scholarly journals Expression of Differentiated Functions in Hepatoma Cell Hybrids: Induction of Mouse Albumin Production in Rat Hepatoma-Mouse Fibroblast Hybrids

1972 ◽  
Vol 69 (3) ◽  
pp. 571-575 ◽  
Author(s):  
J. A. Peterson ◽  
M. C. Weiss
Keyword(s):  
Hepatoma Cell ◽  
Mouse Fibroblast ◽  
Cell Hybrids ◽  
Albumin Production ◽  
Rat Hepatoma

scholarly journals Immunofluorescence analysis of the time-course of extinction, reexpression, and activation of albumin production in rat hepatoma-mouse fibroblast heterokaryons and hybrids.

1981 ◽  
Vol 90 (2) ◽  
pp. 339-350 ◽  
Author(s):  
M Mével-Ninio ◽  
M C Weiss
Keyword(s):  
Messenger Rna ◽  
Time Course ◽  
Gene Dosage ◽  
Mouse Fibroblast ◽  
Chromosome Loss ◽  
No Production ◽  
Embryonic Cells ◽  
L Cell ◽  
Albumin Production ◽  
Rat Hepatoma

We have used a combination of a sensitive immunocytochemical stain for intracellular albumin, and Hoechst 33258 dye for identification of parental nuclei to investigate the time-course of extinction, reexpression, and activation of albumin production in fusion products of 1s (hyperdiploid) or 2s (hypertetradiploid) rat hepatoma cells with mouse fibroblasts (L cells or embryonic cells). In all combinations, the initial event is extinction of albumin production. Extinction occurs immediately after fusion when the mouse fibroblast is a normal embryonic (senescent?) cell. In the case of an L cell, rat albumin is synthesized and secreted during the first 12 h after fusion; no production of mouse albumin occurs. Thereafter, albumin production ceases. 8-12 d after fusion, young hybrid colonies are found to resume the synthesis of rat albumin (reexpression), and several days later the production of mouse albumin begins (activation). The patterns of reexpression and activation indicate (a) that chromosome loss is not necessary for either event to occur and (b) that the cells active in the synthesis of mouse albumin are a subpopulation of those cells already engaged in the production of rat albumin. We conclude that (a) extinction is mediated by diffusible factor(s) from the L-cell parent that act in the hepatoma nucleus to prevent the formation of new albumin messenger RNA; (b) reexpression and activation are gene dosage-dependent but extinction is not; and (c) previously active genes are more rapidly expressed than previously silent ones.

scholarly journals A domain of methylation change at the albumin locus in rat hepatoma cell variants.

1985 ◽  
Vol 5 (1) ◽  
pp. 214-225 ◽  
Author(s):  
A Orlofsky ◽  
L A Chasin
Keyword(s):  
De Novo ◽  
Hepatoma Cell ◽  
Northern Blotting ◽  
Cell Protein ◽  
Hepatoma Cell Line ◽  
Mrna Levels ◽  
Albumin Production ◽  
Albumin Mrna ◽  
Rat Hepatoma ◽  
De Novo Methylation

A well-differentiated rat hepatoma cell line, Fu5-5, yields variant clones whose rate of secretion of serum albumin ranges from 40 to less than 0.08 micrograms of albumin/mg of cell protein per 48 h. Clones were classified as high producers (10 to 40 micrograms/mg per 48 h), intermediate producers (1 to 10 micrograms/mg per 48 h), low producers (0.1 to 1.0 micrograms/mg per 48 h), and null variants (less than 0.1 micrograms/mg per 48 h). Albumin synthetic rates are proportional to secretion rates and range from 0.9 to less than 0.002% of total protein synthesis as measured by pulse-labeling. Steady-state albumin mRNA levels were measured by filter hybridization of fragmented, end-labeled mRNA and by Northern blotting. Message levels are proportional to albumin synthetic rates except for a high producer in which albumin mRNA is less elevated than the synthetic rate. The extent of methylation was quantitated at each of 24 CpG-containing sites or site clusters at the albumin locus. These sites span a region that contains the albumin gene as well as 10 kilobases of the 5' flank and 1 kilobase of the 3' flank. An 8-kilobase region is described, with boundaries in the 5' flank and in the middle of the gene, within which all 11 sites examined showed a correlation of undermethylation with the high-producer phenotype. In contrast, 12 of 13 sites outside of this region showed no phenotype correlation. Null variants derived from a high producer underwent de novo methylation of this domain. Six independent hybrid clones derived from the cross of a high producer with a null variant showed extinction of albumin production and hypermethylation of the domain. Apparently these cells retain the capacity for the de novo methylation of these specific sites.

A domain of methylation change at the albumin locus in rat hepatoma cell variants

1985 ◽  
Vol 5 (1) ◽  
pp. 214-225
Author(s):  
A Orlofsky ◽  
L A Chasin
Keyword(s):  
De Novo ◽  
Hepatoma Cell ◽  
Northern Blotting ◽  
Cell Protein ◽  
Hepatoma Cell Line ◽  
Mrna Levels ◽  
Albumin Production ◽  
Albumin Mrna ◽  
Rat Hepatoma ◽  
De Novo Methylation

A well-differentiated rat hepatoma cell line, Fu5-5, yields variant clones whose rate of secretion of serum albumin ranges from 40 to less than 0.08 micrograms of albumin/mg of cell protein per 48 h. Clones were classified as high producers (10 to 40 micrograms/mg per 48 h), intermediate producers (1 to 10 micrograms/mg per 48 h), low producers (0.1 to 1.0 micrograms/mg per 48 h), and null variants (less than 0.1 micrograms/mg per 48 h). Albumin synthetic rates are proportional to secretion rates and range from 0.9 to less than 0.002% of total protein synthesis as measured by pulse-labeling. Steady-state albumin mRNA levels were measured by filter hybridization of fragmented, end-labeled mRNA and by Northern blotting. Message levels are proportional to albumin synthetic rates except for a high producer in which albumin mRNA is less elevated than the synthetic rate. The extent of methylation was quantitated at each of 24 CpG-containing sites or site clusters at the albumin locus. These sites span a region that contains the albumin gene as well as 10 kilobases of the 5' flank and 1 kilobase of the 3' flank. An 8-kilobase region is described, with boundaries in the 5' flank and in the middle of the gene, within which all 11 sites examined showed a correlation of undermethylation with the high-producer phenotype. In contrast, 12 of 13 sites outside of this region showed no phenotype correlation. Null variants derived from a high producer underwent de novo methylation of this domain. Six independent hybrid clones derived from the cross of a high producer with a null variant showed extinction of albumin production and hypermethylation of the domain. Apparently these cells retain the capacity for the de novo methylation of these specific sites.

scholarly journals Bioanalytical screening of low levels of dioxins and dioxin-like PCBs in pig meat (pork) for checking compliance with EU maximum and action levels using highly sensitive “third generation” recombinant H4L7.5c2 rat hepatoma cells

2021 ◽  
Vol 33 (1) ◽  
Author(s):  
Johannes Haedrich ◽  
Claudia Stumpf ◽  
Michael S. Denison
Keyword(s):  
European Union ◽  
Hepatoma Cell ◽  
Animal Origin ◽  
The European Union ◽  
Highly Sensitive ◽  
Commission Regulation ◽  
Pig Meat ◽  
Low Levels ◽  
Action Levels ◽  
Rat Hepatoma

Abstract Background Low maximum and action levels set by the European Union for polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) and dioxin-like polychlorinated biphenyls (DL-PCBs) in pig meat (pork) have led to a demand for reliable and cost-effective bioanalytical screening methods implemented upstream of gas chromatography/high-resolution mass spectrometry confirmatory technology, that can detect low levels of contamination in EU-regulated foods with quick turn-around times. Results Based on the Chemically Activated LUciferase gene eXpression (CALUX) bioassay, extraction and clean-up steps were optimized for recovery and reproducibility within working ranges significantly lower than in current bioassays. A highly sensitive “3rd generation” recombinant rat hepatoma cell line (H4L7.5c2) containing 20 dioxin responsive elements was exposed to pork sample extracts, and their PCDD/Fs and DL-PCBs levels were evaluated by measuring luciferase activity. The method was validated according to the provisions of Commission Regulation (EU) 2017/644 of 5 April 2017 with spiking experiments performed selectively for PCDD/Fs and DL-PCBs and individual calibration for PCDD/Fs, DL-PCBs and the calculated sum of PCDD/Fs and DL-PCBs. The resulting performance parameters met all legal specifications as confirmed by re-calibration using authentic samples. Cut-off concentrations for assessing compliance with low maximum levels and action levels set for PCDD/Fs and DL-PCBs within a range of 0.50–1.25 pg WHO-TEQ/g fat were derived, ensuring low rates of false-compliant results (ß-error < 1%) and keeping the rate of false-noncompliant results well under control (α-error < 12%). Conclusions We present a fast and efficient bioanalytical routine method validated according to the European Union’s legal requirements on the basis of authentic samples, allowing the analyst to reliably identify pork samples and any other EU-regulated foods of animal origin suspected to be noncompliant with a high level of performance and turn-around times of 52 h. This was facilitated in particular by a quick and efficient extraction step followed by selective clean-up, use of a highly sensitive “3rd generation” H4L7.5c2 recombinant rat hepatoma cell CALUX bioassay, and optimized assay performance with improved calibrator precision and reduced lack-of-fit errors. New restrictions are proposed for the calibrator bias and the unspecific background contribution to reportable results. The procedure can utilize comparably small sample amounts and allows an annual throughput of 840–1000 samples per lab technician. The described bioanalytical method contributes to the European Commission's objective of generating accurate and reproducible analytical results according to Commission Regulation (EU) 2017/644 across the European Union.

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