Birgitte Lindeman

Immunocytochemical Localization of Shc and Activated EGF Receptor in Early Endosomes After EGF Stimulation of HeLa Cells

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.

Paternal Benzo[a]pyrene Exposure Modulates MicroRNA Expression Patterns in the Developing Mouse Embryo

Little attention has been given to how microRNA expression is affected by environmental contaminants exposure. We investigate the effects of paternal exposure to benzo[a]pyrene (B[a]P) on miRNA expression in the developing mouse embryo. Male mice were exposed to B[a]P (150 mg/kg i.p.), and their sperm was used four days later in in-vitro fertilization experiments. Twenty embryos each from 2-, 8-cell and the blastocyst stage were used for genome-wide miRNA expression profiling. Paternal exposure to B[a]P affected the expression of several miRNAs, and the target genes for some of the dysregulated miRNAs were enriched in many different pathways that are likely to be relevant for the developing mouse embryo. By linking the miRNA target genes to publicly available databases, we identified some miRNA target genes that may serve as global markers of B[a]P-mediated genotoxic stress. The dysregulated miRNAs may provide valuable knowledge about potential transgenerational effects of sublethal exposure to chemicals.

Impaired sperm chromatin integrity in obese mice.

An increased global prevalence of obesity coincides with an apparent decline in male sperm quality and a possible association between these pathologies has been suggested. In this study, we examined the effects of obesity on sperm chromatin integrity using two mouse models of obesity. In one group of mice, obesity was induced by a high‐fat diet (HFD) (diet‐induced obesity; DIO model), whereas in the other group, leptin deficiency was used to study the effects of obesity independently of the influence of dietary factors. Sperm chromatin integrity is recognized as an important measure of male infertility, and was analysed by the sperm chromatin structure assay. We found increased sperm DNA fragmentation in both groups of obese mice compared to lean mice, whereas the percentage of immature spermatozoa was not increased by obesity. The DIO model reflects the human condition more closely than the leptin‐deficient model and was therefore selected for examination of the transcriptional response of a selection of marker genes in the testis by quantitative real‐time PCR. The analysis of transcript levels of the selected testicular marker genes showed moderate, but significant, up‐regulation of the Cyp2e1, Cyp19a1, Tnf and Pparg genes in DIO mice compared to lean mice. In conclusion, a clear positive correlation between body mass index and sperm DNA fragmentation was found in two mouse models of obesity. However, the variability in sperm DNA fragmentation within the two groups of obese animals was high. The observed changes in the transcript level of the marker genes suggest that there may be a local response in testicular cells to the HFD regimen with a potential impact on intratesticular signalling and spermatogenesis.

Genotoxicity and gene expression modulation of silver and titanium dioxide nanoparticles in mice.

Abstract Recently, we showed that silver nanoparticles (AgNPs) caused apoptosis, necrosis and DNA strand breaks in different cell models in vitro. These findings warranted analyses of their relevance in vivo. We investigated the genotoxic potential and gene expression profiles of silver particles of nano- (Ag20, 20 nm) and submicron- (Ag200, 200 nm) size and titanium dioxide nanoparticles (TiO2-NPs, 21 nm) in selected tissues from exposed male mice including the gonades. A single dose of 5 mg/kg bw nanoparticles was administered intravenously to male mice derived from C57BL6 (WT) and 8-oxoguanine DNA glycosylase knock-out (Ogg1−/− KO). Testis, lung and liver were harvested one and seven days post-exposure and analyzed for DNA strand breaks and oxidized purines employing the Comet assay with Formamidopyrimidine DNA glycosylase (Fpg) treatment, and sperm DNA fragmentation by the sperm chromatin structure assay (SCSA). Based on an initial screening of a panel of 21 genes, seven genes were selected and their expression levels were analyzed in all lung and testis tissues sampled from all animals (n = 6 mice/treatment group) using qPCR. AgNPs, in particular Ag200, caused significantly increased levels of DNA strand breaks and alkali labile sites in lung, seven days post-exposure. Fpg-sensitive lesions were significantly induced in both testis and lung. The transcript level of some key genes; Atm, Rad51, Sod1, Fos and Mmp3, were significantly induced compared to controls, particularly in lung samples from Ag200-exposed KO mice. We conclude that the Ag200 causes genotoxicity and distinct gene expression patterns in selected DNA damage response and repair related genes.

Impaired Nuclear Accumulation and Shortened Phosphorylation of ERK After Growth Factor Stimulation in Cultured Hepatocytes From Rats Exposed to 2-Acetylaminofluorene

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.

Activation of the p53–p21 Cip1 pathway is required for CDK2 activation and S-phase entry in primary rat hepatocytes

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 p21Cip1 in primary rat hepatocytes. p53 inactivation with a dominant-negative mutant (p53V143A) attenuated EGF-induced DNA synthesis and was associated with reduced CDK2 phosphorylation and retinoblastoma protein hyperphosphorylation. When p21Cip1 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 p21Cip1 expression.

Paternal Benzo(a)pyrene Exposure Affects Gene Expression in the Early Developing Mouse Embryo

The health of the offspring depends on the genetic constitution of the parental germ cells. The paternal genome appears to be important; e.g., de novo mutations in some genes seem to arise mostly from the father, whereas epigenetic modifications of DNA and histones are frequent in the paternal gonads. Environmental contaminants which may affect the integrity of the germ cells comprise the polycyclic aromatic hydrocarbon, benzo[a]pyrene (B[a]P). B[a]P has received much attention due to its ubiquitous distribution, its carcinogenic and mutagenic potential, and also effects on reproduction. We conducted an in vitro fertilization (IVF) experiment using sperm cells from B[a]P-exposed male mice to study effects of paternal B[a]P exposure on early gene expression in the developing mouse embryo. Male mice were exposed to a single acute dose of B[a]P (150mg/kg, ip) 4 days prior to isolation of cauda sperm, followed by IVF of oocytes from unexposed superovulated mice. Gene expression in fertilized zygotes/embryos was determined using reverse transcription-qPCR at the 1-, 2-, 4-, 8-, and blastocyst cell stages of embryo development. We found that paternal B[a]P exposure altered the expression of numerous genes in the developing embryo especially at the blastocyst stage. Some genes were also affected at earlier developmental stages. Embryonic gene expression studies seem useful to identify perturbations of signaling pathways resulting from exposure to contaminants, and can be used to address mechanisms of paternal effects on embryo development.

CDK2 regulation through PI3K and CDK4 is necessary for cell cycle progression of primary rat hepatocytes

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 G1 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 G1 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 G1 phase.

Effects of per- and polyfluorinated compounds on adult rat testicular cells following in vitro exposure

Testicular toxicity is observed following exposure of rats to per- and polyfluorinated compounds (PFCs). Such compounds were also shown to induce oxidative stress and changes in ABC efflux transporters e.g. P-gp, implying two mechanisms which may contribute to testicular toxicity. We studied the toxicity of four PFCs (PFOA, PFNA, 8:2 FTOH and 6:2 FTOH) on primary rat testicular cells. DNA damage was studied by the comet assay including Fpg enzyme treatment to detect oxidative lesions. The levels of the ABC efflux transporters Bcrp1, Oat2 and P-gp were studied by real-time RT-PCR or flow cytometry. A PFNA associated increase in DNA SSBs was attributed to a subpopulation of moderately damaged cells possibly associated with cytotoxicity. No significant increase in oxidative DNA damage was measured for any of the PFCs. Expression levels of ABC efflux transporters suggest that PFCs may increase expression levels of the P-gp protein and the Oat2 gene.

The carcinogen 2-acetylaminofluorene inhibits activation and nuclear accumulation of cyclin-dependent kinase 2 in growth-induced rat liver

Growth arrest in G1 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 p21waf1/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. Mol. Carcinog. 27:190–199, 2000.