Crystallographic studies on damaged DNAs IV. N( 4)-methoxycytosine shows a second face for Watson-Crick base-pairing, leading to purine transition mutagenesis.

To investigate the mutation mechanism of purine transitions in DNA damaged with methoxyamine, a DNA dodecamer with the sequence d(CGCAAATTmo(4)CGCG), where mo(4)C is 2'-deoxy-N(4)-methoxycytidine, has been synthesized and the crystal structure determined by X-ray analysis. The duplex structure is similar to that of the original undamaged B-form dodecamer, indicating that the methoxylation does not affect the overall DNA conformation. Electron density maps clearly show that the two mo(4)C residues form Watson-Crick-type base pairs with the adenine residues of the opposite strand and that the methoxy groups of mo(4)C adopt the anti conformation to N(3) around the C(4)-N(4) bond. For the pair formation through hydrogen bonds the mo(4)C residues are in the imino tautomeric state. Together with previous work, the present work establishes that the methoxylated cytosine residue can present two alternate faces for Watson-Crick base-pairing, thanks to the amino<-->imino tautomerism allowed by methoxylation. Based on this property, two gene transition routes are proposed.

Crystallographic studies on damaged DNAs: III. N(4)-methoxycytosine can form both Watson-Crick type and wobbled base pairs in a B-form duplex.

To investigate the mutation mechanism of purine transition in DNA damaged with methoxyamine, a DNA dodecamer with the sequence d(CGCGAATTmo(4)CGCG), where mo(4)C is 2'-deoxy-N(4)-methoxycytidine, has been synthesized and its crystal structure determined. Two dodecamers form a B-form duplex. Electron density maps clearly show that one of the two mo(4)C residues forms a pair with a guanine residue of the opposite strand, the geometry being the canonical Watson-Crick type, and that the other mo(4)C residue forms a wobble pair with the opposite guanine residue. These two pairings are ascribed to the tautomerization of the methoxylated cytosine moieties between the amino and imino forms.

X-ray analyses of d(GCGAAAGC) and d(GXGAAAGCT), where X = 2'-deoxy-5-iodocytidine.

DNA fragments containing a sequence d(GCGAAAGC) are known to be highly thermostable. To investigate the structural basis for such a specific property, crystallographic studies of the DNA octamer and a nanomer d(GXGAAAGCT) (X = 2'-deoxy-5-iodocytidine) have been performed. The present higher resolution X-ray analyses have shown that both DNA oligomers are stabilized respectively to form a zipper-type duplex homodimer. PAGE of these oligomers, however, indicates that they are monomeric even when their crystals were dissolved at room temperature.

X-ray analysis of d(GCGAACGC); intra-duplex and inter-duplex hand-in-pocket motifs.

The structure of DNA octamer with the sequence d(GCGAACGC) has been determined by the X-ray crystallography to investigate the specific DNA structural motifs that are useful for designing various functional DNA molecules. Two kinds of interaction motifs for the bulged out adenosine residues have been found. One is an intra-duplex and the other is an inter-duplex hand-in-pocket motif. These interactions consist of the novel A:G pairs that have never been found so far in any crystal and solution studies on DNA molecules.

X-ray analyses of DNA dodecamers containing 2'-deoxy-5-formyluridine.

It is known that formylation of thymine base induces purine transition in DNA replication. In order to establish the structural basis for such mutagenesis, crystal structures of two kinds of DNA dodecamers d(CGCGRATf5UCGCG) with f5U = 2'-deoxy-5-formyluridine and R = A or G have been determined. The f5U residues form a Watson-Crick-type pair with A and two types of pairs (wobble and reversed wobble) with G, the latter being the first example. Structural modeling suggests that the DNA polymerase can accept the reversed wobble pair with G, as well as the Watson-Crick pair with A.

ICAM-1 (intercellular adhesion molecule-1) gene transfection inhibits lymph node metastasis by human gastric cancer cells.

Lymph node metastasis is one of the prognostic factors in gastric cancer. We have previously reported that decreased intercellular adhesion molecule-1 (ICAM-1) expression on cancer cells is associated with lymph node metastasis using a gastric cancer cell. In this study, we transfected ICAM-1 gene into a gastric cancer cell line, 2MLN, and analyzed the effect on lymph node metastasis in vitro and in vivo. A significantly greater amount of peripheral blood mononuclear cells (PBMC) adhered to ICAM-1 transfected 2MLN cells, 2MLN / ICAM cells, than to 2MLN / Vector cells. The lysis of 2MLN / ICAM cells by PBMC was significantly increased compared with that of 2MLN / Vector cells. The tumor growth rate of 2MLN / ICAM cells was significantly decreased in vivo. Lymph node metastases caused by 2MLN / ICAM cells were recognized as being fewer in number and smaller, while many lymph node metastases were caused by 2MLN cells. Histologic findings showed that leukocytes were heavily infiltrated in both the 2MLN / ICAM tumors and metastatic lesions, while only a few leukocytes were observed in the lesions associated with 2MLN cells. The above findings indicate that ICAM-1 gene transduction could prove to be an effective gene therapy for lymph node metastasis of gastric cancer.

Clinical significance of serum soluble intercellular adhesion molecule 1 in gastric cancer.

We studied the correlation between serum soluble intercellular molecule 1 (sICAM-1) and clinicopathological features in patients with gastric cancer. The impact of sICAM-1 on prognosis was also evaluated. The sera from 224 patients with gastric cancer, 44 healthy individuals, and 35 patients with benign gastrointestinal diseases (4 patients with submucosal stomach tumors, 6 patients with gastric ulcers, 1 patient with Crohn disease, 2 patients with ulcerative colitis, 7 patients with gall stones, 5 patients with chronic pancreatitis, and 10 patients with liver cirrhosis) were measured for sICAM-1 titer using a sandwich enzyme immunoassay method. There was no correlation between the serum titer of sICAM-1 and the age or gender of healthy controls. Among patients with benign gastrointestinal diseases, the patients with liver cirrhosis had a significantly higher mean serum sICAM-1 titer than that of healthy controls (P < 0.0001). The mean serum sICAM-1 titer of all patients with gastric cancer was not significantly different from that of healthy controls. However, among the patients with stage IV and recurrent disease, the serum sICAM-1 titer of those with hematogenous metastasis was significantly higher than that of patients without hematogenous metastasis (P = 0.001). The patients with a high serum sICAM-1 titer of more than 304 ng/ml (mean of healthy controls plus SD) showed a significantly worse prognosis than patients with a low serum sICAM-1 titer (P = 0.010). Nevertheless, serum sICAM-1 titer was not an independent predictor of prognosis by multivariate analysis. In conclusion, serum sICAM-1 cannot be used as a tumor marker for early diagnosis. However, sICAM-1 in sera may still be worthwhile to measure for monitoring hematogenous metastasis.

[Autopsy case of adenoid cystic carcinoma of the trachea: endobronchial treatment improves quality of life].

A 67-year-old man presented with dyspnea on exertion. Bronchoscopic examination revealed a tumor arising from the middle portion of the trachea and extending to the right main bronchus. The pathological diagnosis was adenoid cystic carcinoma. Radiotherapy and subsequent endobronchial electrocautery were performed, and elicited a partial response. In the clinical course. Dumon and Ultraflex stents were placed in the trachea asynchronically. Brachytherapy and esophageal stent placement were also performed for tumor control in the trachea and esophagus. Autopsy revealed that the tumor had invaded the trachea and esophagus, and bacterial mediastinitis was also demonstrated. Because the tumor was successfully controlled during the following 4 years and 9 months, we concluded that endobronchial therapy such as stent placement or electrocautery is useful for maintaining good quality of life.

Solitary squamous papilloma of the bronchus associated with human papilloma virus type 11.

A 79-year-old female presented with persistent dry cough, and a chest radiograph showed a mass shadow in the right upper lung. Bronchoscopic examination revealed that the right main bronchus was severely obstructed by a polypoid tumor, which was diagnosed pathologically as squamous papilloma. After the failure of the attempted endobronchial snare to remove the tumor, right upper lobectomy was performed. The polymerase chain reaction (PCR) examination showed the presence of human papilloma virus type 11 DNA in the resected tumor, suggesting that this virus was the cause of this solitary squamous papilloma of the lung.

In vitro interactions of a new derivative of spicamycin, KRN5500, and other anticancer drugs using a three-dimensional model.

PURPOSE: KRN5500 is a new derivative of spicamycin produced by Streptomyces alanosinicus and is known to have a wide range of antitumor activities against human cancer cell lines. Because of its unique structure, this compound seems to have a different mode of action from other antitumor drugs and nonoverlapping toxicities. Therefore, KRN5500 is expected to be a suitable candidate for combination chemotherapy. METHODS: We investigated the effects of combinations of KRN5500 and other anticancer drugs on the growth of a human non-small-cell lung cancer cell line, PC14, using a revised three-dimensional model. RESULTS: Synergism was observed when KRN5500 and cisplatin were combined at concentrations in the ranges 0.005 to 0.25 microg/ml and 0.025 to 0.25 microg/ml, respectively. In combination with carboplatin, an analog of cisplatin, and etoposide, a marked synergistic interaction was also found. CONCLUSION: These results suggest the usefulness of combinations of KRN5500 with cisplatin, carboplatin or etoposide for chemotherapy for non-small-cell lung cancer.

Combination effects of TAS-103, a novel dual topoisomerase I and II inhibitor, with other anticancer agents on human small cell lung cancer cells.

PURPOSE: TAS-103 [6-((2-(dimethylamino) ethyl)amino)-3-hydroxy-7H-iindeno(2,1-c)quinolin-7-one dihydrochloride] is a newly synthesized dual inhibitor of topoisomerase I and II. Since anticancer drugs are used in combination with other drugs for effective chemotherapy, we investigated the cytotoxic effect of TAS-103 in combination with other conventional anticancer agents, such as cisplatin, vindesine, doxorubicin, 5-fluorouracil, and the antitopoisomerase inhibitors SN-38 and etoposide in vitro. METHODS: Inhibition of the growth of the human small-cell lung cancer cell line SBC-3 was evaluated using the tetrazolium dye (MTT) assay. Drug interactions were evaluated by isobologram analysis and the determination of combination indices supplemented by a three-dimensional model. RESULTS: Simultaneous use of TAS-103 and cisplatin had a supraadditive effect, but combinations of TAS-103 with other drugs had an additive or marginally subadditive effect. Three-dimensional model analysis added more information about the synergistic concentration ranges of two drugs (cisplatin 200-400 nM and TAS-103 7 10 nM). Sequential use of TAS-103 and cisplatin had only an additive effect. CONCLUSION: These results suggest that the concomitant use of TAS-103 and cisplatin has a greater cytotoxic effect on cancer cells than single drug use, and may provide a beneficial effect in the treatment of small-cell lung cancer.

Crystallization and preliminary analysis of a DNA dodecamer of d(CGCGmo6AATCCGCG) containing 2'-deoxy-N6-methoxyadenosine: change in crystal packing with different humidity.

The DNA dodecamer of (CGCGmo6AATCCGCG) containing 2'-deoxy-N6-methoxyadenosine has been crystallized for X-ray analysis in order to investigate the effects of the modified adenosine on base pairing. It has been found that the crystal changes from one form to another during data collection in a manner similar to a phase transition. The two crystal structures show that this phenomenon, ascribed to differences in humidity, is correlated with a change in the contact angle between the two duplexes.

Mitogen-activated protein kinase antisense oligonucleotide inhibits the growth of human lung cancer cells.

Mitogen-activated protein kinase (MAPK) pathway is proposed to be a therapeutic target for cancer cells. In order to find the potential therapeutic usefulness of MAPK for cancer cells, the effect of EAS1, an antisense oligonucleotide for an MAPK, on cancer-cell-growth were investigated in vitro. EAS1 effectively inhibited the growth of several human lung cancer cell lines such as PC-14 cells upon exposure to 10-0-10-1 microM of EAS1 determined dye-formation (MTT) assay. The ED50 values were comparable to those obtained for the inhibition of MAPK activity, DNA synthesis. EAS1 arrested the PC-14 cells at the G2/M phase of cell cycle followed by apoptosis in a dose-dependent manner. In order to determine the factors which influence the cellular sensitivity against MAPK inhibition, the effect of EAS1 on H-ras-transformed murine fibroblast cells were compared with that on parental cells. The NIH3T3 cells transformed by the H-ras gene (PT22-3) showed higher sensitivity against the effects of EAS1. Because MAPK activity was activated by H-ras gene transfection in PT22-3, the status of the MAPK cascade in cells was the determining factor for the efficacy of EAS1. In addition, cell permeabilization by digitonin enhanced the growth inhibitory effect of EAS1. Penetration of the cell membrane by EAS1 is also crucial for the growth inhibitory effect of EAS1. In conclusion, MAPK is an important target for cancer treatment and MAPK antisense oligonucleotide is a potentially significant antitumor oligonucleotide.

Churg-Strauss syndrome diagnosed and followed with gastrointestinal fiberscopic studies and electroneuromyography.

Churg-Strauss syndrome (CSS) is a disorder characterized by hypereosinophilia and systematic vasculitis occurring in individuals with bronchial asthma. We present a case of 24-year-old woman with CSS diagnosed by and followed with gastrointestinal fiberscopic examination and electroneuromyography. The duodenal biopsy showed granulomatous angiitis with eosinophilic infiltration, and the electroneuromyography showed peripheral nerve dysfunction. After steroid treatment, the duodenal erosion and ulcer were almost completely resolved. There was no improvement in electroneuromyography, but the patient was able to walk independently after intensive rehabilitative training.

X-ray analysis of DNA dodecamer containing 2'-deoxy-N6-methoxyadenosine.

Oxyamines have been known as a mutagen which attacks amino groups of DNA bases. It is expected that the modified adenine derivative has a tautomer which can form a stable base pair not only with thymine but also with cytosine. For establishing such tautomerization mutagenesis, we have solved a crystal structure of DNA dodecamer containing 2'-deoxy-N6-methoxyadenosine. It is shown that the N6-methoxyadenine takes a imino form to form a Watson-Crick type pairing with cytosine in the DNA duplex.

Regional difference in depolarization-elicited accumulation of cyclic AMP in cobalt-induced epileptic cortex of the rat.

The appearance of epileptiform discharges in electrocorticograms was induced by a unilateral injection of CoCl2 solution into the sensorimotor cortex of rats. Accumulation of cyclic AMP elicited by ouabain or a high concentration of potassium ions was determined in slices from different cortical areas of rats 9 or 10 days after the injection. In the anterior cortex, the depolarization-elicited accumulation of cyclic AMP was significantly higher in the cortical area ipsilateral to the injection site than in the contralateral cortical area. In the posterior cortex, a similar but not significant difference in the accumulation of cyclic AMP was noted.

The role of the corpus callosum in interhemispheric propagation of iron-induced epileptogenic activity in the rat.

In iron-induced epilepsy, partial section of the corpus callosum resulted in a decrease in rats showing dominant spike activity on the side of secondary focus and an increase in rats showing almost equal spike activity on the sides of primary and secondary focus in electrocorticograms (ECoGs). Changes in ECoG spike frequencies caused by sectioning the corpus callosum included an increase in unilateral spikes on the side of primary focus, a decrease in unilateral spikes on the side of secondary focus, and an increase in bilateral spikes appearing almost synchronously on both sides.

Diazepines. 5. Synthesis and biological action of 6-phenyl-4H-pyrrolo[1,2-a][1,4]benzodiazepines.

A series of 6-phenyl-4H-pyrrolo[1,2-a][1,4]benzodiazepines (2) has been prepared with 2-phthalimidomethylfurans (12) and 1-phthalimidoalkane-2,5-diones (15) or 2,5-dimethoxy-2-phthalimidomethyltetrahydrofurans (16) as the key intermediates and subsequently evaluated for CNS activity. The structure-activity data generated indicate that, in general, introduction of the methyl and/or ethyl group(s) in the pyrrole ring and a chlorine atom at the ortho position of the 6-phenyl group increases the activity and that substitution of the above chlorine atom for a fluorine atom decreases the activity. 8-Chloro-6-(2-chlorophenyl)-1,3-dimethyl-4H-pyrrolo[,2-a][1,4]benzodiazepine (2p), the most potent among the compounds synthesized, was equipotent in taming and sedative activities to diazepam. The acute LD50 of 2p in mice was larger than 3000 mg/kg po.