Effects of protein kinase C activators on phorbol ester-sensitive and - resistant EL4 thymoma cells

Abstract

Phorbol ester-sensitive EL4 murine thymoma cells respond to phorbol 12-myristate 13-acetate with activation of ERK mitogen-activated protein kinases, synthesis of interleukin-2, and death, whereas phorbol ester-resistant variants of this cell line do not exhibit these responses. Additional aspects of the resistant phenotype were examined, using a newly-established resistant cell line. Phorbol ester induced morphological changes, ERK activation, calcium-dependent activation of the c-Jun N-terminal kinase (JNK), interleukin-2 synthesis, and growth inhibition in sensitive but not resistant cells. A series of protein kinase C activators caused membrane translocation of protein kinase C's (PKCs) alpha, eta, and theta in both cell lines. While PKC eta was expressed at higher levels in sensitive than in resistant cells, overexpression of PKC eta did not restore phorbol ester-induced ERK activation to resistant cells. In sensitive cells, PKC activators had similar effects on cell viability and ERK activation, but differed in their abilities to induce JNK activation and interleukin-2 synthesis. PD 098059, an inhibitor of the mitogen activated protein (MAP)/ERK kinase kinase MEK, partially inhibited ERK activation and completely blocked phorbol ester-induced cell death in sensitive cells. Thus MEK and/or ERK activation, but not JNK activation or interleukin-2 synthesis, appears to be required for phorbol ester-induced toxicity. Alterations in phorbol ester response pathways, rather than altered expression of PKC isoforms, appear to confer phorbol ester resistance to EL4 cells.

Effects of Active and Inactive Phospholipase D2 on Signal Transduction, Adhesion, Migration, Invasion, and Metastasis in EL4 Lymphoma Cells

“…PKC␣-mediated phosphorylation may play a role in inactivating PLD2, whereas direct association with PKC␣ may be involved in PLD2 activation (Chen and Exton, 2004). EL4 cells express PKC␣ and other PKC isoforms (Sansbury et al, 1997). Serine/threonine phosphorylation of PLD2 (Chen and Exton, 2004) has been shown to result in mobility shifts on SDS-PAGE.…”

“…7B, with PMA-sensitive clone WT2 included for purposes of comparison. As described previously (Sansbury et al, 1997;Ku and Meier, 2000), PMA-resistant clones (e.g., V7) attach and spread on tissue culture plastic to a greater extent than PMA-sensitive (e.g., WT2) clones. This is reflected by a decrease in the proportion of V7 cells having an axis ratio of 1 to 2 (rounded) and an increase in cells having ratios of greater than 2 (elongated) compared with WT2 cells.…”

“…With respect to EL4 cells, PMA resistance refers to a lack of effect of PMA on long-term growth and survival. Sensitive cells undergo growth arrest and death in response to PMA, whereas resistant cells do not (Sansbury et al, 1997). PMA sensitivity is conferred by expression of RasGRP, a phorbol ester binding protein (Han et al, 2007); PMA-induced cell death is dependent on RasGRP-mediated Erk activation (Sansbury et al, 1997).…”

“…Sensitive cells undergo growth arrest and death in response to PMA, whereas resistant cells do not (Sansbury et al, 1997). PMA sensitivity is conferred by expression of RasGRP, a phorbol ester binding protein (Han et al, 2007); PMA-induced cell death is dependent on RasGRP-mediated Erk activation (Sansbury et al, 1997). Both sensitive and resistant cells activate PKC isoforms in response to PMA (Ku and Meier, 2000).…”

RasGRP1 Confers the Phorbol Ester-Sensitive Phenotype to EL4 Lymphoma Cells

“…Responses of sensitive "wild-type" (WT) EL4 cells to phorbol 12-myristate 13-acetate (PMA) include protein kinase C (PKC) activation (Kramer and Sando, 1986;Meier et al, 1991;Baier-Bitterlich et al, 1996;Sansbury et al, 1997), tyrosine phosphorylation (Richardson and Sando, 1995;Luo and Sando, 1997), Erk mitogen-activated protein kinase activation (Meier et al, 1991;Gause et al, 1993;Sansbury et al, 1997), adhesion (Resnick et al, 1997), IL-2 production (Farrar et al, 1980;Sando et al, 1982;Pearlstein et al, 1983;Harrison et al, 1987;Rayter et al, 1992), and growth arrest (Sando et al, 1982;Harrison et al, 1987;Desrivieres et al, 1997;Sansbury et al, 1997). To study the mechanisms by which PMA elicits these responses, we and other investigators have characterized PMA-resistant EL4 cells, which by definition proliferate in the presence of PMA (Resnick et al, 1997;Sansbury et al, 1997;Ku and Meier, 2000). Resistant EL4 cells activate PKCs in response to PMA but show minimal activation of Ras (Rayter et al, 1992), Erks (Meier et al, 1991), MEK (Gause et al, 1993), pp90…”