Frequent Bax frameshift mutations in gastric cancer with high but not low microsatellite instability

Widespread microsatellite instability (MSI) due to the defective DNA mismatch repair underlies the pathogenesis of the majority of hereditary non-polyposis colorectal cancer and a subset of various sporadic malignant tumors. Using 5 microsatellite markers and the criteria of MSI proposed by the National Cancer Institute (NCI) workshop, we analyzed 205 gastric adenocarcinomas for MSI. Based on the number of markers showing instability per tumor, the tumors were divided into three groups; those with two or more of the five markers displaying instability (high MSI, MSI-H), those with one of five markers displaying instability (low MSI, MSI-L), and those with no instability (microsatellite stable, MSS). Among 205 tumors, 30 (15%) were MSI-H, 15 (7%) were MSI-L, and 160 (78%) were MSS. All of the 30 MSI-H tumors demonstrated instability at BAT26, a sensitive marker for the widespread MSI, while none of the 15 MSI-L tumors did. MSI-H tumors were significantly associated with distal location and well or moderate differentiation, but MSI-L tumors were indistinguishable from MSS tumors. Bax frameshift mutations were detected in 60% of the 30 MSI-H tumors, while not in any of the 15 MSI-L tumors. These results suggest that microsatellite analysis using the criteria proposed by the NCI workshop may be appropriate for gastric cancers because it unveils real differences in genotype and phenotype.     

Distinct methylation pattern and microsatellite instability in sporadic gastric cancer.

Aberrant 5' CpG island methylation is an alternative mechanism of gene inactivation during the development of cancer as demonstrated for several tumor-suppressor genes. Also, marked relationship of microsatellite instability (MSI) and DNA methylation has been reported in sporadic colorectal cancer, which is a result of epigenetic inactivation of hMLH1 in association of promoter hypermethylation. In the present study, we investigated the 5' CpG island hypermethylation of hMLH1, E-cadherin and p16 in 61 primary gastric cancers (GCs) by using combined bisulfite restriction analysis (COBRA) and methylation-specific PCR (MSP), and their MSI status. Of 61 GCs investigated, 5 (8.1%) tumors presented hMLH1 methylation, 16 (26.2%) and 25 (40.9%) showed E-cadherin and p16 methylation respectively, and 8 (13.1%) presented high-frequency MSI (MSI-H). Of the 8 MSI-H patients, 5 presented hMLH1 methylation, whereas no low-frequency MSI (MSI-L) and microsatellite stable (MSS) cases exhibited hMLH1 methylation (5/8 vs. 0/43, p < 0.00001). Furthermore, these patients also presented E-cadherin and p16 hypermethylation. Our data showed a significant correlation between hMLH1 methylation and MSI in GC, and suggested that a common mechanism of aberrant de novo methylation can be postulated in these cancers.     

Changes in levels of 8-hydroxyguanine in DNA, its repair and OGG1 mRNA in rat lungs after intratracheal administration of diesel exhaust particles.

Diesel exhaust particles (DEP), an environmental pollutant, are known to induce lung cancer in experimental animals. To clarify whether reactive oxygen species (ROS) are involved in its carcinogenic mechanism, we examined the levels of 8-hydroxyguanine (8-OH-Gua), its total repair and the repair enzyme OGG1 mRNA in female Fischer 344 rat lungs, as markers of the response to ROS, after DEP was intratracheally instilled. The 8-OH-Gua levels in both DEP-treated groups (2 and 4 mg) were increased during the 2-8 h following exposure to DEP. The 8-OH-Gua repair activities in the DEP-treated groups decreased during the period from 2 h to 2 days following DEP exposure and then recovered to the level of the control group at 5 days after exposure. OGG1 mRNA was induced in rats treated with 4 mg DEP for 5-7 days after administration. In conclusion, the 8-OH-Gua level in rat lung DNA increases markedly at an early phase after DEP exposure, by the generation of ROS and the inhibition of 8-OH-Gua repair activity, and induction of OGG1 mRNA is also a good marker of cellular oxidative stress during carcinogenesis.     

Acute myeloid leukemia with t(5;11): two case reports.

A case of acute monocytic leukemia (AMoL) with t(5;11)(q31;q23) and a case of acute myelomonocytic leukemia (AMMoL) with t(5;11)(q35;q13.1) are reported. The translocation between the long arm of chromosome 11q and that of chromosome 5q with leukemia have been rarely reported. Though breakpoint of both cases were subtlety different, they had morphologically monocytic character and showed hyperleukocytosis and chemoresistance.     

Differential gene expression of human telomerase-associated protein hTERT and TEP1 in human hematopoietic cells

The maintenance of telomere length is crucial for the survival of cells. Recently, genes for proteins that consist of human telomerase have been cloned and the results have indicated a close relationship between telomerase activity and its gene expression. We studied the mRNA expression of the telomerase-associated genes, hTERT and TEP1, in hematopoietic cells in order to clarify the relation between them and telomerase activity using semiquantitative RT-PCR. In polymorphonuclear cells and monocytes isolated from peripheral blood, which had no detectable telomerase activity, no hTERT mRNA expression was seen. On the other hand, lymphocytes and CD34-positive cells both demonstrated hTERT mRNA expression. TEP1 mRNA was detected in all samples, showing no differential expression. We then assessed hTERT and TEP1 mRNA expression in CD34-positive cells cultured in vitro with growth factors. After 4 weeks of culture, all the cells showed myeloid differentiation and the telomerase activity was downregulated. hTERT mRNA was expressed in CD34-positive cells, but was downregulated in 4-week-cultured cells. TEP1 showed no apparent differential expression. We conclude that hTERT mRNA expression is downregulated in accordance with telomerase downregulation during the course of myeloid differentiation, which suggests that it plays a crucial role in the expression of enzyme activity, while TEP1 has a much smaller role to play, if any.     

In vitro and in vivo pharmacological characterization of J-113397, a potent and selective non-peptidyl ORL1 receptor antagonist

1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl -1, 3-dihydro-2H-benzimidazol-2-one (J-113397) was found to be the first potent nonpeptidyl ORL1 receptor antagonist (K(i): cloned human ORL1=1.8 nM) with high selectivity over other opioid receptors (K(i): 1000 nM for human mu-opioid receptor, >10,000 nM for human delta-opioid receptor, and 640 nM for human kappa-opioid receptor). In vitro, J-113397 inhibited nociceptin/orphanin FQ-stimulated [35S]guanosine 5'-O-(gamma-thio)triphosphate (GTP gamma S) binding to Chinese Hamster Ovary (CHO) cells expressing ORL1 (CHO-ORL1) with an IC(50) value of 5.3 nM but had no effect on [35S]GTP gamma S binding by itself. Schild plot analysis of the [35S]GTP gamma S binding assay and cAMP assay using CHO-ORL1 indicated competitive antagonism of J-113397 on the ORL1 receptor. In CHO cells expressing mu-, delta- or kappa-opioid receptors, J-113397 had no effects on [35S]GTP gamma S binding up to a concentration of 100 nM, indicating selective antagonism of the compound on the ORL1 receptor. In vivo, J-113397, when administered subcutaneously (s.c.), dose-dependently inhibited hyperalgesia elicited by intracerebroventricular (i.c.v.) administration of nociceptin/orphanin FQ in a tail-flick test with mice. An in vitro binding study using mouse brains indicated that J-113397 possesses high affinity for the mouse ORL1 receptor (K(i): 1.1 nM) as well as the human receptor. In summary, J-113397 is the first potent, selective ORL1 receptor antagonist that may be useful in elucidating the physiological roles of nociceptin/orphanin FQ.     

SM-20220, a potent Na+/H+ exchange inhibitor, improves consciousness recovery and neurological outcome following transient cerebral ischaemia in gerbils

We studied the cerebroprotective effect of SM-20220 (N-(aminoiminomethyl)-1-methyl-1H-indole-2-carboxamide methanesulphonate), a newly synthesized Na+/H+ exchanger (NHE) inhibitor, in Mongolian gerbil global ischaemia. Transient cerebral ischaemia was induced by clipping both common carotid arteries for 30 min followed by 24h reperfusion. Intravenous administration of SM-20220 (0.3 or 1.0 mg kg(-1)) immediately after reperfusion significantly shortened the consciousness recovery time (P < 0.01). SM-20220 also improved the neurological outcome (McGraw's scale) after reperfusion. At the dose of 1.0 mg kg(-1), the mortality rate was significantly reduced at 24 h after reperfusion (P < 0.01). This study shows that NHE is involved in the aggravation of cerebral function, represented by consciousness recovery, and neurological outcome following transient forebrain ischaemia, and that its inhibitor may exert protective effects on post-ischaemic brain damage.     

Relationship between effects of alcohol on psychomotor performances and blood alcohol concentrations

Ethanol is a social drug and has been generally known to be a CNS depressant. A large fluctuation of blood alcohol concentration (BAC) is well-known to occur due to main factors such as the genetic polymorphism of the main alcohol metabolizing enzymes and the effect of blood. Few studies have substantially discussed the relationship between impaired CNS activities and BAC. In this study, focusing on the correlation of BAC, we investigated the acute effects of alcohol intake on cognitive performance in humans by objective evaluation methods consisting of the attention-demanding cognitive tasks. Tasks were administered to ten healthy male volunteers before and after ingesting established amounts of alcohol. With increased BAC, we observed prolongation of reaction time performances and lowering of a coordination performance. From the results, we concluded that cognitive performance deteriorates with an increase of BAC. Additionally, the BAC threshold that causes significant impairment of cognitive performance was estimated to be approximately 50 mg/dl (ca. 10 mM).