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c-Jun activation-dependent tumorigenic transformation induced paradoxically by overexpression or block of S-adenosylmethionine decarboxylase.

Paasinen-Sohns A, Kielosto M, Kääriäinen E, Eloranta T, Laine A, Jänne OA, Birrer MJ, Hölttä E - J. Cell Biol. (2000)

Bottom Line: The phenotype of the AdoMetDC sense transformants was reversed by expression of dominant-negative mutants of SEK1 (MKK4), JNK1, and c-Jun (TAM-67), which were also found to impair cytokinesis.Similarly, TAM-67 reverted the morphology of the AdoMetDC-antisense expressors.In addition, we show that the polyamine biosynthetic enzymes require c-Jun activation for eliciting their biological effects.

View Article: PubMed Central - PubMed

Affiliation: Haartman Institute, Department of Pathology, FIN-00014 University of Helsinki, Helsinki, Finland.

ABSTRACT
All mammalian cells absolutely require polyamines (putrescine, spermidine, and spermine) for growth. Here we show that the overexpression of cDNA for S-adenosylmethionine decarboxylase (AdoMetDC), the main regulatory enzyme in the biosynthesis of higher polyamines, induces transformation of rodent fibroblasts when expressed in the sense or the antisense orientation. Both transformants were able to induce invasive tumors in nude mice. Neither transformation was associated with activation of the mitogen-activated protein kinases Erk1 and Erk2. Instead, the AdoMet DC sense, but not antisense, transformants displayed constitutive activation of the c-Jun NH(2)-terminal kinase (JNK) pathway. However, both transformations converged on persistent phosphorylation of endogenous c-Jun at Ser73. The phenotype of the AdoMetDC sense transformants was reversed by expression of dominant-negative mutants of SEK1 (MKK4), JNK1, and c-Jun (TAM-67), which were also found to impair cytokinesis. Similarly, TAM-67 reverted the morphology of the AdoMetDC-antisense expressors. This report is the first demonstration of a protein whose overexpression or block of synthesis can induce cell transformation. In addition, we show that the polyamine biosynthetic enzymes require c-Jun activation for eliciting their biological effects.

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Erk1 and Erk2 are not constitutively activated in AdoMetDC transformants. (A) Western blot analysis of the phosphorylation status of Erks. The electrophoretic mobilities of Erk1 and Erk2 in Amdc-s or Amdc-as+spd cells did not display upshifts of protein bands (in 12.5% SDS-PAGE) that were seen in control cells (4N and Rat-1) stimulated with PDGF-BB. (B) In vitro immunocomplex kinase assays showed a clear stimulation of MAPK activity in PDGF-BB–stimulated control cells, but no constitutive upregulation was detected in AdoMetDC transformants. Gray bars, cells starved for 24 h in 0.5% serum; white bars, starved cells stimulated with PDGF-BB for 15 min at 37°C; black bars, cells grown normally in 5% serum.
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Figure 3: Erk1 and Erk2 are not constitutively activated in AdoMetDC transformants. (A) Western blot analysis of the phosphorylation status of Erks. The electrophoretic mobilities of Erk1 and Erk2 in Amdc-s or Amdc-as+spd cells did not display upshifts of protein bands (in 12.5% SDS-PAGE) that were seen in control cells (4N and Rat-1) stimulated with PDGF-BB. (B) In vitro immunocomplex kinase assays showed a clear stimulation of MAPK activity in PDGF-BB–stimulated control cells, but no constitutive upregulation was detected in AdoMetDC transformants. Gray bars, cells starved for 24 h in 0.5% serum; white bars, starved cells stimulated with PDGF-BB for 15 min at 37°C; black bars, cells grown normally in 5% serum.

Mentions: Numerous reports indicate that activation of the Erk1/Erk2 MAPK cascade is necessary for cell growth. Since MAPK is activated in cells transformed by many oncogenes (Howe et al. 1992; Samuels et al. 1993; Okazaki and Sagata 1995), we tested whether MAPK might also be constitutively activated and responsible for the transformed phenotype in Amdc-s and Amdc-as+spd transformants. To distinguish between the inactive and active states of Erk1 and Erk2 proteins, we starved the normal cells (4N and Rat-1) with 0.5% FBS for 20 h, and then stimulated the cells with PDGF-BB (30 ng/ml) for 15 min at 37°C. Exposure to PDGF-BB resulted in distinct shifts of Erk1 and Erk2 migration, as expected (Fig. 3 A). No shifts of Erks, indicative of constitutive activation, were detected in the transformants that were grown normally in the presence of serum (Fig. 3 A). Additionally, MAPK activities were measured by in vitro immunocomplex kinase assays, which used a synthetic peptide of myelic basic protein as a substrate. The normal 4N cells responded to PDGF-BB with a threefold activation, and Rat-1 cells exhibited a 15-fold increase in MAPK activity (Fig. 3 B). Again, neither the NIH3T3 nor Rat-1 transformants displayed a constitutive increase in MAPK activity (Fig. 3 B).


c-Jun activation-dependent tumorigenic transformation induced paradoxically by overexpression or block of S-adenosylmethionine decarboxylase.

Paasinen-Sohns A, Kielosto M, Kääriäinen E, Eloranta T, Laine A, Jänne OA, Birrer MJ, Hölttä E - J. Cell Biol. (2000)

Erk1 and Erk2 are not constitutively activated in AdoMetDC transformants. (A) Western blot analysis of the phosphorylation status of Erks. The electrophoretic mobilities of Erk1 and Erk2 in Amdc-s or Amdc-as+spd cells did not display upshifts of protein bands (in 12.5% SDS-PAGE) that were seen in control cells (4N and Rat-1) stimulated with PDGF-BB. (B) In vitro immunocomplex kinase assays showed a clear stimulation of MAPK activity in PDGF-BB–stimulated control cells, but no constitutive upregulation was detected in AdoMetDC transformants. Gray bars, cells starved for 24 h in 0.5% serum; white bars, starved cells stimulated with PDGF-BB for 15 min at 37°C; black bars, cells grown normally in 5% serum.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169445&req=5

Figure 3: Erk1 and Erk2 are not constitutively activated in AdoMetDC transformants. (A) Western blot analysis of the phosphorylation status of Erks. The electrophoretic mobilities of Erk1 and Erk2 in Amdc-s or Amdc-as+spd cells did not display upshifts of protein bands (in 12.5% SDS-PAGE) that were seen in control cells (4N and Rat-1) stimulated with PDGF-BB. (B) In vitro immunocomplex kinase assays showed a clear stimulation of MAPK activity in PDGF-BB–stimulated control cells, but no constitutive upregulation was detected in AdoMetDC transformants. Gray bars, cells starved for 24 h in 0.5% serum; white bars, starved cells stimulated with PDGF-BB for 15 min at 37°C; black bars, cells grown normally in 5% serum.
Mentions: Numerous reports indicate that activation of the Erk1/Erk2 MAPK cascade is necessary for cell growth. Since MAPK is activated in cells transformed by many oncogenes (Howe et al. 1992; Samuels et al. 1993; Okazaki and Sagata 1995), we tested whether MAPK might also be constitutively activated and responsible for the transformed phenotype in Amdc-s and Amdc-as+spd transformants. To distinguish between the inactive and active states of Erk1 and Erk2 proteins, we starved the normal cells (4N and Rat-1) with 0.5% FBS for 20 h, and then stimulated the cells with PDGF-BB (30 ng/ml) for 15 min at 37°C. Exposure to PDGF-BB resulted in distinct shifts of Erk1 and Erk2 migration, as expected (Fig. 3 A). No shifts of Erks, indicative of constitutive activation, were detected in the transformants that were grown normally in the presence of serum (Fig. 3 A). Additionally, MAPK activities were measured by in vitro immunocomplex kinase assays, which used a synthetic peptide of myelic basic protein as a substrate. The normal 4N cells responded to PDGF-BB with a threefold activation, and Rat-1 cells exhibited a 15-fold increase in MAPK activity (Fig. 3 B). Again, neither the NIH3T3 nor Rat-1 transformants displayed a constitutive increase in MAPK activity (Fig. 3 B).

Bottom Line: The phenotype of the AdoMetDC sense transformants was reversed by expression of dominant-negative mutants of SEK1 (MKK4), JNK1, and c-Jun (TAM-67), which were also found to impair cytokinesis.Similarly, TAM-67 reverted the morphology of the AdoMetDC-antisense expressors.In addition, we show that the polyamine biosynthetic enzymes require c-Jun activation for eliciting their biological effects.

View Article: PubMed Central - PubMed

Affiliation: Haartman Institute, Department of Pathology, FIN-00014 University of Helsinki, Helsinki, Finland.

ABSTRACT
All mammalian cells absolutely require polyamines (putrescine, spermidine, and spermine) for growth. Here we show that the overexpression of cDNA for S-adenosylmethionine decarboxylase (AdoMetDC), the main regulatory enzyme in the biosynthesis of higher polyamines, induces transformation of rodent fibroblasts when expressed in the sense or the antisense orientation. Both transformants were able to induce invasive tumors in nude mice. Neither transformation was associated with activation of the mitogen-activated protein kinases Erk1 and Erk2. Instead, the AdoMet DC sense, but not antisense, transformants displayed constitutive activation of the c-Jun NH(2)-terminal kinase (JNK) pathway. However, both transformations converged on persistent phosphorylation of endogenous c-Jun at Ser73. The phenotype of the AdoMetDC sense transformants was reversed by expression of dominant-negative mutants of SEK1 (MKK4), JNK1, and c-Jun (TAM-67), which were also found to impair cytokinesis. Similarly, TAM-67 reverted the morphology of the AdoMetDC-antisense expressors. This report is the first demonstration of a protein whose overexpression or block of synthesis can induce cell transformation. In addition, we show that the polyamine biosynthetic enzymes require c-Jun activation for eliciting their biological effects.

Show MeSH
Related in: MedlinePlus