Immunofluorescent localization of type II insulin-like growth factor receptor in rat liver and hepatoma cells

1989 ◽  
Vol 121 (2) ◽  
pp. 221-NP ◽  
Author(s):  
M. A. Hartshorn ◽  
C. D. Scott ◽  
R. C. Baxter
Keyword(s):  
Rat Liver ◽  
Hepatoma Cell ◽  
Rat Hepatocytes ◽  
Hepatoma Cells ◽  
Type Ii ◽  
Adult Rat ◽  
Hepatoma Cell Lines ◽  
Igf Receptors ◽  
Immunofluorescent Technique ◽  
Igf Receptor

ABSTRACT We have used an immunofluorescent technique to localize type II insulin-like growth factor (IGF) receptors in rat liver, rat hepatocytes and three rat hepatoma cell lines (HTC, H-35 and 5123) using a polyclonal antibody (C-1) raised to purified rat liver type II IGF receptor. Specificity of the antiserum was confirmed by Western blotting of microsomal membranes prepared from hepatocytes and hepatoma cells which showed a single class of receptor in all cells, of Mr approximately 210 000 for hepatocytes, HTC and H-35 cells and approximately 220 000 for 5123 cells, on non-reduced, 4–15% polyacrylamide gradient gels. The specificity of the immunofluorescent technique was also verified by abolition of labelling after preincubation of antiserum with purified type II IGF receptor. Rat liver cryosections contained areas of juxtanuclear labelling in hepatocytes, consistent with the presence of type II IGF receptor in the Golgi region. Brightest immunofluorescence was seen in sections from fetal and neonatal rats with adult rat hepatocytes staining brightly only around central veins. Areas of labelling were also seen in connective tissue surrounding larger veins. Cultured adult rat hepatocytes and rat hepatoma cell lines also showed bright areas of juxtanuclear immunofluorescence, with HTC and H-35 cells staining more than 5123 and adult hepatocytes. Fetal rat hepatocytes in culture also labelled very brightly both in a juxtanuclear location and in small clusters over the cell, possibly on the cell surface. These observations indicate that type II IGF receptors are located predominantly on intracellular membranes and are most abundant in rapidly growing cells and tissues (such as fetal liver and hepatoma cells). Journal of Endocrinology (1989) 121, 221–227

Negative regulation of catalase gene expression in hepatoma cells

1992 ◽  
Vol 12 (6) ◽  
pp. 2525-2533
Author(s):  
K Sato ◽  
K Ito ◽  
H Kohara ◽  
Y Yamaguchi ◽  
K Adachi ◽  
...  
Keyword(s):  
Rat Liver ◽  
Catalase Activity ◽  
Hepatoma Cell ◽  
Nuclear Extract ◽  
Hepatoma Cells ◽  
Negative Regulation ◽  
Regulation Of Transcription ◽  
Catalase Gene ◽  
Core Sequence ◽  
Silencer Element

For an understanding of the molecular basis of the marked decrease in catalase activity of various tumor cells, expression of the catalase gene was studied in rat and human hepatoma cell lines and in rat liver, which was used as a control with high activity. RNA blot hybridization profiles and run-on assays indicated that the decrease in catalase activity was due to depression of catalase gene transcription. Chloramphenicol acetyltransferase (CAT) assays for the fragments with various lengths of the 5'-flanking region (up to -4.5 kb from the ATG codon) of the catalase gene revealed the presence of several cis-acting elements involved in the negative regulation of transcription. The most-upstream element with the strongest activity (-3504 to -3364 bp), when linked to the catalase promoter region (-126 bp) of the CAT construct and subjected to an in vitro transcription assay, did not yield transcripts in experiments with the hepatoma nuclear extract, whereas the unlinked template did yield transcripts. A gel shift competition assay using hepatoma nuclear extract showed the core sequence of the silencer element to be 5'-TGGGGGGAG-3'. A homology search found that the same core sequence was also present in 5'-flanking regions of the albumin gene and of some other liver enzyme genes, the expression of which has been reported to be down regulated in some hepatoma cells. Southwestern (DNA-protein) analysis demonstrated that an approximately 35-kDa nuclear protein bound to the silencer element was present in hepatoma cells but not in rat liver cells.

Different Expression of Esterase Variants in Rat Hepatic and Hepatom a-Derived Cell Lines Detected by Electrophoresis

1995 ◽  
Vol 50 (9-10) ◽  
pp. 664-668 ◽  
Author(s):  
Adel S. Afify ◽  
Yoshimitsu Yamazaki ◽  
Yu-ichi Kageyama ◽  
Shiro Yusa ◽  
Yoshikatsu Ogawa ◽  
...  
Keyword(s):  
Cell Lines ◽  
Rat Liver ◽  
Hepatoma Cell ◽  
Polyacrylamide Gel Electrophoresis ◽  
Liver Homogenate ◽  
Normal Liver ◽  
Liver Parenchyma ◽  
Hepatoma Cell Lines ◽  
Normal Rat ◽  
Transformed Cell Lines

Abstract Esterases in nine rat hepatic and hepatoma-derived cell lines and normal rat liver homogenate were detected by polyacrylamide gel electrophoresis coupled with active staining with a-naphthyl acetate or butyrate as a substrate. The esterase band patterns of the non-cancerous and oncogene-transformed cell lines were alike, but different from those of hepatoma cell lines and normal rat liver homogenate. The former groups of cells might have completely lost the characteristics of rat liver parenchymal cells, or else they might have their origin at cells other than liver parenchyma. The esterase patterns of the hepatoma cell lines (e.g., McA-RH7777) and the normal rat liver highly resembled with each other, exemplifying the slight biochemical deviation of cancer from normal cells. However, two-dimensional electrophoretogram for the McA-RH7777 cell line showed a prominent esterase spot {p/ 6.0-Mr 110 kDa) that was lacking in the normal liver. This result indicates that there is invariably some change in esterase expression between the cancer cells and the normal liver cells

Comparison of cytochrome P-450 levels in adult rat liver, postnatal rat liver, and primary cultures of postnatal rat hepatocytes

Life Sciences ◽  
1979 ◽  
Vol 25 (16) ◽  
pp. 1413-1418 ◽  
Author(s):  
Daniel Acosta ◽  
David C. Anuforo ◽  
Reagan McMillin ◽  
William H. Soine ◽  
Robert V. Smith
Keyword(s):  
Rat Liver ◽  
Primary Cultures ◽  
Rat Hepatocytes ◽  
Adult Rat ◽  
Cytochrome P 450

scholarly journals Biosynthesis, surface expression and function of the fibronectin receptor after rat liver cell transformation to tumorigenicity

1993 ◽  
Vol 291 (1) ◽  
pp. 247-255 ◽  
Author(s):  
M Decastel ◽  
M A Doyennette-Moyne ◽  
E Gouet ◽  
M Aubery ◽  
P Codogno
Keyword(s):  
Molecular Mass ◽  
Hepatoma Cell ◽  
Rat Hepatocytes ◽  
Hepatoma Cells ◽  
Rat Hepatocyte ◽  
Fibronectin Receptor ◽  
Zajdela Hepatoma ◽  
Sds Page ◽  
Normal Rat ◽  
And Function

Zajdela hepatoma cells are poorly-adherent cells derived from an undifferentiated tumour and transplanted into rat. We compared the biosynthesis, structure and function of the fibronectin receptor in normal rat hepatocytes with that in Zajdela hepatoma cells. The rat hepatocyte fibronectin receptor has been isolated. It is composed of two subunits: alpha 5 (molecular mass 155 kDa) and beta 1 (molecular mass 115 kDa). However, its biosynthesis has not yet been described. Using polyclonal antibodies raised against each of the subunits of the receptor, we observed that the alpha 5-subunit was synthesized as a 155-kDa polypeptide in normal rat hepatocytes and Zajdela hepatoma cells. In contrast, the molecular mass of the beta 1-subunit was 130 kDa in Zajdela hepatoma cells versus 115 kDa in normal rat hepatocytes. Pulse-chase experiments showed that the apparent transition time from the 100-kDa beta 1-precursor to the 130-kDa mature form was abnormally prolonged in Zajdela hepatoma cells since the latter was not detected until 24 h, while the transition from the 100-kDa precursor to the 115-kDa mature form began within 3 h in normal rat hepatocytes. Digestion of both the normal rat hepatocytes and Zajdela hepatoma cells 100-kDa beta 1-precursors with endo-beta-N-acetylglucosaminidase H and peptide N-glycosidase yielded products from 100 kDa to 84 kDa and 82 kDa, respectively, as judged by SDS/PAGE, suggesting that the same polypeptide chain is synthesized in normal rat hepatocytes and in Zajdela hepatoma cells. Incubation of the mature normal rat hepatocyte beta 1-subunit with peptide N-glycosidase reduced its molecular mass from 115 kDa to 82 kDa, as judged by SDS/PAGE, while the molecular mass of the abnormal mature Zajdela hepatoma cell beta 1-subunit decreased from 130 to 110 kDa. Thus, in addition to alterations in the Asn-linked oligosaccharide processing, ‘ascitic growth’ induced other post-translational modifications in the Zajdela hepatoma cell beta 1-subunit. Furthermore, both the abnormal mature 130-kDa and precursor 100-kDa beta 1-subunits were detected on the surface of Zajdela hepatoma cells, associated with the alpha 5-subunit. The relationship between these structural alterations in the fibronectin receptor and the impaired Zajdela hepatoma cell binding to soluble fibronectin or to a coated fibronectin matrix that was observed in this study is discussed.

scholarly journals Differential toxicities of albendazole and its two main metabolites to Balb/c 3T3, HepG2, and FaO lines and rat hepatocytes

2016 ◽  
Vol 60 (4) ◽  
pp. 495-500 ◽  
Author(s):  
Lidia Radko ◽  
Maria Minta ◽  
Sylwia Stypuła-Trębas
Keyword(s):  
Hepatoma Cell ◽  
Rat Hepatocytes ◽  
Neutral Red ◽  
In Vitro Toxicity ◽  
Isolated Rat Hepatocytes ◽  
Hepatoma Cell Lines ◽  
Cellular Models ◽  
The Impact ◽  
Isolated Rat

Abstract Introduction: The cytotoxicity of anthelmintic agent, albendazole (ABZ) and its two major metabolites, sulfoxide (ABZSO) and sulfone (ABZ-SO2), on non-hepatic Balb/c 3T3 line, two hepatoma cell lines (FaO, HepG2), and isolated rat hepatocytes was investigated. Material and Methods: Cell cultures were exposed for 24, 48, and 72 h to eight concentrations of the compounds ranging from 0.05 to 100 μg/mL (ABZ) and from 0.78 to 100 μg/mL (ABZ-SO and ABZ-SO2). Three different assays were applied in which various biochemical endpoints were assessed: lysosomal activity - neutral red uptake (NRU) assay, proliferation - total protein contents (TPC) assay and lactate dehydrogenase (LDH) leakage assay. Results: The most toxic was albendazole whose EC50 values calculated from the concentration effect curves ranged from 0.2 to 0.5 μg/mL (Balb/c 3T3 ) and from 0.4 to 73.3 μg/mL (HepG2). Rat hepatoma line and isolated rat hepatocytes were less sensitive to the impact of ABZ. Toxic action expressed as EC50 was recorded after 72 h exposure only in LDH release assay at 0.8 μg/mL and 9.7 μg/mL respectively. The toxicity of metabolites was much lower. The most sensitive to ABZ-SO were fibroblasts and EC50-72h values were similar in all three assays used, i.e. NRU (14.1 μg/mL), TPC (15.8 μg/mL), and LDH (20.9 μg/mL). In the case of ABZ-SO2 the mean effective concentrations were the highest, and could be reached only in one LDH assay. These values (μg/mL) were as follows: 65.3 (FaO), 65.4 (HepG2), 75.8 (hepatocytes), and 77.4 (Balb/c 3T3). Conclusion: The differences in in vitro toxicity of albendazole depend on metabolic ability of the cellular models. Primary cultured rat hepatocytes represent a valuable tool to study the impact of biotransformation on the cytotoxicity of drugs.

scholarly journals Elimination of Cancer Stem-Like “Side Population” Cells in Hepatoma Cell Lines by Chinese Herbal Mixture “Tien-Hsien Liquid”

2012 ◽  
Vol 2012 ◽  
pp. 1-10 ◽  
Author(s):  
Chih-Jung Yao ◽  
Chi-Tai Yeh ◽  
Liang-Ming Lee ◽  
Shuang-En Chuang ◽  
Chuan-Feng Yeh ◽  
...  
Keyword(s):  
Side Population ◽  
Hepatoma Cell ◽  
Hepatoma Cells ◽  
Human Hepatoma Cells ◽  
Herbal Mixture ◽  
Hepatoma Cell Lines ◽  
Chinese Herbal ◽  
Huh7 Cells ◽  
Complementary And Integrative Medicine ◽  
Small Subpopulation

There are increasing pieces of evidence suggesting that the recurrence of cancer may result from a small subpopulation of cancer stem cells, which are resistant to the conventional chemotherapy and radiotherapy. We investigated the effects of Chinese herbal mixtureTien-Hsien Liquid(THL) on the cancer stem-like side population (SP) cells isolated from human hepatoma cells. After sorting and subsequent culture, the SP cells from Huh7 hepatoma cells appear to have higher clonogenicity and mRNA expressions of stemness genes such asSMO, ABCG2, CD133,β-catenin,andOct-4than those of non-SP cells. At dose of 2 mg/mL, THL reduced the proportion of SP cells in HepG2, Hep3B, and Huh7 cells from 1.33% to 0.49%, 1.55% to 0.43%, and 1.69% to 0.27%, respectively. The viability and colony formation of Huh7 SP cells were effectively suppressed by THL dose-dependently, accompanied with the inhibition of stemness genes, e.g.,ABCG2, CD133,andSMO. The tumorigenicity of THL-treated Huh7 SP cells in NOD/SCID mice was also diminished. Moreover, combination with THL could synergize the effect of doxorubicin against Huh7 SP cells. Our data indicate that THL may act as a cancer stem cell targeting therapeutics and be regarded as complementary and integrative medicine in the treatment of hepatoma.

Molecular cloning of cDNA for rat argininosuccinate lyase and its expression in rat hepatoma cell lines

1986 ◽  
Vol 6 (5) ◽  
pp. 1722-1728
Author(s):  
M A Lambert ◽  
L R Simard ◽  
P N Ray ◽  
R R McInnes
Keyword(s):  
Cell Lines ◽  
Rat Liver ◽  
Transcriptional Activity ◽  
Hepatoma Cell ◽  
Cdna Clones ◽  
Argininosuccinate Lyase ◽  
Hepatoma Cell Lines ◽  
Liver Cdna Library ◽  
Nuclear Processing ◽  
Rat Hepatoma

Using antibody and plaque hybridization screening, we isolated rat argininosuccinate lyase (AS lyase) cDNA clones from a liver cDNA library prepared in the phage expression vector lambda gt11. Five overlapping cDNAs covering 1.7 kilobases of the estimated 2.0-kilobase AS lyase mRNA were characterized and confirmed as AS lyase sequences by hybrid selection. We examined the differential expression of AS lyase in rat liver and four rat hepatoma cell lines (7800C1, H4, HTC, and MH1C1). These cells exhibited a 60-fold range of AS lyase enzyme activity, with a direct correlation between activity, amount of AS lyase immunoreactive protein, and quantity of specific AS lyase mRNA. These observations suggest that the differences in AS lyase expression between rat liver and the hepatoma cell lines result from variations in AS lyase transcriptional activity or alterations in nuclear processing of AS lyase RNA.

scholarly journals Liver-enriched transcription factors uncoupled from expression of hepatic functions in hepatoma cell lines.

1997 ◽  
Vol 17 (11) ◽  
pp. 6311-6320 ◽  
Author(s):  
D Chaya ◽  
C Fougère-Deschatrette ◽  
M C Weiss
Keyword(s):  
Transcription Factors ◽  
Hepatoma Cell ◽  
Hepatoma Cells ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Hepatic Differentiation ◽  
Hepatoma Cell Lines ◽  
Differentiated Cells ◽  
Heritable Phenotype ◽  
Nuclear Factor 1

Among the liver-enriched transcription factors identified to date, only expression of hepatocyte nuclear factor 4 (HNF4) and hepatocyte nuclear factor 1 (HNF1) is in strict correlation with hepatic differentiation in cultured rat hepatoma cells. Indeed, differentiated hepatoma cells that stably express an extensive set of adult hepatic functions express liver-enriched transcription factors, while dedifferentiated cells that have lost expression of all these hepatic functions no longer express HNF4 and HNF1. We describe a new heritable phenotype, designated as uncoupled, in which there is a spontaneous dissociation between the expression of these transcription factors and that of the hepatic functions. Cells presenting this phenotype, isolated from differentiated hepatoma cells, cease to accumulate all transcripts coding for hepatic functions but nevertheless maintain expression of HNF4 and HNF1. Transitory transfection experiments indicate that these two factors present in these cells have transcriptional activity similar to that of differentiated hepatoma cells. Characterization of the appropriate intertypic cell hybrids demonstrates that this new phenotype is recessive to the dedifferentiated state and fails to be complemented by differentiated cells. These results indicate the existence of mechanisms that inhibit transcription of genes coding for hepatocyte functions in spite of the presence of functional HNF4 and HNF1. Cells of the uncoupled phenotype present certain properties of oval cells described for pathological states of the liver.

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