Lymphoid Lines Research Articles

Methylation of Herpesvirus saimiri DNA in lymphoid tumor cell lines

Several continuous lymphoid cell lines have been established from tumors induced by Herpesvirus saimiri. At least a portion of the viral DNA in the marmoset lymphoid cell line 1670, which does not produce detectable virus, is present as covalently closed circular episomal DNA. The use of restriction endonuclease digestion, transfer to nitrocellulose filters, and hybridization of the virus-specific DNA has produced strong evidence that viral DNA sequences present in total 1670 cell DNA and in isolated episomes are extensively methylated. The restriction endonuclease Hpa II has the same recognition sequence as Msp I but, unlike Msp I, fails to cleave when the C of the C-G dinucleotide is methylated. Viral DNA sequences of 1670 cells are refractory to cleavage by Hpa II but not Msp I; greater than 80% of the Hpa II cleavage sites appear to be methylated. Similarly, viral DNA sequences of 1670 cells are refractory to cleavage by Sma I (C-C-C-G-G-G) and Sac II (C-C-G-C-G-G) but not Sac I, Pvu II, or Pst I, which lack the dinucleotide C-G in their recognition sequences. Methylation of mammalian DNA has been previously found exclusively at C residues in the dinucleotide C-G. H. saimiri DNA sequences of another nonproducer cell line, 70N2, also appeared to be extensively methylated, but analysis of total cell DNA extracted from three virus-producing lymphoid lines revealed no evidence of methylation of viral DNA sequences. It remains to be seen if methylation of viral DNA plays a role in the lack of complete expression of H. saimiri genome information in nonproducing lymphoid cell lines.

The Immune Response to Vasectomy and its Relation to the HLA System

We examined 188 men who had undergone vasectomy up to 6 years previously and, for comparative purposes, 100 men who were about to undergo the operation. Blood specimens were available from a total of 283 men. Sperm antibody assays using immunofluorescence, microagglutination, and microimmobilization confirmed that the prevalance of several types of antibody is higher after vasectomy. Immobilizing and agglutinating antibodies appeared to be the most important. Trends in antibody prevalence according to the time after the operation were analyzed. Screening against lymphocytes and lymphoid lines showed that the anti-sperm activity of era was not related to anti-HLA or anti-Ia activity. Associations were examined between different types of sperm antibody, and between these antibodies and autoantibodies to other antigens. When antibody prevalence was studied in relation to HLA types, the HLA antigen A28 was found to be strongly associated with production of head agglutinating antibody (and immobilizing and immunofluorescent equatorial antibodies) after vasectomy. This is one of the first clear-cut examples of antibody production associated with the HLA system.

Phosphofructokinase in human blood cells

The subunit composition of phosphofructokinase from normal and malignant blood cells has been investigated by means of immunologic, electrophoretic, and chromatographic methods. Immunoprecipitation tests were performed with three specific antisera recognizing each of the basic subunits of human phosphofructokinase: muscle, M-type; liver, L- type; and fibroblast, F-type. Mature polymorphonuclear cells contain mainly L-subunits, while lymphocytes and platelets contain hybrids formed of L and F subunits; these hybrids can be electrophoretically separated. Red cell phosphofructokinase is composed of L and M subunits, as judged by its reactivity with anti-L and anti-M-type antisera. The various M-L hybrids composing red cell phosphofructokinase could be only separated by chromatography on DEAE- Cellulose. Lymphocytes from patients with chronic lymphocytic leukemia and lymphoblasts from patients with acute lymphoblastic leukemia contain phosphofructokinase forms similar to those from normal lymphocytes, while the immature granulocytic cells (leukemic myeloblasts and myeloid cells of chronic myeloid leukemia) are characterized by a reinforcement of enzyme inhibition by anti-F-type antiserum. Lymphoid lines in culture (Epstein-Barr virus (EBV)-induced or malignant lymphoma-derived lines) are characterized by the indistinctive expression of all three basic subunits, similar to that found in some fetal tissues. This article represents the first description of the isozymic nature of phosphofructokinase in platelets and white blood cells and of its changes with malignancy and cell culture. This enzyme might represent a useful marker in the characterization of the leukemic cells.

Phosphofructokinase in Human Blood Cells

The subunit composition of phosphofructokinase from normal and malignant blood cells has been investigated by means of immunologic, electrophoretic, and chromatographic methods. Immunoprecipita-tion tests were performed with three specific antisera recognizing each of the basic subunits of human phosphofructokinase: muscle, M-type; liver, L-type; and fibroblast, F-type.Mature polymorphonuclear cells contain mainly L-subunits, while lymphocytes and platelets contain hybrids formed of L and F subunits; these hybrids can be electropho-retically separated. Red cell phosphofructokinase is composed of L and M subunits, as judged by its reactivity with anti-L and anti-M-type antisera. The various M-L hybrids composing red cell phosphofructokinase could be only separated by chromatography on DEAE-Cellulose. Lymphocytes from patients with chronic lymphocytic leukemia and lymphoblasts from patients with acute lymphoblastic leukemia contain phosphofructokinase forms similar to those from normal lymphocytes, while the immature granulocytic cells (leukemic myeloblasts and myeloid cells of chronic myeloid leukemia) are characterized by a reinforcement of enzyme inhibition by anti-F-type antiserum. Lymphoid lines in culture (Epstein-Barr virus (EBV)-induced or malignant lymphoma-derived lines) are characterized by the indistinctive expression of all three basic subunits, similar to that found in some fetal tissues. This article represents the first description of the isozymic nature of phosphofructokinase in platelets and white blood cells and of its changes with malignancy and cell culture. This enzyme might represent a useful marker in the characterization of the leukemic cells.

The expression of creatine kinase isozymes in human cultured cells.

The BB isozyme of creatine kinase is consistently present in cultured human fibroblasts and shows great variation in activity in long-term lymphoid lines. One mouse line tested, PG 19, had strong activity, but all other rodent lines tested did not express CK BB. Human and mouse CK BB can be expressed independently of each other in human--rodent somatic cell hybrids. There is some evidence that the structural locus for CK BB may be on chromosome 14, but the involvement of other chromosomes, expecially no.17, cannot be excluded. The MM and MB isozymes of creatine kinase were not seen in any human cultured cells.

Genetic Nature of Regression of Rous sarcoma Virus-Induced Tumors in Crosses of Regional Poultry Research Laboratory Lines 6 and 72 ,

Regional Poultry Research Laboratory (RPRL) inbred lines 61, 63, and 72 and F1, F2, and reciprocal backcross progenies of these lines were used to investigate host-gene effects upon the regression of Rous sarcoma virus (RSV)-induced tumors. Lines 61 and 63 were susceptible to subgroup A and C lymphoid leukosis (LL) viruses and line 72 was resistant to subgroup A but was segregating for susceptibility to subgroup C LL virus. Viruses of RSV subgroups A and C were used. Lines 61, 63, and 72 were homozygous for shared blood group alleles B2, C5, L1, and r.The incidence of tumor regression was higher in line 63 than in 72, and in reciprocal F1 crosses of these lines was of the same order of magnitude as in line 63. Progeny from F1, generation parents mated to line 63 had a higher frequency of regression than offspring from F1 generation parents mated to line 72. The frequency of regression in F2 generation offspring was intermediate between the two backcrosses.The data suggest that either a locus (or loci) other than L and B has a role in Rous tumor regression in this species or the immune response region of the B blood group-major histocompatibility complex differs in the two lines even though the serological and/or graft vs. host regions have not been shown to differ.

Differential inhibition of adenosine deaminase deficient peripheral blood lymphocytes and lymphoid line cells by deoxyadenosine and adenosine

The response to mitogens of peripheral blood lymphocytes (PBL) from adenosine deaminase deficient (ADA −) patients with Severe Combined Immunodeficiency (SCID), but not from normals, was more sensitive to inhibition by deoxyadenosine than by adenosine. In contrast, proliferation of long-term lymphoid line (B) cells from these patients was essentially equally inhibited by adenosine and deoxyadenosine. Deoxycytidine could “rescue” ADA − PBLs from deoxyadenosine toxicity.

Establishment and Characterization of BALB/c Lymphoma Lines with B Cell Properties

Abstract Five spontaneously derived BALB/c tumors and one 1-ethyl-1-nitrosourea induced BALB/c tumor (BALENLM 17), have been established in culture and characterized according to their surface markers. K46, X16C, L10A, and BALENLM 17 are IgM+, IgG-, IgA-, Ig+ (staining with polyvalent anti-Ig), Ia+, Fc receptor+ and complement receptor-. A20 is IgM-, IgG-, IgA-, Ig+, Ia+, Fc receptor+, and complement receptor-. M12 is IgM-, IgG-, IgA-, Ig±, Ia-, Fc receptor+ and complement receptor-. All these lines are Thy 1.2 antigen negative, do not phagocytize latex particles, and grow as stationary suspension cultures. Therefore these six BALB/c lymphoid lines are presumably of B cell origin at various stages of differentiation. All these lines except X16C are tumorigenic in mice within a short period of time and have relatively short generation times in the range of 18 to 26 hr. Karyotype studies show that L10A, K46, X16C, and BALENLM 17 are hyperdiploid to hypotetraploid. A20 and M12 have near diploid chromosome numbers.

Somatic cell fusion in the study of glucocorticoid action.

The basic phenomena of cell fusion and hybrid cell formation are briefly described and the potential of somatic cell hybridization in studies on the expression of differentiated cellular functions is discussed. The technique of cell hybridization has been applied to two types of cellular responses to glucocorticoids. The induction of specific proteins has been investigated in hybrids of inducible cells with uninducible cells. Most studies dealt with the liver-specific enzyme tyrosine aminotransferase, whose inducibility was extinguished in the majority of the hybrids between hepatoma and nonliver cells. However, upon chromosome segregation, inducibility reappeared in some of these hybrid cells. The current ideas about cellular control of inducibility are discussed. The other major glucocorticoid-responsive system investigated in cell hybridization studies consists of lymphoid cells which are killed when exposed to the steroid. Such sensitive cells were hybridized with several types of glucocorticoid-resistant lymphoid lines, and sensitivity was found to be dominant over resistence. Hybrids between sensitive and resistant lymphoid cells, however, showed an increase in the frequency at which resistance occurred as compared to the rate observed with the wild-type parental cells. No complementation to steroid sensitivity was found in hybrids between different types of resistant cells with defects in the glucocorticoid-specific receptor system.

Inhibition of human erythropoietic colony formation in culture by treatment with Ia antisera.

Incubation with Ia antiserum, followed by complement, markedly inhibited erythroid colonies arising from hematopoietic cells present in the nonadherent low density fractions of normal bone marrow. Both erythropoietin-dependent colonies and bursts were eliminated at dilutions of antiserum equivalent to, or greater than the dilutions required to abolish the granulocyte-macrophage colony formation. The inhibitory effect of the Ia antiserum was abolished by absorption with B but not T cells from lymphoid lines. Available evidence suggested that Ia determinants are expressed on the erythropoietin-sensitive progenitors of the erythroid series in precise analogy to their sequence of expression on the granulocyte lineage. In both lineages, as shown previously, the Ia determinants become undetectable during subsequent stages of differentiation.

Calcitonin and calcium ionophores: cyclic AMP responses in cells of a human lymphoid line.

Receptors for calcitonin, as assayed by the specific binding of 125I-labeled salmon calcitonin and stimulation of cyclic AMP formation, were found in 8866 cells derived from a human lymphoid line. The affinity of calcitonin from different species and of various analogues of human calcitonin for the binding sites and their ability to stimulate cyclic AMP formation were closely related to their hypocalcemic activity and presumably reflected biological properties of the hormones. Besides calcitonin, prostaglandin E1 and beta-adrenergic catecholamines stimulated cyclic AMP formation in these cells. The calcium ionophores, A23187 and Br-X-573A, did not influence the specific binding of 125I-labeled salmon calcitonin. A23187, however, suppressed basal and calcitonin-stimulated formation of cyclic AMP in the presence of at least 0.6 mM calcium in the incubation medium. Br-X-537A did not require extracellular calcium to suppress basal and calcitonin-stimulated formation of cyclic AMP, suggesting that the release of calcium from internal stores may regulate adenylyate cyclase activity in 8866 cells.

Association of the H-Y male antigen with beta2-microglobulin on human lymphoid and differentiated mouse teratocarcinoma cell lines.

The expression of the H-Y antigen has been tested on several human lymphoid lines and mouse teratocarcinoma cell lines during differentiation. The human male lymphoid cell line Raji is a very useful target for studies of the H-Y antigen by lymphocytotoxicity test with rat anti-H-Y sera. With a few exceptions, all cells carrying the Y chromosome were H-Y positive. One of the exceptions is the human Daudi cell line which, besides lacking H-Y antigen, also lacks beta2-microglobulin. We have studied a possible association between the H-Y antigen, beta2-microglobulin, and HLA antigen with redistribution experiments. The results strongly suggest that H-Y antigen is not associated with HLA antigens but with beta2-microglobulin.

Location of the genes for human heavy chain immunoglobulin to chromosome 6.

Immunoglobulin synthesis was examined in 31 man-mouse hybrid clones produced by fusing RAG mouse cells with human lymphoid cells. Cells were grown in serum-free medium containing [(14)C]leucine and a (14)C-labeled amino acid mixture. Spent medium was dialyzed, concentrated, and subjected to radioimmunoelectrophoresis. Eighteen clones were found to produce material that gave a radiolabeled precipitin line with anti-human IgG (gamma-chain specific). Production of material which was indistinguishable on radioimmunoelectrophoresis from human Ig gamma heavy chain, was dependent on the presence in hybrid clones of human chromosome 6. The material was found to have the ion-exchange elution characteristics of human IgG. When radiolabeled spent medium from human lymphoid lines and from chromosome 6-positive hybrid clones was exposed to protein A-Sepharose and bound material eluted with 8 M urea was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, three radiolabeled peaks occurred with molecular weights of approximately 55,000 (coinciding with that of Ig gamma heavy chain), 40,000 and 25,000 (coinciding with that of Ig light chains). No similar peaks were detected in experiments where spent medium from RAG cells was treated identically. These studies lead us to conclude that certain RAG-human lymphoid hybrid clones produce human IgG and that the structural genes for gamma heavy chains are located on human chromosome 6. These results also imply that the locus coding for human alpha,-antitrypsin (Pi) is located on chromosome 6.

Identification of an Abelson murine leukemia virus-encoded protein present in transformed fibroblast and lymphoid cells.

Extracts from lymphoid and fibroblast cell lines transformed by Abelson murine leukemia virus (A-MuLV) contain a protein of molecular weight 120,000 (P120). Immunoprecipitation with specific sera shows that P120 contains regions homologous to the 5'-terminal segment of the MULV gag gene complex--p15, p12, and at least part of p30--but lacks detectable determinants of p10, reverse transcriptase, and the envelope glycoprotein. P120 is phosphorylated and has an intracellular half-life of 3--6 hr. In vitro translation of virion RNA from A-MuLV, with Moloney MuLV as helper, yields a product of molecular weight 120,000 with serological reactivity similar to that of the cellular P120. Translation of the RNA from the helper gave no P120. P120 is expressed in all lymphoid and fibroblastic cell lines we have tested that were transformed by A-MuLV but is not detectable in a lymphoid line in which the A-MuLV genome was established by infection but was not responsible for the transformation. Expression of P120 is selectively retained in clones of A-MuLV-transformed lymphocytes that convert to a nonproducer state after loss of expression of helper MuLV intracellular precursors. These results suggest that the P120 product of the A-MuLV genome may be responsible for maintenance of the transformed phenotype of lymphoid and fibroblast cells transformed by the virus.

Mouse natural killer (NK) cell activity against human cell lines is not influenced by superinfection of the target cell with xenotropic murine c‐type virus

Mouse natural killer (NK) cells can lyse a variety of syngeneic, allogeneic and xenogeneic target cells in short-term 51Cr release assays. The target specificity of NK cells is not known, but endogenous C-type viral antigens have been suggested as possible target structures. To test this hypothesis, human lymphoid lines were superinfected with xenotropic mouse C-type virus either by repeated dosage through nude mice or by in vitro superinfection with the supernatants of nude-mouse-passaged lines. The appearance of surface-associated MuLV antigens after superinfection was confirmed in a complement-dependent cytotoxicity test. Subsequently, the NK sensitivity of each infected line was compared with its non-infected counterpart in direct cytolytic and competition assays. None of these two assay systems showed a consistent difference in NK sensitivity of infected and non-infected cell lines. These findings do not lend support to the concept tht murine C-type viral antigens are responsible for NK sensitivity.

553 EFFECT OF POKEWEED MITOGEN AND RICIN ON PROTEIN SYNTHESIS IN CULTURED HUMAN LYMPHOID LINES

Pokeweed mitogen (PWM) and ricin are both lectins derived from plant seeds. They are glycoproteins and share the ability to agglutinate a variety of animal cells particularly erythrocytes. Preliminary to development of a system to screen for the effect of environmental mutagens on the frequency of lectin resistant mutants we studied the effect of these 2 lectins on protein synthesis in long-term lymphoid lines. Ricin, a highly toxic compound is a known inhibitor of protein synthesis in other cell systems. PWM has primarily been evaluated for its mitogenic effect. Four lymphoid lines were studied: 8866 and GM1531 which are B cell lines, and CCRC/CEM and MOLT 4 which are considered to be T cell lines. Ricin (50 ug/ml) completely inhibited protein synthesis by 2 hours in all 4 lines as measured by the uptake of 3H leucine. PWM appeared more specific and at a concentration of 500 ug/ml inhibited protein synthesis only in the B lines (8866 and GM1531). This effect was maximal at 5 hours. To investigate the reason for the differential effect of PWM on T & B cells, 125I labeled PWM was incubated with 8866 and MOLT 4 to see if an increased binding to B cells could be demonstrated. A significantly greater number of counts bound to 8866 compared to MOLT 4. As lectins bind to cell surface carbohydrates, the possibility that this may represent a difference in the composition or arrangement of the surface glycoproteins is being investigated.

Cystathionase Deficiency: Evidence for Genetic Heterogeneity in Primary Cystathioninuria

Summary: Optimal conditions are described for measurement of cystathionase activity in long term lymphoid cell lines. In 21 control lines established from normal subjects, cystathionase (EC 4.4.1.1) specific activity was 25.8 ± 1.7 (mean ± SE) nmole cysteine/hr/mg protein. Extracts of three lymphoid lines (NB-36, NB-95, and NB-77) established from three vitamin B6-responsive patients with primary cystathioninuria had activity of 3.6–7.3; from a B6-unresponsive patient (NB-68) had no detectable activity; from five obligate heterozygotes for cystathioninuria had activity of 11.2–18.9.Two, or possibly three, different modifications of the cystathionase molecule could be demonstrated in cultured cells from the patients with primary cystathioninuria, based on the effects of added pyridoxal phosphate (PLP) in the enzyme assay, on the extent of reaction with rabbit antihuman hepatic cystathionase, and on the ability to compete with normal lymphoid cell line enzyme extract for the antibody combining sites. When PLP is not added to the assay system, the normal enzyme extract still had 89% of its activity in 1.0 mM PLP; on agar double diffusion analysis it gave a band of identity with normal human hepatic cystathionase; the precipitin band had cystathionase activity, but inhibition by antibody could be shown in solution. Lymphoid line extract from the B6-unresponsive patient had no detectable activity in the absence or presence of PLP, did not form a precipitin band, and did not compete with normal enzyme extract for the antibody combining sites. Thus, synthesis of apocystathionase is absent or significantly reduced; alternatively, the protein produced has lost its antigenic determinants as well as catalytic activity. Extracts of the three cell lines established from the three B6-responsive patients had no activity in the absence of added PLP, but progressively greater activity was found with increasing concentrations of PLP: at 1.0 mM PLP 37, 26, and 16% of the mean normal value, respectively was attained. Cell extract from each of the B6-responsive patients formed a band of identity with normal enzyme on agar double diffusion and cystathionase activity could be demonstrated on the precipitin band only if PLP was added to the extracts before immunodiffusion. Extract of the B6-responsive lymphoid cell line NB-36 combined with the antibody sites for cystathionase, thereby blocking inhibition of normal enzyme, whereas extract of the line NB-95 had only a slight blocking effect and extract of the line NB-77 had no blocking ability. Thus, the lymphoid cell lines from the three B6-responsive patients produce a cyctsthionase with an altered capacity to combine with PLP; the enzyme in lines NB-95 and NB-77 also may have been affected in a manner which changes as well the ability to compete for the antibody combining site. These altered cystathionases, both B6-responsive and B6-unresponsive, also may represent forms more readily degradable than the normal enzyme.Antigenic identity was shown for cystathionase from various organs within a given vertebrate species, but only partial identity was observed among cystathionases from different vertebrate species.Speculation: Primary cystathioninuria is caused by different mutations affecting the cystathionase molecule. One form of cystathioninuria, vitamin B6-unresponsive, appears to result from absence of synthesis of the enzyme protein. B6-responsive forms appear to result from production of cystathionase molecules altered in ability to combine with coenzyme, but with antigenic indentity on agar double diffusion analysis maintained; in some cases, there may be changes in antibody binding capacity as well, although immunologic cross-reactivity remains.

Sister chromatid exchanges in human lymphoid cell lines cultured in diffusion chambers in mice: A host-mediated assay system for mutagenicity

A host-mediated assay system for evaluating mutagenic potential of a compound was described. It involved culturing cells from human lymphoid lines in diffusion chambers and implanted into mice. Induction of sister chromatid exchanges (SCE) in the human target cells by injection of the model compound cyclophosphamide to the hosts was used as an index. The numbers of SCE/cell was significantly increased in a dose-dependent manner.

Mutation in cultures human lymphoid lines

The lag in phenotype expression of methylnitrosourea(MNU)-induced mutation to 6-thioguanine (6TG) resistance has been studied in a diploid human lymphoblastoid cell line. We find that a considerable period (8–12 days) elapses before new mutants appear in treated cultures; after 2 weeks, however, a stable maximum fraction is attained, as would be expected for a genetic mutation. We present preliminary data linking this phenotypic lag to the slow degradation rate of hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and to an apparent requirement for very low (<0.2% normal) cellular HGPRT content in order for cells to be resistant to 10 μg 6TG/ml. A series of reconstruction experiments are presented, the results of which support the conclusion that selective pressures in the assay procedure do not bias the quantitative estimates of induced mutant fraction.

Assignment of the gene locus for human alpha-L-fucosidase to chromosome 1 by analysis of somatic cell hybrids.

The alpha-L-fucosidases (EC 3.2.1.51) from human and mouse cells could be separated by isoelectric focusing of neuraminidase-treated cell extracts in acrylamide slab gels. Fourteen hybrid clones derived from the fusion of mouse and human cultured fibroblasts and 37 hybrid clones derived from the fusion of human long-term lymphoid lines with mouse RAG cells were tested for expression of human alpha-L-fucosidase. A strong correlation between the expression of the human enzyme and the presence or absence of human chromosome 1 was found. The presence of human alpha-L-fucosidase in clones scored as positive by isoelectric focusing was confirmed by Ouchterlony double immunodiffusion against IgG from rabbits immunized with purified human alpha-L-fucosidase. It is concluded that the structural gene locus for human alpha-L-fucosidase is located on chromosome 1.