Lysyl oxidase (LOX) and HRAS-like suppressor (HRASLS) are silenced in human gastric cancers and are reported to have growth-suppressive activities in ras-transformed mouse/rat fibroblasts. Here, we analyzed whether or not LOX and HRASLS are tumor suppressor genes in human gastric cancers. Loss of heterozygosity and promoter methylation of LOX were detected in 33% (9 of 27) and 27% (26 of 96) of gastric cancers, respectively. Biallelic methylation and loss of heterozygosity with promoter methylation were also demonstrated in gastric cancers. Silencing of LOX was also observed in colon, lung, and ovarian cancer cell lines. As for mutations, only one possible somatic mutation was found by analysis of 96 gastric cancer samples and 58 gastric and other cancer cell lines. When LOX was introduced into a gastric cancer cell line, MKN28, in which LOX and HRASLS were silenced, it reduced the number of anchorage-dependent colonies to 57 to 61%, and the number of anchorage-independent colonies to 11 to 23%. Sizes of tumors formed in nude mice were reduced to 19 to 26%. Growth suppression in soft agar assay was also observed in another gastric cancer cell line, KATOIII. On the other hand, neither loss of heterozygosity nor a somatic mutation was detected in HRASLS, and its introduction into MKN28 did not suppress the growth in vitro or in vivo. These data showed that LOX is a tumor suppressor gene inactivated by methylation and loss of heterozygosity in gastric cancers, and possibly also in other cancers.

Aberrantly methylated DNA fragments were searched for in human pancreatic cancers, using the genome scanning technique: methylation-sensitive-representational difference analysis (MS-RDA). MS-RDA isolated 111 DNA fragments derived from CpG islands (CGIs), and 35 of them were from CGIs in the 5 0 regions of known genes. Methylation-specific PCR (MSP) of the CGIs in seven pancreatic cancer cell lines and two pancreatic ductal epithelial cell lines showed that 27 CGIs in the 5 0 regions were aberrantly methylated in at least one of the cancer cell lines. Quantitative reverse-transcription-PCR analysis showed that downstream genes of all the CGIs were either not expressed or only very weakly expressed in cancer cell lines with the aberrant methylation. In the pancreatic ductal epithelial cell lines, 18 genes were expressed at various levels, and nine genes were not expressed at all. Treatment of a cancer cell line with a demethylating agent, 5-aza-2 0 -deoxycytidine, restored the expression of 13 genes, RASGRF2, ADAM23, NEF3, NKX2-8, HAND1, EGR4, PRG2, FBN2, CDH2, TLL1, NPTX1, NTSR1 and THBD, showing their silencing by methylation of their 5 0 CGIs. MSP of 24 primary pancreatic cancers showed that all these genes, except for THBD, were methylated in at least one cancer. Some of those were suggested to be potentially involved in pancreatic cancer development and progression.

Alteration in the methylation status of a gene is often associated with its altered expression. Based on a genome scanning technique for differences in CpG methylations, methylation-sensitive representational difference analysis, DNA fragments hypermethylated in a human breast cancer were isolated. A DNA fragment was isolated from intron 1 of guanine-nucleotide-binding protein α-11 (GNA11). mRNA expression of GNA11 was shown to be decreased in 10 of 16 breast cancers by RT-PCR analysis, and the immunoreactivity of the GNA11 product, Gα11 subunit of heterotrimeric G-protein, was observed to be reduced in 14 of the 16 cancers by immunohistochemistry. Methylation of a CpG island (CGI) in the 5′ region of GNA11 or that of intron 1 did not show a clear correlation with its decreased expression. Another DNA fragment was isolated from a CGI in the 5′ upstream region of monocarboxylate transporter 1 (MCT1), and was methylated in 4 of 20 breast cancers. The CGI was also methylated in a human breast cancer cell line, MDA-MB-231, and quantitative RT-PCR showed that its expression was almost lost in the cell line. By treatment of the cells with a demethylating agent, 5-aza-2′-deoxycytidine, the methylation was removed and the expression was restored. GNA11 is involved in signalling of gonadotropin-releasing hormone receptor, which negatively regulates cell growth. MCT1 is involved in cellular transportation of butyrate, which induces cellular differentiation. Downregulation of these two genes was suggested to be involved in human breast cancers.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is known to induce a characteristic mutation, G deletion at the 5'-GGGA-3' site, preferentially in the lacI transgene of the colonic mucosa of Big Blue((R)) rats (BBR) and mice and specifically in the Apc gene of rat colon tumors. In this study, lacI mutations of the mammary glands in PhIP-treated rats were investigated. Six-week-old female (BBRxSprague-Dawley)F(1) rats were administered 10 gavages of 65 mg/kg/day PhIP. Mammary ducts were collected from the macroscopically normal mammary tissue of PhIP-treated and untreated rats at 56-69 weeks of age by collagenase treatment. The mutant frequencies were 25 +/- 2.1x10(-6) in control rats and 323 +/- 44x10(-6) in the PhIP-treated rats. By sequencing 40 and 177 mutants in the control and PhIP-treated groups, respectively, 34 and 149 mutations were considered independent mutations. In the control group, G:C-->A:T transitions at CpG sites dominated and no G:C deletions were detected. In the PhIP-treated group, G:C-->T:A transversions were most frequent (43%), followed by single base pair deletions of G:C (21%). A total of nine deletions were at 5'-GGGA-3' sites, accounting for 29% of the G:C deletions and 6% of the 149 total mutations. Clusters of more than three mutations at one nucleotide position were observed at 12 positions and two were G deletions at 5'-GGGA-3' sites. Comparison of the PhIP-induced mutations in the mammary glands with those previously reported in the colon revealed that G:C-->T:A transversions occurred at a significantly higher frequency in the mammary glands and that G:C deletions occurred at a significantly lower frequency. However, the signature mutation, G deletion at the 5'-GGGA-3' site, was commonly observed in both tissues.

Chronic myelogenous leukemia (CML) begins with an indolent chronic phase but inevitably progresses to a fatal blast crisis. Although the Philadelphia chromosome, which generates p210bcr/abl, is a unique chromosomal abnormality in the chronic phase, additional chromosomal abnormalities are frequently detected in the blast crisis, suggesting that superimposed genetic events are responsible for disease progression. To investigate whether loss of p53 plays a role in the evolution of CML, we crossmated p210bcr/abl-transgenic (BCR/ABLtg/−) mice with p53-heterozygous (p53+/−) mice and generated p210bcr/abl-transgenic, p53-heterozygous (BCR/ABLtg/−p53+/−) mice, in which a somatic alteration in the residual normal p53 allele directly abrogates p53 function. TheBCR/ABLtg/−p53+/− mice died in a short period compared with their wild-type (BCR/ABL−/−p53+/+), p53 heterozygous (BCR/ABL−/−p53+/−), and p210bcr/abl transgenic (BCR/ABLtg/−p53+/+) litter mates. They had rapid proliferation of blast cells, which was preceded by subclinical or clinical signs of a myeloproliferative disorder resembling human CML. The blast cells were clonal in origin and expressed p210bcr/abl with an increased kinase activity. Interestingly, the residual normal p53 allele was frequently and preferentially lost in the tumor tissues, implying that a certain mechanism facilitating the loss of p53 allele exists in p210bcr/abl-expressing hematopoietic cells. Our study presents in vivo evidence that acquired loss of p53 contributes to the blastic transformation of p210bcr/abl-expressing hematopoietic cells and provides insights into the molecular mechanism for blast crisis of human CML.

Aberrantly hypermethylated genes in human lung cancers were searched for by a genome scanning technique, methylation-sensitive-representational di erence analysis (MS-RDA). A total of 59 DNA fragments were isolated as those methylated more heavily in either/both of two lung squamous cell carcinoma cell lines, EBC-1 and LK-2, than in a primary culture of normal human bronchial epithelium, NHBE. Thirty-four DNA fragments, whose hypermethylation was con®rmed in primary squamous cell carcinomas, were sequenced. By database searches, 17 of them were shown to be located within 2 kb of putative CpG islands, and ®ve of the 17 DNA fragments had transcribed regions of known genes in their vicinities. By RT ± PCR of the ®ve genes in the carcinoma cell lines and NHBE, decreased expression of HTR1B (5-hydroxytryptamine receptor 1B) and EDN1 (endothelin-1) was observed. Sequencing after bisul®te modi®ca-tion showed that the CpG island in the promoter region of HTR1B was hypermethylated, while that of EDN1 was not. Demethylation and re-expression of HTR1B were observed after treatment of LK-2 cells with 5-aza-2'-deoxycytidine. In primary lung cancers, decreased mRNA expression of HTR1B was observed in 11 of 20 cases, and that of EDN1 was in 16 of 20 cases. Immunohistochemical analysis of endothelin-1 con®rmed that its immunoreactivity was reduced in squamous cell carcinoma cells compared with that in normal bronchial epithelial cells. Considering that endothelin-1 induces apoptosis in melanoma cells and that silencing of endothelin receptor B is observed in prostate cancers, its reduced expression was speculated to confer a growth advantage to lung cancer cells. MS-RDA was shown to isolate DNA fragments that are hypermethylated and silenced, such as HTR1B, and those whose expressions are altered and the methylation statuses outside the promoter region are altered, such as EDN1. Oncogene (2001) 20, 7505 ± 7513.

Differential expression of mRNA among animal strains is one of the mechanisms for their diversity. cDNA microarray analysis of the prostates of BUF/Nac (BUF) and ACI/N (ACI) rats, which show different susceptibility to prostate cancers, found 195 differentially expressed genes. To identify loci that control differential expression of 13 genes with diverse expression levels, their expression levels were measured by quantitative RT-PCR in 89 backcross rats, and expression quantitative trait locus (eQTL) analysis was performed. Nine genes [Aldh1a1, Aldr1, Bmp6, Cdkn1a (p21), Cntn6, Ghr, Jund, Nupr1, and RT1-M3] were controlled by cis-acting loci. Cdkn1a, a cell cycle regulator and a candidate for a prostate cancer susceptibility gene, was mapped to its own locus and had polymorphisms, including a 119-bp insertion in the 59 upstream region in BUF rats. Four genes (Kclr, Pbsn, Psat1, and Ptn) were controlled by trans-acting loci. Pbsn, a prostate-specific gene on chromosome X, was controlled by a QTL on chromosome 8. Depending upon which gene that we selected from the genes widely used for normalization (Actb, Gapd, or Ppia), different QTL were mapped for Kclr, Psat1, and Ptn. Normalization using Actb most appropriately explained the expression levels in a congenic strain for chromosome 3. eQTL analysis with precise measurement of expression levels and appropriate normalization was shown to be effective for mapping loci that control gene expression in vivo.

Rat stomach cancers induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) have been widely used as a model for human stomach cancers of the differentiated type. However, there has been little information regarding their molecular basis. In this study, we examined the genetic alterations reported in human stomach cancers in 10 rat stomach cancers that had been induced in male ACI/N rats by administering MNNG in the drinking water. One of the 10 cancers had a mutation of the p53 gene at the second position of codon 171 (Val --> Glu). However, none of the 10 cancers had mutations in codons 12, 13, or 61 of Ki-ras or in the N-terminal phosphorylation sites of the beta-catenin gene. Southern blot analysis showed no amplification of K-sam or c-erbB-2 in the seven cancers examined. Finally, we searched for microsatellite alterations in 12 loci in nine cancers, but no alterations were observed. As these genetic alterations are observed in only a minor fraction of human stomach cancers, further analysis of genetic and epigenetic alterations in MNNG-induced rat stomach cancers is needed to disclose the major mechanisms of stomach carcinogenesis.