Susumu Ohwada

Cloning of a human homolog of the yeast OGG1 gene that is involved in the repair of oxidative DNA damage

We report the cloning of a human homolog of the yeast OGG1 gene, which encodes a DNA glycosylase that excises an oxidatively damaged form of guanine, 8-hydroxyguanine (also known as 7,8-dihydro-8-oxoguanine). Since the deduced amino acid sequence (68 amino acids) of a human expressed sequence tag, N55394, matched a short stretch of yeast OGG1 protein with greater than 40% amino acid identity, a full length cDNA clone was isolated from a HeLa cell cDNA library with the N55394 clone as a probe. The cDNA clone encodes a predicted protein of 345 amino acids which is homologous to yeast OGG1 protein throughout the entire polypeptide sequence and shares 38% amino acid identity with yeast OGG1 protein. Moreover, we found that both a human homolog and yeast OGG1 protein possess two distinct DNA binding motifs, a helix-hairpin-helix (HhH) motif and a C2H2 zinc finger like motif, and a domain homologous to human and E. coli MutY proteins. Expression of a human homolog suppressed spontaneous mutagenesis of an E. coli (mutM mutY) mutant as in the case of yeast OGG1 protein. The gene was ubiquitously expressed in a variety of human organs and mapped to chromosome 3p26.2. These results strongly suggest that the gene isolated here is a human counterpart of the yeast OGGI gene and is involved in the repair of oxidative DNA damage in human cells.

NOTCH3 Signaling Pathway Plays Crucial Roles in the Proliferation of ErbB2-Negative Human Breast Cancer Cells

ErbB2-negative breast tumors represent a significant therapeutic hurdle because of a lack of effective molecular targets. Although NOTCH proteins are known to be involved in mammary tumorigenesis, the functional significance of these proteins in ErbB2-negative breast tumors is not clear. In the present study, we examined the expression of activated NOTCH receptors in human breast cancer cell lines, including ErbB2-negative and ErbB2-positive cell lines. Activated NOTCH1 and NOTCH3 proteins generated by gamma-secretase were detected in most of the cell lines tested, and both proteins activated CSL-mediated transcription. Down-regulation of NOTCH1 by RNA interference had little or no suppressive effect on the proliferation of either ErbB2-positive or ErbB2-negative cell lines. In contrast, down-regulation of NOTCH3 significantly suppressed proliferation and promoted apoptosis of the ErbB2-negative tumor cell lines. Down-regulation of NOTCH3 did not have a significant effect on the ErbB2-positive tumor cell lines. Down-regulation of CSL also suppressed the proliferation of ErbB2-negative breast tumor cell lines, indicating that the NOTCH-CSL signaling axis is involved in cell proliferation. Finally, NOTCH3 gene amplification was detected in a breast tumor cell line and one breast cancer tissue specimen even though the frequency of NOTCH3 gene amplification was low (<1%). Taken together, these findings indicate that NOTCH3-mediated signaling rather than NOTCH1-mediated signaling plays an important role in the proliferation of ErbB2-negative breast tumor cells and that targeted suppression of this signaling pathway may be a promising strategy for the treatment of ErbB2-negative breast cancers.

Fibrin Glue Sandwich Prevents Pancreatic Fistula following Distal Pancreatectomy

Pancreatic fistula is a major form of morbidity following pancreatic resection. We conducted a nonrandomized clinical trial comparing the sealing and sandwich techniques of spraying fibrin glue to prevent pancreatic fistula following distal pancreatectomy. The pancreas was transected with a scalpel to identify and suture the main pancreatic duct and its small branches. In the sealing group, fibrin glue was sprayed over the closed pancreatic stump and sutures. Alternatively, in the sandwich group fibrin glue was sprayed so as to cover and join the cut surface of the pancreatic remnant, which was then held closed with sutures. Altogether 111 patients were included in the study (90 with gastric cancer, 10 with esophageal cancer, and 11 with pancreatic cancer). Patients were nonrandomly assigned to the sandwich or the sealing group. Morbidity was 21.8% for the patients in the sandwich group versus 33.9% in the sealing group. Pancreatic fistulas occurred in 9.0% of the sandwich group versus 26.8% of the sealing group. The incidence of fistula was thus significantly lower in the sandwich group. The incidence of fistula was also significantly lower in the sandwich group for gastric malignancy patients undergoing extended radical lymphadenectomy down to the paraaortic lymph nodes combined with left adrenalectomy. Of the patients with gastric malignancy, pancreatic fistulas occurred in 9.3% of the sandwich group versus 25.5% of the sealing group. The fibrin glue sandwich technique is simple and reliable and should be valuable for complementing other prophylactic methods of preventing pancreatic fistula.

The autocrine motility factor receptor gene encodes a novel type of seven transmembrane protein1

Autocrine motility factor receptor (AMFR) is a cell surface glycoprotein of molecular weight 78 000 (gp78), mediating cell motility signaling in vitro and metastasis in vivo. Here, we cloned the full‐length cDNAs for both human and mouse AMFR genes. Both genes encode a protein of 643 amino acids containing a seven transmembrane domain, a RING‐H2 motif and a leucine zipper motif and showed a 94.7% amino acid sequence identity to each other. Analysis of the amino acid sequence of AMFR with protein databases revealed no significant homology with all known seven transmembrane proteins, but a significant structural similarity to a hypothetical protein of Caenorhabditis elegans, F26E4.11. Thus, AMFR is a highly conserved gene which encodes a novel type of seven transmembrane protein.

Increased Choline Kinase Activity and Elevated Phosphocholine Levels in Human Colon Cancer

Nuclear magnetic resonance spectroscopy has detected elevated phosphocholine levels in human tumor tissues and cells, and in cells that were transformed with the activated Ha‐ras gene and stimulated in vitro with growth‐promoting factors such as platelet‐derived growth factor, epidermal growth factor, and phorbol ester. However, the mechanism of the elevation and the function of the increased phosphocholine levels have not been clearly demonstrated. We studied phosphocholine levels enzymatically and analyzed the activity of choline kinase, which catalyzes the phosphorylation of choline to produce phosphocholine, in human colon cancer and adenoma. Both choline kinase activity and phosphocholine levels were increased in colon cancer and adenoma tissue. The activation of choline kinase and the increased levels of choline kinase α were partly responsible for the elevated phosphocholine levels. This study suggests that choline kinase might play a role in growth promotion or signal transduction in carcinogenesis.

Amplification of c-myc in Hepatocellular Carcinoma: Correlation with Clinicopathologic Features, Proliferative Activity and p53 Overexpression

Expression of the proto-oncogene c-myc has been implicated in liver regeneration and hepatocarcinogenesis. The biologic significance of c-myc gene amplification in human hepatocellular carcinoma, however, is unconfirmed. We correlated c-myc gene amplification with clinicopathologic features, proliferative activity, and p53 expression in 42 resected tumors. c-myc amplification in tumor tissue was determined using a differential polymerase chain reaction, a useful procedure for the evaluation of gene amplification in archival formalin-fixed paraffin-embedded tissues, in comparison with a dopamine D2 receptor gene. Proliferative activity was estimated by numbers of argyrophilic nucleolar organizer regions and immunohistochemical nuclear labeling rates using a monoclonal antibody against Ki-67. The c-myc gene was amplified in 14 of 42 tumors (33.3%). Amplification of c-myc was more frequent in younger patients and in larger tumors, and less differentiated tumors. No correlation was noted with α-fetoprotein level or viral hepatitis state. The amplification showed positive correlation with both proliferative activity and p53 overexpression. Disease-free survival in patients showing c-myc amplification was significantly shorter than in those without amplification. These results suggest that c-myc amplification is an indicator of malignant potential and poor prognosis in hepatocellular carcinoma. c-myc amplification and p53 alteration may be coparticipating events in the progression of these tumors.

Biological properties and gene expression associated with metastatic potential of human osteosarcoma

Lung metastasis has a great influence on the prognosis of patients with osteosarcoma. We previously established two high-metastatic sublines, M112 and M132, from the HuO9 human osteosarcoma cell line by in vivo selection. In this study, we newly isolated a high-metastatic subline, H3, and three low-metastatic sublines, L6, L12 and L13, from HuO9 by the dilution plating method. Three high-metastatic sublines produced more than 200 metastatic nodules in the lung, while three low-metastatic sublines produced no or few nodules after injection of 2 × 106 cells into the tail vein of nude mice. There were significant differences in the motility and invasiveness between high- and low-metastatic sublines, whereas the growth rates in vitro and the tumorigenicity in vivo showed no correlation with their metastatic abilities. Early adherence to culture plates was significantly lower in two of three low-metastatic sublines, which occupied smaller surface areas on the culture plates than other sublines did. Comparison of the expression of 637 cancer-related genes by cDNA microarray revealed that seven genes were differentially expressed between high- and low-metastatic sublines. Among them, five genes (AXL, TGFA, COLL7A1, WNT5A, and MKK6) were associated with adherence, motility, and/or invasiveness. These results suggest that the differences in motility/invasiveness and adhesive abilities are key determinants of lung metastasis in osteosarcoma.

Low-Dose Erythromycin Reduces Delayed Gastric Emptying and Improves Gastric Motility After Billroth I Pylorus-Preserving Pancreaticoduodenectomy

ObjectiveTo test the hypothesis that early and low doses of erythromycin reduce the incidence of early delayed gastric emptying (DGE) and induce phase 3 of the migratory motor complex in the stomach after Billroth I pylorus-preserving pancreaticoduodenectomy (PPPD). Summary Background DataDelayed gastric emptying is a leading cause of complications after PPPD, occurring in up to 50% of patients. High doses of erythromycin (200 mg) accelerate gastric emptying after pancreaticoduodenectomy and reduce the incidence of DGE, although they induce strong contractions that do not migrate to the duodenum. MethodsThirty-one patients were randomly assigned to either the erythromycin or control groups. The patients received erythromycin lactobionate (1 mg/kg) every 8 hours, or H2-receptor antagonists and gastrokinetic drugs from days 1 to 14 after surgery. On postoperative day 30, gastroduodenal motility was recorded in 14 patients. ResultsPreoperative, intraoperative, and postoperative factors were comparable in the erythromycin and control groups. The erythromycin group had a shorter duration of nasogastric drainage, earlier resumption of eating, and a 75% reduction in the incidence of DGE. Erythromycin was an independent influence on nasogastric tube removal, and preservation of the right gastric vessels was a significant covariate. Low doses of erythromycin induced phase 3 of the migratory motor complex and phase 3-like activity, with the same characteristics as spontaneous phase 3, in 86% of patients: two had quiescent stomachs and the others had spontaneous phase 3 or phase 3-like activity. ConclusionsLow doses of erythromycin reduced the incidence of DGE by 75% and induced phase 3 of the migratory motor complex after Billroth I PPPD. Low doses of erythromycin are preferable to high doses in the unfed period after PPPD.

Effects of a dual inhibitor of tumor necrosis factor-α and interleukin-1 on lipopolysaccharide-induced lung injury in rats : Involvement of the p38 mitogen-activated protein kinase pathway

ObjectiveSepsis is a major cause of adult respiratory distress syndrome. In this study, we evaluated the effect of FR167653, which is a potent suppressant of tumor necrosis factor (TNF)-&agr; and interleukin (IL)-1 production, on lipopolysaccharide (LPS)-induced lung injury and lethality in rats, and we examined the involvement of p38 mitogen-activated protein (MAP) kinase in the action of FR167653. DesignProspective, randomized study. SettingAnimal research facility in a university. SubjectsMale Sprague-Dawley rats weighing 200–270 g. InterventionsAll the animals were assigned to one of the following four groups: control group, FR-only group, LPS-only group, and LPS/FR group. Animals in the LPS-only and LPS/FR groups received 6 mg/kg of LPS intravenously. The animals in the FR-only and LPS/FR groups also received an infusion of FR167653 at 0.2 mg·kg−1·hr−1, commencing 30 mins before the LPS (or vehicle) injection and continuing for 5.5 hrs. Measurements and Main Results LPS significantly induced the accumulation of pulmonary neutrophils and lung edema, both of which were significantly attenuated by treatment with FR167653. FR167653 also significantly decreased the LPS-induced lethality. Histologically, tissue damage was milder in the LPS/FR group than in the LPS-only group. Serum concentrations of TNF-&agr; and IL-1&bgr; and plasma concentrations of thromboxane B2 were all suppressed in the LPS/FR group compared with the LPS-only group. Western blot analysis revealed that FR167653 inhibited the phosphorylation of p38 MAP kinase in lung tissues. ConclusionsFR167653 administration decreased serum TNF-&agr; and IL-1&bgr; concentrations, which was associated with decreased lung injury and lethality. The mechanism responsible for the decreased TNF-&agr; and IL-1 may be related to the inhibitory effect of FR167653 on p38 MAP kinase activation.

Role of p38 mitogen-activated kinase and c-Jun terminal kinase in migration response to lysophosphatidic acid and sphingosine-1-phosphate in glioma cells

A potential role for 1-oleoyl-sn-glycero-3-phosphate or lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) in the regulation of malignant diseases has been widely considered. In this study, we found that in transformed astroglial cells, the expression profile of lysophospholipid receptor mRNA and the action modes of LPA and S1P on cell motility were changed: there was a change in the acquisition of the ability of LPA to stimulate cell migration and a change in the migratory response to S1P from stimulation through S1P1 to inhibition through S1P2. LPA-induced cell migration was almost completely inhibited by either pertussis toxin, LPA1 receptor antagonists including Ki16425 (3-(4-[4-([1-(2-chlorophenyl)ethoxy]carbonyl amino)-3-methyl-5-isoxazolyl] benzylsulfonyl)propanoic acid) or an inhibitor of phosphatidylinositol 3-kinase (PI3K) wortmannin. The LPA-induced action was also suppressed, although incompletely, by several specific inhibitors for intracellular signaling pathways including Rac1, Cdc42, p38 mitogen-activated protein kinase (p38MAPK) and c-Jun terminal kinase (JNK), but not extracellular signal-regulated kinase. Nearly complete inhibition of migration response to LPA, however, required simultaneous inhibition of both the p38MAPK and JNK pathways. Inhibition of Rac1 suppressed JNK but not p38MAPK, while the activity of p38MAPK was abolished by a dominant-negative form of Cdc42. These findings suggest that, in glioma cells, the PI3K/Cdc42/p38MAPK and PI3K/Rac1/JNK pathways are equally important for LPA1 receptor-mediated migration.