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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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ERK and JNK knockdown inhibits colony formation of v-Rel transformed cells. (a–b) The v-Rel transformed cell line, 160/2, was electroporated with siRNA targeting ERK (3 or 5 μg) or with negative control siRNA (3 μg). Cells from the same electroporation population were plated into soft agar 16 hours after transfection and harvested at 48 hours for protein. (a) The levels of total ERK, as well as the levels of phosphorylation of a downstream target, p90-RSK were examined by Western blot. (b) Soft agar colonies were scored microscopically 7–8 days after plating. Colony numbers from cells electroporated with negative control siRNA were standardized to 100. The average for four independent experiments is shown with standard error. (c–d) 160/2 cells were electroporated with negative control siRNA or with siRNA (1 μg) targeting JNK1, JNK2, or both. Cells were plated into soft agar and harvested for protein 16 hours after transfection. (c) Western blot analysis examined the levels of phosphorylated and total JNK1 and JNK2. (d) Soft agar colonies were scored microscopically 7–8 days after plating. Colony numbers from cells electroporated with negative control siRNA were standardized to 100. The average for four independent experiments is shown with standard error. Statistically significant differences in colony formation of cells electroporated with specific siRNA relative to negative control are indicated (**P<0.01, ***P<0.001).
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Figure 3: ERK and JNK knockdown inhibits colony formation of v-Rel transformed cells. (a–b) The v-Rel transformed cell line, 160/2, was electroporated with siRNA targeting ERK (3 or 5 μg) or with negative control siRNA (3 μg). Cells from the same electroporation population were plated into soft agar 16 hours after transfection and harvested at 48 hours for protein. (a) The levels of total ERK, as well as the levels of phosphorylation of a downstream target, p90-RSK were examined by Western blot. (b) Soft agar colonies were scored microscopically 7–8 days after plating. Colony numbers from cells electroporated with negative control siRNA were standardized to 100. The average for four independent experiments is shown with standard error. (c–d) 160/2 cells were electroporated with negative control siRNA or with siRNA (1 μg) targeting JNK1, JNK2, or both. Cells were plated into soft agar and harvested for protein 16 hours after transfection. (c) Western blot analysis examined the levels of phosphorylated and total JNK1 and JNK2. (d) Soft agar colonies were scored microscopically 7–8 days after plating. Colony numbers from cells electroporated with negative control siRNA were standardized to 100. The average for four independent experiments is shown with standard error. Statistically significant differences in colony formation of cells electroporated with specific siRNA relative to negative control are indicated (**P<0.01, ***P<0.001).

Mentions: To investigate the importance of individual MAPK isoforms, we used a siRNA knockdown approach. In chicken, only one isoform of ERK is present, which shares the greatest homology with mammalian ERK2. In our experiments, the T-cell line (160/2) was electroporated with negative control siRNA or with increasing amounts of siRNA targeting ERK. Cells from the same electroporation population were plated into soft agar and harvested for protein. Western blot analysis showed a clear decrease in ERK protein levels (3 μg and 5 μg of ERK siRNA resulted in a 60% and 75% decrease in total ERK levels, respectively) (Figure 3a). This reduced level of protein corresponded with diminished ERK activity, as demonstrated by lowered phosphorylation of its downstream target, RSK. Moreover, cells transfected with ERK siRNA formed 2–3-fold fewer colonies than those receiving negative control siRNA (Figure 3b).


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

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

ERK and JNK knockdown inhibits colony formation of v-Rel transformed cells. (a–b) The v-Rel transformed cell line, 160/2, was electroporated with siRNA targeting ERK (3 or 5 μg) or with negative control siRNA (3 μg). Cells from the same electroporation population were plated into soft agar 16 hours after transfection and harvested at 48 hours for protein. (a) The levels of total ERK, as well as the levels of phosphorylation of a downstream target, p90-RSK were examined by Western blot. (b) Soft agar colonies were scored microscopically 7–8 days after plating. Colony numbers from cells electroporated with negative control siRNA were standardized to 100. The average for four independent experiments is shown with standard error. (c–d) 160/2 cells were electroporated with negative control siRNA or with siRNA (1 μg) targeting JNK1, JNK2, or both. Cells were plated into soft agar and harvested for protein 16 hours after transfection. (c) Western blot analysis examined the levels of phosphorylated and total JNK1 and JNK2. (d) Soft agar colonies were scored microscopically 7–8 days after plating. Colony numbers from cells electroporated with negative control siRNA were standardized to 100. The average for four independent experiments is shown with standard error. Statistically significant differences in colony formation of cells electroporated with specific siRNA relative to negative control are indicated (**P<0.01, ***P<0.001).
© Copyright Policy
Related In: Results  -  Collection

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Figure 3: ERK and JNK knockdown inhibits colony formation of v-Rel transformed cells. (a–b) The v-Rel transformed cell line, 160/2, was electroporated with siRNA targeting ERK (3 or 5 μg) or with negative control siRNA (3 μg). Cells from the same electroporation population were plated into soft agar 16 hours after transfection and harvested at 48 hours for protein. (a) The levels of total ERK, as well as the levels of phosphorylation of a downstream target, p90-RSK were examined by Western blot. (b) Soft agar colonies were scored microscopically 7–8 days after plating. Colony numbers from cells electroporated with negative control siRNA were standardized to 100. The average for four independent experiments is shown with standard error. (c–d) 160/2 cells were electroporated with negative control siRNA or with siRNA (1 μg) targeting JNK1, JNK2, or both. Cells were plated into soft agar and harvested for protein 16 hours after transfection. (c) Western blot analysis examined the levels of phosphorylated and total JNK1 and JNK2. (d) Soft agar colonies were scored microscopically 7–8 days after plating. Colony numbers from cells electroporated with negative control siRNA were standardized to 100. The average for four independent experiments is shown with standard error. Statistically significant differences in colony formation of cells electroporated with specific siRNA relative to negative control are indicated (**P<0.01, ***P<0.001).
Mentions: To investigate the importance of individual MAPK isoforms, we used a siRNA knockdown approach. In chicken, only one isoform of ERK is present, which shares the greatest homology with mammalian ERK2. In our experiments, the T-cell line (160/2) was electroporated with negative control siRNA or with increasing amounts of siRNA targeting ERK. Cells from the same electroporation population were plated into soft agar and harvested for protein. Western blot analysis showed a clear decrease in ERK protein levels (3 μg and 5 μg of ERK siRNA resulted in a 60% and 75% decrease in total ERK levels, respectively) (Figure 3a). This reduced level of protein corresponded with diminished ERK activity, as demonstrated by lowered phosphorylation of its downstream target, RSK. Moreover, cells transfected with ERK siRNA formed 2–3-fold fewer colonies than those receiving negative control siRNA (Figure 3b).

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