In rats, partial hepatectomy induces reasonably synchronized DNA replication in the remaining liver after approximately 20 h. Events occurring during the earlier stages of liver regeneration are of interest because they may tell us how cells in vivo respond when they move from a differentiated resting state (G0 phase) to a proliferative state. We report here that the expression of the c-myc oncogene is increased up to 10-15-fold of the normal level within 1-3 h after partial hepatectomy. This expression begins to decrease rapidly after 4 h and has returned to less than double the normal level after 8 h, at which time replicative DNA synthesis has still not begun. A still larger increase in c-myc transcription (approximately 600-fold) is observed in the liver when protein synthesis is inhibited by an injection of cycloheximide. These findings suggest the existence of a short-lived protein that is synthesized soon after partial hepatectomy, and which suppresses the expression of c-myc.

Purpose: Gastric and intestinal phenotypic cell markers are expressed in gastric carcinomas, irrespective of their histologic type. In the present study, we determined the clinicopathologic significance of phenotypic marker expression in early-stage gastric differentiated-type tumors and the association between marker expression and genetic alterations. Experimental Design: Phenotypic marker expression was determined by examining the expressions of human gastric mucin (HGM), MUC6, MUC2, and CD10 in 63 gastric adenomas, 133 early differentiated-type carcinomas, and 24 follow-up cases with gastric adenoma. Tumors were classified into gastric, gastric and intestinal mixed, or intestinal phenotypes according to the immunopositivity of the above markers. The presence of mutations in APC, K-ras, and p53 and the microsatellite instability status were also determined in all tumors. Results: The expressions of HGM and MUC6, representing gastric or gastric and intestinal mixed phenotypes, were significantly associated with high-grade atypia in the 63 gastric adenomas. Among the 133 early differentiated-type carcinomas, HGM expression was significantly associated with mixed-type (with an undifferentiated-type component) tumors and lymph node metastasis. MUC2 expression was inversely associated with submucosal invasion. A multivariate analysis revealed that gastric adenomas were significantly associated with the intestinal phenotype and were inversely associated with p53 mutation compared with early differentiated-type carcinomas. Among all 196 tumors, APC mutation was significantly associated with CD10 expression and the intestinal phenotype and was inversely associated with the expressions of HGM and MUC6. The microsatellite instability status was significantly associated with MUC6 expression. Malignant transformation from gastric adenoma to carcinoma was shown in 5 of the 24 follow-up cases of gastric adenoma. The malignant transformation was significantly associated with the gastric and intestinal mixed phenotype and was inversely associated with APC mutation. No malignant transformation was found in intestinal phenotype gastric adenomas with APC mutation. Conclusions: Our present findings show that phenotypic marker expression is associated with tumor aggressiveness during the early stage of gastric differentiated-type tumors. Differences in the biological behavior of tumors with different phenotypes may result from differences in the genetic backgrounds during the incipient phase of gastric tumorigenesis.Gastric carcinoma is histologically classified into two types, differentiated and undifferentiated or intestinal and diffuse type, based on the gland formation tendency (1, 2). With respect to the histogenesis of these two types of gastric carcinoma, differentiated-type tumors have generally been considered to arise from gastric mucosa with intestinal metaplasia and undifferentiated-type tumors from ordinary gastric mucosa without intestinal metaplasia; these two tumor types are believed to follow different g...

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Purpose:The purpose is to compare the molecular characteristics of serrated adenomas (SAs) with those of conventional adenomas (CADs) and hyperplastic polyps (HPs).Experimental Design: We evaluated the proliferative activity and molecular alterations in 47 SAs (25 pure-type and 22 mixed-type), 71 CADs, and 23 HPs.Results: The proliferative activity of SAs, as evaluated by Ki-67 expression, was intermediate between CADs and HPs. There was no significant difference in the incidence of KRAS or p53 mutations between the three histological groups. In the microsatellite instability (MSI) analysis, 21% of SAs (9 of 43) showed MSI at two or more loci (MSI-H); corresponding values were 5% of CADs (3 of 64) and 8% of HPs (1 of 13; SAs versus CADs, P ‫؍‬ 0.0125). MSI-H was more likely to be found in pure-type SAs (36%; 8 of 22) than in mixed-type SAs (5%; 1 of 21; P ‫؍‬ 0.0212). Loss of hMLH-1 expression was found in 8 of 9 SAs with MSI-H. The incidence of BRAF or KRAS mutations was 36 and 15% of SAs, respectively; the combined incidence of BRAF and KRAS mutations occurred in 49% of SAs. However, there was no significant difference in the incidence of BRAF or KRAS mutations between SAs with and without MSI-H.Conclusions: Genetic instability is more frequently implicated in the tumorigenesis of SAs, especially pure-type SAs, than in that of CADs. In contrast, activation of the Ras/Raf/MEK/MAP kinase cascade by BRAF or KRAS mutation, independently of the genetic instability, may be associated with the progression of about half of SAs.

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A fluorescence-based method for polymerase chain reaction-singlestrand conformation polymorphism (PCR-SSCP) analysis, F-SSCP, was developed in which the target sequence is amplified by the PCR using fluorescent primers. The amplified products are then heat-denatured and applied to a water-jacket controlled gel in an automated DNA sequencer. The separated strands are detected as laserexcited fluorescence at the bottom of the gel, and mutations are detected as shifts in the position of the peaks in the fluorogram. The system does not involve radioactivity, and the conditions of electrophoresis are more strictly controlled than in the previous system, which relied on ambient air-cooling to maintain the gel at a constant temperature. The nature of the output data allows direct quantitative interpretation, and so the relative abundance of each allele in a mixture of two or more alleles can easily be estimated. The application of F-SSCP for detection of mutations and loss of heterozygositles of p$3 in tumor tissues is reported.P o l y m e r a s e chain reaction-singlestrand conformation polymorphism analysis (PCR-SSCP) is a rapid and efficient method for the detection of mutations and polymorphisms in genomic or cDNA sequences, cl) In the original method, the target sequence is amplified and radioisotopically labeled simultaneously and then separated by polyacrylamide gel electrophoresis in a singlestranded state. (2) Mutations are detected as mobility shifts caused by a change in the conformation of single-stranded amplification products. Potential uses for this method in clinical DNA diagnosis include detection of germ-line mutations of various genes responsible for genetic diseases and detection of somatic mutations of oncogenes/anti-oncogenes in cancerous tissues, c]) Use of a radioisotope is, however, a disadvantage, especially in clinical tests or in analyses of large numbers of samples. Several groups have demonstrated that SSCP analysis can be performed by electrophoresis in small gels with detection of the bands by silver-staining, c3-s) These nonradioactive methods are convenient for detecting some mutations but are not likely to be sensitive enough for detection of mutations in general because of the small size of the gels.We describe here a nonradioactive high-resolution PCR-SSCP method, F-SSCP, in which fluorescence-labeled primers are used in the PCR. A procedure for fluorescence-labeling of the PCR product and detection of bands of double-stranded DNA in polyacrylamide gel illuminated with UV-light has been described, c6) However, the sensitivity of this method is not high enough for detecting amounts of DNA that are suitable for SSCP analysis. Therefore, we used an automated DNA sequencer for the detection. Use of this automated sequencer also permitted strict control of the gel at any desired temperature, and thus separation could be reproduced at any ambient condition. Moreover, the results can be interpreted quantitatively because the bands are detected as peaks in the fluorogram and their heights a...

The 5'-flanking region of the gene for a Ca(2+)-binding protein regucalcin was cloned from a rat genomic library which was constructed in lambda EMBL3 SP6/T7 vector. The genomic library was screened by using the radiolabeled probe with the 5' region (0.5 kb) of rat regucalcin complementary deoxyribonucleic acid (cDNA). Positive clone had the 5.5 kb fragment which was hybridized with the 5'-probe. This fragment contained three exons (I-III) of the gene coding for a rat regucalcin. The nucleotide sequence of exons completely agreed with that of a rat regucalcin cDNA clone. A supposed translational initiation site existed in the exon II. Homology analysis showed that a putative transcription start site in the rat regucalcin gene was located at position 26 downstream from a TATA-box. Another upstream element, a CCAAT box-like sequence, was located at -170. Moreover, there were many regulatory elements (Hox, AP-1, AP-2 and AP-4) in the 5'-flanking region of the rat regucalcin gene. The organization of rat regucalcin gene seemed to be about 18 kb in size and consisted of seven exons and six introns.

The cDNA for a c-myc intron 1 binding protein 1 (MIBP1) in the rat was isolated from lambda gt11 and lambda ZAPII cDNA libraries. Sequencing of the cDNA clones revealed a long ORF which encoded a putative protein of 2437 amino acid residues. This protein has two widely separated zinc finger regions, each of which carries C2H2 motifs. When expressed in E. coli as a fusion protein, part of the MIBP1 showed sequence-specific binding to the target sequence, i.e., a 9-bp sequence in the rat c-myc intron 1. MIBP1 is most likely the rat counterpart of human MHC binding protein-2 (MBP-2/HIV-EP2), based on the 86% similarity in nucleotide sequence and 93% similarity in amno acid sequence. Northern blotting revealed a high level of MIBP1 mRNA in the brain.