Response of Jurkat T cells to phorbol ester and bryostatin. Development of sublines with distinct functional responses and changes in protein kinase C activity.
T lymphocyte activation is initiated as a result of the interaction between the TCR complex and Ag as seen in the framework of a membrane-bound MHC molecule. Receptor stimulation results in a rise in free intracellular Ca2+ and the activation of protein kinase C (PKC). Bryostatin (Bryo) and phorbol esters (e.g., 12-O-tetradecanoylphorbol 13-acetate (TPA] are PKC activators with somewhat different immunologic effects. We compared the effect of Bryo and TPA on the T cell tumor line Jurkat and derivatives of Jurkat cells grown in media supplemented with 100 nM Bryo ("BR100" cells) or 100 nM TPA ("TP100" cells). In untreated Jurkat cells, there is a dose- and time-dependent decrease in proliferation, compared to media controls, after the administration of as little as 10 nM TPA. This can be reversed in a dose- and time-dependent manner by Bryo. Interestingly, the expression of the transferrin receptor parallelled this effect on proliferation. Furthermore, Jurkat cells grown continuously in 100 nM TPA regained full proliferative capacity after several weeks in culture and transferrin receptor expression returned to near the level seen in untreated Jurkat cells. The chromatographic separation of PKC activity in these three cell lines showed that total PKC activity was dramatically decreased in both the TP100 and BR100 cells when compared to untreated Jurkat cells. However, in the TP100 cells there exists a peak of activity that is activated by Bryo, but not TPA. Western blots of whole cell lysates of the three cell lines showed that PKC-alpha and PKC-beta II were both down-regulated in BR100 and TP100 cells compared to untreated Jurkat cells. PKC-gamma was not detected in any of the cell lines. Therefore, the Bryo-specific peak seen in TP100 cells may be PKC-delta, -epsilon, -zeta, -eta, or a novel PKC isoform. This could provide the basis for a molecular characterization of the differences in PKC activation between phorbol esters and Bryo.
Levine
B L
BL
Johns Hopkins Oncology Center, Baltimore, MD 21205.
May
W S
WS
Tyler
P G
PG
Hess
A D
AD
D015703:Antigens, CD / Q000378:metabolism; D000945:Antigens, Differentiation, T-Lymphocyte / Q000378:metabolism; D054713:Bryostatins; D017252:CD3 Complex; D002999:Clone Cells; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D066298:In Vitro Techniques; D007783:Lactones / Q000494:pharmacology*; D008213:Lymphocyte Activation / Q000187:drug effects*; D018942:Macrolides; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D011493:Protein Kinase C / Q000502:physiology*; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism; D011990:Receptors, Transferrin / Q000378:metabolism; D013601:T-Lymphocytes / Q000187:drug effects*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte; D054713:Bryostatins; D017252:CD3 Complex; D007783:Lactones; D018942:Macrolides; D011948:Receptors, Antigen, T-Cell; D011990:Receptors, Transferrin; D015240:Phorbol 12,13-Dibutyrate; C046785:bryostatin 1; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate