<i>p53</i> mutations are absent from carcinogen‐induced mouse liver tumors but occur in cell lines established from these tumors

Kress, Stefan; König, Jörg; Schweizer, Jürgen; Löhrke, Heinz; Bauer-Hofmann, R.; Schwarz, Michael

doi:10.1002/mc.2940060210

Cited by 81 publications

(64 citation statements)

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“…The human hepatoblastoma cell line HepG2 (American Type Culture Collection) was cul-tured in alpha-minimum essential medium (Life Technologies), the human renal adenocarcinoma cell line ACHN (American Type Culture Collection) was cultured in minimum essential medium (Life Technologies), and mouse hepatoma cell lines 53.2b and 56.1c [30] were cultured in MX-82 medium. All media were supplemented with 10% fetal bovine serum and cells were maintained at 37°C in a humidified atmosphere of 5 % CO 2 :air.…”

Section: Methodsmentioning

confidence: 99%

Functional analysis of the human cytochrome P4501A1 (CYP1A1) gene enhancer

Kress

Reichert

Schwarz

1998

European Journal of Biochemistry

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The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin) induces gene transcription, a process that requires binding of the activated aryl hydrocarbon receptor (AhR) to dioxinresponsive elements (DREs) within the enhancer region of responsive genes. Most of what is known about the molecular mechanism of AhR-dependent gene activation results from studies on the murine prototype TCDD-responsive gene cytochrome P4501A1 (CYP1A1). Much less is known, however, about the regulation of human TCDD-responsive genes. We have therefore conducted a detailed analysis of the enhancer region of the human CYP1A1 gene. From the ten DRE core motifs investigated within a stretch of 1400 bp in two human tumor cell lines using a ligation-mediated PCR technique, five motifs displayed a TCDD-inducible in vivo footprint. Four of these sites were functional enhancer sequences as demonstrated by a transient expression assay. Based on these data, a distinct functional consensus sequence for DRE motifs within the human CYP1A1 gene is suggested. After introduction of the four functional sites into various mouse hepatoma cell lines, only three exhibited a functional response, suggesting some species differences in CYP1A1 gene regulation. In addition to the footprints at DRE sites, we also detected protein-DNA interactions at three G-rich domains located within the enhancer region of the human CYP1A1 gene. Our data show that, besides some similarities in the regulation of the human and mouse CYP1A1 genes, there also exist some distinct differences, including number, location, and functional consensus sequences of DRE motifs, as well as quantity and location of footprinted G-rich domains.Keywords : CYP1A1; 2,3,7,8-tetrachlorodibenzo-p-dioxin ; Ah receptor; dioxin-responsive elements; gene regulation.2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD or dioxin) is a prototype for a large class of halogenated aromatic hydrocarbons that are widespread and persistent environmental contaminants. In rodents, dioxin produces a broad spectrum of toxic responses and is a potent tumor-promoting agent, while its relevance as a toxicant in humans is less clear (for review see [1]). The diversity of effects are assumed to reflect the ability of TCDD to alter gene expression in a species-specific and tissue-specific manner [2Ϫ4]. Many of the actions of TCDD have been shown to be mediated through an intracellular binding protein, designated the Ah receptor (AhR). This receptor is a member of a distinct class of basic helix-loop-helix (bHLH) transcription factors [5]. The inactive Ah receptor resides in the cytoplasm of target cells in a complex with heat-shock protein Hsp90 and possibly other proteins [6Ϫ9]. Upon binding of a ligand, the receptor is released from Hsp90, and heterodimerizes with a second bHLH protein named Ah-receptor nuclear translocator or ARNT [10]. The resultant AhR/ARNT/ligand complex is able to bind to specific DNA enhancer sequences, termed dioxin-responsive elements (DREs), located in the 5′ regulatory region of respon...

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Section: Methodsmentioning

confidence: 99%

Functional analysis of the human cytochrome P4501A1 (CYP1A1) gene enhancer

Kress

Reichert

Schwarz

1998

European Journal of Biochemistry

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“…We have developed in our laboratory a series of different mouse hepatoma lines, which were molecularly characterized with respect to their p53 genotype. 7 Cells of the p53 wild-type line 55.1c, used in the present study, respond typically to UV-irradiation with an increase in P53 and nuclear accumulation of the protein. The accumulation of P53 in these cells is believed to result from a decrease in interaction of the tumor suppressor protein with its negative regulator MDM2, which is mainly caused by post-translational modifications of P53 (for a review see Ref.…”

Section: Discussionmentioning

confidence: 99%

“…The mouse hepatoma lines have been described in detail in Ref. 7; their p53 genotyope is as follows: 55.1c, wildtype; 56.1b (R277T/wildtype), 70.4 (A135P/C138W) and 56.1d (C132W/del). In addition, HUH7 (Y220C/del) were used.…”

Section: Cell Linesmentioning

confidence: 99%

“…However, mutations in the tumor suppressor gene become frequently apparent upon brief in vitro culturing of tumor cells, and inactivation of both p53 alleles has been observed in certain mouse hepatoma cell lines. 7 In this study, we investigated the stability of P53 in stressed p53 wild-type and p53 defective hepatoma cells, and detected P53 degradation in cell lines with defective P53. This finding was unexpected and may have implications for tumor therapy.…”

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Regulation of P53 stability in p53 mutated human and mouse hepatoma cells

Hailfinger

Jaworski

Marx‐Stoelting

et al. 2007

Intl Journal of Cancer

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The tumor suppressor p53 is frequently mutated in cancer. We have investigated the regulation of P53 in p53 wild type mouse hepatoma cells (line 55.1c), in p53 heterozygeously mutated cells (56.1b) and in p53 defective cells (lines 56.1d, 70.4 and HUH7) under various experimental settings. The basal levels of P53 were low in 55.1c cells, but nuclear accumulation occurred upon UVirradiation. Similarly, UV-exposure induced stabilization of P53 in the heterozygeously p53 mutated 56.1b hepatoma cells. By contrast, the 3 hepatoma lines, which lack transcriptionally active P53, demonstrated high basal nuclear concentrations of P53 protein and, unexpectedly, showed loss of P53 upon UV-irradiation. Expression of p53 mRNA was also decreased in p53 defective cells after 24 hr post UV-irradiation, which may be linked to induction of apoptosis of the irradiated cells under these conditions. Other stressors like H 2 O 2 also mediated a decrease in P53 concentration in p53 defective cells. This effect occurred at very low concentrations and was already detectable 1-2 hr after exposure of cells. There were no signs of apoptosis of H 2 O 2 -exposed cells at this time point and no significant changes in p53 mRNA or MDM2 level. These unexpected findings indicate a new aspect related to regulation of P53 stability in cells with a defect in the tumor suppressor protein. ' 2007 Wiley-Liss, Inc.Key words: p53 mutation; hepatoma cells; MDM2; DFX; UV irradiation; ROS The p53 tumor suppressor protein is a powerful cellular caretaker, which protects cells from malignant transformation by means of transcriptional upregulation of proapoptotic, DNA repair and cell cycle arrest related proteins. In unstressed cells the P53 level is kept low by the E3 ubiquitin ligase MDM2, which transfers activated ubiquitin residues to P53. 1 Polyubiquitination of P53 leads to a rapid proteasomal degradation of the tumor suppressor, preventing P53 from affecting the cells' viability. P53 itself transcriptionally upregulates MDM2 expression, forming a negative feedback loop.Stress signals, caused by UV-light, DNA-damaging agents or hypoxia lead to an activation of several kinases, of which the most prominent members are the ATM (ataxia-telangiectasia mutated), ATR (ataxia-telangiectasia and Rad3-related), DNA-dependent protein kinase and casein kinase II, which phosphorylate the P53 protein at different sites (for review see Ref.2). Polyphosphorylation at the N-terminus of P53 reduces MDM2-dependent degradation, leading to an accumulation of the P53 protein in the cell followed by upregulation of apoptosis or cell cycle arrest related proteins. Exposure of cells to UV light correlates with post-translational modifications of P53, such as phosphorylation of serine 15 (mouse serine 18) and serine 20 (mouse serine 23). Desferrioxamine (DFX) treatment, which mimics hypoxia, results in phosphorylation of serine 15. Both sites are known to reduce MDM2-binding when phosphorylated. 3

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“…[36][37][38][39][40] However, a mechanism involving alterations in p53 is not ated tumor still resemble the neoplasm growing in vivo.…”

Section: Aidmentioning

confidence: 99%

Cell lines with heterogeneous phenotypes result from a single isolation of albumin-sv40 T-antigen transgenic rat hepatocytes

et al. 1997

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Transgenic animals and the cell lines derived from these Several immortalized cell lines were established from transgenic animals have become important tools in the study the livers of two transgenic rats expressing the simian of many diseases. [1][2][3][4][5][6] The construction of transgenic animals virus protein large T antigen under the control of the involves the introduction of a specific gene into the germ line albumin promoter. Hepatocytes from transgenic rats of an animal, resulting in one or more copies of the transgene were isolated by a two-step perfusion procedure from a in every cell of the adult animal. Expression of the gene may normal-appearing liver and from a liver that contained be regulated through the use of tissue-specific promoters, a single neoplasm. Cells were also isolated from the diswhich allow the gene to be expressed in specific tissues and sociated liver neoplasm. After 5 weeks in culture, cell not to be active in other tissues. colonies were isolated and subcloned as individual cellIn our laboratory, we have developed a line of transgenic lines. Electron microscopy revealed that all cell lines rats to study the process of hepatocellular carcinogenesis in had a high nuclear-to-cytoplasmic ratio compared with this species. 7 These animals were produced by microinjection normal hepatocytes. The cytoplasm contained numerinto fertilized rat eggs of the DNA coding sequence for the ous organelles, including smooth and rough endoplassimian virus large T antigen (SV40) under the control of the mic reticulum, Golgi, mitochondria, peroxisomes, and 5 regulatory region of the mouse albumin gene. 8 These aniannulate lamellae. However, the lines exhibited a varimals develop hepatocellular carcinomas, adenocarcinomas, ety of different cell morphologies. All cell lines, including and hepatoblastomas at about 3 to 5 months of age. Focal those derived from neoplastic cells, exhibited a similar lesions and nodules are observed as early as 2 months, with doubling time of 26 hours and the ability to grow in soft metastases observed in some animals later in life. By 6 to 8 agar. Northern blot analysis revealed that the cell lines months of age, the animals either die of the disease or from differentially expressed hepatocyte markers. Large T other pathological abnormalities that can be attributed to the antigen was expressed in the cultured cell lines at much expression of the transgene. 7 higher levels than was observed in transgenic hepatoThis report presents data characterizing a number of cell cytes in vivo. This suggests that the viral protein is relines derived from isolated alb-SV40 T-antigen transgenic rat quired to maintain cell viability in culture. In addition, hepatocytes. It is important to note that, although the cell the tumor suppressor proteins, p53 and Rbp105, were lines were derived from a single population of isolated hepatodetected in all cultured cell lines. In contrast, these same cytes, the established cell lines exhibit very different cellular proteins were not detected by West...

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p53 mutations are absent from carcinogen‐induced mouse liver tumors but occur in cell lines established from these tumors

Cited by 81 publications

References 47 publications

Functional analysis of the human cytochrome P4501A1 (CYP1A1) gene enhancer

Functional analysis of the human cytochrome P4501A1 (CYP1A1) gene enhancer

Regulation of P53 stability in p53 mutated human and mouse hepatoma cells

Cell lines with heterogeneous phenotypes result from a single isolation of albumin-sv40 T-antigen transgenic rat hepatocytes

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