4583

The c-Jun N-terminal protein kinase (JNK) is a member of the mitogen-activated protein kinase (MAPK) superfamily and plays a critical role in many cellular activities, from growth control to programmed cell death. Despite intensive studies, the role of JNK in normal and malignant heamatopoiesis is incompletely understood. In this report, we investigated the potential role of JNK in tumorigenesis, using interleukin-3 (IL-3)-dependent hematopoietic FL5.12 cells as a model system. FL5.12 cells depend on IL-3 for proliferation and survival. FL5.12 cells rapidly undergo apoptosis in the absence of IL-3. JNK was inhibited by IL-3 withdrawal but stimulated by IL-3 re-addition. FL5.Bcl-xL cells underwent growth arrest and subsequent apoptosis when treated with a specific JNK inhibitor SP600125 even in the presence of IL-3. Furthermore, inhibition of JNK by SP600125 promoted IL-3 withdrawal-induced apoptosis. Thus, JNK is required for survival of IL-3-dependent FL5.12 cells. FL5.12 cells stably transfected with an expression vector containing the anti-apoptotic Bcl-2 family protein Bc1-xL (FL5.Bcl-xL) were less sensitive to apoptosis induced by IL-3 withdrawal, but still remained dependent on IL-3 for proliferation. Repeated IL-3 withdrawal produces IL-3-independent FL5.Bcl-xL cells (FL5.Bcl-xLI), which were able to proliferate in media lacking IL-3, form colonies in soft agar cloning assay in vitro, and cause leukemogenesis in vivo. Interestingly, JNK was constitutively activated in FL5.Bcl-xLI cells. Inhibition of JNK by SP600125 induced growth arrest, apoptosis, and reduced ability of forming colonies in soft agar in FL5.Bcl-xLI cells. Thus, constitutive activation of JNK may contribute to proliferation, survival and transformation of IL-3-independent FL5.12 cells. We further tested whether constitutive JNK activation is sufficient for IL-3-independent growth and transformation. FL5.Bcl-xL cells were transfected with mammalian expression vectors encoding HA-JNKK2-JNK1, which has constitutive Jun kinase activity and were then subjected to IL-3 withdrawal. Cells expressing the constitutively active JNKK2-JNK1 were much more resistant to IL-3 withdrawal-induced apoptosis but failed to grow in the absence of IL-3. Thus, constitutive activation of JNK suppressed IL-3 withdrawal-induced apoptosis but was not enough to support IL-3-independent proliferation. Furthermore, both FL5.12 Bcl-xL/BAD and FL5.Bcl-xL cells expressing the constitutively active JNKK2-JNK1 alone or in combination with Akt did not form colonies in soft agar cloning assay. Thus, JNK activation is not sufficient for IL-3-independent growth and insufficient for transformation. Taken together, JNK activity is necessary, but not sufficient, for the transformation of FL5.12 pro-B cells.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]