scholarly journals Transcriptional Activation of Chac1 and Other Atf4-Target Genes Induced by Extracellular l-Serine Depletion is negated with Glycine Consumption in Hepa1-6 Hepatocarcinoma Cells

Nutrients ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 3018
Author(s):  
Momoko Hamano ◽  
Shozo Tomonaga ◽  
Yusuke Osaki ◽  
Hiroaki Oda ◽  
Hisanori Kato ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Target Genes ◽  
De Novo ◽  
Hepatoma Cell ◽  
Cell Types ◽  
Hepatoma Cell Line ◽  
Rapid Reduction ◽  
Altered Expression ◽  
Hepatocarcinoma Cells ◽  
Expression Program

Mouse embryonic fibroblasts lacking D-3-phosphoglycerate dehydrogenase (Phgdh), which catalyzes the first step of de novo synthesis of l-serine, are particularly sensitive to depletion of extracellular L-serine. In these cells, depletion of l-serine leads to a rapid reduction of intracellular L-serine, cell growth arrest, and altered expression of a wide variety of genes. However, it remains unclear whether reduced availability of extracellular l-serine elicits such responses in other cell types expressing Phgdh. Here, we show in the mouse hepatoma cell line Hepa1-6 that extracellular l-serine depletion transiently induced transcriptional activation of Atf4-target genes, including cation transport regulator-like protein 1 (Chac1). Expression levels of these genes returned to normal 24 h after l-serine depletion, and were suppressed by the addition of l-serine or glycine in the medium. Extracellular l-serine depletion caused a reduction of extracellular and intracellular glycine levels but maintained intracellular l-serine levels in the cells. Further, Phgdh and serine hydroxymethyltransferase 2 (Shmt2) were upregulated after l-serine depletion. These results led us to conclude that the Atf4-mediated gene expression program is activated by extracellular l-serine depletion in Hepa1-6 cells expressing Phgdh, but is antagonized by the subsequent upregulation of l-serine synthesis, mainly from autonomous glycine consumption.

scholarly journals Stimulation of de novo glutathione synthesis by nitrofurantoin for enhanced resilience of hepatocytes

2021 ◽  
Author(s):  
Lukas S. Wijaya ◽  
Carina Rau ◽  
Theresa S. Braun ◽  
Serif Marangoz ◽  
Vincent Spegg ◽  
...  
Keyword(s):  
De Novo ◽  
Hepatoma Cell ◽  
Proteasome Inhibitors ◽  
Essential Element ◽  
Hepatoma Cell Line ◽  
Related Factor ◽  
Human Hepatoma Cell ◽  
Intact Cells ◽  
Intracellular Glutathione ◽  
Endogenous Antioxidant

AbstractToxicity is not only a function of damage mechanisms, but is also determined by cellular resilience factors. Glutathione has been reported as essential element to counteract negative influences. The present work hence pursued the question how intracellular glutathione can be elevated transiently to render cells more resistant toward harmful conditions. The antibiotic nitrofurantoin (NFT) was identified to stimulate de novo synthesis of glutathione in the human hepatoma cell line, HepG2, and in primary human hepatocytes. In intact cells, activation of NFT yielded a radical anion, which subsequently initiated nuclear-factor-erythroid 2-related-factor-2 (Nrf2)-dependent induction of glutamate cysteine ligase (GCL). Application of siRNA-based intervention approaches confirmed the involvement of the Nrf2-GCL axis in the observed elevation of intracellular glutathione levels. Quantitative activation of Nrf2 by NFT, and the subsequent rise in glutathione, were similar as observed with the potent experimental Nrf2 activator diethyl maleate. The elevation of glutathione levels, observed even 48 h after withdrawal of NFT, rendered cells resistant to different stressors such as the mitochondrial inhibitor rotenone, the redox cycler paraquat, the proteasome inhibitors MG-132 or bortezomib, or high concentrations of NFT. Repurpose of the antibiotic NFT as activator of Nrf2 could thus be a promising strategy for a transient and targeted activation of the endogenous antioxidant machinery.

A distinct glucocorticoid hormone response regulates phosphoprotein maturation in rat hepatoma cells

1986 ◽  
Vol 6 (2) ◽  
pp. 574-585
Author(s):  
K Karlsen ◽  
A K Vallerga ◽  
J Hone ◽  
G L Firestone
Keyword(s):  
Receptor Binding ◽  
De Novo ◽  
Hepatoma Cell ◽  
Hepatoma Cell Line ◽  
Hormone Response ◽  
Absolute Level ◽  
Chase Period ◽  
Glucocorticoid Hormone ◽  
Tumor Virus ◽  
Rat Hepatoma

Glucocorticoid hormone-dependent maturation of the mouse mammary tumor virus (MMTV) phosphorylated polyprotein (Pr74) allows experimental access to certain posttranslational regulatory circuits under steroid control in M1.54 cells, an MMTV-infected rat hepatoma cell line. Pulse-chase experiments revealed that [35S]methionine-labeled Pr74 synthesized in uninduced cells could be converted posttranslationally into p24, a stable phosphorylated maturation product, only after 4 h of exposure to 1 microM dexamethasone, a synthetic glucocorticoid. This regulated processing could be prevented by prior exposure, during the chase period, to inhibitors of RNA (actinomycin D) or protein (cycloheximide or puromycin) synthesis. Moreover, half-maximal production of p24 occurred at 10 nM dexamethasone, a concentration that approximated half-maximal receptor binding and stimulation of MMTV transcript synthesis. Kinetic, hormonal, and genetic evidence suggest that p24 expression did not require or result from the overall glucocorticoid-dependent increase in polyprotein concentration. First, 20 h after dexamethasone withdrawal, Pr74 maturation was completely deinduced, whereas the absolute level of this MMTV precursor remained 10-fold over its basal level. Second, progesterone, which competes with dexamethasone for receptor binding, facilitated the regulated production of p24 but prevented the steroid-mediated accumulation of functional MMTV mRNA. Lastly, certain glucocorticoid-responsive variants, derived from M1.54 cells by resistance to complement cytolysis, expressed p24 in the presence or absence of glucocorticoid-induced levels of Pr74. Taken together, our results suggest that the glucocorticoid-regulated maturation of MMTV phosphopolyproteins resulted from an independent hormone response that required normal receptor function and de novo RNA and protein synthesis.

Homeotic response elements are tightly linked to tissue-specific elements in a transcriptional enhancer of the teashirt gene

Development ◽  
1995 ◽  
Vol 121 (9) ◽  
pp. 2799-2812 ◽  
Author(s):  
A. McCormick ◽  
N. Core ◽  
S. Kerridge ◽  
M.P. Scott
Keyword(s):  
Transcriptional Activation ◽  
Target Genes ◽  
Hox Genes ◽  
Zinc Finger Protein ◽  
Cell Types ◽  
Transcriptional Enhancer ◽  
Hox Proteins ◽  
Tissue Specific ◽  
Conserved Sequences

Along the anterior-posterior axis of animal embryos, the choice of cell fates, and the organization of morphogenesis, is regulated by transcription factors encoded by clustered homeotic or ‘Hox’ genes. Hox genes function in both epidermis and internal tissues by regulating the transcription of target genes in a position- and tissue-specific manner. Hox proteins can have distinct targets in different tissues; the mechanisms underlying tissue and homeotic protein specificity are unknown. Light may be shed by studying the organization of target gene enhancers. In flies, one of the target genes is teashirt (tsh), which encodes a zinc finger protein. tsh itself is a homeotic gene that controls trunk versus head development. We identified a tsh gene enhancer that is differentially activated by Hox proteins in epidermis and mesoderm. Sites where Antennapedia (Antp) and Ultrabithorax (Ubx) proteins bind in vitro were mapped within evolutionarily conserved sequences. Although Antp and Ubx bind to identical sites in vitro, Antp activates the tsh enhancer only in epidermis while Ubx activates the tsh enhancer in both epidermis and in somatic mesoderm. We show that the DNA elements driving tissue-specific transcriptional activation by Antp and Ubx are separable. Next to the homeotic protein-binding sites are extensive conserved sequences likely to control tissue activation by different homeodomain proteins. We propose that local interactions between homeotic proteins and other factors effect activation of targets in proper cell types.

Analysis of MLL Amplification and 5q Deletions in AML/MDS Cell Lines Identifies Novel Targets.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1424-1424
Author(s):  
Bjoern Schneider ◽  
Stefan Nagel ◽  
Maren Kaufmann ◽  
Hilmar Quentmeier ◽  
Yoshinobu Matsuo ◽  
...  
Keyword(s):  
Cell Lines ◽  
Transcriptional Activation ◽  
Myeloid Leukemia ◽  
Target Genes ◽  
Histone Acetyltransferase ◽  
Cell Types ◽  
Signalling Pathways ◽  
Specific Cell ◽  
Genomic Amplification ◽  
The Common

Abstract Genomic amplifications of the 11q23 region occur in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) where MLL and a few neighboring genes, notably DDX6, are deemed salient targets. However, the extent to which amp(lified)-MLL and translocated MLL share effector targets remains to be established. Even less is known about the target(s) of deletions affecting the long arm of chromosome 5 (5q-) which reportedly partner amp-MLL. We analyzed three AML/MDS cell lines by cytogenetics (conventional and FISH) in parallel with real time q(uantitative)-PCR at both 11q23 and 5q2 to measure copy number and expression of salient target genes together with putative downstream targets. The cell lines comprised: MOLM-17 (transforming-MDS), SAML-2 (therapy-related AML), and UOC-M1 (AML-M1). All three cell lines exhibited approximately four-fold genomic amplification of 11q23 including MLL and DDX6, while the amplicon extended telomerically to include FLI1 (11q24) and HNT (11q25) in MOLM-17 and UOC-M1 only. Expression, quantified relative to AML/MDS cell lines without MLL rearrangement, revealed that of the genomically amplified genes only MLL was generally overexpressed, namely by 9.5x (MOLM-17), 5.1x (UOC-M1), and 4.6x (SAML-2). In addition to the highest MLL expression, in MOLM-17 FLI1 (3.8x) and DDX6 (2.8x) were significantly upregulated. Expression was also quantified among reputed MLL target genes, and showed that in the three cell lines MEIS1 was upregulated in MOLM-17 only (by 6x), and CDKN2C in all cell lines (by about 2x), while HOXA9 and CDKN1B showed near-normal levels of expression. All three cell lines carried 5q- with a common deleted region at 5q31 extending from 134.2–137.5 Mbp. Of a panel of genes recently identified as 5q- deletion targets (centromere-TIGA1, CAMLG, C5orf15, C5orf14, BRD8, HARS, KIAA0141, CSNK1A1, RBM22-telomere), only C5orf15 (function unknown) and BRD8 (a component of the nua4 histone acetyltransferase complex involved in transcriptional activation) were generally downregulated - to about 0.25x, and about 0.4x normalized expression levels, respectively. Both genes lie within the common deleted region. In summary, we have characterized amp-MLL and 5q- in MOLM-17, the first MDS cell line to be described with these rearrangements, together with two AML cell lines with similar cytogenetic profiles. Our data suggest that MLL is the only clear object of 11q23 amplification hitherto identified and CDKN2C its sole unequivocal target in AML/MDS cell lines. It is possible that MEIS1 is also targeted for activation in specific cell types or disease phases in MDS. These findings also highlight C5orf15 and/or BRD8 as possible leukemogenic accomplices targeted for downregulation in accompanying 5q-. These findings may point to differences in signalling pathways targeted by amp-MLL in AML and MDS.

scholarly journals Differences in the endosomal distributions of the two mannose 6-phosphate receptors.

1993 ◽  
Vol 121 (5) ◽  
pp. 997-1010 ◽  
Author(s):  
J Klumperman ◽  
A Hille ◽  
T Veenendaal ◽  
V Oorschot ◽  
W Stoorvogel ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Hepatoma Cell ◽  
Cell Types ◽  
Hepatoma Cell Line ◽  
Human Hepatoma Cell ◽  
Intracellular Targeting ◽  
Cytoplasmic Vesicles ◽  
Mannose 6 Phosphate ◽  
Double Immunolabeling ◽  
Lateral Segregation

Multiple immunolabeling of cryosections was performed to compare the subcellular distributions of the two mannose 6-phosphate receptors (MPRs) involved in the intracellular targeting of lysosomal enzymes: the cation-dependent (CD) and cation-independent (CI) MPR. In two cell types, the human hepatoma cell line HepG2 and BHK cells double transfected with cDNA's encoding for the human CD-MPR and CI-MPR, we found the two receptors at the same sites: the trans-Golgi reticulum (TGR), endosomes, electron-dense cytoplasmic vesicles, and the plasma membrane. In the TGR the two receptors colocalized and were concentrated to the same extent in the same HA I-adaptor positive coated buds and vesicles. Endosomes were identified by the presence of exogenous tracers. The two MPR codistributed to the same endosomes, but semiquantitative analysis showed a relative enrichment of the CI-MPR in endosomes containing many internal vesicles. Two endosomal subcompartments were discerned, the central vacuole and the associated tubules and vesicles (ATV). We found an enrichment of CD-MPR over CI-MPR in the ATV. Lateral segregation of the two receptors within the plane of membranes was also detected on isolated organelles. Double immunolabeling for the CD-MPR and the asialoglycoprotein receptor, which mainly recycles between endosomes and the plasma membrane, revealed that these two receptors were concentrated in different subpopulations of endosomal ATV. The small GTP-binding protein rab4, which has been shown to mediate recycling from endosomes to the plasma membrane, was localized at the cytosolic face of many endosomal ATV. Quantitative analysis of double-immunolabeled cells revealed only a limited codistribution of the MPRs and rab4 in ATV. These data suggest that the two MPRs exit the TGR via the same coated vesicles, but that upon arrival in the endosomes CD-MPR is more rapidly than CI-MPR, segregated into ATV which probably are destined to recycle MPRs to TGR.

scholarly journals The extracellular matrix coordinately modulates liver transcription factors and hepatocyte morphology.

1991 ◽  
Vol 11 (9) ◽  
pp. 4405-4414 ◽  
Author(s):  
C M DiPersio ◽  
D A Jackson ◽  
K S Zaret
Keyword(s):  
Extracellular Matrix ◽  
Transcription Factors ◽  
Cell Line ◽  
Cell Morphology ◽  
Transcriptional Activation ◽  
Hepatoma Cell ◽  
Collagen Gel ◽  
Hepatoma Cell Line ◽  
Transcriptional Enhancer ◽  
Mrna Accumulation

The extracellular matrix (ECM) promotes tissue morphogenesis, cell migration, and the differentiation of a variety of cell types. However, the mechanisms by which ECM causes differentiated gene expression have been unknown. In this report, we show that culturing the hepatocyte-derived cell line H2.35 on an ECM gel changes cell morphology and selectively stimulates the transcription of a subset of liver-specific genes, including serum albumin. Transcriptional activation by ECM also occurs with transfected plasmids bearing the transcriptional enhancer of the albumin gene. ECM substrates of different composition activated the albumin enhancer only when the ECM promoted a cuboidal, differentiated cell morphology. Enhancer activation by the ECM was mediated by two liver transcription factors, HNF3 alpha and eH-TF, which appear to be regulated differently by matrix. Specifically, we found that a collagen gel substratum caused a selective increase in the factor HNF3 alpha at the levels of mRNA accumulation and DNA-binding activity in nuclear extracts, both in H2.35 cells and in the hepatoma cell line HepG2. We conclude that the ECM can stimulate cell differentiation by selectively activating transcriptional regulatory factors and that such regulation occurs coordinately with ECM-promoted changes in cell shape.

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.

scholarly journals A distinct glucocorticoid hormone response regulates phosphoprotein maturation in rat hepatoma cells.

1986 ◽  
Vol 6 (2) ◽  
pp. 574-585 ◽  
Author(s):  
K Karlsen ◽  
A K Vallerga ◽  
J Hone ◽  
G L Firestone
Keyword(s):  
Receptor Binding ◽  
De Novo ◽  
Hepatoma Cell ◽  
Hepatoma Cell Line ◽  
Hormone Response ◽  
Absolute Level ◽  
Chase Period ◽  
Glucocorticoid Hormone ◽  
Tumor Virus ◽  
Rat Hepatoma

Glucocorticoid hormone-dependent maturation of the mouse mammary tumor virus (MMTV) phosphorylated polyprotein (Pr74) allows experimental access to certain posttranslational regulatory circuits under steroid control in M1.54 cells, an MMTV-infected rat hepatoma cell line. Pulse-chase experiments revealed that [35S]methionine-labeled Pr74 synthesized in uninduced cells could be converted posttranslationally into p24, a stable phosphorylated maturation product, only after 4 h of exposure to 1 microM dexamethasone, a synthetic glucocorticoid. This regulated processing could be prevented by prior exposure, during the chase period, to inhibitors of RNA (actinomycin D) or protein (cycloheximide or puromycin) synthesis. Moreover, half-maximal production of p24 occurred at 10 nM dexamethasone, a concentration that approximated half-maximal receptor binding and stimulation of MMTV transcript synthesis. Kinetic, hormonal, and genetic evidence suggest that p24 expression did not require or result from the overall glucocorticoid-dependent increase in polyprotein concentration. First, 20 h after dexamethasone withdrawal, Pr74 maturation was completely deinduced, whereas the absolute level of this MMTV precursor remained 10-fold over its basal level. Second, progesterone, which competes with dexamethasone for receptor binding, facilitated the regulated production of p24 but prevented the steroid-mediated accumulation of functional MMTV mRNA. Lastly, certain glucocorticoid-responsive variants, derived from M1.54 cells by resistance to complement cytolysis, expressed p24 in the presence or absence of glucocorticoid-induced levels of Pr74. Taken together, our results suggest that the glucocorticoid-regulated maturation of MMTV phosphopolyproteins resulted from an independent hormone response that required normal receptor function and de novo RNA and protein synthesis.

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