Showing papers on "Cell culture published in 2002"

Identification of Genes Periodically Expressed in the Human Cell Cycle and Their Expression in Tumors

Abstract: The genome-wide program of gene expression during the cell division cycle in a human cancer cell line (HeLa) was characterized using cDNA microarrays. Transcripts of >850 genes showed periodic variation during the cell cycle. Hierarchical clustering of the expression patterns revealed coexpressed groups of previously well-characterized genes involved in essential cell cycle processes such as DNA replication, chromosome segregation, and cell adhesion along with genes of uncharacterized function. Most of the genes whose expression had previously been reported to correlate with the proliferative state of tumors were found herein also to be periodically expressed during the HeLa cell cycle. However, some of the genes periodically expressed in the HeLa cell cycle do not have a consistent correlation with tumor proliferation. Cell cycle-regulated transcripts of genes involved in fundamental processes such as DNA replication and chromosome segregation seem to be more highly expressed in proliferative tumors simply because they contain more cycling cells. The data in this report provide a comprehensive catalog of cell cycle regulated genes that can serve as a starting point for functional discovery. The full dataset is available at http://genome-www.stanford.edu/Human-CellCycle/HeLa/.

The human scavenger receptor class B type I is a novel candidate receptor for the hepatitis C virus

Abstract: We discovered that the hepatitis C virus (HCV) envelope glycoprotein E2 binds to human hepatoma cell lines independently of the previously proposed HCV receptor CD81. Comparative binding studies using recombinant E2 from the most prevalent 1a and 1b genotypes revealed that E2 recognition by hepatoma cells is independent from the viral isolate, while E2–CD81 interaction is isolate specific. Binding of soluble E2 to human hepatoma cells was impaired by deletion of the hypervariable region 1 (HVR1), but the wild-type phenotype was recovered by introducing a compensatory mutation reported previously to rescue infectivity of an HVR1-deleted HCV infectious clone. We have identified the receptor responsible for E2 binding to human hepatic cells as the human scavenger receptor class B type I (SR-BI). E2–SR-BI interaction is very selective since neither mouse SR-BI nor the closely related human scavenger receptor CD36, were able to bind E2. Finally, E2 recognition by SR-BI was competed out in an isolate-specific manner both on the hepatoma cell line and on the human SR-BI-transfected cell line by an anti-HVR1 monoclonal antibody.

Direct Recognition of Cytomegalovirus by Activating and Inhibitory NK Cell Receptors

Abstract: Natural killer (NK) cells express inhibitory receptors for major histocompatibility complex (MHC) class I antigens, preventing attack against healthy cells Mouse cytomegalovirus (MCMV) encodes an MHC-like protein (m157) that binds to an inhibitory NK cell receptor in certain MCMV-susceptible mice In MCMV-resistant mice, this viral protein engages a related activating receptor (Ly49H) and confers host protection These activating and inhibitory receptors are highly homologous, suggesting the possibility that one evolved from the other in response to selective pressure imposed by the pathogen

High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells

Abstract: A scalable transfection procedure using polyethylenimine (PEI) is described for the human embryonic kidney 293 cell line grown in suspension. Green fluorescent protein (GFP) and human placental secreted alkaline phosphatase (SEAP) were used as reporter genes to monitor transfection efficiency and productivity. Up to 75% of GFP-positive cells were obtained using linear or branched 25 kDa PEI. The 293 cell line and two genetic variants, either expressing the SV40 large T-antigen (293T) or the Epstein–Barr virus (EBV) EBNA1 protein (293E), were tested for protein expression. The highest expression level was obtained with 293E cells using the EBV oriP-containing plasmid pCEP4. We designed the pTT vector, an oriP-based vector having an improved cytomegalovirus expression cassette. Using this vector, 10- and 3-fold increases in SEAP expression was obtained in 293E cells compared with pcDNA3.1 and pCEP4 vectors, respectively. The presence of serum had a positive effect on gene transfer and expression. Transfection of suspension-growing cells was more efficient with linear PEI and was not affected by the presence of medium conditioned for 24 h. Using the pTT vector, >20 mg/l of purified Histagged SEAP was recovered from a 3.5 l bioreactor. Intracellular proteins were also produced at levels as high as 50 mg/l, representing up to 20% of total cell proteins.

Mammalian cell size is controlled by mTOR and its downstream targets S6K1 and 4EBP1/eIF4E

Abstract: The coordinated action of cell cycle progression and cell growth (an increase in cell size and cell mass) is critical for sustained cellular proliferation, yet the biochemical signals that control cell growth are poorly defined, particularly in mammalian systems. We find that cell growth and cell cycle progression are separable processes in mammalian cells and that growth to appropriate cell size requires mTOR- and PI3K-dependent signals. Expression of a rapamycin-resistant mutant of mTOR rescues the reduced cell size phenotype induced by rapamycin in a kinase-dependent manner, showing the evolutionarily conserved role of mTOR in control of cell growth. Expression of S6K1 mutants that possess partial rapamycin-resistant activity or overexpression of eIF4E individually and additively partially rescues the rapamycin-induced decrease in cell size. In the absence of rapamycin, overexpression of S6K1 or eIF4E increases cell size, and, when coexpressed, they cooperate to increase cell size further. Expression of a phosphorylation site-defective mutant of 4EBP1 that constitutively binds the eIF4E-Cap complex to inhibit translation initiation reduces cell size and blocks eIF4E effects on cell size. These data show that mTOR signals downstream to at least two independent targets, S6K1 and 4EBP1/eIF4E, that function in translational control to regulate mammalian cell size.

Characterization and Enrichment of Cardiomyocytes Derived From Human Embryonic Stem Cells

Abstract: Cell replacement therapy is a promising approach for the treatment of cardiac diseases, but is challenged by a limited supply of appropriate cells. We have investigated whether functional cardiomyocytes can be efficiently generated from human embryonic stem (hES) cells. Cardiomyocyte differentiation was evaluated using 3 parent (H1, H7, and H9) hES cell lines and 2 clonal (H9.1 and H9.2) hES cell lines. All cell lines examined differentiated into cardiomyocytes, even after long-term culture (50 passages or approximately 260 population doublings). Upon differentiation, beating cells were observed after one week in differentiation conditions, increased in numbers with time, and could retain contractility for over 70 days. The beating cells expressed markers characteristic of cardiomyocytes, such as cardiac alpha-myosin heavy chain, cardiac troponin I and T, atrial natriuretic factor, and cardiac transcription factors GATA-4, Nkx2.5, and MEF-2. In addition, cardiomyocyte differentiation could be enhanced by treatment of cells with 5-aza-2'-deoxycytidine but not DMSO or retinoic acid. Furthermore, the differentiated cultures could be dissociated and enriched by Percoll density centrifugation to give a population containing 70% cardiomyocytes. The enriched population was proliferative and showed appropriate expression of cardiomyocyte markers. The extended replicative capacity of hES cells and the ability to differentiate and enrich for functional human cardiomyocytes warrant further development of these cells for clinical application in heart diseases.

Expression of Programmed Death 1 Ligands by Murine T Cells and APC

Abstract: Programmed death 1 (PD-1) is a new member of the CD28/CTLA-4 family, which has been implicated in the maintenance of peripheral tolerance. Two ligands for PD-1, namely, B7-H1 (PD-L1) and B7-DC (PD-L2), have recently been identified as new members of the B7 family but their expression at the protein level remains largely unknown. To characterize the expression of B7-H1 and B7-DC, we newly generated an anti-mouse B7-H1 mAb (MIH6) and an anti-mouse B7-DC mAb (TY25). MIH6 and TY25 immunoprecipitated a single molecule of 43 and 42 kDa from the lysate of B7-H1 and B7-DC transfectants, respectively. Flow cytometric analysis revealed that B7-H1 was broadly expressed on the surface of mouse tumor cell lines while the expression of B7-DC was rather restricted. PD-1 was expressed on anti-CD3-stimulated T cells and anti-IgM plus anti-CD40-stimulated B cells at high levels but was undetectable on activated macrophages or DCs. B7-H1 was constitutively expressed on freshly isolated splenic T cells, B cells, macrophages, and dendritic cells (DCs), and up-regulated on T cells by anti-CD3 stimulation on macrophages by LPS, IFN-gamma, GM-CSF, or IL-4, and on DCs by IFN-gamma, GM-CSF, or IL-4. In contrast, B7-DC expression was only inducible on macrophages and DCs upon stimulation with IFN-gamma, GM-CSF, or IL-4. The inducible expression of PD-1 ligands on both T cells and APCs may suggest new paradigms of PD-1-mediated immune regulation.

Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells.

Abstract: Previous reports have demonstrated the growth of undifferentiated human embryonic stem (HES) cells on mouse embryonic fibroblast (MEF) feeders and on laminin- or Matrigel-coated plastic surfaces supplemented with MEF-conditioned medium. These xenosupport systems run the risk of cross-transfer of animal pathogens from the animal feeder, matrix, or conditioned medium to the HES cells, thus compromising later clinical application. Here we show that human fetal and adult fibroblast feeders support prolonged undifferentiated HES cell growth of existing cell lines and are superior to cell-free matrices (collagen I, human extracellular matrix, Matrigel, and laminin) supplemented with human or MEF feeder-conditioned medium. Additionally, we report the derivation and establishment of a new HES cell line in completely animal-free conditions. Like HES cells cultured on MEF feeders, the HES cells grown on human feeders had normal karyotypes, tested positive for alkaline phosphatase activity, expressed Oct-4 and cell surface markers including SSEA-3, SSEA-4, Tra 1-60, and GCTM-2, formed teratomas in severely combined immunodeficient (SCID) mice, and retained all key morphological characteristics. Human feeder#150;supported HES cells should provide a safer alternative to existing HES cell lines in therapeutic applications.

Preferential transformation of human neuronal cells by human adenoviruses and the origin of HEK 293 cells.

Abstract: The 293 cell line was derived by transformation of primary cultures of human embryonic kidney (HEK) cells with sheared adenovirus (Ad)5 DNA. A combination of immunostaining, immunoblot, and microarray analysis showed that 293 cells express the neurofilament (NF) subunits NF-L, NF-M, NF-H, and a-internexin as well as many other proteins typically found in neurons. Three other independently derived HEK lines, two transformed by Ad5 and one by Ad12, also expressed NFs, as did one human embryonic retinal cell line transformed with Ad5. Two rodent kidney lines transformed with Ad12 were also found to express NF proteins, although several rodent kidney cell lines transformed by Ad5 DNA and three HEK cell lines transformed by the SV40 early region did not express NFs. These results suggest that human Ads preferentially transform human neuronal lineage cells. We also demonstrate that the widely used HEK293 cells have an unexpected relationship to neurons, a finding that may require reinterpretation of many previous studies in which it was assumed that HEK293 cells resembled more typical kidney epithelial cells.

Cytoplasmic relocalization and inhibition of the cyclin-dependent kinase inhibitor p27 Kip1 by PKB/Akt-mediated phosphorylation in breast cancer

Abstract: The cyclin-dependent kinase inhibitor p27(kip1) is a putative tumor suppressor for human cancer. The mechanism underlying p27(kip1) deregulation in human cancer is, however, poorly understood. We demonstrate that the serine/threonine kinase Akt regulates cell proliferation in breast cancer cells by preventing p27(kip1)-mediated growth arrest. Threonine 157 (T157), which maps within the nuclear localization signal of p27(kip1), is a predicted Akt-phosphorylation site. Akt-induced T157 phosphorylation causes retention of p27(kip1) in the cytoplasm, precluding p27(kip1)-induced G1 arrest. Conversely, the p27(kip1)-T157A mutant accumulates in cell nuclei and Akt does not affect p27(kip1)-T157A-mediated cell cycle arrest. Lastly, T157-phosphorylated p27(kip1) accumulates in the cytoplasm of primary human breast cancer cells coincident with Akt activation. Thus, cytoplasmic relocalization of p27(kip1), secondary to Akt-mediated phosphorylation, is a novel mechanism whereby the growth inhibitory properties of p27(kip1) are functionally inactivated and the proliferation of breast cancer cells is sustained.

Enumeration of the Simian Virus 40 Early Region Elements Necessary for Human Cell Transformation

Abstract: While it is clear that cancer arises from the accumulation of genetic mutations that endow the malignant cell with the properties of uncontrolled growth and proliferation, the precise combinations of mutations that program human tumor cell growth remain unknown. The study of the transforming proteins derived from DNA tumor viruses in experimental models of transformation has provided fundamental insights into the process of cell transformation. We recently reported that coexpression of the simian virus 40 (SV40) early region (ER), the gene encoding the telomerase catalytic subunit (hTERT), and an oncogenic allele of the H-ras gene in normal human fibroblast, kidney epithelial, and mammary epithelial cells converted these cells to a tumorigenic state. Here we show that the SV40 ER contributes to tumorigenic transformation in the presence of hTERT and oncogenic H-ras by perturbing three intracellular pathways through the actions of the SV40 large T antigen (LT) and the SV40 small t antigen (ST). LT simultaneously disables the retinoblastoma (pRB) and p53 tumor suppressor pathways; however, complete transformation of human cells requires the additional perturbation of protein phosphatase 2A by ST. Expression of ST in this setting stimulates cell proliferation, permits anchorage-independent growth, and confers increased resistance to nutrient deprivation. Taken together, these observations define the elements of the SV40 ER required for the transformation of human cells and begin to delineate a set of intracellular pathways whose disruption, in aggregate, appears to be necessary to generate tumorigenic human cells.

Myeloid Suppressor Lines Inhibit T Cell Responses by an NO-Dependent Mechanism

Abstract: CD11b(+)Gr-1(+) myeloid suppressor cells (MSC) accumulate in lymphoid organs under conditions of intense immune stress where they inhibit T and B cell function. We recently described the generation of immortalized MSC lines that provide a homogeneous source of suppressor cells for dissecting the mechanism of suppression. In this study we show that the MSC lines potently block in vitro proliferation of T cells stimulated with either mitogen or antigenic peptide, with as few as 3% of MSC cells causing complete suppression. Inhibition of mitogenic and peptide-specific responses is not associated with a loss in IL-2 production or inability to up-modulate the early activation markers, CD69 and CD25, but results in direct impairment of the three IL-2R signaling pathways, as demonstrated by the lack of Janus kinase 3, STAT5, extracellular signal-regulated kinase, and Akt phosphorylation in response to IL-2. Suppression is mediated by and requires NO, which is secreted by MSC in response to signals from activated T cells, including IFN-gamma and a contact-dependent stimulus. Experiments with inducible NO synthase knockout mice demonstrated that the inhibition of T cell proliferation by CD11b(+)Gr-1(+) cells in the spleens of immunosuppressed mice is also dependent upon NO, indicating that the MSC lines accurately represent their normal counterparts. The distinctive capacity of MSC to generate suppressive signals when encountering activated T cells defines a specialized subset of myeloid cells that most likely serve a regulatory function during times of heightened immune activity.

Structure and function in rhodopsin: High-level expression of rhodopsin with restricted and homogeneous N-glycosylation by a tetracycline-inducible N-acetylglucosaminyltransferase I-negative HEK293S stable mammalian cell line

Abstract: An HEK293S cell line resistant to ricin was prepared by mutagenesis by using ethyl methanesulfonate. It was shown to lack N-acetylglucosaminyltransferase I (GnTI) activity, and consequently unable to synthesize complex N-glycans. The tetracycline-inducible opsin expression system was assembled into this GnTI− HEK293S cell line. Stable cell lines were isolated that gave tetracycline/sodium butyrate-inducible expression of the WT opsin gene at levels comparable with those observed in the parent tetracycline-inducible HEK293S cell line. Analysis of the N-glycan in rhodopsin expressed by the HEK293S GnTI− stable cell line showed it to be Man5GlcNAc2. In a larger-scale expression experiment (1.1 liter) a WT opsin production level of 6 mg/liter was obtained. Further, the toxic constitutively active rhodopsin mutant, E113Q/E134Q/M257Y, previously shown to require inducible expression, has now been expressed in an HEK293S GNTI−-inducible cell line at levels comparable with those obtained with WT rhodopsin.

Expression of hypoxia-inducible factor-1alpha and -2alpha in hypoxic and ischemic rat kidneys.

Abstract: Oxygen tensions in the kidney are heterogeneous, and their changes presumably play an important role in renal physiologic and pathophysiologic processes A family of hypoxia-inducible transcription factors (HIF) have been identified as mediators of transcriptional responses to hypoxia, which include the regulation of erythropoietin, metabolic adaptation, vascular tone, and neoangiogenesis In vitro, the oxygen-regulated subunits HIF-1alpha and -2alpha are expressed in inverse relationship to oxygen tensions in every cell line investigated to date The characteristics and functional significance of the HIF response in vivo are largely unknown High-amplification immunohistochemical analyses were used to study the expression of HIF-1alpha and -2alpha in kidneys of rats exposed to systemic hypoxia bleeding anemia, functional anemia (01% carbon monoxide), renal ischemia, or cobaltous chloride (which is known to mimic hypoxia) These treatments led to marked nuclear accumulation of HIF-1alpha and -2alpha in different renal cell populations HIF-1alpha was mainly induced in tubular cells, including proximal segments with exposure to anemia/carbon monoxide, in distal segments with cobaltous chloride treatment, and in connecting tubules and collecting ducts with all stimuli Staining for HIF-1alpha colocalized with inducible expression of the target genes heme oxygenase-1 and glucose transporter-1 HIF-2alpha was not expressed in tubular cells but was expressed in endothelial cells of a small subset of glomeruli and in peritubular endothelial cells and fibroblasts The kidney demonstrates a marked potential for upregulation of HIF, but accumulation of HIF-1alpha and HIF-2alpha is selective with respect to cell type, kidney zone, and experimental conditions, with the expression patterns partly matching known oxygen profiles The expression of HIF-2alpha in peritubular fibroblasts suggests a role in erythropoietin regulation

Insect Cells as a Factory to Produce Adeno-Associated Virus Type 2 Vectors

Abstract: Recombinant adeno-associated viruses (rAAV) are produced transiently in mammalian cells usually by cotransfecting two or three plasmids containing AAV genes, adenovirus helper genes, and a vector genome. Expansion and transfection of adherent cells limit the scale of rAAV production. Efficient transfection is performed with cells on solid support media such as tissue culture plates. A large animal study or a human clinical trial may require 1015 particles of vector, depending on dose. To generate this quantity of rAAV by transfection, more than 1011 HEK293 cells may be needed, which would require about 5000 × 175 cm2 flasks. The ability to scale up rAAV production by these methods severely restricts the commercialization and use of AAV vectors. A recombinant baculovirus derived from the Autographa californica nuclear polyhedrosis virus is widely employed for large-scale production of heterologous proteins in cultured insect cells and may provide an attractive alternative. Toward this goal, we have explore...

The multidrug resistance transporter ABCG2 (breast cancer resistance protein 1) effluxes Hoechst 33342 and is overexpressed in hematopoietic stem cells.

Abstract: The human ATP-binding cassette superfamily G (White) member 2 (ABCG2) gene and its murine homologue breast cancer resistance protein 1 (Bcrp1) are recently described ATP-binding cassette transporters associated with drug resistance in tumor cell lines, including the MCF-7 cell line, selected for its resistance to mitoxantrone (MCF-7/MitoR). Infection of MCF-7 cells with the retroviral vector containing ABCG2 cDNA (G1-ABCG2) resulted in cells (MCF-7/ABCG2) that were resistant to mitoxantrone at levels similar to those observed in MCF-7/MitoR cells. Previous studies have shown that pluripotent hematopoietic stem cells overexpress the multidrug-resistant transport (MDR1) gene and efflux rhodamine, a substrate for the MDR1 transporter. Other studies have identified a primitive hematopoietic stem cell population, or side population (SP) cells, which are identified by their efflux of the fluorescent dye, Hoechst 33342. In an attempt to identify the transport genes responsible for this phenotype, we examined the uptake of Hoechst 33342 into MCF-7, MCF-7/MitoR, and MCF-7 cells infected with a retroviral vector expressing the ABCG2 gene (MCF-7/ABCG2). MCF-7/MitoR cells as well as MCF-7/ABCG2 cells demonstrated lower levels of Hoechst 33342 uptake compared with the parental MCF-7 cells. We also examined the level of the mouse Bcrp1 RNA in SP cells and non-SP cells isolated from mouse hematopoietic cells. Mouse SP cells expressed relatively high levels of Bcrp1 mRNA relative to non-SP cells. These results suggest that Hoechst 33342 is a substrate for the ABCG2 transporter and that ABCG2/Bcrp1 expression may serve as a marker for hematopoietic stem cells in hematopoietic cells.

Amplification of PPM1D in human tumors abrogates p53 tumor-suppressor activity

Abstract: Expression of oncogenic Ras in primary human cells activates p53, thereby protecting cells from transformation. We show that in Ras-expressing IMR-90 cells, p53 is phosphorylated at Ser33 and Ser46 by the p38 mitogen-activated protein kinase (MAPK). Activity of p38 MAPK is regulated by the p53-inducible phosphatase PPM1D, creating a potential feedback loop. Expression of oncogenic Ras suppresses PPM1D mRNA induction, leaving p53 phosphorylated at Ser33 and Ser46 and in an active state. Retrovirus-mediated overexpression of PPM1D reduced p53 phosphorylation at these sites, abrogated Ras-induced apoptosis and partially rescued cells from cell-cycle arrest. Inactivation of p38 MAPK (the product of Mapk14) in vivo by gene targeting or by PPM1D overexpression expedited tumor formation after injection of mouse embryo fibroblasts (MEFs) expressing E1A+Ras into nude mice. The gene encoding PPM1D (PPM1D, at 17q22/q23) is amplified in human breast-tumor cell lines and in approximately 11% of primary breast tumors, most of which harbor wildtype p53. These findings suggest that inactivation of the p38 MAPK through PPM1D overexpression resulting from PPM1D amplification contributes to the development of human cancers by suppressing p53 activation.

Persistent and Transient Replication of Full-Length Hepatitis C Virus Genomes in Cell Culture

Abstract: The recently developed subgenomic hepatitis C virus (HCV) replicons were limited by the fact that the sequence encoding the structural proteins was missing. Therefore, important information about a possible influence of these proteins on replication and pathogenesis and about the mechanism of virus formation could not be obtained. Taking advantage of three cell culture-adaptive mutations that enhance RNA replication synergistically, we generated selectable full-length HCV genomes that amplify to high levels in the human hepatoma cell line Huh-7 and can be stably propagated for more than 6 months. The structural proteins are efficiently expressed, with the viral glycoproteins E1 and E2 forming heterodimers which are stable under nondenaturing conditions. No disulfide-linked glycoprotein aggregates were observed, suggesting that the envelope proteins fold productively. Electron microscopy studies indicate that cell lines harboring these full-length HCV RNAs contain lipid droplets. The majority of the core protein was found on the surfaces of these structures, whereas the glycoproteins appear to localize to the endoplasmic reticulum and cis-Golgi compartments. In agreement with this distribution, no endoglycosidase H-resistant forms of these proteins were detectable. In a search for the production of viral particles, we noticed that these cells release substantial amounts of nuclease-resistant HCV RNA-containing structures with a buoyant density of 1.04 to 1.1 g/ml in iodixanol gradients. The same observation was made in transient-replication assays using an authentic highly adapted full-length HCV genome that lacks heterologous sequences. However, the fact that comparable amounts of such RNA-containing structures were found in the supernatant of cells carrying subgenomic replicons demonstrates a nonspecific release independent of the presence of the structural proteins. These results suggest that Huh-7 cells lack host cell factors that are important for virus particle assembly and/or release.

Validation of FLT uptake as a measure of thymidine kinase-1 activity in A549 carcinoma cells

Abstract: The thymidine analog 18F-3′-deoxy-3′ -fluorothymidine (FLT) is being used clinically for PET imaging of tumor proliferation. Appropriate use of this tracer requires validating the mechanisms by which it accumulates in dividing cells. We tested the accuracy with which FLT uptake predicted the activity of cytosolic thymidine kinase-1 (TK1), an enzyme that is upregulated before and during DNA synthesis. Methods: Cultured A549 human lung carcinoma cells were manipulated to a range of proliferation rates from actively dividing to growth arrested. Uptake of radiolabeled FLT was compared with cell cycle activity, which was expressed as the percentage of cells in S phase, and with activity of cytosolic TK1. We also compared uptake of FLT and deoxyglucose. We genetically manipulated A549 cells by transfecting them with human papillomavirus type 16 E6 (designated A549-E6) to abrogate function of the tumor suppressor gene, p53. Although radiation typically inhibits progression of mammalian cells through the cell cycle, abrogation of p53 function eliminates this inhibition. We then compared FLT uptake with the percentage of cells in S phase and TK1 activity in irradiated A549-E6 cells and in irradiated control cells having normal p53 function and the expected radiation-induced growth delay. Results: A549 cells with only 3%–5% cells in S phase took up little FLT and had low levels of TK1 activity. When cells were stimulated to grow by being placed into fresh medium, we observed a strong correlation between increased FLT uptake and increased TK1 activity. As expected, FLT uptake varied much more as a function of growth than did uptake of deoxyglucose. Nonproliferating A549 cells did not enter the cell cycle if they were irradiated before being placed into fresh medium, and they did not accumulate FLT or show elevated TK1 activity. In contrast, radiation did not inhibit the cell cycle progression of A549-E6 cells. When subcultured, they began to grow and showed increased uptake of FLT commensurate with greater TK1 activity. Conclusion: In cultured A549 cells FLT uptake is positively correlated with cell growth and TK1 activity. Inhibition of cell cycle progression prevents FLT uptake and increased TK1 activity. These results suggest that FLT images reflect TK1 activity and the percentage of cells in S phase.

Isolation, immortalization, and characterization of a human breast epithelial cell line with stem cell properties

Abstract: The epithelial compartment of the human breast comprises two distinct lineages: the luminal epithelial and the myoepithelial lineage. We have shown previously that a subset of the luminal epithelial cells could convert to myoepithelial cells in culture signifying the possible existence of a progenitor cell. We therefore set out to identify and isolate the putative precursor in the luminal epithelial compartment. Using cell surface markers and immunomagnetic sorting, we isolated two luminal epithelial cell populations from primary cultures of reduction mammoplasties. The major population coexpresses sialomucin (MUC+) and epithelial-specific antigen (ESA+) whereas the minor population has a suprabasal position and expresses epithelial specific antigen but no sialomucin (MUC−/ESA+). Two cell lines were further established by transduction of the E6/E7 genes from human papilloma virus type 16. Both cell lines maintained a luminal epithelial phenotype as evidenced by expression of the tight junction proteins, claudin-1 and occludin, and by generation of a high transepithelial electrical resistance on semipermeable filters. Whereas in clonal cultures, the MUC+/ESA+ epithelial cell line was luminal epithelial restricted in its differentiation repertoire, the suprabasal-derived MUC−/ESA+ epithelial cell line was able to generate itself as well as MUC+/ESA+ epithelial cells and Thy-1+/α-smooth muscle actin+ (ASMA+) myoepithelial cells. The MUC−/ESA+ epithelial cell line further differed from the MUC+/ESA+ epithelial cell line by the expression of keratin K19, a feature of a subpopulation of epithelial cells in terminal duct lobular units in vivo. Within a reconstituted basement membrane, the MUC+/ESA+ epithelial cell line formed acinus-like spheres. In contrast, the MUC−/ESA+ epithelial cell line formed elaborate branching structures resembling uncultured terminal duct lobular units both by morphology and marker expression. Similar structures were obtained by inoculating the extracellular matrix-embedded cells subcutaneously in nude mice. Thus, MUC−/ESA+ epithelial cells within the luminal epithelial lineage may function as precursor cells of terminal duct lobular units in the human breast.

Stability, Cellular Uptake, Biotransformation, and Efflux of Tea Polyphenol (−)-Epigallocatechin-3-Gallate in HT-29 Human Colon Adenocarcinoma Cells

Abstract: The biological effects of (-)-epigallocatechin-3-gallate (EGCG) have been extensively investigated in cell lines, but its stability and interactions with cells under culture conditions are unclear. In the present study, the stability, uptake, biotransformation, and efflux of [(3)H]EGCG in HT-29 human colon adenocarcinoma cells were investigated. EGCG was unstable in McCoy's 5A culture media with a half-life of less than 30 min, and the half-life increased to 130 min in the presence of cells. The major oxidative products were theasinensin (M(r) 914) and another dimer with M(r) 884. Addition of EGCG (50 micro M) to cell culture media caused the production of H(2)O(2) (up to 25 micro M), and the amount was lower and gradually decreased in the presence of cells. The uptake of EGCG was concentration dependent and did not plateau, even at 640 micro M, suggesting a passive diffusion process. Approximately 75% of the [(3)H]EGCG was found in the cytoplasmic fraction when the cells were incubated with 0.5-20 micro M [(3)H]EGCG for 15 min. The membrane-associated radioactivity increased with time, apparently because of the binding of dimers to the membrane. The accumulation of [(3)H]EGCG in the cells was significantly higher at 4 degrees C than at 37 degrees C. Multidrug-resistant protein inhibitors, such as indomethacin and probenecid, effectively increased the accumulation of EGCG 4"-glucuronide and 4"-methyl EGCG in the cell. These results suggest that EGCG is metabolized in the cell and that the metabolites are pumped out by MRPs. The present study provides fundamental information on the stability, uptake, biotransformation, and efflux of EGCG under cell culture conditions and suggests the need for careful interpretation of related results on the biological activities of EGCG.

Cytochrome P450 expression in human hepatocytes and hepatoma cell lines: molecular mechanisms that determine lower expression in cultured cells.

Abstract: 1. Cultured hepatic cells have reduced cytochrome P450 (CYP) activities in comparison with human liver, but the mechanism(s) that underlies this circumstance is not clear. We investigated the causes of this low CYP activity by analysing the activity, protein, mRNA and heterologous nuclear RNA contents of the most important CYPs involved in drug metabolism (1A1, 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5) in cultured human hepatocytes, and in HepG2 and Mz-Hep-1 hepatoma cell lines. 2. After 24 h of culture, hepatocytes retained most of their CYP activities and protein contents, but the mRNA decreased 20-fold. However, the mRNA content of most CYPs in 24-h hepatocytes was still 400-fold higher than in hepatoma cells. When we examined the transcriptional activity of the CYP genes, this decreased during culture time in hepatocytes and it was poor in hepatoma cell lines. 3. We investigated the abundance of key hepatic transcription factors that govern CYP transcription (C/EBP-beta: LAP and LIP, HNF-3alpha, HNF-4alpha, RXR-alpha) and observed that the expression of some factors was altered in the hepatoma cells. 4. In conclusion, the loss of biotransformation activity in cultured hepatic cells is caused by a decrease in CYP transcription, which correlates with an alteration in the expression of key transcription factors.

Cellular transcriptional profiling in influenza A virus-infected lung epithelial cells: The role of the nonstructural NS1 protein in the evasion of the host innate defense and its potential contribution to pandemic influenza

Abstract: The NS1 protein of influenza A virus contributes to viral pathogenesis, primarily by enabling the virus to disarm the host cell type IFN defense system. We examined the downstream effects of NS1 protein expression during influenza A virus infection on global cellular mRNA levels by measuring expression of over 13,000 cellular genes in response to infection with wild-type and mutant viruses in human lung epithelial cells. Influenza A/PR/8/34 virus infection resulted in a significant induction of genes involved in the IFN pathway. Deletion of the viral NS1 gene increased the number and magnitude of expression of cellular genes implicated in the IFN, NF-κB, and other antiviral pathways. Interestingly, different IFN-induced genes showed different sensitivities to NS1-mediated inhibition of their expression. A recombinant virus with a C-terminal deletion in its NS1 gene induced an intermediate cellular mRNA expression pattern between wild-type and NS1 knockout viruses. Most significantly, a virus containing the 1918 pandemic NS1 gene was more efficient at blocking the expression of IFN-regulated genes than its parental influenza A/WSN/33 virus. Taken together, our results suggest that the cellular response to influenza A virus infection in human lung cells is significantly influenced by the sequence of the NS1 gene, demonstrating the importance of the NS1 protein in regulating the host cell response triggered by virus infection.

Activation of Protease-Activated Receptor (PAR)-1, PAR-2, and PAR-4 Stimulates IL-6, IL-8, and Prostaglandin E2 Release from Human Respiratory Epithelial Cells

Abstract: Epithelia from many tissues express protease-activated receptors (PARs) that play a major role in several different physiological processes In this study, we examined their capacity to modulate IL-6, IL-8, and PGE 2 production in both the A459 and BEAS-2B cell lines and primary human bronchial epithelial cells (HBECs) All three cell types expressed PAR-1, PAR-2, PAR-3, and PAR-4, as judged by RT-PCR and immunocytochemistry Agonist peptides corresponding to the nascent N termini of PAR-1, PAR-2, and PAR-4 induced the release of cytokines from A549, BEAS-2B, and HBECs with a rank order of potency of PAR-2 > PAR-4 > PAR-1 at 400 μM PAR-1, PAR-2, and PAR-4 also caused the release of PGE 2 from A549 and HBECs The PAR-3 agonist peptide was inactive in all systems tested PAR-1, PAR-2, or PAR-4, in combination, caused additive IL-6 release, but only the PAR-1 and PAR-2 combination resulted in an additive IL-8 response PAR peptide-induced responses were accompanied by changes in intracellular calcium ion concentrations However, Ca 2+ ion shutoff was ∼2-fold slower with PAR-4 than with PAR-1 or PAR-2, suggesting differential G protein coupling Combined, these data suggest an important role for PAR in the modulation of inflammation in the lung