Received for publication July 17, 2006.
Revised October 4, 2006.
Accepted for publication October 20, 2006.

RasGRP1 confers the phorbol ester-sensitive phenotype to EL4 lymphoma cells

Abstract

The murine EL4 lymphoma cell line exists in variants that are either sensitive or resistant to the tumor promoter phorbol 12-myristate 13-acetate (PMA). In sensitive EL4 cells, PMA causes robust Erk MAPK activation that results in growth arrest. In resistant cells, PMA induces minimal Erk activation, without growth arrest. PMA stimulates IL-2 production in sensitive, but not resistant, cells. The role of RasGRP1, a PMA-activated guanine nucleotide exchange factor for Ras, in EL4 phenotype was examined. Endogenous RasGRP1 protein is expressed at much higher levels in sensitive than in resistant cells. PMA-induced Ras activation is observed in sensitive cells, but not in resistant cells lacking RasGRP1. PMA induces down-regulation of RasGRP1 protein in sensitive cells, but increases RasGRP1 in resistant cells. Transfection of RasGRP1 into resistant cells enhances PMA-induced Erk activation. In the reverse experiment, introduction of siRNA for RasGRP1 suppresses PMA-induced Ras and Erk activations in sensitive cells. Sensitive cells incubated with siRNA for RasGRP1 exhibit the PMA-resistant phenotype, in that they are able to proliferate in the presence of PMA, and do not secrete IL-2 when stimulated with PMA. These studies indicate that the PMA sensitive phenotype, as previously defined for the EL4 cell line, is conferred by endogenous expression of RasGRP1 protein.

Key words: IP3/DAG, Ras, Raf family, MAP Kinase

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