The APC gene, responsible for familial adenomatous polyposis, is mutated in human gastric cancer.

Although gastric cancer is the most common cancer in the world, genetic changes during its carcinogenesis are not well understood. Since some gastric cancers are considered to originate from the intestinal metaplasia, it is likely that the adenomatous polyposis coli (APC) gene, the mutation of which causes adenomatous polyps in the colon, is associated with carcinogenesis of gastric cancer. Based on this idea, DNAs isolated from gastric cancers were examined by means of a RNase protection analysis coupled with polymerase chain reaction followed by sequencing of the polymerase chain reaction products. By screening nearly one-half of the coding region of the APC gene in 44 tumors, somatic mutations were detected in three tumors: a missense mutation, a nonsense mutation, and a 5-base pair deletion resulting in a frame shift which causes truncation of the gene product. These results suggest that the mutation of the APC gene also plays an important role during the carcinogenesis of at least some gastric cancers.

Frequent somatic mutations of the APC gene in human pancreatic cancer.

The APC (adenomatous polyposis coli) gene is responsible for familial adenomatous polyposis and is also associated with the development of sporadic tumors of the colon and stomach. To investigate whether or not mutations of APC play any role in tumors arising in other organs, we examined somatic mutations of this gene in sporadic (nonfamilial) renal cell carcinomas, hepatocellular carcinomas, and cancers of the lung and pancreas. DNAs isolated from tumors were examined by means of a RNase protection analysis, coupled with the polymerase chain reaction followed by DNA sequencing of the polymerase chain reaction products. By screening a part of the APC coding region, we detected somatic mutations in four of ten pancreatic cancers; each of these mutations would yield a truncated APC product due to a 1- or 5-base pair deletion. These results imply that mutations in APC contribute to carcinogenesis in the pancreas.

Frequent association of alternative splicing of NER, a nuclear hormone receptor gene in cancer tissues.

We have detected frequent alternative splicing of a gene that encodes NER, a protein homologous to the retinoic acid receptors, in cancer cells. Western and immunohistochemical analyses disclosed accumulation of a large amount of the aberrant NER product, generated by alternative splicing that caused skipping of an exon corresponding to the DNA-binding domain, in the nucleoli of cells of cancer cell lines and primary cancer tissues. The aberrant protein was detected in 116 of 228 primary cancers developed in various tissues including breast and colon, but was absent in the corresponding normal tissues; it was also detected in 31 of 39 cancer cell lines. This observation may imply that the aberrant NER product has some relation to the development and/or progression of cancers in a variety of human tissues.

Detailed analysis of genetic alterations in colorectal tumors from patients with and without familial adenomatous polyposis (FAP).

To examine early genetic events during colorectal carcinogenesis, we searched for genetic alterations in 75 adenomas from seven patients with familial polyposis coli (FAP) and in 64 sporadic colorectal tumors (63 carcinomas and one adenoma). We investigated germ-line and somatic mutations in the APC gene, somatic mutations in the K-ras and p53 genes, and loss of heterozygosity (LOH) on chromosome 8p21-22. Thirty-two FAP adenomas carried detectable somatic mutations in the APC gene. The frequency of somatic APC mutations among adenomas was the same regardless of differences in size or histopathological classification. On the other hand, K-ras mutation was very rare in small adenomas where dysplasia was mild or moderate but frequent in large adenomas with severe dysplasia. Mutation of the p53 gene was observed in only two adenomas and LOH on 8p22 was detected in none. These results imply that a second 'hit' in the APC gene, but not necessarily mutation in K-ras or p53, is an important and critical event for formation of a colorectal adenoma.

Effect of mercurials on lymphocyte functions in vitro.

The effect of in vitro treatment with mercurials on several functions of mouse lymphocytes was studied. When lymphocytes were cultured in the presence of mercurials, DNA synthesis induced by mitogen (concanavalin A, phytohemagglutinin-P, lipopolysaccharide) and polyclonal B cell activation induced by lipopolysaccharide were strongly inhibited by methylmercuric chloride at the concentration of 10(-6) M, but mercuric chloride inhibited these functions by 50% at 10(-5) M. Furthermore, 10(-7) M methylmercuric chloride and 10(-6) M mercuric chloride inhibited mixed lymphocyte reaction by 80%. Thus, the inhibitory effect of methylmercuric chloride was 10 times stronger than that of mercuric chloride when the mercurials were present in the culture throughout the incubation. On the other hand, DNA synthesis once induced by mitogens was not significantly affected when 10(-6) M methylmercury was added during the last 3 h of the incubation. Pretreatment with 10(-6) M methylmercury for 1 h, however, showed 50% inhibition of thymidine incorporation into DNA and also reduced the rate of metabolism of phosphatidyl inositol by 50%. These results indicate that methylmercury may act on mouse lymphocytes at an early stage of transformation induced by the mitogens, while inorganic mercury failed to cause the pronounced difference in its potency of inhibition on the functions of lymphocytes by the different treatments of the cells under the various conditions used.

Genome analysis of collagen disease in MRL/lpr mice: polygenic inheritance resulting in the complex pathological manifestations.

MRL/MpJ-lpr/lpr (MRL/lpr) mice develop collagen disease involving vasculitis, glomerulonephritis, arthritis and sialoadenitis, each of which has been studied as a model for polyarteritis, lupus nephritis, rheumatoid arthritis and Sjögren's syndrome, respectively. In the previous studies, we observed genetic segregation of these complex pathological manifestations throughout the genome recombination with a C57Bl/6-lpr/lpr or a C3H/HeJ-lpr/lpr (C3H/lpr) strain of mice which rarely develops such lesions, indicating that development of collagen disease is dependent on an MRL host genetic background. To clarify the mode of inheritance and the gene loci affecting four types of the lesions in MRL/lpr mice; vasculitis, glomerulonephritis, arthritis and sialoadenitis, a genetic dissection of the lesions was carried out by using MRL/lpr, C3H/lpr, (MRL/lprxC3H/lpr) F1 intercross, and MRL/lprx(MRL/lprxC3H/lpr) F1 backcross mice. Definition of each lesion was performed by histopathology under light microscopy, and genomic DNA of the backcross mice were subjected to association studies by chi-square analysis for determining which polymorphic microsatellite locus occurs at higher frequency among affected compared to unaffected individuals for each lesion. We observed that gene loci recessively associated with each lesion were mapped on different chromosomal positions. We conclude that each of four types of the lesions in MRL/lpr mice is under the control of different set of genes, suggesting the complex pathological manifestations of collagen disease result from polygenic inheritance.

Isolation and mapping of a human gene (MCM2) encoding a product homologous to yeast proteins involved in DNA replication.

From a human fetal-lung cDNA library, we isolated a gene, human MCM2, whose nucleotide sequence predicts a protein product homologous to the yeast nuclear proteins MCM2, MCM3, CDC21, and CDC46. Determination of cDNA sequences covering the entire coding region demonstrated an open reading frame of 1,629 nucleotides encoding 543 amino acids. Sequence comparison revealed 30-40% amino acid identity with these four yeast proteins, all of which are considered to play important roles in DNA replication. Analysis by Northern blotting revealed that the human MCM2 gene is expressed ubiquitously in normal tissues. Furthermore, we localized this gene to chromosomal bands 7q21.3-->q22.1 by fluorescence in situ hybridization.

Molecular cloning of a novel human cDNA homologous to CDC10 in Saccharomyces cerevisiae.

We isolated a novel human cDNA, termed hCDC10, whose predicted product showed a high degree of homology to the CDC10 protein of Saccharomyces cerevisiae. This cDNA contained an open reading frame of 1254 nucleotides encoding 418 amino acids, which included a GTP-binding motif, GX4GKS--DX2G--KXD. The predicted peptide sequence also revealed partial amino-acid identity (40-50%) with Diff 6 in Drosophila and with H5 in mouse. Each of these sequence homologues, including Saccharomyces cerevisiae CDC10, contains the GTP-binding motif. Northern blot analyses indicated that the hCDC10 gene is expressed ubiquitously in normal tissues.

Somatic mutation of the APC gene in gastric cancer: frequent mutations in very well differentiated adenocarcinoma and signet-ring cell carcinoma.

We searched for somatic mutations of the adenomatous polyposis coli (APC) gene in DNA samples isolated from 57 sporadic gastric cancers, by means of a ribonuclease (RNase) protection analysis coupled with DNA amplification by the polymerase chain reaction (PCR). Examining 30% of the APC coding region, including a region where somatic mutations in colorectal tumors are known to be clustered, we detected somatic mutations in 12 tumors; seven in 17 very well differentiated adenocarcinomas, two in 19 well or moderately differentiated adenocarcinomas, and three in ten signet-ring cell carcinomas. So far, no somatic mutations have been identified in 11 poorly differentiated adenocarcinomas. Eight of the 17 somatic mutations found in 12 tumors caused truncation of the gene product due to a nonsense mutation and a 1-, 2- or 5-bp deletion; nine others were point mutations that altered amino acids. Our results suggest that inactivation of APC plays a role in development of some gastric cancers, particularly very well differentiated adenocarcinomas and signet-ring cell carcinomas.

Inactivation of both APC alleles in an early stage of colon adenomas in a patient with familial adenomatous polyposis (FAP).

To examine whether the dosage effect of germ-line mutations in patients with familial adenomatous polyposis (FAP) is sufficient to cause colorectal adenomas, or an additional somatic mutation of the normal allele is required as well, we have investigated somatic mutations of the APC gene in multiple adenomas developed in one FAP patient. In addition to a 5-bp deletion of one allele present constitutionally in this patient, the normal APC allele had been lost in five of seven DNA samples extracted from small adenomas (< 3 mm in diameter) with mild or moderate atypia. This result indicates that the inactivation of both alleles of the APC gene is probably essential for the development of an early-stage adenoma, in agreement with the two-hit mutational model underlying the concept of tumor suppressor genes.

Genetic basis of autoimmune disease in MRL/lpr mice: dissection of the complex pathological manifestations and their susceptibility loci.

MRL/MpJ-lpr/lpr (MRL/lpr) mice spontaneously develop various forms of autoimmune disease in the same individuals, including glomerulonephritis, polyarteritis, arthritis and sialoadenitis. An MRL recombinant congenic strain of mice bearing the gld gene, MRLiMpTn-gld/gld (MRL/gld), also develops lesions similar to those in MRL/lpr mice. The lpr and gld genes are a Fas deletion mutant and a Fas ligand mutant, respectively. Thus, autoimmune disease in these mice seemed to be a single gene disease involving the complex pathological manifestations as pleiotropy. However, comparative studies with C3H/HeJ and C57BL/6J strains of mice bearing lpr or gld revealed that these lesions developed only in mice with an MRL background. Moreover, these lesions were genetically segregated among MRL/lpr x (MRL/lpr x C3H/lpr)F1 mice. This indicates that an MRL strain has particular gene(s) affecting the development of each lesion. Association studies of each lesion with polymorphic microsatellite markers using backcross mice revealed that gene loci responsible for each lesion exist at different chromosomal positions and have additive and hierarchical properties of polygenic inheritance for some of the lesions. We conclude that the complex pathological manifestations of autoimmune disease are under the control of different combinations of polygenes.

Structure, expression and chromosome assignment of the human catenin (cadherin-associated protein) alpha 1 gene (CTNNA1).

We have isolated the human alpha-catenin gene (CTNNA1), which encodes a cadherin-associated protein, and have determined its primary structure and chromosomal localization. The transcript of CTNNA1 is 3.4 kb long and consists of 16 coding exons encoding 906 amino acids and at least one 5' noncoding exon. The 102-kDa predicted protein is the same size as the murine homolog, and the amino acid sequences of the two proteins are 99.2% homologous. Analysis by reverse transcription-PCR revealed that this gene is expressed ubiquitously in normal tissues. It was mapped to chromosome band 5q31 by fluorescent in situ hybridization.

Multiple forms of the APC gene transcripts and their tissue-specific expression.

We have detected multiple forms of RNA transcript from APC, the gene which is responsible for familial adenomatous polyposis (FAP). Transcriptional initiation occurs at three sites in two distinct non-translating exons at the 5' end of the gene. At least five different forms of 5' non-coding sequences, generated by alternative splicing, exist. The splicing mechanism seems to be regulated in a tissue-specific fashion, and one type of transcript contained an additional exon, which was transcribed specifically in brain. Analyses of mRNAs from two colorectal-tumor cell lines by reverse-transcriptase polymerase chain reaction (RT-PCR) revealed that one or another of the transcriptional forms was absent in both cell lines. This observation suggested the presence of mutations in the control region or the first exon of APC, or that mutation(s) could have affected the splicing efficiency or transcriptional initiation of the gene in these tumors. Furthermore, we found that the alternative splicing involving the 19 kDa protein of signal recognition particle (SRP19) gene, that is known to occur at exon 14 of APC, is also controlled in a tissue-specific manner, and one type of transcript lacked in some organs.

Somatic mutations of the APC gene in precancerous lesion of the stomach.

The adenomatous polyposis coli (APC) gene, responsible for familial adenomatous polyposis, is also associated with development of sporadic tumors in digestive system as colon, stomach, or pancreas. In order to investigate whether or not APC mutations occur as an early genetic event during gastric carcinogenesis, we examined somatic mutations of APC in flat adenomas of the stomach. DNAs isolated from flat adenomas were examined by means of an RNase protection analysis coupled with polymerase chain reaction (PCR) followed by DNA sequencing of the PCR products. By screening a mutation cluster region (MCR: codons between 1286 and 1513) of APC in which two-thirds of somatic mutations were detected in colorectal tumors, somatic mutations were found in four of ten flat adenomas: three of which caused truncation of the gene product due to a nonsense mutation or 4-bp deletion; one other was a point mutation that altered amino acid from alanine to threonine. Our results imply that APC plays a crucial role in an early step of gastric carcinogenesis, as was observed in colorectal carcinogenesis.

Genetic basis of autoimmune sialadenitis in MRL/lpr lupus-prone mice: additive and hierarchical properties of polygenic inheritance.

OBJECTIVE: To clarify the mode of inheritance of autoimmune sialadenitis in MRL/MpJ-lpr/lpr (MRL/lpr) lupus-prone mice and identify the susceptibility loci. METHODS: MRL/lpr, C3H/HeJ-lpr/lpr (C3H/lpr), (MRL/lpr x C3H/lpr)F1 intercross, and MRL/lpr x (MRL/lpr x C3H/lpr)F1 backcross mice were prepared, and sialadenitis in individual mice was analyzed by histopathologic grading. The genomic DNA of the backcross mice was examined by simple sequence-length polymorphism analysis, and the highly associated polymorphic microsatellite markers with sialadenitis were determined as sialadenitis susceptibility loci. RESULTS: Four susceptible gene loci recessively associated with sialadenitis were mapped on chromosomes 10, 18, 4, and 1, respectively. These loci manifested additive and hierarchical properties in the development of sialadenitis. CONCLUSION: The results indicate that sialadenitis in MRL/lpr mice is under the control of polygenic inheritance, possibly involving allelic polymorphism.

Somatic mutations of the APC gene in colorectal tumors: mutation cluster region in the APC gene.

We examined somatic mutations of the adenomatous polyposis coli (APC) gene in 63 colorectal tumors (16 adenomas and 47 carcinomas) developed in familial adenomatous polyposis (FAP) and non-FAP patients. In addition to loss of heterozygosity (LOH) at the APC locus in 30 tumors, 43 other somatic mutations were detected. Twenty-one of them were point mutations; 16 nonsense and two missense mutations, and three occurred in introns at the splicing site. Twenty-two tumors had frameshift mutations due to deletion or insertion; nineteen of them were deletions of one to 31 bp and three were a 1-bp insertion. One tumor had a 1-bp deletion in an intron near the splicing site. Hence, 41 (95%) of 43 mutations resulted in truncation of the APC protein. Over 60% of the somatic mutations in the APC gene were clustered within a small region of exon 15, designated as MCR (mutation cluster region), which accounted for less than 10% of the coding region. Combining these data and the results of LOH, more than 80% of tumors (14 adenomas and 39 carcinomas) had at least one mutation in the APC gene, of which more than 60% (9 adenomas and 23 carcinomas) had two mutations. These results strongly suggest that somatic mutations of the APC gene are associated with development of a great majority of colorectal tumors.

Germ-line and somatic mutations of the APC gene in patients with Turcot syndrome and analysis of APC mutations in brain tumors.

The Turcot syndrome (TS) is a rare, probably autosomal recessive, disorder characterized by development of primary neuroepithelial tumors of the central nervous system (CNS) and numerous adenomatous colorectal polyps. To examine the possible involvement of mutations of the APC gene, which is responsible for familial adenomatous polyposis (FAP), in Turcot syndrome, we examined DNAs from TS patients for alterations in this gene by means of ribonuclease protection analysis. Germ-line APC mutations were detected in each of three unrelated cases of TS, and additional (somatic) mutations were observed in colonic adenomas that had developed in one of these patients. However, no somatic mutations in APC were found among 91 neuroepithelial tumors (medulloblastoma, glioblastoma, astrocytoma, and oligodendroglioma), whether sporadic or associated with TS. These results suggest that the APC gene is associated with pathogenesis of one feature of TS, but that at least one other gene is responsible for the genesis of neuroepithelial tumors in the CNS.

Genetic dissection of vasculitis in MRL/lpr lupus mice: a novel susceptibility locus involving the CD72c allele.

An MRL/MpJ strain of mice bearing the Fas deletion mutant gene, lpr (MRL/lpr), composed of genomes derived from LG/J, AKR/J, C3H/Di and C57BL/6J mice, develops systemic vasculitis coincidentally with other collagen diseases, but a C3H/HeJ-lpr/lpr (C3H/lpr) strain does not. In a genome-wide screening of the MRL background genes mediating susceptibility to collagen diseases using N2 progeny mice MRL/lpr x (MRL/lpr x C3H/lpr)F1, we previously found that each collagen disease is controlled by a different set of genes. To clarify the candidate genes for vasculitis, we extended the linkage analysis of renal vasculitis to a larger number of N2 mice and to F2 intercross mice. Two distinct recessive susceptibility loci for vasculitis were mapped on chromosome (Chr) 4 at D4Mit89 and D4Mit147 in both progenies. The former was a novel locus for lupus phenotypes, which involved the MRL allele CD72(c) in contrast to the C3H allele CD72(b). The one on Chr 3 was a recessive locus which had an inhibitory effect on vasculitis. From their composition these loci seemed to be derived from AKR/J (for one) and LG/J (for another two) strains, and appeared to act in an additive manner on the development of vasculitis, indicating that vasculitis in MRL/lpr mice is inherited in a polygenic manner.

Genetic dissection of the complex pathological manifestations of collagen disease in MRL/lpr mice.

An MRL strain of mice bearing a Fas-deletion mutant gene, lpr, MRL/MpJ-lpr/lpr (MRL/lpr) develops collagen disease involving vasculitis, glomerulonephritis, arthritis and sialoadenitis, each of which has been studied as a model for polyarteritis, lupus nephritis, rheumatoid arthritis and Sjögren's syndrome, respectively. Development of such lesions seems dependent on host genetic background since the congenic C3H/HeJ-lpr/lpr (C3H/lpr) mice rarely develop them. To identify the gene loci affecting each lesion, a genetic dissection of these complex pathological manifestations was carried out. First, histopathological features in MRL/lpr, C3H/lpr, (MRL/lpr x C3H/lpr) F1 intercross, and MRL/lpr x (MRL/lpr x C3H/lpr) F1 backcross mice were analyzed. Genomic DNA of the backcross mice were subjected to association studies by Chi-squared analysis for determining which polymorphic microsatellite locus occurs at higher frequency among affected compared to unaffected individuals for each lesion. As a result, gene loci recessively associated with each lesion were mapped on different chromosomal positions. We concluded that each of these lesions in MRL/lpr mice is under the control of a different set of genes, suggesting that the complex pathological manifestations of collagen disease result from polygenic inheritance.