Mouse Embryonic Cell Line Research Articles

Death receptors and p53 dependent impairment of UV-induced apoptosis in FADD knockouts cells

Ultraviolet (UV) irradiation is the cause of many adverse biological effects including development of cancer and aging. UV light targets both membrane receptors and nuclear DNA, thus evoking signals triggering apoptosis. In UV mediated apoptosis different molecular pathways are involved including DNAdamage, activation of tumor suppressor gene p53, triggering of cell death receptors either directly or by autocrine release of death ligants, mitochondrial damage and cytochrome C release. Detailed knowledge about the interplay between these pathways will increase our understanding of photo-carcinogenesis. To investigate comparatively the role of death receptors apoptotic signaling pathway and participation of the p53 mutation in the signaling cascade of UV induced apoptosis we used mouse embryonic cell lines from knockout mice deficient for death-domain-containing adaptor molecules FADD (Fas-associated protein with death domain). FADD is responsible for downstream signal transduction of death receptors belonging to the tumor necrosis factor (TNF) superfamily. Survival, apoptosis, and p53 mutations studies revealed that exposure of two cell lines, knockout and wild type, to UV-C radiation and TNF. As expected, FADD knockout cells were protected completely from death induced by TNF. The results indicate that apoptosis induced by UV-C light does not require FADD protein. The knockout cells were more sensitive than wild-type cells with respect to cell death. Allele- specific PCR detection of p53 in genomic DNAfrom UV-C irradiated knockout and wild type cells were analyzed by gel electrophoresis. The results show that UV-C induced apoptosis is independent of functional p53 for which the FADD knockout cells showed to be mutated. We challenge the hypothesis that UV carcinogenesis in wild type cells includes a loss of FADD function and generation of p53 mutations.

Establishment of Mouse Embryonic Stem Cell-Derived Erythroid Progenitor Cell Lines Able to Produce Functional Red Blood Cells

BackgroundThe supply of transfusable red blood cells (RBCs) is not sufficient in many countries. If erythroid cell lines able to produce transfusable RBCs in vitro were established, they would be valuable resources. However, such cell lines have not been established. To evaluate the feasibility of establishing useful erythroid cell lines, we attempted to establish such cell lines from mouse embryonic stem (ES) cells.Methodology/Principal FindingsWe developed a robust method to obtain differentiated cell lines following the induction of hematopoietic differentiation of mouse ES cells and established five independent hematopoietic cell lines using the method. Three of these lines exhibited characteristics of erythroid cells. Although their precise characteristics varied, each of these lines could differentiate in vitro into more mature erythroid cells, including enucleated RBCs. Following transplantation of these erythroid cells into mice suffering from acute anemia, the cells proliferated transiently, subsequently differentiated into functional RBCs, and significantly ameliorated the acute anemia. In addition, we did not observe formation of any tumors following transplantation of these cells.Conclusion/SignificanceTo the best of our knowledge, this is the first report to show the feasibility of establishing erythroid cell lines able to produce mature RBCs. Considering the number of human ES cell lines that have been established so far, the intensive testing of a number of these lines for erythroid potential may allow the establishment of human erythroid cell lines similar to the mouse erythroid cell lines described here. In addition, our results strongly suggest the possibility of establishing useful cell lines committed to specific lineages other than hematopoietic progenitors from human ES cells.

Генетична нестабільність ембріональних гермінативних клітин лінії G1 миші та порушення функцій контрольної точки мітозу і білка р53

Генетична нестабільність ембріональних гермінативних клітин лінії G1 миші та порушення функцій контрольної точки мітозу і білка р53

Analysis of progenitor cell–scaffold combinations by in vivo non-invasive photonic imaging

Recent developments in stem cell research have promoted a flourishing of new biomaterials and scaffolds for tissue repair. However, there is a scarcity of procedures to monitor the performance of scaffold-stem cell combinations implanted in live animals, avoiding the inherent artefacts associated with in vitro assay conditions. We report the implementation of a procedure based on the use of the luciferase gene as a cell proliferation tracer to monitor, by in vivo non-invasive imaging, the performance of stem cell–biomaterial combinations used for tissue regeneration. In a model system using immunodepressed mice we show preference of a mouse embryonic mesenchymal cell line (C3H/10T1/2) for specific implantation sites and biomaterials during a prolonged in vivo growth period (3 months). Moreover, we analyzed the safety of implanted cells using a sensitive luminometric procedure and showed that the implanted cells did not spread to other organs. Our results demonstrate the utility of this simple and resource-saving procedure in the development and screening of biomaterials for tissue engineering.

Autophagy for Cancer Therapy through Inhibition of Pro-apoptotic Proteins and Mammalian Target of Rapamycin Signaling

Autophagy is an alternative cell death pathway that is induced by mammalian target of rapamycin (mTOR) inhibitors and up-regulated when apoptosis is defective. We investigated radiation-induced autophagy in the presence or absence of Bax/Bak with or without an mTOR inhibitor, Rad001. Two isogenic cell lines, wild type (WT) and Bak/Bak(-/-) mouse embryonic fibroblasts and tumor cell lines were used for this study. Irradiated Bak/Bak(-/-) cells had a decrease of Akt/mTOR signaling and a significant increase of pro-autophagic proteins ATG5-ATG12 COMPLEX and Beclin-1. These molecular events resulted in an up-regulation of autophagy. Bax/Bak(-/-) cells were defective in undergoing apoptosis but were more radiosensitive than the WT cells in autophagy. Both autophagy and sensitization of Bak/Bax(-/-) cells were further enhanced in the presence of Rad001. In contrast, inhibitors of autophagy rendered the Bak/Bax(-/-) cells radioresistant, whereas overexpression of ATG5 and Beclin-1 made the WT cells radiosensitive. When this novel concept of radiosensitization was tested in cancer models, small interfering RNAs against Bak/Bax also led to increased autophagy and sensitization of human breast and lung cancer cells to gamma radiation, which was further enhanced by Rad001. This is the first report to demonstrate that inhibition of pro-apoptotic proteins and induction of autophagy sensitizes cancer cells to therapy. Therapeutically targeting this novel pathway may yield significant benefits for cancer patients.

Notch Activation in Cord Blood Progenitors Induces a CD7+ Common NK/T Precursor Capable of NK Cell Commitment without Full Maturation and Differentiation of Lineage Committed Pre-T Lymphocytes with a Th1 Phenotype.

Activation of Notch signaling regulates differentiation and homeostasis of hematopoetic stem cells. After stimulation, intracellular Notch is proteolytically released and by binding the CSL complex and co-activator MAML, and initiates transcription of downstream genes. We hypothesize that Notch is important for distinct stages of lymphoid development. Human cord blood CD34+ progenitor cells were transduced with retrovirus based eGFP-control, eGFP-Notch and Notch Dominant Negative/MAML (eGFP-DN) constructs. CD34+/eGFP+ were sorted and then co-cultured with the mouse embryonic liver cell line EL08.1D2 and exogenous human cytokines (IL-3. IL-7, IL-15, Flt3 ligand and c-kit ligand). As early as 48 hours after transduction, CD34+/Notch+ cells gave rise to population of lymphoid precursors CD34+CD7+CD10- (42±5% of all cells) while essentially no cells with this phenotype were detected with the control or DN construct. Proliferation of eGFP-Notch transduced cells in a 6-day thymidine incorporation assay was higher compared to eGFP-DN transduced cells (8410±839 vs. 1103±209 cpm; n=3; p=0.00005). Within 7 days 11±1.5% NK emerged from CD34+/Notch+cells compared to 0.8±0.2% of CD34+/eGFP+ control cells (n=5, p=0.0001). NK cell generation peaked at day 28 with a significantly higher expression of CD7 on NK cells (Notch: 75±5% vs. eGFP: 4.5±1%, n=5, p=0.00004), and no B lymphocytes were seen. Analysis of Notch induced NK cells demonstrated early expression of L-selectin and increased expression of CD45RA on all lymphoid progenitors. At 4 weeks, functional testing revealed reduced cytotoxicity against K562 (Notch: 37±0.5% vs. eGFP: 63.5±1.3%; n=7, p=0.007) suggesting immature function. CD34+/Notch+ derived NK lymphocytes also showed diminished acquisition of the lectin-type receptor NKG2A (Notch: 8.3±3% vs. eGFP: 27.4±4.5%; p=0.04) and killer immunoglobulin receptors (Notch: 2.2±0.5% vs. eGFP: 10.8±4: p=0.05). We next asked whether the Notch induced CD7+ precursor was NK restricted or a common NK/T cell precursor. After 5 weeks in culture, a distinct population of CD3+ T-cells emerged (Notch: 18±5% vs. eGFP: 1.6±0.2; n=5, p<0.001%) which were CD4 and CD8 negative and did not express surface TCR a/b or g/d, but expressed high levels pre-T-alpha mRNA. These Notch activated cells were bona fide T-cells based on their capacity to produce IL-2 after PMA/Ca/I stimulation (62.4±4% by intracellular staining) while essentially no IL-2 production occurred from eGFP control cells (1.6±0.2%; p<0.0001). T-cell development was dependent on both Notch and the EL08.1D2 as no T-cells resulted from CD34+/eGFP-Notch in the absence of stroma. These findings suggest that in addition to Notch and exogenous cytokines, other soluble factors are required for T cell development. In conclusion, our data showed that activated Notch pathway leads to differentiation of a common CD7+ lymphoid precursor capable of both early NK cell and T-cell differentiation. This suggests that differences in Notch ligands in local microenvironments (marrow, thymus, lymph node) may be an important mechanism to orchestrate NK and T cell development.

Concentration-Dependent Modulation of Amyloid-β in Vivo and in Vitro Using the γ-Secretase Inhibitor, LY-450139

LY-450139 is a gamma-secretase inhibitor shown to have efficacy in multiple cellular and animal models. Paradoxically, robust elevations of plasma amyloid-beta (Abeta) have been reported in dogs and humans after administration of subefficacious doses. The present study sought to further evaluate Abeta responses to LY-450139 in the guinea pig, a nontransgenic model that has an Abeta sequence identical to that of human. Male guinea pigs were treated with LY-450139 (0.2-60 mg/kg), and brain, cerebrospinal fluid, and plasma Abeta levels were characterized at 1, 3, 6, 9, and 14 h postdose. Low doses significantly elevated plasma Abeta levels at early time points, with return to baseline within hours. Higher doses inhibited Abeta levels in all compartments at early time points, but elevated plasma Abeta levels at later time points. To determine whether this phenomenon occurs under steady-state drug exposure, guinea pigs were implanted with subcutaneous minipumps delivering LY-450139 (0.3-30 mg/kg/day) for 5 days. Plasma Abeta was significantly inhibited at 10-30 mg/kg/day, but significantly elevated at 1 mg/kg/day. To further understand the mechanism of Abeta elevation by LY-450139, H4 cells overexpressing the Swedish mutant of amyloid-precursor protein and a mouse embryonic stem cell-derived neuronal cell line were studied. In both cellular models, elevated levels of secreted Abeta were observed at subefficacious concentrations, whereas dose-responsive inhibition was observed at higher concentrations. These results suggest that LY-450139 modulates the gamma-secretase complex, eliciting Abeta lowering at high concentrations but Abeta elevation at low concentrations.

Telomeres and chromosome instability

Genomic instability has been proposed to play an important role in cancer by accelerating the accumulation of genetic changes responsible for cancer cell evolution. One mechanism for chromosome instability is through the loss of telomeres, which are DNA–protein complexes that protect the ends of chromosomes and prevent chromosome fusion. Telomere loss can occur as a result of exogenous DNA damage, or spontaneously in cancer cells that commonly have a high rate of telomere loss. Mouse embryonic stem cells and human tumor cell lines that contain a selectable marker gene located immediately adjacent to a telomere have been used to investigate the consequences of telomere loss. In both cell types, telomere loss is followed by either the addition of a new telomere on to the end of the broken chromosome, or sister chromatid fusion and prolonged breakage/fusion/bridge (B/F/B) cycles that result in DNA amplification and large terminal deletions. The regions amplified by B/F/B cycles can then be transferred to other chromosomes, either through the formation of double-minute chromosomes that reintegrate at other sites, or through end-to-end fusions between chromosomes. B/F/B cycles eventually end when a chromosome acquires a new telomere by one of several mechanisms, the most common of which is translocation, which can involve either nonreciprocal transfer or duplication of all or part of an arm of another chromosome. Telomere acquisition involving nonreciprocal translocations results in the loss of a telomere on the donor chromosome, which subsequently becomes unstable. In contrast, translocations involving duplications do not destabilize the donor chromosome, although they result in allelic imbalances. Thus, the loss of a single telomere can generate a wide variety of chromosome alterations commonly associated with human cancer, not only on the chromosome that originally lost its telomere, but other chromosomes as well. Factors promoting spontaneous telomere loss and the resulting B/F/B cycles are therefore likely to be important in generating the karyotypic changes associated with human cancer.

Instability of expanded simple tandem repeats is induced in cell culture by a variety of agents: N-Nitroso- N-ethylurea, benzo( a)pyrene, etoposide and okadaic acid

Expanded simple tandem repeat (ESTR) sequences have proven useful biomarkers to detect genotoxicity in vivo. Their high sensitivity has been used to assess environmentally relevant doses of mutagens such as ionizing radiation, DNA alkylating agents and airborne particulate pollution, for germline mutations in mouse assays. The mutagenic response involves size alteration of these ESTR loci induced by agents causing a variety of cellular damage. The mechanistic aspects of this induced instability remain unclear and have not been studied in detail. Mechanistic knowledge is important to help understand the relevance of increased ESTR mutation frequencies. In this study, we applied a murine cell culture system to examine induced response to four agents exhibiting different modes of toxic action including: N-nitroso- N-ethylurea (ENU), benzo( a)pyrene (BaP), okadaic acid and etoposide at slightly sub-toxic levels. We used single-molecule-polymerase chain reaction (SM-PCR) to assess the relative mutant frequency after 4-week chemical treatments at the Ms6- hm ESTR sequence of cultured C3H/10T1/2 cells (a mouse embryonic cell line). Increased mutation was observed with both 0.64 mM ENU (1.95-fold increase, P < 0.0001), 1 μM benzo( a)pyrene (1.87-fold increase, P = 0.0006) and 3 nM etoposide (1.89-fold increase, P = 0.0003). The putative ESTR mutagen okadaic acid (1.27-fold increase, P = 0.2289), administered at 0.5 nM, did not affect the C3H/10T1/2 Ms6- hm locus. Therefore, agents inducing small and bulky adducts, and indirectly causing strand breaks through inhibition of topoisomerase, caused similar induction of instability at an ESTR locus at matched toxicities. As size spectra for induced mutations were identical, the data indicate that although these chemicals exhibit distinct modes of action, a similar indirect process is influencing ESTR instability. In contrast, a potent tumour promoter that is a kinase inhibitor does not contribute to induced ESTR instability in cell culture.

Mitochondrial Proteomic Analysis of a Cell Line Model of Familial Amyotrophic Lateral Sclerosis

Mutations in copper-zinc superoxide dismutase (SOD1) have been linked to a subset of familial amytrophic lateral sclerosis (fALS), a fatal neurodegenerative disease characterized by progressive motor neuron death. An increasing amount of evidence supports that mitochondrial dysfunction and apoptosis activation play a critical role in the fALS etiology, but little is known about the mechanisms by which SOD1 mutants cause the mitochondrial dysfunction and apoptosis. In this study, we use proteomic approaches to identify the mitochondrial proteins that are altered in the presence of a fALS-causing mutant G93A-SOD1. A comprehensive characterization of mitochondrial proteins from NSC34 cells, a motor neuron-like cell line, was achieved by two independent proteomic approaches. Four hundred seventy unique proteins were identified in the mitochondrial fraction collectively, 75 of which are newly discovered proteins that previously had only been reported at the cDNA level. Two-dimensional gel electrophoresis was subsequently used to analyze the differences between the mitochondrial proteomes of NSC34 cells expressing wild-type and G93A-SOD1. Nine and 36 protein spots displayed elevated and suppressed abundance respectively in G93A-SOD1-expressing cells. The 45 spots were identified by MS, and they include proteins involved in mitochondrial membrane transport, apoptosis, the respiratory chain, and molecular chaperones. In particular, alterations in the post-translational modifications of voltage-dependent anion channel 2 (VDAC2) were found, and its relevance to regulating mitochondrial membrane permeability and activation of apoptotic pathways is discussed. The potential role of other proteins in the mutant SOD1-mediated fALS is also discussed. This study has produced a short list of mitochondrial proteins that may hold the key to the mechanisms by which SOD1 mutants cause mitochondrial dysfunction and neuronal death. It has laid the foundation for further detailed functional studies to elucidate the role of particular mitochondrial proteins, such as VDAC2, in the pathogenesis of familial ALS.

DNA methyltransferase Dnmt1 and mismatch repair.

We have examined the relationship between DNA mismatch repair and deficiency of DNA methylation in a mouse embryonic cell line, Dnmt1-/- ES, with homologous deletion of the gene coding for the maintenance DNA methyltransferase Dnmt1. With the use of a sensitive PCR for the assay of two mononucleotide- and three dinucleotide microsatellite markers that exhibited instabilities in mismatch repair-deficient cells, significantly higher frequencies of instability were detected at three of the five markers in Dnmt1-/- ES than the wild-type ES. The data suggest that Dnmt1 enzyme plays an integrating role in DNA replication and/or repair. The implication that Dnmt1 enzyme and/or cytosine methylation may participate in the strand discrimination of mismatch repair during eukaryotic DNA replication is discussed.

Generation of hybrid cell lines with endothelial potential from spontaneous fusion of adult bone marrow cells with embryonic fibroblast feeder.

We have previously isolated mouse embryonic cell lines with endothelial potential using a simple empirical approach. In an attempt to isolate similar cell lines from adult mouse bone marrow (BM), BM cells were cultured on mitotically inactive mouse embryonic fibroblast (MEF) feeder cells. Several cell lines with putative endothelial potential were generated. They expressed endothelial-specific genes and formed vascular-like structures when plated on matrigel. When transplanted into appropriate mouse models, they incorporated into the endothelium of the vasculature. Similar cell lines were also obtained using human or porcine BM. None of these lines induced tumor formation when transplanted into immunodeficient Rag1-/- mice. However, all the lines were aneuploid with genetic markers from BM samples and the MEF feeder, suggesting that they resulted from a non-species-specific fusion of a BM cell and mitotically inactive MEF. Together, these lines demonstrated for the first time that BM cells can also undergo fusion with commonly used mitotically inactive feeder cells. Although these fusion cell lines were culture artifacts, their derivation would be useful in understanding fusion of BM cells with other cell types, and their endothelial potential will also be useful in characterizing endothelial differentiation.

Characterization of mutant spectra generated by a forward mutational assay for gene A of Phi X174 from ENU-treated transgenic mouse embryonic cell line PX-2.

The sensitivity of in vivo transgenic mutation assays benefits from the sequencing of mutations, although the large number of possible mutations hinders high throughput sequencing. A forward mutational assay exists for Phi X174 that requires an altered, functional Phi X174 protein and therefore should have fewer targets (sense, base-pair substitutions) than forward assays that inactivate a protein. We investigated this assay to determine the number of targets and their suitability for detecting a known mutagen, N-ethyl-N-nitrosourea (ENU). We identified 25 target sites and 33 different mutations in Phi X174 gene A after sequencing over 350 spontaneous and ENU-induced mutants, mostly from mouse embryonic cell line PX-2 isolated from mice transgenic for Phi X174 am3, cs70 (line 54). All six types of base-pair substitution were represented among both the spontaneous and ENU-treated mutant spectra. The mutant spectra from cells treated with 200 and 400 microg/ml ENU were both highly different from the spontaneous spectrum (P < 0.000001) but not from each other. The dose trend was significant (P < 0.0001) for a linear regression of mutant frequencies (R(2) = 0.79), with a ninefold increase in mutant frequency at the 400 microg/ml dose. The spontaneous mutant frequency was 1.9 x 10(-5) and the spontaneous spectrum occurred at 11 target base pairs with 15 different mutations. Thirteen mutations at 12 targets were identified only from ENU-treated cells. Seven mutations had highly significant increases with ENU treatment (P < 0.0001) and 15 showed significant increases. The results suggest that the Phi X174 forward assay might be developed into a sensitive, inexpensive in vivo mutagenicity assay.

Involvement of the phosphoinositide 3-kinase/protein kinase B signaling pathway in insulin/IGF-I-induced chondrogenesis of the mouse embryonal carcinoma-derived cell line ATDC5

The embryonal carcinoma-derived cell line, ATDC5, differentiates into chondrocytes in response to insulin/insulin-like growth factor-I (IGF-I) stimulation. In the present study, we examined whether insulin/IGF-I stimulation caused activation of the phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB) pathway in ATDC5 cells. We also determined whether the insulin-stimulated differentiation of ATDC5 cells into chondrocytes could be mimicked by activation of the PKB pathway alone. ATDC5 cells produced phosphatidylinositol 3,4,5-trisphosphate and the pleckstrin homology domain of PKB was recruited to the plasma membrane in response to insulin stimulation. This was probably a result of activation of PI3K because the PI3K inhibitors, wortmannin and LY294002, inhibited both responses, although the effective concentrations were as high as 10 μM. Insulin stimulation caused the chondrogenic differentiation of ATDC5 cells as assessed by chondrogenic nodule staining with alcian blue. The addition of wortmannin or LY294002, PI3K inhibitors, suppressed the staining, and the suppression was reversible, indicating the effect of the inhibitors is not toxic. Finally, we exogenously expressed a constitutively-activated from of PKB (myristoylated PKB, myr-PKB) in ATDC5 cells, and found the chondrogenic differentiation of ATDC5 cells to form nodules occurred in the absence of insulin stimulation. The kinase-negative mutant of myr-PKB did not caused differentiation, indicating that kinase activity is required. These results support the hypothesis that the PI3K/PKB signaling pathway is involved in the chondrogenic differentiation of ATDC5 cells in response to insulin/IGF-I stimulation. This is the first report that demonstrates the involvement of phosphoinositide signaling in the induction of chondrogenesis from undifferentiated cells.

Activation of calpain precedes morphological alterations during hydrogen peroxide-induced apoptosis in neuronally differentiated mouse embryonal carcinoma P19 cell line

In order to reveal neurodegeneration elicited by reactive oxygen intermediates (ROI), neuronally differentiated cells from mouse embryonal carcinoma P19 cell line were exposed to hydrogen peroxide (H 2O 2). Enhanced protein-phosphorylation on tyrosine residues was detectable within 5 min of exposure to H 2O 2, and gradual rises in intracellular free Ca 2+ level and in calpain activity were observed. Furthermore, H 2O 2 stimulation of differentiated P19 cells for 24 h resulted in morphological alterations in somas as well as neurites. Also, within 6 h of H 2O 2 treatment DNA fragmentation has been detected. Taken together, these results suggest that oxidative stress induces degradation of cytoskeletal proteins presumably resulting from increased intracellular Ca 2+ concentration and subsequent activation of calpain.

Membrane-bound transferrin-like protein (MTf): structure, evolution and selective expression during chondrogenic differentiation of mouse embryonic cells

Mouse membrane-bound transferrin-like protein (MTf) cDNA was cloned to examine its expression during chondrogenic differentiation in the mouse embryonic cell line ATDC5, and to analyze the phylogenetic relationships among the MTfs of four animal species and 23 other transferrin members. Phylogenetic analysis indicated that the MTf gene diverged from the common ancestor gene earlier than the genes of the other transferrins such as serum transferrin, lactoferrin and ovotransferrin, and that the divergence occurred after the divergence of vertebrates and invertebrates. MTf, as well as the other transferrins, consists of two repeated domains. The similarity between the N-terminal and the C-terminal domains of MTf is much higher than that of the other transferrins, although the five amino acid residues required for iron binding were not conserved in the C-terminal domain of MTf in contrast to the conservation of these residues in both domains of the other transferrins. Among various adult mouse tissues, MTf mRNA was expressed at the highest level in cartilage and at a moderate level in the testis. MTf mRNA was expressed only at very low levels in the brain, spleen, thymus, muscle, lung, skin and intestine, and hardly detected in the heart, kidney, stomach and liver. In cultures of the mouse ATDC5 cell line, MTf is developmentally expressed in parallel with the expression of type II collagen and aggrecan, in the pattern commensurate with the onset of chondrogenesis to form cartilage nodules. The structural characteristics and the expression pattern suggest that during development and in adult tissues, MTf has some functions that are different from those of other transferrins.

Feedback regulation of DNA methyltransferase gene expression by methylation.

This paper tests the hypothesis that expression of the DNA methyltransferase, dnmt1, gene is regulated by a methylation-sensitive DNA element. Methylation of DNA is an attractive system for feedback regulation of DNA methyltransferase as the final product of the reaction, methylated DNA, can regulate gene expression in cis. We show that an AP-1-dependent regulatory element of dnmt1 is heavily methylated in most somatic tissues and in the mouse embryonal cell line, P19, and completely unmethylated in a mouse adrenal carcinoma cell line, Y1. dnmt1 is highly over expressed in Y1 relative to P19 cell lines. Global inhibition of DNA methylation in P19 cells by 5-azadeoxycytidine results in demethylation of the AP-1 regulatory region and induction of dnmt1 expression in P19cells, but not Y1 cells. We propose that this regulatory region of dnmt1 acts as a sensor of the DNA methylation capacity of the cell. These results provide an explanation for the documented coexistence of global hypomethylation and high levels of DNA methyltransferase activity in many cancer cells and for the carcinogenic effect of hypomethylating diets.

Identification of megalin/gp330 as a receptor for lipoprotein(a) in vitro.

Lipoprotein(a) [Lp(a)] is an atherogenic lipoprotein of unknown physiological function. The mechanism of Lp(a) atherogenicity as well as its catabolic pathways are only incompletely understood at present. In this report, we show that the low density lipoprotein receptor (LDLR) gene family member megalin/glycoprotein (gp) 330 is capable of binding and mediating the cellular uptake and degradation of Lp(a) in vitro. A mouse embryonic yolk sac cell line with native expression of megalin/gp330 but genetically deficient in LDLR-related protein (LRP) and a control cell line carrying a double knockout for both LRP and megalin/gp330 were compared with regard to their ability to bind, internalize, and degrade dioctadecyltetramethylindocarbocyanine perchlorate (DiI)-fluorescence-labeled Lp(a) as well as equimolar amounts of 125I-labeled Lp(a) and LDL. Uptake and degradation of radiolabeled Lp(a) by the megalin/gp330-expressing cells were, on average, 2-fold higher than that of control cells. This difference could be completely abolished by addition of the receptor-associated protein, an inhibitor of ligand binding to megalin/gp330. Mutual suppression of the uptake of 125I-Lp(a) and of 125I-LDL by both unlabeled Lp(a) and LDL suggested that Lp(a) uptake is mediated at least partially by apolipoprotein B100. Binding and uptake of DiI-Lp(a) resulted in strong signals on megalin/gp330-expressing cells versus background only on control cells. In addition, we show that purified megalin/gp330, immobilized on a sensor chip, directly binds Lp(a) in a Ca2+-dependent manner with an affinity similar to that for LDL. We conclude that megalin/gp330 binds Lp(a) in vitro and is capable of mediating its cellular uptake and degradation.

A role for the epithelial-cell-specific tyrosine kinase Sik during keratinocyte differentiation.

Sik, the mouse homologue of the breast tumor kinase Brk, is expressed in differentiating cells of the gastrointestinal tract and skin. We examined expression and activity of Sik in primary mouse keratinocytes and a mouse embryonic keratinocyte cell line (EMK). Calcium-induced differentiation of these cells has been shown to be accompanied by the activation of tyrosine kinases and rapid phosphorylation of a 65-kDa GTPase-activating protein (GAP)-associated protein (GAP-A.p65). We demonstrate that Sik is activated within 2 min after calcium addition in primary keratinocytes and EMK cells. In EMK cells, Sik binds GAP-A.p65, and this interaction is mediated by the Sik Src homology 2 domain. Although Sik directly complexes with GAP-A.p65, overexpression of wild-type or kinase defective Sik in EMK cells does not lead to detectable changes in GAP-A.p65 phosphorylation. These data suggest that Sik is not responsible for phosphorylation of GAP-A.p65. GAP-A. p65 may act as an adapter protein, bringing Sik into proximity of an unidentified substrate. Overexpression of Sik in EMK cells results in increased expression of filaggrin during differentiation, supporting a role for Sik in differentiation.

Cellular uptake of lipoprotein[a] by mouse embryonic fibroblasts via the LDL receptor and the LDL receptor-related protein

The sites and precise mechanisms of the catabolism of the atherogenic lipoprotein[a] (Lp[a]) are unknown. It has been proposed that the low density lipoprotein receptor (LDL-R) and the low density lipoprotein receptor-related protein (LRP) are involved in the catabolism of Lp[a]. To address the question whether and to what extent the LDL-R and/or LRP are involved in the catabolism of Lp[a], we studied the cellular uptake of Lp[a] via those two receptors using mouse embryonic fibroblast (MEF) cell lines lacking either the LDL-R, the LRP, or both receptors due to disruption of the respective mouse genes. 125I-labeled LDL and 125I-labeled Lp[a] uptake by wild-type fibroblasts (MEF1) was compared with that by fibroblasts homozygous for the disrupted LRP allele (MEF2), fibroblasts with two defective alleles for the LDL-R (MEF3), and fibroblasts homozygous for defects both in the LDL-R and LRP gene (MEF4). Compared with MEF1, 125I-labeled LDL uptake by MEF2 was 77%, by MEF3 30%, and by MEF4 24% of that by MEF1. However, no significant differences in the specific 125I-labeled Lp[a] uptake by the four mouse embryonic cell lines was observed. In comparison with MEF1, the 125I-labeled Lp[a] uptake by MEF2 was 98%, by MEF3 111%, and 73% by MEF4. Approximately 50% of the total cellular uptake of 125I-labeled Lp[a] was nonspecific. In conclusion, our results suggest that Lp[a] is a poor ligand for the LDL receptor and the LRP. The data of the displacement studies, however, indicated that the nonspecific uptake of Lp[a] constitutes a major route for the cellular Lp[a] catabolism in this study.