Interaction of putrescine with nuclear oligopeptides in the enterocyte-like Caco-2 cells.
Intestinal cells are able both to synthesize and take up putrescine, the main compound of the metabolic polyamine pathway. Polyamine binding to nuclear macromolecules is thougth to modulate DNA synthesis and transcription. Our aim was to study the fate of putrescine when taken up from the medium in the enterocyte-like Caco-2 cells and to analyze its binding to nuclear proteins. After having incubated the cells with 14C-putrescine (0.8 microM), during cell replication and differentiation, the nuclei were separated by sequential centrifugations in a sucrose gradient. About 20% of the putrescine taken up by Caco-2 cells resulted in the nuclei in both proliferating and differentiated cells. The binding of polyamines to nuclear proteins was studied on nuclear extracts, separated by both alkaline polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) and gel permeation chromatography (GPC). No radioactivity was found in nuclear protein extracts when using the SDS-PAGE method. Conversely, in replicating cells, GPC showed that the greatest amount of radioactivity was present in the nuclear peaks corresponding to oligopeptides with a molecular weight of 4,800-8,000 daltons. High-performance liquid chromatography analysis showed the presence of putrescine and spermidine in the 8, 000-dalton protein peak, whereas in the 4,800-dalton peak spermine was found in addition to putrescine. The radioactive count in the HPLC separated polyamines showed that a small percentage of the radioactivity present in the 8,000 and 4,800-dalton GPC peaks was linked to spermine and spermidine, suggesting an interconversion of the supplemented putrescine. Conversely, in differentiated cells, the nuclear oligopeptides did not reveal any radioactivity or any polyamines, suggesting that the binding of polyamines to nuclear oligopeptides is exclusively concerned with replicating cells.
Pignata
S
S
Divisione di Oncologia Medica B, Istituto Nazionale Tumori, Università 'Federico II', Napoli, Italia.
Di Luccia
A
A
Lamanda
R
R
Menchise
A
A
D'Agostino
L
L
D001665:Binding Sites; D018938:Caco-2 Cells / Q000378:metabolism* / Q000648:ultrastructure; D002455:Cell Division / Q000502:physiology; D002467:Cell Nucleus / Q000378:metabolism*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism*; D011700:Putrescine / Q000378:metabolism*
D009687:Nuclear Proteins; D009842:Oligopeptides; D004247:DNA; D011700:Putrescine