Toshihiko Tsukamoto

Involvement of a Cellular Glycolytic Enzyme, Phosphoglycerate Kinase, in Sendai Virus Transcription

In vitro mRNA synthesis of Sendai virus is almost entirely dependent on the addition of cellular proteins (host factors). Previous studies indicated that the host factor activity from bovine brain was resolved into at least two complementary fractions, one of which may be tubulin. In this study, the host factor activity that stimulates the transcription in the presence of tubulin was further purified from bovine brain. This fraction was found to contain at least two complementary factors, and one of them was purified to a single polypeptide chain with an apparentM r of 46,000 (p46). From the amino acid sequence, biochemical, and immunological analyses, p46 was identified as a glycolytic enzyme, phosphoglycerate kinase (PGK). Purified native PGK from rabbit and yeast, and a recombinant human PGK substituted for p46. Although, as previously suggested, tubulin was involved in the transcription initiation complex formation by being integrated into the complex, p46 and its complementary factor had little effect on the complex formation. On the other hand, when p46 and the complementary factor were added to the RNA chain elongation reaction from the isolated initiation complex formed with tubulin, mRNA synthesis was dramatically stimulated. The enzymatic activity per se of PGK did not seem to be required for its activity. West-Western blot analysis showed that PGK could directly interact with tubulin. These data suggest that PGK stimulates Sendai virus mRNA synthesis at the elongation step, probably through its interaction with tubulin in the initiation complex.

Densely methylated MLH1 promoter correlates with decreased mRNA expression in sporadic colorectal cancers

It has been reported that MLH1 is silenced by promoter methylation, and that this phenomenon is associated with microsatellite instability (MSI) in sporadic colorectal cancer (CRC). To clarify the significance of MLH1 promoter methylation in sporadic CRC, we examined the correlation between methylation status over the entire promoter region and mRNA expression in cases showing high‐frequency MSI (MSI‐H). MLH1 promoter methylation was analyzed using the bisulfite modification sequencing in 48 MSI‐H cases. We also screened for somatic mutation, loss of heterozygosity, and immunohistochemical staining of MLH1. The results showed that methylation patterns could be subdivided into three types: methylation of more than 80% of the CpG sites analyzed (type 1 methylation), methylation of less than 20% (type 2 methylation), and methylation mainly in the region 500 to 921 bases upstream from the translation start site (type 3 methylation). Of the three types, only type 1 methylation correlated with decreased mRNA expression. The frequency of type 1 methylation was significantly higher in cases involving the proximal colon (66.7%, 18/27) compared to that of the distal colon and rectum (23.8%, 5/21, P = 0.004). Immunohistochemical staining of MSI‐H cases showed that decreased MLH1 was found in 77.1% (37/48). Of the cases with decreased MLH1, type 1 methylation was present in 59.5% (22/37). Overall, our data suggested that the type 1 methylation pattern may affect MLH1 mRNA expression, such that the majority of MSI‐H cases in sporadic CRC, especially proximal colon cancer, exhibited type 1 methylation.

Frequent activation of the ?-catenin-Tcf signaling pathway in nonfamilial colorectal carcinomas with microsatellite instability

It has been reported that wild‐type APC protein forms a complex with β‐Catenin and GSK3β, inducing degradation of β‐Catenin in normal cells. Both β‐Catenin and APC gene mutations have recently been shown to activate the same signaling pathway. Frequent mutations of β‐Catenin in hereditary nonpolyposis colorectal carcinomas have also been reported. It was, however, controversial whether the mutation of the β‐Catenin gene was frequent in nonfamilial colorectal carcinomas with high‐frequency microsatellite instability (MSI‐H). We analyzed the mutations of the APC and β‐Catenin genes in 56 nonfamilial colorectal carcinomas stratified according to the presence or absence of microsatellite instability (MSI). APC mutations were identified in 11 of 22 (50%) cases of MSI‐H and 14 of 34 (41%) cases of microsatellite‐stable (MSS)/low‐frequency microsatellite instability (MSI‐L). In contrast, the frequency of β‐Catenin mutations was significantly higher in MSI‐H (6/22; 27%) than in MSS/MSI‐L (1/34; 3%) (P = 0.01). β‐Catenin mutations were not detected in carcinomas with APC mutation. APC mutation occurred irrespective of MSI status. β‐Catenin mutation, however, occurred frequently in MSI‐H carcinomas. Our data suggest that activation of the β‐Catenin‐Tcf signaling pathway, through either β‐Catenin or APC mutation, frequently contributes to MSI‐H nonfamilial colorectal carcinomas (17/22; 77%).

Ki -ras point mutation in different types of colorectal carcinomas in early stages

PURPOSE: The aim of this study was to elucidate pathways of carcinogenesis in the colon and rectum by investigating Ki-raspoint mutation in different types of colorectal carcinomas in the early stage. METHODS: We analyzed rates of Ki-rascodon 12 mutations in 34 small, polypoid-type carcinomas (Tis or T1), 21 superficial-type carcinomas (Tis or T1), and 42 advanced carcinomas (T2, T3, and T4). RESULTS: Frequency of Ki-rasmutations in superficial-type carcinomas was 14.3 percent (3/21), which was significantly lower than 50 percent (17/34) in small polypoid carcinomas and 40.5 percent (17/42) in advanced carcinomas. These data suggest that another pathway of colorectal carcinogenesis that does not involve Ki-raspoint mutation might exist. Among the 17 small polypoid carcinomas with Ki-raspoint mutation in which both adenomatous and carcinomatous tissue were examined, 12 showed a mutation of the same type in both carcinomatous and adenomatous tissues. In two cases, mutation was present only in carcinomatous tissue and not in adenomatous tissue; in the other three cases, Ki-raspoint mutation was present only in adenomatous tissue but not in carcinomatous tissue. CONCLUSIONS: These data suggest that carcinoma in a small polypoid lesion does not always develop from pre-existing adenoma with Ki-raspoint mutation; in a small number of the polypoid-type early carcinomas, polyclonal composition concerning the Ki-rasgene may exist.

Clinicopathological characteristics of colorectal cancers with loss of imprinting of insulin-like growth factor 2

Loss of imprinting (LOI), the biallelic expression of an imprinting gene, of insulin‐like growth factor 2 (IGF2) has been reported to be associated with colorectal carcinogenesis because of its high prevalence in normal colorectal mucosa as well as cancerous tissue in patients with colorectal cancer. However, the characteristics of colorectal cancer associated with IGF2 LOI have not been clearly demonstrated. In this study, we investigated the IGF2 LOI status of tumor and normal mucosa in 255 consecutive patients with colorectal cancer. Of these, 95 were informative for IGF2 LOI, by direct sequencing of mRNA of IGF2. Regarding the LOI status in each patient, the prevalence of LOI in nontumorus normal mucosa was significantly higher in cases with LOI‐positive cancer than in those with LOI‐negative cancer (p < 0.001). Concerning the clinicopathological characteristics of LOI‐positive cancer, the prevalence of poorly differentiated or mucinous carcinoma (p = 0.016) and of right‐sided locations (p = 0.009) were significantly higher than those of LOI‐negative cancer. Contrary to past reports that revealed a significant correlation between microsatellite instability (MSI) and IGF2 LOI in a relatively small series of noncohort patients, our study did not find a statistically significant difference in LOI‐positive rate between MSI‐positive and ‐negative cases. Our results suggested the presence of a particular type of colorectal cancer associated with the proximal colon and poor differentiation, but independent of MSI. These results may contribute to clarification of the mechanism of colorectal tumorigenesis and to determining an appropriate screening strategy for colorectal carcinoma.

Significance of microsatellite instability in different types of early-stage nonfamilial colorectal carcinomas.

PURPOSE: The aim of this study was to investigate the genetic alterations of early-stage nonfamilial colorectal carcinomas regarding microsatellite instability, with special reference to the shape of the tumors and the site of the lesions. METHODS: Formalin-fixed, paraffin-embedded specimens of 44 early-stage nonfamilial colorectal carcinomas were examined for microsatellite instability with use of polymerase chain reaction. RESULTS: The 44 carcinomas consisted of 16 flat carcinomas and 28 polypoid carcinomas. Nineteen carcinomas were located in the proximal colon (9 flat type and 10 polypoid type), whereas 25 were in the distal colon and rectum (7 flat type and 18 polypoid type). Ten (22.7 percent) of the 44 carcinomas had at least one positive locus, whereas five (11.4 percent) of them had two or more positive loci. In the proximal colon the percentage of flat carcinomas with at least one positive locus was significantly greater than that of the polypoid carcinomas (4/9 (44 percent)vs. 0/10;P=0.04). Six patients had synchronous or metachronous colorectal carcinomas or both. They harbored microsatellite instability more frequently than patients with single colorectal carcinomas, and the differences were statistically significant (P<0.02). CONCLUSIONS: These data suggest that in nonfamilial carcinomas in the proximal colon, the genetic pathway in flat carcinomas may be different from that in polypoid carcinomas.

cDNA array analysis for prediction of hepatic metastasis of colorectal carcinoma

PurposeDistant metastasis is a significant prognostic factor of colon carcinoma. Adjuvant chemotherapy has been shown to decrease its recurrence. However, there are no definitive methods for the diagnosis of hepatic recurrence after potentially curative surgery. The aim of this study was to evaluate the accuracy of mRNA expression profiling using samples obtained from primary tumors to predict hepatic recurrence.MethodsPatients with stage III colorectal carcinoma without any recurrence for at least 5 years (group A: n = 9) and patients with stage IV carcinoma with hepatic metastasis (group B: n = 10) were included in this study. Tissue samples were collected from the primary tumor and adjacent normal colonic mucosa at the time of surgery in each patient. Total RNA was extracted and the mRNA expression profile was examined using a cDNA macroarray.ResultsA hierarchical clustering analysis revealed a dendrogram in which the patients were divided into two clusters. One cluster consisted of seven patients in group A and two in group B. The other consisted of two patients in group A and eight in group B. Therefore, the positive and negative predictive value of hierarchical clustering analysis for hepatic metastasis was 80.0% and 78.8%, respectively. Fifteen genes were revealed to be upregulated and 12 were downregulated in group B. The upregulated genes included CCNA2, TP53, and MDM2, while the downregulated genes included CDH1, GADD45A, and BCL2L2.ConclusionsmRNA expression profiling by a cDNA array analysis of specimens obtained from primary tumors was found to be useful for distinguishing patients with and without hepatic metastasis. This method is expected to contribute to the identification of patients at high risk for hepatic recurrence, while also helping in the administration of intensive adjuvant chemotherapy for such high risk patients.

Overexpression in colorectal carcinoma of two lysosomal enzymes, CLN2 and CLN1, involved in neuronal ceroid lipofuscinosis

Lysosomal proteases are implicated in cancer progression and metastasis. In the current study, using subtraction cloning for genes that are differentially expressed in metastasis, the authors isolated a clone encoding ceroid lipofuscinosis, neuronal 2 (CLN2), which is a lysosomal serine protease defective in neuronal ceroid lipofuscinosis (NCL). Increased CLN2 activity has been reported in breast carcinoma and the antiapoptotic effect of another causative gene of NCL, ceroid lipofuscinosis, neuronal 1 (CLN1), is known.