Activation of the Jun N-terminal kinase pathway by friend spleen focus-forming virus and its role in the growth and survival of friend virus-induced erythroleukemia cells

Abstract

Members of the mitogen-activated protein kinase (MAPK) family, including Jun amino-terminal kinase (JNK) and extracellular signal-related kinase (ERK), play an important role in the proliferation of erythroid cells in response to erythropoietin (Epo). Erythroid cells infected with the Friend spleen focus-forming virus (SFFV) proliferate in the absence of Epo and show constitutive activation of Epo signal transduction pathways. We previously demonstrated that the ERK pathway was constitutively activated in Friend SFFV-infected erythroid cells, and in this study JNK is also shown to be constitutively activated. Pharmacological inhibitors of both the ERK and JNK pathways stopped the proliferation of primary erythroleukemic cells from Friend SFFV-infected mice, with little induction of apoptosis, and furthermore blocked their ability to form Epo-independent colonies. However, only the JNK inhibitor blocked the proliferation of erythroleukemia cell lines derived from these mice. The JNK inhibitor caused significant apoptosis in these cell lines as well as an increase in the fraction of cells in G(2)/M and undergoing endoreduplication. In contrast, the growth of erythroleukemia cell lines derived from Friend murine leukemia virus (MuLV)-infected mice was inhibited by both the MEK and JNK inhibitors. JNK is important for AP1 activity, and we found that JNK inhibitor treatment reduced AP1 DNA-binding activity in primary erythroleukemic splenocytes from Friend SFFV-infected mice and in erythroleukemia cell lines from Friend MuLV-infected mice but did not alter AP1 DNA binding in erythroleukemia cell lines from Friend SFFV-infected mice. These data suggest that JNK plays an important role in cell proliferation and/or the survival of erythroleukemia cells.

FIG. 1.

Expression of phosphorylated MEK and JNK in erythroleukemia cells from Friend SFFV- and Friend MuLV-infected mice. JNK and MEK phosphorylation was determined in uninfected and Friend SFFV-infected HCD-57 cells grown in the presence or absence of Epo (A), primary erythroleukemia cells derived from the spleens of two separate Friend SFFV-infected mice (B), erythroleukemia cell lines (lane 1, NP1; lane 2, NP4; lane 3, NP5; lane 4, NP7; lane 5, NP13) derived from mice infected with helper-free Friend SFFV (C), and erythroleukemia cells lines derived from mice infected with Friend MuLV (lane 1, TP1; lane 2, TP3; lane 3, CB7; lane 4, HB22.2; lane 5, HB9.2ED) (D). Western blot analysis of total cell lysates was carried out using anti-phospho-JNK and anti-phospho-MEK antibodies. The filters were stripped and then incubated with anti-JNK or anti-MEK antibodies to determine total kinase levels.

FIG. 2.

Effect of MEK and JNK inhibitors on the proliferation of uninfected and Friend SFFV-infected HCD-57 cells. Epo-dependent HCD-57 cells (□) and Epo-independent HCD-57/SFFV cells (▴) were cultured with and without Epo, respectively, for 48 h in the presence of various concentrations of the MEK inhibitor PD98059 (A) or the JNK inhibitor SP600125 (B), and proliferation was measured using the WST-1 reagent. Graphs represent results with triplicate samples, with bars showing standard errors. Cells were also analyzed using flow cytometry for PI-stained cells 48 h after treatment with the DMSO control, PD98059 (50 μM), or SP600125 (10 μM) (C). The sub-G₁ fraction was assessed as apoptotic cells, and the percentages of the total were determined. OD, optical density.

FIG. 3.

Effect of MEK and JNK inhibitors on the proliferation of primary erythroleukemia cells from Friend SFFV-infected mice. Primary erythroleukemic cells from the spleens of Friend SFFV-infected mice were cultured in Epo-free medium with various concentrations of the MEK inhibitor PD98059 (A) or the JNK inhibitor SP600125 (B) for 24 (□) or 48 (▴) hours, and proliferation was measured using the WST-1 reagent. Cells were also plated in methylcellulose in the absence of Epo, and benzidine-positive erythroid cells (numbers of SFFV CFU) were determined after 72 h (C). Graphs represent mean results from triplicate samples, with bars showing standard errors. OD, optical density.

FIG. 4.

Effects of MEK and JNK inhibitors on the proliferation of erythroleukemia cell lines from Friend SFFV-infected mice. Erythroleukemia cell lines from Friend SFFV-infected mice were cultured with different concentrations of the MEK inhibitor PD98059 (A) or the JNK inhibitor SP600125 (B) for 24 or 48 h. Proliferation was then measured using the WST-1 reagent. Cell lines analyzed were NP1 (□), NP4 (⋄), NP5 (○), NP7 (Δ), and NP13 (▪). Graphs represent mean results from triplicate samples. The standard error was less than 0.06. OD, optical density.

FIG. 5.

Effects of MEK and JNK inhibitors on the proliferation of erythroleukemia cell lines from Friend MuLV-infected mice. Erythroleukemia cell lines from Friend MuLV-infected mice were cultured with different concentrations of the MEK inhibitor PD98059 (A) or the JNK inhibitor SP600125 (B) for 24 or 48 h. Proliferation was then measured using the WST-1 reagent. Cell lines analyzed were TP1 (□), TP3 (⋄), CB7 (○), HB22.2 (Δ), and HB9.2ED (▪). Graphs represent mean results from triplicate samples. The standard error was less than 0.06. OD, optical density.

FIG. 6.

Effects of MEK and JNK inhibitors on the induction of apoptosis in erythroleukemia cell lines from Friend SFFV- and Friend MuLV-infected mice. Erythroleukemia cell lines from Friend SFFV-infected mice (A) and Friend MuLV-infected mice (B) were cultured for 48 h with the DMSO control, the MEK inhibitor PD98059 (50 μM), or the JNK inhibitor SP600125 (10 μM). Numbers of apoptotic cells were then determined by flow cytometry analysis using annexin V-fluorescein isothiocyanate staining. Graphs represent mean results from triplicate samples, with bars showing standard errors.

FIG. 7.

Effect of a JNK inhibitor on the cell cycle of transformed erythroleukemia cell lines from Friend SFFV- and Friend MuLV-infected mice. Erythroleukemia cells from Friend SFFV-infected mice (A) or Friend MuLV-infected mice (B) were cultured for 12 or 24 h in the presence of DMSO (control) or 10 μM SP600125. Cells were analyzed by flow cytometry of PI-stained cells. Results shown are representative of two separate experiments.

FIG. 8.

Effect of MEK and JNK inhibitors on AP1 binding in primary and transformed erythroleukemia cells from Friend SFFV- and Friend MuLV-infected mice. Cells were treated with the DMSO control (lanes 1 and 2), the MEK inhibitor PD98059 (50 μM) (lanes 3), or the JNK inhibitor SP600125 (10 μM) (lanes 4) for 48 h, and nuclear extracts were prepared. Extracts were then subjected to electrophoretic mobility shift assays using an AP1 consensus probe (lanes 2 to 4) or an AP1 mutant probe (lanes 1). Cells examined were (A) HCD-57 cells grown in Epo or Friend SFFV-infected HCD-57 cells grown in the absence of Epo, (B) a primary leukemic spleen from a Friend SFFV-infected mouse, (C) erythroleukemia cell lines from mice infected with Friend SFFV, and (D) erythroleukemia cell lines from mice infected with Friend MuLV. Arrows indicate the AP1 band. The asterisk indicates a nonspecific band.