Stability of von Willebrand Factor secretion in different human endothelial hybrid cell lines

Endothelial cells form a highly differentiated tissue on the inner surface of blood vessels. One of the typical characteristics is the expression of von Willebrand Factor, a protein that participates in blood coagulation. Thein vitro cultivation of endothelial cells is limited by the fact that primary cells become senescent after 40 generation doublings. We have immortalized human endothelial cells by somatic cell hybridization. Primary cells were fused to different tumor cell lines of murine and human origin. The degree of differentiation of the resulting hybrids was analyzed by characterizing the expression of von Willebrand Factor. This protein was identified intracellularly and in the culture supernatant. During long-term cultivation the hybrid cells showed a tendency to lose this differentiated property even after several subcloning steps. However by fusing them with primary endothelial cells a second time, cell lines expressing von Willebrand Factor for more than 180 population doublings were generated.     

Rituximab induces different but overlapping sets of genes in human B-lymphoma cell lines

The therapeutic unconjugated anti-CD20 Mab rituximab is used for the treatment of B-non-Hodgkins lymphomas. We have studied the direct biological effects, signalling and gene expression profiles induced by rituximab in two human B-lymphoma cell lines, DHL4 and BJAB, using microarray, quantitative PCR and gel shift analysis. Rituximab alone inhibited thymidine uptake and induced homotypic adhesion in DHL4 only, but not BJAB. Analysis of Affymetrix microchips carrying probes for about 10,000 human cDNAs, allowed us to identify 16 genes in DHL4 and 12 in BJAB induced by rituximab at 4 h. Eleven and seven of these genes were specific for DHL4 and BJAB, respectively; whereas the remaining five were up-regulated in both cell lines. Mean induction ranged from 2- to 16-fold. Real time PCR analysis allowed us to confirm up-regulation of all genes identified, except one in BJAB. Time course of induction of eight genes was studied, showing peak induction in most cases at 4 h. The up-regulation of 5/5 genes was also observed with the F(ab)₂ fragment of rituximab. Analysis of three further B-cell lymphoma lines showed that gene induction is not restricted to BJAB and DHL4. Finally, we show that rituximab alone can induce AP1 activation in both cell lines and provide evidence that the ERK1/2 pathway is involved in the rituximab-mediated up-regulation of gene expression. These data demontrate that rituximab alone has direct signalling capacity in different B-lymphoma lines, inducing distinct but overlapping sets of genes which may play a role in the biological and/or therapeutic effect of the antibody.     

Comparison of different hepatocyte cell lines for use in a hybrid artificial liver model

Selection of a cell line suitable for a hybrid artificial liver model employing cellulose porous beads (CPBs) was investigated. Hep G2 cells grown in a culture dish exhibited appreciably higher ureogenesis and gluconeogenesis activities than those grown in CPBs. SEM observation of CPBs revealed marked difference in the distribution of attached cells from one bead to another, and showed that almost all the cell-bearing micropores were completely packed with cells. With the aim of selecting a cell line not prone to excessive aggregation and which grows moderately so as not to fill up the micropores, cells of 6 cell lines, HLE, HLF, Hep 3B, PLC/PRF/5, Huh 7 and Hep G2, were cultivated in dishes. Hep G2, HLE, and HLF increased to 5 × 10⁵ cells/cm², whereas PLC/PRF/5 grew only to 5 × 10⁴, and Hep 3B and Huh 7 up to 2 × 10⁴ cells/cm². The specific activities of ureogenesis and gluconeogenesis of Huh 7 were the highest among the lines tested - 42- and 7-fold those of Hep G2, respectively. When the 6 cell lines were grown in a submerged culture with 0.6 g/l of CPBs, Huh 7 had the lowest cell concentration of 0.54 × 10⁶ cells/ml, and the highest activities of ammonia consumption and urea and glucose production (1.38 μ mol NH₃, 99 nmol urea, and 14.5 nmol glucose/10⁶cells/h). Consequently, Huh 7 is considered to be a suitable cell line for use in the development of an artificial liver model employing porous beads. This revised version was published online in July 2006 with corrections to the Cover Date.     

Restriction endonuclease sensitivity of DNA containing globin genes in different murine erythroleukemia cell lines

The arrangement of the globin structural genes has been examined in murine erythroleukemia cells, strain DS19, and several related inducer-resistant variant cell lines. One fragment larger than 20 kilobases and six globin gene-containing fragments between 10 and 1.9 kilobases in size are detected in EcoRI-cleaved purified DNA prepared from strain DS19. By comparison, when isolated nuclei are digested with EcoRI, only two globin gene-containing fragments are detected, one greater than 20 kilobases and the other 1.9 kilobases. Of seven cell lines derived from DS19 and resistant to inducers, six had similar patterns to DS19 of globin gene-containing EcoRI-generated DNA fragments from nuclei and from purified DNA. One cell line, DR10, a DMSo-resistant cell line, lacks the 1.9 kilobase fragment after digestion of either nuclei or purified DNA. The 1.9 kilobase fragment hybridizes with alpha-globin cDNA but not with the beta-globin cDNA, suggesting either rearrangement or deletion of an alpha-globin gene-like fragment in DR10 DNA.     

Ammonium toxicity in different cell lines

The toxic effect of ammonium upon a variety of cell lines of lymphoid (Jurkat), pituitary (GH(4)), and renal (LLC-PK(1)) origin was studied. Millimolar concentrations of the ion mildly affected the growth of GH(4) cells and prevented the growth of LLC-PK(1) cells. The ion did not lead to the death of LLC-PK(1) cells but it produced morphologic changes in these cells. The effects of ammonium upon Jurkat cells were different because cells died after accumulating at S phase. Cell death was due to apoptosis and might be related to ammonium-induced calcium mobilization from intracellular stores. These results indicate that the toxic effects caused by ammonium accumulation are different depending upon the cell type. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 530-537, 1997.     

Differential repair and replication of damaged DNA in ribosomal RNA genes in different CHO cell lines

We studied the repair of psoralen adducts in the pol I-transcribed ribosomal RNA (rRNA) genes of excision repair competent Chinese hamster ovary (CHO) cell lines, their UV sensitive mutant derivatives, and their UV resistant transformants, which express a human excision repair gene. In the parental cell line CHO-AA8, both monoadducts and interstrand crosslinks are removed efficiently from the rRNA genes, whereas neither adduct is removed in the UV sensitive derivative UV5; removal of both adducts is restored in the UV resistant transformant CHO-5T4 carrying the human excision repair gene ERCC-2. In contrast, removal of psoralen adducts from the rRNA genes is not detected in another parental CHO cell line CHO-9, neither in its UV sensitive derivative 43-3B, nor in its UV resistant transformant 83-G5 carrying the human excision repair gene ERCC-1. In contrast to such intergenomic heterogeneity of repair, persistence of psoralen monoadducts during replication of the rRNA genes occurs equally well in all CHO cell lines tested. From these data, we conclude that: 1) the repair efficiency of DNA damage in the rRNA genes varies between established parental CHO cell lines; 2) the repair pathways of intrastrand adducts and interstrand crosslinks in mammalian cells share, at least, one gene product, i.e., the excision repair gene ERCC-2; 3) replicational bypass of psoralen monoadducts at the CHO rRNA locus occurs similarly on both DNA strands.     

Abnormal mutation frequencies in human repair-defective hybrid cell lines

Two intraspecific human cell hybrids, HD2 and HD1A, produced from fusion between HeLa cells and xeroderma pigmentosum fibroblasts, express XPD-like rates of excision repair and hypersensitivity to UV-radiation. In the present paper we describe unusual patterns of UV-induced mutation in both cell lines. Though HD2 very closely resembles XPD both phenotypically and genetically, in UV-dose response it is hypomutable at the loci for ouabain and diphtheria toxin resistance. At equitoxic dose, however, it shows normal mutability, HD1A, by contrast, is hypermutable as a function either of UV dose or in terms of equitoxicity for these genes. HD1A's mutator phenotype is a dominant characteristic and is not associated with grossly abnormal DNA precursor pool imbalance. The possibility remains that DNA polymerase infidelity underlies its hypermutability.     

Cytogenetic and immunological study of mammalian hybrid cell lines

Two hybrid cell lines, KS-RL-3 (hybrid between TK^? sheep kidney cells and rabbit lymphocytes) and CR-KS TK^? (hybrid between rabbit β-cells and TK^? sheep kidney cells), were assayed cytogenetically. It was shown that these hybrid cell lines were characterized by the presence of both sheep and rabbit chromosomes, with a number and structure which varied depending on the cell type and the number of passages. In some cases aberrant chromosomes were identified. The modal chromosome number was 121–135 (40.7%) in the KS-RL-3 cell line and 106–120 (51.6%) in the CR-KS TK^? cell line. CR-KS TK^? and KS-RL-3 cells were identified in the testicles and ear lobes of experimental animals during periods of 7 to 28 days after cell inoculation. Partial immunological tolerance of the hybrid cell lines was suggested.     

Cytogenetic and immunological specificity of mammalian hybrid cell lines

Karyotypes of the hybrid cell lines NS-RL-3 (TK- -sheep kidney cells and rabbit lymphocytes) and betaCR-NS (TK- -rabbit beta-cells and TK- -sheep kidney cells) were investigated. It was shown that both hybrid cell lines were characterized by presence of both sheep and rabbit chromosomes, which number and structure varies depending on the cell type and the number of passages. In some cases the aberrant chromosomes were identified. It was observed, that 40-50% of the NS-RL-3 cells survived in culture in the presence of the human blood serum, and also were identified during 7-28 days after their introduction into the organism of the animal. Thus, the partial immunological tolerance of the hybrid cell lines has been suggested.     

Contact inhibition and gene expression in HTC-L cell hybrid lines

Contact-inhibited somatic cell hybrids were formed between two malignant cell lines lacking contact inhibition of growth. One cell line was HTC-AR1, an azaguanine-resistant subline from the rat hepatoma line HTC +; the other line was the BUDR-resistant mouse L-cell subline L-B82. Hybrids were obtained from selective medium and characterized by chromosomal and enzymic analysis. The hybrids lacked the inducible rat enzyme tyrosine aminotransferase.     

Rapid analysis of mouse-hamster hybrid cell lines by in situ hybridization

In situ hybridization techniques for analyzing the murine DNA complement of mouse-hamster hybrid cells are described. Total genomic mouse DNA is labeled with biotin and hybridized without suppression to metaphase spreads from a mouse-hamster hybrid line containing the mouse fusion chromosome X12. Detection via fluorochrome-conjugated avidin reveals mouse chromosomal DNA with high sensitivity and permits the identification of both normal and aberrant murine chromosomes. Conversely, biotinylated total genomic DNA from a hybrid line can be used as a probe on normal mouse metaphase spreads if suppression techniques are employed, facilitating the analysis of mouse chromosomes present in the hybrid line.     

Three different human tumor cell lines contain different oncogenes

We have obtained foci of transformed mouse cells after transfection of human DNA from colon and bladder carcinoma cell lines and a promyelocytic leukemia cell line. These foci can be shown to contain a large number of human DNA sequences by use of highly repetitive human DNA sequence probes. Cell DNA from primary foci can be used in a subsequent cycle of transfection resulting in secondary foci that contain relatively little human DNA. Secondary foci appear to contain only the human sequences proximal to those responsible for the transformed phenotype. A set of characteristic DNA restriction fragments is found in common among secondary foci derived from each tumor cell line DNA. Comparison of the common DNA fragments found in secondary foci derived from three different human tumor cell lines indicates that these three cell lines contain three different transforming genes.     

T cell receptor genes and T cell development in virus-transformed early T cell lines

We have derived T cell lines from mice inoculated with Gross leukemia virus, which appear to represent early T cell developmental stages and to reflect normal T cell development. These cell lines may provide a breakthrough in the study of T cell development as Abelson transformants have done for the study of B cell development. Analysis of the TCR gene expression in these cell lines reveals that the sequence of rearrangement and expression of each TCR gene is not strictly ordered. Expression of RNA for the TCR alpha and -beta genes appears to be coordinated with rearrangement at the alpha and beta loci. This is not the case for gamma gene expression. Availability of the homogeneous populations of cells represented in these cells lines allows for a more detailed molecular analysis of T cell development than was previously possible.     

Cytogenetic characteristics of 26 polyethylene glycol-induced human-hamster hybrid cell lines

A cytological analysis of 26 polyethylene glycol (PEG) induced human/hamster hybrid lines has shown that such lines are similar to inactivated Sendai virus (ISV) induced hybrids in respect to stability, retention of specific chromosomes, and cell selection. The evolution of stable hybrid cell lines carrying variable human chromosome complements depends upon a balance being established between the retained human and hamster genomes. This balance is a result of random loss of human and hamster chromosomes followed by selection of the fittest stem lines. A major mechanism ofchromosome loss may be fragmentation and elimination of acentric fragments. Twelve of the 26 lines had stabilized by the 30th passage, an incidence similar to that found with ISV-induced hybrids studied in this laboratory. Thus, PEG may be considered to be an ideal chemical for inducing somatic cell hybrids for genetic analysis.