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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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The expression of DN JNK1, DN SEK1, and c-Jun (TAM67) mutant proteins and inhibition of c-Jun phosphorylation by DN JNK1 and DN SEK1. (A and B) Western blot analyses of the cell lysates for the expression of the mutant proteins. Note that there are multiple c-Jun TAM67 bands (marked with a bracket), as reported earlier (Brown et al. 1994). (C) Western blot analysis showing the inhibition of c-Jun phosphorylation in Amdc-s cells transfected with DN JNK1 and DN SEK1.
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Figure 6: The expression of DN JNK1, DN SEK1, and c-Jun (TAM67) mutant proteins and inhibition of c-Jun phosphorylation by DN JNK1 and DN SEK1. (A and B) Western blot analyses of the cell lysates for the expression of the mutant proteins. Note that there are multiple c-Jun TAM67 bands (marked with a bracket), as reported earlier (Brown et al. 1994). (C) Western blot analysis showing the inhibition of c-Jun phosphorylation in Amdc-s cells transfected with DN JNK1 and DN SEK1.

Mentions: Previous studies with Ras-transformed cells have indicated that c-Jun becomes transcriptionally activated by the phosphorylation of Ser63 and Ser73 (Smeal et al. 1991), and the transactivation domain of c-Jun is required for transformation by ras in NIH3T3 cells (Westwick et al. 1994) and for cotransformation of rat embryo fibroblasts (Alani et al. 1991). The cascade leading to these phosphorylations includes consecutive activation of the kinases SEK1/MKK4 or MKK7 (immediate upstream activators of JNKs) and the JNKs 1 and 2 (Su and Karin 1996; Ip and Davis 1998). We tested the effects of dominant-negative mutants of SEK1, JNK1, and TAM-67, lacking the transactivation domain of c-Jun, on AdoMetDC-transformed cells. The plasmids containing the mutants or the control vectors were transfected together with a selection marker (zeo or puro) into the 4N cells and Amdc-s cells (Fig. 5 A, a). Transfections of the control vectors did not elicit any morphological changes in either cell type. In contrast, expression of dominant-negative mutants of SEK1 and JNK1 in Amdc-s cells led to a reversion towards the normal, epithelioid phenotype (Fig. 5 A, b and d) and produced multinucleated cells in subsequent cell divisions (Fig. 5 A, c and e). Expression of TAM67 resulted in the most efficient reversion of the transformed phenotype of Amdc-s cells to normal (Fig. 5 A, f) and formation of flat multinucleated cells, as well (Fig. 5 A, g). The reversal was not due to TAM67 interfering nonspecifically with the expression of AdoMetDC, as verified by the continued elevation in AdoMetDC activity. Also the phenotype of Amdc-as+spd cells was reverted by TAM67 expression (Fig. 5 B, a and b), but DN SEK1 and DN JNK1 mutants did not appear to be effective (data not shown). The mutants had no visible effect on the morphology of normal cells. The reversal of transformation by TAM67 expression was also seen in soft agar growth of the transfected cell lines (Fig. 5 C). In both Amdc-s (Fig. 5 C, a) and Amdc-as+spd cells (Fig. 5 C, c), TAM67 expression markedly reduced the size and amount of the colonies (Fig. 5 C, b and d). To confirm the expression of the mutants, cell lysates were analyzed for mutant proteins by Western blotting (Fig. 6A and Fig. B). It is also notable that the expression of DN JNK1 and DN SEK1 mutants in Amdc-s cells markedly inhibited c-Jun phosphorylation (Fig. 6 C).


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)

The expression of DN JNK1, DN SEK1, and c-Jun (TAM67) mutant proteins and inhibition of c-Jun phosphorylation by DN JNK1 and DN SEK1. (A and B) Western blot analyses of the cell lysates for the expression of the mutant proteins. Note that there are multiple c-Jun TAM67 bands (marked with a bracket), as reported earlier (Brown et al. 1994). (C) Western blot analysis showing the inhibition of c-Jun phosphorylation in Amdc-s cells transfected with DN JNK1 and DN SEK1.
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Figure 6: The expression of DN JNK1, DN SEK1, and c-Jun (TAM67) mutant proteins and inhibition of c-Jun phosphorylation by DN JNK1 and DN SEK1. (A and B) Western blot analyses of the cell lysates for the expression of the mutant proteins. Note that there are multiple c-Jun TAM67 bands (marked with a bracket), as reported earlier (Brown et al. 1994). (C) Western blot analysis showing the inhibition of c-Jun phosphorylation in Amdc-s cells transfected with DN JNK1 and DN SEK1.
Mentions: Previous studies with Ras-transformed cells have indicated that c-Jun becomes transcriptionally activated by the phosphorylation of Ser63 and Ser73 (Smeal et al. 1991), and the transactivation domain of c-Jun is required for transformation by ras in NIH3T3 cells (Westwick et al. 1994) and for cotransformation of rat embryo fibroblasts (Alani et al. 1991). The cascade leading to these phosphorylations includes consecutive activation of the kinases SEK1/MKK4 or MKK7 (immediate upstream activators of JNKs) and the JNKs 1 and 2 (Su and Karin 1996; Ip and Davis 1998). We tested the effects of dominant-negative mutants of SEK1, JNK1, and TAM-67, lacking the transactivation domain of c-Jun, on AdoMetDC-transformed cells. The plasmids containing the mutants or the control vectors were transfected together with a selection marker (zeo or puro) into the 4N cells and Amdc-s cells (Fig. 5 A, a). Transfections of the control vectors did not elicit any morphological changes in either cell type. In contrast, expression of dominant-negative mutants of SEK1 and JNK1 in Amdc-s cells led to a reversion towards the normal, epithelioid phenotype (Fig. 5 A, b and d) and produced multinucleated cells in subsequent cell divisions (Fig. 5 A, c and e). Expression of TAM67 resulted in the most efficient reversion of the transformed phenotype of Amdc-s cells to normal (Fig. 5 A, f) and formation of flat multinucleated cells, as well (Fig. 5 A, g). The reversal was not due to TAM67 interfering nonspecifically with the expression of AdoMetDC, as verified by the continued elevation in AdoMetDC activity. Also the phenotype of Amdc-as+spd cells was reverted by TAM67 expression (Fig. 5 B, a and b), but DN SEK1 and DN JNK1 mutants did not appear to be effective (data not shown). The mutants had no visible effect on the morphology of normal cells. The reversal of transformation by TAM67 expression was also seen in soft agar growth of the transfected cell lines (Fig. 5 C). In both Amdc-s (Fig. 5 C, a) and Amdc-as+spd cells (Fig. 5 C, c), TAM67 expression markedly reduced the size and amount of the colonies (Fig. 5 C, b and d). To confirm the expression of the mutants, cell lysates were analyzed for mutant proteins by Western blotting (Fig. 6A and Fig. B). It is also notable that the expression of DN JNK1 and DN SEK1 mutants in Amdc-s cells markedly inhibited c-Jun phosphorylation (Fig. 6 C).

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