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The incidence of apoptotic and necrotic cell death was compared in CHO, SF9 insect cells and murine plasmacytoma (J558L) and hybridoma (TB/C3) cells during in vitro cultivation in batch cultures. Acridine orange staining and fluorescence microscopy enabled the visualization of a classic morphological feature of apoptotic cell, the presence of condensed and/or fragmented chromatin. DNA gel electrophoresis was employed to show an additional characteristic of the process, the endonuclease-mediated fragmentation of DNA into multiples of 180 base pairs. The levels of apoptosis at the end of batch cultures of plasmacytoma and hybridoma cell lines were found to be 60% and 90% of total dead cells, respectively. However, employing the above-mentioned techniques, the biochemical and morphological features of apoptosis were not found in CHO and SF9 insect cells. Some factors affecting the induction of apoptosis during the batch culture of the hybridoma and plasmacytoma cell lines were identified. The most effective inducer was found to be glutamine limitation, followed by (in order of importance) serum limitation, glucose limitation, and ammonia toxicity. Blockage of the cell cycle of the plasmacytoma and hybridoma cells using thymidine resulted in the induction of apoptosis. This has important implications for the development of cell culture processes that minimize cell division and thereby increase specific productivity. © 1994 John Wiley & Sons, Inc.

The levels of necrosis and apoptosis were quantified in CHO, SF9 insect cells, and murine plasmacytoma and hybridoma cells by acridine orange staining and fluorescence microscopy. This technique enabled the visualisation of a classic morphological feature of apoptotic cells, the presence of condensed and/or fragmented chromatin. DNA gel electrophoresis was also employed to show an additional characteristic of the process, the endonuclease mediated fragmentation of DNA into multiples of 180 base pairs. Necrosis was found to be the dominant mechanism of cell death in the cultures of CHO and SF9. However, significant levels of apoptosis were found in the plasmacytoma and hybridoma cell lines. Around 81% of dead hybridoma cells were apoptotic by day 5 of a batch culture. By day 4 of the plasmacytoma batch culture the level of apoptosis was approximately 60% of dead cells.

Peripheral blood lymphocytes taken from healthy women planning a pregnancy and then at various intervals up to the 16th week of pregnancy were assayed for natural killer (NK) cell activity against K562 target cells both in a 51Cr-release assay and in a single cell cytotoxicity assay. Results indicated a depression in NK activity from the earliest stages of pregnancy. The target binding capacity of the effector cells remained unimpaired up to 16 weeks, but a significant reduction in the post-binding lytic potential was observed, which parallelled the drop in cytotoxicity as assayed by the 51Cr-release method. The ability of individual effector cells to recycle and kill multiple targets remained essentially unimpaired. Analysis of lymphocyte subpopulations using the monoclonal antibodies anti-Leu-7 and anti-Leu-11b, which recognize NK cell-associated antigens, showed a significant reduction in the proportion of the mature, lytically active Leu-7-11+ cells capable of both binding and lysing K562 target cells. The suggestion that the depression in cytotoxicity may be associated with the reduction in the Leu-7-11+ subpopulation is supported by the high correlation levels observed between the proportion of Leu-7-11+ cells and target cell lysis.

Natural killer (NK) cells have the ability to kill a variety of target cell types and the possibility that such cells could mount an effective attack on the developing fetus has not been discounted. The present study extends previous work showing that maternal NK reactivity against K562 target cells (TC) is reduced during pregnancy. Here we demonstrate using cytotoxicity assays at both the population and single cell level that, although depressed in number, maternal NK cells exhibiting the capacity to kill K562 TC are as lytically active in their ability to recycle and destroy multiple TC as NK cells from non-pregnant females. Moreover, two colour immunofluorescence analysis of the NK cell-associated markers Leu-7 and Leu-11b indicates that, in addition to a reduction in the absolute number of TC conjugate-forming cells, pregnant females present in their peripheral blood a larger proportion of TC-binding Leu-7+11- cells. These cells may be lytically immature. Small changes in NK cell profile and activity in maternal peripheral blood may be indicative of much more significant changes at the feto-maternal interface. It is, however, clear that pregnant females retain a population of highly active NK cells, thus minimising the possibility of immunocompromise.