Mammalian homologue of E. coli Ras-like GTPase (ERA) is a possible apoptosis regulator with RNA binding activity.

BACKGROUND: ERA (Escherichia coli Ras-like protein) is an E. coli GTP binding protein that is essential for proliferation. A DNA database search suggests that homologous sequences with ERA exist in various organisms including human, mouse, Drosophila, Caenorhabditis elegans and Antirrhinum majus. However, the physiological function of eukaryotic ERA-like proteins is not known. RESULTS: We have cloned cDNAs encoding the entire coding region of a human homologue (H-ERA) and a mouse homologue (M-ERA) of ERA. The mammalian homologue of ERA consists of a typical GTPase/GTP-binding domain and a putative K homology (KH) domain, which is known as an RNA binding domain. We performed transfection experiments with wild-type H-ERA or various H-ERA mutants. H-ERA possessing the amino acid substitution mutation into the GTPase domain induced apoptosis of HeLa cells, which was blocked by Bcl-2 expression. Deletion of the C-terminus, which contains a part of the KH domain, alleviated apoptosis by the H-ERA mutant, suggesting the importance of this domain in the function of H-ERA. We have also shown the RNA binding activity of H-ERA by pull-down experiments using RNA homopolymer immobilized on beads or recombinant H-ERA proteins. CONCLUSION: Our data suggest that H-ERA plays an important role in the regulation of apoptotic signalling with its GTPase/GTP binding domain.

Requirement for STAT1 in LPS-induced gene expression in macrophages.

This study examines the role of the signal transducer and activator of transcription 1 (STAT1) in induction of lipopolysaccharide (LPS)-stimulated gene expression both in vitro and in vivo. LPS-induced expression of an interferon (IFN)-inducible 10-kDa protein (IP-10), IFN regulatory factor-1 (IRF-1), and inducible nitric oxide synthase (iNOS) mRNAs was severely impaired in macrophages prepared from Stat1-/- mice, whereas levels of tumor necrosis factor alpha and KC (a C-X-C chemokine) mRNA in LPS-treated cell cultures were unaffected. A similar deficiency in LPS-induced gene expression was observed in livers and spleens from Stat1-/- mice. The reduced LPS-stimulated gene expression seen in Stat1-/- macrophages was not the result of reduced activation of nuclear factor kappaB. LPS stimulated the delayed activation of both IFN-stimulated response element and IFN-gamma-activated sequence binding activity in macrophages from wild-type mice. Activation of these STAT1-containing transcription factors was mediated by the intermediate induction of type I IFNs, since the LPS-induced IP-10, IRF-1, and iNOS mRNA expression was markedly reduced in macrophages from IFN-alpha/betaR-/- mice and blocked by cotreatment with antibodies against type I IFN. These results indicate that indirect activation of STAT1 by LPS-induced type I IFN participates in promoting optimal expression of LPS-inducible genes, and they suggest that STAT1 may play a critical role in innate immunity against gram-negative bacterial infection.

Inhibitory effect of M50054, a novel inhibitor of apoptosis, on anti-Fas-antibody-induced hepatitis and chemotherapy-induced alopecia.

M50054, 2,2'-methylenebis (1,3-cyclohexanedione), was identified as a novel inhibitor of apoptosis (programmed cell death) using an in vitro cell death assay system induced in human Fas-expressing WC8 cells by soluble human Fas ligand. Furthermore, M50054 inhibited the apoptotic cell death of U937, a human monocytic leukemic cell line, induced by anticancer agents such as etoposide; it was also confirmed that M50054 inhibited apoptotic features such as DNA fragmentation and phosphatidylserine exposure in these cells. These anti-apoptotic effects were attributable to inhibition of caspase-3 activation. Additionally, M50054 significantly inhibited anti-Fas-antibody-induced elevation of plasma alanine aminotransferase and aspartate aminotransferase. Alopecia (hair loss) symptoms were also significantly improved with topical treatment with M50054. In conclusion, M50054 inhibits apoptosis induced by a variety of stimuli via inhibition of caspase-3 activation, and may thus be effective for hepatitis and chemotherapy-induced alopecia.

Interleukin-4/STAT6 represses STAT1 and NF-kappa B-dependent transcription through distinct mechanisms.

STAT6 mediates interleukin-4 (IL-4)-dependent positive and negative regulation of inflammatory gene expression. In the present report we examined the molecular mechanisms involved in IL-4-induced repression of reporter gene transcription driven by STAT1 and/or NF-kappaB. Transient expression of STAT6 in a STAT6-deficient cell line (HEK 293) conferred sensitivity to IL-4 for STAT6-dependent transcription and for repression of interferon-gamma (IFNgamma)/STAT1- and/or tumor necrosis factor-alpha (TNFalpha)/NF-kappaB-driven reporter gene expression. In cells transfected with a deletion mutant of STAT6 lacking its transactivating domain, IL-4 could not mediate either positive or negative control of reporter gene expression. Overexpression of CREB-binding protein dramatically enhanced IL-4/STAT6-stimulated transcription and overcame IL-4-mediated repression of TNFalpha/NF-kappaB-dependent but not IFNgamma/STAT1-dependent transcription. A single amino acid change in the DNA-binding domain of STAT6 (H415A) selectively reduced the affinity of STAT6 for IL-4-responsive STAT sequence motifs (N4) without affecting the affinity for IFNgamma-responsive (GAS) sequences (N3) and, accordingly, eliminated transcription from an IL-4-responsive promoter. Interestingly, this mutation eliminated IL-4-mediated suppression of reporter gene transcription stimulated by TNFalpha/NF-kappaB but retained nearly full capacity to suppress IFNgamma/STAT1-stimulated transcription. Taken together these results demonstrate that STAT6 mediates suppression of STAT1 and NF-kappaB-dependent transcription by distinct mechanisms. Both processes are dependent upon the STAT6 transactivation domain and may involve sequestration of necessary but different transcriptional coactivator proteins. These two suppressive mechanisms are controlled differentially by the nature of the STAT6 DNA-binding site (i.e. N3 versus N4).

Interleukin-1-mediated stabilization of mouse KC mRNA depends on sequences in both 5'- and 3'-untranslated regions.

mRNA transcribed from the mouse KC chemokine gene accumulated to significantly higher levels in multiple cell types after treatment with interleukin 1alpha (IL-1alpha) as compared with tumor necrosis factor-alpha (TNFalpha). Although TNFalpha and IL-1alpha both signaled the activation of nuclear factor kappaB and enhanced transcription of the KC gene with equal potency, only IL-1alpha treatment resulted in stabilization of KC mRNA. Nucleotide sequences that confer sensitivity for IL-1alpha-mediated mRNA stabilization were identified within the 5'- and 3'-untranslated regions (UTRs) of KC mRNA using transient transfection of chimeric plasmids containing specific portions of KC mRNA linked to the chloramphenicol acetyltransferase (CAT) gene. When plasmids containing either the 3'- or 5'-UTR of KC mRNA were used, the half-life of CAT mRNA was unaltered either in untreated or IL-1alpha-stimulated cells. In contrast, CAT mRNA transcribed from plasmids that contained both the 5'- and 3'-UTRs of the KC mRNA decayed more rapidly than control CAT mRNA, and this enhanced decay was prevented in cells treated with IL-1alpha. A cluster of four overlapping AUUUA motifs within the 3'-UTR was required, whereas the 5'-UTR region exhibited orientation dependence. These findings indicate that cooperative function of the two nucleotide sequences involves a distinct signaling pathway used by IL-1alpha but not TNFalpha.

[Thrombomodulin].

Thrombomodulin (TM), a membrane-bound receptor for thrombin on the endothelial cell surface, contributes to the regulation of the coagulation system. TM is known to exist in human plasma and urine as soluble forms. We purified soluble TM from human urine (MR-33) and investigated the anticoagulant effects of MR-33 in vitro and in vivo. In human plasma, MR-33 inhibited not only the procoagulant activity of thrombin, but also the thrombin generation via accelerating the thrombin-catalyzed protein C activation. In rat disseminated intravascular coagulation (DIC) models, intravenous infusion of MR-33 improved the hematological abnormalities without excessive prolongation of APTT and bleeding time. Benefit (dose required for 50% inhibition of fibrinogen decrease: ED50) to risk (minimum dose required for significant prolongation of bleeding time) ratio was 1:27 for MR-33. Furthermore, the anticoagulant activities of MR-33 was independent of AT III activity, and MR-33 was effective on heparin-resistant DIC models with low AT III level in rats. Intravenous injection of MR-33 prevented the endotoxin-induced increases in TAT, TNF-alpha and IL-6 level and pulmonary vascular permeability in mice. These results indicate that MR-33 may be a clinically useful antithrombotic agent with reduced risk for hemorrhage, and this drug also has anti-inflammatory effects. Clinical trials of MR-33 for the treatment of DIC are now in progress in Japan.

Primary T-cell lymphoma of the duodenum: report of a case.

A case of primary non-Hodgkin's T-cell lymphoma of the duodenum is presented. A 41-year-old man was hospitalized in 1984 complaining of abdominal distention and vomiting. Hypotonic duodenography showed an encircling filling defect in the second portion of the duodenum, and a biopsy specimen revealed features of malignancy suggestive of either undifferentiated carcinoma or malignant lymphoma. Radical surgery (pancreaticoduodenectomy) was performed, after which chemotherapy was administered. A histological evaluation of the duodenal tumor showed it to be non-Hodgkin's lymphoma. It was a diffuse, large-cell type, which immunohistochemically suggested it to be of T-cell origin. Currently the patient is doing well, with no evidence of disease recurrence 13 years after surgery.

Effect of multidisciplinary treatment with high dose rate intraluminal brachytherapy on survival in patients with unresectable esophageal cancer.

BACKGROUND: Since carcinoma of the esophagus is usually diagnosed at an advanced stage, many cases of esophageal cancer are beyond possible radical resection and only palliative treatment can be performed in such cases. Therefore, a great deal of discussion has taken place concerning indications for treatment modality, and various procedures have been performed to palliate such patients. The prognosis for such patients is still poor, even though many kinds of palliation have already been developed and applied. To improve the prognosis of such patients we developed a multidisciplinary treatment which includes high dose rate intraluminal brachytherapy (HDR-ILBRT) and evaluated its effectiveness, especially the HDR-ILBRT component. PATIENTS AND TREATMENT METHODS: Sixty-six patients with unresectable esophageal cancer enrolled in this study. Twenty-seven patients underwent bypass operations. Seven of the 27 patients received external irradiation only (group BE), 11 received external irradiation and HDR-ILBRT (group BEH), while the remaining 9 did not receive radiotherapy (group B). Another 39 patients without bypass operations were all treated with radiotherapy, 22 with external irradiation only (group E) and 17 were treated with external irradiation and HDR-ILBRT (group EH). After completion of radiotherapy, all patients received chemotherapy with 5-FU (2,500 mg/body) and CDDP (100 mg/body). RESULTS: Mean survival time and the 5-year survival rate of group BEH (13.3 +/- 1.5 months and 20%) were significantly improved compared with group BE + B (p < 0.05). In the patients without bypass operations, there were significant differences in the mean survival time and the 3-year survival rate in groups EH and E (group EH 16.5 +/- 2.5 months, 21.8%, group E 9.0 +/- 1.3 months, 0%, p < 0.05). The bypass operation itself and chemotherapy did not significantly affect the prognosis of patients with unresectable esophageal cancer. CONCLUSION: These results strongly suggest the use of HDR-ILBRT, as a component of multidisciplinary treatment for unresectable esophageal cancer, was significantly effective and HDR-ILBRT contributed to improve outcomes in patients with advanced esophageal cancer. HDR-ILBRT should be established as a component of treatments for unresectable esophageal cancer.

Positron emission tomography-based boron neutron capture therapy using boronophenylalanine for high-grade gliomas: part I.

Determination of tumor boron-10 (10B) levels is required for accurate neutron dosimetry during boron neutron capture therapy. We assessed a new method for quantitative measurement of boronated drug uptake in high-grade gliomas. This method uses positron emission tomography (PET) with fluorine-18-labeled L-fluoroborono-phenylalanine (L-18F-10B-FBPA), which was synthesized as an analogue of L-boronophenylalanine. We studied the accumulation of L-18F-10B-FBPA by PET in patients with high-grade gliomas. Dynamic PET studies of brain tumors revealed that L-18F-10B-FBPA accumulated gradually after bolus injection, and the value of PET activity divided by the integrated plasma activity reached a constant level 42 min after injection, which was defined as the incorporation constant (Ic*). This constant reflected the appropriate L-18F-10B-FBPA accumulation in tumor tissue. Based on the Ic* constant, the methods for estimating tumor 10B concentration were devised. With this method, the estimated values of 10B concentration in gliomas were very close to the 10B levels in surgical specimens. This method was based solely on PET and can potentially provide data that would assist in the selection of patients for future treatment with boron neutron capture therapy after surgical resection of their brain tumors.

Positron emission tomography-based boron neutron capture therapy using boronophenylalanine for high-grade gliomas: part II.

Based on pharmacokinetic findings of fluorine-18-labeled L-fluoroboronophenylalanine by positron emission tomography (PET), methods for estimating tumor 10B concentration were devised. In clinical practice of boron neutron capture therapy (BNCT) for high-grade gliomas, a large amount of L-boronophenylalanine (L-10B-BPA)-fructose solution is used. Under these conditions, a slow i.v. infusion of L-10B-BPA-fructose solution should be performed for BNCT; therefore, the changes over time in 10B concentration in the target tissue were estimated by convoluting the actual time course of changes in plasma 10B concentration with a PET-based weight function including the proper rate constants [K1 (ml/g/min), k2 (min(-1)), k3 (min(-1)), and k4 (min(-1))]. With this method, the estimated values of 10B concentration in gliomas were very close to the 10B levels in surgical specimens. This demonstrated the similarity in pharmacokinetics between fluorine-18-labeled L-fluoroboronophenylalanine and L-10B-BPA. This method, using the appropriate rate constant, permits the determination of tumor 10B concentration and is widely suitable for clinical BNCT, because the averaged PET data are enough to use in future patients without individual PET study.

New multi-disciplinary treatment modality with RALS for patients with esophageal cancer.

Esophageal cancer is frequently found when it is already in the advanced stage and curative surgery for such cases is consequently difficult to perform. The new multi-disciplinary treatment for esophageal cancer presented here was, therefore, conceived to improve both the survival rate and quality of life of these patients. This combined treatment modality consists of limited surgery, external irradiation, intracavitary irradiation with remote-controlled after-loading system (RALS) and peri-operative chemotherapy. In the present series, 45 patients with esophageal cancer received esophagectomy and on another 11 patients bypass operation was performed. All patients were treated with this multi-disciplinary treatment after operation. A 3 cm-wide thin gastric tube was made from the greater curvature of the stomach of the patient using an autosuture apparatus (PLC55 or GIA). In the bypass operation, the jejunum was anastomosed to the original esophagus in the Roux-en Y fashion and jejunostomy was performed on the oral side of the Roux loop. A silastic tube of 9 mm inner diameter was inserted from the jejunostomy and placed into the original esophagus for the purpose of postoperative intracavitary irradiation with RALS. For the patients receiving esophagectomy, a similar silastic tube was also placed in the posterior mediastinum for intracavitary irradiation with RALS. The indication of the bypass operation was i) a tumor length longer than 9 cm on the X-ray film and/or ii) direct invasion to the aortic wall evident by CT or MRI examination. Two weeks after the operation, external irradiation to the mediastinum with Linac 10 MV X-ray, and to the bilateral cervical regions with Linac 15 MeV electron beam, was started. The irradiation doses were 30 Gy (2 Gy/day, 5 times/ week) and 48 Gy (4 Gy/day, 3 times/week), respectively. The intracavitary irradiation with RALS was started shortly before the end of the external irradiation period and was delivered from a 60Co source. The total dose was 24 Gy (6 Gy/day, once a week) for the esophagectomized cases, and 18 Gy for the bypassed cases. Two or three weeks after the termination of the radiotherapy, chemotherapy with cisplatinum and 5-fluorouracil was performed and repeated every 6 months for 2 years. All patients could eat normally and were discharged after finishing the first chemotherapy session. The overall 5-year survival rate was 49% for the esophagectomized cases and 11% for the bypassed cases. The longest survival time in the bypassed cases was 5 years and 4 months. Neither operative death nor severe complications were experienced during the treatment period. The results indicate that this newly developed multi-disciplinary treatment with RALS can improve the prognosis and the quality of life not only in the esophagectomized patients but also in the bypassed patients with advanced esophageal cancer.