Overexpression of the p53-inducible brain-specific angiogenesis inhibitor 1 suppresses efficiently tumour angiogenesis.

The brain-specific angiogenesis inhibitor 1 gene has been isolated in an attempt to find fragments with p53 "functional" binding sites. As reported herein and by others, brain-specific angiogenesis inhibitor 1 expression is present in some normal tissues, but is reduced or lost in tumour tissues. Such data and its particular structure prompted the hypothesis that brain-specific angiogenesis inhibitor 1 may act as a mediator in the local angiogenesis balance. We herein demonstrate that brain-specific angiogenesis inhibitor 1 over-expression suppresses tumour angiogenesis, delaying significantly the human tumour growth in immunodeficient mice. The inhibitory effect of brain-specific angiogenesis inhibitor 1 was documented using our intravital microscopy system, strongly implicating brain-specific angiogenesis inhibitor 1 as a mediator in the control of tumour angiogenesis. In contrast, in vitro tumour cell proliferation was not inhibited by brain-specific angiogenesis inhibitor 1 transfection, whereas some level of cytotoxicity was assessed for endothelial cells. Immunohistochemical analysis of tumour samples confirmed a reduction in the microvessel density index in brain-specific angiogenesis inhibitor 1-overexpressing tumours. At messenger level, moderate changes could be detected, involving the down-regulation of vascular endothelial growth factor and collagenase-1 expression. Furthermore, brain-specific angiogenesis inhibitor 1 expression that was lost in a selection of human cancer cell lines could be restored by wild-type p53 adenoviral transfection. Brain-specific angiogenesis inhibitor 1 should be considered for gene therapy and development of efficient drugs based on endogenous antiangiogenic molecules.

Human BAC contig covering the deleted region in pancreatic cancer at 12q21.

In sporadic human primary pancreatic cancer tissues, loss of heterozygosity is frequently observed in the 1-cM region between D12S81 and D12S1719 at 12q21. Loss of this chromosome arm is known to be associate with a poor prognosis in pancreatic cancer patients. Herein we report a complete contig of human bacterial artificial chromosome (BAC) clones covering the deleted region. The region was covered by 21 BAC clones in a minimum tiling path. The clones were confirmed to exist at 12q21 by fluorescence in situ hybridization. We identified novel 40 sequence tagged sites and mapped 10 expressed sequence tags in this region. The BAC contig reported here provides an avenue for determining the complete nucleotide sequence and mining putative tumor suppressor genes in the deleted region of pancreatic cancer at 12q21.

Isolation and characterization of the human gene homologous to the Drosophila headcase (hdc) gene in chromosome bands 6q23-q24, a region of common deletion in human pancreatic cancer.

Deletions of the long arm of chromosome 6 (6q) are one of the most common chromosomal abnormalities in multiple human malignancies. Previously, we have identified three commonly deleted regions on 6q (6q21, 6q23-q24, and 6q26) in pancreatic cancer by loss of heterozygosity studies, suggesting the presence of one or more tumor suppressor genes on this chromosome arm. Using a combination of database search and cDNA library screening, we successfully isolated a transcript from 6q24. This mRNA encodes a protein consisting of 543 amino acids with homology to the Drosophila headcase (hdc) gene and, thus, is designated as hHDC. Northern analysis identified a ubiquitously expressed 5.6-kb transcript. Seventeen (81%) of 21 pancreatic cancer cell lines and four (80%) of five renal cell carcinoma cell lines showed low level expression, suggesting that the hHDC gene may play an important role in some human cancers.

Association of poor prognosis with loss of 12q, 17p, and 18q, and concordant loss of 6q/17p and 12q/18q in human pancreatic ductal adenocarcinoma.

OBJECTIVE: Pancreatic cancer is one of the diseases with the poorest prognosis, but the associated genetic alterations are not yet well understood. The genetic alterations reported to date in pancreatic cancer include frequent mutations of the KRAS, TP53, p16, and SMAD4 genes. Mutation of the TP53 gene was reported to be associated with a poor prognosis. In this study, we analyzed the association of loss of heterozygosity (LOH) with clinicopathological features to attempt to devise effective methods in the future for clinically applying our results to diagnosis and treatment. METHODS: A total of 55 tumors from patients with primary pancreatic ductal carcinomas (34 men and 21 women, mean average age 63.9 yr) in which all the relevant clinical and pathological data were available were analyzed. A total of 46 cases were surgically resected, and nine cases were not. Tumor cells as well as corresponding normal cells were collected by microdissection under a microscope, and DNAs were purified. Allelotype analysis was performed by the PCR-based method, and the results were statistically analyzed. RESULTS: LOH of > or =30% were observed on chromosome arms 17p (47%, 17/36), 9p (45%, 14/31), 18q (43%, 15/35), 12q (34%, 10/29), and 6q (30%, 10/33). LOH of 12q, 17p, and 18q were significantly associated with a poor prognosis. Concordant losses of 6q with 17p and 18q were significantly associated with a poor prognosis. Concordant losses of 6q with 17p and of 12q with 18q were also found. CONCLUSIONS: Because LOH of 12q, 17p, and 18q are significantly associated with a poor prognosis, mutation of the putative tumor suppressor genes on these chromosome arms may play significant roles in the disease progression. Concordant losses of 6q with 17p and of 12q with 18q suggest that protein products of putative tumor suppressor genes on 6q and 12q may function in association with TP53 and SMAD4, respectively.

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.

Cloning and characterization of the human UDP-N-acetylglucosamine: alpha-1,3-D-mannoside beta-1,4-N-acetylglucosaminyltransferase IV-homologue (hGnT-IV-H) gene.

A novel human gene determining a polypeptide product of 478 residues with an estimated molecular mass of 55 kDa, which has significant homology and structural similarity to Bos UDP-N-acetylglucosamine: alpha-1,3-D-mannoside beta-1,4-N-acetylglucosaminyltransferase (GnT-IV), was cloned from the commonly deleted region in pancreatic cancer at 12q21. The gene is composed of at least six exons, and the last three exons cover the open reading frame. Different sized transcripts, 3.8-kb in the heart, brain, and fetal brain and 2.8-kb and 1.7-kb in the testis were observed by Northern blot analysis. By reverse transcription-polymerase chain reaction, expression was also observed in the adult brain, liver, and adrenal gland, but not in pancreas. Because of its significant homology and structural similarity to Bos GnT-IV, it is potentially the gene encoding human GnT-IV or its homologue, which had been one of two genes remaining to be cloned in the human GnT family.

Genomic analysis of the thymine-DNA glycosylase (TDG) gene on 12q22-q24.1 in human pancreatic ductal adenocarcinoma.

CONCLUSION: Abnormality of the thymine-DNA glycosylase (TDG) gene on 12q22-q24.1 appears to play a limited role in pancreatic ductal carcinogenesis. BACKGROUND: Recently, a human G/T-specific TDG gene was identified. This protein acts in a system correcting G/T mispairs to G/C pairs. TDG was mapped to chromosome bands 12q22-q24.1, one of the regions frequently lost in pancreatic cancer. Therefore, there is the possibility that the TDG gene on 12q is one of the genes responsible for pancreatic ductal carcinogenesis. METHODS: Nucleotide sequences of the entire coding region of the TDG gene were analyzed in 21 human pancreatic cancer cell lines. mRNA expression of the TDG gene was also analyzed by Northern hybridization in several human tissues and 21 human pancreatic cancer cell lines. RESULTS: Decreased levels of mRNA expression were detected in the pancreatic cancer cell lines, but no somatic mutations were observed.

Identification of three commonly deleted regions on chromosome arm 6q in human pancreatic cancer.

Pancreatic cancer has one of the poorest prognoses among malignant diseases. To understand its molecular mechanisms, we studied allelic losses on the long arm of chromosome 6. Using 55 paired DNAs of tumors and their corresponding normal tissues and 30 microsatellite markers that spanned the entire 6q chromosome arm, we found three distinct regions of common allelic loss: region A, a less than 500-kb region bordered by D6S449 and D6S283 on 6q21 with a loss of heterozygosity (LOH) frequency of 69% (38/55); region B, a 7-cM region bordered by D6S292 and D6S308 on 6q23-q24 with a LOH frequency of 60% (33/55); and region C, a 13-cM region bordered by D6S305 and D6S264 with a LOH frequency of 51% (28/55). We further focused on region A and constructed a physical map using yeast artificial chromosome (YAC) clones, their derived cosmid clones, and bacterial artificial chromosome (BAC) clones. Region A was completely covered by three overlapping BAC clones. Our results in the present study should shed light on the cloning and characterization of tumor suppressor genes in pancreatic carcinogenesis.

Genomic analysis of DUSP6, a dual specificity MAP kinase phosphatase, in pancreatic cancer.

DUSP6 (alias PYST1), one of the dual-specificity tyrosine phosphatases, is localized on 12q21, one of the regions of frequent allelic loss in pancreatic cancer. This gene is composed of three exons, and two forms of alternatively spliced transcripts are ubiquitously expressed. Although no mutations were observed in 26 pancreatic cancer cell lines, reduced expressions of the full-length transcripts were observed in some cell lines, which may suggest some role for DUSP6 in pancreatic carcinogenesis.

Identification of two common regions of allelic loss in chromosome arm 12q in human pancreatic cancer.

Using the method of microsatellite analysis, we studied 40 tissues with pancreatic ductal adenocarcinoma and identified two commonly deleted regions on the long arm of chromosome 12. One (region A) was found between D12S81 and D12S1719 at 12q21 at a frequency of 67.5%, and the other (region B) was located between D12S360 and D12S78 at 12q22-q23.1 at a frequency of 60%; the latter was reported previously (M. Kimura, et al. Genes Chromosomes Cancer, 17: 88-93, 1996). The results of microsatellite analyses were verified by fluorescence in situ hybridization. We further analyzed 19 pancreatic cancer cell lines by fluorescence in situ hybridization and found that 10 of them showed allelic loss at D12S81 and 6 showed allelic loss at D12S360. Yeast artificial chromosome contigs were constructed to cover the deleted regions. Region B was completely covered by a 650-kb yeast artificial chromosome clone. The frequently deleted regions in chromosome 12q in pancreatic cancer that were identified here may provide new avenues for isolating novel tumor suppressor genes.