Hypoxanthine-aminopterin-thymidine Research Articles

Generation of bovine intraspecies hybridomas with initial suppressed growth

Bovine B-cell hybrids were generated by fusing pokeweed mitogen (PWM) activated normal bovine peripheral blood lymphocytes (PBL) with an aminopterin-sensitive bovine B-cell line BL20. The fusion partner was derived by irradiation and growth in medium containing increasing concentrations of 6-thioguanine (6TG). Resultant cells were designated ATS/BL20. Polyethylene glycol-induced (PEG) fusion was used and hybrids were selected in hypoxanthine-aminopterin-thymidine (HAT) medium. Hybrid cell growth was noticed after 4 to 6 weeks of fusion following a period of quiescence. Hybrid formation was confirmed by selection in HAT medium, expression of cytoplasmic IgM (cIgM) and surface antigens and karyotype analysis.

Establishment of 8-azaguanine-resistant mutants from rat thyroid cell line FRTL-5

8-Azaguanine (AG)-resistant mutant clones were isolated from FRTL-5 rat thyroid cells, by treating them with ethylmethane sulfonate for 48 h and then, after the recovery period, by selecting them in AG-containing medium. Isolated mutants were further selected in 6-thioguanine-containing medium, and two mutant clones were finally obtained (AGI and AG2). The mutants retained several thyroid stimulating hormone (TSH) responsiveneses following acquisition of AG resistance: they responded to bovine TSH (bTSH), by producing cyclic 3',5'-adenosine monophosphate (cAMP) and thyroglobulin, and taking up iodine. However, TSH-responsive profiles were slightly different from each other in several points. They were killed in hypoxanthine-aminopterin-thymidine (HAT)-selective medium and applied to cell hybridization study with human thyrocyte from a Graves' patient: the hybrid turned out to produce both rat and human thyroglobulin even 12 months after the hybridization.

Retention of cyclic AMP response to TSH in a cloned human thyrocyte/ T cell hybridoma (HY 2–15)

Our observation of a human T cell leukemia cell (Molt 4) demonstrating low affinity thyroid-stimulating hormone (TSH) responses, as evidenced by generation of cyclic AMP, led us to test Molt 4 cells as a suitable partner for immortalizing high affinity TSH receptors present on human thyroid cells. Therefore, we generated a hybridoma (HY 2–15) by a fusion between thyroid monolayer cells from a patient with Graves' disease, and a hypoxanthine-aminopterin-thymidine (HAT)-sensitive variant of this human T cell leukemia line, Molt 4–8 AG R. The hybrid nature of HY 2–15 was confirmed by DNA histograms using propidium iodide and flow cytometry. Karyotyping showed the HY 2–15 cells to have five sets of chromosomes and human leukocyte antigen (HLA) class I determination revealed the presence of an additional HLA class I antigen (A2) not present on the Molt 4 partner cells. The established, cloned, hybridoma cells showed a > 30-fold increase in cyclic AMP release after stimulation with bovine TSH (bTSH, 1 mU/ml) with a minimum detectable stimulating dose of < 10 μU/ml bTSH. However, no other thyroid-specific functions could be detected. Furthermore, HY 2–15 cells failed to express HLA class II antigens either constitutively or in response to recombinant human gamma Interferon (IF) and a variety of other stimuli, data similar to the Molt 4 partner cells but in contrast to human thyroid cells which show high sensitivity to gamma IF. The preservation of highly sensitive TSH responsiveness in a proliferating cell offers a unique approach to the study of human TSH receptor function

Production of murine hybrid-hybridomas secreting bispecific monoclonal antibodies for use in urease-based immunoassays.

To produce bispecific antibodies (BiAbs) for enzyme immunoassay (EIA) to replace antibody-enzyme conjugates, we developed a panel of 8-azaguanine/ouabain-resistant anti-urease variant hybridoma cell lines for use in hybridoma-hybridoma fusions. These variants represent mouse immunoglobulin subclasses IgM, IgG1, IgG2a, and IgG2b and have growth rates equal to those of the parental hybridomas. We fused an anti-urease-secreting variant hybridoma with an anti-human choriogonadotropin (hCG)-secreting hybridoma (both of IgG1 subclass) and selected the desired product with growth media containing hypoxanthine-aminopterin-thymidine (HAT) and ouabain. Over 95% of the resulting hybrids secreted anti-urease, and 60% of these secreted anti-hCG. The bispecific nature of secreted antibodies was demonstrated in a simultaneous EIA where BiAbs, hCG, and urease (EC 3.5.1.5) were incubated together in anti-hCG-coated microwells. As little as 25 int. units of hCG per liter could be reliably detected, which is equivalent to that for antibody-enzyme conjugates in EIA.

Phenotypic cyclophosphamide resistance in mouse myeloma hybridomas.

We have analyzed the expression of cyclophosphamide (CY) resistance in somatic cell hybrids of mouse LPC-1/CY-R plasmacytoma and P3-X63-Ag8.653 (Ag8) myeloma cells. LPC-1/CY-R tumor is resistant to curative doses (60-250 mg/kg body weight) of CY. The median survival time (MST) of drug treated LPC-1/CY-R tumor bearing mice is 25 days, similar to that of untreated mice. LPC-1/CY-R tumor cells secrete an IgG 2a kappa M component and do not survive in tissue culture. Ag8 tumor cells are CY sensitive, are selected out in hypoxanthine-aminopterin-thymidine (HAT) media and do not secrete any immunoglobulin. Hybrids formed between these two cell lines survived in HAT and secreted IgG 2a kappa. Hybrid cells exhibited greater ploidy than that of their parents and contained the metacentric marker chromosome of the Ag8 cell line. Hybrid cells exhibited the same growth characteristics in BALB/c mice as that of their LPC-1/CY-R parent and were resistant to curative doses of CY. These studies demonstrate that, CY resistance is a somatic cell dominant trait which can be transferred to daughter cells via somatic cell hybridization. Availability of somatic cell hybrids produced between CY sensitive and resistant tumor cells may provide useful tools to study the biochemical nature and the somatic cell genetics of this drug resistant trait.

Exaggerated triglyceride accretion in human preadipocyte-murine renal line hybrids composed of cells from massively obese subjects.

To learn about adipose differentiation of precursors from postnatal adipose tissue of lean and massively obese subjects, human omental adipocyte precursor-murine renal adenocarcinoma cell (RAG) hybrids were formed by fusion with polyethylene glycol, and cultured selectively with 50 microM ouabain in hypoxanthine aminopterin thymidine (HAT) medium. Under conditions in which the parent cells did not differentiate, a number of hybrids, which were cloned, revealed morphologic and biochemical evidence of differentiation. In addition to activation of human genes within the common nucleus of the hybrids, murine cytoplasmic activators are probably also involved because heterocaryons (fused cells with two interspecific nuclei) revealed the same phenomenon. Hybrids composed of precursors from massively obese subjects disclosed more frequent and prominent differentiation. Since these hybrids, in contrast to those from the lean, recapitulate this phenomenon in subcultures, they provide the potential system for mapping the human gene(s) responsible for adipose differentiation and its exaggeration in massive obesity.

Lack of complementation in somatic cell hybrids between fibroblasts from patients with different forms of cystinosis.

Cystinosis is an autosomal recessive disease in which three clinical forms are recognized: infantile nephropathic, with renal tubular damage by 1 year of age and progressive glomerular insufficiency; intermediate, with tubular and glomerular insufficiency beginning at a later age; benign, with no kidney damage. Skin fibroblasts cultured from patients with all types of cystinosis show increased intralysosomal free (nonprotein) cystine; however, fibroblasts from heterozygotes have normal free-cystine values. To determine whether genetic complementation occurs between the different forms, somatic cell hybrids were constructed between cells from a patient with infantile nephropathic cystinosis and cells from patients with other types of cystinosis. If complementation occurred, the hybrids would be expected to have normal cystine levels. To construct hybrid cells, a "universal parent" cell type (TG1-neo), which was hypoxanthine/aminopterin/thymidine (HAT) sensitive and G418 resistant was constructed from an infantile nephropathic cystinosis fibroblast strain. Polyethylene glycol fusion of TG1-neo with other cells that are not HAT sensitive or G418 resistant allowed for selection of hybrid cells in a medium containing HAT and the aminoglycoside G418. As indicated by elevated cystine levels, complementation did not occur between TG1-neo and two different benign cystinosis strains, an intermediate cystinosis strain, or another nephropathic cystinosis cell strain. When a normal fibroblast strain was fused with TG1-neo, all 15 hybrid clones studied contained normal amounts of intracellular free cystine.

Spontaneous fusion between metastatic mammary tumor subpopulations.

This study describes a differential frequency of spontaneous fusion between metastatic and nonmetastatic subpopulations derived from a single mouse mammary tumor. Subpopulations 66, 66c14 (a variant of 66 which is resistant to both thioguanine and ouabain), 410.4, and 44FTO (a thioguanine-resistant, ouabain-resistant derivative of 410.4) spontaneously metastasize from subcutaneous and mammary fatpad sites. Subpopulations 168, 168FARO (a diaminopurine-resistant, ouabain-resistant derivative of 168), 67, 68H, and 410 do not. The ability of these subpopulation lines to fuse spontaneously in vitro was determined after coculturing a drug-resistant line with a wild-type line in nonselective media. After 16-20 h of coculture, cells were plated in the appropriate media to select for fusion products--either HAT (hypoxanthine, aminopterin, thymidine) plus ouabain or AA (alanosine, adenine) plus ouabain--to determine the number of colony-forming cells (fusion products) present per 10(4) cells plated. When both subpopulations of the pair in the fusion mixture were metastatic, a significantly greater number of fusion products was recovered than if one or both of the subpopulations in the fusion mixture was nonmetastatic, with one exception: line 410 readily fused with both 66c14 and 44FTO. Subline 410 was highly metastatic when originally isolated but lost its metastatic competence after a brief time in tissue culture.

Electrical fusion of protoplasts from sycamore mutants and hybrid selection on hypoxanthine-aminopterin-thymidine (HAT) medium

An electrical fusion method has been used to form somatic hybrids between protoplasts of two mutant cell lines of sycamore tissue culture cells. Both mutants will not grow in a hypoxanthine-aminopterin-thymidine (HAT) medium. It was possible to select the fused hybrids from homospecific fusion products and nonfused protoplasts by the use of HAT medium. In this way the viability and regeneration of the fused cells during the first few weeks of culture could be evaluated. An electron microscopic examination of the fusion process showed that it occurred at a series of points along the surface of the plasmalemma. Cytoplasmic bridges between the two cells were formed separated by vesicles which later dispersed to give complete cytoplasmic continuity between the cells.

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.

Cloned cell lines from a transplantable islet cell tumor are heterogeneous and express cholecystokinin in addition to islet hormones.

A liver metastasis (MSL) with a remarkable in vitro proliferation potential has been identified in an NEDH rat carrying a transplantable x-ray-induced islet cell tumor. Two insulin-secreting cell lines, MSL-G and MSL-H, with doubling times of 3-5 d were established by repeated limiting dilution cloning. In vivo inoculation of MSL-G cells induced severe hypoglycemia caused by a small but highly heterogeneous tumor as revealed by immunocytochemistry. Whereas most cells stained for the islet hormones, insulin, glucagon, and somatostatin, clustered cells were discovered to contain cholecystokinin (CCK). Additional in vitro-limiting dilution cloning, followed by immunocytochemical characterization, clearly demonstrated the capacity of single cell clones to simultaneously express the same four hormones. Radioimmunoassays with a panel of site-specific antisera of culture supernatants and purified cell extracts showed the MSL-G2 cells to produce, store, and secrete readily detectable amounts of processed and unprocessed CCK. Gastrin was not detected while coexpression of glucagon and CCK were demonstrated. Mutant clones selected for resistance to 6-thioguanine (frequency, 2 X 10(-7] and checked for HAT (hypoxanthine, aminopterin, thymidine) sensitivity retained the capacity for multi-hormone expression. We propose that the MSL tumor contains pluripotent endocrine stem cells. The MSL tumor and the MSL-G2 cells in particular will allow studies of not only CCK biosynthesis and processing but also of mechanisms involved in tumor and islet cell differentiation.

Enhanced formation of mouse hybridomas without hat treatment in a serum-free medium.

A newly developed, serum-free medium (NYSF-404) selects for antibody-producing hybridomas after fusion of antigen-sensitized mouse spleen cells with myeloma cell lines P3-X63-Ag8-U1 (P3-U1), P3-X63-Ag8-6.5.3 (Ag8.653), or P3-NSI/1-Ag4-1 (NS-1). Without the need for hypoxanthine-aminopterin-thymidine (HAT) selection of hybrid cells, frequency of hybridoma formation in medium NYSF-404 is higher (twice) than that in serum- and HAT-containing medium. Colonies developed upon limiting dilution in the presence of the mortal parent myeloma cells in medium NYSF-404 and pure culture of antibody-secreting cells could be subsequently established. The results suggest that fusions can be done in serum-free medium and that the clonal growth of hybridomas is dependent on factors produced by parent myeloma cells under serum-free culture conditions. Such factors seem deficient in serum- and HAT-containing medium or are masked by serum.

Dissociation of cellular transformation from platelet-derived growth factor independence.

ST2-3T3, a spontaneously transformed BALB/c-3T3 cell line which does not require platelet-derived growth factor (PDGF) for growth, was fused to THO2, a PDGF-responsive non-transformed BALB/c-3T3 cell line, in order to learn whether transformation is expressed coordinately with PDGF independence. Hybrid cells were selected and grown in medium containing both HAT (hypoxanthine-aminopterin-thymidine) and ouabain; unfused cells of each parental type were killed in HAT-ouabain medium. Five independently isolated ST2-3T3xTHO2 hybrid cell lines were established and characterized for both transformation and PDGF responsiveness. All five were transformed, having a disorganized growth pattern and achieving a final cell density similar to that of ST2-3T3 cells. Two of these lines did not respond to a brief treatment with PDGF: the mitogen neither induced the synthesis of a PDGF-modulated lysosomal protein (termed MEP), nor stimulated the cells to enter the S phase; one line responded to PDGF by synthesizing both MEP and DNA, whereas two others synthesized MEP but not DNA. In contrast, four independently isolated cell lines obtained by fusing PDGF-responsive non-transformed BALB/c-3TC cells to the THO2 line were all PDGF-responsive for both MEP and DNA synthesis and were not transformed. It appears that PDGF independence is not required for the transformation of BALB/c-3T3 cells.

Isolation and characterization of rat-mouse somatic cell hybrids secreting growth hormone and prolactin

Interspecific somatic cell hybrid clones have been isolated and characterized in order to study growth hormone (GH) and prolactin (PRL) gene expression. Rat pituitary tumor cells (GH 3, 69 chromosomes) secreting rat GH and PRL were grown for 48 h together with non-hormone secreting, aminopterin-sensitive murine fibroblast cells (LMTK −, 55 chromosomes) and fused using polyethylene glycol. Resultant heterokaryons were selected in hypoxanthine-aminopterin-thymidine (HAT) medium and cloned. Five clones produced rat GH and PRL. Hormone-producing hybrids morphologically resembled the mouse parent fibroblast. Hybrids grew in monolayers and contained 80–142 chromosomes, and marker chromosomes for both rat (small submetacentric) and mouse (bi-armed and large true metacentric) were identified. The interspecific nature of the hybrids was further confirmed by the presence of both rat and mouse adenosine deaminase and superoxide dismutase isozymes. Using specific antisera and indirect immunoperoxidase staining, both hybrid clones and GH 3 rat parental cells stained positively for rat GH and PRL, while the murine fibroblast parental cells were negative. Hormone production by the hybrids has been sustained for over twenty subcultures; secretion rates were initially 150 ng PRL and 321 ng GH/10 6 cells/24 h and are currently 100 ng PRL and 90 ng GH/10 6 cells/24 h. Parental GH 3 rat cells secreted 720 ng PRL and 660 ng GH/10 6 cells/24 h. Exposure of hybrids to KCl (50 mM) resulted in acute stimulation of rat PRL, but not rat GH release, and long-term incubation with thyrotropin-releasing hormone (TRH, 80 nM) stimulated PRL secretion. Hormone-dependent modulation of PRL secretion was transferred to the hybrid cell thus enabling the model to be used in studying regulation of PRL gene expression.

Human monoclonal antibodies against Mycobacterium leprae.

Human hybridomas were constructed which produce antibodies against three different extracts of Mycobacterium leprae. A thioguanine-resistant (Thgr), ouabain-resistant (Ouar), human lymphoblastoid cell line, KR-4, was hybridized with Epstein-Barr virus-transformed cell lines from lepromatous leprosy patients with fusion frequencies of greater than 10(-5). Non-Epstein-Barr virus-transformed donor cells fused at much lower rates (less than 2 X 10(-7]. Hybrids were selected in medium containing hypoxanthine aminopterin thymidine and 10(-5) M ouabain. An enzyme-linked immunosorbent assay was used to screen for antibodies against three crude extracts of armadillo-derived M. leprae, including (i) a soluble sonic extract preparation, (ii) sodium dodecyl sulfate extract of insoluble sonicated M. leprae, and (iii) a purified phenolic glycolipid antigen. Of a total of 2,200 final clones screened, 359 were found to secrete antibody which bound to soluble sonic extracts and the sodium dodecyl sulfate extract (6.7 and 9.6%, respectively), whereas 12.5% (21 out of 168) showed positivity to the glycolipid antigen. Four selected hybridomas also reacted with the deacylated derivative of M. leprae phenolic-glycolipid antigen. The specificity of these monoclonal antibodies was partially determined by screening on a panel of crude extracts from four other mycobacteria. Nine clones of 122 showed reactivity to M. leprae only. The predominant immunoglobulin was immunoglobulin M, and quantities up to 10 micrograms/ml were produced. Antibody production by hybrid clones was stable in more than 75% of the clones grown in continuous culture. By comparison, 10,000 Epstein-Barr virus-transformed lymphocyte clones from lepromatous leprosy patients were screened for anti-M. leprae antibody production, and all of the 42 clones that were initially positive in the enzyme-linked immunosorbent assay lost their antibody-producing capabilities within 6 weeks in culture. These results suggest that a combination of Epstein-Barr virus transformation and hybridization may be an optimal method in producing human monoclonal antibodies from leprosy patients.

The influence of murine macrophage-conditioned medium on cloning efficiency, antibody synthesis, and growth rate of hybridomas

Murine B-cell hybridomas made with the P3X63-AG8.653 myeloma showed increases in cloning efficiency and efficiency of growth in hypoxanthine-aminopterin-thymidine (HAT) medium of 50–100-fold in the presence of medium conditioned by primary mouse peritoneal macrophages (MCM). Similar effects were elicited by MCM from 3 continuous macrophage lines. The J774A.1 line conditioned the medium as efficiently as primary macrophages without induction. Conditioning by the P388D 1 line was several-fold less efficient, but could be increased by treating the cells with Escherichia coli lipopolysaccharide. By contrast, the BJ-1 macrophage line required treatment with the lipopolysaccharide to induce expression of the hybridoma growth factor(s). Four commercially available serum supplements could not substitute for MCM, but addition of MCM and the supplements together stimulated the growth rate of hybridomas in media with 4% or less fetal bovine serum. The rate of antibody synthesis paralleled the growth rate, and the amount of antibody synthesized per cell was approximately the same for hybridomas grown in medium supplemented with MCM or adapted to growth in the absence of MCM. The results indicate that MCM has advantages as an alternative to ‘feeder cells’ and serum supplements in hybridoma cultures, and suggest that MCM may be useful for hybridoma culture at reduced serum concentrations. The nature of the soluble factor(s) in MCM which promote these effects remains unknown.

A simple panning method for the selection of cell surface antigen transfectants

Mouse Ltk − (H-2 k) cells that became reactive with anti-H-2 d monoclonal antibodies (McAb) after transfection with DNA from H-2 d-positive cells were isolated by an indirect panning method. The cells were transfected with DNA from a plasmid containing the selective thymidine kinase (tk) gene in addition to total cell DNA, so that a first selection could be carried out in hypoxanthine/aminopterin/thymidine (HAT) medium. The HAT-selected tk + transfectants were incubated with anti-H-2 d McAb, washed and transferred to dishes coated with purified anti-immunoglobulin (anti-Ig). This 2-step method of panning has the advantage that only the anti-Ig reagent requires purification. Transfected cells clearly reactive with either anti-L d/R d or anti-K d McAb were isolated after only 1 or2 cycles of panning.

Activity of X-linked genes in stem and differentiated Musmusculus × Muscaroli hybrid cells

Hypoxanthine phosphoribosyltransferase-deficient (HPRT −) F9-derived teratocarcinoma stem cells carrying an SV40 genome (12-16TG cells) were fused with Mus caroli (M. car.) spleen cells, and a stem cell hybrid containing reduced numbers of M. car. chromosomes was isolated (BC6 stem cell). The BC6 cells containing an active X chromosome from each parental cell were induced to differentiate in retinoic acid, and differentiated clones were isolated. Most differentiated clones retained both parental X chromosomes in active form. One differentiated clone, BC6-13, grew equally well in hypoxanthine / aminopterin / thymidine (HAT) selective medium (which requires an active M. car. HPRT (E.C.2.4.2.8) locus) or in 6-thioguanine (6TG, which would require either loss or inactivation of the M. car. HPRT locus). Using cDNA probes for HPRT and phosphoglycerate kinase (PGK) (E.C.2.7.2.3) loci and biochemical assays for HPRT and PGK enzymes, it was shown that BC6-13 cells, whether grown in nonselective medium, HAT medium, or 6TG-containing medium, retain the HPRT and PGK genes of both parental cells, but the M. car. forms of HPRT and PGK were inactivated in cells treated with 6TG. 6-Thioguanine seems to act as an inducer, one effect of which is X chromosome inactivation, which seems to be complete and irreversible as early as 24 h after addition of 6TG to BC6-13 cells.

Cell-surface antigens expressed on L-cells transfected with whole DNA from non-expressing and expressing cells.

We have shown previously that transfection of mouse L-cells with DNA from JM, a human T-cell line expressing certain T-cell differentiation antigens, yields stable transfectants expressing one or another of these antigens. The identities of the antigens were confirmed by immunoprecipitation and SDS-polyacrylamide gel electrophoresis. We now report that our procedure--co-transfection with the chicken thymidine kinase gene (tk) and whole cellular DNA, selection with hypoxanthine-aminopterin-thymidine (HAT), and staining of the cells with fluorochrome-conjugated monoclonal antibodies and fluorescence-activated cell-sorter (FACS) selection--yields transfectants expressing a variety of cell-surface molecules (19 of 21 investigated), most at a frequency of about one per 10(3) Tk+ transformants. Of these, 9 of 12 were transferred and expressed as readily using DNA from cells which did not express the cell-surface antigens as from tissues or cells that did express them.

Gene transfer, expression, and molecular cloning of the human transferrin receptor gene

We describe the molecular cloning of the human transferrin receptor gene by a gene transfer approach. Mouse Ltk − cells were cotransformed with the herpes simplex thymidine kinase gene and total human DNA. Transformants expressing human transferrin receptor were isolated by selection on hypoxanthine/aminopterin/thymidine (HAT) medium and fluorescence-activated cell sorting of HAT-resistant cells. Thirty-four kilobases of human DNA was isolated by screening a genomic library constructed from the DNA of a secondary transformant. Gene transfer of the cloned DNA established that 31 kb of DNA was sufficient to encode the receptor. A probe from the 5′ end of the gene was used to isolate a cDNA clone with an insert of 4.9 kb. Hybridization of the cDNA to the cloned genomic DNA revealed a minimum of 12 exons. They extend over the entire 31 kb of expressing DNA and over 2 kb of adjacent 3′ untranslated sequences that are not required for receptor expression in L cells.