Synthesis and secretion of light-chain immunoglobulin in two successive cycles of synchronized plasmacytoma cells.

Abstract

Suspension-cultured mouse plasmacytoma cells (MPC-11) were accumulated in the late G1 phase by exposure to isoleucine-deficient medium for 20-24 h. The arrested culture was fed with complete medium enabling the cells to continue the cell cycle synchronously, undergo mitosis, and enter a second cycle of growth. This method of synchronization left the protein-synthesizing ability intact as judged by the polysome profile and the capacity of the cells to incorporate labeled amino acids into protein after the restoration of isoleucine. After incubation in isoleucine-deficient medium and the addition of isoleucine to the culture, cells entered the S phase after a short lag, as judged by [SH]thymidine incorporation into nucleic acid and by spectrophotometric measurement of nuclear DNA. The cells were in mitosis between 12 and 18 h as judged by the increase in cell count and analysis of cell populations on albumin gradients.Synthesis and secretion of light-chain immunoglobulin were maximal in the late G1/early S phase of the first cycle. During late S phase, G2 phase, and mitosis, both synthesis and secretion were observed to be at a low level; however, immediately after mitosis the cells which then entered the G I phase apparently commenced synthesis of light chain immunoglobulin straight away, although secretion of labeled material remained at a low level.Lymphocytes ordinarily are quiescent cells that produce little or no immunoglobulin. After stimulation by antigen, lymphocytes undergo a transition to active, immunoglobulin-producing cells. Several investigators, using immunoglobulinsecreting, tissue-cultured cells which had been synchronized by different techniques, have suggested that the bulk of immunoglobulin synthesis occurs in the late G l/early S phase of the cell cycle (4,5,16,20), while other observations have not demonstrated any cyclical behavior with respect to immunoglobulin synthesis (6, 13).As part of studies concerned with elucidating the details of immunoglobulin biosynthesis and of membrane biogenesis on a molecular level, it became important to isolate large quantities of 232

p27Kip1‐mediated controlled proliferation technology increases constitutive sICAM production in CHO‐DUKX adapted for growth in suspension and serum‐free media

“…The predictions of the mathematical model were in line with observations that several endogenous genes are synthesized at increased rates in the G1 phase of the mammalian cell cycle (Buell and Fahey, 1969;Chabanas et al, 1983;Chafouleas et al, 1984; [for a review]; Garatun-Tjeldosto et al, 1976;Gu et al, 1993;Herz et al, 1991).…”

A novel cytostatic process enhances the productivity of Chinese hamster ovary cells

“…The fact that p53 can also induce the transcription from certain promoters; e.g., the p21 promoter, as part of its function to arrest the cell cycle, opens perspective for future work which could use synthetic p53-responsive promoters to increase further the productivity of G1-arrested cells (Barak et al, 1993;El-Deiry et al, 1994;Kastan et al, 1991). Other observations also show that foreign as well as host-cell protein production is dependent on the cell-cycle phase (Kubbies and Stockinger, 1990) and that several native genes are synthesized at increased rates in the G1-phase (Buell and Fahey, 1969;Chabanas et al, 1983;Chafouleas et al, 1984;Garatun-Tjeldsto et al, 1976;Gu et al, 1993b;Herz et al, 1991). Further experiments showing increased production predominantly in S-phase used viral promoters which were shown to become particularly active in this cell-cycle phase (Gu et al, 1993b;Kubbies and Stockinger, 1990).…”

Apoptosis-resistant E1B-19K-expressing NS/0 myeloma cells exhibit increased viability and chimeric antibody productivity under perfusion culture conditions

“…Indeed, many mammalian cell lines cultivated in perfusion exhibit an increase in specific MAb productivity under such slow growth conditions (Al-Rubeai et al, 1992;Banik and Heath, 1995;Batt et al, 1990;de la Broise et al, 1991de la Broise et al, , 1992Johnson et al, 1996;Hansen et al, 1993;Mercille et al, 1994a-c;Shi et al, 1993;Tokashiki and Takamatsu, 1993;Trampler et al, 1994). The q MAb has been shown to be cell cycle dependent and optimum in the G 1 (Byars and Kidson, 1970;Cazzador and Mariani, 1993;Garatun-Tjeldsto et al, 1976;Linardos et al, 1992;Mercille and Massie, 1998;Ramirez and Mutharasan, 1990;Richieri et al, 1991;Suzuki and Ollis, 1989) or G 2 /M phase (Cherlet et al, 1995;Kromenaker and Srienc, 1991;Mercille and Massie, 1998;Watanabe et al, 1973). The increase in the perfusion to batch q MAb ratio can therefore be explained by an increase in the percentage of G 1 cells in perfusion from 40 ± 5% for NS/0 control cells to 57 ± 6% for E1B-19K cells.…”