Regulation of PTEN expression by the SWI/SNF chromatin-remodelling protein BRG1 in human colorectal carcinoma cells.

BACKGROUND: Aberrant expression of Brahma-related gene-1 (BRG1), a core component of the SWI/SNF chromatin-remodelling complex, has been implicated in cancer development; however, the biological significance of BRG1 in colorectal carcinoma (CRC) remains unknown. METHODS: In CRC tissues, expression of BRG1 and Brahma (BRM) was investigated immunohistochemically. Colorectal carcinoma-derived DLD-1 cells were used for knockdown of BRG1 and PTEN with small interfering RNA (siRNA) and transduction of Akt. Complementary DNA (cDNA) microarray analysis was performed to explore the genes affected by BRG1. RESULTS: Expression of BRG1, but not BRM, was frequently elevated in CRC specimens, and knockdown of BRG1 suppressed cell proliferation of DLD-1 cells. By cDNA microarray, we determined that PTEN expression was negatively regulated by BRG1 in DLD-1 cells, which subsequently influenced the cyclin D1 levels via the phosphoinositide 3-OH kinase (PI3K)-Akt signalling pathway. The interplay of BRG1 on cyclin D1 expression was confirmed by the introduction of Akt and knockdown of PTEN in the BRG1 siRNA-transduced DLD-1 cells. Interestingly, this positive correlation between BRG1 and cyclin D1 expression was also observed in CRC specimens. CONCLUSION: Brahma-related gene-1 has an important role in the process of CRC development by activating the PI3K-Akt signalling pathway and resultant upregulation of cyclin D1 levels.

Correction: Fhit modulation of the Akt-survivin pathway in lung cancer cells: Fhit-tyrosine 114 (Y114) is essential.

After publication of this Article the authors noticed errors in several figures. In Fig. 2b the Gapdh panels are incorrect. The lysates are identical to those used in Fig. 1b, therefore the Gapdh panels should be the same in both figures. In Fig. 3b the Gapdh panels for Ad-Fhit-wt and Ad-Fhit-Y114F are incorrect and have been replaced with scans from original films. In Fig. 4A the Gapdh panels are incorrect. The lysates are identical to those used in Fig. 3b, therefore the Gapdh panels should be the same in both figures. In Fig. 4Bb the Gapdh panels for Fhit siRNA were incorrect and have been replaced with scans from original films. All resupplied figures are provided below. In Fig. 5C several panels are incorrect. The Authors were unable to locate the original films for all of these panels so Fig. 5c has been deleted. The scientific conclusions of this paper have not been affected.

Hypermethylation-mediated reduction of WWOX expression in intraductal papillary mucinous neoplasms of the pancreas.

We have previously shown that WW domain-containing oxidoreductase (WWOX) has tumour-suppressing effects and that its expression is frequently reduced in pancreatic carcinoma. In this study, we examined WWOX expression in intraductal papillary mucinous neoplasm of the pancreas (IPMN) to assess the function of WWOX in pancreatic duct tumourigenesis using immunohistochemistry and methylation-specific polymerase chain reaction analysis. Among 41 IPMNs including intraductal papillary mucinous adenomas (IPMAs) and intraductal papillary mucinous carcinomas (IPMCs), loss or reduced WWOX immunoreactivity was detected in 3 (15%) of 20 IPMAs and 17 (81%) of 21 IPMCs. In addition, hypermethylation of the WWOX regulatory site was detected in 1 (33%) of 3 WWOX(-) IPMAs and 9 (53%) of 17 WWOX(-) IPMCs, suggesting that hypermethylation may possibly be important in the suppression of WWOX expression. Reduction of WWOX expression was significantly correlated with a higher Ki-67 labelling index but was not correlated with the ssDNA apoptotic body index. Interestingly, decreased WWOX expression was significantly correlated with loss of SMAD4 expression in these IPMNs. The results indicate that downregulation of WWOX expression by the WWOX regulatory region hypermethylation is critical for transformation of pancreatic duct.

Direct cancer-stromal interaction increases fibroblast proliferation and enhances invasive properties of scirrhous-type gastric carcinoma cells.

BACKGROUND: Scirrhous-type gastric carcinoma (SGC) exhibits an extensive submucosal fibrosis and extremely poor patient prognosis. We investigated the importance of the cancer-stromal interaction in the histogenesis of SGC. METHODS: Gastric fibroblasts NF-25 and intestinal fibroblasts NF-j2 were co-cultured with SGC-derived (HSC-39) or non-SGC-derived (HSC-57 and HSC-64) cells. To identify genes that are up- or downregulated in NF-25, complementary DNA (cDNA) microarray analysis was performed. The antibody against vascular-cell adhesion molecule-1 (VCAM-1) was used for cell growth test and immunohistochemistry. Moreover, the impact of interaction with NF-25 fibroblasts on HSC-39 cells was investigated using western blot and reverse transcription-polymerase chain reaction. RESULTS: HSC-39 cells stimulated growth of NF-25 but not NF-j2 when co-cultured. Induction of VCAM-1 in NF-25 fibroblasts was identified, which was specific when co-cultured with HSC-39 but not with non-SGC-derived HSC-57 and HSC-64 cells. Neutralising antibody to VCAM-1 suppressed NF-25 growth in dose-dependent manners. In tissue samples, positive immunoreactivity of VCAM-1 in SGC-derived fibroblasts was significantly higher than that in non-SGC-derived fibroblasts. Furthermore, interaction with NF-25 fibroblasts not only induced the epithelial-mesenchymal transition-like change, but also expressions of matrix metalloproteinase- related genes in HSC-39 cells. CONCLUSION: Direct interaction between SGC cells and gastric fibroblasts establishes the tumour microenvironment and reinforces the aggressiveness of SGC.

Fhit modulation of the Akt-survivin pathway in lung cancer cells: Fhit-tyrosine 114 (Y114) is essential.

The Fhit tumor suppressor binds and hydrolyses diadenosine polyphosphates and the Fhit-substrate complex has been proposed as a proapoptotic effector, as determined by infection of susceptible cancer cells with adenoviruses carrying wild-type fragile histidine triad (FHIT) or catalytic site mutants. The highly conserved Fhit tyrosine 114 (Y114), within the unstructured loop C-terminal of the catalytic site, can be phosphorylated by Src family tyrosine kinases, although endogenous phospho-Fhit is rarely detected. To explore the importance of Y114 and identify Fhit-mediated signaling events, wild-type and Y114 mutant FHIT-expressing adenoviruses were introduced into two human lung cancer cell lines. Caspase-dependent apoptosis was effectively induced only by wild-type but not Y114 mutant Fhit proteins. By expression profiling of FHIT versus mutant FHIT-infected cells, we found that survivin, an Inhibitor of Apoptosis Protein (IAP) family member, was significantly decreased by wild-type Fhit. In addition, Fhit inhibited activity of Akt, a key effector in the phosphatidylinositol 3-OH kinase (PI3K) pathway; loss of endogenous Fhit expression caused increased Akt activity in vitro and in vivo, and overexpression of constitutively active Akt inhibited Fhit-induced apoptosis. The results indicate that the Fhit Y114 residue plays a critical role in Fhit-induced apoptosis, occurring through inactivation of the PI3K-Akt-survivin signal pathway.

High PRL-3 expression in human gastric cancer is a marker of metastasis and grades of malignancies: an in situ hybridization study.

Phosphatase of regenerating liver (PRL)-3, encoding a 22-kD low molecular weight tyrosine phosphatase, has been reported to be associated with metastasis of colorectal carcinoma. We assessed the levels of PRL-3 mRNA expression to know whether its up-regulation was involved in progression and metastasis of gastric carcinoma. Levels of PRL-3 expression in 94 human gastric adenocarcinomas and 54 matched lymph node metastases were detected by in situ hybridization and compared with clinicopathological characteristics including prognosis. High PRL-3 expression was detected in 36.2% of primary gastric carcinoma (with nodal metastasis, 55.6%; without nodal metastasis, 10%; P < 0.001) and in 74.1% of lymph node metastases. The incidence of high PRL-3 expression in lymph node metastasis was significantly higher than in primary tumors (P < 0.044). Moreover, high expression of PRL-3 was closely associated with tumor size, lymphatic invasion, venous invasion, extent of lymph node metastasis, and tumor stage. These results suggest that high PRL-3 expression may participate in the progression and metastasis of gastric carcinoma. PRL-3 might be a novel molecular marker for aggressive gastric cancer.

Rapid and long-term effects of PTH(1-34) on growth plate chondrocytes are mediated through two different pathways in a cell-maturation-dependent manner.

The aims of this study were to clarify the role of cell maturation stage on chondrocyte response to parathyroid hormone (PTH) by examining the effect of PTH(1-34) on alkaline-phosphatase-specific activity (ALPase) of chondrocyte cultures at two distinct stages of maturation, and to determine the signaling pathways used by the cells to mediate this effect. Confluent, fourth passage rat costochondral resting zone (RC) and growth zone (GC) chondrocytes were used. ALPase was measured in the cell layer, as well as in matrix vesicles (MV) and plasma membranes (PM), after the addition of 10(-7) 10(-11) mol/L bovine PTH(1-34), the active peptide, or bovine PTH(3-34), the inactive peptide, to the cultures. PTH(1-34) increased ALPase in the GC cultures at two separate times: between 5 and 180 min, with maximal stimulation at 10 min, and 36 to 48 h. In contrast, PTH(3-34) had no effect. At 10 min and 48 h, PTH(1-34) produced a dose-dependent increase in ALPase of both MV and PM isolated from GC cultures. Addition of forskolin and IBMX to increase cAMP increased ALPase in GC cultures to a level similar to that seen after addition of PTH(1-34). In contrast, the addition of PTH(1-34) to RC cells only increased ALPase between 5 and 60 min, with peak activity at 10 min. As with GC, PTH increased ALPase in both MV and PM. Moreover, the addition of PTH(3-34) or forskolin and IBMX had no effect on ALPase in RC. PTH(1-34) had no effect on GC protein kinase C (PKC) activity; however, the addition of PTH(1-34) to RC caused a dose-dependent increase in PKC activity. H8, an inhibitor of PKA, had no effect on PTH-stimulated ALPase in RC cells, but inhibited the PTH-dependent response in GC cells. In contrast, chelerythrine, an inhibitor of PKC activity, inhibited PTH-stimulated ALPase in RC cells, but had no effect on PTH-stimulated ALPase in GC cells. This study shows that the effect of PTH(1-34) on RC and GC cells is maturation dependent in terms of time course and mechanism. Whereas both cell types exhibit a rapid response to PTH, only GC cells show a long-term response. In GC, the effects of PTH are associated with changes in cAMP and may also involve at least one other pathway, whereas, in RC, the PTH effects appear to be associated with changes in PKC.

Molecular biological observations in gastric cancer.

Alterations in the structure and function of oncogenes and tumor suppressor genes, as well as genetic instability at several other genetic foci may be responsible for stomach carcinogenesis. The particular combination of multiple gene changes found in gastric cancer differs depending on the two histological types, strongly indicating that different genetic pathways exist for well differentiated or intestinal type and poorly differentiated or diffuse type gastric cancers. In general, genetic instability, telomerase activity, CD44 abnormal transcripts, and p53 mutation, all of which are common events of two types of gastric cancer, may be involved mainly in the early stage of stomach carcinogenesis, whereas activation of oncogenes and overexpression of the epidermal growth factor-related growth factor system may chiefly confer progression on gastric cancer. A new strategy of molecular diagnosis of gastrointestinal cancer, which has been implemented as a routine service in the Hiroshima University Clinical Laboratory, may provide new opportunities for early cancer diagnosis and more accurate evaluation of prognosis or grade of malignancy.

Frequent microsatellite instability and loss of heterozygosity in the region including BRCA1 (17q21) in young patients with gastric cancer.

It is known that nearly 5% of gastric carcinomas arise under the age of 40. To elucidate genetic alterations in these patients, we performed studies using microsatellite assay in 27 gastric cancers under 35 years of age, composed of 5 well and 22 poorly differentiated adenocarcinomas. We detected replication errors (RERs) in 18 (67%) of 27 tumors, but no germline mutation in DNA mismatch repair genes (hMLH1 and hMSH2), except fory 3 somatic mutations in the hMLH1 gene. Loss of heterozygosity (LOH) at D17S855, located on chromosome 17q21 (BRCA1), was detected in 8 (40%) of 20 informative cases. In 12 (44%) of 27 cases, LOH on chromosome 17q12-21 including the BRCA1 was found in several neighboring markers in this region, while no mutation was found in the BRCA1 gene. Four (40%) of 10 scirrhous type gastric cancers exhibited wide allelic deletions on chromosome 17q12-21. These results overall suggest that young gastric cancer patients display highly frequent micro-satellite instability that might be due to defect of DNA repair system rather than hMLH1 and hMSH2. In addition, chromosome 17q12-21 including BRCA1 locus may contain a candidate for tumor suppressor gene, particularly in scirrhous type gastric cancers arising in young patients.

Analysis of the candidate target genes for mutation in microsatellite instability-positive cancers of the colorectum, stomach, and endometrium.

Microsatellite instability (MSI) in human carcinoma DNA is a characteristic phenotype observed in hereditary non-polyposis colorectal cancer and also in some human sporadic cancers including multiple primary carcinomas. In this study, we analyzed mutations in the hCHK1, E2F4, hMSH3, and hMSH6 genes in MSI+ human cancers arising in colorectum, stomach and endometrium. The E2F4 and hMSH3 genes were mutated in all tumor types. Interestingly, the hMSH6 gene was mutated in colorectal and gastric cancers but not in endometrial cancer; this is similar to the TGFbetaRII gene. It is notable that the mutation status of the secondary mutators, hMSH3 and hMSH6, did not influence slippage-related frameshift mutations in genes harboring simple tandem-repeats, which suggests that the MSI phenotype may be affected mainly by abnormalities in the primary mutator genes, not by the secondary mutator genes. No mutations were observed in the cell cycle checkpoint gene hCHK1; mutations of this gene are thought to have a limited role, if any, in at least the tumor types analyzed in this study.

Microsatellite instabilities in gastric cancer patients with multiple primary cancers.

To answer whether microsatellite instability (MSI) can serve as a molecular marker for the risk-assessment of the development of multiple cancers, 26 tumors from 10 gastric cancer cases with multiple primary cancers were investigated. Six out of 10 cases revealed MSI in one or more cancer DNA. Significant statistical association was observed between MSI positive gastric cancer and cancer multiplicity (chi2 test, P<0.05). The complicated primary tumors in MSI-positive cases arose in colorectum, urogenital tract and ovary, which mimicked the tumor spectrum of hereditary non-polyposis colorectal cancer (HNPCC). On the other hand, most of the multiple cancers in MSI negative group were found synchronously and limited to the digestive organs. These observations suggest that MSI test on gastric cancer may be considered as a good marker for the assessment of multiple cancer development especially in the sites where tumors of HNPCC usually develop.

Not hMSH2 but hMLH1 is frequently silenced by hypermethylation in endometrial cancer but rarely silenced in pancreatic cancer with microsatellite instability.

Disruption of the DNA mismatch repair (MMR) system has been found to play an important role in sporadic human cancers of several organs such as colorectum, stomach, endometrium, and pancreas. In cancers of the former three organs, disruption of the MMR system is mainly caused by hypermethylation of the hMLH1 gene. We investigated the expression of the hMLH1 and hMSH2 proteins immunohistochemically in pancreatic and endometrial cancers with high frequency microsatellite instability (MSI-H). Loss of expression of hMLH1 was found in none of seven pancreatic cancer, whereas eight (57%) of 14 endometrial cancer showed loss of expression of hMLH1. On the other hand, one (14%) of seven pancreatic cancers and two (14%) of 14 endometrial cancers showed loss of hMSH2 expression. We further analyzed the methylation status at the promoter region of the hMLH1 and hMSH2 genes and found hypermethylation of hMLH1 at the promoter region in the great majority of endometrial cancers with loss of expression. However, no pancreatic cancer showed hypermethylation. We then further analyzed 22 pancreatic cancer cell lines and obtained similar results. These results suggested that MSI-H in pancreatic cancer is probably caused by different mechanisms from those of other sporadic cancers with MSI-H.

Expression of cyclin E in colorectal adenomas and adenocarcinomas: correlation with expression of Ki-67 antigen and p53 protein.

The expression of cyclin E in human colorectal adenomas and adenocarcinomas was examined immunohistochemically to elucidate the role of cyclin E in the colorectal carcinogenesis. The expression of cyclin E was detected in 25% (91/358) of the adenomas and 56% (149/267) of the adenocarcinomas. The incidence of strongly positive cases was significantly higher in the adenocarcinomas (20%) than in the adenomas (5%) (P < 0.01). Among adenomas, a significant correlation was noticed between the expression of cyclin E and the grade of atypia. The incidence of cyclin E expression was significantly higher in the adenocarcinomas without an adenoma component (62%; 104/169) than in those with this component (46%; 45/98) (P < 0.05). Furthermore, the incidence of the cyclin E expression was higher in stages 1 and 2 carcinoma than in stage 0 and stages 3 and 4 carcinoma. The expression of cyclin E was the most prominent in tumors invading the submucosa and muscularis propria. The expression of cyclin E was significantly correlated with the proliferative activity of the tumor cells measured by Ki-67 antigen expression (P < 0.01). It was also correlated with the expression of p53 protein in the tumor cells (P < 0.01). Overexpression of cyclin E and subsequent deregulation of cell cycle may contribute to the development and early progression of the colorectal carcinomas.

Altered microsatellites in incomplete-type intestinal metaplasia adjacent to primary gastric cancers.

AIM: To investigate the presence of genetic instability in precancerous lesions of the stomach. METHODS: Fifteen cases of sporadic gastric cancers with a background of intestinal metaplasia were studied by microsatellite assay at nine loci. Altered metaplastic mucosa was microdissected, reconstructed topographically, and examined immunohistochemically with an anti-p53 antibody, comparing its positive area with foci of microsatellite instability in each individual. RESULTS: Alterations at one or more loci were observed in seven of 15 cancers (46.7%) and four of 15 intestinal metaplasias (26.7%). Two cases of replication error positive phenotype had no microsatellite alterations in their metaplastic mucosa. All the microsatellite alterations in the metaplastic mucosa were restricted to incomplete-type intestinal metaplasia around the respective cancers. Moreover, in one case, an identical pattern of microsatellite alteration was detected in the cancer tissue and in the adjacent metaplastic mucosa, suggesting the sequential development of gastric cancer from intestinal metaplasia. Frequent alteration was found at the locus D1S191 (1q), indicating that this locus might be altered early in the development of intestinal-type gastric cancer. No significant association between microsatellite instability and p53 immunoreactivity was observed in the cases examined. CONCLUSION: These results indicate that microsatellite instability may be an early event in stomach carcinogenesis, especially in intestinal-type cancers.

Expression of cyclin D1, CDK4 and p27KIP1 is associated with the p16MTS1 gene status in human esophageal carcinoma cell lines.

p16MTS1/INK4A negatively regulates cell cycle progression by inhibiting the cyclin D/CDK4 complex that phosphorylates pRb. Frequent homozygous deletions of the p16 gene were recently found in various tumor cell lines. We examined the relationship between the genetic status of p16 and the expression of the cell cycle regulating molecules in human esophageal carcinoma cell lines. Out of eight human esophageal carcinoma cell lines, seven (67.5%) and six (75%) cell lines showed homozygous deletions of the p16 and p15 genes, respectively. All the p16-negative cell lines expressed high levels of cyclin D1, CDK4 and p27KIP1 proteins. Interestingly, the expression level of cyclin D1 was closely correlated to the levels of not only CDK4 but also p27KIP1 protein in p16-negative cell lines. Furthermore, all the p16-negative cell lines expressed Rb protein of approx 110 kDa which corresponds to the phosphorylated form, whereas the cell line with intact p15 and p16 genes did not express pRb. These results suggest that the expression of cyclin D1, CDK4, Rb and p27 is associated with the p16 gene status in esophageal carcinoma cell lines. Alternatively, loss of the p16 gene and subsequent over-expression of cyclin D1 and CDK4 might be involved in autonomous growth of esophageal carcinoma cells.

Expression of cyclin E in human gastric adenomas and adenocarcinomas: correlation with proliferative activity and p53 status.

The expression of cyclin E in human gastric adenomas and adenocarcinomas was examined immunohistochemically to elucidate the role of cyclin E in stomach carcinogenesis. The expression of cyclin E was detected in 49% (90/182) of the adenomas and 59% (260/439) of the adenocarcinomas. The incidence of strongly positive cases (overexpression of cyclin E) was significantly higher in the adenocarcinomas (29%; 128/439) than in the adenomas (4%; 8/182) (p < 0.01). The incidence of the cyclin E expression showed a tendency to be higher in deeply invasive carcinomas and in the cases with lymph node metastasis, while the incidence did not differ among histological types. The expression of cyclin E was significantly correlated with the proliferative activity of the tumor cells measured by KI-67 antigen expression (p < 0.01). It was also correlated with the abnormal accumulation of p53 protein in the tumor cells (p < 0.01). These results suggest that overexpression of cyclin E and subsequent deregulation of the cell cycle may confer the development and progression of the gastric carcinomas.

A splice variant of the c-met proto-oncogene is the predominant population expressed in human gastric mucosa and carcinoma.

Difference in the expression of the c-met protooncogene transcription variants in human gastric mucosa and cancer was studied by deoxynucleotide sequencing and cloning-restriction fragment length assay of the PCR products amplified with the specific primer set to membrane proximal extracellular domain of the c-met c-DNA. Fight gastric cancer cell lines, five gastric carcinoma tissues as well as their corresponding non-neoplastic gastric mucosas had the 54 bp- form splice variant of the c-met which encodes mature 190 kDa alpha beta heterodimeric protein as the major transcript population and 54 bp+ form variant which encodes 170 kDa protein, which is distinct from the met precursor protein, could not be detected. These results indicate that 190 kDa alpha beta heterodimeric protein encoded by major 54 bp- form c-met transcript mainly mediates the effect of HGF/SF in the stomach.

Mutational analysis of the CTNNB1 (beta-catenin) gene in human endometrial cancer: frequent mutations at codon 34 that cause nuclear accumulation.

Recently, CTNNB1 (beta-catenin) has been found to function as an oncoprotein that works in the Wnt signaling pathway, and mutation of this gene has been reported in various human cancers. In this study, we analyzed 44 endometrial cancers and found somatic missense mutations in five (11%) tumors. Interestingly, four (80%) of the five tumors with mutations would cause amino acid alterations at residues next to Ser 33, one of the targets for phosphorylation of glycogen synthase kinase (GSK)-3beta. The tumors with mutations showed accumulation of the CTNNB1 protein in cytoplasm and nucleus. This is the first report of frequent somatic mutation of the CTNNB1 gene at codons adjacent to those encoding to Ser/Thr residues in endometrial cancer.

Infrequent somatic mutations of the p73 gene in various human cancers.

AIMS: It has already been reported that loss of heterozygosity (LOH) on chromosome 1p is frequent in a variety of human cancers. This finding implies the presence of some important tumour suppressor genes in this region. p73, a candidate tumour suppressor gene identified recently in chromosome band 1p36.33, encodes a protein highly homologous to p53. To investigate the role of the p73 gene in human carcinogenesis, we studied genetic alterations of this gene in various human cancers. METHODS: We analysed the entire coding exons as well as their surrounding exon-intron boundaries of the p73 gene in 185 cases of various types of tumours (47 breast cancers, 43 colorectal cancers, 31 gastric cancers, 23 neuroblastomas, 21 lung cancer cell lines, and 20 pancreatic cancer cell lines); they are known as a group of tumours with frequent LOHs in the 1p region. PCR-SSCP analysis was performed and tumours in which aberrant migrating sized bands were observed were subjected to direct sequencing analyses. RESULTS: Of the 185 cases, only one somatic mis-sense mutation of glutamine from arginine at codon 269 in exon 7 was found in one breast cancer. In addition, several polymorphisms were found at codons 137, 336, 349, and 610, as well as in introns 6, 8, and 9. Monoallelic expression was also observed in pancreatic cancer cell lines. CONCLUSIONS: Our results suggest that inactivation of the p73 gene does not play a major role in the tumour types analysed in the present study.