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 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.     

Cytogenetic analysis of lymphoblastoid cell lines

Cytogenetic abnormalities were discovered in more than half of 16 lymphoblastoid cell lines established from fragile X individuals and their relatives upon routine cytogenetic analysis of early passage cultures. Subsequently, a second series of lymphoblastoid lines was analyzed to determine if the aneuploidy was a characteristic of lymphoblastoid cell lines derived from fragile X families or a result of the use of cyclosporin A in the establishment of these lines. In the second series of 33 lymphoblastoid cultures, no aneuploid clones were found in the fragile X group, while two were detected in the control cultures, one in a line initiated with cyclosporin A and the other in a line established without cyclosporin A. We conclude that the abnormal clones in our preliminary series were not a characteristic of lines derived from fragile X families and probably not due to the use of cyclosporin A. However, the finding of chromosome abnormalities in a large proportion of lines during the first 3 mo of culture contrasts with previous reports of chromosome stability for the first 12-18 mo of cultivation and indicates that the chromosomes of lymphoblastoid lines should be monitored in any experiment in which a normal diploid complement is critical.     

Cytogenetic harvesting of commonly used tumor cell lines

Tumor cell lines are widely used both as disease models and, increasingly, as genomic resources for the ascertainment of new cancer genes. Cytogenetic analysis remains a major route to uncovering the cancer genome. However, cancer cell lines vary inexplicably in their harvesting preferences, which must, therefore, be determined by trial and error. This article describes harvesting protocols optimized empirically for 550 commonly used, mainly human, cancer cell lines together with evidence-based procedures to assist in determining conditions for unlisted cell lines and subsidiary protocols for cytogenetic analysis using G-banding and fluorescence in situ hybridization.     

Cytogenetic instability of dental pulp stem cell lines

Human adult stem cells (hASCs) offer a potentially renewable source of cell types that are easily isolated and rapidly expanded for use in regenerative medicine and cell therapies without the complicating ethical problems that are associated with embryonic stem cells. However, the eventual therapeutic use of hASCs requires that these cells and their derivatives maintain their genomic stability. There is currently a lack of systematic studies that are aimed at characterising aberrant chromosomal changes in cultured ASCs over time. However, the presence of mosaicism and accumulation of karyotypic abnormalities within cultured cell subpopulations have been reported. To investigate cytogenetic integrity of cultured human dental stem cell (hDSC) lines, we analysed four expanded hDSC cultures using classical G banding and fluorescent in situ hybridisation (FISH) with X chromosome specific probe. Our preliminary results revealed that about 70% of the cells exhibited karyotypic abnormalities including polyploidy, aneuploidy and ring chromosomes. The heterogeneous spectrum of abnormalities indicates a high frequency of chromosomal mutations that continuously arise upon extended culture. These findings emphasise the need for the careful analysis of the cytogenetic stability of cultured hDSCs before they can be used in clinical therapies.     

Sialic acid of mammalian cell lines

Approximately two-thirds of the total sialic acid (S.A.) per cell of a number of cell lines (L-929, L5178Y, HeLa, C13, P183, and CHO) was located at the cell surface but was inaccessible to the action of trypsin, pronase, lysozyme, β-glucuronidase, or hyaluronidase. The mean surface density of S.A. ranged from 5.4 × 10⁵ molecules/μ² surface area for the L5178Y cell to 16.1 × 10⁵ molecules/μ² for the P183 cell. The P183 cell line, which is a polyoma virus-transformed derivative of Stoker's C13 line, consistently contained more S.A. per cell than the latter under a variety of growth conditions, although the two lines did not differ in mean cell volume. When mean cell volume of C13, P183, or CHO cells was experimentally manipulated by thymidine or colcemide blockade, S.A. content per cell followed size changes closely. No evidence could be found for a shift in total S.A. per unit cell volume accompanying the period of maximum mitotic activity of partially synchronized CHO suspension cultures. Comparisons between cells grown on glass and the same cells grown in suspension, or between cells grown to different densities on glass, indicated no differences in the characteristic S.A. content per cell.     

Genetic complementation in hybrid cells derived from two metabolic co-operation defective mammalian cell lines

A series of hybrid clones have been isolated following the somatic cell fusion of two mammalian cell lines, each defective in junctional transfer of metabolites. One of these parental lines is a variant isolated by selection from the metabolic co-operation competent embryonal carcinoma line PC13TG8. The other parent is LMTK⁻ in which inability to transfer was found to be a pre-existing property. Hybrids between these two cell lines are restored in their ability to co-operate, indicating the existence of at least two genetically distinct lesions affecting metabolic co-operation, each of which is recessive. This is the first demonstration that more than one locus is involved in junctional communication.     

Genetic complementation in hybrid cells derived from two metabolic co-operative defective mammalian cell lines

A series of hybrid clones have been isolated following the somatic cell fusion of two mammalian cell lines, each defective in junctional transfer of metabolites. One of these parental lines is a variant isolated by selection from the metabolic co-operation competent embryonal carcinoma line PC13TG8. The other parent is LMTK^? in which inability to transfer was found to be a pre-existing property. Hybrids between these two cell lines are restored in their ability to co-operate, indicating the existence of at least two genetically distinct lesions affecting metabolic co-operation, each of which is recessive. This is the first demonstration that more than one locus is involved in junctional communication.     

Cytogenetic studies in mammalian cells after treatment with insect pathogenic viruses [Baculoviridae]. II.In vitro studies with mammalian cell lines

Mammalian cell-lines from Chinese hamster, Indian muntjac and mouse were inoculated with infectious supernatant ofAutographa californica (Speyer) nuclear polyhedrosis virus (Ac NPV) replicated inMamestra brassicae (L.) cell cultures (IZD-Mb-0503). There was no adverse effect on cell proliferation, nor was a cytopathogenic effect (CPE) induced in such cultures. Cytogenetic data indicate that uptake ofAc-NPVs into the cytoplasm of mammalian cells, as shown by an electron microscopic study, induced neither numerical or structural chromosome aberrations nor sister chromatid exchange (SCE) events.     

The use of an immunological probe to measure the kinetics of DNA repair in normal and UV-sensitive mammalian cell lines

Chinese hamster ovary cells and human fibroblasts were used to study UV-light-induced repair replication and removal of antibody-binding sites. Whereas repair replication still continued 8 h post irradiation, removal of antibody-binding sites was 80% complete within 2 h and reached a plateau by 4 h. This was found to be independent of the method of DNA isolation. UV-hypersensitive CHO cells exhibited reduced levels of repair synthesis that closely correlated with the extent of removal of antibody-binding sites. XP group A, C and D cells, each of which had less than 15% of the level of repair synthesis found in the control fibroblasts, removed less than 30% of the antibody-binding sites. Group E cells demonstrated intermediate levels of DNA-repair capacity in both assays.     

Selection methods for high-producing mammalian cell lines

The selection of high-producing mammalian cell lines represents a bottleneck in process development for the production of biopharmaceuticals. Traditional methods are time consuming (development times often exceed six months) and significantly limited by the number of clones that can be feasibly screened. The market for therapeutic proteins is set to double by 2010, so there is an increasing need to develop methods for the selection of mammalian cell lines stably expressing recombinant products at high levels in an efficient, cost-effective and high-throughput manner. Alternatives include higher throughput methods based on flow-cytometric screening and recently developed automated systems for the selection of high-producing cell lines.     

Colchicine resistance in mammalian cell lines.

Colchicine-resistant variants derived from mouse and Syrian hamster lines are described. The resistant cells do not appear to be true mutants, since they appear at a high frequency, unaffected by treatment with ethyl methyl sulphonate, and are unstable in the absence of the drug. They are cross-resistant to other drugs, show a reduced rate of binding of colchicine in monolayer, and give extracts with colchicine-binding properties identical to those of the wild type. Thus the resistance is due to a permeability barrier. The naturally occurring resistance of the Syrian hamster line is specific for colchicine, and may be due to a specific permeability barrier. The Syrian hamster line is also shown to have an extra colchicine-binding pool.     

Comparing N-glycan processing in mammalian cell lines to native and engineered lepidopteran insect cell lines

In the past decades, a large number of studies in mammalian cells have revealed that processing of glycoproteins is compartmentalized into several subcellular organelles that process N-glycans to generate complex-type oligosaccharides with terminal N -acetlyneuraminic acid. Recent studies also suggested that processing of N-glycans in insect cells appear to follow a similar initial pathway but diverge at subsequent processing steps. N-glycans from insect cell lines are not usually processed to terminally sialylated complex-type structures but are instead modified to paucimannosidic or oligomannose structures. These differences in processing between insect cells and mammalian cells are due to insufficient expression of multiple processing enzymes including glycosyltransferases responsible for generating complex-type structures and metabolic enzymes involved in generating appropriate sugar nucleotides. Recent genomics studies suggest that insects themselves may include many of these complex transferases and metabolic enzymes at certain developmental stages but expression is lost or limited in most lines derived for cell culture. In addition, insect cells include an N -acetylglucosaminidase that removes a terminal N -acetylglucosamine from the N-glycan. The innermost N -acetylglucosamine residue attached to asparagine residue is also modified with alpha(1,3)-linked fucose, a potential allergenic epitope, in some insect cells. In spite of these limitations in N-glycosylation, insect cells have been widely used to express various recombinant proteins with the baculovirus expression vector system, taking advantage of their safety, ease of use, and high productivity. Recently, genetic engineering techniques have been applied successfully to insect cells in order to enable them to produce glycoproteins which include complex-type N-glycans. Modifications to insect N-glycan processing include the expression of missing glycosyltransferases and inclusion of the metabolic enzymes responsible for generating the essential donor sugar nucleotide, CMP- N -acetylneuraminic acid, required for sialylation. Inhibition of N -acetylglucosaminidase has also been applied to alter N-glycan processing in insect cells. This review summarizes current knowledge on N-glycan processing in lepidopteran insect cell lines, and recent progress in glycoengineering lepidopteran insect cells to produce glycoproteins containing complex N-glycans.     

Introduction of estrogen-responsiveness into mammalian cell lines.

We introduced estrogen responsiveness as a new characteristic into rat hepatoma, mouse Ltk- and human HeLatk-cells by transfecting the human estrogen receptor (ER) cDNA. To measure the estrogen response we used Xenopus vitellogenin gene A2 constructs linked to the bacterial CAT gene. Transient cotransfections of the ER cDNA and the vitellogenin gene-CAT constructs containing the estrogen responsive element (ERE) lead to a hormone dependent induction of CAT activity whereas cotransfected vitellogenin gene constructs lacking the ERE are not inducible. Stable transfections of ER cDNA into Ltk- cells give rise to cell clones that are estrogen responsive as shown by transfection of various vitellogenin gene-CAT constructs. These results prove that the transfected ER is biologically active and is sufficient to make a cell estrogen responsive.     

Human lymphoid cell lines: Models for immunological analysis

Summary Investigations with human long term lymphoid cell lines have amply demonstrated the versatility of these tissue culture systems for the detection, definition, and solution of current problems in cell biology, biochemistry, genetics, and immunology. These systems are contributing much to our understanding of the multiple functions of lymphoid cells in the immune response. Human lymphoid cell lines produce, in large quantities, the putative extracellular mediators of cell-mediated immunity, including migration inhibitory factor (MIF), lymphotoxin, interferon, and a specific, reversible inhibitor of lymphocyte biosynthetic activity. The MIF released by human lymphoid cell lines is similar to that produced by phytomitogen- or antigen-stimulated human peripheral lymphocytes. Human lymphoid cells from lines producing MIF mimic the capillary migration patterns of guinea pig peritoneal macrophages, and are more sensitive than the guinea pig cells to human MIFs. Studies with these migrating cells indicate that MIF is not solely a lymphoid cell product, but is synthesized by a wide variety of activated cell types. Extracts of cultured human lymphoid cells inhibit the synthesis of RNA, protein, and DNA by established lymphoid cell lines and by phytomitogen-stimulated human peripheral lymphocytes, but have no inhibitory effects on human nonlymphoid cells. The reversible inhibition is produced with physiological quantities of extract, suggesting a functional immunoregulatory activity for this material in lymphocyte-mediated immunological reactions. Initial findings indicate that these mediators are multiple and distinct molecular species. The remarkable proliferative and synthetic potential of human lymphoid cell systems provides a most useful resource for the purification and characterization of these immunological substances. This invited paper was presented at the Hematopoietic Systems Sessions in Depth section of the 24th Annual Meeting of the Tissue Culture Association, Inc., Boston, June 4, 1973. The work was supported by Grants RO1-AI10422 and TO1-AI00445 from the National Institute of Allergy and Infectious Diseases and PO1-GM19443 from the National Institute of General Medical Sciences of the National Institute of Health, United States Public Health Service. Recipient of Research Career Development Award AI46371 from the National Institutes of Allergy and Infectious Diseases, National Institutes of Health.