Comparative ultrastructural morphometrical studies in acute leukemia.

Four hundred paramyeloblasts (from meyloblastic, promyelocytic, monoblastic and lymphoblastic types), isolated from peripheral blood of untreated leukemia patients, were studied by planimetric ultrastructural morphometry. The data of 19 parameters for these four paramyeloblastic types were compared with statistical methods. More central "scattered" heterochromatin was found from this morphometric investigation, i.e. early prophases in the lymphoblastic type of acute leukemia (these cases are more sensitive to therapy). The absolute mean values of the areas of whole cells, areas of the nucleus and nucleolus, areas of the heterochromatin and other indices show that different cell clones will undergo leukaemic transformation.     

Transferrin and iron uptake by human lymphoblastoid and K-562 cells

Two human lymphoblastoid cell lines and K-562 cells were found to take up radioiodinated transferrin and transferrin-bound iron in amounts comparable to reticulocytes. These cell lines were also shown to possess transferrin receptors whose numbers and affinity for transferrin were similar to those of reticulocytes. However, unlike reticulocytes, in which at least 90% of the iron taken up is incorporated into heme, in the lymphoblastoid and K-562 cells only around 10% of the incorporated iron is found in heme. In addition, in contrast to the hemoglobin synthesizing cells, excess heme does not inhibit the removal of iron from transferrin by the lymphoblastoid and K-562 cells, suggesting that only during erythroid differentiation do cells acquire a specific mechanism for removing iron from transferrin which is subject to feedback inhibition by heme.     

Subcellular action of Neocarzinostatin. Intracellular incorporation, DNA breakdown and cytotoxicity

The subcellular site of action of a proteinaceous antitumor antibiotic neocarzinostatin (NCS) was studied using normal human lymphocytes, Epstein-Barr virus transformed lymphoblastoid cells, osmotically burst lymphoblastoid cells, and colicin E1 plasmid DNA. The rate of DNA strand break in these different types of DNA was found to be in the following order: Colicin DNA > burst cell DNA > lymphoblastoid cell DNA > normal lymphocyte DNA. Furthermore, fluorescence microscopy revealed that lymphoblastoid cells incorporated more fluorescein isothiocyanate labeled NCS than normal cells. High uptake of NCS in lymphoblastoid cells coincided with a high killing rate; low uptake of NCS in lymphocytes resulted in very little cell killing. Uptake velocity using fluorescein diacetate (FDA) also showed that the lymphoblastoid cells exhibited a higher uptake of FDA coinciding with a higher killing rate. The cell killing activity of NCS appears to be closely associated with the rate of intracellular uptake of NCS and subsequent direct degradation of DNA by the drug. This notion is reinforced by the reported finding that the dose required for DNA strand scission is only about 1/100 of that for the inhibition of cap formation. Thus DNA strand scission, rather than the cell membrane, appears to be the primary target of NCS. Enhanced incorporation of many substances is commonly observed upon transformation of cells by viruses, and our present results may provide an important clue toward the explanation of the selective toxicity toward tumor cells of NCS.     

Human plasma accelerates immortalization of B lymphocytes by Epstein-Barr virus

Abstract.   Objective : Serum and plasma contain species-specific factors that modulate cell population growth and function, and that are required for proliferation of most cell cultures. Foetal calf serum (FCS) is the most common source of these growth factors. We studied the effect of human plasma (HP) on the immortalization process of B lymphocytes by Epstein–Barr virus (EBV) was studied. Materials and methods : The effect of HP as compared to FCS was done through assessment of cell proliferation. Results : It was found that HP (autologous and non-autologous plasma) is more effective than FCS in generating lymphoblastoid cell lines, regardless of EBV status of the donors: 65% of HP-supplemented cultures developed into lymphoblastoid cell lines by 7–14 culture days, as compared to 16% of cultures with FCS. In addition, 6% of HP-supplemented cultures did not achieve becoming lymphoblastoid cell lines by day 35 in comparison to 94% of cultures with FCS. The higher proliferative effect of HP was not altered by heat inactivation or filtration. HP maintained its proliferative activity at 4 °C over 8 months, thus indicating that HP contains a stable growth factor(s), which accelerates B-lymphocyte immortalization. Conclusion : The results support other studies that recommend the use of autologous plasma for tissue culture, mainly in the case of autologous transplantation. Furthermore, the use of HP allows preparation of lymphoblastoid cell lines from a small amount of peripheral blood in a shorter period of time and with a higher rate of success.     

Common copy number variations in fifty radiosensitive cell lines

Hypersensitivity to radiation exposure is a major challenge to radiotherapy in the treatment of cancer patients. Copy number variations (CNVs) are believed to identify genomic regions of functional significance for radiosensitivity (RS) but have yet to be systematically investigated. We used Affymetrix 6.0 SNP arrays to survey common CNVs in a cohort of 50 radiosensitive lymphoblastoid cell lines (RS-LCLs) derived from patients with undiagnosed diseases. A total of 317 CNVs that were present in at least 10% of the studied cell lines were identified. Three hundred and eight CNVs overlapped with polymorphic CNVs, 13 of which were significantly enriched in the RS-LCLs compared to the reference. The remaining 9 CNVs were novel. The majority of these enriched and novel CNVs were chromosomal gains. The dominance of the chromosomal gains over losses is inconsistent with the traditional concept of molecular basis of RS and suggests more complex genetic mechanisms for RS.     

Evidence for the existence of an actin-derived protein in ataxia-telangiectasia lymphoblastoid cell lines

In a number of lymphoblastoid cell lines from individuals with the genetic disease ataxia-telangiectasia (A-T), a decrease in the levels of actin and a concomitant increase in the levels of a protein species of 37K has been observed to occur following high cell density. In this paper we describe ataxia-telangiectasia lymphoblastoid lines that show this response constitutively, and demonstrate that this 37K protein is most likely an actin-derived peptide. This is because of extensive similarity between the two proteins in the spectrum of peptides produced by proteolytic fingerprinting. It appears likely that this 37K protein is similar to the well documented protease-resistant actin fragments produced by protease digestion of actin in vitro. Examination of a number of possible initiators found no clear signal that promoted this response, although the response is clearly very dependent on cell culture conditions. The possible involvement of a cytoskeletal anomaly in ataxia-telangiectasia is discussed.     

Surface immunoglobulins as targets for anti-immunoglobulin-dependent cell-mediated lysis of B cells

Human K cells are able to lyse human lymphoblastoid B cells in the presence of specific anti-human immunoglobulin isotype antibodies. The human lymphoblastoid B-cell line DAUDI (surface mu- and kappa-chains positive) was lysed in the presence of anti-mu and anti-kappa antisera, or IgG fraction of these antisera, and B-cell-depleted human lymphocytes. Lysis was not induced by anti-isotypic antisera alone or human lymphocytes alone. Lysis was not induced by antisera directed at isotypes which were not expressed on the DAUDI cells. The human lymphoblastoid B-cell line RAJI, which does not express surface membrane isotypes, could not be lysed by the anti-isotype-dependent cell-mediated mechanism. Lysis of DAUDI cells by this mechanism was mediated by Fc gamma receptor-bearing human non-B lymphocytes and required an intact Fc piece in the inducing anti-isotypic antibody. These observations are discussed as a possible role for K cells in the regulation of immune responses by interaction between anti-idiotypic antibodies and idiotype-bearing cells.     

Similar repair of O6-methylguanine in normal and ataxia-telangiectasia fibroblast strains. Deficient repair capacity of lymphoblastoid cell lines does not reflect a genetic polymorphism

The ability of human fibroblast strains to repair the mutagenic DNA adduct O6-methylguanine (O6-MeG) induced by brief exposure to N-methyl-N'-nitroso-N-nitrosoguanidine (MNNG) was investigated. The repair reaction proceeded rapidly during the first hour after alkylation, followed by a slow, continuous phase of repair, and both processes were saturated by low doses of carcinogen. This was similar to what had previously been found in human lymphoblastoid lines. Three fibroblast strains from healthy donors and six strains from patients with ataxia telangiectasia were all proficient in their capacity to repair O6-MeG and had the same sensitivity to the cytotoxicity of MNNG and methyl methanesulphonate as normal cells. Three of these cell strains were derived from individuals whose lymphoblastoid lines were deficient in their ability to repair O6-MeG. These lymphoblastoid lines were also extremely hypersensitive to killing by methylating carcinogens. Because non-transformed cells from the same donors behaved normally with regard to both parameters, we concluded that the repair deficiency accompanied by carcinogen hypersensitivity of the lymphoblastoid lines does not indicate a genetic deficiency in the donor. These findings imply that lymphoblastoid lines may not always be the appropriate cell type for investigating genetic susceptibility to chemical mutagens.     

Serological analysis of xenogeneic anti-lymphoblastoid cell-line sera with specificity against HLA-B12

In view of the importance of potent anti-HLA sera with narrow reaction patterns against defined HLA antigens, two xenogeneic antisera were raised in rabbits following immunization with human lymphoblastoid cell lines from HLA-nonidentical donors homozygous for HLA-B12. After absorption with lymphoblastoid cell lines of an appropriate HLA phenotype, the antisera were purified over DEAE-cellulose ion exchange chromatography and reconcentrated. Both antisera recognized HLA-B12-positive peripheral blood cells of unrelated donors tested in the microcytotoxicity assay. The two rabbit antisera revealed a high degree of similarity in their anti-HLA-B12 antibody specificity. One antiserum showed some cross reactivity with HLA-B13 as has been reported in allo-anti-HLA-B12 sera. The other antiserum revealed some activity against HLA-DRw7-positive donors. Antibody activity could be removed completely from two further rabbit anti-HLA antisera by absorption with lymphoblastoid cell lines from related and unrelated HLA-identical donors. The advantages of using lymphoblastoid cell lines as immunogens and absorption material for the production of heterologous anti-HLA typing sera are discussed.     

Complementation analysis of measles virus mutants isolated from persistently infected lymphoblastoid cell lines.

Human lymphoblastoid cell lines persistently infected with measles virus release a heterogeneous population of virions. At least 80% of the infectious particles were temperature sensitive for plaque formation at 39 degrees C. Plaque-purified temperature-sensitive mutants from four persistently infected human lymphoblastoid cell lines were shown to be heterogeneous with respect to efficiency of plating at 31 and 39 degrees C, as well as to antigen and RNA production at 39 degrees C. The heterogeneity was confirmed by complementation analysis in which 21 temperature-sensitive isolates were found to represent at least four of the five previously described complementation groups of measles virus. Two isolates complemented four reference temperature-sensitive mutants. These isolates either represent new complementation groups or are members of the fifth complementation group, group E. The majority of isolates were found to have multiple mutations, and group B mutants (RNA-) predominated. Two temperature-sensitive isolates were able to interfere with production of parental measles virus at both permissive and nonpermissive temperatures.     

Binding of human recombinant 125I-interferon gamma to receptors on human cells

Recombinant human interferon gamma (rIFN-gamma) produced in Escherichia coli was labeled with 125I to study its binding to receptors of HeLa and lymphoblastoid cells. All the cell lines examined had receptors for rIFN-gamma, although the binding varied considerably among different cell lines. The binding of 125I-rIFN-gamma was competed up to 90% by the addition of unlabeled rIFN-gamma, although not by the addition of IFN-alpha or -beta. By adding increasing concentrations of unlabeled rIFN-gamma to binding assays containing a constant amount of 125I-rIFN-gamma, we determined a KD of 3.7 and 6.3 X 10(-10) M for its binding to Daudi and HeLa cells, respectively. About 13,000 receptors per cell were present in Daudi and 5,000 in HeLa cells. The Mr of the IFN-gamma/receptor complex was determined by cross-linking experiments to be about 125,000. This complex is smaller than the IFN-alpha/receptor complex that has a Mr of about 140,000. The rIFN-gamma receptor was down-regulated when HeLa cells were treated with this interferon, but not when these cells were treated with IFN-beta. These findings suggest that the receptors for IFN-alpha and -gamma differ in several characteristics. The turnover of the rIFN-gamma receptor was measured by inhibiting protein synthesis with cycloheximide and the half-life of this receptor was found to be 2 h. The unglycosylated rIFN-gamma was bound to cellular receptors with an affinity similar to that previously reported for natural IFN-gamma. The lymphoblastoid cell lines examined had high affinity receptors for rIFN-gamma, but did not respond to treatment with this IFN with an induction of the synthesis of the enzyme (2'-5')oligo(A) synthetase, whereas HeLa cells responded to rIFN-gamma. The reason for the lack of response of lymphoblastoid cells is presently unknown.     

A study of the ability of Namalwa interferon to induce sister-chromatid exchanges in vitro

Heparinized cord blood from a newborn child and peripheral venous blood from three other individuals have been cultured in vitro to investigate whether the frequency of sister-chromatid exchange (SCE) can be affected by the presence of human Namalwa lymphoblastoid interferon (Namalwa IFN). The results demonstrated that both purified and crude Namalwa IFN failed to show higher yields of SCE than normal controls (P greater than 0.05). No Namalwa IFN-dependent variation was found in the frequencies of SCE as well. Our findings seem not to support the view that Namalwa IFN, produced by Epstein-Barr virus (EBV)-transformed lymphoblastoid cells, is a carcinogen or mutagen.     

In vitro metabolism of deoxycoformycin in human T lymphoblastoid cells. Phosphorylation of deoxycoformycin and incorporation into cellular DNA

The biochemical and metabolic effects of deoxycoformycin, a potent inhibitor of adenosine deaminase, were investigated using two human T lymphoblastoid cell lines. A dose-response analysis demonstrated that the concentration of deoxycoformycin at which there was 50% inhibition of growth was greater than 1 X 10(-3) M in lymphoblastoid cells. Uptake of deoxycoformycin was biphasic and occurred much more slowly than for natural nucleosides, and lower saturation levels were reached. The intracellular concentration of deoxycoformycin achieved was 0.4 to 0.5 microM when the extracellular concentration was 1 microM. At 10 microM extracellular concentration, the intracellular concentration was 3-4 microM. Although deoxycoformycin at very low concentrations (1 or 10 microM) did not have any detectable effects on the growth of these cells, the nucleoside was found to be metabolized, and was phosphorylated to give the mono-, di-, and triphosphate derivatives. The triphosphate derivative was incorporated into cellular DNA with little incorporation into cellular RNA. Metabolism of deoxycoformycin in several mutant lymphoblastoid cells deficient in adenosine kinase and/or deoxycytidine kinase was found to be unchanged from wild-type cells, indicating that these major nucleoside kinases do not play a significant role in the phosphorylation of deoxycoformycin. These results may account, at least in part, for the differences that are observed between the pharmacologic inhibition of adenosine deaminase, and the inherited deficiency of adenosine deaminase.     

Serological analysis of xenogeneic anti-lymphoblastoid cell-line sera with specificity against HLA-B12

In view of the importance of potent anti-HLA sera with narrow reaction patterns against defined HLA antigens, two xenogeneic antisera were raised in rabbits following immunization with human lymphoblastoid cell lines fromHLA-nonidentical donors homozygous for HLA-B12. After absorption with lymphoblastoid cell lines of an appropriate HLA phenotype, the antisera were purified over DEAE-cellulose ion exchange chromatography and reconcentrated. Both antisera recognized HLA-B12-positive peripheral blood cells of unrelated donors tested in the microcytotoxicity assay. The two rabbit antisera revealed a high degree of similarity in their anti-HLA-B12 antibody specificity. One antiserum showed some cross reactivity with HLA-B13 as has been reported in allo-anti-HLA-B12 sera. The other antiserum revealed some activity against HLA-DRw7-positive donors. Antibody activity could be removed completely from two further rabbit anti-HLA antisera by absorption with lymphoblastoid cell lines from related and unrelatedHLA-identical donors. The advantages of using lymphoblastoid cell lines as immunogens and absorption material for the production of heterologous anti-HLA typing sera are discussed.     

Expression of a particular beta-N-acetylglucosaminidase isoenzyme in human haematopoietic leukemic cell-lines

N-acetyl-beta-D-glucosaminidase (NAG) activity and isoenzyme profiles were studied in myeloid, histiocytic, B-lymphoid, T-lymphoid and lymphoblastoid continuous cell lines in order to determine if N-acetyl-beta-D-glucosaminidase isoenzyme expression may help to distinguish among various types of leukemic proliferation. Total NAG activity in myeloid, histiocytic, erythroleukemic cell lines were higher than Burkitt's lymphoma derived cell lines (B-lymphoid), T- or lymphoblastoid cell lines. On chromatofocusing by PBE 94 coupled with an automated enzyme assay an intermediate (I) beta-N-acetyl-glucosaminidase form, eluting between forms B and A, was found in all leukemic and in Epstein-Barr virus infected lymphoblastoid cell lines analysed. The different profiles recorded, the expression of the I form and the different I/B ratios may be useful as markers of tumour proliferation.     

Synthesis, isolation, and characterization of endogenous beta-galactoside-binding lectins in human leukocytes

Galaptin, a beta-galactoside-binding lectin, was isolated from human buffy coat cells (peripheral leukocytes) and spleen by affinity chromatography. The molecular weight (32K) of the native buffy coat galaptin was similar to that for splenic galaptin. Their subunit molecular weight (14.5K), pI (4.60-4.85), and amino acid composition were identical. Both galaptins showed the presence of a single polypeptide when subjected to reversed-phase HPLC. Monospecific rabbit polyclonal antiserum raised against the 14.5-kDa subunit of splenic galaptin reacted with a 14.5-kDa polypeptide present in buffy coat cells, Epstein-Barr virus-immortalized B lymphoblastoid cells, and HL-60 promyelocytic leukemia cells. However, galaptin was not synthesized in vitro by buffy coat cells. Rather, a monomeric beta-galactoside-binding protein of Mr 15.5-16.5K that is immunologically distinct from galaptin was synthesized. This galactoside-binding protein was separable from galaptin by polyacrylamide gel electrophoresis and by anion-exchange chromatography. In contrast, immunoprecipitation experiments confirmed that galaptin was synthesized by the B lymphoblastoid cells. cDNA corresponding to the B lymphoblastoid cell mRNA encoding galaptin was amplified by the polymerase chain reaction. The amplified product was partially sequenced, and 299 nucleotides were identified. The derived amino acids corresponded to residues 6-65, 84-114, and 118-126 found to be present in human splenic galaptin. Immunohistochemical analyses revealed that galaptin was distributed throughout the cytoplasm of B lymphoblastoid cells rather than being localized to the cell surface.(ABSTRACT TRUNCATED AT 250 WORDS)