Search

A novel rat model of hereditary renal cell carcinoma (RC) was found in a rat colony of the Sprague-Dawley strain in Japan, and named the rising "Nihon" rat. In this strain, RCs develop from early preneoplastic lesions, which begin to appear at 4 weeks of age, forming adenomas by the age of 16 weeks. The RCs are predominantly of clear cell type. Southern blot, northern blot and SSCP analyses revealed no change in the Tsc1, Tsc2, VHL, and c-Met genes. Thus, the Nihon rat should be a valuable experimental model for understanding renal carcinogenesis, especially clear cell type, which is common among human RCs.

A modified AFLP (amplified fragment length polymorphism) method was employed to isolate genes differentially expressed in renal carcinogenesis of Tsc2 gene mutant (Eker) rats. One gene, selected for further investigation, was named “Niban” (“second” in Japanese), because it is the second new gene to be found after Erc (expressed in renal carcinoma) in our laboratory. Importantly, “Niban” is well expressed even in small primary rat Eker renal tumors, more than in progressed cell lines, and is also expressed in human renal carcinoma cells, but not in normal human or rat kidneys. Chromosome assignment was to RNO 13 in the rat, and HSA 1. This “Niban” gene is a candidate as a marker for renal tumor, especially early‐stage renal carcinogenesis.

We searched for the rat homologue of the human tuberous sclerosis (TSC2) gene mutations in loss of heterozygosity (LOH)-negative Eker rat renal carcinomas (RCs) by polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP) analysis using 45 primer sets covering all 41 coding exons and one leader exon including splicing donor/acceptor sites. We have identified intragenic somatic mutations in 7 of 21 spontaneous RCs, including one cell line (33%), and in 3 of 9 (33%) N-ethyl-N-nitrosourea (ENU)-induced LOH-negative RCs. Interestingly, five mutations in the spontaneous RCs were either deletion or duplication (5/7 = 71%). In contrast, all three in ENU-induced RCs were base substitutions (3/3 = 100%), as expected. Thus, a qualitative difference in the second hit might exist between spontaneous and ENU-induced mutations (e.g., deletion or duplication versus point mutation). By a direct cloning approach utilizing the restriction length difference caused by germline insertional mutation or reverse transcriptase-PCR analysis in two applicable cases, we could clearly show the presence of intragenic somatic mutations in the second copy (wild-type) of the Tsc2 gene. This is the first demonstration at the DNA sequence level of the validity of Knudson's two-hits hypothesis in the Tsc2 gene.

Hereditary renal carcinomas (RCs) develop in virtually all Eker rats by the age of one year. Investigation of extra‐renal primary tumors co‐occurring in Eker rats late in life (at 2 years) additionally revealed enhanced development of hemangiosarcomas of the spleen, uterine leiomyosarcomas and pituitary adenomas, although the demonstrated predilection for these extra‐renal tumors was not as complete as with RCs. We identified the germline mutated tuberous sclerosis (Tsc2) gene as the predisposing Eker gene and revealed the tumor suppressor nature of Tsc2 gene function in renal carcinogenesis. In the present study, we examined allelic loss at the Tsc2 gene locus in uterine leiomyosarcomas and pituitary adenomas developing in hybrid F1 rats carrying the Eker mutation as well as in pituitary adenomas from non‐carrier rats. We detected loss of heterozygosity in 4 of 11 uterine leiomyosarcomas (36%) and 11 of 31 pituitary adenomas (35%) from Eker rats but in none of 9 pituitary adenomas from non‐carrier rats (P

The Yoshida sarcoma (YS) is characterized by growth as "free cells" in ascites. Long-survival Yoshida sarcoma (LY) variants, which develop after transplantation of YS into immunologically conditioned Donryu rats, in contrast, form "islands" in ascites. A representational difference analysis (RDA) approach was adopted to isolate genes differentially expressed between YS and LY variants to elucidate the molecular mechanism of their development. Fifteen clones presenting differences in expression were characterized. Nine genes (those encoding for the high-affinity IgE receptor gamma chain, pJG116 repetitive sequence, non neuronal enolase, proteasome subunit RC1, cytotoxic T lymphocyte-associated gene transcript CTLA-1, interleukin-2 receptor gamma chain, and three unknown sequences) expressed mRNA in YS, but showed lower or no expression of mRNA in LYs. The mRNAs of the other six genes (those encoding for cytokeratin 8, cytokeratin18 (Endo B), TIMP2 and three unknown sequences) were not found in YS, but were present in LYs. Interestingly, CTLA-1 is a non-epithelial (hematopoietic) cell-specific gene in terms of transcription, while cytokeratin 8 and cytokeratin 18 are both epithelium-specific genes. Immunohistochemically, YS expressed T-cell specific antigens CD2 and CD3, and T cell receptor beta and gamma chain genes were rearranged in YS, but not in LYs. Moreover, using restriction fragment length polymorphism probes, we found that LYs exhibited different cell lineage from YS. Thus, our present findings, unexpectedly, raise fundamental questions concerning the cellular origins of YS and LY variants rather than pointing to any specific mechanism to explain the LY phenomenon.

Hereditary renal cell carcinoma (RC) in the rat, originally reported by Eker in 1954, is an example of Mendelian dominant predisposition to a specific cancer in an experimental animal. We previously reported that this predisposing inherited gene is a tumor suppressor gene fitting Knudson's "two-hit" model. This study was designed to map the RC susceptibility gene in the Eker rat using backcross animals. Our present data clearly show that the RC gene is genetically linked to the protamine-1 gene (Lod score = 11.65) and the interleukin-3 gene (Lod score = 4.13), both of which are located on the proximal part of rat chromosome 10. Rat chromosome 10 is currently believed to have no syntenic relationship to human chromosome 3p, the presumed site of the putative tumor suppressor gene for human RC and the locus of von Hippel Lindau disease (affected patients develop multiple RCs). Thus, the Eker rat might have a mutation of a novel tumor suppressor gene related to renal carcinogenesis.

A novel rat model of hereditary renal cell carcinoma (RC) was found in a rat colony of the Sprague-Dawley (SD) strain in Japan, and named the "Nihon" rat in 2000. This study was designed to map the RC susceptibility gene in the Nihon rat using 113 backcross animals. Our present data clearly show that the Nihon gene is genetically linked to interleukin-3 (IL3) gene (chi(2) = 93.6, Lod score = 25.16), lethal (2) giant larvae (LLGL1) locus (chi(2) = 109.0, Lod score = 31.56) and myosin heavy chain, embryonic skeletal muscle (MYHSE) gene (chi(2) = 90.6, Lod score = 23.87), which are located on the distal part of rat chromosome 10. The order of the genes is the Eker (Tsc2) gene (located on the proximal part of rat chromosome 10; human chromosome 16p 13.3)--21.3 cM--IL3 gene (human 5q23-31)--4.4 cM--Nihon gene--0.9 cM--LLGL1 locus (human 17p11.2)--4.4 cM--MYHSE gene (human 17p13.1). We also detected loss of the wild-type allele at the MYHSE locus, fitting Knudson's "two hit" model. Thus, the Nihon rat should have a mutation of a novel tumor suppressor gene related to renal carcinogenesis.

We have developed transgenic mice that inherit albumin promoter-regulated simian virus 40 (SV40) large T antigen gene, expressed specifically in hepatocytes. These mice all develop multifocal hepatocellular carcinomas at around 5 months and die of liver insufficiency by 7 months. Sequential morphological observation of hepatocarcinogenesis revealed 5 distinct stages: (I) newborn to 2 weeks of age, neither recognizable histological changes nor cellular replication in spite of T antigen expression; (II) between 3 and 7 weeks, diffuse cytomegalic change of hepatocytes with numerous abnormal mitoses, usually resulting in cell death; (III) from 7 weeks onwards, quasi-regenerative small hepatocyte foci with a decreased tendency for cytomegaly in spite of T antigen expression, rapidly replacing the hepatic tissue; (IV) 11 weeks of age and thereafter, neoplastic foci and nodules with enzymatic alteration; (V) 20 weeks of age and thereafter, gross hepatocellular carcinomas with occasional pulmonary metastases. Considerable variation existed both in morphological and enzymatic features and T antigen expression among neoplastic lesions, including carcinomas. Thus, these transgenic mice clearly show a multistep process in hepatocarcinogenesis with remarkable synchrony and provide a promising model for analyzing the essential events of carcinogenesis at different stages.