Activation of transient receptor potential ankyrin 1 by eugenol.

Eugenol is a bioactive plant extract used as an analgesic agent in dentistry. The structural similarity of eugenol to cinnamaldehyde, an active ligand for transient receptor potential ankyrin 1 (TRPA1), suggests that eugenol might produce its effect via TRPA1, in addition to TRPV1 as we reported previously. In this study, we investigated the effect of eugenol on TRPA1, by fura-2-based calcium imaging and patch clamp recording in trigeminal ganglion neurons and in a heterologous expression system. As the result, eugenol induced robust calcium responses in rat trigeminal ganglion neurons that responded to a specific TRPA1 agonist, allyl isothiocyanate (AITC), and not to capsaicin. Capsazepine, a TRPV1 antagonist failed to inhibit eugenol-induced calcium responses in AITC-responding neurons. In addition, eugenol response was observed in trigeminal ganglion neurons from TRPV1 knockout mice and human embryonic kidney 293 cell lines that express human TRPA1, which was inhibited by TRPA1-specific antagonist HC-030031. Eugenol-evoked TRPA1 single channel activity and eugenol-induced TRPA1 currents were dose-dependent with EC50 of 261.5µM. In summary, these results demonstrate that the activation of TRPA1 might account for another molecular mechanism underlying the pharmacological action of eugenol.

Aqueous extract of Schizandra chinensis fruit causes endothelium-dependent and -independent relaxation of isolated rat thoracic aorta.

An aqueous extract of Schizandra chinensis fruit (ScEx) has long been used to promote the vascular health of postmenopausal women in Korea. This study investigated the ability of ScEx to relax rat aorta constricted with norepinephrine (NE) and the mechanism(s) of such relaxation. ScEx induced partial, endothelium-dependent relaxation. In particular, the relaxation induced by lower concentrations of ScEx (0.1 and 0.3 mg/ml) was largely endothelium-dependent, and was essentially abolished by NG-nitro-L-arginine, methylene blue, 1H-[1,2,3] oxadiazole [4,4-a] quinoxalin-1-one, indomethacin, or ICI 182,780. The results indicate that the response to ScEx involves enhancement of the nitric oxide (NO)-cGMP system, and that it occurs via estrogen receptors. The magnitude of the inhibition with these treatments decreased with increasing ScEx concentration, however, indicating that other vasorelaxation mechanisms are involved, which depend on the ScEx concentration. Calcium concentration-dependent contraction curves in high potassium depolarization medium were shifted significantly to the right and downward after incubation with ScEx (0.3 and 1.0 mg/ml), implying that ScEx is also involved in inhibition of the extracellular calcium influx to vascular smooth muscle. These data demonstrate that ScEx caused both endothelium-dependent and -independent vasorelaxation, which may contribute to understanding the cardiovascular protective effect of ScEx.

Nitric oxide-mediated vasorelaxation by Rhizoma Ligustici wallichii in isolated rat thoracic aorta.

The vasorelaxant effect of Rhizoma Ligustici wallichii and its possible mechanism of action on the vasomotor tone of the rat thoracic aortic rings were examined in an organ bath. Chloroform extracts of Rhizoma Ligustici wallichii (Ch1LW) elicited a dose-dependent, transient, relaxing response in endothelium-intact rat aorta contracted with norepinephrine (NE). This relaxant effect was abolished by removal of the endothelium and also by pretreatment with nitric oxide synthase inhibitors. Neither a muscarinic receptor antagonist nor a cyclooxygenase inhibitor altered the Ch1LW-induced relaxation. Tetramethylpyrazine, derived from Rhizoma Ligustici wallichii as a potent vasodilating component, induced a complete relaxation in both endothelium-intact and denuded rat aortas contracted by NE, but nitric oxide synthase inhibitors did not affect the relaxation. Ch1LW-induced endothelium-dependent relaxation was mediated by nitric oxide released from the endothelium, and could be caused by component(s) other than tetramethylpyrazine.

CsRn1, a novel active retrotransposon in a parasitic trematode, Clonorchis sinensis, discloses a new phylogenetic clade of Ty3/gypsy-like LTR retrotransposons.

We screened the genome of a trematode, Clonorchis sinensis, in order to identify novel retrotransposons and thereby provide additional information on retrotransposons for comprehensive phylogenetic study. Considering the vast potential of retrotransposons to generate genetically variable regions among individual genomes, randomly amplified polymorphic DNAs (RAPDs) detected by arbitrarily primed polymerase chain reactions were selected as candidates for retrotransposon-related sequences. From RAPD analysis, we isolated and characterized a novel retrotransposon in C. sinensis as the first member of uncorrupted long-terminal-repeat (LTR) retrotransposons in phylum Platyhelminthes. The retrotransposon, which was named Clonorchis sinensis Retrotransposon 1 (CsRn1), showed a genomewide distribution and had a copy number of more than 100 per haploid genome. CsRn1 encoded an uninterrupted open reading frame (ORF) of 1,304 amino acids, and the deduced ORF exhibited similarities to the pol proteins of Ty3/gypsy-like LTR retrotransposons. The mobile activity of master copies was predicted by sequence analysis and confirmed by the presence of mRNA transcripts. Phylogenetic analysis of Ty3/gypsy-like LTR retrotransposons detected a new clade comprising CsRn1, Kabuki of Bombyx mori, and an uncharacterized element of Drosophila melanogaster. With its high repetitiveness and preserved mobile activity, it is proposed that CsRn1 may play a significant role in the genomic evolution of C. sinensis.

CpG island methylation in premalignant stages of gastric carcinoma.

There are limited reports on methylation analysis of the premalignant lesions of gastric carcinoma thus far. This is despite the fact that gastric carcinoma is one of the tumors with a high frequency of CpG island hypermethylation. To determine the frequency and timing of hypermethylation during multistep gastric carcinogenesis, non-neoplastic gastric mucosa (n = 118), adenomas (n = 61), and carcinomas (n = 64) were analyzed for their p16, human Mut L homologue 1 (hMLH1), death-associated protein (DAP)-kinase, thromobospondin-1 (THBS1), and tissue inhibitor of metalloproteinase 3 (TIMP-3) methylation status using methylation-specific PCR. Three different classes of methylation behaviors were found in the five tested genes. DAP-kinase was methylated at a similar frequency in all four stages, whereas hMLH1 and p16 were methylated in cancer samples (20.3% and 42.2%, respectively) more frequently than in intestinal metaplasia (6.3% and 2.1%, respectively) or adenomas (9.8% and 11.5%, respectively). However, hMLH1 and p16 were not methylated in chronic gastritis. THBS-1 and TIMP-3 were methylated in all stages but showed a marked increase in hypermethylation frequency from chronic gastritis (10.1% and 14.5%, respectively) to intestinal metaplasia (34.7% and 36.7%, respectively; P < 0.05) and from adenomas (28.3% and 26.7%, respectively) to carcinomas (48.4% and 57.4%, respectively: P < 0.05). The hMLH1, THBS1, and TIMP-3 hypermethylation frequencies were similar in both intestinal metaplasia and adenomas, but the p16 hypermethylation frequency tended to be higher in adenomas (11.5%) than in intestinal metaplasia (2.1%; P = 0.073). The average number of methylated genes was 0.6, 1.1, 1.1, and 2.0 per five genes per sample in chronic gastritis, intestinal metaplasia, adenomas, and carcinomas, respectively. This shows a marked increase in methylated genes from non-metaplastic mucosa to intestinal metaplasia (P = 0.001) as well as from premalignant lesions to carcinomas (P = 0.002). These results suggest that CpG island hypermethylation occur early in multistep gastric carcinogenesis and tend to accumulate along the multistep carcinogenesis.

Evidence for two modes of allelic loss: multifocal analysis on both early and advanced gastric carcinomas.

To assess the extent and the timing of allelic loss required for the progression of gastric carcinoma, the intratumoral distribution of loss of heterozygosity (LOH) was compared in early and advanced tumors: early loss is uniformly observed in all tumor areas and late loss is localized in parts of tumor tissue. Tumor sites (167 sites) obtained from 42 gastric carcinoma tissues (26 advanced cancers and 16 early cancers) were examined for LOH on chromosomes 5q, 9p, 13q, 17p, and 18q. By using two or three microsatellite markers for each chromosome arm, it was shown that of 29 tumors showing LOH in at least one tumor site, 15 (51.7%, 12 advanced and three early cancers) harbored multiple losses on three or more chromosome arms, and 89.4% (84 of 94) of these losses was uniformly found in all tumor sites tested. In the remaining 14 tumors (48.3%, eight advanced and six early tumors) with sporadic losses on one or two chromosome arms, 44% (11 of 25) of the losses were commonly shared among the sites tested. Such marked difference (P<0.001, Fisher's exact test) in the intratumoral distribution of multiple and sporadic LOH patterns proposes two distinct LOH subtypes: multiple losses (high LOH), occurring at an early stage with a few additional losses, and sporadic losses (low LOH), taking place relatively late during tumor progression. The multifocal LOH findings imply that, rather than being gradual, the allelic losses take place in two manners that are already determined at an early stage.

Prognostic implications of microsatellite genotypes in gastric carcinoma.

Microsatellite alterations such as loss of heterozygosity (LOH) and microsatellite instability (MSI) are observed in most (70% to 80%) gastric carcinomas. To determine whether the microsatellite genotypes are correlated with clinicopathological features, 118 patients with gastric carcinomas were examined by using polymorphic microsatellite markers for LOH on 5 gastric cancer-associated chromosome arms and non-polymorphic BAT markers for MSI. Microsatellite genotypes were categorized as high-frequency MSI (MSI-H), high-level LOH (LOH-H), low-level LOH (LOH-L) and LOH non-detectable (LOH-N). A significant fraction of the MSI-H, LOH-H and LOH-L types was observed in intestinal-type gastric carcinomas, whereas the LOH-N type was highly associated with diffuse-type tumors (p = 0.00162). There was a close relationship between microsatellite genotype and TNM (tumor-node-metastasis) stage (p = 0. 001). Univariate analysis showed that patients of LOH-H or LOH-N types and those of MSI-H or LOH-L types correlated with poor and favorable survival, respectively, not only in all tumor stages (p = 0.0001) but also in stages II and III (p = 0.0271). It is likely that the major genotypes of gastric carcinomas can be placed into at least 4 microsatellite categories, thus allowing the construction of a comprehensive genetic classification useful for the prediction of diverse clinical courses.

Nitric oxide-mediated endothelium-dependent relaxation of rat thoracic aorta induced by aqueous extract of red rice fermented with Monascus ruber.

Vasodilatory effects of aqueous extract of red rice fermented with Monascus ruber IFO32318 were examined on the isolated rat aorta. The water phase of fermented rice with Monascus (WP/FRM, 0.1-10 mg/ml) caused a transient relaxation of the endothelium-intact rat aorta precontracted with norepinephrine (NE, 300 nM). The WP/FRM-induced relaxation was abolished by removal of endothelium or in the presence of N(G)-nitro-L-arginine (L-NNA, 10 microM), a nitric oxide (NO) synthase inhibitor. Neither atropine, a muscarinic receptor antagonist (10 microM), nor indomethacin, a cyclooxygenase inhibitor (10 microM), altered the WP/FRM-induced endothelium-dependent relaxation. gamma-Aminobutyric acid (GABA), one of the principle components of the extract, did not affect the muscle tension of the aorta with intact endothelium. In addition, WP/FRM increased the production of NO in primary cultured endothelial cells from human umbilical vein. The enhanced production of NO by WP/FRM was diminished by pretreatment with L-NNA (10 microM). In conclusion, WP/FRM induces relaxation of rat aorta by releasing NO from endothelium. There seem to be some unknown factor(s) other than acetylcholine (Ach) and GABA, in the aqueous extract of red rice, which stimulate vascular endothelial cells to produce and/or release NO leading to endothelium-dependent relaxation by WP/FRM.

Relationship between intratumor histological heterogeneity and genetic abnormalities in gastric carcinoma with microsatellite instability.

Microsatellite instability (MSI)-mutator phenotype variably targets microsatellite-like sequences in coding regions of cancer-related genes. Intratumor histological heterogeneity of gastric carcinoma with MSI was evaluated and found to be linked with the topographical distribution of MSI-associated mutations. One hundred fifty tumor sites derived from 51 gastric cancer patients were microdissected with respect to histological and topographical clonality. We found 11 gastric carcinomas with a high frequency of MSI, which were characterized by marked intratumor genetic heterogeneity arising from the progressive MSI-phenotype that was associated with frameshift mutations on multiple cancer-related genes. The 11 MSI-tumor cases manifested the MSI-phenotype in 34 of 36 tumor sites tested, but not in the remaining 2 sites. Most (88.2%, 30 of 34) MSI-positive sites and most (96.2%, 25 of 26) tumor sites harboring the frameshift mutations in transforming growth factor-beta receptor type II gene exhibited intestinal-type histology, whereas the 2 MSI-negative sites were found to be of diffuse-type histology without accompanying frameshift mutations. In 2 of 5 cases harboring E2F-4 frameshift mutations, glandular structures of intestinal-type tumor were likely to be variably differentiated in relation to the extent of the mutation, i.e., the number of mutated alleles and the size of deleted or inserted base pairs. Overall, the intratumor histological heterogeneity of gastric carcinoma with MSI was associated with the progressive frameshift mutations in transforming growth factor-beta receptor type II and E2F-4 genes.

The relationship between cisplatin-induced apoptosis and p53, bcl-2 and bax expression in human lung cancer cells.

OBJECTIVES: Given the roles of bcl-2, bax and p53 in apoptosis, we investigated the effect of their expression on the response to cisplatin in order to understand the molecular events of cisplatin-resistance in lung cancers. METHODS: Three parental human lung cancer cell lines (PC9, PC14 and H69) and their in vitro selected cisplatin-resistant sublines were examined. Cells treated with cisplatin were processed for acridine orange and ethidium bromide staining and DNA gel electrophoresis for the morphologic detection of apoptosis. The endogenous levels of bcl-2, bax and p53 protein expression in lung cancer cells were assessed by Western blot analysis and DNA of polymerase chain reaction-amplified exon 5 to 8 of p53 gene was directly sequenced. RESULTS: H69, which had bcl-2 expression, p53 mutation and decreased expression of p53 and bax, was relatively resistant to cisplatin and delayed and reduced apoptosis. Although apoptosis was markedly reduced in cisplatin-resistant sublines compared to their parental cells, there were no significant differences in the expression of p53, bcl-2 and bax. CONCLUSIONS: Cisplatin-resistance was associated with the reduced cellular susceptibility to apoptosis. Cancer cells with the natural expression of bcl-2 and p53 mutation may be more resistant to cisplatin and less susceptible to apoptosis.

Hypermethylation of the hMLH1 gene promoter in human gastric cancers with microsatellite instability.

Human gastric carcinoma shows a higher prevalence of microsatellite instability (MSI) than does any other type of sporadic human cancer. The reasons for this high frequency of MSI are not yet known. In contrast to endometrial and colorectal carcinoma, mutations of the DNA mismatch repair (MMR) genes hMLH1 or hMSH2 have not been described in gastric carcinoma. However, hypermethylation of the hMLH1 MMR gene promoter is quite common in MSI-positive endometrial and colorectal cancers. This hypermethylation has been associated with hMLH1 transcriptional blockade, which is reversible with demethylation, suggesting that an epigenetic mechanism underlies hMLH1 gene inactivation and MMR deficiency. Therefore, we studied the prevalence of hMLH1 promoter hypermethylation in a total of 65 gastric tumors: 18 with frequent MSI (MSI-H), 8 with infrequent MSI (MSI-L), and 39 that were MSI negative. We found a striking association between hMLH1 promoter hypermethylation and MSI; of 18 MSI-H tumors, 14 (77.8%) showed hypermethylation, whereas 6 of 8 MSI-L tumors (75%) were hypermethylated at hMLH1. In contrast, only 1 of 39 (2.6%) MSI-negative tumors demonstrated hMLH1 hypermethylation (P<0.0001 for MSI-H or MSI-L versus MSI-negative). Moreover, hypermethylated cancers demonstrated diminished expression of hMLH1 protein by both immunohistochemistry and Western blotting, whereas nonhypermethylated tumors expressed abundant hMLH1 protein. These data indicate that hypermethylation of hMLH1 is strongly associated with MSI in gastric cancers and suggest an epigenetic mechanism by which defective MMR occurs in this group of cancers.

Fractional allelic loss in gastric carcinoma correlates with growth patterns.

To gain an insight into the genetic events underlying morphological phenotypes, we analysed 58 gastric carcinoma tissues for the genome-wide allelotype study using microsatellite markers. Based on a binomial distribution, loss of heterozygosity (LOH) that was significantly more frequent than expected (P<0.05) thus interpreted as nonrandom LOH selected during tumorigenesis. The overall extent of chromosomes undergoing LOH i.e. fractional allelic loss (FAL, the ratio of LOH-positive markers to the total number of informative markers) was measured in each tumor patient. Nonrandom LOH was found on 17p (48.0%), 18q (38.4%), 13q (38.1%) and 9p (36.4%). Overall, there were no significant phenotypes correlated with allelic loss on specific chromosome regions. Based on a bimodal distribution of FAL values with two peaks bordered by a mean of 0.233, tumors were classified into LOH-related (>0.233) and LOH-unrelated (<0.233) types. Among 24 patients with LOH-related tumors, increase in the infiltrative type of growth pattern was found to correspond with a significant trend of increasing FAL values. This study shows that the growth pattern of gastric carcinoma is correlated with FAL, suggesting that a malignant phenotype is influenced by LOH event.

Genetic events underlying morphological complexity of gastric carcinoma.

Cancer is a genetic disorder in which gene alterations are selected to provide growth advantage by oncogene activation and/or tumor suppressor gene inactivation. Even marked intra-tumor variation in the histologic pattern, which is common in gastric carcinoma, is considered a result of distinct oncogenic pathways coexisting together. The present review describes that most gastric carcinomas arise through two distinct genetic pathways: microsatellite instability targeting the mononucleotide tracts within coding regions of cancer-related genes and chromosomal deletion involving tumor suppressor genes. With regard to malignant phenotypes, microsatellite instability is associated with the intestinal histological type and chromosomal deletion is correlated with the growth pattern of gastric carcinoma. Moreover, the genetic instability would in turn lead to an increase in alterations of cancer-related genes. The corresponding cells gradually manifest diverse neoplastic properties, thus bringing about consecutive subclonal evolution of more malignant cells. We now have some dues leading to the characterization of phenotypic complexity of gastric carcinoma based on gene-inactivation mechanisms.

Frequent mutation of the E2F-4 cell cycle gene in primary human gastrointestinal tumors.

The E2F group of transcription factors transactivates genes that promote progression through the G1-S transition of the cell cycle. Members of the retinoblastoma (Rb) family of proteins bind to E2Fs and inhibit this function. E2F-4, one example of the E2F group, functions as an oncogene when transfected into nontransformed cells in vitro. On the other hand, mice that are homozygously lacking a normal E2F-1 gene develop cancers, consistent with a tumor-suppressive role for this gene. The exact function of E2Fs has thus been unclear; moreover, direct involvement of this gene in primary human tumorigenesis has not been shown. We, therefore, investigated mutation within the E2F-4 coding region in 16 primary gastric adenocarcinomas, 12 ulcerative colitis-associated neoplasms, 46 sporadic colorectal carcinomas, 9 endometrial cancers, and 3 prostatic carcinomas. We limited our investigation to the serine repeat within E2F-4, reasoning that this tract might be altered in genetically unstable tumors (replication error-positive, or RER+). All tumors were RER+, with the exception of a control group of 15 RER- sporadic colorectal carcinomas. PCR with incorporation of [32P]dCTP was performed using primers flanking the serine trinucleotide (AGC) repeat. Twenty-two of 59 gastrointestinal tumors (37%) contained E2F-4 mutations; these comprised 5 of 16 gastric tumors (31%), 4 of 12 ulcerative colitis-associated neoplasms (33%, including 1 dysplastic lesion), and 13 of 31 sporadic colorectal cancers (42%). No mutation was present in any of the endometrial, prostate, or RER- colorectal tumors. Of note, homozygous mutations occurred in three cases, and two of seven informative patients showed loss of one E2F-4 allele in their tumors. Furthermore, the RER+ sporadic colorectal tumors were evaluated at trinucleotide repeats within the genes for N-cadherin and B-catenin; no tumors demonstrated mutation of these genes. These data suggest that E2F-4 is a target of defective DNA repair in these tumors.

Apparent protection from instability of repeat sequences in cancer-related genes in replication error positive gastrointestinal cancers.

Genomic instability at simple repeated sequences has been observed in various types of human cancers and is considered an important mechanism in tumorigenesis. Alterations at microsatellite loci have been reported scattered throughout the genome. Recently, the transforming growth factor-beta receptor type II (TGF-beta RII) and the insulin-like growth factor II receptor (IGF-IIR) genes were shown to have inactivating mutations within coding microsatellite sequences. The demonstration of mutations in two growth regulatory genes supports the idea that other regulatory genes with repeat sequences may also be targets in tumours with defective mismatch repair. We examined genes involved in tumour suppression, cell adhesion and cell cycle regulation for mutations at small repeat sequences in replication error positive gastrointestinal cancers. Several polymorphisms were found which exhibited instability, but no other instability was present in the regions examined.

Nucleotide sequencing of a part of the 5'-noncoding region of echovirus type 9 and rapid virus detection during the acute phase of aseptic meningitis.

A part of the 5'-noncoding region of echovirus type 9 isolates was sequenced, and an attempt was made for rapid virus detection in clinical samples obtained from 22 subjects hospitalized with aseptic meningitis. The sequence identity of 440-bp products amplified from the region by RT-PCR was 87.7% between the standard echovirus type 9(Hill strain) and the isolates. Specific IgM antibodies to Hill strain were positive in 45.5% by immunofluorescent antibody staining of virus-infected cells. A high detection rate of PCR products was observed in cerebrospinal fluids (CSFs; 54.5%) at admission, and in peripheral mononuclear cells (PMCs; 72.7%) at the end of hospitalization. Viral genomes were detectable for 2 days in serum samples, and for 6 days in PMC samples after onset of disease. When specific IgM antibody titers were less than 1:40, the amplification rate of viral genome from serum samples was 50.0%. These results indicate that the combination of specific IgM determination and viral genome amplification from CSFs will be a rapid and reliable method for early diagnosis.

A transforming growth factor beta 1 receptor type II mutation in ulcerative colitis-associated neoplasms.

BACKGROUND & AIMS: Numerous gastrointestinal tumors, notably sporadic and ulcerative colitis (UC)-associated colorectal carcinomas and dysplasias, gastric cancers, and esophageal carcinomas, manifest microsatellite instability. Recently, a transforming growth factor beta 1 type II receptor (TGF-beta 1RII) mutation in a coding microsatellite was described in colorectal carcinomas showing instability. One hundred thirty-eight human neoplasms (61 UC-associated, 35 gastric, 26 esophageal, and 16 sporadic colorectal) were evaluated for this TGF-beta 1RII mutation. METHODS: Whether instability was present at other chromosomal loci in these lesions was determined. In lesions manifesting or lacking instability, the TGF-beta 1RII coding region polydeoxyadenine (poly A) microsatellite tract was polymerase chain reaction amplified with 32P-labeled deoxycytidine triphosphate. Polymerase chain reaction products were electrophoresed on denaturing gels and exposed to radiographic film. RESULTS: Three of 18 UC specimens with instability at other chromosomal loci (17%) showed TGF-beta 1RII poly A tract mutation, including 2 cancers and 1 dysplasia; moreover, 2% of UC specimens without instability (1 of 43) (1 cancer), 81% of unstable sporadic colorectal cancers (13 of 16), and none of the 61 stable or unstable gastric or esophageal cancers contained TGF-beta 1RII mutations. CONCLUSIONS: Mutational inactivation of the poly A microsatellite tract within TGF-beta 1RII occurs early and in a subset of unstable UC neoplasms and commonly in sporadic colorectal cancers but may be rare in unstable gastric and esophageal tumors.

Evidence of genetic progression in human gastric carcinomas with microsatellite instability.

Mutator phenotype tumors provide unique opportunities to unravel malignant progression because of various gene alterations acquired during clonal tumor evolution. Gastric carcinomas, which have been known to show frequent genetic instability, would be composed of initial gene alterations shared by most tumor areas and subsequent alterations restricted to particular tumor sites. To analyse the timing of genetic events, we examined separate sites of tumor tissue obtained from a given gastric carcinoma patient with microsatellite instability (MSI). Our study included 95 normal/tumor area pairs from 25 patients. Six of the 25 patients (24%) demonstrated various levels of MSI ranging from 7% (two of 30) to 97% (28 of 29) of markers tested in multiple tumor sites. Of the six patients, five manifested frameshift mutations in a tract of ten deoxyadenosines within transforming growth factor beta receptor type II and four demonstrated frameshift mutations in a tract of eight deoxyguanosines within BAX. These mutations were common to all tumor sites regardless of the various level of MSI phenotype, indicating initial events. Two of the six patients exhibited frameshift mutations in mononucleotide repeats of mismatch repair genes, hMSH3 and hMSH6, and the insulin-like growth factor II receptor in restricted tumor areas, indicating additional alterations. Insulin-like growth factor II receptor mutations appear to be caused by hMSH3 and hMSH6 mutations because the former mutations were confined to tumor portions with the latter two mismatch repair lesions. These results provide genetic progression evidence for gastric carcinomas of the mutator pathway. In this pathway, mismatch repair insufficiency initially targets mononucleotide tracts of transforming growth factor beta receptor type II and BAX. During tumorigenesis, primary mismatch repair failure may give rise to the secondary mismatch repair lesions, frameshift mutations of hMSH3 and hMSH6, which result in another tumorigenic mutation in the insulin-like growth factor II receptor.

Microsatellite instability-associated mutations associate preferentially with the intestinal type of primary gastric carcinomas in a high-risk population.

Most colon cancers exhibiting microsatellite instability (MI), a mutator phenotype of mismatch repair failure, are associated with mutations of the transforming growth factor-beta receptor type II genes (TGF-beta RII). Of intestinal- and diffuse-type gastric carcinomas, the former have been thought to arise from intestinal metaplasia in which gastric mucosa resembles intestinal mucosa. To evaluate the preferential histological type of MI-associated mutations in the development of gastric carcinoma, mutations of TGF-beta RII, p53, and p16 were analyzed for the two types of primary gastric carcinomas showing MI. Of 50 primary gastric carcinomas, including 33 intestinal types and 17 diffuse types, 15 cases (30%) demonstrated MI at 1 or more of the 11 microsatellite markers tested. The 15 MI cases were classified into two groups, widespread MI and low-level MI, based on the number of markers exhibiting the instability. Eleven were widespread MIs, and the remaining four cases were low-level MIs. Ten of the 11 (91%) widespread MIs were of the intestinal type, and 1 case (9%) was of the diffuse type. Of the 11 widespread MIs, 10 cases (91%) demonstrated frameshift mutations within the polyadenylate tract of the TGF-beta RII. The frameshift mutation was rarely detected at p53 and p16 (1 of 11, 9%). In contrast, the four low-level MI cases had no frameshift mutations within the repeat sequences of TGF-beta RII, p53, and p16, but two of the four cases demonstrated base substitution mutations within p53. Our results suggest that mismatch repair failure can mutate the TGF-beta RII and may provide one of the pathways for the development of the intestinal-type gastric carcinoma in high-risk populations.

Alterations of transforming growth factor-beta 1 receptor type II occur in ulcerative colitis-associated carcinomas, sporadic colorectal neoplasms, and esophageal carcinomas, but not in gastric neoplasms.

BACKGROUND & AIMS: Gastric cancers, sporadic colorectal cancers, and ulcerative colitis (UC)-associated colorectal carcinomas and dysplasias manifest microsatellite instability (MI); however, esophageal carcinomas rarely exhibit MI. Recently, a transforming growth factor-beta 1 type II receptor (TGF-beta 1RII) mutation in a coding microsatellite was described in primary colorectal carcinomas demonstrating MI. No previous studies of TGF-beta 1RII have addressed mechanisms of inactivation other than MI in human tumors; furthermore, MI-negative tumors have not been examined for TGF-beta 1RII mutation. We evaluated 138 primary human neoplasms for mutation in the poly-A microsatellite tract of TGF-beta 1RII. Additionally, a group of esophageal tumors was evaluated for the expression of TGF-beta 1RII messenger RNA (mRNA). METHODS: First, we determined whether MI was present at other chromosomal loci in these lesions. The poly-deoxyadenine (poly-A) microsatellite tract within the TGF-beta 1RII coding region was then PCR-amplified. In a group of MI-negative esophageal tumors, RT-PCR was performed to determine the expression of TGF-beta 1RII mRNA. RESULTS: Among 17 MI+ UC specimens, 3 (18%) demonstrated TGF-beta 1RII poly-A tract mutation (2 cancers and 1 dysplasia), while 2 (4%) of 44 MI-negative UC specimens (1 dysplasia and 1 tumor), and 13 (81%) of 16 MI+ sporadic colorectal cancers, contained TGF-beta 1RII poly-A mutation. No gastric or esophageal tumors contained TGF-beta 1RII mutation. Among 21 MI-negative esophageal carcinomas. 6 cases (28.5%) had TGF-beta 1RII transcripts that were low or undetectable by RT-PCR. CONCLUSIONS: Mutation within the poly-A microsatellite tract of TGF-beta 1RII occurs early in a subset of UC-neoplasms and commonly in sporadic colorectal cancers, but may be rare in MI+ gastric tumors. Diminished expression of TGF-beta 1RII mRNA in esophageal tumors suggests that mechanisms of inactivation in this gene other than MI play a role in esophageal carcinogenesis.