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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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Expression of human AdoMetDC cDNA, the activities of AdoMetDC and ODC, and polyamine contents in NIH3T3 transfectants are shown. (A) The Northern blot shows the expression levels of AdoMetDC mRNA in transfected cells. The integrity and loading of RNA was controlled by hybridizing the blot with human β-actin c-DNA. Note, the consistent lower β-actin signal in the Amdc-as cells may also reflect their retarded growth rate. The size of the mRNAs are indicated in kb on the left. (B) AdoMetDC (left) and ODC (right) activities were measured as described in Materials and Methods. Dotted bars, cells grown without spermidine addition; striped bars, cells grown with spermidine. (C) The amount of polyamines is expressed as pmol/μg DNA. Acetylated polyamines were not detected. Black bars, putrescine; gray bars, spermidine; striped bars, spermine. The enzymatic activities and the polyamine contents were determined from parallel dishes after 2 d of culture. The results are representative of four independent experiments.
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Figure 2: Expression of human AdoMetDC cDNA, the activities of AdoMetDC and ODC, and polyamine contents in NIH3T3 transfectants are shown. (A) The Northern blot shows the expression levels of AdoMetDC mRNA in transfected cells. The integrity and loading of RNA was controlled by hybridizing the blot with human β-actin c-DNA. Note, the consistent lower β-actin signal in the Amdc-as cells may also reflect their retarded growth rate. The size of the mRNAs are indicated in kb on the left. (B) AdoMetDC (left) and ODC (right) activities were measured as described in Materials and Methods. Dotted bars, cells grown without spermidine addition; striped bars, cells grown with spermidine. (C) The amount of polyamines is expressed as pmol/μg DNA. Acetylated polyamines were not detected. Black bars, putrescine; gray bars, spermidine; striped bars, spermine. The enzymatic activities and the polyamine contents were determined from parallel dishes after 2 d of culture. The results are representative of four independent experiments.

Mentions: Northern blot analyses showed that the normal 4N cells expressed low levels of two endogenous mouse Ado MetDC mRNAs (Pajunen et al. 1988). The 3.1-kb mRNA was expressed at a higher level than the 2.1-kb species, which was visible only upon longer exposure (Fig. 2 A). The Amdc-s cells carrying the human AdoMetDC sense construct expressed high levels of two AdoMetDC mRNAs of 1.65 kb and 2.65 kb, which are the sizes predicted for the use of polyadenylation signals in AdoMetDC cDNA (Pajunen et al. 1988) and in the pLTRpoly vector, respectively. The Amdc-as cells carrying the antisense construct also expressed the 2.65-kb mRNA of expected size, but the most prominent band was of a slightly smaller size (Fig. 2 A). This smaller mRNA likely arises as a result of splicing out 0.3-kb vector sequences, as there is a consensus splice donor site at the end of the AdoMetDC antisense cDNA and a splice acceptor site in the vector. The Amdc-as+spd cells had higher levels of AdoMetDC antisense mRNAs, as expected (see above). No appreciable changes in the expression level of the AdoMetDC mRNAs were detected in normal or Amdc-s cells cultured with spermidine.


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)

Expression of human AdoMetDC cDNA, the activities of AdoMetDC and ODC, and polyamine contents in NIH3T3 transfectants are shown. (A) The Northern blot shows the expression levels of AdoMetDC mRNA in transfected cells. The integrity and loading of RNA was controlled by hybridizing the blot with human β-actin c-DNA. Note, the consistent lower β-actin signal in the Amdc-as cells may also reflect their retarded growth rate. The size of the mRNAs are indicated in kb on the left. (B) AdoMetDC (left) and ODC (right) activities were measured as described in Materials and Methods. Dotted bars, cells grown without spermidine addition; striped bars, cells grown with spermidine. (C) The amount of polyamines is expressed as pmol/μg DNA. Acetylated polyamines were not detected. Black bars, putrescine; gray bars, spermidine; striped bars, spermine. The enzymatic activities and the polyamine contents were determined from parallel dishes after 2 d of culture. The results are representative of four independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Expression of human AdoMetDC cDNA, the activities of AdoMetDC and ODC, and polyamine contents in NIH3T3 transfectants are shown. (A) The Northern blot shows the expression levels of AdoMetDC mRNA in transfected cells. The integrity and loading of RNA was controlled by hybridizing the blot with human β-actin c-DNA. Note, the consistent lower β-actin signal in the Amdc-as cells may also reflect their retarded growth rate. The size of the mRNAs are indicated in kb on the left. (B) AdoMetDC (left) and ODC (right) activities were measured as described in Materials and Methods. Dotted bars, cells grown without spermidine addition; striped bars, cells grown with spermidine. (C) The amount of polyamines is expressed as pmol/μg DNA. Acetylated polyamines were not detected. Black bars, putrescine; gray bars, spermidine; striped bars, spermine. The enzymatic activities and the polyamine contents were determined from parallel dishes after 2 d of culture. The results are representative of four independent experiments.
Mentions: Northern blot analyses showed that the normal 4N cells expressed low levels of two endogenous mouse Ado MetDC mRNAs (Pajunen et al. 1988). The 3.1-kb mRNA was expressed at a higher level than the 2.1-kb species, which was visible only upon longer exposure (Fig. 2 A). The Amdc-s cells carrying the human AdoMetDC sense construct expressed high levels of two AdoMetDC mRNAs of 1.65 kb and 2.65 kb, which are the sizes predicted for the use of polyadenylation signals in AdoMetDC cDNA (Pajunen et al. 1988) and in the pLTRpoly vector, respectively. The Amdc-as cells carrying the antisense construct also expressed the 2.65-kb mRNA of expected size, but the most prominent band was of a slightly smaller size (Fig. 2 A). This smaller mRNA likely arises as a result of splicing out 0.3-kb vector sequences, as there is a consensus splice donor site at the end of the AdoMetDC antisense cDNA and a splice acceptor site in the vector. The Amdc-as+spd cells had higher levels of AdoMetDC antisense mRNAs, as expected (see above). No appreciable changes in the expression level of the AdoMetDC mRNAs were detected in normal or Amdc-s cells cultured with spermidine.

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