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At least 145 records · Page 8Linked to original sources
PubMed1993

Characterization of peanut-lectin (+) cells derived from the RCCT-28A cell line.

Cells from RCCT-28A cell line, which exhibited the highest peanut-lectin binding capacity [RCCT-28A(P+)] were separated and cultured attempting to obtain a population of HCO3- secreting cells. The mechanisms of apical solution acid or base extrusion were studied in confluent monolayers. In RCCT-28A (P+) cells net H+ flux (JH+, nmol.min-1.cm-2) was significantly lower (JH+ = 13 +/- 3) than that observed in the RCCT-28A cells (21 +/- 2). N-ethylmaleimide, Bafilomycin-A, omeprazole and Schering 28080 decreased JH+. Cl- removal from the basolateral side, in the presence or absence of SCH in the apical side, decreased JH+. Removal of apical Cl- abolished apical extrusion of base-equivalents. Incubation in a high bicarbonate solution results in base-equivalent flux to the apical side (JH+ = -7.0 +/- 1.0). Preincubation in a hyperosmotic medium (mannitol addition) at pH 7.5 decreased JH+ to -3.0 +/- 1.0. We conclude that, functionally, RCCT-28A(P+) cells are only quantitatively different from RCCT-28A cells. Acid secretion by these cells can be modulated by alkaline and by hyperosmolar preincubation and is most likely mediated by coexistent of apical H(+)-ATPase and H+,K(+)-ATPase. The main mechanism of net OH- secretion appears to be Cl-/HCO3- exchange at the apical side.

Acid-Base Equilibrium↗
PubMed2001

Use of helper-free replication-defective simian immunodeficiency virus-based vectors to study macrophage and T tropism: evidence for distinct levels of restriction in primary macrophages and a T-cell line.

Cell tropism of human and simian immunodeficiency viruses (HIV and SIV, respectively) is governed in part by interactions between the viral envelope protein and the cellular receptors. However, there is evidence that envelope-host cell interactions also affect postentry steps in viral replication. We used a helper-free replication-defective SIV macaque (SIVmac)-based retroviral vector carrying the enhanced jellyfish green fluorescent protein inserted into the nef region (V1EGFP) to examine SIV tropism in a single cycle of infection. Vector stocks containing envelope proteins from three different SIVmac clones, namely, SIVmac239 (T-lymphocyte tropic [T-tropic]), SIVmac316 (macrophage tropic [M-tropic]), and SIVmac1A11 (dualtropic), were tested. SIVmac239 replicates efficiently in many human T-cell lines, but it does not efficiently infect primary rhesus macrophages. Conversely, SIVmac316 efficiently infects primary macrophages, but it does not replicate in Molt4-Clone8 (M4C8) T cells. SIVmac1A11 replicates efficiently in both cell types. When primary macrophages were infected with V1EGFP pseudotyped by SIVmac316 or SIVmac1A11 envelopes, the infection was substantially (ca. 200- to 300-fold) more efficient than for the SIVmac239 pseudotype. Thus, in primary macrophages, a major component of M versus T tropism involves relatively early events in the infection cycle. Quantitative PCR studies indicated that synthesis and transport of vector DNA into the nucleus were similar for macrophages infected with the clone 239 and 316 pseudotypes, suggesting that the restriction for SIVmac239 infection is after reverse transcription and nuclear import of viral DNA. When the same vector pseudotypes were used to infect M4C8 cells, they all showed approximately equivalent infectivities, even though replication-competent SIVmac316 does not continue to replicate in these cells. Therefore, in M4C8 cells, restriction involves a late step in the infection cycle (after proviral integration and expression). Thus, depending on the cell type infected, envelope-dependent cell interactions that govern SIV M and T tropism may involve different steps in infection.

Animals↗
PubMed1998

Cellular characterization of pituitary adenoma cell line (AtT20 cell) transfected with insulin, glucose transporter type 2 (GLUT2) and glucokinase genes: insulin secretion in response to physiological concentrations of glucose.

We investigated the mechanisms of insulin secretion by transfecting into a pituitary adenoma cell line (AtT20) a combination of genes encoding human insulin (HI), glucose transporter type 2 (GLUT2) and glucokinase (GK), followed by studying the characteristics of these cells. In static incubation, a cell line transfected with insulin gene alone (AtT20HI) secreted mature human insulin but this was not in a glucose-dependent manner. Other cell lines transfected with insulin and GLUT2 genes (AtT20HI-GLUT2-3) or with insulin and GK genes (AtT20HI-GK-1) secreted insulin in response to glucose concentrations of only less than 1 mmol/l. In contrast, cell lines transfected with insulin, GLUT2 and GK genes (AtT20HI-GLUT2-GK-6, AtT20HI-GLUT2-GK-7 and AtT20HI-GLUT2-GK-10) showed a glucose-dependent insulin secretion up to 25 mmol/l glucose. Glucose utilization and oxidation were increased in AtT20HI-GLUT2-GK cell lines but not in AtT20HI, AtT20HI-GLUT2-3 and AtT20HI-GK-1 cells at physiological glucose concentrations, compared with AtT20 cells. Diazoxide, nifedipine and 2-deoxy glucose suppressed (p < 0.05) glucose stimulated insulin secretion in AtT20HI-GLUT2-GK-6 cells. Glibenclamide, KCl or corticotropin releasing factor (CRF) stimulated (p < 0.05) insulin secretion both in AtT20HI and AtT20HI-GLUT2-GK-6 cells. Insulin secretion stimulated by glibenclamide, KCl or CRF was further enhanced by the addition of 25 mmol/l glucose in AtT20HI-GLUT2-GK-6 cells but not in AtT20HI cells. In perifusion experiments, a stepwise increase in glucose concentration from 5 to 25 mmol/l stimulated insulin secretion in AtT20HI-GLUT2-GK cell lines but the response lacked a clear first phase of insulin secretion. Our results suggest that both GLUT2 and glucokinase are necessary for the glucose stimulated insulin secretion in at least rodent cell lines, and that other element(s) are necessary for a biphasic insulin secretion typically observed in beta cells.

Adenoma↗
PubMed1978

Human cell membrane components bound to beta2-microglobulin in T cell-type cell lines.

Cell membrane components bound to beta2-microglobulin were isolated from Renex 30 (a nonionic detergent)-solubilized membrane materials of two human T cell-type cell lines, MOLT-4 and CCRF-CEM, by gel filtration and lectin affinity chromatography. The isolation was carried out by following the beta2-microglobulin activity by radioimmune inhibition assay. The T cell membrane components bound to beta2-microblogulin had a uniform molecular size of about 200,000 daltons and most of them showed an affinity to lentil lectin. The isolated membrane components were radioiodinated and examined for identity to HLA antigens by sequential precipitation with rabbit anti-HLA antiserum (specific to HLA large components) and with rabbit anti-beta2-microblogulin antiserum. In addition to HLA antigens, the beta2-microglobulin-bound components obtained from the MOLT-4 cells were found to contain certain membrane components that are the same in molecular size as the HLA large components but that are different antigenically from the HLA large components. On the other hand, the beta2-microglobulin-bound membrane components obtained from the CCRF-CEM cells were all HLA antigens. No other membrane components were involved in the binding.

Cell Line↗
PubMed1986

Presentation of Candida albicans and purified protein derivative soluble antigens by Epstein-Barr virus-transformed human lymphoblastoid B-cell lines.

Cells other than the macrophage can function as antigen-presenting cells (APCs). These class II-bearing accessory cells include dendritic cells, epidermal Langerhans cells, B cells, murine B-cell tumors, and human Epstein-Barr virus-transformed lymphoblastoid cell lines (EBV-LCL). We investigated the ability of EBV-LCL to present two soluble antigens, Candida albicans and purified protein derivative of tuberculin (PPD). The EBV-LCL derived from B cells of two different individuals can present both antigens to bulk cultures of autologous antigen-primed peripheral blood lymphocytes. The responses of PPD-reactive T-cell clones were weaker to PPD when presented by EBV-LCL than by PBL-APCs, with some clones responding only to PPD presented by PBL-APCs. This suggests that EBV-LCL are not equivalent to PBL monocytes in APC function, and that expression of class II major histocompatibility complex antigen is not sufficient in enabling antigen-presenting capability.

Antigen-Presenting Cells↗
PubMed1992

Characterization and comparison of two newly established Epstein-Barr virus (EBV)-negative and EBV-positive Burkitt's lymphoma cell lines. EBV-negative cell line with a low level of expression of ICAM-1 molecule and EBV-positive cell line with a high level of expression of ICAM-1 molecule.

Two human Burkitt's lymphoma cell lines (HBL-4 and HBL-5) were established individually from two patients with small noncleaved cell lymphoma (Burkitt's type). The HBL-4 cell line is Epstein-Barr virus (EBV)-negative, and the HBL-5 cell line is EBV-positive. Cytogenetically, both cell lines had the same chromosomal translocation, t(8;14)(q24;q32) as those observed in the primary malignant cells from individual patients. Morphologic, immunophenotypic, cytogenetic, and molecular studies confirmed that both cell lines were derived from the primary lymphoma cells in vivo. HBL-4 cells lacked CD23(H107), CD11a(LFA-1), and latent membrane protein (LMP) but expressed CD54(ICAM-1) at low levels, whereas HBL-5 cells showed the high level of expression of CD54 and faint expression of LMP but lacked CD11a. In addition, the EBV-positive lymphoblastoid cell line (LCL) expressed CD11a, CD23, CD54, and LMP at high levels. Therefore, an HBL-5 phenotype with expression of CD54 and LMP tends toward an LCL phenotype, and the augmentation of CD54 on the HBL-5 cells in comparison with primary lymphoma cells is likely to be upregulated by LMP, probably resulting from the EBV infection. There was little difference in the BrdUrd uptake in vivo and in vitro, doubling time, tumorigenicity, and dynamics of tumor growth in athymic nude mice between both cell lines. These findings indicate that the potentiality of cell growth and tumorigenicity of these two cell lines are unlikely to be related with EBV.

Adult↗
PubMedDate not supplied

Expression of HLA-DR molecules in human gestational choriocarcinoma cell lines and malignant cell lines.

In order to determine whether trophoblast (or gestational choriocarcinoma) expresses the HLA-DR antigen or not, it was analysed using human gestational choriocarcinoma cell lines (GCH-1, GCH-1(m) and TAK-N) by the Northern hybridization method. TYK-nu (human undifferentiated ovarian carcinoma cell line), M-14 (human malignant melanoma cell line), L-14 (human B lymphocyte cell line), and KKNS-1, KKNS-2 and KKNS-3 (three human endometrial carcinoma cell lines) were also examined. Messenger ribonucleic acids (mRNAs) were prepared from 2 X 10(7) cells in each cell line and hybridized by HLA-DR alpha-chain complementary deoxyribonucleic acid (cDNA) probe (pDR alpha-1) and beta-chain cDNA probe (DK-10). Northern hybridization analysis revealed that GCH-1 transcribed at least two kinds of alpha-chain (16s and 23s) and beta-chain mRNA (15s and 23s). TYK-nu transcribed alpha- and beta-chain mRNA, L-14 transcribed only alpha-chain mRNA, and TAK-N appeared to transcribe only a little alpha-chain mRNA. The HLA-DR molecules were not, however, expressed in the other cell lines (GCH-1(m), M-14, KKNS-1, -2 and -3). These results are compared with those obtained by immunocytochemical methods in our laboratory.

Cell Line↗
PubMed1985

A small phospholipase inhibitory factor released by cultured cell lines.

Cells of the mouse macrophage-like cell line RAW264 release a dialysable inhibitor of phospholipase activity into their culture medium. This inhibitor can be detected in saline solution, Hanks solution and a variety of tissue culture media in the presence or absence of serum. The inhibitor is stable at 4 degrees C, unaffected by trypsin, nucleases, or boiling, and partially extractable with chloroform/methanol. The release of both arachidonic acid and prostaglandins from mouse macrophages or human monocytes is inhibited by this material. A variety of other cell types release the inhibitor, which is effective against stimulation of arachidonic acid release from cultured macrophages by zymosan, serum, immune complexes and the calcium ionophore A23187.

Animals↗
PubMed1985

Cell bank characterization for recombinant DNA mammalian cell lines.

Cell banks were created by controlled rate freezing of pooled cell suspensions. Each line was characterized for freedom from adventitious agents, identity and biological behavior in immunosuppressed animals. Pharmacopeal culture methods were employed to search for bacterial, fungal and mycoplasma contamination. Conditioned medium was inoculated into a battery of 6 cell lines to search for acute viral contamination. Recombinant cell lines were also examined for retroviruses by electron microscopy, reverse transcriptase assay, XC plaque assay, tritiated uridine labelling, chemical induction techniques, and co-cultivation with three indicator cell lines. Further characterization was obtained from inoculation of 4 animal species in vivo, mouse antibody production test, and inoculation of immunosuppressed rodents. Finally, cytogenetic evaluation including banding methods and isoenzyme analysis were employed to verify cell bank identity. These methods have generated useful data concerning the identity, safety, and biological purity of recombinant DNA mammalian cell lines.

Animals↗
PubMed1986

Increased efficacy in selective elimination of leukemic cell line clonogenic cells by a combination of monoclonal antibodies BA-1, BA-2, BA-3 + complement and mafosfamid (ASTA Z 7557).

Studies were undertaken to examine the efficacy of combining monoclonal antibodies BA-1, BA-2, BA-3 + complement with a stabilized derivative of cyclophosphamide (mafosfamid) for elimination of leukemic cell line clonogenic cells. In a series of experiments conducted with leukemic cell lines a combination of two cycles of treatment with BA-1, 2, 3 + complement combined with 25 micrograms/ml-1 mafosfamid was superior to either modality alone in killing clonogenic leukemic cells. Identical treatment of normal bone marrow had no effect on the monopotent stem cells CFU-E and CFU-GM, or the pluripotent stem cell CFU-MIX, but some inhibition of the monopotent stem cell CFU-MK did occur. These results suggest that consideration be given to incorporation of BA-1, 2, 3 + complement and mafosfamid combination treatment in autologous bone marrow transplantation for acute lymphoblastic leukemia.

Antibodies, Monoclonal↗
PubMed1992

Secretion of gelatinases and tissue inhibitors of metalloproteinases by human lung cancer cell lines and revertant cell lines: not an invariant correlation with metastasis.

Numerous studies have reported a correlation between production of 72-kDa (MMP-2) and 92-kDa (MMP-9) type-IV collagenases/gelatinases and the metastatic potential of cancer cells. An abrogating effect of tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) on metastases has also been noted. In this report we have used sensitive enzyme-linked immunoassays to measure MMP-2, MMP-9, TIMP-1 and TIMP-2 levels in eight human lung-cancer cell lines which were characterized for biological behavior in nude mice. We demonstrated that the Calu-6 and A549 cell lines with the highest metastatic, invasive and tumorigenic potential secreted the highest levels of MMP-2. MMP-9 and TIMP-1 secretions were comparatively low in all cell lines. TIMP-2 secretion, which exceeded MMP-2 secretion for all cell lines, did not correlate with metastatic potential. To further explore these correlations, the metastatic Calu-6 cell line was transfected with a K-rev-1 cDNA expression construct. The K-rev revertant cell lines demonstrated a more differentiated phenotype and were less tumorigenic, invasive and metastatic in nude mice. Nonetheless, the Calu-6 revertant cell lines secreted higher levels of MMP-2 than the parent cell line. In conclusion, invasion and metastasis by lung-cancer cells requires not only enhanced MMP production, but also other less well-understood tumorigenic characteristics. The multiplicity of factors required by cancer cells for dissemination helps to explain the minute fraction of cancer cells from a primary tumor that ever develop into a metastasis.

Animals↗
PubMed1980

Characterization of lymphoma-derived cell lines: comparison of cell lines positive and negative for Epstein-Barr virus nuclear antigen. I. Physical, cytogenetic, and growth characteristics.

Sixteen lymphoid cell lines were derived from patients with undifferentiated lymphoma of Burkitt's or non-Burkitt's type. They were obtained directly from tumor biopsies, from serous effusions, or from bone marrow. In 10 of the cell lines, the Epstein-Barr virus (EBV) nuclear antigen (EBNA) was undetectable; the remaining 6 lines were EBNA-positive (EB-pos). Of the 16 lines, 15 were aneuploid, with detectable chromosome "14q+ markers (11 had +8;14 translocations). These 15 lines, which included the EBNA-negative (EB-neg) lines, were believed to be of tumor cell origin. The remaining line consisted predominantly of diploid cells derived from normal lymphocytes, but some cells of tumor origin were present. Four EB-pos cell lines derived from EB-neg tumors had an aneuploid karyotype consistent with an origin from tumor cells (including no.8;14 translocation in two), which suggested that either tumor cells were infected with EBV in vitro or a tiny fraction of EB-pos tumor cells (or potential tumor cells) present in vivo gave rise to the predominant cell of the line. EB-neg B-cell lines and EB-pos cell lines established from undifferentiated lymphomas differed greatly. EB-neg lines had consistently smaller electronic mean cell volumes and narrow-angle light scatter than did EB-pos lines. This finding correlated with a lower nuclear:cytoplasmic ratio in EB-pos lines. EB-neg lines also had higher saturation cell densities than did EB-pos lines under standard culture conditions. The data indicate either that EBV influences the morphologic and physiologic characteristics of lymphoid cell lines or that EB-neg B-cell lines and EB-pos cell lines are derived ultimately from different lymphocyte subpopulations or that both may apply.

Antigens, Viral↗
PubMed1996

Bcr/Abl expression stimulates integrin function in hematopoietic cell lines.

Cell adhesion to the extracellular matrix is largely mediated by adhesion molecules of the integrin family and is often diminished upon oncogenic transformation. However, we show here that the chronic myelogenous leukemia oncogene Bcr/Abl has positive effects on VLA-4 and VLA-5 integrin function. The presence of Bcr/Abl in the GM-CSF- or IL-3-dependent hematopoietic cell lines MO7e, 32D, and BaF/3 enhanced cell binding to both soluble and immobilized fibronectin. The effect was due to enhanced function of the VLA-5 integrin fibronectin receptor and not to increased surface expression. In parallel, Bcr/Abl stimulated cell adhesion to the VLA-4 integrin ligand VCAM-1. Stimulation of VLA-5 function directly correlated with induction of Bcr/Abl tyrosine kinase activity in a temperature-sensitive kinase mutant. Thus, Bcr/Abl stimulates integrin-dependent cell adhesion, by a mechanism involving increased ligand binding, with the tyrosine kinase activity of Bcr/Abl likely playing a key role. Consistent with these results, hematopoietic precursor cells from chronic myelogenous leukemia patients also showed increased adhesion to fibronectin.

Animals↗
PubMed1979

Surface adhesive properties of baboon lymphocyte and derived lymphoblastoid cell lines.

Cell surface adhesiveness of lymphocytes isolated from the peripheral blood of Papio hamadryas monkeys was compared to the adhesiveness of lymphoblastoid cell lines derived by transformation of these lymphocytes with herpesvirus Papio. For the quantitative comparison of the cell surface adhesive properties, a recently developed latex particle adherence assay was utilized. It was found that cell surface adhesiveness of the suspension lines was substantially lower than that of their precursor lymphocytes.

Adhesiveness↗
PubMed2005

Restoration of plakoglobin expression in bladder carcinoma cell lines suppresses cell migration and tumorigenic potential.

The reduction or loss of plakoglobin expression in late-stage bladder cancer has been correlated with poor survival where upregulation of this catenin member by histone deacetylase inhibitors has been shown to accompany tumour suppression in an in vivo model. In this study, we directly addressed the question of the role of plakoglobin in bladder tumorigenesis following restoration, or knockdown of expression in bladder carcinoma cell lines. Restoration of plakoglobin expression resulted in a reduction in migration and suppression of tumorigenic potential in vivo. Immunocytochemistry revealed cytoplasmic and membranous localisation of plakoglobin in transfectants with < 1% of cells displaying detectable nuclear localisation of plakoglobin. siRNA knockdown experiments targeting plakoglobin, revealed enhanced migration in all cell lines in the presence and absence of E-cadherin expression. In bladder cell lines expressing low levels of plakoglobin and desmoglein-2, elevated levels of desmoglein-2 were detected following restoration of plakoglobin expression in transfected cell lines. Analysis of wnt signalling revealed no activation event associated with plakoglobin expression in the bladder model. These results show that plakoglobin acts as a tumour suppressor gene in bladder carcinoma cells and the silencing of plakoglobin gene expression in late-stage bladder cancer is a primary event in tumour progression.

Animals↗
PubMed2001

Acute activation of gp130 gene expression in bone marrow stromal cells by contact with myeloma-derived lymphoblastic cell line ARH77 cell membranes.

Cell-cell contact of myeloma-derived cell lines (MDCL) or fresh myeloma cells with bone marrow stromal cells (BMSC) is known to induce interleukin-6 (IL-6) and matrix metalloproteinase-1 (MMP-1) production by a marrow stromal cell line. To determine if other BMSC transcripts are altered during cell-cell contact between BMSC and tumor cells, we have used cell lines ARH77 and U266 in an in vitro model. Using mRNA differential display and reverse transcriptase-polymerase chain reaction (RT-PCR), it was determined that a total of 141 transcripts were either upregulated or downregulated in the BMSC on contact with cell membrane from cell lines ARH77 and U266. Induction of two of these transcripts, interleukin-6 (IL-6) and gp130 in the BMSC by ARH77 cell membranes was studied in greater detail. Real-time PCR was used to quantitate transcript levels of gp130, IL-6, and 36b4, a housekeeping gene. Cycloheximide (CHX) alone increased both gp130 and IL-6 transcripts in the BMSC. In addition, CHX caused a superinduction of these transcripts in BMSC exposed to ARH77 cell membranes. The induction of gp130 was independent of the increase in IL-6 mRNA. Upregulation of gp130, a component of the membrane receptors for the IL-6 superfamily, can have profound effects on the response of BMSC to the IL-6 superfamily of cytokines.

Antigens, CD↗
PubMedDate not supplied

Stability of producer hybridoma cell lines after cell sorting: a case study.

Flow cytometry was used in combination with immunofluorescent staining for intracellular and surface-associated antibody contents to identify a significant non-producer cell fraction in a murine hybridoma cell line that had shown a decline in monoclonal antibody productivity with passaging. Viable producer cells stained for surface-associated antibody content were isolated by cell sorting on the basis of surface fluorescence intensity. The fraction of nonproducers was initially reduced from 85% to 20%. Sorting a second time, after these cells were cultivated for 2 weeks, further reduced the fraction of nonproducers to less than 4%. The stability of this purified producer hybridoma cell line during passaging and after a freeze-thaw cycle was investigated. This cell line was found to be highly unstable. A simple batch-growth model simulation was used as follows: (i) to demonstrate that nonproducers appear in hybridoma cell lines after a rare, random mutation event that results in the loss of the heavy-chain gene and/or light-chain gene expression; (ii) to show that a high rate of conversion of producer hybridomas to nonproducer hybridomas cannot entirely explain the population dynamics; (iii) to estimate the rate of conversion of producers to nonproducers to be 8.7 x 10(-5) h-1; (iv) to show that, for this conversion rate, the nonproducer cells' specific growth rates need only be 9% higher than those of the producer cells to dominate the hybridoma culture after only 25 passages; and (v) to predict that producer cells are preferentially lost during cell freezing and thawing procedures. The data suggest that particularly unstable cell lines should be analyzed and purified frequently to prevent overgrowth by nonproducers.

Animals↗