Ornithine decarboxylase and extracellular polyamines regulate microvascular sprouting and actin cytoskeleton dynamics in endothelial cells

The polyamines are essential for cancer cell proliferation during tumorigenesis. Targeted inhibition of ornithine decarboxylase (ODC), i.e. a key enzyme of polyamine biosynthesis, by α-difluoromethylornithine (DFMO) has shown anti-neoplastic activity in various experimental models. This activity has mainly been attributed to the anti-proliferative effect of DFMO in cancer cells. Here, we provide evidence that unperturbed ODC activity is a requirement for proper microvessel sprouting ex vivo as well as the migration of primary human endothelial cells. DFMO-mediated ODC inhibition was reversed by extracellular polyamine supplementation, showing that anti-angiogenic effects of DFMO were specifically related to polyamine levels. ODC inhibition was associated with an abnormal morphology of the actin cytoskeleton during cell spreading and migration. Moreover, our data suggest that de-regulated actin cytoskeleton dynamics in DFMO treated endothelial cells may be related to constitutive activation of the small GTPase CDC42, i.e. a well-known regulator of cell motility and actin cytoskeleton remodeling. These insights into the potential role of polyamines in angiogenesis should stimulate further studies testing the combined anti-tumor effect of polyamine inhibition and established anti-angiogenic therapies in vivo.     

Reduced tyrosine phosphorylation in polyamine-starved cells.

Onset of cell proliferation is associated with enhanced turnover of the polyamines putrescine, spermidine, and spermine, particularly evident in the massive increase in the activity of the rate-limiting enzyme in their production, ornithine decarboxylase (ODC). The physiological functions of these polyamines, however, have remained unclear. Here we report that treatment of LSTRA cells for 2-18 h with alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, decreased the amount of phosphotyrosine in several cellular substrates including the T cell protein tyrosine kinase p56lck. No reductions in the amount of p56lck, overall synthesis of protein and DNA, or cell viability were observed until much later. DFMO did not affect the catalytic activity of p56lck in vitro and the activity of p56lck immunoprecipitated from DFMO-treated cells was unaltered. Addition of putrescine, the reaction product of ODC, completely reversed the effect of DFMO on tyrosine phosphorylation. Finally, we provide evidence that polyamines reduce the activity of cellular protein tyrosine phosphatases toward endogenous substrates. Our results suggest that polyamines may influence the extent of tyrosine phosphorylation during cell proliferation and malignant transformation, perhaps by modulating the rate of dephosphorylation of specific target proteins.     

Cell-cycle-dependent regulation of ornithine decarboxylase activity in the unicellular green alga Chlamydomonas reinhardtii

Polyamines play an important role in the control of cell growth and cell division. In the unicellular green alga Chlamydomonas reinhardtii as in animal cells, biosynthesis of the 3 commonly occurring polyamines (putrescine, spermidine and spermine) is dependent on the activity of ornithine decarboxylase (ODC, EC 4.1.1.17) catalyzing the formation of putrescine, which is the precursor of the other two polyamines. Therefore, we have investigated the regulation of ODC activity during the cell cycle of Clamydomonas reinhardtii using synchronized cultures. A 2.5–3-fold increase in ODC activity was observed during the transition to the cell division phase. This up-regulation of ODC activity was not due to an increased level of ODC-mRNA as revealed by northern-blot analyses, but correlated with an increased half-life of this particular enzyme (from 1.1 to 3.2 h). Addition of the DNA topoisomerase II inhibitor nalidixic acid during the second half of the growth period caused a transient decrease of ODC activity and a considerable delay of cell divisions. After cell division, a down-regulation of ODC activity was observed which was faster in the dark than in the light and also correlated with changes of the ODC half-life.     

Primary mesenchymal cells isolated from SPARC-null mice exhibit altered morphology and rates of proliferation

SPARC (secreted protein acidic and rich in cysteine)/BM 40/osteonectin is a matricellular protein shown to function as a counteradhesive factor that induces cell rounding and as an inhibitor of cell proliferation. These activities have been defined in cell culture, in which interpretation has been complicated by the presence of endogenous SPARC. We therefore sought to determine whether cell shape and proliferation would be affected by the absence of SPARC. Mesangial cells, fibroblasts, and aortic smooth muscle cells were isolated from SPARC-null and age-matched, wild-type mice. In contrast to wild-type cells, SPARC-null mesangial cells exhibited a flat morphology and an altered actin cytoskeleton. In addition, vinculin-containing focal adhesions were distributed over the center of SPARC-null cells, whereas in wild-type cells, the number of focal adhesions was reduced, and these structures were restricted largely to the cell periphery. Although the SPARC-null fibroblasts did not display overt differences in cell morphology, the cells responded to exogenous recombinant SPARC by rounding up in a manner similar to that of wild-type fibroblasts. Thus, the expression of endogenous SPARC is not required for the response of cells to SPARC. Additionally, SPARC-null mesangial cells, fibroblasts, and smooth muscle cells proliferated faster than their respective wild-type counterparts. Null cells also showed a greater sensitivity to the inhibition of cell cycle progression by the addition of recombinant SPARC. The increased proliferation rate of SPARC-null cells appeared to be mediated, at least in part, by an increase in the cell cycle regulatory protein cyclin A. We conclude that the expression of SPARC influences the cellular architecture of mesangial cells and that SPARC plays a role in the regulation of cell cycle in mesangial cells, fibroblasts, and smooth muscle cells.     

Inhibition of ornithine decarboxylase by retinoic acid and difluoromethylornithine in relation to their effects on differentiation and proliferation

Murine embryonal carcinoma F9 cells can be induced to differentiate by 2-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC). The differentiated phenotype is similar to that of retinoic acid (RA)-treated F9 cells. In contrast to F9 cells the differentiated cells secrete plasminogen activator and express keratin intermediate filaments. Both DFMO and RA reduce ornithine decarboxylase activity, polyamine levels and inhibit cell proliferation of F9 cells. These compounds also reduce ODC, polyamine levels and proliferation of mouse BALB/c 3T6 fibroblasts. RA inhibits the induction of ODC by insulin, serum and to a lesser extent that of epidermal growth factor (EGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA). The action of DFMO and RA can be distinguished by their response to putrescine. The induction of differentiation and the inhibition of cell proliferation by DFMO can be totally abolished upon the addition of putrescine, whereas the actions of RA are not affected at all. These results suggest that the inhibition of ODC and reduction of polyamines are not causal in the induction of differentiation and the inhibition of proliferation by RA.     

Comparative effects of TGFβ on proliferation of 7- and 14-day-old chick embryo fibroblasts and lack of involvement of the ODC/PA system in the TGFβ signaling pathway

The growth regulatory activity of transforming growth factor β (TGFβ) on chick embryo skin fibroblasts was compared in two developmental ages, days 7 and 14. The time course of ³H-thymidine incorporation, an S-phase marker of replication, was determined during 36 hr of TGFβ treatment. Seven-day-old cells showed a prereplicative phase of 6 hr, and 14-day-old cells showed a prereplicative phase of 12 hr. DNA synthesis peaked at 24 hr in 7-day-old fibroblasts and was 10 times higher than that in 14-day-old fibroblasts. Ornithine decarboxylase (ODC) activity and content of the natural polyamines spermine (Spm), spermidine (Spd), and putrescine (Put) differed during cell cycle. ODC activity peaked at 12 hr in 7-day-old cells and at 6 hr in 14-day-old cells. Its level was two times higher at day 7 and was associated with a greater content of ODC mRNA. The maximum of polyamine (PA) concentration was determined after 12 hr of treatment in 7-day-old cells and after 36 hr in 14-day-old cells. These findings indicate that the TGFβ proliferative response of embryo fibroblasts changes during development and is associated with activation of the ODC/PA system. Cotreatment with α-difluoromethylornithine, an enzyme-activated irreversible inhibitor of ODC, did not reduced growth rate. Inhibition of ODC resulted in levels of Put and Spd comparable to that of quiescent fibroblasts, whereas Spm concentration remained higher. Because an altered ODC metabolism does not convey the effects of TGFβ on DNA synthesis, the ODC/PA system may not play a role in the pathway of TGFβ signaling. J. Cell. Physiol. 178:304–310, 1999. © 1999 Wiley-Liss, Inc.     

Growth factors induce early pre-replicative changes in senescent human fibroblasts.

As human fibroblasts in culture senesce their response to platelet-derived growth factor (PDGF) becomes attenuated. To clarify at which level such cells are blocked in the pre-replicative part of the cell cycle, we have analysed PDGF-induced pre-replicative events in senescent (phase III) cultures. We found that phase III cells retain a normal number of PDGF receptors and that these are functional with regard to PDGF-induced receptor autophosphorylation. Phase III cells also respond to PDGF by rapid actin reorganization and increased levels of c-fos and c-myc mRNA, similar to growth-arrested phase II fibroblasts. However, the expression of the nuclear antigen K-67, which in phase II cell is induced in S-phase and continues to be expressed throughout the cell cycle, is not induced in phase III cells in response to PDGF. We conclude that phase III human fibroblasts, although blocked with regard to proliferation, still retain a functional growth factor receptor system, and display early responses when exposed to growth factors, such as changes in the cytoskeleton and the expression of proto-oncogenes.     

Induction of apoptotic cell death by putrescine

The polyamines are essential for cellular growth and differentiation. Ornithine decarboxylase (ODC), which catalyses the first step in the biosynthesis of the polyamines, has a very fast turnover and is subject to a strong feedback control by the polyamines. In the present study, we show that overexpression of a metabolically stable ODC in CHO cells induced a massive cell death unless the cells were grown in the presence of the ODC inhibitor alpha-difluoromethylornithine (DFMO). Cells overexpressing wild-type (unstable) ODC, on the other hand, were not dependent on the presence of DFMO for their growth. The induction of cell death was correlated with a dramatic increase in cellular putrescine levels. Analysis using flow cytometry revealed perturbed cell cycle kinetics, with a large accumulation of cells with sub-G1 amounts of DNA, which is a typical sign of apoptosis. Another strong indication of apoptosis was the finding that one of the key enzymes in the apoptotic process, caspase-3, was induced when DFMO was omitted from the growth medium. Furthermore, inhibition of the caspase activity significantly reduced the recruitment of cells to the sub-G1 fraction. In conclusion, deregulation of polyamine homeostasis may negatively affect cell proliferation and eventually lead to cell death by apoptosis if putrescine levels become too high.     

Peroxynitrite inhibits the activity of ornithine decarboxylase

Polyamines are required during cell proliferation, whereas NO has anti-proliferative properties. Ornithine decarboxylase (ODC) is a critical enzyme for the synthesis of polyamines. We tested the hypothesis that the modification of ODC by peroxynitrite (OONO-), a short-lived free radical formed from NO and superoxide produces a fall in ODC activity, and therefore polyamine synthesis and cell proliferation. The treatment of a rat recombinant ODC (rODC) with OONO- resulted in a dose-dependent inhibition of rODC activity with an IC50 of approximately 100 microM. A Western blot employing a specific antibody to nitrotyrosine revealed a dose-dependent nitration of rODC tyrosine residues. When intact IEC-6 cells were treated with ONOO-, ODC activity decreased by 49%. These data suggest a correlation between ODC activity and nitration, and a possible mechanism by which NO synthesis may modulate polyamine synthesis.     

Ornithine decarboxylase- and ras-induced cell transformations: reversal by protein tyrosine kinase inhibitors and role of pp130CAS.

We have found that overexpression of human ornithine decarboxylase (ODC) induces cell transformation in NIH 3T3 and Rat-1 cells (M. Auvinen, A. Paasinen, L. C. Andersson, and E. Hölttä, Nature (London) 360:355-358, 1992). The ODC-transformed cells display increased levels of tyrosine phosphorylation, in particular of a cluster of 130-kDa proteins. Here we show that one of the proteins with enhanced levels of tyrosine phosphorylation in ODC-overexpressing cells is the previously described p130 substrate of pp60v-src, known to associate also with v-Crk and designated p130CAS. We also studied the role of protein tyrosine phosphorylation in the ODC-induced cell transformation by exposing the cells to herbimycin A, a potent inhibitor of Src-family kinases, and to other inhibitors of protein tyrosine kinases. Treatment with the inhibitors reversed the phenotype of ODC-transformed cells to normal, with an organized, filamentous actin cytoskeleton. Coincidentally, the tyrosine hyperphosphorylation of p130 was markedly reduced, while the level of activity of ODC remained highly elevated. A similar reduction in pp130 phosphorylation and reversion of morphology by herbimycin A were observed in v-src- and c-Ha-ras-transformed cells. In addition, we show that expression of antisense mRNA for p130CAS resulted in reversion of the transformed phenotype of all these cell lines. An increased level of tyrosine kinase activity, not caused by c-Src or c-Abl, was further detected in the cytoplasmic fraction of ODC-transformed cells. Preliminary characteristics of this kinase are shown. These data indicate that p130CAS is involved in cell transformation by ODC, c-ras, and v-src oncogenes, raise the intriguing possibility that p130CAS may be generally required for transformation, and imply that there is at least one protein tyrosine kinase downstream of ODC that is instrumental for cell transformation.     

Visualization of actin cytoskeletal dynamics during the cell cycle in tobacco (Nicotiana tabacum L. cv Bright Yellow) cells

BACKGROUND INFORMATION: The actin cytoskeleton forms distinct actin arrays which fulfil their functions during cell cycle progression. Reorganization of the actin cytoskeleton occurs during transition from one actin array to another. Although actin arrays have been well described during cell cycle progression, the dynamic organization of the actin cytoskeleton during actin array transition remains to be dissected. RESULTS: In the present study, a GFP (green fluorescent protein)-mTalin (mouse talin) fusion gene was introduced into suspension-cultured tobacco BY-2 (Nicotiana tabacum L. cv Bright Yellow) cells by a calli-cocultivation transformation method to visualize the reorganization of the actin cytoskeleton in vivo during the progression of the cell cycle. Typical actin structures were indicated by GFP-mTalin, such as the pre-prophase actin band, mitotic spindle actin filament cage and phragmoplast actin arrays. In addition, dynamic organization of actin filaments was observed during the progression of the cell from metaphase to anaphase. In late metaphase, spindle actin filaments gradually shrank to the equatorial plane along both the long and short axes. Soon after the separation of sister chromosomes, actin filaments aligned in parallel at the cell division plane, forming a cylinder-like structure. During the formation of the cell plate, one cylinder-like structure changed into two cylinder-like structures: the typical actin arrays of the phragmoplast. However, the two actin arrays remained overlapping at the margin of the centrally growing cell plate, forming an actin wreath. When the cell plate matured further, an actin filament network attached to the cell plate was formed. CONCLUSIONS: Our results clearly describe the dynamic organization of the actin cytoskeleton during mitosis and cytokinesis of a plant cell. This demonstrates that GFP-mTalin-transformed tobacco BY-2 cells are a valuable tool to study actin cytoskeleton functions in the plant cell cycle.     

IL-4 and IL-13 upregulate ornithine decarboxylase expression by PI3K and MAP kinase pathways in vascular smooth muscle cells

Ornithine decarboxylase (ODC) is the first and rate-controlling enzyme in the synthesis of polyamines, which are essential for normal cell growth. We have previously demonstrated that IL-4 and IL-13 can stimulate rat aortic smooth muscle cell (RASMC) proliferation. The objective of this study was to determine whether IL-4 and IL-13 induce cell proliferation by upregulating ODC expression in RASMC. The results revealed that incubation of RASMC with IL-4 and IL-13 for 24 h caused four- to fivefold induction of ODC catalytic activity. The increased ODC catalytic activity was attributed to the increased expression of ODC mRNA. Moreover, these observations were paralleled by increased production of polyamines. We further investigated the signal transduction pathways responsible for ODC induction by IL-4 and IL-13. The data illustrated that PD-98059, a MEK (MAPK kinase) inhibitor, LY-294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, and H-89, a protein kinase A (PKA) inhibitor, substantially decreased the induction of ODC catalytic activity and ODC mRNA expression induced by IL-4 and IL-13, suggesting positive regulation of the ODC gene by ERK, PI3K, and PKA pathways. Interestingly, dexamethasone, a known inhibitor of cell proliferation, completely abrogated the response of RASMC to IL-4 and IL-13. Furthermore, the inhibition of ODC by these inhibitors led to the reduced production of polyamines and decreased DNA synthesis as monitored by [(3)H]thymidine incorporation. Our data indicate that upregulation of ODC by IL-4 and IL-13 might play an important role in the pathophysiology of vascular disorders characterized by excessive smooth muscle growth.     

Role of p38alpha kinase in activation of premature senescence program in transformed mouse fibroblasts

We investigated the role of p38alpha stress-kinase in regulation of premature senescence program, stimulated by histone deacetylase inhibitor--sodium butyrate (NaB)--after application to rodent transformed cell lines. Investigation was performed on the E1A + cHa-ras transformants selected from mice embryonic fibroblasts null at the p38alpha kinase gene or null fibroblasts at the PPM1D gene, which encoded phosphatase Wip1. Absence of Wip1 led to constitutive activation of p38alpha kinase. It was revealed that after NaB treatment both cell lines completely stopped proliferation due to irreversible cell cycle arrest in G1/S phase. In both cell lines sodium butyrate induced sustained block of prolifaration due to irreversible cell cycle arrest in G1/S phase. Following sodium butyrate treatment cells expressed marker of senescence--beta-galactosidase activity (SA-beta-Gal). Long-term (during several days) NaB treatment of cells led to partial restoration of actin cytoskeleton, focal adhesion contacts and heterochromatin focus formation (SAHF) in the nucleus of senescent cells. Obtained data allow us to suppose that irreversible process of cellular senescence activated by sodium butyrate can occur in the absence of functionally active p38 kinase by means of other ways of cell cycle suppression.     

Role of ornithine decarboxylase in the regulation of cell growth by IL-1 and tumor necrosis factor

Ornithine decarboxylase (ODC) is a rate-limiting enzyme in polyamine synthesis, and polyamines are required for cell growth. As an approach to clarifying the mechanism of action IL-1, the effects of IL-1 on ODC activity were examined in various cell lines whose proliferation was either suppressed or enhanced by IL-1. The proliferation of all cell types used in these experiments was markedly suppressed by a specific ODC inhibitor, alpha-difluoromethyl ornithine (DFMO), substantiating the crucial role of ODC activity for cell proliferation. ODC activity also was considerably suppressed by IL-1 in those cells on which IL-1 exerts an antiproliferative effect, such as a human melanoma cell line (A375) and malignant human mammary cell lines (MCF-7 and T-47D). On the other hand, ODC activity was stimulated in cells that are stimulated to proliferate in response to IL-1, such as a mouse helper T cell line (D10.G4.1), a NK cell-like cell line (YT), and a human glioblastoma cell line (U373 MG). The effect of IL-1 on ODC activity preceded and directly correlated in a dose-dependent manner with its effect on DNA synthesis. Furthermore, putrescine, a product of the ODC reaction and a precursor of polyamines, was able to overcome most, but not all, the antiproliferative action of IL-1 in A375 melanoma cells, which were the most sensitive to suppression by IL-1. However, putrescine did not reverse the cytostatic effect of IL-1 on MCF-7 and T-47D cell lines. In contrast, putrescine, like IL-1, exhibited some co-mitogenic activity on D10.G4.1 cells. Because the biological activities of TNF and IL-1 show considerable overlap, the effect of TNF on ODC activity also was examined. TNF had an antiproliferative effect on A375 cells and stimulated the proliferation of U373 MG cells. The ODC activity in A375 cells was suppressed by TNF, and the ODC activity in U373 MG cells was stimulated by TNF. Putrescine also partially overcame the inhibitory effect of TNF. These results suggest that the regulation of ODC activity may be a key component in the antiproliferative and proliferative action of IL-1 and TNF in some tumor cell types.     

Polyamines are essential for cell transformation by pp60v-src: delineation of molecular events relevant for the transformed phenotype

Ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis, becomes upregulated during cell proliferation and transformation. Here we show that intact ODC activity is needed for the acquisition of a transformed phenotype in rat 2R cells infected with a temperature-sensitive mutant of Rous sarcoma virus. Addition of the ODC inhibitor alpha-difluoromethyl ornithine (DFMO) to the cells (in polyamine-free medium) before shift to permissive temperature prevented the depolymerization of filamentous actin and morphological transformation. Polyamine supplementation restored the transforming potential of pp60v-src. DFMO did not interfere with the expression of pp60v-src or its in vitro tyrosine kinase activity. The tyrosine phosphorylation of most cellular proteins, including ras GAP, did not either display clear temperature- or DFMO-sensitive changes. A marked increase was, however, observed in the tyrosine phosphorylation of phosphatidylinositol 3-kinase and proteins of 33 and 36 kD upon the temperature shift, and these hyperphosphorylations were partially inhibited by DFMO. A DFMO-sensitive increase was also found in the total phosphorylation of calpactins I and II. The well-documented association of GAP with the phosphotyrosine-containing proteins p190 and p62 did not correlate with transformation, but a novel 42-kD tyrosine phosphorylated protein was complexed with GAP in a polyamine- and transformation-dependent manner. Further, tyrosine phosphorylated proteins of 130, 80/85, and 36 kD were found to coimmunoprecipitate with pp60v-src in a transformation-related manner. Altogether, this model offers a tool for sorting out the protein phosphorylations and associations critical for the transformed phenotype triggered by pp60v- src, and implicates a pivotal role for polyamines in cell transformation.