Analytical fractionation of cultured hepatoma cells (HTC cells)

Rat liver hepatoma cells (HTC) which express liver-specific gene products were assayed for the expression of the rat growth hormone (rGH) gene, which is normally expressed in anterior pituitary somatotrophs. The combination of immunoprecipitation and two-dimensional gel electrophoresis provided a highly sensitive assay for rGH synthesis at levels as low as one part in 10(9) of cell protein synthesis (or four molecules of rGH per cell). No rGH expression was detected at this level. The lack of expression in HTC cells did not derive from a deletion of the rGH gene, as shown by Southern hybridization analysis of genomic DNA. Because the gene is expressed at greater than 30% of anterior pituitary protein synthesis, differentiation regulated rGH expression by over 10(8)-fold between the two cell types. Additionally, DNA-excess solution hybridization was used to measure the level of rGH mRNA sequences. A novel and general method for preparing single-strand probes from recombinant plasmids was developed. Hybridization analyses with a sensitivity of detection of 1 part in 10(8) failed to detect any rGH RNA sequences in either the nucleus or cytoplasm of HTC cells. It is concluded, therefore, that the restriction in rGH expression in the liver tumor cells is likely to occur at the level of the transcription of the gene, and that for all practical purposes, the rGH gene is completely shut off in the hepatoma cells.

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Analytical characterization and purification of plasma membrane from cultured hepatoma cells (HTC cells)

Biochimica et Biophysica Acta (BBA) - Biomembranes ◽

10.1016/0005-2736(81)90056-0 ◽

1981 ◽

Vol 644 (1) ◽

pp. 41-52 ◽

Cited By ~ 3

Author(s):

P. Sauvage ◽

P. Lopez-Saura ◽

M.-A. Leroy-Houyet ◽

P. Tulkens ◽

A. Trouet

Keyword(s):

Plasma Membrane ◽

Hepatoma Cells ◽

Analytical Characterization ◽

Htc Cells

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Biosynthesis and processing of mitochondrial glutaminase in HTC hepatoma cells

Biochemical Journal ◽

10.1042/bj2730265 ◽

1991 ◽

Vol 273 (2) ◽

pp. 265-270 ◽

Cited By ~ 10

Author(s):

S Y Perera ◽

D M Voith ◽

N P Curthoys

Keyword(s):

Oxidative Phosphorylation ◽

Structural Similarity ◽

Immunoblot Analysis ◽

Hepatoma Cells ◽

Specific Antibodies ◽

Carbonyl Cyanide ◽

Htc Cells

Rat HTC hepatoma cells were used to characterize the biosynthesis and processing of the renal isoenzyme of the mitochondrial glutaminase. Immunoblot analysis indicated that mitochondria isolated from HTC cells contained two prominent glutaminase peptides of 68 and 65 kDa and two minor peptides of 61 and 58 kDa. When the cells were labelled with [35S]methionine, the glutaminase-specific antibodies precipitated the same four polypeptides. However, when labelled in the presence of 5 microM-carbonyl cyanide m-chlorophenylhydrazone, an uncoupler of oxidative phosphorylation, only a 72 kDa cytoplasmic precursor of the mitochondrial glutaminase was immunoprecipitated. A comparison of the peptides generated by partial proteolysis of the precursor and the fully processed peptides indicates significant structural similarity. A 71 kDa form of the glutaminase was also observed when HTC cells were pulse-labelled for 2-6 min with [35S]methionine. Pulse-chase experiments indicate that the cytoplasmic precursor is quantitatively converted into the mature forms of the glutaminase. In addition, the observed kinetics established that the 71 kDa peptide is a true intermediate in the import of the mitochondrial glutaminase.

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The level of expression of the rat growth hormone gene in liver tumor cells is at least eight orders of magnitude less than that in anterior pituitary cells.

Molecular and Cellular Biology ◽

10.1128/mcb.3.8.1460 ◽

1983 ◽

Vol 3 (8) ◽

pp. 1460-1467 ◽

Cited By ~ 38

Author(s):

R D Ivarie ◽

B S Schacter ◽

P H O'Farrell

Keyword(s):

Growth Hormone ◽

Protein Synthesis ◽

Tumor Cells ◽

Liver Tumor ◽

Anterior Pituitary ◽

Hepatoma Cells ◽

Cell Protein ◽

Pituitary Cells ◽

Rat Growth ◽

Htc Cells

Rat liver hepatoma cells (HTC) which express liver-specific gene products were assayed for the expression of the rat growth hormone (rGH) gene, which is normally expressed in anterior pituitary somatotrophs. The combination of immunoprecipitation and two-dimensional gel electrophoresis provided a highly sensitive assay for rGH synthesis at levels as low as one part in 10(9) of cell protein synthesis (or four molecules of rGH per cell). No rGH expression was detected at this level. The lack of expression in HTC cells did not derive from a deletion of the rGH gene, as shown by Southern hybridization analysis of genomic DNA. Because the gene is expressed at greater than 30% of anterior pituitary protein synthesis, differentiation regulated rGH expression by over 10(8)-fold between the two cell types. Additionally, DNA-excess solution hybridization was used to measure the level of rGH mRNA sequences. A novel and general method for preparing single-strand probes from recombinant plasmids was developed. Hybridization analyses with a sensitivity of detection of 1 part in 10(8) failed to detect any rGH RNA sequences in either the nucleus or cytoplasm of HTC cells. It is concluded, therefore, that the restriction in rGH expression in the liver tumor cells is likely to occur at the level of the transcription of the gene, and that for all practical purposes, the rGH gene is completely shut off in the hepatoma cells.

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Endocytosis and chloroquine accumulation during the cell cycle of hepatoma cells in culture.

The Journal of Cell Biology ◽

10.1083/jcb.82.3.644 ◽

1979 ◽

Vol 82 (3) ◽

pp. 644-653 ◽

Cited By ~ 40

Author(s):

J Quintart ◽

M A Leroy-Houyet ◽

A Trouet ◽

P Baudhuin

Keyword(s):

Cell Cycle ◽

Experimental System ◽

Hepatoma Cells ◽

Cell Fractionation ◽

Dividing Cells ◽

Htc Cells ◽

Morris Hepatoma ◽

Extracellular Substances ◽

Peroxidase Uptake ◽

Cyclic Changes

Variations of endocytic and of lysosomal functions during the cell cycle have been investigated in synchronized hepatoma cells (derived from Morris hepatoma 7288c) by following the cellular uptake of horseradish peroxidase, dextran (mol wt. 70,000), and chloroquine. Cell fractionation and cytochemistry show that in asynchronously growing cells exposed for 1 h to 5 mg/ml peroxidase, the bulk of the enzyme taken up by the cells is found in phagosomes. By using the same experimental system with synchronized HTC cells, large variations of endocytosis are observed during the cell cycle. Peroxidase uptake is lowest during mitosis, increases 5--10 times during G1 phase, reaches a plateau, and finally decreases at the end of S phase and during G2 phase. A similar evolution is observed for the uptake of dextran (0.5 or 1 mg/ml), but it is likely that a significant part of the polysaccharide is still associated with the pericellular surface after 1 h. Moreover, dextran is transferred more slowly than peroxidase to lysosomes. Cellular accumulation of chloroquine is related to intralysosomal pH or to the buffering capacity of lysosomes. Our results show that this drug is taken up more rapidly during G1 and S phases while the rate of accumulation is lowest in mitotic cells. The results are discussed in relation to the modifications of the physical properties of lysosomes during the cell cycle observed previously by cell fractionation and electron microsocopy, and to the possible role of lysosomes in the initiation of mitosis. Cyclic changes of endocytosis in actively dividing cells are demonstrated by our observations and may induce large differences in the uptake rate of extracellular substances.

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