Identification and characterization of a human cell line with dendritic cell features

CORP-4 is a cell line obtained in our laboratory from an explanted human bladder carcinoma. This cell line shows certain dendritic cell features such as adherence to the culture plate surface, a doubling time of 24 h and an enzymatic profile typical of cells involved in antigen presentation (non-specific esterases, lysozyme and alpha-1-antitrypsin). Its phenotypic analysis revealed CD 15 and Fc receptor expression, S-100 surface protein and the presence of positive reactivity to different lectins such as Concanavalin A (Con A) and Peanut agglutinin (PNA). CORP-4 was found to be a non-phagocytic cell line after it was assayed with latex, and FcR- and C3bR-mediated phagocytosis. Furthermore, CORP-4 produced interleukin-1 (IL-1) as determined by thymocyte proliferation assays and also fixes immune complexes in a non-complement dependent fashion. HLA class I and class II antigens were inducible by both 5 azacytidine and gamma interferon.     

Calcium in epithelial cell contraction

Epithelial morphogenesis in many organs involves asymmetric microfilament-mediated cellular contractions. Similar contractions, in terms of ultrastructure and cytochalasin B sensitivity, can be induced in the carcinoma cell line C-4II in culture. This line was used to compare total intracellular calcium levels ([Ca]i) in contracted monolayer fragments and in control cultures, and to determine whether epithelial cell contraction depends on influx of extracellular Ca. [Ca]i, defined as Ca not displaceable by lanthanum, was measured by atomic absorption spectrophotometry. Degrees of contraction were determined from shape changes of monolayer fragments. Detachment from the growth surface initiated cellular contractions and caused an immediate increase in [Ca]i, from 1.0 to 4.0-5.0 micrograms Ca/mg protein in early confluent cultures, and from 0.3 to 1.0-2.0 micrograms Ca/mg protein in crowded cultures. This increase was followed by a gradual decline in [Ca]i, though Ca levels remained higher than in controls and contraction progressed for 30 min. Contraction was inhibited completely by cold (7 degrees C) and by Ca-free medium, and in a dose-dependent manner by papaverine (2.5 x 10(-6) M-2.5 x 10(-4) M), lanthanum (1.0 x 10(-6) M-1.0 x 10(-4) M); and D-600 (1.0-2.0 x 10(- 4) M). The Ca ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 had no effect at 5.0 x 10(-6) M and was inhibitory at higher concentrations. The results provided direct evidence for increased [Ca]i in contracting epithelial cells, and suggest that Ca influx is required for such contraction to take place.     

Apoptotic events induced by synthetic naphthylchalcones in human acute leukemia cell lines

Acute leukemia is a disorder of the hematopoietic system characterized by the expansion of a clonal population of cells blocked from differentiating into mature cells. Recent studies have shown that chalcones and their derivatives induce apoptosis in different cell lines. Since new compounds with biological activity are needed, the aim of this study was to evaluate the cytotoxic effect of three synthetic chalcones, derived from 1-naphthaldehyde and 2-naphthaldehyde, on human acute myeloid leukemia K562 cells and on human acute lymphoblastic leukemia Jurkat cells. Based on the results, the most cytotoxic compound (A1) was chosen for further analysis in six human acute leukemia cells and in a human colon adenocarcinoma cell line (HT-29). Chalcone A1 significantly reduced the cell viability of K562, Jurkat, Kasumi, U937, CEM and NB4 cells in a concentration and time-dependent manner when compared with the control group (IC₅₀ values between ∼1.5 μM and 40 μM). It was also cytotoxic to HL-29 cells. To further examine its effect on normal cells, peripheral blood lymphocytes collected from healthy volunteers were incubated with the compound. It has also been incubated with human fibroblasts cultured from bone marrow (JMA). Chalcone A1 is non-cytotoxic to PBL cells and to JMA cells. A1 caused significant cell cycle arrest in all phases according to the cell line, and increased the proportion of cells in the sub G0/G1 phase. To evaluate whether this chalcone induced cell death via an apoptotic or necrotic pathway, cell morphology was examined using fluorescence microscopy. Cells treated with A1 at IC₅₀ demonstrated the morphological characteristic of apoptosis, such as chromatin condensation and formation of apoptotic bodies. Apoptosis was confirmed by externalization of phosphatidylserine, which was detected by the Annexin V-FITC method, and by DNA fragmentation. The results suggest that chalcone A1 has potential as a new lead compound for cancer therapy.     

Use of carbohydrate probes in conjunction with fluorescence-activated cell sorting to select mutant cell lines of Chlamydomonas with defects in cell surface glycoproteins

Two carbohydrate-binding probes (the lectin concanavalin A and the anti-carbohydrate monoclonal antibody FMG-1) have been utilized in conjunction with fluorescence-activated cell sorting to select cell lines of Chlamydomonas reinhardtii that contain defects in cell surface-exposed glycoproteins. Two very different selection strategies (sorting cells with the lowest binding for the FMG-1 monoclonal antibody or the highest binding of concanavalin A) yield a class of mutant cells that exhibit a total lack of binding of the monoclonal antibody to cell wall and plasma membrane glycoproteins along with an increased affinity for concanavalin A. Detailed characterization of one such mutant cell line, designated L-23, is provided. The subtle glycosylation defect exhibited by this cell line does not alter the ability of the affected glycoproteins to be targeted to the flagellar membrane and does not affect the expression of flagellar surface motility, a phenomenon that appears to involve the major concanavalin A-binding glycoprotein of the flagellar membrane. This approach has general applicability for dissecting the role of carbohydrate epitopes in the targeting and function of any cell surface glycoprotein for which suitable carbohydrate probes are available.     

A novel adamantane thiadiazole derivative induces mitochondria-mediated apoptosis in lung carcinoma cell line

The interaction of organic compounds with apoptosis regulatory proteins is an attractive field of research because of its relevance in the development of new chemotherapeutic agents for cancer treatment. Our group designed four new adamantane thiadiazole derivatives (ATDs). The four ATDs were theoretically tested for their binding affinities to a model of an apoptosis inhibitor protein using molecular modeling. ATD-4 which interacted with the highest binding affinity was synthesized and characterized. The in vitro cytotoxicity of ATD-4 against different cancer cell lines as well as normal cell line was determined and compared with 5-fluorouracil as a standard positive control. The lung carcinoma cell line that showed the highest cytotoxic activity due to ATD-4 treatment was chosen to further study if ATD-4 can perform its cytotoxic activity through the induction of apoptosis as expected from molecular modeling. Inducing apoptosis by ATD-4 in lung carcinoma cell line was assessed by various biochemical and morphological characteristics. Biochemically: The effect of ATD-4 on cell cycle and its ability to induce apoptosis were checked through flow cytometry. Caspase-3 activity was detected by a colorimetric method. Real time-polymerase chain reaction (q-PCR) was used to detect p53, caspase-3, bcl-2 and bax gene expression. Morphologically: Changes in cell surface morphology, granulation and average surface roughness were detected using atomic force microscopy (AFM). Cell shrinkage, increase in cytoplasmic organelles, changes in mitochondrial number and morphology, chromatin condensation, membrane blebbing and formation of apoptotic bodies were detected using transmission electron microscopy (TEM). The obtained results suggest that ATD-4 exerted its antitumor activity against A549 cells through the induction of the intrinsic (mitochondrial) apoptotic pathway.     

Induction of class I and class II MHC antigen expression on murine bone marrow-derived macrophages by IL-4 (B cell stimulatory factor 1)

We have studied the effects of IL-4 (B cell stimulatory factor 1) on the expression of MHC gene products in normal bone marrow-derived macrophages, peritoneal macrophages, and the myelomonocytic cell line WEHI-3. Using both IL-4-containing T cell supernatant and rIL-4, we have observed significant induction of both class I and class II MHC surface expression (about 1.5- to 4-fold increase) in 2-, 3-, and 4-day cultures of bone marrow-derived macrophages. This induction was also apparent at the mRNA level as assessed by Northern blot analysis using A beta, E alpha, and class I probes. Kinetic analysis revealed that induction of class II mRNA by IL-4 was slower than induction by IFN-gamma, requiring 48 h before a significant increase was noted. The magnitude of MHC induction by IL-4 was not as great as that seen with IFN-gamma, which was found to increase surface expression of MHC antigens two- to eightfold. IL-4 also differs from IFN-gamma in the repertoire of macrophages responsive to it. IL-4 was unable to induce class I or class II expression in either thioglycolate-elicited peritoneal macrophages or WEHI-3 cells whereas IFN-gamma induced MHC antigen expression on both cell types under the same conditions. These data demonstrate that IL-4 is capable of inducing both class I and class II MHC gene products in some, but not all, macrophages.     

Pluripotency of bovine embryonic cell line derived from precompacting embryos

We report herein the establishment of three bovine pluripotent embryonic cell lines derived from 8-16-cell precompacting embryos. Two cell lines were cultured for 10 passages and underwent spontaneous differentiation. One cell line (Z2) has been cultured continuously for over 3 years and has remained undifferentiated. These cells express cell surface markers that have been used routinely to characterize embryonic stem (ES) and embryonic germ (EG) cells in other species such as stage-specific embryonic antigens SSEA-1, SSEA-3, and SSEA-4, and c-Kit receptor. In the absence of a feeder layer, these cells differentiated into a variety of cell types and formed embryoid bodies (EBs). When cultured for an extended period of time, EBs differentiated into derivatives of three EG layers - mesoderm, ectoderm, and endoderm - which were characterized by detection of specific cell surface markers. Our results indicate that the Z2 cell line is pluripotent and resembles an ES cell line. To our knowledge, this is the first bovine embryonic cell line that has remained pluripotent in culture for more than 150 passages.     

Differential glycosylation requirements for the cell surface expression of class I molecules

The importance of asparagine-linked glycosylation in the cell surface expression of several class I molecules was examined. C57BL/6 (B6) T cell blasts were treated with tunicamycin (TM), an antibiotic that inhibits N-linked glycosylation. The levels of various class I molecules on these cells were examined by flow cytometry and were compared to the levels of the same molecules on untreated cells. A 12-hr TM treatment did not significantly alter the levels of H-2Kb, Db, or Qa-2; however, such treatment decreased the surface expression of the Qa-1b allelic product to undetectable levels. A time-course study indicated that a decrease in the level of Qa-1.2 expression was apparent after only 4 hr of TM treatment. An examination of T cell blasts prepared from mouse strains possessing the Qa-1a, Qa-1c, and Qa-1d alleles indicated that all allelic products of this locus demonstrated a marked decrease in cell surface expression on TM treatment, whereas other class I molecules (H-2Ks, TL) exhibited slight or no decrease. Two-dimensional polyacrylamide gel electrophoresis analysis of immunoprecipitates from detergent lysates of surface-iodinated TM-treated B6 blasts revealed the presence of the unglycosylated form of the H-2Kb molecule on the cell surface. No such form of the Qa-1.2 molecule could be detected by similar analysis. To establish that the above observations were not simply a result of the inability of the Qa-1-specific alloantisera to react with the unglycosylated Qa-1 molecule, lysates of surface-iodinated B6 blasts were digested with endoglycosidase F, which cleaves N-linked carbohydrate moieties. Immunoprecipitation analysis indicated that the antisera could react with the unglycosylated form of the Qa-1 molecule. These results indicate that N-linked glycosylation has differential importance in the cell surface expression of class I molecules.     

Role of the extracellular domain of human herpesvirus 7 glycoprotein B in virus binding to cell surface heparan sulfate proteoglycans.

In an attempt to identify the human herpesvirus 7 (HHV-7) envelope protein(s) involved in cell surface binding, the extracellular domain of the HHV-7 glycoprotein B (gB) homolog protein was cloned and expressed as a fusion product with the Fc domain of human immunoglobulin G heavy chain gamma1 (gB-Fc) in an eukaryotic cell system. Indirect immunofluorescence followed by flow cytometric analysis revealed specific binding of gB-Fc to the membrane of SupT1 cells but not to other CD4+ T-lymphoblastoid cell lines, such as Jurkat or PM1, clearly indicating that gB-Fc did not bind to the CD4 molecule. This was also suggested by the ability of gB-Fc to bind to CD4-negative fibroblastoid Chinese hamster ovary (CHO) cells. The binding was abrogated by enzymatic removal of cell surface heparan sulfate proteoglycans by heparinase and heparitinase but not by treatment with condroitinase ABC. In addition, binding of the gB-Fc fusion protein to CHO cells was severely impaired in the presence of soluble heparin, as well as when heparan sulfate-deficient mutant CHO cells were used. Consistent with these findings, soluble heparin was found to block HHV-7 infection and syncytium formation in the SupT1 cell line. Although the CD4 antigen is a critical component of the receptor for the T-lymphotropic HHV-7, these findings suggest that heparin-like molecules also play an important role in HHV-7-cell surface interactions required for infection and that gB represents one of the HHV-7 envelope proteins involved in the adsorption of virus-to-cell surface proteoglycans.     

Establishment and characterization of a novel ovarian clear cell carcinoma cell line,TU-OC-2, with loss of ARID1A expression

A new cell line of human ovarian clear cell carcinoma (CCC), TU-OC-2, was established and characterized. The cells were polygonal in shape, grew in monolayers without contact inhibition and were arranged in islands like pieces of a jigsaw puzzle. The chromosome numbers ranged from 41 to 96. A low rate of proliferation was observed and the doubling time was 37.5 h. The IC₅₀ values of cisplatin, 7-ethyl-10-hydroxycamptothecin (SN38), which is an active metabolite of camptothecin, and paclitaxel were 7.7 μM, 17.7 nM and 301 nM, respectively. The drug sensitivity assay indicated that TU-OC-2 was sensitive to SN38, but resistant to cisplatin and paclitaxel. Mutational analysis revealed that TU-OC-2 cells have no mutations of PIK3CA in exons 9 and 20 and of TP53 in exons 4–9. We observed the loss of ARID1A protein expression in TU-OC-2 cells by western blot analysis and in the original tumor tissue by immunohistochemistry. This cell line may be useful for studying the chemoresistant mechanisms of CCC and exploring novel therapeutic targets such as the ARID1A-related signaling pathway.     

Establishment and characterization of a malignant lymphoid cell line from a chronic lymphocytic leukemia patient

A long-term culture Epstein-Barr virus (EBV)-negative malignant lymphoid cell line (NAK) was established from a lymph node biopsy of a chronic lymphocytic leukemia patient. This cell line is of particular interest because it grows as an adherent cell line and depends on the presence of autologous conditioned medium for growth. After 6 months of growth in vitro, doubling time and cell cycle parameters were derived. Doubling time was 48 hours with over 45% cycling cells. Cell viability was over 90%. Expression of B-cell markers (CD19 and CD20) and surface immunoglobulin of the original tumor cell biopsy were roughly the same as in passage 14 (3 months in culture), including the expression of the original patient idiotype and IgM-lambda. Furthermore, binding of antiidiotypic antibodies was only slightly decreased at passage 14. Cytogenetic studies of chromosomal abnormalities in the primary tumor tissue and in later passages indicated similar abnormalities, with no translocations t(8;14), t(14;22), or t(2;8). However, frequent trisomies, deletions, and t(1;4) translocations were observed. Negative results for EBV nuclear antigen indicate that this cell line is an EBV-negative cell line.     

Fed-batch bioreactor performance and cell line stability evaluation of the artificial chromosome expression technology expressing an IgG1 in Chinese hamster ovary cells

The artificial chromosome expression (ACE) technology system uses an engineered artificial chromosome containing multiple site-specific recombination acceptor sites for the rapid and efficient construction of stable cell lines. The construction of Chinese hamster ovary(CHO) cell lines expressing an IgG1 monoclonal antibody (MAb) using the ACE system has been previously described (Kennard et al., Biotechnol Bioeng. 2009;104:540-553). To further demonstrate the manufacturing feasibility of the ACE system, four CHO cell lines expressing the human IgG1 MAb 4A1 were evaluated in batch and fed-batch shake flasks and in a 2-L fed-batch bioreactor. The batch shake flasks achieved titers between 0.7 and 1.1 g/L, whereas the fed-batch shake flask process improved titers to 2.5–3.0 g/L. The lead 4A1 ACE cell line achieved titers of 4.0 g/L with an average specific productivity of 40 pg/(cell day) when cultured in a non optimized 2-L fed-batch bioreactor using a completely chemically defined process. Generational stability characterization of the lead 4A1-expressing cell line demonstrated that the cell line was stable for up to 75 days in culture. Product quality attributes of the 4A1 MAb produced by the ACE system during the stability evaluation period were unchanged and also comparable to existing expression technologies such as the CHO-dhfr system. The results of this evaluation demonstrate that a clonal, stable MAb-expressing CHO cell line can be produced using ACE technology that performs competitively using a chemically defined fed-batch bioreactor process with comparable product quality attributes to cell lines generated by existing technologies.     

In vitro activities of novel 4-HPR derivatives on a panel of rhabdoid and other tumor cell lines

Background

Natural killer (NK) cells are an important resource of the innate immune system directly involved in the spontaneous recognition and lysis of virus-infected and tumor cells. An exquisite balance of inhibitory and activating receptors tightly controls the NK cell activity. At present, one of the best-characterized activating receptors is NKG2D, which promotes the NK-mediated lysis of target cells by binding to a family of cell surface ligands encoded by the MHC class I chain-related (MIC) genes, among others. The goal of this study was to describe the expression pattern of MICA and MICB at the molecular and cellular levels in human cervical cancer cell lines infected or not with human papillomavirus, as well as in a non-tumorigenic keratinocyte cell line.

Results

Here we show that MICA and MICB exhibit differential expression patterns among HPV-infected (SiHa and HeLa) and non-infected cell lines (C33-A, a tumor cell line, and HaCaT, an immortalized keratinocyte cell line). Cell surface expression of MICA was higher than cell surface expression of MICB in the HPV-positive cell lines; in contrast, HPV-negative cells expressed lower levels of MICA. Interestingly, the MICA levels observed in C33-A cells were overcome by significantly higher MICB expression. Also, all cell lines released higher amounts of soluble MICB than of soluble MICA into the cell culture supernatant, although this was most pronounced in C33-A cells. Additionally, Real-Time PCR analysis demonstrated that MICA was strongly upregulated after genotoxic stress.

Conclusions

This study provides evidence that even when MICA and MICB share a high degree of homology at both genomic and protein levels, differential regulation of their expression and cell surface appearance might be occurring in cervical cancer-derived cells.     

A172 and T98G cell lines characteristics

During prolonged cultivation, cell lines may lose a number of innate characteristics or acquire new ones. In this work, we compared growth and phenotypic characteristics of human glioblastoma А172 and Т98G cell lines received from the cell culture collection of the Research Institute of Influenza (St. Petersburg, Russia). The activity of genes encoding intracellular proteins that define belonging of these cell lines to mesenchymal type, as well as activity of several growth factor genes and extracellular matrix genes was evaluated. Cell lines A172 and T98G varied in morphology and surface markers expression. High level of mesenchymal markers CD90 and CD105, fibroblast activation protein, and tenascin C was detected for A172 cell line. Both cell lines expressed high level of α2 smooth muscle actin gene. Data demonstrating high activity of genes encoding major angiogenesis inductors (VEGF, FGF2(b), TGFβ1) and thrombospondin-1 in cell lines under study are in agreement with published data. Reduction of fetal serum content in culture medium from 10 to 5% increased the number of cells with CD73 and CD105 surface antigens in both cell lines. A172 and T98G cell lines maintain the main features of glioblastomas and therefore can be used as research objects in investigation of this type of neoplasms.     

A newly established human acute lymphoblastic leukemia cell line with characteristics of the earliest B-cell maturation

A new human acute lymphoblastic leukemia (ALL) cell line, designated HBL-3, was established from the bone marrow of a patient with non-T-ALL. The HBL-3 cell line expressed B4 (CD 19), BA-1 (CD 24) and HLA-DR antigens, but not surface immunoglobulin (SIg) or cytoplasmic immunoglobulin (CIg). The cell line lacked the common acute lymphoblastic leukemia antigen (CALLA) and antigenic markers characteristic of T-cell and myeloid cell lineages. The HBL-3 cells had structural rearrangements of both the homologous chromosome 9s, including a translocation with chromosome 1 which has been reported in a patient with common ALL. The cell line had rearranged immunoglobulin heavy chain genes but retained germ-line κ light chain genes and germ-line T-cell receptorβ- and γ-chain genes. The HBL-3 cell line was strongly positive for terminal deoxynucleotidyl transferase (TdT). These findings indicate that the HBL-3 cell line is derived from the earliest B-cell committed to B-cell lineage.     

A novel ganglioside expressed by mouse hematopoietic cell lines.

Mouse progenitor T cell-derived cell lines were established by fusion of cells of hematopoietic organs such as bone marrow and fetal liver with T lymphoma (BW5147) to determine their characteristic cell-surface components. The hybridomas with the phenotype of Thy-1+, CD3-, CD4-, CD8- and expression of T cell receptor gene mRNA (BM216 and FL339) were selected for progenitor T cell-derived cell lines, and their ganglioside compositions were studied. A ganglioside component with a mobility slightly faster than that of bovine brain GD1a on high-performance thin-layer chromatography was found in the cell extracts of these cell lines as one of the most abundant components and was absent in the extract of the parental cell line (BW5147). The structure of the ganglioside was determined to be: NeuAc alpha-Gal beta-Gal beta-Gal alpha-Gal beta-Glc beta-ceramide. Gangliosides with such a sequence have never been found before, suggesting the possibility that the ganglioside is expressed as a surface marker of the cells in hematopoietic organs committed to a specific cell lineage, presumably to T cell lineage. cells in hematopoietic organs committed to a specific     

Decreased transforming growth factor-beta response and binding in insulin-independent teratoma-derived cell lines with increased tumorigenic properties.

The mouse C3H teratoma-derived cell line 1246 is an adipogenic cell line which stringently requires insulin to proliferate and differentiate in defined medium. From this cell line an insulin-independent cell line called 1246-3A was isolated. It was found that, in contrast to 1246 cells, 1246-3A cells had lost the ability to differentiate and became tumorigenic when injected at a density of 10(6) cells/mouse into syngeneic host C3H mice. In addition, they produce in their culture medium transforming growth factor alpha- and beta-like polypeptides which stimulate their proliferation. Highly tumorigenic insulin-independent cell lines able to give rise to tumor when injected at a density of 10(4) cells/mouse were isolated by using an in vitro-in vivo shuttle technique. The highly tumorigenic cell lines have lost the response to TGF-beta 1. The binding of TGF-beta 1 to the nontumorigenic parent cell line or to cells displaying increased tumorigenic properties was investigated. The data presented here indicate that the increased tumorigenicity is accompanied by a progressive decrease of specific binding of TGF-beta 1 to the cells. However, the decreased number of cell surface TGF-beta 1 binding sites in the highly tumorigenic cells did not correlate with an increase in the secretion of TGF-beta protein by the tumorigenic cells, as all of TGF-beta produced by the cells was in a latent form. Affinity cross-linking experiments indicated that the 1246 cell line displayed several TGF-beta cross-linked molecular species (MW 140, 92, and 70 kDa). Increase of tumorigenicity was accompanied by a marked decrease in the intensity of the cross-linked bands, particularly of the 92 and 70 kDa species.     

A novel, carbohydrate signal-mediated cell surface protein phosphorylation: ganglioside GQ1b stimulates ecto-protein kinase activity on the cell surface of a human neuroblastoma cell line, GOTO

A ganglioside-stimulated ecto-type protein phosphorylation system (ecto-Gg-kinase) was detected on the cell surface of a human neuroblastoma cell line (GOTO). When intact cells were incubated with [gamma-32P]ATP, at least 28 cell surface proteins were phosphorylated, as evident on SDS-PAGE (4-20%) analysis. Exogenously added gangliosides specifically stimulated the phosphorylation of at least three cell surface associated proteins of Mr = 64,000, 60,000, and 54,000. Phosphorylation was directed toward Thr and Ser residues, respectively, as revealed on acid hydrolysis followed by electrophoresis. GQ1b, at 5 nM, was the most potent among the several gangliosides tested and was more effective when added to cells before [gamma-32P]ATP administration. The simultaneous addition of an excess amount of the saccharide portion of GQ1b (oligo-GQ1b) inhibited the GQ1b-stimulated phosphorylation, indicating the necessity of the sialosaccharide moiety. These results strongly suggest that phosphorylation of the three proteins may be closely associated with the highly specific neuritogenic effect of GQ1b previously reported.