Presence on human chromosome 10 of omeprazole-sensitivity gene whose product mediates CYP1A1 induction.

Previously, we showed that CYP1A1 expression can be induced by omeprazole (OP) in the human cell line HepG2, but not in the mouse cell line Hepa-1. Now we show induction of CYP1A1 by alpha-naphthoflavone (alphaNF) in Hepa-1 cells. This induction was inhibited by the tyrosine kinase inhibitor herbimycin A, but not by the aromatic hydrocarbon (Ah)-receptor antagonist PD98059, suggesting the presence of a ligand-independent signal-transduction pathway in the mouse cell line too. We utilized the lack of CYP1A1 induction by OP in Hepa-1 cells to map a putative human gene for OP-respon- siveness in cell hybrids produced by fusion of Hepa-1 and HepG2 cells. OP-induced CYP1A1 expression was detected in four out of the 32 Hepa-1 x HepG2 cell hybrids analyzed. To help identify the gene locus, a radiation-hybrid cell (E11) was constructed. Use of reverse-fluorescence in situ hybridization revealed that these five cell lines commonly retained human chromosome 10p. These results suggest that the human gene for OP-responsiveness is present on chromosome 10p.

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.

The interacting domains of three MutL heterodimers in man: hMLH1 interacts with 36 homologous amino acid residues within hMLH3, hPMS1 and hPMS2.

In human cells, hMLH1, hMLH3, hPMS1 and hPMS2 are four recognised and distinctive homologues of MutL, an essential component of the bacterial DNA mismatch repair (MMR) system. The hMLH1 protein forms three different heterodimers with one of the other MutL homologues. As a first step towards functional analysis of these molecules, we determined the interacting domains of each heterodimer and tried to understand their common features. Using a yeast two-hybrid assay, we show that these MutL homologues can form heterodimers by interacting with the same amino acid residues of hMLH1, residues 492-742. In contrast, three hMLH1 partners, hMLH3, hPMS1 and hPMS2 contain the 36 homologous amino acid residues that interact strongly with hMLH1. Contrary to the previous studies, these homologous residues reside at the N-terminal regions of three subdomains conserved in MutL homologues in many species. Interestingly, these residues in hPMS2 and hMLH3 may form coiled-coil structures as predicted by the MULTICOIL program. Furthermore, we show that there is competition for the interacting domain in hMLH1 among the three other MutL homologues. Therefore, the quantitative balance of these three MutL heterodimers may be important in their functions.

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.

Frequent loss of copy number on the long arm of chromosome 21 in human esophageal squamous cell carcinoma.

To understand the molecular pathogenesis of human esophageal cancer, we performed a comparative genomic hybridization (CGH) analysis using 10 esophageal squamous cell carcinomas. Frequent gains of 1q, 3q, 7p, 7q, 8q, 11q, and 20q and losses of 3p, 4p, 4q, 5q, 9p, 11p, 11q, 13q, 18q, 21q, and Y were observed. Among these regions, 21q has not yet been investigated in detail. We performed an allelotype study using 55 squamous cell carcinomas of the esophagus and 20 microsatellite markers on 21q and found LOH in 36 cases (65%): 22 (61%) of 36 cases with LOH indicated allelic loss in all informative loci, suggesting loss of the whole chromosome arm 21q, and five smallest regions of overlap were found. Our present results suggest the existence of a tumor suppressor gene(s) that plays a role in the genesis of squamous cell carcinoma of the esophagus.

p24/ING1-ALT1 and p47/ING1-ALT2, distinct alternative transcripts of p33/ING1.

p33/ING1s (the growth inhibitor ING1 and candidate tumor suppressor ING1) are key players in the suppressive pathways for human tumorigenesis. We analyzed their complete transcripts, primary structures, and expression. The results led us to discover two novel and related alternatively spliced transcripts encoding p24/ING1-ALT1 and p47/ING1-ALT2. They share C-terminal residues with the candidate tumor suppressors p33/ING1. The candidate tumor suppressors p33/ING1 and p24/ING1-ALT1 were coexpressed in a variety of fetal and adult human tissues, but p47/ING1-ALT2 was minimally expressed.

Adenovirus-mediated delivery of the PTEN gene inhibits cell growth by induction of apoptosis in endometrial cancer.

PTEN, a gene encoding a dual specificity phosphatase, is frequently altered in endometrial carcinoma. Moreover, these alterations are observed even in atypical hyperplasia of the endometrium. This evidence suggests that mutation of PTEN is an early genetic alteration involved in endometrial carcinogenesis. Adenovirus-mediated gene transfer was carried out using Ishikawa 3 H 12 and RL95-2, the endometrial cancer cell lines with completely inactivated PTEN, together with endometrial cancer cell lines HEC1-A and KLE expressing wild-type PTEN as the control. The PTEN transgene significantly suppressed cell growth in vitro through induction of apoptosis in cells lacking wild-type PTEN. Furthermore, the ex vivo tumor formation by Ishikawa 3 H 12 cells was completely inhibited by the introduction of wild-type PTEN. However, neither regression nor progression was observed in inoculated tumors of either cell line by in vivo introduction of the PTEN gene. These results suggest that PTEN may be a good candidate for gene therapy in patients with endometrial carcinoma.

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.

Chromosome band 16q24 is frequently deleted in human gastric cancer.

We have analysed the loss of heterozygosity (LOH) on chromosome bands 16q22-q24 in 24 primary gastric cancer tissues and found three regions of frequent allelic loss (16q22, 16q24.1-q24.3 and 16q24.3). The region for the most frequent allelic loss (63%) was in 16q24.1-q24.3. LOH of this region had no relationship with histological subtype, but a significant association between LOH and microscopic lymphangial invasion was observed. Although not significant, vascular and gastric wall invasions are also associated with LOH. The region includes the locus for the H-cadherin gene. Therefore we examined the genetic and epigenetic alterations of this gene. Markedly reduced expression was observed in gastric cancer cell lines compared with that of normal gastric mucosa. However, no mutation was found in this gene in any of the gastric cancer tissues or the gastric cancer cell lines. Furthermore, we analysed the methylation status of the 5'-flanking region of the gene, but no significant association was found. We suggest that some other tumour suppressor gene(s) in 16q24.1-q24.3 may be responsible for gastric carcinogenesis.

Identification of a 5-cM region of common allelic loss on 8p12-p21 in human breast cancer and genomic analysis of the hEXT1L/EXTR1/EXTL3 gene in this locus.

The short arm of chromosome 8 is frequently lost in many human carcinomas including breast cancer, suggesting the presence of a tumor suppressor gene(s) in this region. We identified a gene termed hEXT1L/EXTR1/EXTL3 (hEXT1L hereinafter) that was mapped to chromosome bands 8p12-p21 where frequent LOHs of this region was reported in breast cancer. The existence of the third breast cancer susceptibility gene was also suggested in this region by linkage analysis. We further performed LOH analysis in 8p12-p21 in 34 breast cancers and identified a 5-cM region of common allelic loss that overlapped with the locus for positive lod score in familial breast cancer. We further analyzed genomic alterations of hEXT1L in tumors in which frequent LOHs of 8p were reported. A total of 327 cancers (313 primary tumors and 14 cancer cell lines) including 22 primary breast cancers were analyzed, but none of the tumors had somatic mutations: only one thyroid cancer patient without any family history of cancer had a 9-bp insertion in the constitutional DNA. These results suggest that mutations of hEXT1L do not play a major role in the development of sporadic cancers including breast cancer, and that other tumor suppressor gene(s) exists in the 5-cM region identified in this study.

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.

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.

The human PMS2L proteins do not interact with hMLH1, a major DNA mismatch repair protein.

The human PMS2 gene encodes one of the bacterial mutL homologs that is associated with hereditary nonpolyposis colorectal cancer (HNPCC). One of the interesting features of the hPMS2 gene is that it is part of a multiple gene family which is localized on chromosome bands 7p22, 7p12-p13, 7q11, and 7q22. Here we report four newly identified hPMS2-like (PMS2L) genes. All four novel members of the PMS2L gene family encode relatively short polypeptides composed of the amino-terminal portion of hPMS2 and are expressed ubiquitously except in the heart. To clarify whether the PMS2L polypeptides contribute to the DNA mismatch repair (MMR) pathway through an interaction with hMLH1, we have performed a yeast two-hybrid assay and an immunoprecipitation study using an hPMS2 mutant cell line, HEC-1-A. Our results clearly indicate that hMLH1 does not interact with two representative PMS2Ls, whereas the carboxyl-terminal portion of hPMS2, not the amino-terminal portion, does interact with hMLH1. Thus, PMS2Ls are not likely to participate in the MMR pathway through association with hMLH1; they must play some other roles in the living cells.

Isolation and characterization of the novel gene, TU3A, in a commonly deleted region on 3p14.3-->p14.2 in renal cell carcinoma.

We previously identified a 700-kb region of common allelic loss on 3p14.3-->p14.2 in renal cell carcinoma (RCC). We further analyzed this region and constructed a sequence ready bacterial artificial chromosome (BAC) contig. This region was totally covered by six overlapping BAC clones and was roughly estimated to be 700 kb. Furthermore, we isolated a gene in this region that we termed TU3A. This gene encodes a protein consisting of 144 amino acids. Homology search did not show any significant similarities with known genes or proteins. Northern analysis with normal tissue identified a 3.0-kb transcript that was expressed ubiquitously. Although our mutation search using 37 primary RCCs as well as five RCC cell lines failed to detect any somatic alterations in the TU3A gene, two of five RCC cell lines had totally lost its expression. Considering the fact that we found no genetic alterations in TU3A, it is possible that some epigenetic alteration may have suppressed its expression.

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.

Infrequent genetic alterations of the PTEN gene in Japanese patients with sporadic prostate cancer.

Prostate cancer is a major cause of cancer death among elderly men in America, Europe, and Japan. However, the molecular mechanism of carcinogenesis is not yet well characterized. Frequent loss of heterozygosity (LOH) on chromosome 10q was reported in prostate cancer, and a candidate tumor suppressor gene, PTEN, was isolated on chromosome band 10q23.3. To investigate the genetic alterations of PTEN, we examined 45 primary prostate cancer specimens. LOH at the PTEN locus was observed in two (11.1%) of 18 tumors. However, no mutations were observed in any of the primary prostate cancers. These data suggest that mutation of the PTEN gene does not play a major role in prostate carcinogenesis of Japanese patients.

Loss of chromosome 18q is an early event in pancreatic ductal tumorigenesis.

Cytogenetic and molecular studies demonstrated that pancreatic cancer frequently shows specific chromosomal abnormalities, such as losses of 9p, 17p, and 18q, and gains of 8q and 20q. We have analyzed alterations in the copy number of specific chromosomal regions in cells from the pancreatic juices of 32 patients with various pancreatic disorders by fluorescence in situ hybridization (FISH) technique to pursue the possible clinical use of early diagnosis of pancreatic cancer. None of the chromosomal abnormalities were found in 13 specimens from individuals who had no neoplastic lesions. On the other hand, 12 specimens (63%) derived from the remaining 19 patients who had neoplastic lesions showed at least one chromosomal abnormality. Ten of these specimens were from pancreatic cancer patients; 7 cases (70%) showed chromosomal abnormalities. All but one of the 12 tumors with chromosomal abnormalities had loss of 18q. Furthermore, we detected a tumor in one patient in whom the routine cytological method and endoscopic retrograde chorangiopancreatography found nothing. Based on the results by FISH, we performed endoscopic ultrasonography and found a small serous cystadenoma in this patient. These results indicate that: (a) FISH analysis of cells from pancreatic juices obtained during endoscopic retrograde chorangiopancreatography is quite useful for detecting pancreatic ductal tumors; and (b) loss of chromosome 18q is one of the early genetic changes that provide very useful information in diagnosing pancreatic neoplasias.

Identification of a 910-kb region of common allelic loss in chromosome bands 16q24.1-q24.2 in human lung cancer.

To understand the molecular pathogenesis of human lung cancer, we analyzed allelic deletions on the long arm of chromosome 16 by PCR amplification of microsatellite markers. A total of 203 lung cancer specimens (78 squamous cell carcinomas and 125 adenocarcinomas) were analyzed. In both cell types, a common region of allelic loss was identified in 16q24.1-q24.2; it is flanked by the two markers D16S534 and D16S422 that spanned at most 910 kb. These results were confirmed by fluorescence in situ hybridization. There was no correlation between allelic loss and histopathologic diagnosis or clinical stage. These results suggest the existence of a tumor-suppressor gene that plays an important role in the course of carcinogenesis in both squamous cell carcinoma and adenocarcinoma of the lungs.

Frequent gains on chromosome arms 1q and/or 8q in human endometrial cancer.

Comparative genomic hybridization (CGH) was employed to survey genomic regions with increased and decreased copy number of the DNA sequence in 15 endometrial cancers [10 cases with microsatellite instability positive (MI+) and 5 cases with MI-]. Twelve of these 15 tumors (80%) showed abnormalities in copy number at one or more of the chromosomal regions. There were no regions with frequent chromosomal losses. Conversely, 11 of 15 cases (73%) showed gains on chromosome arms 1q (8/15; 53%) and/or 8q (6/15; 40%). Concordant gains of both chromosome arms 1q and 8q were observed in all three endometrial cancers of histological grade 3. These results suggest that these two chromosomal regions may contain genes whose increased expression contributes to development and/or progression of endometrial carcinogenesis. Two cases were further analyzed by fluorescence in situ hybridization (FISH) using three probes on chromosome 1 and two probes on chromosome 8 to more accurately determine increases in copy number. We found gains of chromosome 1q to 2.9-3.6 copies per cell and on 8q to 4.4 copies per cell.