Depletion of polyamines and increase of transforming growth factor-beta1, c-myc, collagen-type I, matrix metalloproteinase-1, and metalloproteinase-2 mRNA in primary human gingival fibroblasts.
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.
Stabellini
Giordano
G
Department of Human Morphology-Interdisciplinary Laboratories of Advanced Technologies, Segrate, University of Milan, Milan, Italy. giordano.stabellini@unimi.it
Moscheni
Claudia
C
Gagliano
Nicoletta
N
Dellavia
Claudia
C
Calastrini
Carla
C
Ferioli
Maria Elena
ME
Gioia
Magda
M
D000328:Adult; D002452:Cell Count; D049109:Cell Proliferation; D002478:Cells, Cultured; D024042:Collagen Type I / Q000032:analysis*; D000518:Eflornithine / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005109:Extracellular Matrix / Q000187:drug effects; D005260:Female; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism*; D005881:Gingiva / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D020781:Matrix Metalloproteinase 1 / Q000032:analysis*; D020778:Matrix Metalloproteinase 2 / Q000032:analysis*; D009955:Ornithine Decarboxylase / Q000032:analysis; D065108:Ornithine Decarboxylase Inhibitors; D011073:Polyamines / Q000037:antagonists & inhibitors*; D016271:Proto-Oncogene Proteins c-myc / Q000032:analysis*; D011700:Putrescine / Q000037:antagonists & inhibitors; D012333:RNA, Messenger / Q000032:analysis; D013095:Spermidine / Q000037:antagonists & inhibitors; D013096:Spermine / Q000037:antagonists & inhibitors; D013997:Time Factors; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000032:analysis; D016212:Transforming Growth Factor beta / Q000032:analysis*
D024042:Collagen Type I; D004791:Enzyme Inhibitors; D065108:Ornithine Decarboxylase Inhibitors; D011073:Polyamines; D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger; D019715:Tissue Inhibitor of Metalloproteinase-1; D016212:Transforming Growth Factor beta; D013096:Spermine; D020778:Matrix Metalloproteinase 2; D020781:Matrix Metalloproteinase 1; D009955:Ornithine Decarboxylase; D013095:Spermidine; D011700:Putrescine; D000518:Eflornithine