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PubMed1976

Control of expression of differentiated functions in neuroblastoma cell hybrids.

Cell hybrids have been extensively utilized for gene mapping; more than 50 enzymes and nonenzyme proteins have been assigned to individual human chromosomes. Hybrids have also been used in the study of differentiation; fusions involving mouse or human neuroblastoma cells and various nonneuronal lines resulted in hybrid cells that continued to express neuronspecific functions. The expression of the differentiated state is, however, not an all-or-none phenomenon: One neuronal trait may be evident in such hybrids, in the absence of others. The potential usefulness of the human neuroblastoma hybrids for the assignment of genes involved in the expression of differentiated functions to specific chromosomes is discussed.

Animals↗
PubMed2004

A dendritic cell based hybrid cell vaccine generated by electrofusion for immunotherapy strategies in HNSCC.

OBJECTIVE: Hybrid cells generated from dendritic cells (DC) and tumor cells provide tumor-associated antigens (TAA) in a polyvalent mode and therefore they have aroused interest in cancer immunotherapy. The present study was designed to investigate the hybrid cell generation and optimize its implementation for a TAA-target treatment of head and neck squamous cell carcinoma (HNSCC). METHODS: Hybrid cells from mature DC and laryngeal carcinoma cell line UTSCC-19A were generated by electrofusion. Fusion efficiency and viability were determined by flow cytometry, light and fluorescence microscopy analyses. RESULTS: The gradual electrofusion process constituted real human tumor and dendritic cell hybrids characterized by polynuclear cells and double staining as a result of overlay of red (HLA-DR:R-PE) and green (HEA:FITC) fluorescence. Furthermore, analyses have proven viability of fusion results, and factors influencing fusion yield were determined. CONCLUSION: Physical fusion of mature dendritic cells with laryngeal carcinoma cells provides a dendritic cell based hybrid cell vaccine as a quantitative prerequisite for anti-cancer vaccination. Specific cytotoxic T-lymphocytes need to be induced before hybrid cell application in clinical studies.

Antigens, Neoplasm↗
PubMed1986

Isolation of antigen-binding membrane molecules from an antigen-specific T-cell hybrid.

Cell surface-radioiodinated proteins of a murine T-cell hybrid specific for and able to bind azobenzenearsonate were isolated by adsorption to Sepharose beads conjugated with a rabbit antiserum to murine T-cell antigen-binding molecules. These isolated proteins, Mr 72,000, were found to bind specifically azobenzenearsonate while proteins isolated in this manner from the tumor parent BW5147 did not bind azobenzenearsonate. Similar cell surface proteins were isolated by affinity for antigen and immunoprecipitated with an antiserum to T-cell antigen-binding molecules. The results suggest that antigen-binding T cells express T-cell antigen-binding molecules as membrane receptors for antigen.

Animals↗
PubMed1980

Independent regulation of H-2K and H-2D gene expression in murine teratocarcinoma somatic cell hybrids.

Cells of two teratocarcinoma stem cell lines (PCC4 azaguanine [aza] 1 and F9 5-bromodeoxyuridine [BrdU]) were fused with normal mouse spleen cells and mouse thymoma-derived cells (BW 5147), respectively. Hybrid clones were tested for the expression of molecules coded by the H-2K and -2D genes both by absorption analysis of conventional H-2 sera and by indirect antibody-binding radioimmunoassay with monoclonal antibodies. Somatic cell hybrids between PCC4 aza 1 and spleen cells morphologically resemble teratocarcinoma stem cells and do not express H-2 antigens. However, after differentiation in vitro, one of these hybrid clones expresses the H-2K and -2D gene products of both parental cell lines, one close expresses H-2-D- but not H-2K-coded antigenic determinants, and one clone remains H-2 negative. Somatic cell hybrids between F9 BrdU and BW 5147 resemble fibroblasts. Analysis of a series of hybrid clones revealed some clones that express both the H-2K- and H-2D-coded antigenic specificities of both parental alleles, some that express H-2D gene products strongly and the H-2K gene products very weakly, and some that express H-2D- but not H-2K-coded molecules. These results imply independent regulation of expression of the H-2K and -2D genes. The H-2D gene products appear to be preferentially expressed if the hybrid cells are capable of expressing H-2. The results suggest complex regulatory mechanisms that are H-2K and H-2D specific.

Animals↗
PubMed1987

A genetic analysis of extinction: trans-regulation of 16 liver-specific genes in hepatoma-fibroblast hybrid cells.

Hybrid cells formed by fusing different cell types generally fail to express the tissue-specific products of either parent, a phenomenon termed extinction. We have investigated the generality of this effect by assaying hepatoma-fibroblast hybrids for expression of 16 different liver-specific mRNAs. Fourteen of the mRNAs failed to accumulate in karyotypically complete hybrid clones, and quantitative measurements indicated that steady-state mRNA levels were depressed by a factor of at least 500-1000. However, all 14 liver-specific mRNAs were reexpressed in the hybrids following fibroblast chromosome loss. These data indicate that expression of whole sets of tissue-specific genes is affected in trans in intertypic hybrids and suggest that negative regulation of heterologous functions may be common form of gene control.

Alcohol Dehydrogenase↗
PubMed1993

The use of methylthioadenosine phosphorylase activity to select for human chromosome 9 in interspecies and intraspecies hybrid cells.

Methylthioadenosine phosphorylase (MTAP) is an enzyme that functions in a salvage pathway for adenine synthesis. The locus that encodes MTAP activity has been mapped to human chromosome 9 (9q12-9pter) by analysis of mouse x human somatic cell hybrids. Cells that have MTAP activity will stop proliferating, and eventually die in the presence of azaserine, an inhibitor of de novo purine synthesis, but can be rescued by the addition of methylthioadenosine (MTA) to the culture medium. Some mouse and human tumor cells lack MTAP activity and can not grow in the presence of azaserine and MTA. We fused MTAP competent human fibroblast cells to MTAP deficient mouse L-cells and selected for somatic cell hybrids, containing MTAP activity, in medium containing azaserine and MTA. In a separate experiment, a CHO cell x human fibroblast somatic cell hybrid, containing a normal copy of human chromosome 9, was used to prepare microcells, which were fused to an MTAP-deficient human leukemic cell line, CCRF-CEM. Somatic cell and microcell hybrids were shown to retain human chromosome 9 by fluorescence in situ hybridization using probes that hybridize to the interferon-alpha and -beta 1 genes on human chromosome 9 (9p21), and the centromere of human chromosome 9. This is the first report of complementation for MTAP activity being used to select for somatic cell hybrids and microcell hybrids that retain a human chromosome 9.

Animals↗
PubMed1983

Evidence for an indirect effect of radiation on mammalian chromosomes. II. UV-induced isochromosome formation in cell hybrids.

Cell fusion has been used to test the hypothesis that chromosomes may be damaged indirectly by UV light, as well as by direct interaction with DNA. Hybrids were found to contain isochromosomes of mouse origin, whose frequency increased as the dose of UV delivered to the hamster cell parent was increased. This unusual type of aberration was shown to be induced also by direct UV irradiation of mouse or hybrid cells. These results suggest that UV light has a hitherto undiscovered indirect effect on chromosome integrity, mediated by some irradiated cell component.

Animals↗
PubMed1971

Biochemical and cytological evidence for triple hybrid cell line formed from fusion of three different cells.

Hybrids between two parental lines of somatic cells have been observed in various genomic multiplicities by ourselves and others. Possible explanations for the higher multiplicities include the fusion of cells in a 1:1 ratio with one genomic set undergoing an asynchronous replication either before or after fusion or the fusion of two or more cells. We now provide evidence for multiple genomic hybrids arising from the fusion of more than two cells in a mixture of three different cell lines. This proof is based on unique chromosomal and biochemical markers characteristic of the three parental cell lines. The distinctive phenotypes of all three lines are expressed in a clonally derived hybrid. Thus, we conclude that long-term, proliferating somatic cell hybrids can arise from the fusion of three or more cells.

Adenoma↗
PubMed1979

Induction of secretion of IgM from cells of the B cell line 38c-13 by somatic cell hybridization.

Cells of the murine B cell line 38C-13 possess immunoglobulins of the IgM class on their surface but do not secrete them. Upon hybridization of 38C-13 cells with murine myeloma cells, hybridoma clones were obtained that secreted both pentameric IgM of 38C-13 origin and the myeloma protein. All hybridoma clones synthesized and secreted large amounts of homogeneous IgM with a half disappearance time of about 2 hr, typical of mature plasma cells. Concomitantly with the induction of IgM secretion, the hybridoma cells lost their surface IgM. The possibility of separate pathways for the synthesis of membrane and secreted IgM is discussed.

Animals↗
PubMed1981

Electrophoretic mobility of mouse T-cell hybrids.

The hybrid cell line BH2 was derived by fusion between an AKR thymoma BW5147 (HGPRT-) and C57BL thymoma EL-4R (TK-). The hybrid cells showed a near-tetraploid modal number of chromosomes, in contrast to the near-diploid stem-lines of both parental cell populations; most of the BH2 hybrid cells acquired marker chromosomes from both parental cell lines. Inoculation of the parental and hybrid cells into C57BL and AKR mice revealed that the possible admixture of revertant parental cells in the hybrid cell population was less than 10(-4). Anodic electrophoretic mobilities (AEM) of the mouse thymoma lines and their hybrids were compared with each other and with normal mouse lymphoid cells. The AEM of the parental and hybrid T-cell lines was slower than that of mouse T LNC and comparable with AEM of some thymocyte subsets. The mean AEM of parental and hybrid cell lines was 0.69 micrometers/sec/V/cm for BW5147 cells, 0.96 for EL-4R cells and 0.83 for BH2 cells, the mean AEM of the hybrid cell population being identical with the mean of the parental AEM values. The mean AEM was found to be a relatively stable characteristic of each cell line.

Animals↗
PubMed1979

Genetic and adaptive differences in the expression of drug resistance in hybrid cells.

Hybrids between Chinese hamster cells were isolated and maintained in media that were selective or nonselective for markers present in the parent cells (HGPRT and TK deficiencies, respectively). Segregation frequencies for resistance to azaguanine (AZG), thioguanine (THG), or bromodeoxyuridine (BrdU) could be enhanced for some groups of hybrids if the stock cells were maintained under nonselective conditions rather than in HAT medium. In these populations the expression of resistance was dominant or codominant even though marker patterns were recessive for the same cells in HAT. Clonal analysis showed that enhancement took place by adaptive shifts rather than by variation and selection. Segregation frequencies in hybrids were also found to differ significantly between clones isolated by replicate fusions of any two parental cell types. The basis for this heterogeneity is unknown and deserves further study.

Animals↗
PubMed1999

Deletion of p16INK4A/CDKN2 and p15INK4B in human somatic cell hybrids and hybrid-derived tumors.

Deletion or epigenetic inactivation of the tumor suppressor gene p16INK4/CDKN2 (p16) has been observed in multiple human tumors. We assayed hybrid cell lines between human diploid fibroblasts and fibrosarcoma cells for p16 allelic status and expression and found that p16 was expressed in the parental diploid fibroblast cell lines used, whereas the parental fibrosarcoma cell line HT1080.6TG exhibited homozygous deletion of p16. Most immortalized hybrid cell lines derived from these parent cell lines, whether tumorigenic or nontumorigenic, exhibited loss of fibroblast-derived p16 alleles. All p16-negative hybrid cell lines also exhibited deletion of p15INK4B (p15). Hybrid cell lines yielded tumors upon s.c. injection into athymic nude mice regardless of p16/p15 status. Tumors derived from six p16/p15-positive hybrid cells, however, revealed deletions of both p16 and p15. When human diploid fibroblasts were fused with A388.6TG squamous cell carcinoma cells, which exhibit aberrant methylation of p16, the resulting hybrids again exhibited deletion of the unmethylated fibroblast-derived p16 alleles. Transfection of both HT1080.6TG and A388.6TG cells with wild-type p16 expression vector resulted in decreased clonogenicity in culture. Although the determinants directing genetic versus epigenetic inactivation of p16 and p15 remain unclear, these results demonstrate that p16-mediated growth suppression could be abrogated by either mechanism in somatic cell hybrids.

Animals↗
PubMed1998

Loss of suppressor loci on chromosomes 11 and 14 may be required for radiation-induced neoplastic transformation of HeLa x skin fibroblast human cell hybrids.

We have previously reported a linkage between radiation-induced damage to a putative tumor suppressor locus on fibroblast chromosome 11 and the re-expression of tumorigenicity in a hybrid cell line (HeLa x human skin fibroblast) used to study neoplastic transformation. Further investigation into the molecular basis of radiation-induced neoplastic transformation of the hybrid cell, CGL1, indicates that loss of fibroblast chromosome 11 appears to be necessary but not sufficient for neoplastic transformation. Previous analysis had suggested, though not clearly demonstrated, a possible role for loss of alleles on fibroblast chromosome 14 in the neoplastic transformation of the hybrid cells. Therefore, the status of chromosome 14 in the gamma-ray-induced, neoplastically transformed (GIM) hybrid cell lines and in nontumorigenic control (CON) hybrid cell lines isolated from irradiated populations has been investigated. Chromosome painting and molecular studies using restriction fragment length polymorphisms and tetranucleotide repeat polymorphism analysis were performed. As an additional control, the status of chromosome 12 was also examined. We report that five of the eight GIM cell lines have lost one complete copy of a fibroblast chromosome 14 while only one of the five CON cell lines has lost a complete copy of a fibroblast chromosome 14. No evidence of large-scale loss of chromosome 12 was detected in the GIM or CON cells. The data further suggest that both copies of fibroblast chromosome 14 contain an active tumor suppressor locus and that radiation-induced loss of either fibroblast chromosome 14 is associated with neoplastic transformation in this system. We now conclude that loss of alleles on both fibroblast chromosome 11 and 14 may be required for the radiation-induced neoplastic transformation of these human hybrid cells.

Alkaline Phosphatase↗
PubMed1996

Coordinate extinction of melanocyte-specific gene expression in hybrid cells.

Whole cell hybrids and microcell hybrids between mouse fibroblasts and pigmented Syrian hamster melanoma cells were analyzed for coordinate regulation of melanocyte-specific gene products. Extinction of pigmentation was observed in whole-cell hybrids and in a microcell hybrid containing a single mouse chromosome (mouse chromosome 1). Analysis of melanocyte-specific transcripts using reverse transcription, combined with the polymerase chain reaction (RT-PCR), demonstrated that tyrosinase, TRP-1, TRP-2, and microphthalmia transcripts were all absent in unpigmented whole-cell hybrids and in the monochromosomal unpigmented microcell hybrid. A pigmented subclone of this microcell hybrid, however, re-expressed the tyrosinase, TRP-1, TRP-2, and microphthalmia genes. These data suggest that all of these genes are coordinately extinguished by a single fibroblast locus. Since the only fibroblast chromosome detected in the unpigmented microcell hybrid was mouse chromosome 1, these results also suggest that the extinguisher locus affecting the expression of the tyrosinase, TRP-1, TRP-2, and microphthalmia genes in hybrid cells is located on that mouse chromosome (or on a fragment of another chromosome present in the unpigmented monochromosomal microcell hybrid but undetected in our analyses). In contrast to the results with the melanocyte-specific genes mentioned above, transcripts for the melanocortin 1 receptor gene (MC1R) were present in the monochromosomal unpigmented microcell hybrid (although absent in the whole-cell hybrids). This suggests that regulation of MC1R gene expression is distinct from regulation of the other melanocyte-specific genes.

Animals↗
PubMed1991

Transactivation of pancreas-specific gene sequences in somatic cell hybrids.

Enhancer/promoter elements from two pancreas-specific genes, those encoding amylase and elastase, were ligated to the bacterial GPT gene. The resulting construct can be used to select for expression of gene products which activate these pancreas-specific promoters in hybrid cells. The selectable GPT construct was stably transferred into several cell lines either directly or by cotransfection with pSV2Neo. GPT was expressed when transferred to pancreatic cell lines but not when transferred to GPT-fibroblast (L) cells or hepatoma cells. When the transformed L cells and hepatoma cells were fused with pancreatic cell lines, GPT was activated in the hybrid cells. Endogenous pancreas-specific genes from the L-cell and hepatoma parents were also activated in the hybrids. In addition, a pancreas-specific nuclear protein, PTF1, was produced in pancreatic and hybrid cells, correlating with GPT expression. The transformed L cells and hepatoma cells thus contained a nonexpressed construct which could be activated in trans by factors present in pancreatic cells. The hepatoma hybrid also continued to produce albumin, demonstrating the coexpression of liver and pancreas-specific genes in the hybrid-cell population. Cell lines carrying the amylase/elastase/GPT construct may be useful as a selection system for cloning of pancreatic transcription activators.

Albumins↗
PubMed1992

Extinction of alpha 1-antitrypsin gene expression in somatic cell hybrids: evidence for multiple controls.

Expression of the liver-specific alpha 1-antitrypsin (alpha 1AT) gene is extinguished in hepatoma/fibroblast hybrids. To define the mechanism of extinction, we identified DNA sequences involved in this process by transiently transfecting mutant alpha 1AT promoters into parental and hybrid cells. The wild-type alpha 1AT promoter (-554 to +44 bp) was highly expressed in rat hepatoma cells, but activity was 100-fold less in fibroblasts or cell hybrids. Mutations in this region failed to activate alpha 1AT expression in nonhepatic cells, but mutations in the binding site for liver factor B1 (LF-B1) reduced hepatic-specific expression greater than 100-fold. Furthermore, the hybrid cells failed to express LF-B1-binding activity and mRNA. This suggested that alpha 1AT extinction in hybrids might be an indirect, lack-of-activation phenotype mediated primarily through repression of LF-B1. To test this possibility, we stably transfected an LF-B1 expression cassette into parental and hybrid cells and monitored expression of transfected and endogenous alpha 1AT genes. Surprisingly, although constitutive LF-B1 expression could activate alpha 1AT-CAT transgenes in these cells, it neither prevented nor reversed extinction of the chromosomal alpha 1AT genes. We conclude that although extinction of the LF-B1 trans-activator accompanies alpha 1AT extinction in cell hybrids, it does not play a causal role in this process.

Animals↗