Fumio Itoh

p53-inducible Wip1 phosphatase mediates a negative feedback regulation of p38 MAPK-p53 signaling in response to UV radiation

The stress‐responsive p38 MAPK, when activated by genotoxic stresses such as UV radiation, enhances p53 activity by phosphorylation and leads to cell cycle arrest or apoptosis. Here we report that a member of the protein phosphatase type 2C family, Wip1, has a role in down‐regulating p38‐p53 signaling during the recovery phase of the damaged cells. Wip1 was originally identified as a gene whose expression is induced following γ or UV radiation in a p53‐dependent manner. We found that Wip1 is also inducible by other environmental stresses, such as anisomycin, H2O2 and methyl methane sulfonate. UV‐induction of Wip1 requires p38 activity in addition to the wild‐type p53. Wip1 selectively inactivates p38 by specific dephosphorylation of its conserved threonine residue. Furthermore, Wip1 expression attenuates UV‐induced p53 phosphorylation at Ser33 and Ser46, residues previously reported to be phosphorylated by p38. Wip1 expression also suppresses both p53‐mediated transcription and apoptosis in response to UV radiation. These results suggest that p53‐dependent expression of Wip1 mediates a negative feedback regulation of p38‐p53 signaling and contributes to suppression of the UV‐induced apoptosis.

Epigenetic inactivation of CHFR in human tumors

Cell-cycle checkpoints controlling the orderly progression through mitosis are frequently disrupted in human cancers. One such checkpoint, entry into metaphase, is regulated by the CHFR gene encoding a protein possessing forkhead-associated and RING finger domains as well as ubiquitin–ligase activity. Although defects in this checkpoint have been described, the molecular basis and prevalence of CHFR inactivation in human tumors are still not fully understood. To address this question, we analyzed the pattern of CHFR expression in a number of human cancer cell lines and primary tumors. We found CpG methylation-dependent silencing of CHFR expression in 45% of cancer cell lines, 40% of primary colorectal cancers, 53% of colorectal adenomas, and 30% of primary head and neck cancers. Expression of CHFR was precisely correlated with both CpG methylation and deacetylation of histones H3 and H4 in the CpG-rich regulatory region. Moreover, CpG methylation and thus silencing of CHFR depended on the activities of two DNA methyltransferases, DNMT1 and DNMT3b, as their genetic inactivation restored CHFR expression. Finally, cells with CHFR methylation had an intrinsically high mitotic index when treated with microtubule inhibitor. This means that cells in which CHFR was epigenetically inactivated constitute loss-of-function alleles for mitotic checkpoint control. Taken together, these findings shed light on a pathway by which mitotic checkpoint is bypassed in cancer cells and suggest that inactivation of checkpoint genes is much more widespread than previously suspected.

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. Int. J. Cancer 83:309–313, 1999.

Smad‐dependent GADD45β expression mediates delayed activation of p38 MAP kinase by TGF‐β

Transforming growth factor‐β (TGF‐β), when bound to its specific receptor, activates the transcription factor Smad by phosphorylation. TGF‐β also activates the p38 MAPK pathway, but there seem to be disparate mechanisms for the early p38 activation and delayed p38 activation. In this report, we demonstrate that Smad‐dependent expression of GADD45β is responsible for the delayed activation of p38 by TGF‐β. The GADD45β protein binds and activates MTK1 (= MEKK4), which is a member of the MAPKKK family kinases and an upstream activator of the p38 MAPK cascade. Both TGF‐β‐induced GADD45β expression and the delayed p38 activation require functional Smad proteins. Antisense inhibition of GADD45β expression suppresses the TGF‐β‐induced delayed p38 activation, whereas overexpression of GADD45β activates the p38 MAPK via MTK1. Expression of the angiogenesis inhibitor thrombospondin‐1 (TSP‐1) is induced by TGF‐β via Smad‐dependent p38 activation. Thus TGF‐β‐induced p38 activation, mediated by GADD45β expression, may play an important role in the biological effects of TGF‐β.

Association of trypsin expression with recurrence and poor prognosis in human esophageal squamous cell carcinoma

Overexpression of the matrix serine proteinase (MSP) trypsin has been implicated in tumor growth, invasion, and metastasis. The objective of this study was to clarify the clinicopathologic and prognostic significance of trypsin expression in esophageal squamous cell carcinomas (SCC).

Relation of enhanced secretion of active matrix metalloproteinases with tumor spread in human hepatocellular carcinoma

BACKGROUND & AIMSnMatrix metalloproteinases have been implicated in invasion and metastasis of various human malignant tumors, but its role in human hepatocellular carcinoma has not been characterized in detail. The aim of the present study was to examine the secretion and activation of metalloproteinases in liver tissues from patients with hepatocellular carcinoma and evaluate its relationship with clinicopathologic characteristics.nnnMETHODSnActivity of metalloproteinases was measured in 30 surgical specimen pairs of human primary hepatocellular carcinoma and adjacent nontumoral liver and in five cultured human hepatoma cell lines using zymography. A comparison of this activity with clinicopathological features was made.nnnRESULTSnIn the liver tissues, enhanced secretion of active forms of gelatinase A and matrilysin was associated with portal venous invasion (P < 0.05, respectively), intrahepatic metastasis (P < 0.05, respectively), and recurrence within the first postoperative year (P < 0.01 and P < 0.05, respectively). Enhanced messenger RNA expression for membrane type 1-matrix metalloproteinase was observed in 22 of 30 cases and associated with capsule invasion and the activation of progelatinase A (P < 0.05, respectively).nnnCONCLUSIONSnActive gelatinase A, active matrilysin, and membrane type 1-matrix metalloproteinase may play an important role in tumor spread of human hepatocellular carcinoma.

Inactivation of p57KIP2 by regional promoter hypermethylation and histone deacetylation in human tumors

To clarify the role of DNA methylation in the silencing of the expression of cyclin-dependent kinase inhibitor p57KIP2 seen in certain tumors, we investigated the methylation status of its 5′ CpG island in various tumor cell lines and primary cancers. Dense methylation of the region around the transcription start site was detected in 1 out of 10 colorectal, 2 out of 8 gastric, and 6 out of 14 hematopoietic tumor cell lines and in 5 out of 35 (14%) gastric, 6 out of 20 (30%) hepatocellular, and 2 out of 18 (11%) pancreatic cancers; 7 out of 25 (28%) acute myeloid leukemia cases also showed methylation of the p57KIP2 gene, which strongly correlated with the CpG island methylator phenotype (P<0.001). Detailed mapping revealed that dense methylation of the region around the transcription start site (−300 to +400), but not of the edges of the CpG island, was closely associated with gene silencing. 5-aza-2′-deoxycytidine, a methyltransferase inhibitor, restored expression of p57KIP2, and chromatin immunoprecipitation using anti-histone H3 and H4 antibodies showed histone to be deacetylated in cell lines where p57KIP2 was methylated at the transcription start site. Regional methylation and histone deacetylation thus appear to be crucially involved in the silencing of p57KIP2 expression in human tumors.

Clinicopathologic and prognostic significance of matrilysin expression at the invasive front in human colorectal cancers

Matrix metalloproteinases (MMPs) have been implicated in tumor progression. Matrilysin, one of the matrix metalloproteinases, is frequently overexpressed in gastrointestinal cancers. The aim of our study was to assess the validity of matrilysin as a prognostic marker of colorectal cancers. Matrilysin expression was immunohistochemically analyzed using formalin‐fixed, paraffin‐embedded specimens from 113 colorectal cancer patients who had undergone curative surgery. The lumenal surface of neoplastic glands in the superficial layer was apically stained, while the cytoplasm of cancer cells at the invasive front was diffusely stained for matrilysin. Sections with immunostaining signals in more than 30% of carcinoma cells at the invasive front, which were observed in 47 (42%) cases, were judged as being positive for matrilysin. Matrilysin positivity was significantly correlated with the depth of invasion, lymph node metastasis, lymphatic invasion, advanced Dukes stage and poor outcome. Patients with matrilysin‐positive cancer had a significantly shorter overall survival time than those with matrilysin‐negative cancer. For patients with intermediate invasive tumor (T2 or T3), only matrilysin was a significant prognostic variable for predicting overall survival in multivariate analysis. Matrilysin expression at the invasive front could be an important marker, predicting an unfavorable prognosis after surgical treatment in patients with colorectal cancer.

Association of trypsin expression with tumour progression and matrilysin expression in human colorectal cancer

Overexpression of the matrix serine protease (MSP) trypsin has been implicated in tumour growth, invasion, and metastasis. The objective of this study was to clarify the clinicopathological and prognostic significance of trypsin expression in colorectal cancer. This study analysed the association between immunohistochemically detected trypsin expression in colorectal cancer and clinicopathological characteristics, and investigated whether trypsin is a predictor of recurrence and/or survival. Trypsin immunoreactivity was more intense at the invasive front than in the superficial part of the tumour. Sections with immunostaining signals in more than 30% of carcinoma cells at the invasive front, which were observed in 48 cases (48%), were judged to be positive for trypsin. Trypsin positivity was significantly correlated with depth of invasion, lymphatic and venous invasion, lymph node and distant metastasis, advanced pathological tumour–node–metastasis (TNM) stage, and recurrence. Patients with trypsin‐positive carcinoma had significantly shorter overall and disease‐free survival periods than did those with trypsin‐negative carcinoma. Trypsin retained its significant predictive value for overall and disease‐free survival in multivariate analysis that included conventional clinicopathological factors. It is well known that trypsin activates matrilysin (matrix metalloproteinase‐7), which plays an important role in colorectal cancer progression. Patients with concordant overexpression of trypsin and matrilysin at the invasive front, in which they were often co‐localized, had the worst prognosis. Trypsinogen‐1‐transfected HCT116 colon cancer cells showed not only trypsin activity, but also active matrilysin activity and were more invasive in vitro than mock‐transfected HCT116 cells. These results suggest that trypsin plays a key role in the progression of colorectal cancer. Detection of trypsin expression as well as matrilysin is useful for the prediction of recurrence in and poor prognosis of colorectal cancer patients. Copyright

EXPRESSION OF CD10/NEUTRAL ENDOPEPTIDASE IN NORMAL AND MALIGNANT TISSUES OF THE HUMAN STOMACH AND COLON

The expression of common acute lymphoblastic leukemia antigen (CALLA; CD10), which is identical to neutral endopeptidase (NEP, EC3.424.11), was examined in the malignant and adjacent noninvaded tissues of the human stomach and colon (n = 27). All of 27 normal and 18 well or moderately differentiated adenocarcioma tissue specimens were positive for monoclonal antibody (mAb) NL-1 against CD10/NEP, whereas the expression level was clearly decreased in all of 9 specimens of poorly differentiated adenocarcinoma. In addition, all of 7 gastric or colorectal carcinoma cell lines tested showed decreased expression of CD10/NEP. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis of the crude antigen J5 from the normal colon tissue lysate by mAb J5 detected a single band of approximately 100 kDa that was consistent with that of NALM-6 cells used as a positive control. These findings suggest that CD10/NEP is expressed in normal epithelial cells of the human stomach and colon, whereas the expression level is decreased in the poorly differentiated type of adenocarcinoma.