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Pulsed electromagnetic field (PEMF) stimulation promotes the healing of fractures in humans, though its effect is little known. The processes of tissue repair include protein synthesis and cell differentiation. The polyamines (PA) are compounds playing a relevant role in both protein synthesis processes and cell differentiation through c-myc and c-fos gene activation. Since several studies have demonstrated that PEMF acts on embryonic bone cells, human osteoblast-like cells and osteosarcoma TE-85 cell line, in this study we analyzed the effect on cell PAs, proliferation, and c-myc and c-fos gene expression of MG-63 human osteoblast-like cell cultures exposed to a clinically useful PEMF. The cells were grown in medium with 0.5 or 10% fetal calf serum (FCS). c-myc and c-fos gene expressions were determined by RT-PCR. Putrescine (PUT), spermidine (SPD), or spermine (SPM) levels were evaluated by HPLC. [(3)H]-thymidine was added to cultures for DNA analysis. The PEMF increased [(3)H]-thymidine incorporation (P < or = .01), while PUT decreased after treatment (P < or = .01); SPM and SPD were not significantly affected. c-myc was activated after 1 h and downregulated thereafter, while c-fos mRNA levels increased after 0.5 h and then decreased. PUT, SPD, SPM trends, and [(3)H]-thymidine incorporation were significantly related to PEMF treatment. These results indicate that exposure to PEMF exerts biological effects on the intracellular PUT of MG-63 cells and DNA synthesis, influencing the genes encoding c-myc and c-fos gene expression. These observations provide evidence that in vitro PEMF affects the mechanisms involved in cell proliferation and differentiation.

Background: The polyamines spermidine, spermine, and putrescine are known to be deeply linked with growth processes, gene expression, and extracellular matrix synthesis. Their cellular content depends primarily on the activity of the enzyme ornithine decarboxylase. High levels of ornithine decarboxylase and polyamines have been found in proliferative, inflammatory, and neoplastic pathologies of the oral cavity and in gingival fluid. Difluoromethylornithine (DFMO) selectively inhibits ornithine decarboxylase, thus depleting polyamine content and preventing cell proliferation and synthesis activity. The aim of this study was to investigate whether DFMO treatment could modify the genes involved in cell proliferation and extracellular matrix turnover., Methods: Fibroblasts derived from non-inflamed gingiva were maintained in Dulbecco's modified Eagle's medium (DMEM) plus alpha-difluoromethylornithine for 4 days. At 0, 24, 48, 72, and 96 hours cell number was assessed, polyamine levels were quantified with high performance liquid chromatography (HPLC) method, and transforming growth factor-beta1 (TGF-beta1), c-myc, matrix metalloproteinases (MMP)-1 and 2, collagen type I (COL-I) and tissue inhibitor of matrix metalloproteinases (TIMP)-1 were evaluated by reverse transcription polymerase chain reaction (RT-PCR)., Results: Fibroblasts treated with DFMO significantly decreased cell proliferation, ornithine decarboxylase activity, and putrescine levels at all treatment times, spermidine after 72 and 96 hours, and spermine after 96 hours of culture. Total polyamines decreased (P < or =0.01) at 96 hours after DFMO treatment, while c-myc, TGF-beta1, MMP-1 and 2, COL-I mRNA significantly increased. Conversely, TIMP-1 did not show any significant change. The polyamines trend was not correlated to c-myc, TGF-beta1, MMP-1 and -2, and TIMP-1 mRNA levels. Transforming growth factor-beta1 and c-myc mRNA expression were related and correlated to MMP-1 and 2, COL-I and TIMP-1 mRNA trend after DFMO treatment., Conclusions: Our data show that as the polyamine content decreases, TGF-beta1, c-myc, MMP-1 and -2, and COL-I mRNA levels increase, therefore a negative regulatory role of the polyamines on the mRNA expression could be suggested.

Polyamines (PA) and retinoic acid affect mammalian cell growth, differentiation and apoptosis. Retinoic acid induces granulocytic differentiation of mieloid cell lines and, during this process, is responsible for the expression of CD11b, a surface antigen. In this study we investigate the effects of retinoic acid on HL-60 cells, monitoring ornithine decarboxylase (ODC) activity (enzyme rate of PA), putrescine (PUT), spermidine (SPD), spermine (SPM) levels, CD11b myeloid surface marker differentiation, cell cycle, and apoptosis. ODC activity and PUT levels are correlated with mieloid cell differentiation induced by retinoic acid treatment. Only the ODC/PUT ratio is connected with retinoic acid treated HL-60 cells. Treated cultures show a decrease of proliferation and a cell block in the G0/G1 phase, with consequent diminished S phase. The G0/G1 and S phases are significantly related to ODC activity and to PUT and SPD behavior, whereas in differentiating condition only the decrease of PUT is related to the S phase. CD11b expression, stimulated by retinoic acid treatment, is associated with the SPM trend. Total PA behavior agrees with apoptotic cell increase after 96 h of stimulation. Our data show that retinoic acid treatment modifies ODC activity and the turnover of PA. PUT, SPD and SPM, therefore, have a different role, and may be involved in the differentiative/apoptotic program of retinoic acid treated HL-60 cells.

Serum and erythrocyte levels of the polyamines spermine, spermidine and putrescine, as well as ornithine decarboxylase in erythrocytes, were studied in patients with different neoplasms (breast, lung and colon cancer) and in those with a nonmalignant proliferative disease (familial polyposis). The blood levels of polyamines and the spermine/putrescine ratio were significantly higher in all tumors and in nonmalignant colon polyposis. In erythrocyte ornithine decarboxylase activity, spermine and spermidine levels, as well as spermidine/putrescine and spermine/putrescine ratios showed a significant decrease after surgery and chemotherapy. Our data suggest that high levels of blood polyamines and erythrocyte ornithine decarboxylase activity are related to cell proliferation and cancer treatment, but that levels of polyamines in serum and erythrocytes are still significantly high after cancer treatment and are similar to those in polyposis disease. Polyamines are related to nuclear activity during differentiation; therefore, the altered turnover of polyamines could be a sign of abnormal nuclear function. Since polyamines stimulate protooncogene expression, their high levels could be considered an important cofactor in malignant cell transformation.

Background: Polyamines are involved in normal and pathological cell proliferation and differentiation. Like acid radicals, they play an important role in remodelling the extracellular matrix and are considered "uremic toxins". Proteins and glycosaminoglycans are essential components of the extracellular matrix, and contribute to normal mature organ functions. The aim of this study was to analyse the effects of free polyamines, dialysate fluid components and dialysis fluid on protein and extracellular glycosaminoglycan synthesis in VERO cell cultures., Methods: The dialysate fluid components were separated with a Sephadex G15 column and the cultures were analysed after incorporation of 3H-leucine and 3H-glucosamine. Cultures were run at pH 7.0 and pH 7.4. The glycosaminoglycan classes were separated with a DEAE column, and polyamines were determined by high-performance liquid chromatography. Proteins and single glycosaminoglycan classes were quantified by a scintillator. DNA gel electrophoresis was done to detect chromatin fragmentation., Results: Dialysate contained putrescine, spermidine and spermine, chromatography showing four peaks; only peaks I and II indicated polyamines at respectively Da 5000 and 1500. Polyamines are therefore linked to different carriers. There was an increase of protein and glycosaminoglycan synthesis with dialysis fluid and polyamines, but inhibition with peak II or dialysate. DNA gel electrophoresis showed no chromatin fragmentation. Findings at pH 7.0 and 7.4 were similar., Conclusions: It would appear that in uremic patients polyamines are conjugated to protein carriers of different molecular weights with different biological actions. As polyamines and dialysis fluid affect changes in extracellular matrix, they could be related to physiological organ functions. However, these in vitro data must be considered with the appropriate limitations when we try to extrapolate them to the in vivo situation.

Lung branching morphogenesis is a result of epithelial-mesenchymal interactions, which are in turn dependent on extracellular matrix composition and cytokine regulation. Polyamines have recently been demonstrated as able to modify chick embryo skin differentiation. In this work we have examined the effects of putrescine and spermidine during chick embryo lung morphogenesis in organotypic cultures by morphological, histochemical and biochemical examination. To verify the role of polyamines, we used specific inhibitors, such as bis-cyclohexylammonium sulphate and alfa-difluoromethylornithine, and transforming growth factor beta1, an ornithine decarboxylase and polyamine stimulator. Our data show that lung morphogenesis is significantly altered following the induced mesenchymal glycosaminoglycan changes. The increase of mesenchymal glycosaminoglycans is correlated with a stimulation of lung development in the presence of polyamines, and with its inhibition when transforming growth factor beta1 is added to the culture medium. The morphometric data show a uniform increase of both the mesenchyme and epithelial branching with spermidine and putrescine stimulus, whereas the mesenchymal substance alone is significantly increased in apical-median lung sections with transforming growth factor beta1 and transforming growth factor beta1 + spermidine lung cultures. Transforming growth factor beta1 and transforming growth factor beta1 + spermidine confirm the blocking of epithelial branching formations and fibroblast activation, and show that polyamines are unable to prevent the blocking of epithelial cells due to the inhibitory effect of transforming growth factor beta1.

In this study we have examined the relationship between growth factor-induced proliferation and ODC/polyamine levels. TGF beta promotes cell growth and enhances [3H]-thymidine incorporation in chick embryo fibroblasts maintained in a serum-depleted medium. The action on DNA synthesis declines in the second day of treatment. IL-1 does not affect proliferation or [3H]-thymidine incorporation either when it is added alone or in combination with TGF beta. The response of the cells to TGF beta is associated with a significant stimulation of ODC activity and Put, Spd levels together with an enhancement of the Spd/polyamines ratio. IL-1, which does not act on cell proliferation, fails to activate ODC and to increase polyamine levels, thus indicating that the ODC/polyamine system is most likely to be an important link in the chain of events that leads to growth factor-induced proliferation.

The dialysate fluid of uremic patients exhibits, in vitro, an inhibitory effect on cell growth, owing to urea, guanidino compounds, and substances named middle molecules. The polyamines are compounds which exhibit high levels in biological fluids during either normal development or disease such as psoriasis, uremia, and tumors. Dialysate and middle molecules show toxicity and degeneration of the organotype cultures, whereas the free polyamines and nonrecirculated dialysate do not have any toxic effect. The aim of this study is to analyze the effects of polyamines, nonrecirculated dialysate, and middle molecules of uremic patients in periodic hemodialysis on cultured VERO (fibroblast-like cells) growth. These cells show an inhibition of growth in middle molecules or 2 x 10(-4) M putrescine and a stimulation with nonrecirculated dialysate and 2 x 10(-8) M putrescine. The effect is different because the cultures with middle molecules begin growth again after 24 hr, whereas in the presence of 2 x 10(-4) M putrescine no further growth is observed. Cells maintained in middle molecules + 2 x 10(-8) M putrescine show an irreversible degeneration, attesting a toxic effect due to the low molarities of putrescine. The electron microscopy shows alteration of cytoplasmic, mitochondrial, and nuclear membranes, but no chromatin fragmentation with either middle molecules or 2 x 10(-4) M putrescine: this suggests that the cells do not die of apoptosis. In conclusion, during uremia the polyamines could cause toxic effects, even at low concentrations, on cells stressed by other toxic stimuli.

The aliphatic polyamines, putrescine, spermine and spermidine belong to a category of molecules implicated in DNA replication. Their synthesis is strongly activated during the G1 period and they have been implicated in the regulation of cell proliferation and differentiation. Terminal transferase is a DNA polymerase present in pre-T and pre-B cells and its expression can be modulated by phorbol ester treatment. In this study we have monitored the relationship of intracellular polyamine levels with terminal deoxynucleotidyl transferase down-regulation induced by 12-O-tetradecanoyl phorbol myristate 13-acetate treatment in the human pre-B KM-3 cell line. Phorbol myristate acetate can cause an increase, at 4 and 8 hours of differentiation, of intracellular levels of putrescine as well as a decrease in terminal deoxynucleotidyl transferase synthesis showing the probable involvement that polyamines have in the differentiation process.

We studied the ornithine decarboxylase (ODC) activity and polyamines (PAs) content in the red cells lysate before and after haemodialysis of 15 uremic patients and 15 healthy subjects. ODC activity is significantly increased after haemodialysis but the PAs concentrations (particularly spermine and spermidine) do not increase. This could be because of a minimal loss of PAs surplus production. On the contrary the increase of ODC activity could be explained in several ways, e.g. by stimuli during haemodialysis (hypoxia of the first hour of treatment, decrease of plasmatic osmolarity and biological stimulation of the blood during its passage through the filter).

We have isolated peak II, which is the peak of the middle molecules containing polyamines, from dialysate of uremic patients in recirculating dialysis. We investigated the effect of total dialysate, chromatographic peaks, I, II, III, IV and commercial polyamines on polymorphonuclear leukocyte chemotaxis and superoxide production (i.e. phagocytic activity) in healthy and uremic patients. Polymorphonuclear chemotaxis was inhibited by total dialysate and peak II but not by polyamines; polyamines, total dialysate and peak II had no activity on polymorphonuclear phagocytosis.

The aim of our study is to evaluate the eventual activity of the total dialysate of two uremic nephrectomized patients in recirculating dialysis and the chromatographic peak of the dialysate fractionated by Sephadex column G 15 on PMN chemotaxis. Only the total dialysate and the chromatographic peak B showed inhibition of chemotaxis. On the contrary the commercial polyamines in the same concentration range, and the other chromatographic peaks, containing polyamines too, did not revealed inhibition. Our data show, therefore, that the chemotaxis inhibition could be due to the middle-molecules present in the peak B, rather than the polyamines itself. Polyamines were determined by dansylation method, separated by thin layer chromatography and quantified by spectrofluorimeter. Chemotaxis was evaluated using the modified Boyden chamber.

We studied possible effects of uremic toxins and polyamines (PAs) on organ cultures of chick embryo. We added on culture media the lyophilized of total dialysate and its chromatographic peak II obtained with chromatography with Sephadex G 15. The total dialysate and peak II showed toxicity with degeneration of the culture, whereas the free PAs, we added, did not effects.