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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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(A) Morphology, actin filaments, and soft agar growth of NIH3T3 cells overexpressing human AdoMetDC cDNA in sense (Amdc-s) and antisense (Amdc-as) orientations are shown. (a, d, and g) Parental NIH3T3 cells transfected with the neomycin resistance gene and the empty pLTRpoly vector (control, 4N), (b, e, and h) Amdc-s cells and (c, f, and i) Amdc-as cells grown with 1 μM spermidine (Amdc-as+spd) in tissue cultures (a–c), stained for actin filaments (d–f), and grown in soft agar (g–i). (B) Morphology of Rat-1 cell transfectants. (a–c) Rat-1 control cells and transfectants expressing AdoMetDC sense and antisense constructs. Rat-1 Amdc-as cells were grown in the presence of spermidine, as described above.
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Figure 1: (A) Morphology, actin filaments, and soft agar growth of NIH3T3 cells overexpressing human AdoMetDC cDNA in sense (Amdc-s) and antisense (Amdc-as) orientations are shown. (a, d, and g) Parental NIH3T3 cells transfected with the neomycin resistance gene and the empty pLTRpoly vector (control, 4N), (b, e, and h) Amdc-s cells and (c, f, and i) Amdc-as cells grown with 1 μM spermidine (Amdc-as+spd) in tissue cultures (a–c), stained for actin filaments (d–f), and grown in soft agar (g–i). (B) Morphology of Rat-1 cell transfectants. (a–c) Rat-1 control cells and transfectants expressing AdoMetDC sense and antisense constructs. Rat-1 Amdc-as cells were grown in the presence of spermidine, as described above.

Mentions: To study the consequences of overexpression of AdoMetDC on growth regulation, we cloned the human AdoMetDC cDNA (truncated at the 5′-untranslated region to remove sequences that inhibit translation) into a pLTRpoly expression vector, both in sense and antisense orientations. The constructs were transfected with a neo selection marker (pSV2neo) into mouse NIH3T3 cells and Rat-1 fibroblasts, and stable cell lines were generated. The control NIH3T3 cells transfected with pLTRpoly and pSV2neo vectors (4N) exhibited an epithelioid morphology, stringent density-dependent growth (Fig. 1 A, a), normal actin filaments (Fig. 1 A, d), and did not grow in soft agar (only 1.7% of the cells formed tiny colonies) (Fig. 1 A, g). Transfection of NIH3T3 cells with the AdoMetDC sense construct resulted in a complete morphological transformation. The transfectants displayed an elongated morphology, grew without contact inhibition in a criss-cross manner, and formed innumerable foci in tissue culture (Fig. 1 A, b). They also showed disintegrated actin filaments (Fig. 1 A, e) and acquired the ability to grow in soft agar (15.2% of the cells formed large foci) (Fig. 1 A, h). Surprisingly, the same was true for the AdoMetDC antisense construct–expressing cells when they were cultured in the presence of spd (1 μM) (Fig. 1 A, c). Exogenous spermidine had to be added to keep alive the high Amdc-as expressors, which are blocked in the synthesis of spermidine. However, the Amdc-as+spd cells displayed a less elongated morphology than the Amdc-s cells, but showed a similar degree of disintegration of the actin filaments (Fig. 1 A, f) and growth in soft agar (19.1%). The Amdc-as cells grown in the absence of spermidine (except what is provided by serum in the growth media) exhibited a much lower degree of morphological transformation with partial disintegration of the actin filaments. Similar inductions of transformation by the AdoMetDC sense and antisense constructs were also seen in Rat-1 cells. Fig. 1 B, a shows the morphology of normal Rat-1 control cells, and Fig. 1 B, b and c, show the transformed phenotype induced by the AdoMetDC sense and antisense constructs (with spermidine supplementation), respectively. Spermidine had no effect on the morphology of normal 4N cells or Amdc-s cells. Overexpression of AdoMetDC was not associated with increased proliferation or cell death (data not shown), thus AdoMetDC is specifically associated with morphological transformation. The data presented below are mostly from experiments with NIH3T3 cells, but also hold true for Rat-1 cells.


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)

(A) Morphology, actin filaments, and soft agar growth of NIH3T3 cells overexpressing human AdoMetDC cDNA in sense (Amdc-s) and antisense (Amdc-as) orientations are shown. (a, d, and g) Parental NIH3T3 cells transfected with the neomycin resistance gene and the empty pLTRpoly vector (control, 4N), (b, e, and h) Amdc-s cells and (c, f, and i) Amdc-as cells grown with 1 μM spermidine (Amdc-as+spd) in tissue cultures (a–c), stained for actin filaments (d–f), and grown in soft agar (g–i). (B) Morphology of Rat-1 cell transfectants. (a–c) Rat-1 control cells and transfectants expressing AdoMetDC sense and antisense constructs. Rat-1 Amdc-as cells were grown in the presence of spermidine, as described above.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2169445&req=5

Figure 1: (A) Morphology, actin filaments, and soft agar growth of NIH3T3 cells overexpressing human AdoMetDC cDNA in sense (Amdc-s) and antisense (Amdc-as) orientations are shown. (a, d, and g) Parental NIH3T3 cells transfected with the neomycin resistance gene and the empty pLTRpoly vector (control, 4N), (b, e, and h) Amdc-s cells and (c, f, and i) Amdc-as cells grown with 1 μM spermidine (Amdc-as+spd) in tissue cultures (a–c), stained for actin filaments (d–f), and grown in soft agar (g–i). (B) Morphology of Rat-1 cell transfectants. (a–c) Rat-1 control cells and transfectants expressing AdoMetDC sense and antisense constructs. Rat-1 Amdc-as cells were grown in the presence of spermidine, as described above.
Mentions: To study the consequences of overexpression of AdoMetDC on growth regulation, we cloned the human AdoMetDC cDNA (truncated at the 5′-untranslated region to remove sequences that inhibit translation) into a pLTRpoly expression vector, both in sense and antisense orientations. The constructs were transfected with a neo selection marker (pSV2neo) into mouse NIH3T3 cells and Rat-1 fibroblasts, and stable cell lines were generated. The control NIH3T3 cells transfected with pLTRpoly and pSV2neo vectors (4N) exhibited an epithelioid morphology, stringent density-dependent growth (Fig. 1 A, a), normal actin filaments (Fig. 1 A, d), and did not grow in soft agar (only 1.7% of the cells formed tiny colonies) (Fig. 1 A, g). Transfection of NIH3T3 cells with the AdoMetDC sense construct resulted in a complete morphological transformation. The transfectants displayed an elongated morphology, grew without contact inhibition in a criss-cross manner, and formed innumerable foci in tissue culture (Fig. 1 A, b). They also showed disintegrated actin filaments (Fig. 1 A, e) and acquired the ability to grow in soft agar (15.2% of the cells formed large foci) (Fig. 1 A, h). Surprisingly, the same was true for the AdoMetDC antisense construct–expressing cells when they were cultured in the presence of spd (1 μM) (Fig. 1 A, c). Exogenous spermidine had to be added to keep alive the high Amdc-as expressors, which are blocked in the synthesis of spermidine. However, the Amdc-as+spd cells displayed a less elongated morphology than the Amdc-s cells, but showed a similar degree of disintegration of the actin filaments (Fig. 1 A, f) and growth in soft agar (19.1%). The Amdc-as cells grown in the absence of spermidine (except what is provided by serum in the growth media) exhibited a much lower degree of morphological transformation with partial disintegration of the actin filaments. Similar inductions of transformation by the AdoMetDC sense and antisense constructs were also seen in Rat-1 cells. Fig. 1 B, a shows the morphology of normal Rat-1 control cells, and Fig. 1 B, b and c, show the transformed phenotype induced by the AdoMetDC sense and antisense constructs (with spermidine supplementation), respectively. Spermidine had no effect on the morphology of normal 4N cells or Amdc-s cells. Overexpression of AdoMetDC was not associated with increased proliferation or cell death (data not shown), thus AdoMetDC is specifically associated with morphological transformation. The data presented below are mostly from experiments with NIH3T3 cells, but also hold true for Rat-1 cells.

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