Fusion and hybridization of marsupial and eutherian cells. Heterokaryon formation

Marsupial x eutherian cell hybrids would be very useful for studies of mammalian genetics and cell biology. A critical step in the formation of such hybrids is the fusion of cells to form heterokaryons. We have examined many different combinations of marsupial and eutherian cells for their ability to fuse, and we have found that all combinations yielded heterokaryons, but with different frequencies, depending on the cell types used. Ranked in order of decreasing ability to fuse with eutherian cells, the marsupial cell types were; established lines, primary diploid fibroblasts and lymphocytes. In all fusion experiments there was a marked preference for the formation of homokaryons compared with heterokaryons. It was possible to control the numbers and types of heterokaryons formed by varying the input ratio of parental cells.     

Association of the herpes simplex-1 viral gene for thymidine kinase with the human gene for adenylate kinase-1 in biochemically transformed cells

The herpes simplex type 1 biochemically transformed human cell line, HB-1, was fused with thymidine kinase deficient rodent cells, and 18 hybrids were isolated using the HAT-ouabain selection system. The selected enzyme, viral thymidine kinase, was present in all 18 hybrids. In 16 of 18 hybrids the viral gene for thymidine kinase cosegregated with the human gene for adenylate kinase-1 (AK-1). Thirty-six bromodeoxyuridine (BrdUrd) resistant sublines were isolated from the 16 human AK-1 positive hybrids. Each BrdUrd-resistant subline was examined for the presence of the viral TK gene by back-selection in HAT medium, and for human AK-1. In all 36 BrdUrd-resistant sublines the viral TK gene cosegregated with the human AK-1 gene. These results indicate that the transforming viral DNA fragment was associated with a specific human chromosomal region in HB-1 cells.     

Use of enzyme-deficient cell lines as feeder layers

Summary Enzyme-deficient cell lines, lacking TK or HPRT and therefore unable to grow in HAT medium, may be used as feeder layers to enhance clonal growth of wild-type cells. Low numbers of wild-type test cells may be plated in HAT medium with 5×10⁵ HAT-sensitive feeder cells per Petri dish. The feeder cells remain attached and metabolizing for 1 to 2 weeks, but ultimately die and detach, leaving colonies of test cells. This feeder layer technique is very simple and flexible and could have wide applicability. This work was a byproduct of a project on fusion and hybridization of marsupial and eutherian cells supported by the Australian Research Grants Committee.     

Reversion in expression of hypoxanthine-guanine phosphoribosyl transferase following cell hybridization.

Hybridization of mutant cell lines deficient in hypoxanthine-guanine phosphoribosyl transferase (HGPRT; E.C.: 2.4.2.8) from a variety of established rodent sources with HGPRT plus human cells yielded progeny cells which grew in selective medium containing hypoxanthine, aminopterin and thymidine (HAT). The same result was obtained when the human cell used was an HGPRT minus transformed line derived from a patient with the Lesch-Nyhan syndrome. Electrophoretic analysis indicated that all HAT-resistant progeny clones contained an active HGPRT enzyme which was indistinguishable from the wild type enzyme of the corresponding normal rodent cells. In contrast, no HAT-resistant cells have been obtained when the same HGPRT minus rodent cells were subjected to fusion processes in the absence of human cells or when they fused with similarly derived HGPRT minus mutant cells of other rodents. Reversion in expression of the rodent gene for HGPRT was detected in clones which retained one or more human chromosomes and in clones which contained no detectable human chromosomal material. The observed re-expression of rodent HGPRT in HAT-resistant clones suggests that HGPRT plus as well as HGPRT minus human cells contributed a factor which determined the expression of respective rodent structural genes for HGPRT. In contrast, HGPRT minus rodent cells were unable to induce the synthesis or normal HGPRT in the cells derived from the patient with the Lesch-Nyhan syndrome.     

Selection of mouse X hamster hybrids using HAT medium and a polyene antibiotic.

In the present study we tested the feasibility of utilizing a structurally modified polyene antibiotic, amphotericin B methyl ester (AME), as a half-selection agent for isolating somatic cell hybrids. By using HAT medium supplemented with AME we have isolated interspecific mouse-hamster hybrids from mixed cultures of mouse (TK-C1 ID or HPRT-A9) and hamster (BHK/C 13) cells fused with Sendai virus, lysolecithin or polyethylene glycol. Hybrid cells proliferated and clones were isolated after 2 to 3 weeks growth in three changes of HAT-AME medium and subsequent growth in HAT medium alone. In contrast, genetically deficient parental C1 1D or A9 cells and AME-sensitive BHK/C 13 cells were killed using a similar growth protocol. The described technique is simple, efficient and permits one to use a cell line without a genetic defect in combination with a genetically deficient cell type in hybrid formation.     

Genome Instability in interspecific cell hybrids II. Aminopterin resistance and gene amplification in lines arising from fusions of cells from divergent mammalian species

In order to investigate instances of genetic instability in divergent cell hybrids, we studied several RAT-resistant colonies recovered from fusions between HPRT or TK-deficient rodent cells and marsupial or monotreme cells. Most of these colonies proved to lack HPRT or TK activity and to have survived by acquiring resistance to aminopterin; such aminopterin-resistant lines were never recovered from parent cells subjected to HAT selection. Two of the aminopterin-resistant hybrids over-produced DHFR, and possessed either double minutes or an abnormally banded region, the cytological manifestations of gene amplification. Selection in higher aminopterin concentrations yielded a highly resistant line with 100X wild-type DHFR activity and a large homogeneously staining region. We suggest that interspecific cell hybrids are predisposed to gene amplification and may also show many other types of genetic and chromosomal instability, possibly thein vitro equivalent of the “genomic shock” phenomena described for interstrain or interspecies hybrids of plants or animals. This paper, no. II in a series by these authors, reached the Editorial Office on the date given, although it had been mailed earlier than paper no. III; the latter paper also appears in this number — Eds.     

Studies on 1-beta-D-arabinofuranosyl-cytosine (Ara-C) resistant mutants of Chinese hamster fibroblasts. II. High resistance to Ara-C as a genetic marker for cellular hybridization

From a variety of independent Chinese hamster cell lines, stable variants resistant to 5 μg/ml of Ara-C were isolated via a single step selection; in contrast to variants selected at lower drug concentrations, the resistant clones appear to be uniformly deficient in Ara-C phosphorylation, an activity previously shown [14] to be carried out in hamster cells by a cytoplasmic dC-kinase (dC-kinase 2). These dC-kinase deficient (dCK^?) variants can be selected against because they are unable to divide in a medium containing dT (0.8 mM) and dC (0.01 mM), which supports the growth of wild type dCK⁺ cells. Plating of dCK^? cells in medium supplemented with both nucleosides yields only rare clones of pseudorevertants which escape the thymidine block through a secondary unknown defect; the growth of these clones can be prevented by further addition of dA to the selective medium. As expected from the complementation pattern for the deficient enzyme activities, hybrids between a dCK^? hamster line and TK^? lines of either mouse or hamster could be isolated in a modified HAT medium (HAT₅₀dC) containing dC and an increased dT concentration. In principle, the same selection can be used to isolate interspecific and intraspecific hybrids between Ara-C resistant variants obtained from a variety of mammalian species and azaguanine resistant lines deficient in HGPRT. The potential interest of this sytem for genetic mapping is discussed.     

Establishment of hybridoma secreting human monoclonal antibody against hepatitis B virus surface antigen

The HAT (hypoxanthine, aminopterin, thymidine) sensitive and ouabain resistant human B lymphoblastoid cell line TAW-925 was obtained from 6-thioguanine resistant B lymphoblastoid cell line WI-L2. Hybridomas were obtained at a high frequency (10(-4)-10(-5) when TAW-925 was hybridized with cells transformed with Epstein-Barr virus. Using TAW-925 as a parental cell line, we have obtained a hybridoma which stably secretes human monoclonal antibody against hepatitis B virus surface antigen.     

Characterization of hypoxanthine-guanine phosphoribosyl transferase in man-mouse somatic cell hybrids by an improved electrophoretic method

The hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity in a group of man-mouse somatic cell hybrids, produced by Sendai virus-mediated cell fusion and HAT selection, has been analyzed by a new electrophoretic technique. Evidence is presented which shows that the hybrid lines derived from fusion of a mouse fibroblast deficient in HGPRT with various human cell strains have an HGPRT activity that is characteristic of the human enzyme, whereas a hybrid line derived from a mouse fibroblast which is deficient in thymidine kinase has an HGPRT activity characteristic of the mouse. This new technique involves electrophoresis of cell extracts on cellulose acetate gel, followed by the localization of the enzyme activity by autoradiography.This research was supported in part by a research grant from the U.S. National Institutes of Health (No. GM-13415).     

Codominance of cisplatin resistance in somatic cell hybrids

Intrinsic or acquired resistance to cisplatin in cancer cells remains a major obstacle to successful chemotherapy. The clinically relevant genetic and molecular mechanisms of resistance have not yet been identified. Cisplatin-resistant (CP-r) human KB epidermoid carcinoma cell lines (HeLa) resistant to varying levels of cisplatin after single and multiple selection steps are cross-resistant to other platinum compounds and to methotrexate. Intraspecies hybrids of the sensitive and KB CP-r cells were fused with HeLa D98(OR) CP-s, hypoxanthine-aminopterin-thymidine (HAT) sensitive, ouabain resistant, to determine whether cisplatin resistance is dominant or recessive. Cell-cell hybridization between the sensitive cells and single-step or two-step KB CP-r cells both indicated codominance of cisplatin resistance compared to hybrids between sensitive cell lines (D98(OR)xKB). The hybrids between sensitive cell lines (D98xKB) and a single-step CP-r KB cell line (D98xKB-CP.5) also were cross-resistant to carboplatin and methotrexate. In addition, the relatively slower growth rate of CP-r cells appears to be dominant. In the two-step CP-r KB cell line, KB-CP1, resistance is no more dominant than in the single-step CP-r KB cell line, KB-CP.5, suggesting that one of the two steps of resistance in KB-CP1 may not be dominant. These dominance data suggest that it might be possible to identify one or more genes responsible for cisplatin resistance by gene transfer from a resistant cell line to a sensitive cell line.     

Selection of mouse x hamster hybrids using hat medium and a polyene antibiotic

Summary In the present study we tested the feasibility of utilizing a structurally modified polyene antibiotic, amphotericin B methyl ester (AME), as a half-selection agent for isolating somatic cell hybrids. By using HAT medium supplemented with AME we have isolated interspecific mouse-hamster hybrids from mixed cultures of mouse (TK⁻C1ID or HPRT⁻ A9) and hamster (BHK/C 13) cells fused with Sendai virus, lysolecithin or polyethylene glycol. Hybrid cells proliferated and clones were isolated after 2 to 3 weeks growth in three changes of HAT-AME medium and subsequent growth in HAT medium alone. In contrast, genetically deficient parental C1 1D or A9 cells and AME-sensitive BHK/C 13 cells were killed using a similar growth protocol. The described technique is simple, efficient and permits one to use a cell line without a genetic defect in combination with a genetically deficient cell type in hybrid formation. This investigation was supported in part by Contract NIH 69-2161, NIH Grant No. AI-2095 and NIH Training Grant No. GM 507 from the National Institute of General Medical Sciences.     

Studies on 1-β- -arabinofuranosyl-cytosine (Ara-C) resistant mutants of Chinese hamster fibroblasts : II. High resistance to Ara-C as a genetic marker for cellular hybridization

From a variety of independent Chinese hamster cell lines, stable variants resistant to 5 μg/ml of Ara-C were isolated via a single step selection; in contrast to variants selected at lower drug concentrations, the resistant clones appear to be uniformly deficient in Ara-C phosphorylation, an activity previously shown [14] to be carried out in hamster cells by a cytoplasmic dC-kinase (dC-kinase 2). These dC-kinase deficient (dCK⁻) variants can be selected against because they are unable to divide in a medium containing dT (0.8 mM) and dC (0.01 mM), which supports the growth of wild type dCK⁺ cells. Plating of dCK⁻ cells in medium supplemented with both nucleosides yields only rare clones of pseudorevertants which escape the thymidine block through a secondary unknown defect; the growth of these clones can be prevented by further addition of dA to the selective medium. As expected from the complementation pattern for the deficient enzyme activities, hybrids between a dCK⁻ hamster line and TK⁻ lines of either mouse or hamster could be isolated in a modified HAT medium (HAT₅₀dC) containing dC and an increased dT concentration. In principle, the same selection can be used to isolate interspecific and intraspecific hybrids between Ara-C resistant variants obtained from a variety of mammalian species and azaguanine resistant lines deficient in HGPRT. The potential interest of this sytem for genetic mapping is discussed.     

Retention of mitochondrial DNA species in somatic cell hybrids using antibiotic selection

Interspecific cell hybrids were constructed by fusion of an antimycin-resistant, thymidine kinase- (TK-) Chinese hamster cell line with a chloramphenicol-resistant, hypoxanthine-guanine phosphoribosyl transferase- (HPRT-) mouse cell line. Hybrids were selected in HAT medium alone, or HAT supplemented with chloramphenicol, antimycin, or both antibiotics. Analysis of the mitochondrial DNA (mtDNA) of these hybrids indicates that antibiotic selection directed at the mitochondrial populations results in retention of the resistant parental genome and loss of the sensitive parental genome.     

Amphibian cells in culture. II. Isolation of drug-resistant variants and an asparagine-independent variant.

With L-15 as the base medium, drug-resistant variants were isolated from two amphibian tissue culture strains: the Xenopus laevis A8 diploid cell line and the ICR 2A cell line of Rana pipiens. Four different classes of variants were obtained: (1) A8 cells resistant to chloramphenicol, an inhibitor of mitochondrial protein synthesis; (2) A8 cells resistant to ouabain, an inhibitor of the Na+/K+-activated ATPase of the plasma membrane;(3) ICR 2A cells resistant to low (20 microgram/ml) and high (300 microgram/ml) levels of bromodeoxyuridine (BUdR), a thymidine analog which interferes with the pyrimidine salvage pathway; and (4) ICR 2A cells resistant to 2,6-diaminopurine (DAP), an adenine analog which interferes with the purine salvage pathway. Unlike the other variants, isolation of BUdR resistant cells is a 2-step process. Resistance to low levels of BUdR is phenotypically expressed by a reduction in thymidine transport activities while resistance to high levels of this compound is evidenced by greatly reduced levels of thymidine kinase activity. DAP-resistant cells, which are characterized by reduced levels of adenine phosphoribosyl transferase (APRT) activity, do not die in AAT (adenine, aminopterin, thymidine) selection medium. This suggests that these cells utilize adenine efficiently. With MEM as the base medium, an asparagine independent clone was isolated from the ICR 2A cell line. When compared with the wild type, this variant exhibited a slightly reduced growth rate in the presence or absence of asparagine.     

Enhanced thymidine uptake causes the lowered thymidine requirement of D. discoideum auxotroph HPS 401

Dictyostelium discoideum strain HPS 401 contains a spontaneous mutation that lowers the amount of thymidine required for cell growth relative to that of the auxotrophic parental strain HPS 400. Growth studies in defined medium show that as little as 8 micrograms thymidine/ml supports maximal growth of HPS 401, whereas 50 micrograms/ml is required by HPS 400. In contrast, both strains require over 40 micrograms thymidylate/ml to achieve maximal growth. HPS 401 exhibits thymidineless death when grown without thymidine; relative viability decreases to less than 0.01% after 190 h incubation. Assays for enzymes related to thymidine metabolism reveal that none of the strains tested (HPS 401, HPS 400, and prototrophic HPS 83 cells) contain detectable thymidine phosphorylase activity and that the specific activity of thymidine kinase is the same in these three strains. Thin-layer chromatography of extracts from cells grown on radiolabeled thymidine shows that there is no detectable conversion of thymidine to thymine in any of these strains. These analyses show that HPS 401 has rapid intracellular accumulation of thymidine, while only slight uptake is observed with HPS 400 or wild-type strains. HPS 401 also shows greater uptake of uridine in comparison to HPS 400 and wild-type cells. Thymidylate uptake was the same for all three strains. Thus, the mutation giving rise to the HPS 401 phenotype selectively increases the uptake of thymidine into the cell, where it can be efficiently utilized for DNA synthesis by the "salvage" pathways of nucleotide metabolism.     

Gene mapping in marsupials and monotremes. II. Assignments to the X chromosome of dasyurid marsupials

Somatic cell hybrids have been obtained between HPRT Chinese hamster cells and cells from several dasyurid marsupial species. These hybrids show the extensive loss of marsupial chromosomes characteristic of the majority of marsupial-eutherian somatic cell hybrids. Although all of the hybrids expressed the selected marsupial marker, HPRT, the only other markers observed were PGK, GLA, and G6PD, consistent with the conservation of X-linked genes extending to this major group of marsupials. Counterselection confirmed the synteny of PGK and GLA with HPRT, whereas G6PD showed decreased concordance.     

Cytoplasmic inheritance in mammalian tissue culture cells.

A series of intraspecific, interspecific and interorder somatic cell cybrids and hybrids have been prepared by fusions in which one of the parents contained the cytoplasmically inherited marker for chloramphenicol (CAP) resistance. A clear relationship has been established between the expression of the CAP-resistant (CAP-R) determinants in the fusion products and the genetic homology of the parents. With increased genetic divergence, the acceptability of the CAP-R mitochondria decreased. Intraspecific cybrids and hybrids of the same strain were stable for the CAP-R marker, while those between strains were stable only in CAP. Intergeneric mouse-hamster cybrids occurred at a high frequency but were unstable in CAP, while CAP suppressed hybrid formation 100-fold. Interorder cybrids (CAP-R human X CAP-S mouse) occurred either at a moderate frequency and were stable at a low frequency and were unstable in CAP. Interorder hybrids could only be formed by challenging HAT-selected hybrids with CAP or by direct selection in ouabain and CAP. Reciprocal interorder crosses between CAP-R mouse and CAP-S human cells were unsuccessful. Interspecific cybrids contain only the chromosomes of the CAP-S parent. Interspecific hybrids selected directly in CAP segregated the chromosomes of the CAP-S parent, while hybrids selected in HAT and then CAP segregated those of the CAP-R parent. The mitochondrial DNA(mtDNA) of all mouse-human cybrids and most HAT and then CAP-selected hybrids contain only the mtDNA of the CAP-S mouse parent. However, preliminary evidence suggests that one of these hybrids contains both mouse and human mtDNA sequences.     

Thymidine Utilization by tut Mutants and Facile Cloning of Mutant Alleles by Plasmid Conversion in S. CEREVISIAE

Plasmid pJM81 contains a Herpes simplex virus thymidine kinase (TK) gene that is expressed in yeast. Cells containing the plasmid utilize thymidine (TdR) and the analogue 5-bromodeoxyuridine (BUdR) for specific incorporation into DNA. TdR auxotrophs, harboring plasmid pJM81 and a mutation in the yeast gene TMP1 require high concentrations of TdR (300 micrograms/ml) to support normal growth rates and the wild-type mitochondrial genome (rho+) cannot be maintained. We have identified a yeast gene, TUT1, in which recessive mutations allow efficient utilization of lower concentrations of TdR. Strains containing the mutations tmp1 and tut1, as well as plasmid pJM81, form colonies at 2 micrograms/ml TdR, grow at nearly normal rates and maintain the rho+ genome at 50 micrograms/ml TdR. These strains can be used to radiolabel DNA specifically and to synchronize DNA replication by TdR starvation. In addition, the substitution of BUdR for TdR allows the selective killing of DNA-synthesizing cells by 310-nm irradiation and allows the separation of replicated and unreplicated forms of DNA by CsCl equilibrium density banding. We also describe a unique, generally applicable system for cloning mutant alleles that exploits the fact that Tk+ yeast cells are sensitive to 5-fluorodeoxyuridine (FUdR) and that gene conversions can occur between a yeast chromosome and a TK-containing plasmid.     

Co-dominant markers for the selection of somatic dihybrids and polyhybrids

Resistance to ouabain and puromycin are shown to represent very useful co-dominant characters for the selection of somatic hybrids between mammalian cells, after fusion with polyethylene glycol. We therefore used, with success, a number of Chinese hamster and mouse cell lines carrying these markers in association with thymidine kinase and hypoxanthine-guanine-phosphoribosyl transferase deficiency for selection of hybrids of triparental origin.     

State of nucleolus organizers in hybrids of pluripotent and somatic mouse cells cultivated under different conditions

In the present work, we studied the state of chromosomal nucleolar organizing regions (NOR) in hybrid cells obtained by fusion of cells of embryonal carcinoma of a murine line PCC4aza1 and of cells of adult mouse spleen at cultivation of hybrids under different conditions. The obtained results have shown that long-term cultivation of hybrid cells in a selective medium containing HAT (hypoxanthine, aminopterin, thymidine) promotes preservation of nucleolar organizing chromosomes (NO chromosomes), whereas in nonselective medium predominant elimination of NO chromosomes was revealed. Under nonselective conditions, an increased number of active, i.e., Ag-positive, NORs was observed as compared to under selective conditions. These observations directly show that reprogramming of parent cell genomes in hybrids includes changes in the state of NO chromosomes. The number of active NORs depends on the conditions of cultivation of hybrid cells and can change in two main ways, i.e., by the elimination of NO chromosomes (under nonselective conditions) or by the inactivation of some NORs with maintenance of NO chromosomes (under selective conditions).