglia (DRG). After the repeated administration of IL‐31 was discontinued, IL‐31RA expression decreased and reached the baseline level 2 days after the last dose of IL‐31. LLS changed along with DRG IL‐31RA expression. Moreover, IL‐31‐induced IL‐31RA protein expression was confirmed by Western blotting analysis. These data suggest that IL‐31 upregulates IL‐31RA expression in DRG neuron cell bodies, and cutaneous‐injected IL‐31‐induced itching is enhanced by DRG IL‐31RA expression in mice.

Characterization of mutations induced by 300 and 320 nm UV radiation in a rat fibroblast cell line.

The cytotoxic and mutagenic activities of monochromatic ultraviolet light (UV) at four wavelengths (254, 290, 300 and 320 nm) were determined using a rat fibroblast cell line CREF stably infected with a retroviral vector carrying the neo and HSV-tk markers. In this system, mutations can be positively detected as acyclovir-resistant colonies. Although the action spectra for these activities closely fit some of the previously reported spectra for photochemical DNA modifications, erythema, cell killing and mouse skin carcinogenesis, they diverge at 320 nm from the absorption spectrum for DNA and the action spectrum for bacterial inactivation and mutagenesis. Structural comparison of the HSV-tk mutants detected after irradiation with 300 and 320 nm UV revealed (1) CC dimers and C oligomers as predominant targets at both wavelengths; (2) increased incidence of relatively large deletions at 300 nm; and (3) greatly increased frequency of tandem double mutations at both wavelengths and of clustered multiple mutations at 320 nm. These results suggest the involvement of distinct mechanisms specifically operating, or becoming evident, in UV-mediated mutagenesis at these different wavelengths in mammalian cells.

Conjugation of Quantum Dots and JT95 IgM Monoclonal Antibody for Thyroid Carcinoma Without Abolishing the Specificity and Activity of the Antibody

Among the immunoglobulins, IgM class-antibodies are now considered to be potent immunological reagents for anticancer remedies. However, only a few reports are available about the effective labeling of IgM with enzymes, fluorescence, or other bioreactive reagents. Here, we report an effective application of luminescent semiconductive nanoparticles, quantum dots (QDs), as a labeling material of the IgM antibody. The CdSe carboxyl QDs were reacted with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysulfo- succinimide in 2-(morpholino) ethanesulfonic acid. The reacted QDs were then coupled to JT95 IgM antibody, which recognizes thyroid carcinoma associated antigen. The specificity and activity of the conjugates were tested by immunoblot, immunoquantitive assay and immunohistological imaging. The QDs were firmly conjugated with JT95 IgM monoclonal antibody. In immunoblot assay, QD-JT95 conjugates directly detected the target molecules without obstructing the binding site. In immunoquantitive assay, the conjugates could quantify the antigen in the range of 1.56-100 μg/mL. Also, QDs-labeled antibody detected the antigen on plasma membrane. Our results demonstrate that labeling of JT95 and other IgM class antibodies with QDs is feasible. This approach may be an important method for the medical application of IgM in the diagnosis and treatment of cancers.

Role of TGF‐β in EGF‐induced transformation of NRK cells is sustaining high‐level EGF‐signaling1

We have been isolating and analyzing NRK cell mutants, which fail to transform by epidermal growth factor (EGF) and transforming growth factor (TGF)‐β. One such mutant, R14, can respond to the growth inhibitory signal of TGF‐β to the same extent as parental NRK but fail to respond to the growth stimulatory signal of EGF. This mutant has a defect in EGF receptor (EGFR) expression. When R14 mutant expressed a high level of EGFR, however, EGF not only induced proliferation in this mutant but also induced transformation without the aid of TGF‐β. These findings suggest that the major role of TGF‐β in this transformation system should be to counteract the ligand‐dependent down‐regulation of EGFR, thereby sustaining high‐level EGF‐signaling.

Regulation of Th2 responses by different cell types expressing the interleukin-31 receptor

BackgroundInterleukin-31 (IL-31) is a recently identified cytokine produced by Th2 cells that is involved in the development of atopic dermatitis-induced skin inflammation and pruritus. Its receptor, IL-31RA, is expressed by a number of cell types, including epithelial cells, eosinophils, and activated monocytes and macrophages. To date, however, the regulation of Th2 responses by distinct cell types and tissues expressing IL-31RA has not been well studied.MethodsIn this study, Cry j 2, one of the major allergens of Japanese cedar pollen, was administered to IL-31RA-deficient or wild-type (WT) mice via nasal or intraperitoneal injection for induction of specific Th2 responses.ResultsAfter nasal administration of Cry j 2, IL-31RA-deficient mice showed lower Cry j 2-specific CD4+ T cell proliferation, Th2 cytokine (IL-5 and IL-13) production, and Th2-mediated (IgE, IgG1, and IgG2b) antibody responses than WT mice. In contrast, IL-31RA-deficient mice administered Cry j 2 intraperitoneally showed stronger Th2 immune responses than WT mice.ConclusionsThese results indicate that IL-31R signaling positively regulates Th2 responses induced by nasal administration of Cry j 2, but negatively regulates these responses when Cry j 2 is administered intraperitoneally. Collectively, these data indicate that the induction of antigen-specific Th2 immune responses might depend on tissue-specific cell types expressing IL-31RA.