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1.
Photochem Photobiol Sci ; 17(5): 539-551, 2018 May 16.
Article in English | MEDLINE | ID: mdl-29565434

ABSTRACT

Triple-negative breast cancer (TNBC) and malignant melanoma are highly aggressive cancers that widely express the cell surface chondroitin sulfate proteoglycan 4 (CSPG4/NG2). CSPG4 plays an important role in tumor cell growth and survival and promotes chemo- and radiotherapy resistance, suggesting that CSPG4 is an attractive target in cancer therapy. In the present work, we applied the drug delivery technology photochemical internalization (PCI) in combination with the novel CSPG4-targeting immunotoxin 225.28-saporin as an efficient and specific strategy to kill aggressive TNBC and amelanotic melanoma cells. Light-activation of the clinically relevant photosensitizer TPCS2a (fimaporfin) and 225.28-saporin was found to act in a synergistic manner, and was superior to both PCI of saporin and PCI-no-drug (TPCS2a + light only) in three TNBC cell lines (MDA-MB-231, MDA-MB-435 and SUM149) and two BRAFV600E mutated malignant melanoma cell lines (Melmet 1 and Melmet 5). The cytotoxic effect was highly dependent on the light dose and expression of CSPG4 since no enhanced cytotoxicity of PCI of 225.28-saporin compared to PCI of saporin was observed in the CSPG4-negative MCF-7 cells. The PCI of a smaller, and clinically relevant CSPG4-targeting toxin (scFvMEL-rGel) validated the CSPG4-targeting concept in vitro and induced a strong inhibition of tumor growth in the amelanotic melanoma xenograft A-375 model. In conclusion, the combination of the drug delivery technology PCI and CSPG4-targeting immunotoxins is an efficient, specific and light-controlled strategy for the elimination of aggressive cells of TNBC and malignant melanoma origin. This study lays the foundation for further preclinical evaluation of PCI in combination with CSPG4-targeting.


Subject(s)
Antineoplastic Agents/pharmacology , Chondroitin Sulfate Proteoglycans/antagonists & inhibitors , Immunotoxins/pharmacology , Melanoma/drug therapy , Membrane Proteins/antagonists & inhibitors , Triple Negative Breast Neoplasms/drug therapy , Animals , Antineoplastic Agents/chemistry , Cell Proliferation/drug effects , Cell Survival/drug effects , Chondroitin Sulfate Proteoglycans/metabolism , Dose-Response Relationship, Drug , Drug Delivery Systems , Drug Screening Assays, Antitumor , Humans , Immunotoxins/chemistry , Light , Melanoma/metabolism , Melanoma/pathology , Membrane Proteins/metabolism , Mice , Photochemical Processes , Structure-Activity Relationship , Triple Negative Breast Neoplasms/metabolism , Triple Negative Breast Neoplasms/pathology , Tumor Cells, Cultured
2.
Oncogene ; 27(19): 2763-71, 2008 Apr 24.
Article in English | MEDLINE | ID: mdl-18026139

ABSTRACT

p53 plays a major role in the prevention of tumor development. It responds to a range of potentially oncogenic stresses by activating protective mechanisms, most notably cell-cycle arrest and apoptosis. The p53 gene is also induced during normal liver regeneration, and it has been hypothesized that p53 serve as a proliferative 'brake' to control excessive proliferation. However, it has lately been shown that p53 inhibition reduces hepatocyte growth factor-induced DNA synthesis of primary hepatocytes. Here we show that epidermal growth factor (EGF) activated p53 in a phosphatidylinositol-3 kinase-dependent way, and thus induced the cyclin-dependent kinase inhibitor p21(Cip1) in primary rat hepatocytes. p53 inactivation with a dominant-negative mutant (p53(V143A)) attenuated EGF-induced DNA synthesis and was associated with reduced CDK2 phosphorylation and retinoblastoma protein hyperphosphorylation. When p21(Cip1) was ectopically expressed in p53-inactivated cells, these effects were neutralized. In conclusion, our results demonstrate that in normal hepatocytes, EGF-induced expression of p53 is involved in regulating CDK2- and CDK4 activity, through p21(Cip1) expression.


Subject(s)
Cyclin-Dependent Kinase 2/metabolism , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Hepatocytes/cytology , Hepatocytes/metabolism , S Phase/physiology , Signal Transduction/physiology , Tumor Suppressor Protein p53/metabolism , Animals , Cells, Cultured , Cyclin-Dependent Kinase 4/metabolism , Cyclin-Dependent Kinase Inhibitor p21/physiology , Enzyme Activation/physiology , Epidermal Growth Factor/antagonists & inhibitors , Epidermal Growth Factor/physiology , Male , Phosphatidylinositol 3-Kinases/physiology , Rats , Rats, Wistar , Tumor Suppressor Protein p53/antagonists & inhibitors , Tumor Suppressor Protein p53/physiology
3.
Cell Prolif ; 40(4): 475-87, 2007 Aug.
Article in English | MEDLINE | ID: mdl-17635516

ABSTRACT

INTRODUCTION/OBJECTIVES: Cell cycle progression is driven by the coordinated regulation of cyclin-dependent kinases (CDKs). In response to mitogenic stimuli, CDK4 and CDK2 form complexes with cyclins D and E, respectively, and translocate to the nucleus in the late G(1) phase. It is an on-going discussion whether mammalian cells need both CDK4 and CDK2 kinase activities for induction of S phase. METHODS AND RESULTS: In this study, we have explored the role of CDK4 activity during G(1) progression of primary rat hepatocytes. We found that CDK4 activity was restricted by either inhibiting growth factor induced cyclin D1-induction with the PI3K inhibitor LY294002, or by transient transfection with a dominant negative CDK4 mutant. In both cases, we observed reduced CDK2 nuclear translocation and reduced CDK2-Thr160 phosphorylation. Furthermore, reduced pRb hyperphosphorylation and reduced cellular proliferation were observed. Ectopic expression of cyclin D1 alone was not sufficient to induce CDK4 nuclear translocation, CDK2 activity or cell proliferation. CONCLUSIONS: Thus, epidermal growth factor-induced CDK4 activity was necessary for CDK2 activation and for hepatocyte proliferation. These results also suggest that, in addition to regulating cyclin D1 expression, PI3K is involved in regulation of nuclear shuttling of cyclin-CDK complexes in G(1) phase.


Subject(s)
Cell Cycle , Cyclin-Dependent Kinase 2/metabolism , Cyclin-Dependent Kinase 4/metabolism , Hepatocytes/enzymology , Phosphatidylinositol 3-Kinases/metabolism , Active Transport, Cell Nucleus , Animals , Cell Nucleus/enzymology , Cell Proliferation , Cells, Cultured , Cyclin D1/metabolism , Cyclin E/biosynthesis , Cyclin-Dependent Kinase 4/chemistry , Epidermal Growth Factor/pharmacology , Hepatocytes/drug effects , Male , Rats , Rats, Wistar , Threonine/metabolism
4.
J Cell Physiol ; 202(3): 707-16, 2005 Mar.
Article in English | MEDLINE | ID: mdl-15389569

ABSTRACT

We have studied epidermal growth factor receptor (EGFR) phosphorylation and localization in the pre-replicative phase of liver regeneration induced by a 70% partial hepatectomy (PH), and how a PH affects EGFR activation and trafficking. When Western blotting was performed on livers after PH with antibodies raised against activated forms of EGFR autophosphorylation sites, no marked increase in EGFR tyrosine phosphorylation was observed. However, events associated with attenuation of EGFR signals were observed. Two hours after PH, we found increased EGFR ubiquitination and internalization, followed by receptor downregulation. Furthermore, EGFR phosphorylation following an injection of EGF was reduced after PH. This reduction correlated with an increased activation of PKC and a distinct augmentation in the phosphorylation of the PKC-regulated T654-site of EGFR. When primary cultured hepatocytes were treated with tetradecanoylphorbol acetate (TPA) to induce T654-phosphorylation of EGFR, we found colocalization of a fraction of EGFR with EEA1, downregulation of EGF-mediated EGFR autophosphorylation, altered ligand-induced intracellular sorting of EGFR, and increased mitogenic signaling through the EGFR-Ras-Raf-ERK pathway. Further, we found that both TPA and a PH enhanced EGF-induced proliferation of hepatocytes. In conclusion, our results suggest that hepatocyte priming involves modulation of EGFR that enhances its ability to mediate growth factor responses without an increase in its receptor tyrosine kinase-activity. This may be a pre-replicative competence event that increases growth factor effects during G1 progression.


Subject(s)
ErbB Receptors/metabolism , Hepatectomy , Liver/metabolism , Signal Transduction/physiology , Animals , Cell Cycle , Cell Fractionation , Cells, Cultured , Extracellular Signal-Regulated MAP Kinases/metabolism , Hepatocytes/cytology , Hepatocytes/metabolism , Liver Regeneration , Male , Membrane Proteins/metabolism , Phosphorylation , Proliferating Cell Nuclear Antigen/metabolism , Rats , Rats, Inbred F344 , Tetradecanoylphorbol Acetate/metabolism , Vesicular Transport Proteins
5.
Eur J Cell Biol ; 80(4): 285-94, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11370743

ABSTRACT

The rapid internalization of receptor tyrosine kinases after ligand binding has been assumed to be a negative modulation of signal transduction. However, accumulating data indicate that signal transduction from internalized cell surface receptors also occurs from endosomes. We show that a substantial fraction of tyrosine-phosphorylated epidermal growth factor receptor (EGFR) and Shc, Grb2 and Cbl after internalization relocates from early endosomes to compartments which are negative for the early endosomes, recycling vesicle markers EEA1 and transferrin in EGF-stimulated cells. These compartments contained the multivesicular body and late endosome marker CD63, and the late endosome and lysosome marker LAMP-1, and showed a multivesicular morphology. Subcellular fractionation revealed that activated EGFR, adaptor proteins and activated ERK 1 and 2 were located in EEA1-negative and LAMP-1-positive fractions. Co-immunoprecipitations showed EGFR in complex with both Shc, Grb2 and Cbl. Treatment with the weak base chloroquine or inhibitors of lysosomal enzymes after EGF stimulation induced an accumulation of tyrosine-phosphorylated EGFR and Shc in EEA1-negative and CD63-positive vesicles after a 120-min chase period. This was accompanied by a sustained activation of ERK 1 and 2. These results suggest that EGFR signaling is not spatially restricted to the plasma membrane, primary vesicles and early endosomes, but is continuing from late endocytic trafficking organelles maturing from early endosomes.


Subject(s)
Adaptor Proteins, Signal Transducing , Endosomes/metabolism , Epidermal Growth Factor/pharmacology , ErbB Receptors/metabolism , Signal Transduction/physiology , Ubiquitin-Protein Ligases , Antigens, CD/analysis , Antigens, CD/metabolism , Antimalarials/pharmacology , Cell Compartmentation/physiology , Chloroquine/pharmacology , Endosomes/chemistry , Endosomes/ultrastructure , ErbB Receptors/analysis , GRB2 Adaptor Protein , HeLa Cells , Humans , Intramolecular Transferases/metabolism , Lysosomal Membrane Proteins , Lysosomes/drug effects , Lysosomes/metabolism , Membrane Glycoproteins/analysis , Membrane Glycoproteins/metabolism , Membrane Proteins/analysis , Microscopy, Electron , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3 , Mitogen-Activated Protein Kinases/metabolism , Phosphorylation , Platelet Membrane Glycoproteins/metabolism , Proteins/metabolism , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-cbl , Signal Transduction/drug effects , Tetraspanin 30 , Tyrosine/metabolism , Vesicular Transport Proteins
6.
Mol Carcinog ; 28(2): 84-96, 2000 Jun.
Article in English | MEDLINE | ID: mdl-10900465

ABSTRACT

The hepatic carcinogen 2-acetylaminofluorene (AAF) exerts its effect as a tumor promoter by mitoinhibition of normal hepatocytes. Initiated cells proliferate selectively and develop into preneoplastic foci and subsequently into carcinomas. To study whether some of the mitoinhibitory effects of AAF could be attributed to an influence on intracellular signal transduction, growth factor signaling was studied in cultured hepatocytes from rats fed AAF for 7 d. Activation through the epidermal growth factor receptor (EGFR) was used to probe possible changes in downstream mitogenic signaling mechanisms. The proliferative response to epidermal growth factor (EGF), measured as proliferating cell nuclear antigen expression and thymidine incorporation, was almost completely inhibited in hepatocytes exposed to AAF. Neither EGFR protein levels nor EGF binding was notably altered in AAF-exposed hepatocytes as opposed to normal hepatocytes. The initial tyrosine phosphorylation of EGFR and downstream activation of Sos, Raf-1, and extracellular signal-regulated protein kinase (ERK) were similar in AAF-treated and control hepatocytes. Even though ERK phosphorylation was unaffected, a remarkable (80%) reduction of ERK nuclear accumulation was observed in AAF-exposed hepatocytes immediately after mitogen stimulation. EGFR tyrosine phosphorylation and downstream signaling lasted 6 h in control cells versus 2 h in AAF-exposed hepatocytes. We previously demonstrated that AAF inhibits the growth factor-dependent induction of cyclin D1 and arrests hepatocyte cell-cycle progression before the p21/CIP1-controlled DNA-damage check point. The present data indicate that the DNA-damaging carcinogen AAF induces growth inhibition by a distinct inhibition of ERK nuclear accumulation after mitogen stimulation. Inhibition of intracellular signal transduction may represent a novel mechanism of growth arrest. Mol. Carcinog. 28:84-96, 2000.


Subject(s)
2-Acetylaminofluorene/pharmacology , Carcinogens/pharmacology , Cell Nucleus/enzymology , Epidermal Growth Factor/pharmacology , Liver/drug effects , Mitogen-Activated Protein Kinases/metabolism , Animals , Cell Division/drug effects , Cells, Cultured , ErbB Receptors/metabolism , Liver/enzymology , Phosphorylation , Protein Binding , Rats
7.
Exp Lung Res ; 26(5): 383-99, 2000.
Article in English | MEDLINE | ID: mdl-10914335

ABSTRACT

CCAAT/enhancer-binding proteins (C/EBP) constitute a family of transcription factors that are involved in regulation of proliferation and differentiation in several cell types. In epithelial lung cells the C/EBP alpha isoform seems to play a role in the regulation of surfactant proteins (SP) and Clara cell specific protein (CCSP), whereas the roles of C/EBP beta and C/EBP delta are unclear. We have examined the protein levels of C/EBP delta in bronchiolar Clara cells and alveolar type 2 cells, and its relation to the expression of lung specific proteins and cell proliferation. The protein expression of C/EBP delta was high in freshly isolated Clara cells compared to type 2 cells. In both cell types C/EBP delta levels increased during culture. Alterations of the levels of C/EBP delta did not correspond with the proliferation levels of Clara cells, but seemed to correspond in type 2 cells. Clara cell secretory protein (CCSP) was highly expressed in freshly isolated Clara cells, in contrast to type 2 cells. SP-D and CYP2B1 were expressed at somewhat higher levels in Clara cells than in type 2 cells, whereas SP-A exhibited highest expression in type 2 cells. During culture the levels of all these lung proteins were strongly reduced. However, compared to with serum we found an increase in CCSP in Clara cell cultures without serum, and this correlated with an increase in C/EBP delta. Overall our in vitro data suggest that C/EBP delta alone is not related to the maintenance of proteins involved in differentiation.


Subject(s)
Bronchi/metabolism , CCAAT-Enhancer-Binding Proteins/metabolism , Interleukin-6/metabolism , Pulmonary Alveoli/metabolism , Transcription Factors , Uteroglobin , Animals , Bronchi/cytology , CCAAT-Enhancer-Binding Protein-delta , Cell Differentiation , Cell Division , Cells, Cultured , Cytochrome P-450 CYP2B1/metabolism , DNA/analysis , Epithelial Cells/cytology , Epithelial Cells/metabolism , Flow Cytometry , Fluorescent Antibody Technique, Indirect , Glycoproteins/metabolism , Male , Proteins/metabolism , Proteolipids/metabolism , Pulmonary Alveoli/cytology , Pulmonary Surfactant-Associated Protein A , Pulmonary Surfactant-Associated Protein D , Pulmonary Surfactant-Associated Proteins , Pulmonary Surfactants/metabolism , Rats , Rats, Inbred WKY
8.
Mol Carcinog ; 27(3): 190-9, 2000 Mar.
Article in English | MEDLINE | ID: mdl-10708481

ABSTRACT

Growth arrest in G(1) is a common cellular response to DNA damage. In the present study, liver regeneration was combined with continuous exposure for 2-acetylaminofluorene (AAF) to study mechanisms of carcinogen-induced growth arrest in vivo. Growth arrest of uninitiated hepatocytes is central for AAF-induced promotion of premalignant lesions in rat liver. To characterize this growth arrest, we examined the activity of cyclin-dependent kinase (Cdk) 2 in unexposed liver and in AAF-exposed liver after growth induction by partial hepatectomy (PH). Rats were fed either a control diet or an AAF-supplemented diet. After 7 d, a two-third PH was performed and the animals were killed after 0, 12, 18, 24, and 36 h. Kinase assays showed that cyclin E- and Cdk2-associated activities were lower in AAF-exposed liver than in unexposed liver after PH. Although the total cellular levels of cyclin E and Cdk2 were similar, cyclin E-Cdk2 assembly was markedly reduced. In unexposed hepatocytes, Cdk2 translocated to the nuclei after PH. Much of the nuclear Cdk2 was in a rapidly migrating form, presumably representing the Thr160-phosphorylated form of Cdk2. In contrast, in AAF-exposed liver both nuclear Cdk2 accumulation and Thr160-phosphorylation of Cdk2 were reduced. Although p53 and p21(waf1/cip1) were induced by AAF, the binding of p21 to cyclin E and Cdk2 was not increased in growth arrested liver. In conclusion, hepatocyte growth arrest caused by AAF exposure was characterized by a lowered Cdk2 activity that was accompanied by a reduced assembly of cyclin E-Cdk2 complexes but not by binding of p21.


Subject(s)
2-Acetylaminofluorene/pharmacology , CDC2-CDC28 Kinases , Carcinogens/pharmacology , Cell Nucleus/metabolism , Cyclin-Dependent Kinases/metabolism , Liver/drug effects , Protein Serine-Threonine Kinases/metabolism , Animals , Cell Division , Cyclin E/metabolism , Cyclin-Dependent Kinase 2 , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/metabolism , Enzyme Activation , Liver/enzymology , Liver/metabolism , Liver Regeneration , Male , Phosphorylation , Rats , Rats, Inbred F344 , Retinoblastoma Protein/metabolism , Tumor Suppressor Protein p53/metabolism
9.
J Histochem Cytochem ; 48(1): 21-33, 2000 Jan.
Article in English | MEDLINE | ID: mdl-10653583

ABSTRACT

After binding of epidermal growth factor (EGF), the EGF receptor (EGFR) becomes autophosphorylated via tyrosine. The ligand-activated receptor is internalized by endocytosis and subsequently degraded in the lysosomal pathway. To follow EGFR activation after EGF stimulation, we generated antisera to the EGFR phosphotyrosine sites pY992 and pY1173. The SH2 region of Shc binds to both these sites. Both antisera identified EGFR after EGF binding and did not crossreact with the unactivated receptor. The intracellular distribution of phosphorylated EGFR after ligand binding was traced by two-color immunofluorescence confocal microscopy and immunoelectron microscopy. Before EGF stimulation EGFR was primarily located along the cell surface. When internalization of activated EGFR was inhibited by incubation with EGF on ice, Y992- and Y1173-phosphorylated EGFR were located along the plasma membrane. Ten minutes after internalization at 37C, Y992- and Y1173-phosphorylated EGFR were almost exclusively located in early endosomes, as shown by co-localization with EEA1. Immunoelectron microscopy confirmed that phosphorylated EGFR was located in intracellular vesicles resembling early endosomes. After EGF stimulation, the adaptor protein Shc redistributed to EGFR-containing early endosomes. Our results indicate that EGFR activation of Shc via tyrosine-phosphorylated Y992 and Y1173 occurred in early endocytic compartments, and support a role for membrane trafficking in intracellular signaling.


Subject(s)
Adaptor Proteins, Signal Transducing , Adaptor Proteins, Vesicular Transport , Endosomes/ultrastructure , Epidermal Growth Factor/pharmacology , ErbB Receptors/isolation & purification , Phosphoproteins/isolation & purification , Proteins/isolation & purification , Binding, Competitive , Biological Transport , Cell Compartmentation , Cell Membrane/metabolism , Enzyme-Linked Immunosorbent Assay , Fluorescent Antibody Technique , HeLa Cells , Humans , Intracellular Membranes/metabolism , Microscopy, Immunoelectron , Phosphorylation , Shc Signaling Adaptor Proteins , Src Homology 2 Domain-Containing, Transforming Protein 1
10.
Mol Carcinog ; 24(1): 36-46, 1999 Jan.
Article in English | MEDLINE | ID: mdl-10029409

ABSTRACT

2-Acetylaminofluorene (AAF) is a potent tumor promoter in rat liver carcinogenesis models. In the resistant hepatocyte model, AAF is combined with a growth stimulus for efficient promotion of preneoplastic lesions. The promoting property of AAF in this model is closely associated with mito-inhibition of normal hepatocytes, an effect to which initiated cells are resistant. How AAF induces growth arrest is not known, but genotoxic as well as non-genotoxic effects have been implicated. To elucidate the mechanisms of AAF-induced mito-inhibition, we studied the expression of the tumor suppressor protein p53 and the cyclin-dependent kinase (cdk) complexes mediating G1 progression and S-phase entry. Hepatocytes were isolated from male Fisher 344 rats fed either a control diet or a diet supplemented with 0.02% AAF for 1 wk and cultured in a defined serum-free medium containing epidermal growth factor, insulin, and dexamethasone. Thymidine labeling revealed a profound inhibition of DNA synthesis in AAF-exposed cells compared with control cells. The retinoblastoma protein did not become hyperphosphorylated in AAF-exposed cells. Thus, inhibition of G1 cyclin-cdk activity was implied as a cause of growth arrest. Indeed, G1 cell-cycle arrest was accompanied by reduced induction and nuclear accumulation of the cyclin D1-cdk4 complex and inhibited nuclear translocation of cdk2. Furthermore, the growth arrest was not mediated through p21/waf1 upregulation, although nuclear levels of p53 were increased. Thus, carcinogen-induced mito-inhibition may be effected by altered levels and localization of G1 cyclin-cdk complexes, independent of the upregulation of cdk inhibitory proteins.


Subject(s)
2-Acetylaminofluorene/pharmacology , Carcinogens/pharmacology , Cell Cycle/genetics , Cyclins/genetics , Gene Expression Regulation/drug effects , Genes, p53 , Liver/drug effects , Animals , Cell Division/drug effects , Cells, Cultured , Culture Media, Serum-Free , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/biosynthesis , DNA/biosynthesis , Enzyme Inhibitors , G1 Phase , Kinetics , Liver/cytology , Liver/metabolism , Male , Rats , Rats, Inbred F344 , Thymidine/metabolism , Time Factors , Tumor Suppressor Protein p53/biosynthesis
11.
Tidsskr Nor Laegeforen ; 119(29): 4323-8, 1999 Nov 30.
Article in Norwegian | MEDLINE | ID: mdl-10667131

ABSTRACT

The scandal in Belgium last spring has drawn attention to the environmental hazards of dioxins. Previous production of pesticides and widespread combustion of organic material in the presence of chloride have lead to environmental accumulation of these toxicants, which more precisely are termed polychlorinated dibenzo-p-dioxins and dibenzofurans. Their very long biological half-lives in combination with detectable biological effects at very low concentrations have caused health concerns. Chloracne is the only well documented health effect in man, but there are experimental evidence for carcinogenic, teratogenic, reproductive and immunosuppressive effects. In this presentation we review current knowledge about the cellular effects of dioxins. Dioxins bind to and exert their effects through the cytoplasmic aryl hydrocarbon receptor, which acts as a transcription factor and regulates a number of cytokines and microsomal enzymes. Furthermore, dioxins interfere with hormonal signalling, and anti-oestrogenic effects, vitamin A inhibition and thyroxin mimicry have been reported. Recently, effects on intracellular growth factor signalling have been demonstrated. Dioxins inhibit epidermal growth factor receptor, activate protein kinase C and other intracellular signal transducers, and activate transcription factors. As overall understanding of their cellular mechanisms of toxicity is lacking, we do not possess a complete basis for estimating the adverse health effects of this group of environmental toxicants.


Subject(s)
Dioxins/adverse effects , Environmental Pollutants/adverse effects , Polychlorinated Dibenzodioxins/adverse effects , Animals , Apoptosis/drug effects , Benzofurans/adverse effects , Benzofurans/chemistry , Benzofurans/poisoning , Benzofurans/toxicity , Carcinogens , Dioxins/chemistry , Dioxins/poisoning , Dioxins/toxicity , Environmental Pollutants/poisoning , Environmental Pollutants/toxicity , Hormones/metabolism , Humans , Immunosuppressive Agents/adverse effects , Immunosuppressive Agents/chemistry , Immunosuppressive Agents/poisoning , Immunosuppressive Agents/toxicity , Polychlorinated Dibenzodioxins/chemistry , Polychlorinated Dibenzodioxins/poisoning , Polychlorinated Dibenzodioxins/toxicity , Receptors, Cell Surface/drug effects , Receptors, Cell Surface/metabolism , Reproduction/drug effects , Risk Factors , Teratogens
12.
Carcinogenesis ; 19(9): 1559-63, 1998 Sep.
Article in English | MEDLINE | ID: mdl-9771925

ABSTRACT

Exposure of cells to a variety of stresses such as heat, radiation and xenobiotics leads to increased expression of heat-shock proteins (HSPs). HSPs protect cells against irreversible protein damage and are involved in adaptive responses to stress stimuli. Some HSPs are overexpressed in neoplasias, possibly contributing to the increased drug tolerance often observed in such lesions. We have studied HSP expression in two experimental rat hepatocarcinogenesis models. Our aim was to clarify whether they are involved in stress adaptation in hepatocytes during carcinogen exposure, and whether HSPs may contribute to xenobiotic resistance in preneoplastic lesions. The complete carcinogen 2-acetylaminofluorene (AAF) was used in a continuous feeding protocol, and in the resistant hepatocyte model where the growth of diethylnitrosamine initiated lesions is efficiently promoted. Of the HSPs tested, only heat-shock protein 27 (hsp27) was induced during continuous AAF exposure. After 4 weeks of feeding AAF, increased hsp27 expression was noted in hepatocytes in perivenous areas of the liver lobule, possibly mediating an adaptive response to stress caused by reactive AAF metabolites. Enzyme altered preneoplastic foci were not found to overexpress HSPs. Thus, HSP induction does not seem to be a general mechanism underlying the increased stress tolerance observed in such lesions.


Subject(s)
2-Acetylaminofluorene/toxicity , Carcinogens/toxicity , Heat-Shock Proteins/biosynthesis , Liver Neoplasms, Experimental/metabolism , Liver/drug effects , Precancerous Conditions/metabolism , Animals , Liver/metabolism , Liver Neoplasms, Experimental/chemically induced , Male , Precancerous Conditions/chemically induced , Rats , Rats, Inbred F344
13.
Exp Cell Res ; 243(1): 161-72, 1998 Aug 25.
Article in English | MEDLINE | ID: mdl-9716460

ABSTRACT

We investigated the ability of endocytosed activated epidermal growth factor receptors (EGFR) to induce expression of the cyclin-interacting protein p21/CIP1 in A431 cells. Transforming growth factor alpha (TGFalpha) and EGF both induced tyrosine phosphorylation, induction of p21/CIP1, and thereby inhibition of DNA synthesis. TGFalpha is released from the EGFR when the TGFalpha-EGFR complex encounters low pH upon endocytosis. Consistently, we found more rapid dephosphorylation of the EGFR and less induction of p21/CIP1 by TGFalpha than by EGF. This difference was abolished upon neutralizing endosomal pH by the carboxylic ionophore monensin or the proton ATPase inhibitor bafilomycin A1. When surface-bound TGFalpha was removed by acid stripping and endosomal pH was neutralized with bafilomycin A1, TGFalpha stimulated EGFR tyrosine phosphorylation, induced p21/CIP1, and inhibited DNA synthesis. This strongly suggests that p21/CIP1 can be induced by endocytosed, activated EGFR and that endocytosed EGFR can affect cell growth.


Subject(s)
Cyclins/metabolism , Epidermal Growth Factor/pharmacology , ErbB Receptors/metabolism , Macrolides , Anti-Bacterial Agents/pharmacology , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cell Division , Cyclin-Dependent Kinase Inhibitor p21 , DNA/biosynthesis , Endocytosis , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , Humans , Hydrogen-Ion Concentration , Microscopy, Fluorescence , Monensin/pharmacology , Phosphorylation , Time Factors , Transforming Growth Factor alpha/pharmacology , Tumor Cells, Cultured
14.
Histochem Cell Biol ; 109(3): 203-9, 1998 Mar.
Article in English | MEDLINE | ID: mdl-9541468

ABSTRACT

Cyclic AMP (cAMP) is an important regulator of liver growth and differentiation. The main intracellular cAMP receptor, cAMP-dependent protein kinase (PKA), consists of two regulatory (R) and two catalytic (C) subunits. There are two classes, RI and RII, of the regulatory subunit, giving rise to type I (RI2C2) and type II (RII2C2) PKA. The RI/RII ratio generally decreases during organ development, and increases during carcinogenesis. Alterations in this ratio have been implicated as an important factor in experimental and clinical carcinogenesis. We have studied the expression of RIalpha, RIIalpha, Calpha, and an important substrate of PKA, the cAMP-response element binding protein, during rat liver carcinogenesis. Two-color immunofluorescence and confocal laser scan microscopy were used to characterize localization of the cAMP-dependent signal transducers in hepatocytes, bile ducts, oval cells, and preneoplastic lesions. We found that bile ducts and oval cells (putative liver stem cells) contained a higher RI/RII ratio than hepatocytes and preneoplastic lesions. Thus, an altered RI/RII ratio was not detected during early rat liver carcinogenesis, but may contribute to differentiation of putative liver stem cells to hepatocytes.


Subject(s)
Cyclic AMP-Dependent Protein Kinases/analysis , Cyclic AMP/metabolism , Liver Neoplasms/enzymology , Liver/enzymology , Neoplasms, Experimental/enzymology , Signal Transduction , Amino Acid Sequence , Animals , Antibodies/immunology , Cyclic AMP Response Element-Binding Protein/analysis , Cyclic AMP-Dependent Protein Kinase RIIalpha Subunit , Cyclic AMP-Dependent Protein Kinase RIalpha Subunit , Cyclic AMP-Dependent Protein Kinase Type II , Cyclic AMP-Dependent Protein Kinases/immunology , Cyclic AMP-Dependent Protein Kinases/metabolism , Humans , Liver/pathology , Liver Neoplasms/chemically induced , Liver Neoplasms/pathology , Male , Molecular Sequence Data , Neoplasms, Experimental/pathology , Proteins/analysis , Rats , Rats, Inbred F344 , Ubiquitins
15.
J Histochem Cytochem ; 44(3): 227-33, 1996 Mar.
Article in English | MEDLINE | ID: mdl-8648082

ABSTRACT

Transforming growth factor-alpha (TGF-alpha) and hepatocyte growth factor (HGF) are strong hepatocyte mitogens and important regulators of liver regeneration. The TGF-alpha receptor EGFr appears primarily to mediate a proliferative signal, whereas mitogenic, motogenic, and morphogenic effects have been attributed to activation of the HGF receptor Met. We have studied the localization of Met and EGFr in normal and carcinogen-treated rat livers. Oval cells and preneoplastic lesions were induced by diethylnitrosamine initiation, followed by promotion with 2-acetylaminofluorene combined with a partial hepatectomy. Different liver cell populations and their receptor expression were characterized by two-color immunofluorescence and confocal laser scanning microscopy. Hepatocytes were detected by keratin K8 staining, and oval cells and bile ducts were recognized by keratin K19 expression. Enzyme-altered preneoplastic lesions ere identified by expression of placental glutathione S-transferase (GST-pi). Staining for these cellular markers was combined with immunodetection of EGFr and Met. Normal liver exhibited strong staining for EGFr in hepatocytes, whereas blood vessels, bile ducts, and some sinusoidal cells were Met-positive. In carcinogen-treated livers, oval cells showed Met but not EGFr immunostaining. GST-pi-positive foci displayed EGFr immunostaining at a similar intensity as surrounding hepatocytes, whereas Met was not detected. Our data indicate that putative liver cells (oval cells) have a growth receptor phenotype similar to that of bile ducts, whereas preneoplastic live lesions appear hepatocyte-like. These results indicate that the preferential proliferation of preneoplastic liver lesions compared to surrounding hepatocytes is not associated with an altered EGFr or Met phenotype.


Subject(s)
Carcinogens/administration & dosage , Diethylnitrosamine/administration & dosage , ErbB Receptors/analysis , Liver/metabolism , Receptor Protein-Tyrosine Kinases/analysis , Animals , Immunohistochemistry , Liver/pathology , Male , Proto-Oncogene Proteins c-met , Rats , Rats, Inbred F344
16.
Histochem Cell Biol ; 104(4): 287-94, 1995 Oct.
Article in English | MEDLINE | ID: mdl-8548562

ABSTRACT

To elucidate cell differentiation in liver carcinogenesis, we have studied the CCAAT/enhancer-binding protein (C/EBP). C/EBP is a positive-acting transcription factor important for the maintenance of liver-specific functions. It is associated with differentiation and regarded as an anti-proliferative agent. We have studied the expression and localization of C/EBP during sequential rat liver carcinogenesis. Two-color immunohistochemistry and confocal laser scan microscopy demonstrated C/EBP in hepatocyte nuclei and preneoplastic liver lesions, but not in bile ducts, non-parenchymal cells or oval cells. Both western blotting and immunohistochemistry revealed down-regulation of C/EBP during normal regeneration and when regeneration was inhibited by the carcinogen, 2-acetylaminofluorene. A similar down-regulation was shown by western blotting in hepatocytes grown in culture. Our data suggest that the altered metabolic phenotype of preneoplastic liver lesions was not caused by a change in the expression of C/EBP. Furthermore, the data favor a hepatocyte derivation of preneoplastic liver lesions.


Subject(s)
DNA-Binding Proteins/metabolism , Liver Neoplasms, Experimental/metabolism , Nuclear Proteins/metabolism , Transcription Factors/metabolism , 2-Acetylaminofluorene/pharmacology , Animals , Blotting, Western , CCAAT-Enhancer-Binding Proteins , Carcinogens/pharmacology , Cell Separation , DNA-Binding Proteins/biosynthesis , Down-Regulation/physiology , Enhancer Elements, Genetic , Fluorescent Antibody Technique, Direct , Immunohistochemistry , Liver Neoplasms, Experimental/chemically induced , Liver Regeneration/drug effects , Liver Regeneration/physiology , Male , Microscopy, Confocal , Nuclear Proteins/biosynthesis , Precancerous Conditions/metabolism , Rats , Rats, Inbred F344 , Rats, Wistar , Tumor Cells, Cultured
18.
Environ Health Perspect ; 102 Suppl 6: 205-7, 1994 Oct.
Article in English | MEDLINE | ID: mdl-7889849

ABSTRACT

Gene activation can be studied at several levels: transcription (mRNA), translation (proteins), or phenotypical alterations (functional activity or morphology). These levels can be studied in situ or biochemically by the use of specific probes for normal or altered DNA, mRNA, or proteins. Immunological probes are potent tools for studies of alterations induced by xenobiotics in target organs. When the effects of xenobiotics are studied in whole tissue, the cellular heterogeneity of the organ must be taken into account. For this reason, combined in situ and biochemical techniques are necessary. Antibodies to normal or altered cellular constituents are used for identification, quantitation, and cellular localization of proteins and modified DNA. Many xenobiotics alter gene activation by interactions with DNA. After activation, 2-acetylaminofluorene (AAF) forms DNA adducts, which can be identified immunologically. Combined with bromodeoxyuridine (BrdU) pulse labeling, techniques have been developed to demonstrate reduced adduct concentrations in proliferating cells and preneoplastic foci in the livers of AAF-fed rats. Carcinogen-induced DNA modifications are implicated as a major mechanism of altered gene activation in neoplasia, leading to phenotypical alterations. Also, cellular differentiation may be affected by xenobiotics. Differentiation-associated markers can be used for studies of gene activation. In mouse skin, the keratins K1 and K10 are only expressed in suprabasal, differentiating cells. BrdU pulse chase experiments combined with double immunofluorescence have revealed that K1 and K10 are sequentially turned on 18 to 24 hr after DNA synthesis and are followed by suprabasal migration. After a single application of the tumor promotor 12-O-tetradecanoylphorbol-13-acetate (TPA), cell migration starts directly after mitosis.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Gene Expression Regulation/physiology , Blotting, Western , Cell Differentiation/genetics , DNA Damage , Immunohistochemistry , Oncogenes/drug effects , Signal Transduction/genetics , Transcriptional Activation
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