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Ultraviolet light produces pyrimidine dimers in cellular DNA. In mouse cell lines the great majority of these pyrimidine dimers are not excised, but remain in high molecular weight DNA. Hence the DNA used as template for DNA synthesis in the first generation after ultraviolet irradiation must contain such dimers. In two mouse cell lines (L5178Y and 3T3), alkaline sucrose sedimentation studies showed that the DNA pulse-labelled shortly after irradiation contained gaps, which by analogy with the situation in Escherichia coli, are presumed to be opposite the pyrimidine dimers. In contrast such gaps could not be detected in DNA pulse-labelled several hours after irradiation, despite the fact that dimers must have been present on the template strands. A possible explanation is that gaps were still being formed, but were filled in rapidly and were therefore not detected. This hypothesis was tested as follows. Either 1 or 8 h after ultraviolet irradiation cells were pulse-labelled in the presence of theophylline. This purine inhibits the filling in of gaps presumably by binding to the DNA in the gap. Even in the presence of theophylline, the late-synthesized DNA was considerably larger than the early-synthesized DNA. This suggests that at late times after irradiation, gaps were either not formed at all opposite the dimers, or were so transient that theophylline could not bind and inhibit their filling in. A model for synthesis of DNA on templates containing pyrimidine dimers in mammalian cells, based on these findings and other recent results, is discussed. [ABSTRACT FROM AUTHOR]

Continuous lymphoblastoid cell lines (CLC) are more reactive with HL-A antisera in a complement-dependent cytotoxic test than are peripheral blood lymphocytes (PBL). This additional reactivity leads to assignment to a given CLC of more than four HL-A antigens, the maximum allowable under the two locus concept of the genetic control of HL-A. However, absorption of antisera by CLC shows that no more than four HL-A antigens exist on any of the CLC used in this laboratory. The additional reactivity of these cells lines can be explained in three ways. Firstly, it may be due to the presence of sublytic amounts of HL-A antibody in operationally monospecific antisera. Secondly, it may be due to cross-reactivity between HL-A antigens. Both these findings can be explained on the basis of the increased quantity of HL-A antigens on CLC compared to PBL. Thirdly, it may be due to the presence of a heat-labile (56 for 30 min) complement-dependent cytotoxic antibody which is present in 90% of normal human sera, and detects an antigen group tentatively labelled 'D'. [ABSTRACT FROM AUTHOR]

Spent culture media of all the human cell lines tested have been found to contain the antigenic activity present on the ll,000-Dalton HL-A common portion fragment of the HL-A antigen molecule that appears to be a characteristic, invariant portion of HL-A antigen molecules. From the culture medium of one of these lines, RPMI 1788, a lymphoid cell line, the substance carrying HL-A common activity was isolated, which was shown to be identical to the HL-A common portion fragment with respect to molecular size, electrophoretic mobility, isoelectric focusing patterns, and certain antigenic characteristics. By an isolation procedure involving differential ultrafiltration, gel filtration, and column electrophoresis, 8 litres of the culture medium yielded 1.5-2.0 A280 units of the substance representing 15-20 per cent of the HL-A common antigenic activity originally present. A single protein band with a Rf of 0.47 was obtained by disc electrophoresis. The molecular size was shown to be about 11,000 Daltons by gel filtration and by sodium dodecyl sulphate acrylamide gel electrophoresis. Upon isoelectric focusing two bands were obtained which corresponded exactly to those obtained with HL-A common portion fragment prepared from papain-solubilized HL-A antigen preparations by acid dissociation. The isoelectric point of the major band was 5.0. The reactions of this substance with rabbit antisera against human lymphoid cell membrane and against the substance were essentially identical to the reactions of HL-A common portion fragment with these same antisera. [ABSTRACT FROM AUTHOR]

A lymphoblastoid cell line derived from a Chinese case of nasopharyngeal carcinoma carried an Epstein-Barr virus associated membrane antigen complex on its surface. With regard to its reactivity with Epstein-Barr virus positive reference conjugates and the blocking reactions obtained with a number of nasopharyngeal carcinoma, Burkitt's lymphoma and infectious mononucleosis derived sera against these conjugates, the reaction patterns obtained with the nasopharyngeal carcinoma derived line were indistinguishable from corresponding Burkitt's lymphoma-infectious mononucleosis derived lines. [ABSTRACT FROM AUTHOR]

Cells from thirty-three human lymphoid cell lines and sublines have been typed for HL-A antigens by a microcytotoxicity test. Similar patterns of HL-A antigens were found for the cell line cells and for the fresh lymphocytes of the donor of the line (eleven cases). However, certain typing sera gave positive reactions with the cell line cells which were not found with the fresh lymphocytes. No correlation was noted between the pattern of these 'extra' reactions and the HL-A typing of the cells. These same typing sera often gave positive reactions with blood lymphocytes cultured for several days in conventional media. These positive reactions were quantitatively more pronounced and sometimes qualitatively different if the cells had been stimulated. A range of normal sera failed to react with cell line cells suggesting that the HL-A typing sera giving 'extra' reactions are detecting antigens in some way related to a histocompatibility system. Absorption studies performed with two of the lines confirmed the HL-A typing by the direct cytotoxicity test. Two sera giving 'extra' reactions were also tested in the absorption experiments. The results indicated that antibodies other than those of the HL-A specificity designated for these sera were responsible for the 'extra' reactions. It is suggested that 'extra' reactions indicate a change in the apparent antigenic expression of lymphoid cells reflecting altered membrane characteristics as they adapt to a culture environment. [ABSTRACT FROM AUTHOR]

Two Burkitt lymphoma (BL)-derived cell lines, characterized by the accumulation of IgM-κ reactive material in their cell membrane, and one BL-line with no such reactivity were studied. Attempts were made to isolate and further characterize the reactive membrane material. Papain digests of crude membrane preparations from the surface reactive cell lines were capable of blocking the direct membrane fluorescence reaction while the digest obtained from the non-reactive line demonstrated no blocking activity. Gel filtered and papain digested membrane material, from all three cell lines, produced similar elution curves when rechromatographed on a calibrated Sephadex G-150 column. The membrane fluorescence blocking activity of papain digests, derived from cells with μ and κ chain reactivity, appeared to be associated with material of a molecular weight less than 50,000. When this gel filtered material, and the corresponding fraction from the non-reactive cell line, were subjected to polyacrylamide gel electrophoresis a difference in electrophoretic mobility was noted. Electron microscopy revealed small protein fragments, some of linear dimensions compatible with a molecular weight of roughly 50,000 and the majority even smaller in all three preparations, μ and κ chain reactivity was demonstrable by indirect haemagglutination only in papain digested and gel filtered fractions from the two membrane reactive cell lines. Haemagglutation inhibition with Fc μ fragments indicated that these fragments inhibited the anti-IgM reactivity of red cells coated with the proteolytic products of the two membrane reactive cell lines. [ABSTRACT FROM AUTHOR]

Irradiated LCL cells from several sources activated the blood lymphocytes of a panel of donors. Individual differences were present but were small. Stimulated blood lymphocytes were more potent activators (after X-irradiation) than small lymphocytes in a one-way mixed lymphocyte reaction, indicating that the state of metabolic activity of the 'stimulating' lymphocyte affects the level of activation achieved. However, LCL cells incubated for several days after irradiation had not lost stimulatory capacity. Lymphocytes of pigs, rabbits, rats and mice were much less responsive than human lymphocytes. The response of animal lymphocytes generally varied with the species of origin of the serum used in the cultures. [ABSTRACT FROM AUTHOR]

Human peripheral lymphocytes from all donors tested were mitotically activated when cultured in the presence of X-irradiated cells from continuously cultivated lymphoid cell lines (LCL). X-irradiated cells from LCL originating from normal individuals were effective as well as those of Burkitt's tumour origin and cells from LCL reported to be free of herpes-like virus were as effective as those reported to contain virus. The activation was of much greater intensity than the normal mixed lymphocyte reaction but occurred over a similar culture period. The reaction occurred only under conditions permitting direct cell contact between the two populations of cells. Most stimulation was obtained with viable X-irradiated LCL cells. Disrupted LCL cells were ineffective. [ABSTRACT FROM AUTHOR]

Immunological memory and antibody formation appear to be properties of different cell fines. This follows from a demonstration that cells from spleens of mice primed with sheep erythrocytes are capable of inducing normally unresponsive bone marrow ceils to produce specific antibody. Primed spleen cells, taken from CBA or CBAT6T6 mice, were cultured for 5 or 6 days with an excess of bone marrow cells of the opposite chromosome type, and with antigen, in lethally irradiated, histocompatible mice. The tissue of origin of antibody-plaque-forming cells from the spleens of the irradiated mice was then identified from chromosome spreads of individual plaque-forming cells. After 5 days of culture, equal numbers of spleen and bone marrow cells were found producing antibody. At 6 days nearly all of the plaque-forming cells were from the bone marrow. Plaque-forming cells of bone marrow origin were also identified, using an immunogenetic technique, when semi-allogeneic mixtures of spleen and bone marrow cells were cultured together. [ABSTRACT FROM AUTHOR]

Cultured tissue cells from different primates may be identified by means of the mixed agglutination reaction using suitable antisera prepared in rabbits and in Old World monkeys. In this way it has been possible to differentiate human, Rhesus, Paras and Vervet monkey cells. Several ceil lines thought to be derived from Rhesus monkey tissue have been shown to have human species antigens, but not Rhesus monkey antigens, suggesting that the original Rhesus monkey cells have been replaced by human cells. Antigens similar to the human A and B blood group antigens have been identified on Rhesus monkey tissue cells ex vivo, and these have been shown to persist in long-term cultures of Rhesus monkey cells. [ABSTRACT FROM AUTHOR]