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ERK and JNK activation is essential for oncogenic transformation by v-Rel.

Kralova J, Sheely JI, Liss AS, Bose HR - Oncogene (2010)

Bottom Line: The expression of v-Rel results in the strong and sustained activation of the ERK and JNK MAPK pathways.MAPK signaling also has an important role in the initial transformation of primary spleen cells by v-Rel, although distinct requirements for MAPK activity at different stages of v-Rel-mediated transformation were identified.We also show that the ability of v-Rel to induce MAPK signaling more strongly than c-Rel contributes to its greater oncogenicity.

View Article: PubMed Central - PubMed

Affiliation: Section of Molecular Genetics and Microbiology, Institute of Cellular and Molecular Biology, University of Texas at Austin, Austin, TX, USA.

ABSTRACT
v-Rel is the acutely oncogenic member of the NF-κB family of transcription factors. Infection with retroviruses expressing v-Rel rapidly induces fatal lymphomas in birds and transforms primary lymphocytes and fibroblasts in vitro. We have previously shown that AP-1 transcriptional activity contributes to v-Rel-mediated transformation. Although v-Rel increases the expression of these factors, their activity may also be induced through phosphorylation by the mitogen-activated protein kinases (MAPKs). The expression of v-Rel results in the strong and sustained activation of the ERK and JNK MAPK pathways. This induction is critical for the v-Rel-transformed phenotype, as suppression of MAPK activity with chemical inhibitors or small interfering RNA severely impairs colony formation of v-Rel-transformed lymphoid cell lines. However, signaling must be maintained within an optimal range in these cells, as strong additional activation of either pathway beyond the levels induced by v-Rel through the expression of constitutively active MAPK proteins attenuates the transformed phenotype. MAPK signaling also has an important role in the initial transformation of primary spleen cells by v-Rel, although distinct requirements for MAPK activity at different stages of v-Rel-mediated transformation were identified. We also show that the ability of v-Rel to induce MAPK signaling more strongly than c-Rel contributes to its greater oncogenicity.

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Inhibition of ERK and JNK pathways attenuates colony formation of v-Rel transformed cells. Three established v-Rel transformed cell lines of histologically distinct origin were utilized, including a T-cell (160/2), B-cell (123/12), and non-B/non-T (123/6T) cell line. (a) Cell lines were treated for one hour with carrier alone (DMSO), MEK inhibitors (PD98059, U0126), or negative control (U0124) at a 3 μM concentration. Cell lysates were prepared and levels of phosphorylated and total ERK were examined by Western blot. (b) Cell lines were treated for one hour with carrier alone (DMSO), JNK inhibitor (SP600125), or negative control (NC-JNK II) at a 3 μM concentration and cell lysates were examined by Western blot for phosphorylated and total c-Jun. (c) Reporter assays evaluated the effect of MAPK inhibitors on AP-1 activity in cells expressing v-Rel. CEFs were co-transfected with a pGL2 luciferase reporter (1 μg) containing multiple repeats of an AP-1 consensus site (Kralova et al., 1998), the Rc/RSV expression vector (1 μg) encoding v-Rel, and with the pRL-TK reporter vector (0.3 μg). Eight hours later, MAPK inhibitors or their respective negative controls were added to the media at a 10 μM concentration. Luciferase activity in cells treated with carrier alone (DMSO) was standardized to 100. The average and standard deviation for four independent experiments are shown. (d) Cell lines were incubated with inhibitors or negative controls (3 μM) for two days and plated into soft agar in the presence of the same concentration of inhibitors. Colonies were scored microscopically 10 days later and colony numbers for cells treated with carrier alone (DMSO) were standardized to 100. The average and standard deviation for four independent experiments are shown.
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Figure 2: Inhibition of ERK and JNK pathways attenuates colony formation of v-Rel transformed cells. Three established v-Rel transformed cell lines of histologically distinct origin were utilized, including a T-cell (160/2), B-cell (123/12), and non-B/non-T (123/6T) cell line. (a) Cell lines were treated for one hour with carrier alone (DMSO), MEK inhibitors (PD98059, U0126), or negative control (U0124) at a 3 μM concentration. Cell lysates were prepared and levels of phosphorylated and total ERK were examined by Western blot. (b) Cell lines were treated for one hour with carrier alone (DMSO), JNK inhibitor (SP600125), or negative control (NC-JNK II) at a 3 μM concentration and cell lysates were examined by Western blot for phosphorylated and total c-Jun. (c) Reporter assays evaluated the effect of MAPK inhibitors on AP-1 activity in cells expressing v-Rel. CEFs were co-transfected with a pGL2 luciferase reporter (1 μg) containing multiple repeats of an AP-1 consensus site (Kralova et al., 1998), the Rc/RSV expression vector (1 μg) encoding v-Rel, and with the pRL-TK reporter vector (0.3 μg). Eight hours later, MAPK inhibitors or their respective negative controls were added to the media at a 10 μM concentration. Luciferase activity in cells treated with carrier alone (DMSO) was standardized to 100. The average and standard deviation for four independent experiments are shown. (d) Cell lines were incubated with inhibitors or negative controls (3 μM) for two days and plated into soft agar in the presence of the same concentration of inhibitors. Colonies were scored microscopically 10 days later and colony numbers for cells treated with carrier alone (DMSO) were standardized to 100. The average and standard deviation for four independent experiments are shown.

Mentions: The importance of ERK and JNK signaling to the transformed phenotype of established v-Rel transformed cell lines was examined. MAPK activity was reduced through the use of pharmacological inhibitors, including MEK inhibitors (U0126 and PD98059) to block ERK activation and a JNK inhibitor (SP600125) to block JNK activity. Three histologically distinct v-Rel transformed lymphoid cell lines were selected, including a T-cell (160/2), B-cell (123/12), and non-B/non-T (123/6T) cell line. Cells were incubated in the presence of DMSO vehicle alone, MEK or JNK inhibitors, or their respective negative controls. Incubation with either MEK inhibitor caused significant reduction in ERK phosphorylation relative to treatment with the negative control (U0124) or DMSO (Figure 2a). Similarly, incubation with the JNK inhibitor reduced the levels of phosphorylated c-Jun in comparison to treatment with negative controls (Figure 2b). Total levels of ERK and c-Jun were not altered by any treatment. Importantly, inhibitor treatment did not affect the retroviral expression of v-Rel in any of these lineages.


ERK and JNK activation is essential for oncogenic transformation by v-Rel.

Kralova J, Sheely JI, Liss AS, Bose HR - Oncogene (2010)

Inhibition of ERK and JNK pathways attenuates colony formation of v-Rel transformed cells. Three established v-Rel transformed cell lines of histologically distinct origin were utilized, including a T-cell (160/2), B-cell (123/12), and non-B/non-T (123/6T) cell line. (a) Cell lines were treated for one hour with carrier alone (DMSO), MEK inhibitors (PD98059, U0126), or negative control (U0124) at a 3 μM concentration. Cell lysates were prepared and levels of phosphorylated and total ERK were examined by Western blot. (b) Cell lines were treated for one hour with carrier alone (DMSO), JNK inhibitor (SP600125), or negative control (NC-JNK II) at a 3 μM concentration and cell lysates were examined by Western blot for phosphorylated and total c-Jun. (c) Reporter assays evaluated the effect of MAPK inhibitors on AP-1 activity in cells expressing v-Rel. CEFs were co-transfected with a pGL2 luciferase reporter (1 μg) containing multiple repeats of an AP-1 consensus site (Kralova et al., 1998), the Rc/RSV expression vector (1 μg) encoding v-Rel, and with the pRL-TK reporter vector (0.3 μg). Eight hours later, MAPK inhibitors or their respective negative controls were added to the media at a 10 μM concentration. Luciferase activity in cells treated with carrier alone (DMSO) was standardized to 100. The average and standard deviation for four independent experiments are shown. (d) Cell lines were incubated with inhibitors or negative controls (3 μM) for two days and plated into soft agar in the presence of the same concentration of inhibitors. Colonies were scored microscopically 10 days later and colony numbers for cells treated with carrier alone (DMSO) were standardized to 100. The average and standard deviation for four independent experiments are shown.
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Figure 2: Inhibition of ERK and JNK pathways attenuates colony formation of v-Rel transformed cells. Three established v-Rel transformed cell lines of histologically distinct origin were utilized, including a T-cell (160/2), B-cell (123/12), and non-B/non-T (123/6T) cell line. (a) Cell lines were treated for one hour with carrier alone (DMSO), MEK inhibitors (PD98059, U0126), or negative control (U0124) at a 3 μM concentration. Cell lysates were prepared and levels of phosphorylated and total ERK were examined by Western blot. (b) Cell lines were treated for one hour with carrier alone (DMSO), JNK inhibitor (SP600125), or negative control (NC-JNK II) at a 3 μM concentration and cell lysates were examined by Western blot for phosphorylated and total c-Jun. (c) Reporter assays evaluated the effect of MAPK inhibitors on AP-1 activity in cells expressing v-Rel. CEFs were co-transfected with a pGL2 luciferase reporter (1 μg) containing multiple repeats of an AP-1 consensus site (Kralova et al., 1998), the Rc/RSV expression vector (1 μg) encoding v-Rel, and with the pRL-TK reporter vector (0.3 μg). Eight hours later, MAPK inhibitors or their respective negative controls were added to the media at a 10 μM concentration. Luciferase activity in cells treated with carrier alone (DMSO) was standardized to 100. The average and standard deviation for four independent experiments are shown. (d) Cell lines were incubated with inhibitors or negative controls (3 μM) for two days and plated into soft agar in the presence of the same concentration of inhibitors. Colonies were scored microscopically 10 days later and colony numbers for cells treated with carrier alone (DMSO) were standardized to 100. The average and standard deviation for four independent experiments are shown.
Mentions: The importance of ERK and JNK signaling to the transformed phenotype of established v-Rel transformed cell lines was examined. MAPK activity was reduced through the use of pharmacological inhibitors, including MEK inhibitors (U0126 and PD98059) to block ERK activation and a JNK inhibitor (SP600125) to block JNK activity. Three histologically distinct v-Rel transformed lymphoid cell lines were selected, including a T-cell (160/2), B-cell (123/12), and non-B/non-T (123/6T) cell line. Cells were incubated in the presence of DMSO vehicle alone, MEK or JNK inhibitors, or their respective negative controls. Incubation with either MEK inhibitor caused significant reduction in ERK phosphorylation relative to treatment with the negative control (U0124) or DMSO (Figure 2a). Similarly, incubation with the JNK inhibitor reduced the levels of phosphorylated c-Jun in comparison to treatment with negative controls (Figure 2b). Total levels of ERK and c-Jun were not altered by any treatment. Importantly, inhibitor treatment did not affect the retroviral expression of v-Rel in any of these lineages.

Bottom Line: The expression of v-Rel results in the strong and sustained activation of the ERK and JNK MAPK pathways.MAPK signaling also has an important role in the initial transformation of primary spleen cells by v-Rel, although distinct requirements for MAPK activity at different stages of v-Rel-mediated transformation were identified.We also show that the ability of v-Rel to induce MAPK signaling more strongly than c-Rel contributes to its greater oncogenicity.

View Article: PubMed Central - PubMed

Affiliation: Section of Molecular Genetics and Microbiology, Institute of Cellular and Molecular Biology, University of Texas at Austin, Austin, TX, USA.

ABSTRACT
v-Rel is the acutely oncogenic member of the NF-κB family of transcription factors. Infection with retroviruses expressing v-Rel rapidly induces fatal lymphomas in birds and transforms primary lymphocytes and fibroblasts in vitro. We have previously shown that AP-1 transcriptional activity contributes to v-Rel-mediated transformation. Although v-Rel increases the expression of these factors, their activity may also be induced through phosphorylation by the mitogen-activated protein kinases (MAPKs). The expression of v-Rel results in the strong and sustained activation of the ERK and JNK MAPK pathways. This induction is critical for the v-Rel-transformed phenotype, as suppression of MAPK activity with chemical inhibitors or small interfering RNA severely impairs colony formation of v-Rel-transformed lymphoid cell lines. However, signaling must be maintained within an optimal range in these cells, as strong additional activation of either pathway beyond the levels induced by v-Rel through the expression of constitutively active MAPK proteins attenuates the transformed phenotype. MAPK signaling also has an important role in the initial transformation of primary spleen cells by v-Rel, although distinct requirements for MAPK activity at different stages of v-Rel-mediated transformation were identified. We also show that the ability of v-Rel to induce MAPK signaling more strongly than c-Rel contributes to its greater oncogenicity.

Show MeSH
Related in: MedlinePlus