Activation of nuclear factor κb by polyamines in breast cancer cells

Polyamines - putrescine, spermidine, and spermine - are involved in the growth of breast cancer cells. A possible target of polyamine action is at the site of interaction of transcription factors with their response elements. NF-κB is a member of the rel family of transcription factors that regulate transcription of genes in the proliferative/anti-apoptotic pathways. We performed electrophoretic mobility shift assays to study the role of polyamines in NF-κB binding to NF-κB response elements (NREs), the consensus sequence of which is GGGGAATTCCCC. Using cellular extract from MCF- 7 breast cancer cells, we found very little binding of NF-κB to NRE in the absence of polyamines. Addition of 1 mM spermidine or spermine caused a 4- and 6-fold increase in NF-κB-NRE binding, respectively. Putrescine induced a 2-fold increase in the binding at 2 mM concentration. Using antibody supershift assays, we identified the p50 subunit of NF-κB to be a major component in NF-κB-NRE complex formation in the presence of polyamines. However, the decreased intensity of the band corresponding to NF-κB-NRE complex in the presence of anti-p65, c-rel, relB and p52 antibodies suggested the participation of these subunits also. Spermine also stimulated NF-κB-NRE binding using cellular extracts from other breast cancer cell lines and a normal breast epithelial cell line. A differential effect of spermine analogues on NF-κB-NRE binding was observed, with spermine exerting the maximal effect. CD spectra of NRE containing oligonucleotides was asymmetric and distinct from that of a typical B-DNA CD spectrum. A concentration- dependent increase in T(m) of the duplex NRE was seen in the presence of polyamines. In transient transfection experiments using an NF-κB driven secreted alkaline phosphatase (SEAP) reporter, spermine induced NF-κB activity by ~2-fold as compared to controls. Spermine induced activation of NF-κB was also confirmed using an NF-κB-EGFP (enhanced green fluorescent protein) vector in transient transfections in which expression of the green fluorescent protein was visualized by fluorescence microscopy. These data show a gene regulatory function of polyamines involving enhanced binding of NF-κB to NRE and a possible mechanism for the action of polyamines in breast cancer cell proliferation.