Absence of Linkage between the Retinoblastoma Gene and hts Gene in the LEC Rat: A Model of Human Wilson's Disease

The LEC rat is an authentic model of human Wilson's disease (WD) with an autosomal recessively inherited hepatitis. We investigated linkage between the hepatitis gene ( hts ) and the rat retinoblas-toma gene (RB), that is closely linked to the WD gene in humans, to see whether or not the hts gene is located on the syntenic region of WD and is the counterpart of the WD gene. Polymerase chain reaction-single strand conformation polymorphism analysis with backcross progenies from LEC and TM strains showed that the recombination rate between these two loci was 55.6%, demonstrating that the hts and RB genes are not linked to each other. These data indicate that the hts gene is not the counterpart of the WD gene and that the human syntenic region on which the WD locus and human RB gene are located, is not conserved in the rat genome.     

Induction of Aldose Reductase Gene Expression in LEC Rats during the Development of the Hereditary Hepatitis and Hepatoma

We examined age-related changes in the protein and the mRNA expression of aldose reductase in livers of Long-Evans with a cinnamon-like color (LEC) rats, which develop hereditary hepatitis and hepatoma with aging, using Long-Evans with an agouti color rats as controls. The levels of the protein and mRNA of aldose reductase increased after 20 weeks, at the stage of acute hepatitis, and were maintained at 60 weeks of age, while those of aldehyde reductase seemed to be constant at all ages. The expression of aldose reductase was marked in cancerous lesions in hepatoma-bearing LEC rat liver compared to uninvolved surrounding tissues. These results indicated that elevation of aldose reductase accompanied hepatocarcinogenesis and may be related to the acquisition of immortality of the cancer cells through detoxifying cytotoxic aldehyde compounds.     

Accumulation of Abnormally High Ploid Nuclei in the Liver of LEC Rats Developing Spontaneous Hepatitis

Enlarged hepatocytes with huge nuclei were found in LEC rats with hereditary hepatitis. Flow cytometric analysis of the DNA content of nuclei from jaundiced LEC rats revealed the presence of very high polyploids, such as 32n and 64n. At the age of 12 weeks, before the onset of hepatitis, 8n polyploid nuclei were more frequent in LEC rats than in LEA rats, a sibling line of LEC rats. Binucleated hepatocytes were also more frequent in LEC rats than in LEA rats at week 4. Bi-, tri- and tetra-nucleated cells whose nuclei were sometimes different in size were observed when jaundice became manifest. The number of proliferating liver cells, determined by pulse labeling with 5-bromo- 2'-deoxyuridine (BrdU), was higher in LEC rats than in LEA rats at 2, 4, 8, 12 and 14 weeks, with a maximum at week 4. A remarkable increase of BrdU uptake was observed at week 16, when jaundice developed. The possible involvement of abnormal cytokinesis and kariokinesis in the manifestation of hepatitis was suggested.     

Differing Distribution of Hepatocyte Growth Factor-positive Cells in the Liver of LEC Rats with Acute Hepatitis, Chronic Hepatitis and Hepatoma

Using anti-rat hepatocyte growth factor (HGF) antibody, we investigated the distribution of HGF-positive cells in the liver tissues of LEC rats at various phases of liver diseases. During the phase of fulminant hepatitis, HGF-positive cells increased remarkably, and many of them were localized at the portal triads; these cells were identified from their shape as non-epithelial cells. A reduced number of HGF-positive cells was observed during the phase of chronic hepatitis, while no HGF-positive cells were seen in the tissue of cholangiofibrosis. During the phase of carcinoma, staining revealed that both the hepatocellular carcinoma cells and the non-epithelial cells in cancerous liver tissue were HGF-positive. These results suggest that, in LEC rats, HGF may play an important role in the regeneration of hepatocytes as well as in the development of hepatocellular carcinoma.     

Two-dimensional Electrophoretic Analysis of Hepatitis-associated Polypeptides in Liver of LEC Rats Developing Spontaneous Hepatitis

High-resolution two-dimensional polyacrylamide gel electrophoresis in combination with silver staining was used to analyze between 800 and 1000 cytosolic and particulate polypeptides from age-matched livers of normal male Long-Evans rat with Agouti coat color (LEA) and Long-Evans rat with Cinnamon-like coat color (LEC) rats with hereditary trait of hepatitis at ages long before, immediately prior to, and just after the onset of hepatitis. Although the electrophoretic patterns of polypeptide expression were very similar with respect to the overall spot patterns, a number of polypeptides which differed either qualitatively or quantitatively were noted. Two constitutively expressed cytosolic polypeptides, P29.5 (M_r 29.5 kDa/pI 6.73) and P30 (30 kDa/6.70), were not detected in livers of LEC animals at any age. In the normal LEA rats both P29.5 and P30 were detected as early as one day after birth and both were expressed at similar concentrations at all ages. In the LEC rats P30-C (30 kDa/6.68) was constitutively expressed in close proximity to the expected position of P30, and P30-C was not detected in the LEA rats. By means of non-equilibrium pH gradient electrophoresis two relatively basic polypeptides were detected in the LEC rats. P18ne was detected immediately prior to and P27ne immediately after the clinical manifestation of hepatitis. Experiments in F₁ backcross ([LEA × LEC] × LEC) animals, however, failed to demonstrate any genetic link between either the expression or lack of expression of P29.5, P30, P30-C, or P18ne and hepatitis development. P27ne was detected in all backcross animals exhibiting hepatitis, but was never observed in LEC rats prior to the onset of hepatitis. Although we were unable to identify any unique loss of expression of polypeptides which are genetically linked to hepatitis susceptibility in LEC rats, specific subsets of quantitatively modulated polypeptides were detected.     

High Sensitivity of LEC Rats with Chronic Hepatitis to Hepatocarcinogenesis: Decreases in Unscheduled and Replicative DNA Synthesis of the Hepatocytes

We carried out the following three experiments to clarify the mechanism of hepatocarcinogenesis in Long-Evans Cinnamon (LEC) rats. (1) Sensitivity to diethylnitrosamine (DEN): LEC rats (8 and 25 weeks old) without and with hepatitis and age-matched F344 rats were administered an intraperitoneal injection of a low dose of DEN. Eight weeks after the injection, the numbers of glutathione-S-transferase placental-form (GST-P)-positive foci in the 33-week-old LEC rat liver were significantly higher than those in the livers of the other three groups of rats. (2) Potential for unscheduled DNA synthesis (UDS): Isolated hepatocytes of 25-week-old LEC rats with chronic hepatitis showed about one-third the level of UDS induced by UV irradiation, as compared to that of age-matched F344 rats, while no significant difference was found between the UDS of isolated hepatocytes of 8-week-old LEC rats and age-matched F344 rats. (3) Potential for proliferation: Isolated hepatocytes from 8-week-old LEC rats responded well to epidermal growth factor (EGF) in culture, to almost the same degree as F344 rat hepatocytes, while a remarkable decrease in the responsiveness of hepatocytes isolated from 25-week-old LEC rats to EGF was found. These results suggested that LEC rat hepatocellular carcinoma could be naturally initiated after the onset of hepatitis by carcinogens contaminating food and the environment, probably due to the reduction of DNA repair activity, after which initiated hepatocytes selectively proliferate in response to growth stimuli endogenously produced as a result of continuous loss of hepatocytes (chronic hepatitis), because of a decrease in growth activity of non-initiated hepatocytes.     

Gene Expression of Placental Glutathione S-Transferase in Hereditary Hepatitis and Spontaneous Hepatocarcinogenesis of LEC Strain Rats

Liver tissues of LEG rats, which develop fulminant hepatitis and hepatocellular carcinoma (hepatoma), were examined by Northern blot analysis using a cDNA probe of rat placental glutathione S -transferase (GST-P). GST-P gene expression was observed not only during hepatocarcinogenesis but also in fulminant hepatitis before development of chronic hepatitis and hepatoma in LEC rats. Cholangiofibrosis in LEC rats also showed high GST-P expression. A transplantable cell line derived from spontaneous LEC hepatoma exhibited a remarkably high expression. By contrast, very weak expression was observed in the livers of young LEC rats before development of hepatitis and control strain rats. Thus, spontaneous hepatic lesions in LEC rats may provide a new clue to elucidate the mechanism of GST-P gene expression.     

Long-Evans A and C Rat Strains Susceptible to Clastogenic Effects of Chemicals in the Bone Marrow Cells

The Clastogenic responses to direct- and indirect-acting carcinogens in bone marrow cells of LEA, LEC, Wistar and SD rats were compared. The frequency of chromosome aberrations (CA) induced by n -butyl-N-nitrosourea or methylmethanesulfonate (MMS), which does not need metabolic activation, was significantly higher in both LEA and LEC rats than in Wistar or SD rats. When bone marrow cells of each rat strain were exposed to MMS in vitro , they also showed the same tendency in CA frequency. Therefore, the high sensitivity of both LEA and LEC rats to the Clastogenic effects of direct-acting carcinogens seems to result from the sensitivity of the bone marrow cells themselves. On the other hand, the CA frequency induced by 7,12-dimethylbenz[a]anthracene (DMBA) or aflatoxin bi (AFB₁), which requires metabolic activation, was lower in LEC rats than in the other 3 strains. The CA frequency induced by DMBA or AFB₁ in LEC rats fed Cu-free diet since birth (Cu-free LEC rats) was higher than that in LEC rats given normal diet and lower than that in LEA rats, although the difference was statistically significant only between Cu-free LEC rats and LEC rats treated with DMBA. The copper concentrations in the livers of LEA, Cu-free LEC and LEC male rats aged 4 weeks were 5.0 ±0.4, 33 ±7.7 and 106±3.4 μg/g wet weight, respectively. These results suggest that the lower sensitivity of LEC rats to the Clastogenic effects of indirect-acting carcinogens may be due to the effect of the large amount of copper accumulated in LEC rat liver.     

Role of Atp7b Gene in Spontaneous and N-Diethylnitrosamine-induced Carcinogenesis in a New Congenic Strain, WKAH.C-Atp7b Rats

To examine whether Long-Evans Cinnamon (LEC) rats, a mutant rat model of Wilson's disease, have a susceptibility gene(s) to hepatocarcinogenesis in addition to the causative gene, Atp7b , we established a new congenic strain, WKAH.C-Atp7b rats, in which the Atp7b gene of the LEG rats is inserted into the normal Wistar-King Aptekman Hokkaido (WKAH) background. Hepatocellular tumors developed spontaneously in both sexes of WKAH.C-Atp7b rats, their incidence being slightly lower than that in LEG rats. Incidences of spontaneous liver tumors in LEG, WKAH.C- Atp7b and WKAH rats correlated with hepatic copper and iron concentrations. Medium-term liver bioassay showed that LEG rats were more susceptible to the induction of glutathione S-transferase placental form-positive preneoplastic foci than WKAH.C -Atp7b rats, and WKAH . C -Atp7b rats were more susceptible than WKAH rats. In an N -diethylnitrosamine (DEN)-induced long-term carcinogenicity study, 1) LEC rats were similarly or rather less susceptible to hepatocellular tumors than WKAH.C-Atp7b and WKAH rats, indicating that the progression of the preneoplastic foci to liver cancer in LEG rats was worse than that in WKAH.C-Atp7b and WKAH rats, 2) the incidences of kidney tumors in LEG and WKAH.C-Atp7b rats were higher than that in WKAH rats and high copper concentrations in the kidneys were observed in LEG and WKAH.C- Atp7b rats, 3) LEC rats were resistant to lung carcinogenesis. These data indicate that the susceptibility of LEG rats to liver and kidney carcinogenesis could be explained by Atp7b gene mutation and that the susceptibility to lung carcinogenesis is controlled by gene(s) other than Atp7b.     

A Transplantable Cell Line Derived from Spontaneous Hepatocellular Carcinoma of the Hereditary Hepatitis LEC Rat

A serially transplantable rat hepatocellular carcinoma (HCC) line was established. The primary spontaneous HCC which developed in a 506-day-old male hereditary hepatitis LEC rat was inoculated into young LEC rats. Only this HCC of 18 primary HCCs was successful in serial transplantation. The established cell line was histologically identical to the primary HCC showing a well-differentiated type with a trabecular structure of tumorous hepatocytes. The characteristic of albumin production was maintained. Chromosome analysis revealed rather widely dispersed polyploid chromosome numbers with a modal value at 96. Every metaphase contained two to five unusually large marker chromosomes.     

非酒精性脂肪性肝病相关基因的单核苷酸多态性研究

背景与目的：探讨载脂蛋白B(Apolilpoprotein B, ApoB)、β3-肾上腺素能受体（β3-adrenergic receptor,β3-AR）、细胞色素P4502E1(cytochrome P4502E1,CYP2E1)基因单核苷酸多态性与非酒精性脂肪性肝病(Nonalcoholic fatty liver disease, NAFLD)发病的关系,并从分子水平探讨该病的发病机制。材料与方法: 应用聚合酶链反应和基因芯片技术,对194例NAFLD患者和189例健康对照的ApoB、β3-AR、 CYP2E1基因多态性进行了分析。结果: 轻度NAFLD组Apo BMspⅠ位点M+M-基因型频率(18.6%)和等位基因型频率(9.3%)均高于对照组( 分别为10.3%和5.2%),差别有统计学意义（p＜0.05） 。Apo BMspⅠ和CYP2E1基因多态性的联合作用使中重度脂肪肝发病的危险性增加近4倍。结论: Apo BMspⅠ位点多态性与轻度NAFLD有关,CYP2E1和Apo B MspⅠ基因多态性的联合作用分析从分子水平解释了NAFLD发生发展的机制,同时也为研究NAFLD遗传易感性从方法学上提供了新的思路。     

载脂蛋白E基因多态性和帕金森病的相关性

背景与目的:探讨乌鲁木齐地区人群中载脂蛋白E(ApoE)基因多态性与帕金森病(Parkinson＇s disease,PD)的相关性.材料与方法:用PCR-RFLP方法检测51例散发性PD和52例正常对照的ApoE基因型.根据有无痴呆将所有PD患者分为二组,即PDD组和PD组.用x2检验进行关联分析.结果:PD PDD组、PD组和PDD组与对照组间的ApoE基因型频率差异无显著性(P＞0.05).PD组ApoE等位基因频率与对照组相近.PD PDD组和PDD组的ε 2和ε3频率类似于对照组,但是,ε4频率(分别为12.8%和16.7%)明显高于正常对照组(3.9%),组间比较差异有显著性(P＜0.05).结论:ApoEε4等位基因对PD的发生无明显影响,但是,它是伴有PD的痴呆患者的发病危险因素.     

Identification of Changes in Gene Expression Associated with Minimal Residual Disease

Minimal residual disease (MRD), the tumour burden which remains after a course of treatment that has resulted in clinical remission [1], appears to differ in certain characteristics from the primary tumour population. Certainly the cells which comprise MRD have had to escape from the constraints of the primary tumour mass, invade normal tissue and penetrate small vessels in order to enter the circulation in which they then have had to survive. Such activities are the consequence of the expression of specific proteins and these may well be a reflection of alterations in DNA or RNA levels. Identifying the changes in RNA expression levels between related cell groups exhibiting different phenotypes recently has become a great deal easier as a consequence of developments in analytical procedures such as Differential Display (DD) and Serial Analysis of Gene Expression (SAGE). Application of these procedures to MRD cells recovered from blood, bone marrow or lymph node, should identify novel sequences associated with tumour progression and the development of disseminated disease.     

Low Susceptibility of Long-Evans Cinnamon Rats to N-Butyl-N-(4-hydroxybutyl)-nitrosamine-induced Urinary Bladder Carcinogenesis and Inhibitory Effect of Urinary Copper

We studied the susceptibilities to N -butyl- N -(4-hydroxybutyl)nitrosamine (BBN)-induced urinary bladder carcinogenesis of male Long-Evans Cinnamon (LEC), F344 and Long-Evans Agouti (LEA) rats. Male rats ( n =21) were given 0.1% BBN in their drinking water from week 6, 8 and 10 for one week, and killed in week 56. The incidences of transitional cell tumors (papillomas plus carcinomas) in BBN-treated LEC and F344 rats were 12% and 76%, respectively ( P < 0.001, experiment 1), and those in LEC and LEA rats were 11% and 95%, respectively ( P < 0.001, experiment 2). When male LEC and F344 rats were given 0.1% BBN in their drinking water for 7 days, the intake of BBN and the urinary concentration of its active metabolite, N -butyl- N -(3-carboxypropyl)nitrosamine (BCPN), were higher in the LEC rats ( P < 0.01). The urinary pHs of untreated LEC and F344 rats were similar between week 6 and 30. The urinary copper concentration was lower in LEC rats before jaundice than in F344 rats, but its concentrations in 28- and 50-week-old LEC rats were 1.7 and 2.3 times those in F344 rats. In a two-stage carcinogenesis study using F344 rats, i.p. injections of cupric nitrilotriacetate increased urinary copper excretion, and inhibited BBN induced bladder carcinogenesis. In a two-stage carcinogenesis study using LEC rats, oral administration of D-penicillamine decreased urinary copper excretion, and increased BBN-induced bladder cancer, although the difference was not significant. These data show that LEC rats are resistant to bladder carcinogenesis and suggest that urinary copper has a significant role in their resistance.     

Mutation Analysis of the PTEN/MMAC1 Gene in Japanese Patients with Cowden Disease

Cowden disease (CD), also known as multiple hamartoma syndrome, is an autosomal dominant cancer syndrome associated with high risk of breast and thyroid cancer. Recently, germline mutations in PTEN/MMAC1 , which has nine exons encoding a dual specificity phosphatase with homology to tensin and auxilin, have been identified on chromosome 10q23 in some 40 to 80% of CD patients. Our polymerase chain reaction amplification and sequence analysis of all coding regions identified five different mutations including four novel germline mutations among 5 of 12 unrelated Japanese CD patients. The novel findings included a missense mutation (G→T) at nucleotide 1004 in exon 8 resulting in an arginine-to-leucine change at codon 335 (R335L), two novel splice-site mutations (209+1delGT and 209+1delGTAA) in intron 3, and insertion of G at nucleotide 632 in exon 6 (632insG). We also detected a nonsense mutation (C→T) at nucleotide 697 producing R233X in exon 7, which has been reported previously. From reported phenotypic data concerning CD patients from five different families who had the R233X mutation, it may be suggested that R233X mutation correlates with macrocephaly. Although previous reports have implicated exon 5 as a "hot spot," we found no mutation in exon 5.     

Deficiency of β1-6 N-Acetylglucosaminyltransferase Involved in the Biosynthesis of Blood Group I Antigen in the Liver of LEC Rats

The activities of the β1-6 and β1–3 N-acetylglucosaminyltransferases, which synthesize blood group I and i antigens, respectively, were measured in various tissues of hepatitis- and hepatoma-predisposed rats (LEC rats). In LEC rats the β1-6 N-acetylglucosaminyltransferase activity was barely detectable in the liver, while substantial enzyme activity was found in other tissues. In the control LEA rats the enzyme was expressed in most tissues, including the liver. Immunochemical studies using a monoclonal antibody which recognizes I antigen indicated that the expression of I antigen was less prominent in hepatocytes of LEC rats than in hepatocytes of LEA rats. The level of β1–3 N-acetylglucosaminyltransferase activity was constant in most of the tissues during the development. These results indicate that the biosynthesis of I antigen does not occur in the livers of the LEC rats.     

抗肿瘤和乙肝双交IL18—HBV S基因疫苗的构建

目的：探讨IL－18及乙肝病毒S基因（HBVS）的联合功效。方法：从胎肝组织中抽提总RNA，RT－PCR扩增IL－18cDNA基因。从载体pcDNA3．0／S中限制性酶切获取HBV S基因，然后，将其与IL－18cDNA一半亚克隆到真核表达质粒pIRES-EGFP中。结果：构建了具有抗肿瘤及乙肝双重功效的IL－18－HBV S乙肝基因疫苗。结论：含有IL－18基因的HBV基因疫苗的构建为基因疫苗的功能和应用研究提供了基础。     

新城疫病毒HN基因构建的核酸疫苗抗肿瘤作用研究

目的 :探讨HN基因在NDV抗肿瘤上发挥的作用。方法 :以pVAX1为表达载体构建了含NDVHN基因的pVHN核酸疫苗 ,以脂质体介导方法在体外转染OS732细胞 72h后 ,用 3,5 二羟基甲苯法测定OS732细胞膜唾液酸含量的变化。 6周龄BALB/c鼠左后肢皮下接种 2× 10 6个S180肿瘤细胞 ,分别于肿瘤接种后的第 7天 (肿瘤直径为 2～ 3mm)和第 17天瘤内注射 10 0 μg核酸重组体 ,同时设pVAX1对照组和单纯荷瘤对照组。荷瘤鼠经治疗后框窦采血 ,分离血清 ,测定血清唾液酸含量。取脾 ,采用FACS方法测定淋巴细胞亚群数量的变化。结果 :pVHN体外转染OS732细胞后 ,能显著降低细胞表面唾液酸含量 (P <0 .0 5 )。pVHN应用于荷瘤鼠显著降低血清中唾液酸含量 (P <0 .0 5 ) ,引起CD8+ T淋巴细胞亚群数量的增加 (P <0 0 5 )。结论 :单独HN基因在体外转染能够降低肿瘤细胞表面唾液酸含量 ,体内表达后引起T淋巴细胞亚群数量改变。     

A Novel Renal Carcinoma Predisposing Gene of the Nihon Rat Maps on Chromosome 10

A novel rat model of hereditary renal cell carcinoma (RC) was found in a rat colony of the Spra-gue-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 annuals. Our present data clearly show that the Nihon gene is genetically linked to interleukin-3 (IL3 ) gene (χ²=93.6, Lod score=25.16), lethal (2) giant larvae (LLGL1 ) locus (χ²=109.0, Lod score=31.56) and myosin heavy chain, embryonic skeletal muscle (MYHSE ) gene (χ²=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 17pl3.1). 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.     

Detection of Minimal Residual Disease in Adult Acute Lymphoblastic Leukemia by Analysis of Gene Rearrangements and Correlation with Early Relapses

While more than 75% of the adult patients with acute lymphoblastic leukemia (ALL) achieve a complete remission after treatment with intensive chemotherapy, about 40% of them relapse within five years. These relapses are probably due to residual leukemic cells. Gene rearrangements are used as markers of clonality and thereby monoclonal leukemic lymphoid cells can be detected with high sensitivity. In this study, we have applied the analysis of gene rearrangements to detect minimal residual disease in patients considered to be in complete remission. Serial bone marrow samples were studied in 35 patients before and four weeks after initiation of a standardized induction chemotherapy. Gene probes for the joining regions of the human immunoglobulin heavy chain and the constant regions of the human T-cell receptor β-chain were used.

In five of the 35 patients, the same gene rearrangements found before therapy persisted and indicated residual disease. Four of them relapsed within a median time of 10 weeks. Six of the 30 other patients without detectable gene rearrangements after induction therapy also relapsed, but median time to relapse was 30 weeks. Two of them had a relapse in the central nervous system without detectable bone marrow infiltration. Our data suggest that minimal residual disease, detected by analysis of gene rearrangements, is associated with a high early relapse rate. Analysis of gene rearrangements at the time of assessing the response to primary therapy seems to be of prognostic value in ALL and may contribute to a stratification of further therapy.