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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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Endogenous JNK is constitutively activated in AdoMetDC-overexpressing NIH3T3 and Rat-1 cells, resulting in increased phosphorylation of c-Jun at Ser73. (A) The JNKs were purified by virtue of their binding to agarose-conjugated GST-c-Jun. The autoradiograms show the phosphorylation status of GST-c-Jun used as the substrate in the solid-phase kinase assays. (B) Western blots from the nuclear extracts show a strong phosphorylation of c-Jun at Ser73 in AdoMetDC transformants. The bottom rows show the total amount of c-Jun in nuclear extracts. The levels of JunD and ATF-2 remained constant and were used as loading controls (data not shown).
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Figure 4: Endogenous JNK is constitutively activated in AdoMetDC-overexpressing NIH3T3 and Rat-1 cells, resulting in increased phosphorylation of c-Jun at Ser73. (A) The JNKs were purified by virtue of their binding to agarose-conjugated GST-c-Jun. The autoradiograms show the phosphorylation status of GST-c-Jun used as the substrate in the solid-phase kinase assays. (B) Western blots from the nuclear extracts show a strong phosphorylation of c-Jun at Ser73 in AdoMetDC transformants. The bottom rows show the total amount of c-Jun in nuclear extracts. The levels of JunD and ATF-2 remained constant and were used as loading controls (data not shown).

Mentions: As AdoMetDC transformation appeared not to be linked to Erk1/Erk2 activation, we analyzed the activity of the JNKs. These kinases were originally described as stress-induced kinases, but have been recently connected to oncogenesis, as well (Raitano et al. 1995; Rodrigues et al. 1997). Interestingly, the JNK kinase activity was 5–9-fold higher in NIH3T3 Amdc-s cells than in control cells, with the Amdc-as+spd cells displaying no significant increase, as analyzed by the solid-phase assay (Fig. 4 A) or the immunocomplex kinase assay (data not shown). Also the transient transfection of AdoMetDC cDNA into NIH3T3 cells resulted in increased JNK activity (data not shown), indicating that the activation of JNK in Amdc-s cells is not just secondary to transformation. p38 MAPK was not activated in these cells, as confirmed by phosphospecific antibody blots (data not shown).


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)

Endogenous JNK is constitutively activated in AdoMetDC-overexpressing NIH3T3 and Rat-1 cells, resulting in increased phosphorylation of c-Jun at Ser73. (A) The JNKs were purified by virtue of their binding to agarose-conjugated GST-c-Jun. The autoradiograms show the phosphorylation status of GST-c-Jun used as the substrate in the solid-phase kinase assays. (B) Western blots from the nuclear extracts show a strong phosphorylation of c-Jun at Ser73 in AdoMetDC transformants. The bottom rows show the total amount of c-Jun in nuclear extracts. The levels of JunD and ATF-2 remained constant and were used as loading controls (data not shown).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2169445&req=5

Figure 4: Endogenous JNK is constitutively activated in AdoMetDC-overexpressing NIH3T3 and Rat-1 cells, resulting in increased phosphorylation of c-Jun at Ser73. (A) The JNKs were purified by virtue of their binding to agarose-conjugated GST-c-Jun. The autoradiograms show the phosphorylation status of GST-c-Jun used as the substrate in the solid-phase kinase assays. (B) Western blots from the nuclear extracts show a strong phosphorylation of c-Jun at Ser73 in AdoMetDC transformants. The bottom rows show the total amount of c-Jun in nuclear extracts. The levels of JunD and ATF-2 remained constant and were used as loading controls (data not shown).
Mentions: As AdoMetDC transformation appeared not to be linked to Erk1/Erk2 activation, we analyzed the activity of the JNKs. These kinases were originally described as stress-induced kinases, but have been recently connected to oncogenesis, as well (Raitano et al. 1995; Rodrigues et al. 1997). Interestingly, the JNK kinase activity was 5–9-fold higher in NIH3T3 Amdc-s cells than in control cells, with the Amdc-as+spd cells displaying no significant increase, as analyzed by the solid-phase assay (Fig. 4 A) or the immunocomplex kinase assay (data not shown). Also the transient transfection of AdoMetDC cDNA into NIH3T3 cells resulted in increased JNK activity (data not shown), indicating that the activation of JNK in Amdc-s cells is not just secondary to transformation. p38 MAPK was not activated in these cells, as confirmed by phosphospecific antibody blots (data not shown).

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