Quantitative image cytometry of hepatocytes expressing gamma-glutamyl transpeptidase and glutathione S-transferase in diethylnitrosamine-initiated rats treated with phenobarbital and/or phthalate esters.

Image cytometry was used to quantify the volume of liver expressing two histochemical markers associated with neoplasia, gamma-glutamyl transpeptidase (GGT) and the placental isozyme of glutathione S-transferase (GST-P). Rats were treated with diethylnitrosamine (DENA) followed by phenobarbital (PB), di(2-ethylhexyl)phthalate (DEHP), or di-n-octyl-phthalate (DOP) for 26 weeks. In one series, PB-treated rats were given 2.0%, 0.5%, or 0.1% DEHP in the feed. GGT expression was detected diffusely throughout the liver parenchyma in several treatment groups so that any enhanced expression in altered foci (AF) and nodules (N) was not apparent. GST-P was detected only in AF and N. GST-P may represent a second genetic alteration, as GST-P+ AF and N also expressed GGT but not the reverse. The peroxisome proliferator DEHP inhibited expression of GGT or GST-P in livers of either DENA-treated or DENA+PB-treated rats. With GST-P the reduction was correlated to a reduced number of AF and N. In contrast, DEHP's stereoisomer, DOP, was as effective as PB in promoting expression of both markers. We conclude that image cytometry of hepatocytes expressing GST-P can be used in the bioassay of the carcinogenic potential of chemicals that affect liver proliferation.     

The expression of oncogenes in human developing liver and hepatomas

Oncogene expression was examined in the human fetal liver and human hepatomas. Erb (B), erb (A+B), Ha-ras, myc, fos and fms oncogene expression elevated in certain stages of fetal liver development and in hepatoma as compared to the normal adult human liver. In contrast, rel, src, mos, sis, myb, Ki-ras and bas oncogenes showed no apparent change of their mRNA levels during fetal liver development and in hepatoma. Further study of erb B oncogene expression in human cirrhotic liver and hepatoma demonstrated a strong correlation between erb B expression and alteration of its gene structure.     

The expressions of oncogenes and liver-specific genes in Morris hepatomas

The expression of three liver-specific genes and four oncogenes was studied in the Morris hepatomas 8994, 7288c, 7777, 5123tc, and 7800. Total RNA isolated from these tumors was probed with cDNA's for alpha-fetoprotein (AFP), albumin, tyrosine aminotransferase (TAT), and the oncogenes Ha-ras, Ki-ras, myc and src. When compared to mRNA's levels expressed in normal adult liver, we found AFP levels elevated in AFP-producing tumors, albumin and TAT mRNA levels depressed in all tumors, except TAT is elevated in 5123tc and the oncogenes with the exception of src elevated in all tumors. These results argue against a coordinated expression of these genes as a result of transformation, but suggest that oncogene expression is related to tumorigenesis or proliferation.     

In Vitro transformation of LW13 Rat liver epithelial Cells

A rat liver epithelial cell line designated LW 13 was established using a sequential sedimentation method.The cell line retained many normal proerties of liver epithelial cells and showed some structural and functional features resembling those of liver parenchymal cells,LW13 cells became malignant after the intrduction of exogenous transforming EJ Ha ras gene,Tumors produced by inoculation of the transformed cells into baby rats contained areas of poorly differentialted hepatocellular carcinoma,In situ hybridization analysis confirmed the random rather than specific integration of exogenous ras gene into host chromosomes.Furthermore,an at least tenfold increase in the expression of the endogenous c mys gene was detected among transformed cell lines,suggesting the involvement of the c myc proto oncogene in the in vitro transformation of rat liver epithelial cells by EJ Ha ras oncogene.     

C-myc、p53基因和AFP在家兔诱发性肝癌及癌旁组织中的表达

目的获得家兔二乙基亚硝胺诱发肝脏肿瘤的形态学资料,探讨Cmyc、p53基因和甲胎蛋白的表达情况。方法取5例家兔多结节肝癌的肿瘤性结节25个,瘤旁肝组织每例1块,进行一般病理组织学和超微结构观察。同时,以免疫组化方法检查Cmyc、p53基因和AFP的表达情况。结果和结论25个结节中,良性增生10个,高分化肝细胞癌11个,中分化肝细胞癌4个。家兔肝细胞癌的发生与Cmyc基因、AFP的过表达和p53基因的突变有关。参照肿瘤诱发过程中,增生性肝硬化、腺瘤样增生、高分化癌、中分化癌的变化顺序,在所检测的三种标志物中,Cmyc蛋白为最早出现的肿瘤标志物,p53基因突变相对较晚出现,AFP在恶性肿瘤结节中的表达率最高(933%)。     

Rat liver foci study on coexposure with 50 Hz magnetic fields and known carcinogens

A study was performed to investigate possible interactions by magnetic fields (MF) with the processes of initiation and promotion of chemically induced preneoplastic lesions in rat liver. Male Sprague-Dawley rats were subjected to a 70% partial hepatectomy followed after 24 h by i.p. injection of diethylnitrosamine (DENA) as a tumour initiator. Starting one week after the DENA-treatment phenobarbital (PB) was given to promote growth of enzymatically altered foci of liver cells. MF was applied immediately after the partial hepatectomy and continued until sacrifice after 12 weeks of PB exposure. Homogenous horizontal AC magnetic fields with a frequency of 50 Hz and flux densities of 0.5 μT or 0.5 mT were used. The rats coexposed with MF and DENA plus PB did not gain weight as much as the rats exposed to the chemical agents only. The MF-exposure also resulted in a slight reduction in size and numbers of the focal lesions. The results suggest an interaction of MF with the processes of chemical carcinogenesis either as a result of stress or depending on effects on the proliferation of preneoplastic cells. © 1993 Wiley-Liss, Inc.     

豚鼠气道及肺组织原癌基因表达与哮喘的相关研究

目的：为研究原癌基因在哮喘发病中的作用。方法：以卵白蛋白致敏豚鼠建立哮喘模型,用Ｄｏｔ－ｂｌｏｔ、Ｎｏｒｔｈ－ｅｒｎ－ｂｌｏｔ分子杂交及免疫组化技术,分别观察正常及哮喘发作后豚鼠气道上皮及肺组织中原癌基因ｃ－ｆｏｓ、ｃ－ｍｙｃ的表达水平。结果：正常豚鼠气道及肺组织中ｃ－ｆｏｓ及ｃ－ｍｙｃｍＲＮＡ无或很少表达,哮喘发作后豚鼠气道上皮及肺组织中ｃ－ｆｏｓ和ｃ－ｍｙｃｍＲＮＡ表达明显增强,３０ｍｉｎ达高峰,发作后４ｈ降至正常水平。地塞米松对ｃ－ｆｏｓ及ｃ－ｍｙｃｍＲ－ＮＡ有部分抑制作用。结论：原癌基因ｃ－ｆｏｓ及ｃ－ｍｙｃ在哮喘发病过程中可能起一定作用。     

Immunohistochemical localization of epoxide hydratase in rat liver

An antibody produced against epoxide hydratase (EC 4.2.1.63) which had been purified to apparent homogeneity from hepatic microsomes of phenobarbital pretreated rats was employed in an unlabeled antibody peroxidase-antiperoxidase technique to localize the enzyme at the light microscopic level in the livers of untreated rats. Immunohistochemical staining for epoxide hydratase was detected in parenchymal cells throughout the liver lobule. Cells within the centrilobular regions, however, were observed to be stained more intensely than were those within the midzonal and periportal regions of the lobule. The results of this immunohistochemical study thus demonstrate that epoxide hydratase does not exhibit a uniform pattern of distribution within the liver lobule in untreated rats.     

血清miR-21 及AFP 在大鼠肝癌发生过程中的动态表达比较分析

目的:分析血清miR-21在肝癌发生过程中的表达水平并将其与传统肝癌血清标志物甲胎蛋白(AFP)比较,探索其成为肝癌早期诊断血清标志物的可能性。方法:二乙基亚硝胺(DENA)腹腔注射诱导建立大鼠肝癌模型,建模过程中收集造癌各个阶段的血清。Realtime-PCR检测血清miR-21的表达情况,ELISA法检测血清AFP水平。结果:与正常组及纤维化期大鼠相比,miR-21在肝硬化期、肝癌早期、肝癌晚期的大鼠血清均有不同程度上调(P<0.05),AFP在肝癌早、晚期的大鼠血清明显上调(P<0.05);与肝硬化期大鼠比较,肝癌早、晚期大鼠体内的miR-21表达显著上调(P<0.05),AFP在肝癌早期、肝癌晚期均显著上调(P<0.05)。结论:血清miR-21参与了肝癌发生的过程,对于肝癌发生的各个阶段均有很大的指示作用,可能作为肝癌预防和早期诊断的一个潜在标志物。     

Immunohistochemical Detection of Induced Expression of Wild-type p53 Tumor Supressor Protein in the Livers of Rats Treated with Diethylnitrosamine

Paraffin sections of formalin-perfused rat livers were stained immunohistochemically for p53. In livers from untreated rats, no p53 expression was observed. p53 expression was induced in a response to treatment with diethylnitrosamine 24h prior to sacrifice. Staining for p53 was localized in the nucleus of perivenous hepatocytes. In serial sections p53-immunopositive areas were found to co-localize with increased expression of TUNEL-positive cells. Without formalin perfusion, the staining for p53 was uneven and often barely detectable. Perfusion with saline prior to formalin resulted in a rapid decrease in the detectability of p53, indicating rapid degradation of this protein under these conditions. We conclude that rapid fixation by formalin perfusion increases the detectability of p53 by immunohistochemical staining. This provides a convenient procedure for studying the response of wild-type p53 in rodent liver. This procedure is also suitable for in situ investigations on the degradation of p53 protein stabilized by DNA damage.     

Transformation by myc prevents fusion but not biochemical differentiation of C2C12 myoblasts: mechanisms of phenotypic correction in mixed culture with normal cells

To study the effects of myc oncogene on muscle differentiation, we infected the murine skeletal muscle cell line C2C12 with retroviral vectors encoding various forms of avian c- or v-myc oncogene. myc expression induced cell transformation but, unlike many other oncogenes, prevented neither biochemical differentiation, nor commitment (irreversible withdrawal from the cell cycle). Yet, myotube formation by fusion of differentiated cells was strongly inhibited. Comparison of uninfected C2C12 myotubes with differentiated myc- expressing C2C12 did not reveal consistent differences in the expression of several muscle regulatory or structural genes. The present results lead us to conclude that transformation by myc is compatible with differentiation in C2C12 cells. myc expression induced cell death under growth restricting conditions. Differentiated cells escaped cell death despite continuing expression of myc, suggesting that the muscle differentiation programme interferes with the mechanism of myc-induced cell death. Cocultivation of v-myc-transformed C2C12 cells with normal fibroblasts or myoblasts restored fusion competence and revealed two distinguishable mechanisms that lead to correction of the fusion defect.     

Interferon-gamma induces altered oncogene expression and terminal differentiation in A431 cells

In tumor cell lines in which oncogene expression is abnormal, modulation of the expression of the oncogene (myc, src, or ras) by interferons (IFNs) has been observed concurrently with cell growth inhibition or phenotypic reversion. Oncogene expression has also been reported to vary during the differentiation of several neoplastic cell lines. Treatment of monolayer cultures of A431, a human epidermoid carcinoma cell line, with IFN-gamma resulted in rapid morphological alterations and cell death not seen with either IFN-alpha or IFN-beta. These changes were accompanied by elevated expression of mRNA's for p21 (the c-ras gene product) and the epidermal growth factor receptor as well as increases in the biosynthetic rate of their respective proteins. These effects likewise appeared to be specific for IFN-gamma. Growth inhibition by IFN-gamma was also observed when A431 cells were grown in a three dimensional in vitro culture system. Immunohistochemical staining of these "tumoroids" with a differentiation specific, anti-keratin antibody indicated that IFN-gamma enhanced expression of this keratin. This observation suggests that the killing by IFN-gamma of A431 cells may result from an acceleration of terminal differentiation.     

Cytoplasmic changes in level and distribution of glucose-6-phosphatase activities from rat liver during diethylnitrosamine-induced carcinogenesis.

Glucose-6-phosphatase (EC 3.1.3.9) activities were determined in isolated microsomes, cytoplasmic smooth and rough membranes, ribosomes and free cytosol from rat liver undergoing carcinogenesis by diethylnitrosamine (DENA) and compared with cytoplasmic fractions isolated in parallel from healthy animals from the same age.With continuous administration of a low dose of DENA (2.6 mg/kg rat per day for 20 weeks in the drinking water) livers of carcinogen treated rats became heavier than the control livers but the body weight decreased. About 70% of total glucose-6-phosphatase activity could be detected in the microsomal fraction. While there was no significant difference in this activity in both animal groups up to the 4th week, glucose-6-phosphatase of cancerous liver showed a distinct decrease of activity compared with normal liver.During cancer induction this enzyme became more soluble, confirmed by the observation that it was detached from firmer structures of cytoplasm as rough membranes and polysomes and translocated to smooth membranes and the soluble cytoplasmic fraction successively. The corresponding increase in glucose-6-phosphatase activity in the 105 000 g supernatant appears to be due to the loss of enzyme activity in a distinct cytoplasmic membrane fraction. These data strongly suggest that in parallel with alteration of cytoplasmic membrane structures during carcinogen feeding glucose-6-phosphatase is detached from heavier components of the cytoplasm while total activity decreased. Possible mechanisms of these findings are discussed.     

维甲酸对鼻咽癌细胞生长、表型和瘤基因表达的作用

研究了维甲酸（ＲＡ）对鼻咽癌细胞生长、表型和癌基因表达的作用．用ＲＡ诱导鼻咽癌细胞,绘制诱导前后的细胞曲线,观察细胞形态,并用Ｎｏｒｔｈｅｒｎ杂交和ＤＮａｓｅⅠ超敏感区分析法检测基因表达和调控．结果表明,ＲＡ能显著抑制鼻咽癌细胞的生长,前５ｄ下降约５０％．ＲＡ处理后的细胞从典型的多边形形态变成扁平、细长,类似纤维细胞状的形态．ＲＡ诱导前ｃ－ｍｙｃ基因和ｃ－Ｈａ－ｒａｓ基因ＨＮＥ２细胞中高表达,而诱导后ｃ－ｍｙｃ基因表达水平急剧下降,ｃ－Ｈａ－ｒａｓ基因无明显改变．在实验中还发现ＲＡ诱导前后的ｃ－ｍｙｃ基因和ｃ－Ｈａ－ｒａｓ基因中一些重要的超敏感位点和它们的功能．由实验结果可得到如下结论：ＲＡ能促进鼻咽癌细胞分化,通过对染色体上调控位点的作用来抑制ｃ－ｍｙｃ基因的表达,ＤＮａｓｅⅠ超敏感位点与细胞的分化程度、细胞的组织特异性和基因表达状态有关,ｃ－ｍｙｃ基因可通过不同的调控方式而失活．     

myc and src oncogenes have complementary effects on cell proliferation and expression of specific extracellular matrix components in definitive chondroblasts.

The effects of the avian viral oncogenes src and myc were compared for their ability to alter the differentiated phenotype and the proliferative capacity of definitive chondroblasts. As previously demonstrated, viruses carrying the src oncogene suppressed the synthesis of the chondroblast-specific products, type II collagen and cartilage-specific sulfated proteoglycan. In contrast, infection with MC29 and HB1 viruses, which carry the myc oncogene, did not suppress the synthesis of these normal differentiated cell products, but the infected cells exhibited an increased proliferative potential. The MH2 virus, which carries both the myc and mil oncogenes, both induced the suppression of these chondroblast-specific products and increased cell proliferation. The implications of these results for cooperation between oncogenes and the multi-oncogene models for neoplastic transformation are discussed.     

Streptozotocin-induced diabetes increases γ-glutamyltranspeptidase activity but not expression in rat liver

Earlier work describing increased biliary excretion of the acetaminophen-cysteine conjugate advanced the hypothesis that streptozotocin-induceddiabetes increases <γ > -glutamyltranspeptidase (GGT) expression in Sprague–Dawley rats. To test this hypothesis, rats were divided into control, diabetic, and insulin-treated diabetic groups. Diabetes was induced by intravenous injection of 45 mg streptozotocin/kg body weight and was effectively controlled by insulin treatment in the appropriate group. Densitometric quantification demonstrated that hepatic GGT activity in diabetic rats was significantly increased when compared to normal and insulin-treated diabetic controls. Histochemical staining of liver was greater in female than in male rats, and staining increased in female rat liver as the duration of diabetes lengthened from 30 to 90 days. GGT activity was increased by diabetes in liver canalicular-enriched and basolateral-enriched membrane preparations, and it was unchanged in renal brush border-enriched membranes. Total mRNA isolated from diabetic and insulin-treated diabetic rat livers did not conclusively demonstrate an elevation of GGT mRNA relative to normal. Western blot analysis showed no differences in the amount of GGT in diabetic versus normal rat livers. These data indicate that streptozotocin-induced diabetes does not alter the expression of, but does increase the activity of, GGT in liver. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 12: 219–225, 1998     

Characterization of electron transport enzymes in the envelope of rat liver nuclei.

Nuclei and microsomes were prepared from the livers of normal, phenobarbital (PB)-treated and beta-naphthoflavone (beta-NF)-treated rats, and the contents of several enzymes in both subcellular fractions were examined. In normal rats, the enzyme activities in the nuclear fraction were about one-third of those of microsomes on a phospholipid basis. The induction of some particular enzymes by the drugs was observed with nuclei as well as with microsomes. Cytochrome P-450 and NADPH-cytochrome c reductase were increased by PB treatment and cytochrome P-448 was induced by beta-NF treatment both in nuclei and in microsomes. The extents of inhibition of nuclear enzyme activities by the antibodies against corresponding microsomal enzymes were almost the same as those of the microsomal activities. It was concluded that a microsomal type electron transport system exists in rat liver nuclei, and that nuclear drug-oxidizing activities are inducible by PB or beta-NF as their microsomal counterparts are.     

Differential expression and function of three closely related phenobarbital-inducible cytochrome P-450 isozymes in untreated rat liver

The levels of expression of cytochromes P-450b and P-450e (both inducible by phenobarbital (PB) and differing by only 14 of 491 amino acids) in liver microsomes from untreated male rats were separately quantitated by Western blotting with a polyclonal antibody raised against P-450b that is equally effective against P-450e (anti P-450b/e). A protein with mobility identical to P-450e was detected in all microsomal samples. Microsomes from uninduced livers of individual male rats from five different strains exhibited only minor interstrain and interindividual variability in the expression of P-450e (17 +/- 5 pmol P-450e/mg microsomal protein) with the exception of the Brown Norway strain (8.5 +/- 0.5 pmol P-450e/mg). Expression of P-450b varied widely from undetectable levels (less than 2 pmol/mg) in most Sprague-Dawley rats to about 50% of P-450e levels in Fischer and Brown Norway strains. Anti P-450b/e inhibited total metabolism of 7,12-dimethylbenz[a]anthracene (DMBA) by uninduced microsomes, to an extent dependent on rat strain (15-30%), predominantly through inhibition of formation of 12-hydroxymethyl-7-methyl BA (12HOMMBA) (65-85%), the major metabolite of purified P-450e. A specific activity for P-450e-dependent DMBA metabolism was calculated from four sets of microsomes where the P-450b content was either undetectable or very low (0.7-1.0 nmol/nmol P-450e/min-1). Comparable calculated activities were, however, obtained from other untreated rat liver microsomes where P-450b levels were significant. Polymorphism in P-450b was detected but did not affect total P-450b expression or the sensitivity of DMBA metabolism to anti P-450b/e. A fourth band of greater mobility than P-450b (apparent Mr less than 50,000), was also recognized by anti P-450b/e. The intensity of this band did not vary among individual rats or among the different strains and therefore did not correlate with the sensitivity of microsomal DMBA metabolism to anti P-450b/e. A monoclonal antibody (MAb) against P-450b (2-66-3) recognized P-450's b, b2, and e on Western blots but did not react with this higher mobility band. MAb 2-66-3 and two other MAbs produced against P-450b inhibited 12-methylhydroxylation of DMBA by untreated rat liver microsomes to the same extent as anti P-450b/e. Following PB induction, P-450b was induced to about double the level of P-450e in most rat strains examined.(ABSTRACT TRUNCATED AT 400 WORDS)     

Expression of myc, fos and Ha-ras associated with chemically induced cell proliferation in the rat liver

Abstract. Events secondary to induced cell proliferation may play a role in the carcinogenic process. These studies investigated the expression of genes associated with growth control in response to two types of cell proliferation stimuli in the livers of male F344 rats. Regenerative hepatocyte proliferation after partial hepatectomy or a single dose of carbon tetrachloride, and mitogenic liver hyperplasia induced by a single dose of pheno-barbital or WY-14,643 were assessed by thymidine incorporation and quantitative autoradiography. The expression of myc, fos , and Ha- ras was evaluated by Northern blot analysis of liver derived poly(A)⁺ mRNA from these same animals. After each treatment, the level of hepatocyte proliferation (labelling index 4–32%) was observed to peak between 24 and 48 h and return to control values by 8 days. In every case, a peak in myc expression was seen between 0.5 and 18 h depending on the proliferative stimulus treatment. A large peak in fos expression was seen at 0.5–2 h but only with the cytotoxic and regenerative proliferative treatments partial hepatectomy or carbon tetrachloride. A broad peak in Ha -ras expression was observed 12 to 36 h after each treatment. These data demonstrate transient expression of these genes following the synchronous induction of hepatocyte proliferation. The increased expression of fos upon treatment with cytotoxicants, but not mitogens, suggests different modes of growth regulation that may be important in understanding the induction of cell proliferation by these two types of agents.